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Effect of Alpha7 Nicotinic Receptor Activation and Over-Expression in Brain and in PC12 Cells

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PAGE 1

EFFECT OF ALPHA7 NICOTINI C RECEPTOR ACTIVATION AND OVEREXPRESSION IN BRAIN AND IN PC12 CELLS By KE REN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by KE REN

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This document is dedicated to my parents: Xilin Ren and Aijuan Wang and my husband Jiang Liu.

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ACKNOWLEDGMENTS I would like to express my sincere appreciation and grateful thanks to Dr. Jeffrey A. Hughes for guiding me through the four years of Ph.D. study, giving me many excellent suggestions and supporting me in every respect. I am also extremely grateful for Dr. Edwin Meyer for providing a great environment in his laboratory and mentorship. He opened the door for me to explore the opportunities in research areas of nicotinic receptors and gene delivery. I would like to thank the members of my supervisory committee, Dr. Sean Sullivan and Dr. Sihong Song, for their valuable and kind advice throughout my doctoral research. I take this opportunity to express my gratitude to Dr. Mike King, Aaron Hirko, Craig Meyers, Jeffrey Thinshmidt and Dr. Yan Gong for all their help in conducting studies and in understanding them. I also would like to extend my thanks to all the graduate students, exchange students, and post-doctoral fellows in the Department of Pharmaceutics who were my colleagues and friends for their support. Finally, I thank my husband Jiang, my parents and my sister for supporting me in many, many ways. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.......................................................................................................................xi CHAPTERS 1 INTRODUCTION........................................................................................................1 Alzheimers Disease.....................................................................................................1 7 Nicotinic Receptors.................................................................................................5 7 Nicotinic Receptors: Effects On Cell Viability in vitro..........................................7 7 Nnicotinic Receptors: Neuroprotection Properties in vivo......................................9 3-benzylideneand 3-cinnamylidene-anabaseine Compounds....................................9 7 Nicotinic Receptors and Memory Related Behaviors...........................................11 Models Of 7 Nicotinic Receptor Dysfunction: 7 Receptor KO Mice And Septohippocampal Lesions....................................................................................12 7 Nicotinic Receptor And Beta Amyloid.................................................................15 Memory Related Behavioral Tests.............................................................................16 rAAV Mediated Gene Transfer Into Brain.................................................................17 Specific Aims..............................................................................................................20 2 MATERIALS AND METHODS...............................................................................21 Construction of rAAV Plasmids.................................................................................21 Subcloning..................................................................................................................22 Plasmid Preparations..................................................................................................24 Packaging rAAV Vectors And Titration....................................................................26 Cell Transfections.......................................................................................................31 Stereotaxic Surgeries..................................................................................................32 High Affinity [ 3 H] MLA Binding Assay...................................................................32 Immunohistrochemistry..............................................................................................34 FluoroJade Staining....................................................................................................35 Western-blot...............................................................................................................35 Morris Water Task......................................................................................................36 v

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Electrophysiological Recordings................................................................................37 Fimbria Fornix Lesions And 4OH-GTS-21 Injections...............................................38 Differentiation Of PC12 Cells....................................................................................39 Protein Kinase C (PKC) Assay...................................................................................40 Tail DNA Extraction And Genetype..........................................................................41 Statistical Analyses.....................................................................................................42 3 MECHANISMS UNDERLYING ALPHA7 NICOTINIC RECEPTOR NEUROPROTECTION IN PC12 CELLS.................................................................43 Introduction.................................................................................................................43 Results.........................................................................................................................45 Discussion...................................................................................................................54 4 NEUROPRECTIVE AND ANTI-AMYLOIDOGENE EFFECTS OF THE ALPHA7 PARTIAL AGONIST 4OH-GTS-21 IN FIMBRIA FORNIX LESIONED MICE OF DIFFERENT GENOTYPES.................................................61 Introduction.................................................................................................................61 Results.........................................................................................................................66 Discussion...................................................................................................................73 5 RAAV MEDIATED GENE TRANSFER IN VITRO AND IN VIVO........................77 Introduction.................................................................................................................77 Results.........................................................................................................................80 Discussion...................................................................................................................95 6 CONCLUSIONS AND FUTURE STUDIES...........................................................101 Conclusions...............................................................................................................101 Future Studies...........................................................................................................106 LIST OF REFERENCES.................................................................................................107 BIOGRAPHICAL SKETCH...........................................................................................116 vi

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LIST OF TABLES Table page 1-1. Mean SD Pharmacokinetic parameters for GTS-21 after oral adminstration of 25, 75 and 150 mg of GTS-21 for 5 Days................................................................11 1-2. Mean SD Pharmacokinetic parameters for 4OH-GTS-21 after oral adminstration of 25, 75 and 150 mg of 4OH-GTS-21 for 5 Days...........................11 3-1. Effects of BAPTA and GTS-21 on 7 receptor binding density in PC12 cells........48 5-1. Scatchard analyses of MLA Kd and Bmax in mouse with or without 7 knockout mice gene transfer....................................................................................................89 vii

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LIST OF FIGURES Figure page 1-1. Chemical structures of GTS-21 and 4OH-GTS-21....................................................10 2-1. The 7 genotype of mice............................................................................................42 3-1. GTS-21 induced protection of PC12 cells during trophic factor deprivation.............46 3-2. Effects of intracellular calcium chelation on GTS-21 induced protection of PC12 cells during trophic factor deprivation.....................................................................47 3-3. Effects of intracellular calcium chelation on PKC activation by GTS-21 in PC12 cells...........................................................................................................................48 3-4. Effects of 4OH-GTS-21 on PKC isozyme translocation............................................49 3-5. Effects of calcium channel antagonists on GTS-21 induced cytoprotection in NGF-deprived PC12 cells........................................................................................50 3-6. Effects of GTS-21 on the phosphorylation of several MAP kinases in PC12 cells...51 3-7. MLA blocks the ERK1/2 phosphorylation triggered by GTS-21...............................52 3-8. GTS-21 induced cytoprotection is dependent on ERK phosphorylation and PKC activation..................................................................................................................53 3-9. Effects of 4OH-GTS-21 and various kinase inhibotor on PC12 cells........................53 3-10. Effects of Abeta 25-35, 4OH-GTS-21 and various kinase inhibitor on SK N SH cells...........................................................................................................................54 3-11. The potential mechanism of 7 nicotinic receptors mediated cytoprotection..........60 4-1. Aspirative FFX-lesion of the septal hippocampal cholinergic pathway.....................67 4-2. Septal ChAT neuron staining in 9 month old mice....................................................68 4-3. Septal ChAT-staining perilarya in 9 month old PS1, APP/PS1 and wild type C57/B16/J mice two weeks after unilateral aspirative FFX-lesions........................69 4-4. Septal GABAergic neuron staining in 9 month old mice...........................................70 viii

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4-5. Septal GABAergic staining perilarya in 9 month old PS1, APP/PS1 and wild type C57/B16/J mice two weeks after unilateral aspirative FFX-lesions........................71 4-6. The thioflavine S and 6E10 staining in 9 month old APP/PS1 mice.........................72 4-7. APP/PS1 mice (9 months old; N=4-5/gp) had lower hippocampal amyloid density stained with 6E10 antibody after a combination of fimbrial lesion and 4OH-GTS-21 IP X2 for 2 wks than either treatment alone...............................................72 5-1. Schematic diagram of the expression cassettes..........................................................80 5-2. Transfection of GH4Cl cells with rat 7 nicotinic receptors:effect of Abeta 25-35 exposure on cell viability and receptor density........................................................81 5-3. Iodixanol gradient for the purification of rAAV and dot plots for titer.....................82 5-4. The dose response of rAAV2-rat 7 receptor and rAAV-GFP..................................83 5-5. 7 receptor expression was measured in different populations of transiently transfected, stably transfected, transduced, and normally expressing cells.............84 5-6. Effects of GTS-21 on the viability of PC12 cells and 7 transduced GH4C1 cells..85 5-7. Low vector dose: in vivo transduction with 7 vectors in hippocampus...................86 5-8. Effects of a higher vector dose on 7 nicotinic receptor exprrssion in hippocampus.............................................................................................................87 5-9. rAAV2-rat 7 vector gene transfer in 7 KO and 7 heterozygous (+/-) mice........88 5-10. mAB 306 immunohistochemical staining of rat, mice and 7 KO mice.................89 5-11. FITC-staining of knockout mouse septum after injection........................................90 5-12. rAAV8/2 transduction in rat brain............................................................................91 5-13. Electrophysiological responses in wild type mouse hippocampus following 7 gene delivery.............................................................................................................92 5-14. Electrophysiological responses in wild type dentate granule rat cells.....................93 5-15. Evoked 7 nicotinic receptor responses in 7 knockout mice transduced with rat 7 nicotinic receptors...............................................................................................93 5-16. Histology following electrophysiological recording: 2 month old mice that received gene transfer with rAAV8/2 -rat 7 + rAAV8/2-GFP...............................94 ix

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5-17. Electrophysiological recording of a CA1 pyramidal neuron in the hippocampus of a 3 week old 7 receptor-transduced mouse........................................................95 6-1. The potential pathway of 7 nicotinic receptors mediated neuroprotection............104 x

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF ALPHA7 NICOTINIC RECEPTOR ACTIVATION AND OVER-EXPRESSION IN BRAIN AND IN PC12 CELLS By Ke Ren December 2005 Chair: Jeffrey Hughes Major Department: Pharmaceutics Brain 7 nicotinic receptors are implicated in Alzheimers disease through their actions on memory related behaviors, binding to beta-amyloid. 7 nicotinic receptors are calcium permeant and provide neuroprotection against many insults. The mechanism of 7 nicotinic receptors, intracellular calcium ions and downstream calcium channels in the protection were investigated. The 7 agonist GTS-21 prevented PC12 cell death induced by NGF + serum deprivation over a 3 day interval. This effect was blocked by the intracellular calcium chelator BAPTA-AM in a manner that did not appear to involve changes in receptor-density. BAPTA-AM blocked GTS-21 induced PKC activation, a necessary process for protection. The IP3 calcium channel blocker xestospongin C and the phospholipase C inhibitor U-93122 blocked protection and ryanodine receptor blocker ryanodine partially attenuated protection, but the L-type channel antagonist nifedipine had no effect. ERK1/2 but not JNK and p38 were activated by GTS-21, and the ERK phosphorylation inhibitors PD98059 and U0126 blocked protection. In vivo, xi

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GTS-21 appeared to prevent cholinergic cell loss in septum following fimbria/fornix lesions in PS1 mice, but no protection in APP/PS1 and wild type mice. Another approach to investigate the effects of 7 receptors on Alzheimers disease-related processes involves elevating the levels of this receptor in the plasma membrane. We therefore took a somatic gene transfer approach to modify the expression of 7 nicotinic receptors in the brain. Recombinant adeno-associated virus (rAAV) was used. Vector containing rat 7 nicotinic receptors driven by the hybrid chicken beta actin/cytomegalovirus promoter was injected stereotaxically into wild type and 7 knockout mice hippocampus. Within three weeks, receptors were detected through binding assay and western-blots. The functional receptors were observed through electrophysiology response. rAAV 8/2-rat 7 nicotinic receptors gene transfer into wild type mice may improve acquisition performance in a dose-dependend manner. In conclusion 1) IP3 calcium channel and ryanodine receptor are important for 7 nicotinic receptors mediated neuroprotection, but the L-type channel is not necessary for long term protection; 2) GTS-21 could prevent cholinergic cell loss in septum following fimbria/fornix lesions in PS1 mice; 3) rAAV-rat 7 nicotinic receptor could express functional receptors in rat, wild type and 7 knockout mice. xii

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CHAPTER 1 INTRODUCTION Alzheimers Disease Alzheimers disease is an age related neuropathological disorder characterized by the presence of large numbers of neuritic plaques, neurofibrillary tangles and the progressive atrophy and loss of neurons. Aging is a major factor for this and many other neurodegenerative disorders. The United Nations population projections estimate that 50% of people older than 85 years of age are afflicted with AD (Allsop et al., 2001). To date, the cause and progression of AD have not been fully elucidated. The cholinergic hypotheses was initially presented over 20 years age and suggests that a dysfunction of acetylcholine containing neurons in the brain contributes to the cognitive decline in AD (Terry and Buccafusco, 2003). Aging and AD have also been associated with degeneration of neurons of the ascending cholinergic pathway. The cell bodies of these neurons are located in the basal forebrain, and their axons innervate the amygdala, hippocampus and neocortex. Brain neuronal cholinergic transmission has not only been suggested to be affected in these pathologies, but also in human cognitive disorders associated with the normal process of aging. Among these are the age-related deficits in shortand long term memory, impairment of attention, and delayed reaction time. Among several other observations relevant to the cholinergic systems, nicotinic acetylcholine receptors (AchR) binding sites have been reduced in number in the cerebral cortex of patients with AD (London et al., 1989). The only FDA recognized therapies for 1

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2 the disease are acetylcholinesterase (AChE) inhibitors such as aricept, tacrine, donepezil, rivastigmine and galantamine, and the NMDA antagonist memantine. The rationale for using AChE inhibitors is to enhance cholinergic transmission in the brain by decreasing the metabolism of the neurotrasmitter acetylcholine. Basal forebrain cholinergic neurons that are important for memory related behavior are decreased in early AD, and this decrease becomes more evident as the disease progresses (Nordberg and Svensson, 1998). These basal forebrain neurons have their perikarya located primarily in the septum or the nucleus basalis. Septal cholinergic neurons send axons and nerve terminals to the hippocampus, providing the only cholinergic innervation of that region. Nucleus basalis cholinergic neurons send their projections throughout the cerebral cortex. This cerebral cortical innervation from nucleus basalis accounts for over 90% of the cholinergic activity in rodents; in humans, it accounts for about 60% of the cholinergic activity with the remainder due to intrinsic neurons in the neocortex. All of the AChE inhibitor drugs for AD are most effective in the early stages of the disease, presumably because there are more cholinergic neurons remaining for the drugs to act upon. Since AChE inhibitors protect acetycholine in the synapse, they increase all types of cholinergic transmission throughout the brain, including multiple muscarinic and nicotinic receptors. Which of these receptor subtypes collectively or individually are important for the beneficial effects of AChE inhibitors is an active area of study. Several studies have shown that the expression and distribution of AChE in AD patients has changed (Kasa et al., 1997; Talesa, 2001). AChE activity is lost in specific regions of the AD brain, such as the neocortex and hippocampus, which are important for memory-related behaviors. The relative proportions of different forms of AChE also

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3 changed in this disease, suggesting changes in function. AChE has been found to co-localize with Abeta peptides, which appear to be involved in the disease as discussed below. The rationale for using an NMDA antagonist in AD is to block the effects of elevated levels of glutamate that may lead to neuronal dysfunction. Glutamate receptors are already greatly reduced in this disease, and this is thought to be due to their excitotoxic properties. According to this model, excessive glutamate receptor activity in AD, especially that triggered by NMDA receptors, results in the excitotoxic loss of glutamate receptor expressing neurons. Memantine would prevent this toxicity, preventing further glutamate receptor loss and loss of neurons expressing these neurons. Memantine is the only one of the FDA approved drugs found to have efficacy in later stages of AD. The amyloid cascade hypothesis is another of the central trends in AD research community (Allsop et al., 2001). According to this hypothesis, neurodegeneration in AD begins with abnormal processing of the amyloid precursor protein (APP) and results in the production and aggregation of Abeta peptide-forming oligomers and amyloid fibrils that form the senile plaques. Less clear is whether these Abeta species trigger the formation of neurofibrillary tangles or eventually causes neuronal cell atrophy or death. The amyloid cascade hypothesis has received considerable support from genetic studies into the early-onset familial forms of AD, for which mutations in APP or presenilin genes causing AD lead to an increase in Abeta production (Allsop et al., 2001). Human APP mutations can cause amyloid plaques in transgenic in an age-related manner. These plaques are typically not seen until about 10 months of age, and become more

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4 pronounced thereafter, especially in hippocampus and neocortex. Interestingly, transgenic mice with high levels of plaques exhibit only very modest decreases in memory related behaviors and few neuronal deficits. This suggests that AD may require more than just amyloid plaques. Presenilins I and II were originally discovered and cloned through their involvement in genetic forms of AD. Subsequently, it was found that both of these gene products were involved in APP gamma secretase activity that leads to amyloidogenic peptides such as Abeta 1-42 (W., 2001). While once thought to be gamma secretase itself, presenilin was later shown to be a component of a multiprotein complex that has this enzymatic activity. Overexpression of mutant, AD-causing, human presenilin I in transgenic mice does not cause amyloid plaque formation. However, it does increase the levels of APP peptides and also causes modest behavioral changes. Further, double transgenic mice expressing mutant human APP and mutant presenilin I express amyloid plaques much more quickly than do single transgenics making mutant human APP alone. Presenilins also appear to have a variety of additional cellular effects that may not be due to gamma secretase activity. These effects include the activation of multiple protein kinase pathways such as AKT and ERK1/2 that are involved in cell viability. It is possible that increased presenilin expression has complex effects on cell function, some reducing it (e.g., by increasing amyloid expression) and other improving it (increased kinase activity). Important questions that remain are 1) how do mutations in presenilin or Abeta cause any of the symptoms seen in A? And 2) how do these mutations affect our ability to treat the disease pharmacologically? Recent studies suggest that both presenilin and

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5 Abeta peptides affect pathways involved in cholinergic transmission, particularly involving nicotinic receptor mediated transmission. A consistent and significant loss of some subtypes of nAChRs has observed in cortical autopsy brain tissue from AD patients compared to aged-matched healthy subjects. The neocortical nAChRs deficits significantly correlate with cognitive impairment in AD patients (Warpman and Nordberg, 1995). It was suggested that the nAChRs deficits in AD brains probably represent an early phenomenon in the course of this disease (Nordberg, 1994). Of the many nicotinic receptor subtypes found in the brain, the most attention has been focused on 7 nicotinic receptors because of their interactions with Abeta peptides, their importantance for memory related behaviors, their ability to be neuroprotective, and their loss of expression in AD. 7 Nicotinic Receptors AChRs exist as a variety of subtypes. Each of AChR subunits encoded by these genes is thought to have an extensive N-terminal domain positioned extracellularly, four transmembrane domains (MI-M4) that anchor these integral membrane proteins, and an extracellular C-terminus. nAChRs are ligand-gated ion channels that can be divided into two groups: muscle receptors and neuronal receptors. My research focuses on neuronal receptors because of their behavioral importance. Neuronal nicotinic receptors form a family of receptors that are differentially expressed in many regions of the CNS. nAChRs have many functions, such as cognition, sleeping, arousal, feeding behavior, neuronal development, and cell survival, but it remains to be determined which receptor subtypes are involved with which functions. Dysfunctions of nAChRs have been linked to a number of human diseases such as AD, Parkinsons disease and schizophrenia.

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6 Nicotinic receptors consist of pentameric subunits that are either homemeric or heteromeric. Heteromeric brain nicotinic receptors consist generally of multiple (mostly 2-4) and (2-4) subunits. The 42 combination is the most common combination of and subunits in the brain. 7 receptors, in contrast, are homomeric receptors. Mapping of AchR distribution at low resolution based on radioligand binding autoradiography is consistent with expression of some form of AchR in most of these major or minor cholonergic targets. Anatomic analyses also suggest that 42 and 7 (labeled using 3 H-labeled nicotinic agonists and 125 I-labeled Btx, respectively) have largely unique but sometimes overlapping distribution. Btx sites predominate in the septum, hippocampus, neocortex and hypothalamus, with lower density in the striatum. It has been suggested that 7 nicotinic receptors may play an important role in cognitive processes. 7 receptors have a number of unique physiological and pharmacological properties among nicotinic receptors, including high permeability to calcium, rapid and reversible desensitization, and pronounced inward rectification. 7 receptors have a high affinity for the antagonists alpha-bungarotoxin and methyllycaconitine (MLA). The 7-receptor channel is highly permeable to calcium ions. Calcium acts as a second messenger inside the neuron and not only stimulates neurotransmitter release, but also stimulates signal transduction events through stimulation of protein kinases, calcineurins, nitric oxide synthetases and other enzymes (Kem, 2000). 7 nicotinic receptors are located both preand post-synaptically (Albuquerque et al., 1997). Their presynaptic location and ability to promote the conductance of calcium ions are consistent with an important role in the depolarization-dependent and calcium

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7 ion-dependent release of neurotransmitters (Albuquerque et al., 1997). These receptors have a very important postsynaptic location on gamma-aminobutyric acid-ergic (GABAergic) inhibitory tone (Adler et al., 1998). One of the difficulties in studying 7 receptor function is rapid desensitization (Seguela et al., 1993). Selective agonists may act through receptor activation or subsequent desensitization. A standard approach to distinguish these possibilities is to use antagonists such as the non-selective mecamylamine, or the more selective MLA and -bungarotoxin. If receptor-activation is required, these antagonists block the action; alternatively, their effects mimic antagonism if through desensitization. Nicotinic receptor antagonists that block 7 receptors interfere with memory related behaviors and block their neuroprotective properties. This indicates that agonist-activity is needed for these receptor functions. 7 Nicotinic Receptors: Effects On Cell Viability in vitro The pattern of activation of 7 nicotinic receptors by agonists is concentration dependent. Low concentrations of agonists induce a long term, low level and almost steady state increase in net cationic influx (Papke et al., 2000). High concentrations of agonists, in contrast, cause a much larger spike in conductance that is also much shorter lived because of desensitization of the receptors. Only the longer term activation of the receptor by low agonist concentrations is associated with the neuroprotective effects of 7 receptor activation. To date, 7 nicotinic receptor-mediated neuroprotection has been demonstrated in the following models: NGFand serum-withdrawal from differentiated PC12 cells (Li et al., 1999b); glutamate induced excitotoxicity in primary rat brain neuron cultures

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8 (Shimohama et al., 1998); Abeta amyloid exposure (Meyer et al., 1998a); ethanol toxicity in primary neuronal cultures and PC12 cell (Li et al., 1999a). In all these models, pretreatment with, and continued exposure to, agonist is necessary for neuroprotection. An overview of this subject recently concluded that nicotinic receptor mediated neuroprotection was attributable to 7 receptors (O'Neill et al., 2002). For 7 receptors, the density of receptors and the binding affinity of the agonist could affect the extent of neuroprotection (Jonnala and Buccafusco, 2001). Even indirect activation of 7 nicotinic receptors by blocking cholinesterase activity is sufficient to be protective. The mechanisms of 7 nicotinic receptor mediated neuroprotection in vitro have been studied with respect to several intracellular processes. Protein kinase C (PKC) activation, mitochondria membrane stabilization, reduced release of mitochondria cytochrome oxidase and increased BCL-2 expression have all been found to be involved in this neuroprotection (Li et al., 1999a; Dajas-Bailador et al., 2002a; Dajas-Bailador et al., 2002c). 7 receptor mediated neuroprotection has also been found to require the Akt antiapoptic pathway (Shimohama and Kihara, 2001). However, many steps in the neuroprotection pathway are not known, starting with the role of calcium ions themselves, various calcium channels, other intermediary kinase systems, and even the level of 7-receptor density. It is also not known whether the pathways involved in neuroprotection depend on the model system. While low levels of agonist activation are neuroprotective, high concentrations of agonists may be toxic to cells if applied quickly in vitro (Li et al., 1999b). There is no evidence that this occurs in vivo, even at high doses of agonist. This may be due to the relatively slow delivery of drug through the circulation vs. rapid receptor desensitization.

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9 However, it is a potential concern that must be addressed when considering any new approach that involves increased 7 receptor activity, since different approaches may affect the receptors and their interactions with cellular function differently. 7 Nnicotinic Receptors: Neuroprotection Properties in vivo The potential neuroprotective effects of 7 nicotinic receptors in vivo are not well understood. Pretreatment with the selective 7 receptor partial agonist GTS-21 was found to reduce the penumbral damage caused by focal ischemia in the rat cerebral cortex (Shimohama et al., 1998). Dr. Meyers laboratory suggested that GTS-21 could protect rat septal cholinergic neurons from partial fimbria-fornix lesions, though these lesions were minimal (fewer than 15% of the cholinergic neurons were lost even in controls). In a longer-term study of trans-synaptic cell loss, GTS-21 and nicotine were both found to protect cerebral cortical neurons from trans-synaptic loss or atrophy following lesioning of ascending nucleus baslis innervation (Meyer et al., 1998b). This result was also seen in other nicotinic agonists (Socci and Arendash, 1996). However, no study has yet evaluated the neuroprotective effects of nicotinic receptor agonists in genetic models of AD such as the PS1 or APP/PS1 mouse. 3-benzylideneand 3-cinnamylidene-anabaseine Compounds Dr. Meyers laboratory was the first to characterize families of 3-benzylideneand 3-cinnamylidene anabaseine compounds as potential 7 receptor agonists for the treatment of brain disorders. While the 3-benzylidene anabaseine compounds (e.g., GTS-21) are typically selective partial agonists, the cinnamylidene anabaseines (e.g., 3-CA) are full or at least more efficacious agonists. Both types of compounds have been used to demonstrate neuroprotection through 7 nicotinic receptors. GTS-21 (also known as

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10 DMXB) is 2,4dimethoxybenzylidene anabaseine. It has been widely studied in preclinical animal models and evaluated in a Phase I trial for Alzheimers disease (Kitagawa et al., 2003). This drug has much lower efficacy for primate or human 7 nicotinic receptors than for rodent receptors. GTS-21 rapidly enters the brain after oral administration and improves memory related performance in nonhuman primates. The pharmacokinetics properties of GTS-21 and 4OH-GTS-21 have been studied to a limited extent. Less than 1% of orally administered GTS-21 is recovered in the urine. Three-hydroxy metabolites are generated in vitro by rat hepatic microsomal O-dealkylation of the two methoxy substituents on the benzylidene ring. These metabolites are also found in plasma of rats after oral administration, but at significantly lower concentrations relative to the parent compound. However, the principal metabolite, 3-(4-hydroxy, 2-methoxy-benzylidene) anabaseine (4OH-GTS-21), displays a higher efficacy than GTS-21 on human as well as rat 7 nicotinic receptors (Figure 1-1). 4OH-GTS-21 has a similar level of efficacy for both rat and human 7 nicotinic receptors. The hydroxy metabolites are all more polar than GTS-21, derived from their octanol/water partition coefficients, and they enter the brain less readily than GTS-21 (Kem, 2000). However, they are behaviorally active when administered IP, indicating the ability to enter the brain at therapeutic levels. Figure 1-1. Chemical structures of GTS-21 and 4OH-GTS-21.

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11 GTS-21 and 4OH-GTS-21 have been evaluated clinically. The pharmacokinetics of GTS-21 and 4OH-GTS-21 are shown in Table 1-1 and Table 1-2 (Kitagawa et al., 2003). Table 1-1. Mean SD Pharmacokinetic parameters for GTS-21 after oral adminstration of 25, 75 and 150 mg of GTS-21 for 5 Days Parameter 25 mg 75 mg 150 mg Cmax (ng/ml) 3.49 + 0.09 12.8 + 8.23 47.8 + 28.4 Tmax (h) 1.19 + 0.6 1.09 + 0.28 1.02 + 0.38 AUC 4.57 + 2.62 32.7 + 16.4 85.1 + 32.9 t1/2 (h) 0.76 + 0.38 0.93 + 0.32 0.93 + 0.19 Table 1-1. Pharmacokinetic parameters of GTS-21. Table 1-2. Mean SD Pharmacokinetic parameters for 4OH-GTS-21 after oral adminstration of 25, 75 and 150 mg of 4OH-GTS-21 for 5 Days Parameter 25 mg 75 mg 150 mg Cmax (ng/ml) 2.71 + 1.12 9.87 + 5.19 32.9 + 18.3 Tmax (h) 1.3 + 0.58 1.23 + 0.51 1.11 + 0.48 AUC 7.49 + 4.73 26.2 + 5.72 71.3 + 24.9 t1/2 (h) 0.81 + 0.30 1.18 + 0.41 1.15 + 0.37 Table 1-2. Pharmacokinetic parameters of 4OH-GTS-21. 7 Nicotinic Receptors and Memory Related Behaviors 7 selective agonist such as GTS-21 improve several types of memory related behaviors, including spatial memory in the Morris water task (Meyer et al., 1998a), passive and active avoidance behaviors in rats (Meyer et al., 1997), radial arm maze in aged rats (Arendash et al., 1995), delayed eye blink behavior in rabbits (Woodruff-Pak, 2003), hippocampal gating behavior in mice (Simosky et al., 2001), and delayed pair

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12 matching and word recall (Briggs et al., 1997). In contrast, the selective antagonist MLA reduces performance in radial arm maze when injected directly into the hippocampus at low concentrations (Bettany and Levin, 2001). When injected peripherally, MLA also interferes with several memory related behaviors, though this effect is more obvious in ages, cognitively unimpaired rats than young adults rats or aged, impaired rats (personal communication; Dr. Greg Rose, Memory Pharmaceuticals). The less selective antagonist mecamylamine also interferes with memory related behaviors including the Morris water task in FFX-lesioned rats when administered peripherally (Brown et al., 2001). As with many drugs that improve memory related behaviors in rodents, 7 nicotinic receptor agonists increase performance in lesioned or hypofunctional animals. Few reports show improvement in intact animals. However, GTS-21 was found to improve performance in delayed word match behavior in a small cohort of healthy adult humans (Kitagawa et al., 2003). This observation suggests that these receptors may be effective for some behaviors even in normal individuals. Models Of 7 Nicotinic Receptor Dysfunction: 7 Receptor KO Mice And Septohippocampal Lesions 7 receptor knockout mice grow to normal size and show no obvious physical or neurological deficits. These animals do exhibit expected deficits in MLA-sensitive electrophysiological responses in hippocampus. This permits the knockout mouse to be used for 7 receptor gene delivery studies to demonstrate restoration of function. Behavioral responses in 7 knockout mice also appear normal, except for anxiety-related behaviors in the open-field test. These observations may reflect developmental compensatory differences between normal and knockout mice, since, as noted above,

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13 modulating 7 receptors function in wild type adults causes behavioral changes. Alternatively, it may be that some 7 receptors are modulatory but not necessary for neuronal function and thus, when present, are able to affect behaviors that do not depend on their presence. The 7 knockout mice were generated by introducing a 7 kb deletion into embryonic stem cells followed by transmission to the germline. The mutation deletes the last three exons (8-10) of the 7 locus. These exons encode the second transmembrane domain, forming the putative ion channel, and the third and fourth transmembrane domains and cytoplasmic loop (Orr-Urtreger et al., 1997). Neuropathological examination of the brains of knockout mice revealed normal structure and cell layering, including normal neocortical barrel fields. The histochemical assessments of the hippocampus are also normal. In contrast to the 7 knockout mice, mice expressing the 7 Leu250 point mutation show extensive apoptosis throughout the neocortex and most of them die within 24 hours of birth. This is probably due to the relative inability of this mutant to undergo desensitization. As previously suggested, the fast desensitization kinetics of wild type 7 nicotinic receptors, which limits large acetylcholine-elicited Ca 2+ influxes, might protect against extensive neurodegeneration. The lack of additional neuroprotection in this 7 mutation relative to wild type mice suggests that a threshold Ca 2+ level has to be reached for toxicity. Another model of 7-receptor dysfunction involves lesions of the septohippocampal pathway, which reduce the cholinergic input to the hippocampus. The hippocampus has been organized to play a fundamental role in some forms of learning and memory, as shown since the early 1950s. The unusual shape of the human

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14 hippocampus resembles that of a sea house, which is what led to its most common name (in Greek, hippo means horse and kampos means sea monster) (Johnston et al., 2003). It has been shown that damage to certain subregions of hippocampus can result in an enduring amnesic syndrome. Affected patients are incapable of recording everyday events and facts. The hippocampus has been implicated in a number of neurological and psychiatric disorders, including epilepsy, AD and schizophrenia. Because of the important role of the hippocampus in learning and memory, it is not surprising that the hippocampus is functionally disconnected from the rest of the brain in this disease during later stages. Lesioning the septal input to the hippocampus by ablating the fimbria-fornix pathway has recently been shown by Dr. Meyer and his colleagues to reduce 7 nicotinic receptor function as measured electrophysiologically in hippocampus in a manner that is overcome by chronic, twice-per-day injections of GTS-21 (Thinschmidt et al., 2005). This reduction in function is paralleled by reductions in septal cholinergic density, but not changes in receptor binding density in the hippocampus. The effects of these fimbrial-fornix lesions on septal GABA neuronal survival are not known, but may be less than seen in the cholinergic system because only a small fraction of GABA neurons in septum project to the hippocampus. Selective lesions of either the cholinergic neurons or the GABA neurons in the septum are not enough to cause these receptor-function deficits. Instead, both types of neurons must be lesioned together such as by aspiration. Similarly, both types of neuron deficits are necessary for memory related behavioral impairments, with selective lesions in either type of nerve not causing much impairment (Yoder and Pang, 2005).

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15 7 Nicotinic Receptor And Beta Amyloid There are data showing that 1) 7 nicotinic receptors are blocked by low concentrations of soluble Abeta 1-42 and other APP-derived peptides in a manner that is likely to be competitive (Liu et al., 2001); 2) chronic oral treatment with nicotine reduces amyloid plaque accumulation in transgenic Swedish mutation overexpressing mice (Nordberg et al., 2002); and 3) nicotine increases -secretase products (APP soluble) (Efthimiopoulos et al., 1996) while decreasing gamma-secretase products (Utsuki et al., 2002). GTS-21 was also found to increase soluble APP in isolated rat brain nerve terminals, which would be expected to reduce amyloidogenic A expression. These data suggested that a complex reciprocal interaction exists between nicotinic receptors and A 1-42. Nicotinic receptor activation can be attenuated by the APP peptide but the levels of A 1-42 and A -containing plaques in turn can be reduced by nicotinic receptors activation. In my project, a selective 7 nicotinic receptor agonist, 4OH-GTS-21, was evaluated for neuroprotection in fimbria fornix (FFX)-lesioned mice of several genotypes: wild type, PSI only transgenic, and APP/PS1 double transgenic mice. The aspirative lesion used for the study affected both cholinergic and GABAergic neurons projecting to the hippocampus. This was the first time that an 7 nicotinic receptor agonist was tested for neuroprotection in this species, as well as in any combination model system consisting of both genetic changes and lesions. I also investigated whether 4OH-GTS-21 could decrease the amyloid load in these mice. While it had been shown that nicotinic administered orally over a longer time interval was effective in reducing amyloid plaque load, it was not clear which receptor subtype or subtypes were mediated this effect. My study was designed to test the role of

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16 7 nicotinic receptors directly over a shorter interval (2 weeks), thereby testing the hypothesis that 7 nicotinic receptors could be targets for these components of this disease. Memory Related Behavioral Tests Morris water maze (MWM) is primarily designed to measure spatial learning and recall, and has become quite useful for evaluating the effects of aging, experimental lesions and drug effects (Jonasson et al., 2004). MWM is a challenging task that employs a variety of sophisticated processes. These processes include the acquisition and spatial localization of relevant visual cues that are subsequently processed, consolidated, retained and retrieved. Several observations regarding the utility of the MWM are notable: 1) The functional integrity of forebrain cholinergic systems that are critical for efficient performance of that MWM appears to be consistently disrupted in patients who suffer AD (Perry et al., 1999). 2) Neocortical and hippocampal projections from the nucleus basalis magnocellularis and septum are reproducibly devastated in AD. Accordingly, reductions in central cholinergic activity in rodents resulting from brain lesions and age reproducibly impair spatial learning in the MWM (McNamara and Skelton, 1993). 3) Other data implicated the hippocampus as an essential structure for place learning which is commonly atrophic in patients with AD (Terry and Katzman, 1983; Mann, 1991). 4) Anticholinergic agents that are used routinely to impair performance in the MWM also impair memory in humans and worsen the dementia in those with AD (Ebert and Kirch, 1998). 5) Spatial orientation, navigation, learning and recall are quite commonly disrupted in AD patients (Morris, 2003). However, the transgenic mice carrying human APP Swedish mutation and that develop amyloid

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17 plaques, with or without mutant PSI co-expression, do not show large deficits in spatial learning and memory in MWM (Holcomb et al., 1998). The MWM procedure has several advantages as a means of assessing cognitive function in rodents when compared to other methods: 1) It requires no pre-training period and can be accomplished in a short period of time with a relatively large number of animals. 2) Through the use of training as well as probes, learning as well as retrieval processes can be analyzed and compared among groups. 3) Through the use of video tracking devices and the measures of swim speeds, behaviors can be delineated and motoric or motivational deficits can be identified. 4) By changing the platform location, both learning and re-learning experiments can be accomplished. This method is quite useful in drug development studies for screening compounds for potential cognitive enhancing effects. rAAV Mediated Gene Transfer Into Brain One of the approaches to increase receptor function without increasing agonist concentration, which as noted above is not always desirable in the case of 7 nicotinic receptors, is to increase receptor density by gene delivery. And one of the keys to successful gene delivery is the selection of the appropriate therapeutic genes and their molecular vehicle. A recombinant adeno-associated virus (rAAV) vector offers the advantage of the ability to infect non-dividing cells, affording a non-pathogenic, long-term transgene expression without a substantial inflammatory response when combined with appropriate promoters. One goal of my project was to develop 7 nicotinic receptors gene delivery systems for brain. This was hypothesized to provide safe, effective and long-term therapy to counteract 7 deficits.

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18 rAAV is one of the most promising viral vectors for gene therapy due to its wide tropism and persistent transgene expression in vivo, and several clinical trials using rAAV to treat genetic disease have been carried out. rAAV is also a safe and effective means for studying transgene function in the brain. AAV is a parvovirus with a diameter around 25 nm (Berns and Giraud, 1996). It is a single-strand 4.7 kb DNA (ssDNA) genome packaged into three viral capsid proteins: VP1 (87 kDa), VP2 (73 kDa) and VP3 (62 kDa). They form the 60-subunit viral particle in a ratio of 1:1:20 (Muzyczka et al., 1984). The relatively high density of AAV particles allows us to be easily separated by CsCl density centrifugation from adenovirus helper particles whose density is approximately 1.35g/cm 3 The linear ssDNA contains two open reading frames flanked by two inverted terminal repeats of 145 nucleotides each (Sperinde and Nugent, 1998). The upstream open reading frame (ORF) encodes four overlapping nonstructural replication proteins (Rep), Rep78, Rep68, Rep52, and Rep40 (McLaughlin et al., 1998). The downstream ORF codes for the capsid proteins. After entering host cells, the ssDNA genome of AAV is converted to the double-strand template in cell nuclei and finally integrated into the host genome at chromosome 19q13.3 in humans (Leopold et al., 1998). This chromosome-selective integration is lost in rAAV vectors in which the Rep coding sequences are removed. AAV has demonstrated a broad tropism of infection, including lung, brain, eye, liver, muscle, hematopoietic progenitors, joint synovium and endothelial cells (Miao et al., 2000). The brain is a particularly good target for rAAV vector approaches because of the topic maps of neuroanatomical organization. rAAV vectors retain much of this tropism, with significant variations seen among serotypes depending on the tissue. These

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19 serotypes differ in the composition of their capsid protein coat. rAAV serotype 2 (rAAV2) has been the most widely studied and best described among these. It binds to both heparan sulfate proteoglycans and fibroblast growth factor receptors as an essential step for cellular entry (Summerford et al., 1999). Recently, rAAV5 and rAAV8 have also been investigated and found to bind to different cellular receptors. This probably accounts for their different biodistribution properties when injected in brain and other tissues. The mechanisms whereby other AAV serotypes enter host cells are actively being studied. Early studies showed that rAAV2 mediated transgene expression occurred in hippocampal interneurons, but not as strongly in hippocampal CA1 pyramidal neurons or dentate gyrus granul neurons. In my project, the rAAV8 vectors were also studied in hippocampus. rAAV mediated transgene expression has persisted for up to 2 years after in vivo injection in rodent brains. rAAV-mediated expression is dose-dependent, but an accurate dose dependence of expression has not yet been established. It also depends on the kind of promoters chosen. Recently, we used the chick beta actin promoter/CMV enhancer, which provided very high, stable, and long term transgene expression. Staining for astrocytes or microglia following AAV-mediated transduction revealed no sign of gliosis or infiltration relative to vehicle injection. Given the lack of inflammatory response or pathogenicity, the reasonably high transduction efficiencies and the long persistence of transgene expression, rAAV has become a leading candidate vector for somatic gene transfer into brain in vivo. AD is characterized by cholinergic deficits and degeneration of basal forebrain cholinergic neurons. Cholinergic degeneration correlates with loss of memory function.

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20 Therefore, rAAV mediated 7 nicotinic receptor gene transfer may become a potential target for the treatment of AD. Specific Aims The goal of this project is to investigate the mechanism of 7 nicotinic receptors mediated neuroprotection and the potential of these receptors as targets for developing drugs for AD. I propose to evaluate 7 nicotinic receptors using selective 7 nicotinic receptors agonist such as GTS-21 and 4OH-GTS-21. Studies are focusing on three models: 1) cell cultures expressing 7 nicotinic receptors; 2) wild type and 7 knockout mice; 3) wild type, APP/PS1 and PS1 mice that have undergone FFX axotomy of the septohippocampal pathway. The specific aims of these projects are as follows: Determine the roles of calcium, calcium channels, and several kinase-systems in 7 nicotinic receptor-mediated neuroprotection in vitro. 2. Test the hypothesis that the 7 nicotinic receptors agonist GTS-21 is efficacious in protecting septal cholinergic neurons from axotomy in wild type, PS1 overexpressing, and amyloid expressing mice. 3. Test the hypothesis that rAAV-rat 7 nicotinic receptor gene transfer increases receptor expression in vitro and in vivo without toxicity.

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CHAPTER 2 MATERIALS AND METHODS Construction of rAAV Plasmids Vector plasmids used for these studies are pUF12-rat 7 with WPRE, pUF12-GFP, pUF12-rat 7 without WPRE were constructed from pUF12, which contains the chick actin promoter with truncated cytomegalovirus (CMV) enhancer (CBA promoter). All plasmids had wild type AAV-2 terminal repeats (TR) and a poly (A) tail from bovine growth hormone. The mRNA stabilizer woodchuck posttranscriptional regulatory element (WPRE) was attached downstream to the 7 receptors in some rAAV2 vectors preparations. Rat 7 cDNA was subcloned into this plasmid using ClaI and verified using EcoRI and HindIII. GFP was subcloned into this plasmid using HindIII and verified with BamHI and BgIII. The DNA Core of the University of Florida analyzed both of the DNA sequences. The biological activities of the 7 nicotinic receptor-expression vectors were confirmed by western immunolabeling and binding assays following calcium phosphate transfection of rat pituitary tumor derived (GH4C1) cells. GH4C1 cells were incubated in 60 mm cell culture dishes in F-10 nutrient mixture (Ham) (Gibco BRL, Grand Island, NY) supplement with 10% fetal calf serum (FBS, Gibco, Invitrogen Corporation, CA) and 1% penicillin/streptomycin (Gibco, Invitrogen Corporation, CA) in a 5% CO 2 humidified atmosphere at 37 o C until 60% confluence. A mixture of 4 g of plasmid DNA, 12.4 l of 2 M CaCl 2 was prepared in a total volume of 100 l filtered water. This mixture was added dropwise into equal volumes of 2 X HBS (280 mM NaCl, 21

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22 10 mM KCl, 1.5 mM Na 2 HPO 4 12 mM dextrose, 50 mM HEPES, pH 7.1). There were small precipitates at the bottom of dishes after two hours of transfection. The medium was changed after 8 hours of transfection and the cultures were incubated for up to 72 hours. For control groups, GFP was monitored in cells after transfection. Rat 7 expression was detected by western blot and binding assays at day 3 post-transfection. Subcloning A backbone was prepared of rAAV2rat 7 vector. 20 g of pUF12 was digested with Hind III at 37 o C for 1 hour. This digested reaction solution contained 6 l Hind III, 6 l NEB buffer 2, 20 g DNA in 60l water. The digested reaction solution was mixed with 12 l 6 X DNA gel loading dye (30% glycerol, 6 mM EDTA, 0.06% bromophenol blue and 0.06% xylene cyanol FF) and loaded on the 1% agarose gel for a hour electrophorsis at 80 V. The larger size band was cut from the gel and extracted using an agarose gel extraction kit (Qiagen). The backbone was dephosphorylated using calf intestinal alkaline phosphatase (CIP, NEB). After dephosphorylation, the reaction was extracted with phenol-chloroform, followed by chloroform. The upper layer was collected. 2.5 volumes of 100% ethanol and one-tenth volume of 3 M NaAc pH 5.1 were added into the sample and mixed and precipitated at -80 o C for 2 hour. The sample was centrifuged at 5,000 g for 15 min. The supernatant was discarded. One ml of 75% ethanol was added to the pellet. The sample was centrifuged at 5,000 g for 15 min. The supernatant was removed and the pellet was air dried for 15 min and dissolved in 20 l filtered water. The second step was to add HindIII linker to rat 7 insert. Rat 7 insert was flanked by ClaI and blunt end ligated with T4 DNA polymerase. The blunt end reaction

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23 contained 10 l 10x Buffer T4 pol (NEB), 0.5 l 25 mM dNTP (NEB), 3 l T4 pol and 48 l rat 7 insert and 38.5 l water. The reaction was incubated at room temperature for 15 min. The whole reaction was mixed with 12 l of 6-x DNA gel loading dye and loaded on the 1% agarose gel for a 1-hour electrophoresis at 80 V. The rat 7 insert band was cut from the gel and extracted using an agarose gel extraction kit (Qiagen). The insert was extracted by phenol-chloroform, then chloroform. DNA was precipitated by ethanol. The linker ligation reaction solution contained 17.5 l rat 7 insert, 5 l 5x DNA dilution buffer, 2.5 l linkers (2.5 g), 25 l 2x ligation buffer and 2.5 l ligase (Boehringer). The reaction mixture was incubated at room temperature for 5 min. The linker reaction was mixed with 6 l of 6-x DNA gel loading dye and loaded on the 1% agarose gel for 1-hour electrophoresis at 80 V. At the same time rat 7 insert was loaded on the gel. The ligated linker shifted up a bit. The rat 7 insert linker band was cut from the gel and extracted using an agarose gel extraction kit (Qiagen). The fragment was extracted by phenol-chloroform, then chloroform. DNA was precipitated by ethanol. The pellet was dissolved in 10 l water. Gel electrophoresis was run to compare the relative concentrations of vector and insert. The third step was ligation. The ligation reaction contained approximately equimoloar concentration of backbone and insert, 2 l 10 x ligation buffer and 2 l T4 DNA ligase (NEB) and water. The total volume was 20 l which was added to a PCR tube. The ligation mixed was incubated in a PTC-200 DNA Engine Thermal Cycles (MJ Research) for overnight at 16 o C. The next day, 180 l water was added into the ligation reaction. The ligation product was extracted by phenol-chloroform, then chloroform,

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24 finally precipitated by ethanol. The pellet was dissolved in 10 l H 2 O. 2 l of ligation product was used for transformation. The next step was transformation. 2 l of ligation product were transformed into 100 l competent SURE cells (Stratagene, La Jolla, CA) using electroporation transformation methods. The cuvette was placed in the BioRad Gene Pulser II (Bio-Rad Laboratories). A single exponential decay pulse of 2.5 kV, 25 F and 200 were delivered. The whole reaction was added to 1 ml NZY (Fisher Scientific) without antibiotics and incubated in the shaker for 1 hour at 37 o C. 50 l, 100 l, 250 l or 600 l were added to 10 cm NZY/AMP (100 g/ml) dishes and incubated at 37 o C overnight. Next day, six single colonies were selected and grown overnight. Minipreps of 6 single colonies were performed using Miniprep kit (Qiagen). The colonies were selected using restriction digestion and gel electrophoreses. Plasmid Preparations The plasmid was grown overnight in 5 ml of NZY/Amp (100 g/ml) at 37C on a shaking platform by pick a single colony. Next day, the culture inoculated into 2 L NZY/Amp (100 g/ml) and incubated for 16-18 hour. The overnight culture was Spin down in 4 X 250 ml bottles at 5000 g for 15 min in a Sorvall RC-5B refrigerated superspeed centrifuge (DuPont Instruments). The pellets were resupsend in 20 ml of lysozyme buffer (25 ml Tris-HCl pH 8, 20 ml 0.5 M EDTA, 17.115 g of sucrose, dH 2 O for a total volume of 1 liter). 12mg/ml of lysozyme (Sigma) was added, mixed and put on the ice for 5 min. 48 ml of solution II (20 ml 10% SDS, 4ml 10N NaOH, water for a total volume of 200 ml) was added, mixed and stored the bottles at room temperature for 4 min. 36ml of 3M NaAc (pH4.6-5.2) and 0.2 ml chloroform were added and mixed and

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25 iced for 20 min. The mixtures were centrifuged at 5,000 g for 20 min. The clear supernatant was transferred into new 250 ml bottle through gauze. 33 ml of 40% PEG (Fishrs Scientific) was added, mixed and stored at o C overnight. The mixtures were centrifuge at 5, 000g for 10min. The supernatant was removed and dissolved pellet in 10 ml of dH 2 O. 10 ml of 5.5 M LiCl (Fisher Scientific) was added, mixed and iced for 10 min followed by centrifugation at 5,000 g for 10 min. 21 ml of supernatant were collected and transferred into 2 40 ml tubes. 6 ml isopropanol (Fisher Scientific) were added, mixed and iced for 20 min. The mixtures were centrifuged at 5,000 g for 10 min in small rotor. Each pellet was dissolved in 6.7 ml of TE buffer (10 mM Tris pH 8 and 1 mM EDTA). 8 g CsCl (Fisher Scientific) were added and mixed until dissolved. 0.44 ml of ethidium bromide (Fisher Scientific, 10 mg/ml) was added to each bottle. 8.9 ml of the solution were loaded into an Optiseal Beckman tube (Beckman Instruments). All tubes were loaded into a 70.1 Ti rotor and ultracentrifuged at 100,000 g in a Beckman L8-70M Ultracentrifuge for at least 19 hours at 20 o C. A hand held long wave UV 366 nm was used to estimate DNA density. There were two bands in the tube. The lower band was collected with 16 G needle (Becton Dickinson) and transferred to a 15 ml tube. The samples were extracted with equal volume of isoamyl alcohol (Fisher Scientific) until the sample was clear, then mixed and centrifuged at 3000 g for 2 min. 3 ml of the extracted sample were put into Corex tubes and 2.5 volume of H 2 O was added, followed by 2 combined volumes of ethanol. The mixture was iced for 30 min. The mixtures were centrifuged at 5,000 g for 15 min. The supernatant was removed and the pellet dissolved in 400 l of TE buffer in a 15 ml tube. It was washed with 400 l of TE, which was pooled into the same tube. The sample was extracted with an equal volume of

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26 phenol/chloroform (Amresco) until the interphase was clear, followed by chloroform extraction. The extracted sample (400 l each) was transferred into 1.5 ml microfuge tubes. 40 l of 3 M NaAc and 1 ml of ethanol were added, mixed and iced for 10 min, followed by microfuge for 5 min at max speed in a 5414 Centrifuge (Hamburg, German). The pellet was washed with 1 ml of 75 % ethanol, followed by 5 min at max speed. The supernatant was removed. The pellets were air dried for 15 min. Each pellet was dissolved in 200 l sterile TE buffer. The DNA concentration was detected at 260/280 nm in a Beckman DU 650 Spectrophotometer. The plasmid was confirmed by restriction digestion and gel electrophoresis. Packaging rAAV Vectors And Titration The method used to package constructs and purify the rAAV was described by or modified from Zolotukhin et al (Zolotukhin et al., 1999). HEK 293 cells at 70% confluence were transfected with two plasmids by the calcium phosphate method. One plasmid was pUF12-rat 7 or pUF12. The other was helper plasmid. The rAAV2 helper plasmid was pDG, which contained replication-deficient AAV genes for the rAAV protein coat and replication-defective adenovirus genes for helper function in packaging. The rAAV5/2 helper plasmid was pXYZ5. The rAAV8/2 helper plasmid was pXYZ8. These two plasmids were used in equal molar ratios for transfection. 10 cell culture dishes of 15 cm diameter were used. 1.25 ml of 2 M CaCl 2, 0.6 mg helper plasmid, 0.3 mg of pUF12-rat 7 or pUF12 and sterile water were mixed to the total volume of 10 ml for ten dishes. This mixture was added dropwise into equal volumes of 2x HBA while vortexing. This whole transfection mixture was added to 200 ml of warmed DMEM, which contain 10% FBS and 1% penicillin/streptomycin. 22 ml of this medium mixture

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27 were added to each dish of HEK 293 cells. After a 6-hour transfection, the medium was removed and replaced with fresh DMEM. Seventy-two hours after the transfection, cells were harvested using cell a cell scraper (Corning Incorporated) and centrifuged at 3000 g at 4 o C for 20 min. The cells were resuspended in 15 ml of lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.5). The suspension underwent 3 cycles of freeze thawing with dry ice/ethanol (10 min freeze, 15 min thaw, vortexing every 5 min). The samples were treated with benzonase (endonuclease, Sigma) to digest unpackaged DNA. 6 l of 4.82 M MgCl 2 and 750 units of benzonase were added to 15 ml lysate and incubated at 37 o C for 30 min. The lysate was centrifuged for 30 min at 3,000 g at 4 o C. 15 ml of supernatant were collected in a 39 ml Optiseal tube through 16G needle. A Pump Pro (Watson-Marlow, UK) was set up as follows. 200 l glass pipettes were used for intake and 100 l glass pipettes were used for output. Pump speed was set at 37 rpm counterclockwise. The tube was rinsed with 40 ml water and 15 ml 15% iodixanol (IOD). The output pipette was loaded into Optiseal tubes. Pumping was started with 15% IOD (1:47 min), 25% IOD (1:15 min), 40% IOD (1:47 min) and 60% IOD (1:50 min). (180 ml of 15% IOD contained 45 ml of OptiPrep (Axis-Shield Poc AS, Norway), 36 ml of 5 M NaCl, 36 ml of 5x TD (5x PBS, 5 mM MgCl 2, 12.5 mM KCl) and 63 ml water. 120 ml of 25% IOD contained 50 ml of OptiPrep, 24 ml of 5x TD, 46 ml of water and 300 l of 0.5 % phenol red solution (Sigma). 100 ml of 40% IOD solution contained 68 ml of OptiPrep, 20 ml of 5x TD and 12 ml of water; 100 ml of 60% IOD contained 100 ml of OptiPrep and 250 l of 0.5% phenol red solution). Tubes were sealed by heat and were untracentrifuged at 100,000 g in a Beckman L8-70M Ultracentrifuge for 2 hour at 18 o C. The rAAV band was located at the 1 st interface (between 60% and 40%) from the

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28 bottom of tube and up to 0.5 cm below the second interface; it was collected through a 16 G needle. Heparin columns were used to purify AAV stereotype 2. Q sepharose was used for AAV serotypes 5 and 8. The heparin column was made as follows: the bottom of the Bio-Rad Econo-pac disposable chromatography column was snapped off and 6 ml of well-mixed heparin immobilized on cross-linked 4% beaded agarose (Sigma) were added to the column. The heparin column was equilibrated with 15 ml of 1xTD (1x PBS, 1 mM MgCl 2 and 2.5 mM KCl). The rAAV2 lysate was added onto the column, and then the column was washed with 20 ml of 1x TD. The rAAV2 was eluted with 15 ml of 1x TD/1 M NaCl. The 15 ml of eluted solution was collected into a 50 ml conical tube. The Q sepharose column was made as follows: the bottom of the Bio-Rad Econo-pac disposable chromatography column was snapped off and 5 ml of well-mixed Q sepharose (Sigma) were added into the column. The Q sepharose column was equilibrated with 20 ml of solution A (20 mM Tris/15 mM NaCl, pH 8.5) and the column was washed with 20 ml solution B (20 mM Tris/1M NaCl, pH 8.5) and again with 30 ml solution A. rAAV5/2 or rAAV8/2 sample was diluted with two times solution A and loaded to the column. After loading the sample, 50 ml of solution A were added to the column. The sample was eluted with 20 ml of solution C (20 mM Tris/355 mM NaCl, pH 8.5) and collected into a 50 ml conical tube. The sample was added into the concentrator (Minipore: Biomax-100K NMWL membrane) and centrifuged at 1,000 g for 5 min. The lysate was concentrated to 1 ml and diluted with 9 ml of Ringers solution twice. The virus was concentrated to a final volume of 300 l. Virus was collected into siliconized tubes (Fisher) and stored at -20 o C.

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29 A dot-blot assay was used to determine the titer of rAAV virus based on total genomic particles. A 4 l aliquot of the virus was treated with DNAse I (Roche) mixture for 1 hour at 37 o C. This reaction contained 20 l of 10X DNAse buffer (50 mM Tris-HCl, pH 7.5 and 10 mM MgCl 2 ), 2 l of DNAse, 174 l of dH 2 O and 4 l virus. After 1 hour, 22 l Proteinase buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA and 10% sodium dodecyl sulfate) were added and incubated for 1 hour at 37 o C. An equal volume of phenol-chloroform was added to the sample, vortexed for 5 min and microfuged for 5 min at 14,000 g. This was followed by an extraction with an equal volume of chloroform and microfuging for 5 min at 14,000 g. The aqueous layer was transferred into a new microfuge tube to which was added 1/10 volume of 3 M NaAc (pH 5.2) and 2.5 volumes of ethanol. DNA was precipitated overnight at -80 o C. Next day; the sample was microfuged at 14,000 g for 20 min. The supernatant was discarded. 0.5 ml of 75% ethanol was added to the pellet and microfuged at 14,000 g for 10 min. The pellet was air dried for 15 min and dissolved in 40 l of water. The sample was quantified by a DNA slot blot assay using 1.7 kb EcoRI segment of pUF12 and a series of dilutions of pUF12 as standard curve. The dilution series of the pUF12 was started with 20-ng/l and continued for 12 tubes. Each tube received 50 l of water. 50 l of DNA (20ng/l) were pipetted from tube 1 to tube 2. Tube 2 was vortexed and 50 l of DNA removed from tube 2 to tube 3. This dilution procedure was continued for all 12 tubes. A second set of tubes labeled 1-12 were set up behind the DNA dilution set. 200 l of alkaline buffer (0.4 M NaOH, 10mM EDTA pH 8.0) were added to each tube. To each alkaline tube, 10 l of DNA dilution set was added to each tube. For each virus sample, 10 l of 1:10 dilution and 1:1 dilution each were added to 200 l of alkaline buffer tube. The next step

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30 was to set up the dot blotter (Bio-Rad). Two pieces of filter paper (Bio-Rad) were placed in water and then put on the dot blotter. Membranes (Bio-Rad, Zeta-Probe Blotting membranes) were put on the top of filter paper. The dot blotter was closed and 400 l of water was added to each well. Vacuum was used to remove excess water. The standards of all 12 tubes (in alkaline buffer) and samples were boiled for 10 minutes. The standard curve tubes in alkaline buffer and samples were loaded to each well. The samples were aspirated slowly with vacuum until all samples disappeared. The vacuum was disconnected. 400 l alkaline buffer were added to each well and allowed to stand for 5 min, followed by vacuuming remaining solution. The membrane was removed from the dot blotter and crosslinked by a UV crosslinker (UV Statalinker 1800, Strategene). The membrane was placed in a small Biometra bottle. Prehybridyzation buffer (7.5 ml, 7% SDS, 0.25 M NaHPO 4 pH 7.2, 1 mM EDTA pH 8.0) was added into the bottle and incubated at 65C for 1 hour before labeling probe was added. 6 l of biotinylated probe was diluted with 54 l of 10 mM EDTA and denatured at 90C for 10 min. The total 60 l of denatured probe were added to the hybridization buffer and quickly mixed and incubated overnight at 65 C in Biometra oven. A Brightstar Bio Detect kit (Ambion, TX) was used for detection. The membrane was placed into a staining container, then washed twice for 5 min in 1 X wash buffer (Ambion), followed by washing twice in blocking buffer (Ambion) for 5 min each. The membrane was then incubated for 30 min in blocking buffer. The membrane was incubated with 1 l strep-alkaline phosphatase (Strep-AP) mixed with 10 ml blocking buffer for 30 min, followed by a 15-min incubation in blocking buffer. The membrane was washed 3 times for 5 min each in 1 X wash buffer and incubated with 1 X assay buffer for 2 min. The membrane was

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31 incubated for 5 min in 5 ml of 1 X CDP-Star solution. The membrane was wrapped in a single layer of plastic wrap and exposed to film at room temperature in a dark room. The film exposure was for 15 mins. The blot was then scanned into computer and the intensity of the bands analyzed with NIH Scion image software (Scion Corporation, Maryland). The intensity of the bands for standard DNA was used to build a standard curve. The titers was calculated using the coefficients: 1 ng DNA = 4 x 10 11 particles/ml. Vector stocks were ranged from 10 12 -10 13 genomic particles per ml, except for that containing the WPRE sequence (genomic lengths over 5000 kb) was 10 7 genomic particles/ml. Cell Transfections GH4C1 cells were obtained from American Type Culture Collection (ATCC). The cells were grown in F-10 nutrient mixture containing 10% FBS and 1% penicillin/streptomycin at 37 o C in a 5% CO 2 and 90-92% humidity. The cells were split at a 1:3 ratio every 5 days, up to 10 passages. Cell confluence at the time of study was approximately 60%. The cell culture medium was removed. The cells were exposed to a 0.05% trypsin/0.53 mM EDTA solution for 5 min. After 5 min, the trypsin/EDTA was removed from the cells by transferring the cellular suspension to sterile conical tubes. These tubes were centrifuged at 3,000 g for 5 min. The supernatants were removed and the pellets were resuspended in 150 l fresh medium. Increasing concentrations of 1.25 x 10 9 /ml, 2.5 x 10 9 /ml, 5 x 10 9 /ml and 15 x 10 9 /ml genomic particles were added to the medium and incubated for 30 min at 37 o C. After incubation, the cells were plated at the 60 mm dishes and added 2 ml of fresh medium was added to the dishes. 3-5 days after transfection, the cells were harvested for binding assays.

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32 Stereotaxic Surgeries Male Sprague-Dawley rats (~250g, 2 months old) were obtained from the Harlan Sprague Dawley Farm (Indiana). 7 heterozygous mice were obtained from the Jackson laboratory. They were housed and bred the in animal facility at the Health Science Center of the University of Florida. Rats or mice were anesthetized with 4% isoflurane/ oxygen. rAAV2-rat 7, rAAV2-GFP, rAAV8-rat 7 and rAAV8-GFP vectors were injected bilaterally into hippocampus or septum through a 27-gauge cannula connected via 26 gauge I.D. polyethylene tubing to a 10 l syringe mounted to a CMA/100 microinjection pump. The pump delivered 2 l virus (10 10 genomic particles) at a rate of 0.15 l /min. The injection coordinates for the hippocampus were .6 mm bregma, 2.2 mm medial-lateral, and 2.8 mm dorsal-ventral. For the medial septum, they were 0.7 mm bregma, 0.2 mm medial-lateral and 7.0 mm dorsal-ventral. The cannula was removed slowly removed after the injection. The skin was sutured. All animal care and procedures were in accordance with institutional IACUC and NIH guidelines. Two weeks or 6 months after injections, the animals were euthanized and their brains evaluated for receptor binding, immunohistochemistry, electrophysiology, or western blot. High Affinity [ 3 H] MLA Binding Assay Brain tissues or cell culture samples were prepared for nicotine-displaceable, high-affinity [ 3 H] methyllycaconitine (MLA) binding assay as follows. Septum or hippocampus was rapidly dissected from animals euthanized while fewer than 4% isoflurane/oxygen anesthesia and suspended in ice-cold Krebs Ringer buffer (KRH; in mM 118 NaCl, 5 KCl, 10 glucose, 1 MgCl 2, 2.5 CaCl 2, 20 HEPES; pH 7.5). Ice-cold

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33 KRH was also used to wash, and then harvest culture cells. Brain tissues or cell culture samples were homogenized in ice-cold KRH buffer with a Polytron (setting 4 for 15 sec). After 2 1 ml washes with KRH at 20,000 g, the membranes were incubated in 0.5 ml Krebs Ringer with 2.3 nM [ 3 H] MLA (Tocris, Ellisville,MO), unless specified otherwise, for 60 min at 4 o C, plus or minus 5 mM nicotine. For determining the Kd and Bmax values, a range of total MLA was used from 0.5 nM to 50 nM for Scatchard analyses. Tissues were washed 3 times with 5 ml ice-cold KRH buffer by filtration through Whatman GF/C filters that were preincubated for 30 min with 0.5% polyethylenimine (Sigma). Liquid scintillation (EcoLite) counting of radioactivity was conducted in a Beckman LS1800. Nicotine-displaceable binding was calculated for each sample in triplicate in each experiment. Scatchard analyses are evaluated with the Statview program. ANOVA was performed for treatment effects. In Bradford protein assay, 100 leach of 0.1, 0.2, 0.4, 0.6 and 0.8 g/l of bovine serum albumin (BSA, Sigma) were used as standards to measure the protein concentration of the medium, brain samples and cell lysate samples. 50 l of each samples were added into 5ml tubes containing 200 l of 1 x KRH. Five ml of diluted (1:4 with water) BioRad protein assay dye reagent (Bio-Rad) was added to each tube, vortexed and sit for 30 min at room temperature. The O.D. of each tube was detected at 595 nm in a Beckman DU 650 Spectrophotometer (Beckman). A standard curve was prepared according to the concentrations and absorbance of the BSA standards at 595 nm and the protein concentration of each sample was determined using Prism software (GraphPad Software, Inc.CA).

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34 Immunohistrochemistry Animals anesthetized with 4% isoflurane/oxygen were perfused with 100 ml of cold phosphate-buffered saline (PBS), followed by 400 ml of cold 4% paraformaldehyde (Sigma) in PBS. The brain was removed and equilibrated in a cryoprotectant solution of 30% sucrose/PBS and stored at 4 o C. Coronal sections (50 m thick) were cut on a sliding microtome with freezing stage. Antigen detection was conducted on floating sections by incubation in a blocking solution (2% goat serum/ 0.3% Triton X-100/ PBS) for 30 min at room temperature, followed by primary antibody incubation overnight at 4 o C. Primary antibodies used were: anti-7-antibody mAb 306 (1:500, Sigma), 6E10 (1:1000, Signet, Dedham MA), glial fibrillary acidic protein (GFAP) (1:2000, Chemicon, Temecula, CA), NeuN (1:1000, Chemicon), anti-Chat (1:1000, Chemicon) and anti-parvalbumin (1:1000, Chemicon). To optimize 7 immunohistochemistry, 250 l of 50% ethanol were added to each section and incubated for 30 min before added anti-7 antibodies. The sections were washed in PBS 3 times for 5 min each and then incubated with biotinylated anti mouse IgG or anti rabbit IgG (1:1000, Dako, Carpinteria, CA) for 1 hour at room temperature. The sections were then washed with PBS three times for 5 min each and labeled with ExtrAvidin peroxidase (HRP) conjugate (1:1000, Sigma) for 30 min at room temperature. Development of color was conducted with a solution of 0.67 mg/ml diaminobenzidine (DAB, Sigma)/0.1 M sodium acetate/8 mM imidazole/2% nickel sulfate/0.003% H 2 O 2 The sections were mounted on Fisher Superfrost Plus slides and air dried for 30 min. They were passed through water, 70% ethanol, 95% ethanol and 100% ethanol, and then the slides were xylene dehydrated. The slides were coverslipped with Eukitt (Calibrated Instruments,NY).

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35 Some sections were used with fluorescent secondary antibody. After incubating with primary antibody, sections were washed in PBS 3 times and incubated with fluorescein (FITC)-conjugated anti-mouse (Jackson Immunoresearch, 1:1000) secondary antibody overnight at 4 o C. Sections were washed in PBS and then mounted on Fisher Superfrost Plus slides, air-dried and coverslipped with glycerol gelatin (Sigma). FluoroJade Staining FluoroJade staining is a method to stain degenerating neurons. Floating brain sections prepared as describe above were treated with 100% ethanol for 3 min, followed by 70% ethanol and dH 2 0 for 1 min each, 0.06% potassium permanganate for 15 min, and a wash with dH 2 0 for 1 min. The sections were treated in the dark with 0.001% Fluoro-Jade for 30 min and then mounted on slides, air-dried and coverslipped with glycerol gelatin. Western-blot Septum or hippocampus was rapidly dissected from animals euthanized under 4% isoflurane/oxygen anesthesia. Ice-cold sodium phosphate buffer (50 mM sodium phosphate, 50 mM NaCl, 2 mM EDTA, 2 mM EGTA, and 1 mM phenylmethysulfonyl fluoride, pH 7.4) was used to harvest cells. Fresh brain tissues or cell culture samples were homogenized with ice-cold 50 mM sodium phosphate buffer and centrifuged at 60,000 g for 60 min at 4 o C. The supernatant was discarded. The pellets were resuspended in an ice-cold buffer containing 2% Triton X-100 and protease inhibitor cocktail (Sigma). The suspension was mixed for 2 hour at 4 o C and then centrifuged at 100,000 g for 60 min at 4 o C. The supernatants were used for western blot analysis. Protein contents in the fraction were assayed with the Bio-Rad protein assay kit. 25 g each sample protein were mixed with 2x Laemmli sample buffer (Bio-Rad) and fresh 5%

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36 2-mercaptoethanol. Samples were boiled for 5 min. Each sample was separated on 10% sodium dodecyl sulfate-polyacrylamide (SDS) gels by electrophoresis (150 V for 60 min). The proteins in the gel were blotted onto polyvinylidene difluoride membranes using a transfer unit (Bio-Rad) (120 V for 2 hour) at 4 o C. The membrane was blocked with 5% nonfat dry milk/ PBS buffer containing 0.05% Tween 20 for 60 min at room temperature. The membrane was incubated with monoclonal anti-7 antibody (Sigma, mAb 306, 1:500) overnight at 4 o C, then washed three times with PBS/ 0.05% Tween 20 for 5 min each. The membrane was incubated with secondary antibody horseradish peroxidase-conjugated anti-mouse IgG (Amersham, 1:10,000 dilution) for 60 min at room temperature. The membrane was washed 3 times with PBS/ 0.05% Tween 20 for 5 min each. The membrane was exposed to ECL PLUS reagent (Amersham) for 1 min and the membrane was exposed to Hyper Performance Chemiluminescence film (Amersham). 7 nicotinic receptor band (MW 38,000) was quantified with the NIH Image program. Morris Water Task Morris water maze tests were performed to test the spatial learning and memory of mice injected rAAV2-rat 7 vectors. These tests were conducted using a specially designed water tanks (1 m) for mice with a fixed platform hidden just below (~ 1.0 cm) the surface of the water. The platform was rendered invisible by adding powdered milk to render the water opaque. Various geometric images (e.g., circles, squares, triangles) were placed in the testing room or hung on the wall in order that mice can use these visible objects as a means of navigating in the maze. Mice received 2 blocks of training trials (4 trials/block) daily for 8 days. With each subsequent entry into the maze the mice progressively become more efficient at locating the platform. The ninth day was for

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37 probe trials. The mice had 60 seconds to search for the platform and they were hand guided to the platform if they did not reach it during that interval. The mouse was allowed 30 seconds on the platform. For each trial, latency to find the platform, path length to the platform, and swim speed were recorded by a video-tracking/computer-digitizing system (HVS Image, Hampton, UK). On day 9, animals were given a probe test in which the platform was removed and the rats had 60 seconds to search for the platform. The swim distance and percentage of time spent in each quadrant were recorded. The total time required for each animal to reach the platform was determined for the 8 days training period, and these values were compared among the treatment group by ANOVA. ANOVA was also used to compare probe (retention and recall) performance among the treatment groups. Electrophysiological Recordings Fresh horizontal slices (300 m) were prepared using a vibratome (Pelco, Redding, CA). Slices were incubated at 30C for 30 min and then maintained submerged at room temperature. The artificial cerebral spinal fluid (ACSF) used for cutting and incubating slices contained in mM: 124 NaCl, 2.5 KCl, 1.2 NaH 2 PO 4 2.5 MgSO 4 10 D-glucose, 1 CaCl 2 and 25.9 NaHCO 3 saturated with 95% O 2 / 5% CO 2 Following incubation, slices were transferred to a recording chamber where they were superfused at a rate of 2 ml/min with ACSF at 30C containing in mM: 126 NaCl, 3 KCl, 1.2 NaH 2 PO 4 1.5 MgSO 4 11 D-glucose, 2.4 CaCl 2 and 25.9 NaHCO 3 saturated with 95% O 2 / 5% CO 2 Individual neurons were identified with infrared differential interference contrast microscopy (IR DIC) using a Nikon E600FN microscope (Nikon, Inc.). Whole-cell patch-clamp recordings were made with pipettes pulled on a Flaming/Brown electrode puller (Sutter

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38 Instruments, Novato, CA). Pipettes were typically 3-5 M when filled with an internal solution that contained in mM: 140 Cs-MeSO 3 8 NaCl, 1 MgCl 2 0.2 EGTA, 10 HEPES, 2 mg ATP, 0.3 Na 3 GTP, and 5 QX-314. This solution blocked all action potentials and allowed stable voltage-clamp recording at depolarized membrane potentials. All internal solutions were pH adjusted to 7.3 using additional CsOH or KOH and volume adjusted to ~285 mOsm. For experiments involving fluorescence microscopy, GFP-expressing neurons were visualized using light from a mercury lamp filtered at 510-560 nM. For all local application experiments, a picospritzer (General Valve, Fairfield, NJ) was used to apply ACh (1 mM) from pipettes identical to those used for whole-cell recording or from double-barreled pipettes made using theta tubing (Sutter Instruments, Novato, CA). An Axon Multiclamp 700A amplifier (Axon Instruments, Union City, CA) was used to amplify voltage and current records. The data were sampled at 20 kHz, filtered at 2 kHz, and recorded on a computer via a Digidata 1200A or 1321 analogue-to-digital converter using Clampex version 8-8.2 (Axon Instruments). Data were analyzed using Clampfit version 8-8.2 (Axon Instruments), OriginPro v. 7.0 (OriginLab, Boston, MA) and Graphpad Prism v. 3.0 (Graphpad Software, San Diego, CA). Fimbria Fornix Lesions And 4OH-GTS-21 Injections Wild type (Swiss Webster) and APP/PS1 (B6/D2 x Swiss Webster; a gift from Dr.Karen Duff) or PS1 (Taconic Swiss Webster x B6D2F1 crosses) mice are anesthetized with sodium 2-6% isoflurane/oxygen gas. Depth of anesthesia was determined by toe pinch and corneal reflex. Body temperature was maintained at 37 o C with an isothermal pad. The skull was exposed, and the bone from the region overlaying the septal area was

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39 removed. A modified Pasteur pipette (pulled to a tip-diameter of 0.4mm) was lowered under visual control with a surgical microscope and mild vacuum applied to remove the fornix and small amounts of surrounding neocortical tissue. Stereotaxic coordinates for the lesion sites in mice were A.P. 0.1mm, M.L. + 0.5mm, D.V.-2-3.8mm. Following the lesioning procedures, a piece of gel foam was placed in the skull hole, antibiotic powder was sprinkled over the skull, and the scalp was closed with tissue clamps. Animals were returned to their cages after fully reaching consciousness. 4OH-GTS-21 (1mg/kg) or 0.9% saline diluents was injected IP 2x daily (8AM or 6 PM) for two weeks post-lesion, with the first injection 30 min prior to the lesion. Differentiation Of PC12 Cells PC12 cells were obtained from American Type Culture Collection (ATCC). The cells were grown in RPMI Medium 1640 containing 10% heat-inactivated horse serum, 5% FBS and 1% penicillin/streptomycin at 37 o C in a 5% CO 2 and 90-92% humidity. The cells were plated in culture plates that had been pre-coated with collagen (BD Biosciences) for 3 hours. The cells were split at a 1:4 ratio every 3 days, up to 10 passages. Cell confluence at the time of study was approximately 60%. Cultures were maintained for 7 days in serum-supplemented media with 100-ng/ml nerve growth factor (NGF, BD Biosciences), which had been added at day 1 and day 3. On the day 7 of differentiation, the medium was replaced with one of the following conditions: serum medium with 100 ng/ml NGF, serum-free medium with or without NGF or serum-free medium containing specified concentrations of various drugs. 10 M of 1,2-Bis (2-aminophenoxy) ethane-N,N,N ,N tetraacetic acid (BAPTA-AM, Molecular Probles Eugene), 500 nM of nifedipine (Sigma), 10 M of xestospongin C (Calbiochem), 10 M

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40 of ryanodine (Sigma), 10 M of PD98059 (Calbiochem), 10 M of U0126 (Cell Signaling), 10 M of U-73122 (Calbiochem), 100 nM of MLA (RBI, South Natick,MA) were added at specified time point in 100 l sterile water immediately after medium replacement. 10 M GTS-21 was added in 50 l sterile water. Three days after NGF+ serum removal, five random photographs were taken in each plate with a Nikon inverted microscope. Images were analyzed in a blinded manner for cell density using the NIH Immage 1.55 program. There were 6 plates per treatment group. Cell counts are expressed as a mean + SEM. Only the cells attached to the bottom of the dishes were analyzed. Protein Kinase C (PKC) Assay The total PKC activity was measured in membrane and soluble fractions of cell lysates obtained by probe sonication using a kit purchased from Amersham. The medium was removed from each well and replaced with HEPES-PO 4 buffer (137 mM NaCl, 5.4 mM KCl, 0.3 mM Na 2 HPO 4, 0.4 mM KH 2 PO 4, 1 mg/ml dextrose, 20 mM HEPES, 1mM CaCl 2 ,pH 7.2) and incubated for 10 min. The HEPES-PO 4 buffer was removed and 40 l of kinase assay buffer (KAB) was added. 50 g/ml digitonin, 200 M MBP [4-14] substrate peptide, 100 M NaATP and 30 Ci/ml ( 32 P) ATP were added immediately. The enzyme activity was measured for 10 min. The reaction was terminated with 10 l of 25% trichloracetic acid. 45 l of the acidified assay mixture from each well were spotted onto 2 x 2 cm Whatman P-81 phosphocellulose filter paper. Phosphorylated MBP [4-14] was quantified by liquid scintillation spectrophotometry in a Beckman LS1800 counter.

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41 Tail DNA Extraction And Genetype Mice homozygous for the Chrna7 tm1Bay mutation are viable and fertile, but are inconsistent breeders, producing small litters. The heterozygous Chrna7 tm1Bay mutation breeder mice were obtained from Jackson laboratory. Two-week-old mouse-tails were cut off at the tip (2-5 mm) and were transferred to a pre-labeled tube. 700 l of lysis buffer (50 mM Tris, 100 mM EDTA, 0.05% SDS and 20 g/ml proteinase K) were added to the tube. The tubes were incubated at 55 o C overnight. The tubes were removed from 55 o C and 150 l 5 M NaCl were added. An equal volume of phenol-chloroform was added to the sample, vortexed for 5 min and microfuged for 5 min at 14,000 g. This was followed by an extraction with an equal volume of chloroform and microfuging for 5 min at 14,000 g. The aqueous layer was transferred into a new microfuge tube to which was added 2.5 volumes of ethanol. DNA was precipitated at -80 o C for 30min. The sample was microfuged at 14,000 g for 20 min. The supernatant was discarded. 0.5 ml of 75% ethanol was added to the pellet and microfuged at 14,000 g for 10 min. The pellet was air dried for 15 min and dissolved in 50 l of water. 4 l tails DNA were added to the 46 l mix of the components for PCR amplification (9 l 10X PCR buffer, 4 l 2.5 mM dNTP, 2.5 l 20 M 1002 primer, 1.25 l 20 M 1003 primer, 1.875 l 20 M 1004 primer, 1 l Taq DAN polymerase and 31.5 l ddH 2 O). The PCR reaction product was loaded to 1.5% agrose gel (Figure 2-1). The size of the 7 KO mouse band is 750 bp. The sizes of 7 bands for heterozygous is 440 bp and 750 bp. The size of wild type mice is 440 bp.

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42 1 2 3 4 5 Figure 2-1. The 7 genotype of mice. 1: wild type mouse; 2: 7 knockout mouse; 3: 7 heterozygous mouse; 4: negative control; 5: 100 bp marker. Statistical Analyses Statistical analyses involved one way or multiple ANOVA for comparisons of parametric populations, using the Statview program. For non-parametric analysis, such as groups that undergo multiple treatments that may be interactive, resulting in a non-normal population, using a rank order test on the Statview program (acquisition behavior Morris water task).

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CHAPTER 3 MECHANISMS UNDERLYING 7 NICOTINIC RECEPTOR NEUROPROTECTION IN PC12 CELLS Introduction PC12 cells and SK N SH cells were being chosen in this aim study. Both of them express endogenous 7 nicotinic receptors. It had shown that NGF-differentiated PC12 cells would undergo apoptosis following removal of both NGF and serum. NGF-deprivation is hypothesized to occur in AD in septal cholinergic neurons. SK N SH cells line is very sensitivity to exposure Abeta. We therefore used these two models to test the cytoprotective action of 7 nicotinic receptors. Several groups including ours have demonstrated that GTS-21 and 4OH-GTS-21 7 agonists increase intracellular calcium concentrations (Gueorguiev et al., 2000; Li et al., 2002). They also activate the calcium-sensitive transduction processes such as protein kinases A (PKA)(Dajas-Bailador et al., 2002b) and C (PKC), inositol triphosphate (IP-3) kinase (Kihara et al., 2001), ERK (Dajas-Bailador et al., 2002b; Bell et al., 2004), and janus kinase (Salehi et al., 2004). PKC (Li et al., 1999c), IP-3 kinase (Kihara et al., 2001) and janus kinase (Salehi et al., 2004) are each essential for 7 mediated protection against one or more apoptotic insults, while the roles of the other kinase pathways have not been studied. This protection through calcium-sensitive kinases suggests a protective role for the increased intracellular calcium ion concentrations observed following treatment with 7 agonists. However, there remains no direct demonstration that 7 receptor mediated protection or kinase activation depends 43

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44 on intracellular calcium ions. We tested this hypothesis by investigating the effects of intracellular calcium ion chelation on both protection and PKC activation in NGF-differentiated rat pheochromocytoma (PC12) cells. These cells undergo apoptosis following NGFremoval and may provide a model for the dysfunction of ascending basal forebrain neurons associated with decreased NGF-transport seen in Down's syndrome and Alzheimer's disease (Kerwin et al., 1992; Scott et al., 1995; Cooper et al., 2001; Salehi et al., 2004). They also express 7 nicotinic receptors and are protected from apoptosis by prolonged treatment with 7 receptor agonists Activation of 7 receptors can increase calcium accumulation both directly as well as through activation of downstream L-type voltage sensitive channels, IP-3 channels, and ryanodine channels (Vijayaraghavan et al., 1992; Gueorguiev et al., 2000; Shoop et al., 2001; Dajas-Bailador et al., 2002a), analogous to what is seen with metabotropic glutamate receptors (Fagni et al., 2000). L-type channels are activated by depolarization triggered by the influx of sodium ions and calcium ions through these receptors, while the intracellular calcium channels are likely activated through calcium-influx through both 7 receptors and L-type channels. Blocking IP-3 channels with xestospongin C attenuates the long term increase in calcium accumulation following 7 receptor activation almost completely in PC12 cells, implicating these channels in neuroprotection (Gueorguiev et al., 2000). An analysis of the calcium elevations triggered by nicotine in SH-SY5Y cells indicated that xestospongin C-sensitive IP-3 channel activation appeared to be more important than nifedipine-sensitive L-type channels or ryanodine-sensitive channels for the long term effects of 7 receptors (Dajas-Bailador et al., 2002a). We therefore hypothesized that these three channels may be differentially important for the

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45 long term cytoprotective actions of 7 receptors, which we tested in PC12 cells that express each of these calcium channels (Gafni et al., 1997; Tully and Treistman, 2004)). A third goal of this study was to evaluated the role of the calcium-sensitive MAP kinases ERK1/2, p38, and JNK in the 7 receptor mediated neuroprotection, since they are also differentially involved in the cytoprotective effects of other anti-apoptotic agents (Hetman and Xia, 2000; Hsu et al., 2004; Kyosseva, 2004). ERK1/2 phosphorylation has been reported to be essential for the antiapoptotic effects of a wide variety of drugs (Hetman and Xia, 2000; Kyosseva, 2004). It was therefore a likely candidate for involvement in 7 receptor mediated protection, along with the other kinases mentioned above. Two other MAP kinase pathways, p38 and JNK, which are more frequently involved in stress responses than cytoprotection (Hsu et al., 2004; Kyosseva, 2004), were also evaluated following 7 receptor activation to determine their potential involvement in the protective effects of these receptors. Results PC12 cells were plated in 60 mm culture dishes and differentiated for 7 days in the presence of 100 ng/ml NGF (Figure 3-1). NGF and serum were withdrawn from the medium at that time. The cells were treated with different concentrations of GTS-21 (0, 3 M, 10 M and 30 M) for 72 hours. Cell density was measured after 3 days by the NIH Image system program (Figure 3-1). This figure demonstrates that NGF and serum withdrawn caused approximately a 50% cell loss. 3 M and 10 M GTS-21 induced protection and provided 80-90% cell survival. But 30 M GTS-21 cause even more extensive cell loss.

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46 Figure 3-1. GTS-21 induced protection of PC12 cells during trophic factor deprivation. PC12 cells were plated in the 60 mm culture dishes and differentiated with NGF for 7 days. Cells were rinsed and the media was replaced with fresh media containing the treatment indicated above. Cell density was measured 3 days later and expressed as the mean + SEM of 6-8 plates/group from 3 experiments. Three random areas were counted per plate and these 3 values were averaged to yield one value per plate. *p < 0.05 compared to untreated group (one way ANOVA). In order to evaluate the role of intracellular calcium ions and 7 receptors in this cytoprotection. 10 M BAPTA-AM and 100 nM MLA were added either 30 min before (pretreatment) or 30 min after (posttreatment) GTS-21. Treatment with BAPTA alone had no effect on cell survival during NGF + serum removal or in the presence of NGF, though it blocked the cytoprotective action of 10 M GTS-21 when added 30 min prior to or 30 min after the receptor agonist. MLA similarly blocked the GTS-21 induced protection when added at both time points (Figure 3-2). None of the treatments affected the Kd for high affinity MLA binding to 7 receptors (range: 1.6-2.3 nM). Removal of NGF + serum reduced the density of 7 receptors over the 3 day but not 30 min interval (Table 3-1); this reduction was not seen with addition of 100 ng/ml of NGF. Addition of BAPTA to the NGF + serum deprived

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47 medium had no acute effect on 7 nicotinic receptor binding density, but also modestly increased density over the 3 day interval compared to cells without the chelator. Neither GTS-21 nor GTS-21 + BAPTA preserved receptor density in this manner. Figure 3-2. Effects of intracellular calcium chelation on GTS-21 induced protection of PC12 cells during trophic factor deprivation. Cells were differentiated for 7 days with 100 ng/ml mouse NGF and then exposed to serum-free medium containing either BAPTA-AM (10 M), GTS-21, or MLA (100 nM). BAPTA-AM and MLA were added either 30 min before (pretreatment) or 30 min after (posttreatment) the GTS-21. Cell density was measured 3 days later and expressed as the mean + SEM of 6-8 plates/group from 3 experiments, normalized to the NGF-treated values for each experiment. *p < 0.05 compared to untreated group (one way ANOVA). 10 M of BAPTA also blocked the concentration-dependent GTS-21 induced elevation in PKC membrane translocation seen in these NGF and serum withdrawn differentiated PC12 cells (Figure 3-3).

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48 Table 3-1. Effects of BAPTA and GTS-21 on 7 receptor binding density in PC12 cells High affinity MLA binding (Bmax in fmol/mg protein) Treatment 30 min 3 days NGF+serum removal 152 + 11 107 + 10^ + NGF (100 ng/ml) 165 + 18 172 + 15* + BAPTA (10 M) 167 + 13 144 + 13* + GTS-21 (10 M) 155 + 17 101 + 14^ + BAPTA + GTS-21 172 + 16 98 + 12^ Table 3-1. PC12 cells were differentiated for 7 days with 100 ng/ml NGF and then exposed to NGF + serum withdrawal. Specified concentrations of NGF, BAPTA, or GTS-21 were added and high affinity [ 3 H] MLA was measured 30 min or 3 days later. Each value is the mean + SEM of three samples, each assayed in tripicate. *p < 0.05 compared to same time point, no drug-treatment (two way ANOVA); ^p < 0.05 compared to 30 min interval, same treatment. Figure 3-3. Effects of intracellular calcium chelation on PKC activation by GTS-21 in PC12 cells. Cells differentiated as in Figure 3-1 were simultaneously exposed to serum and NGF removal, as well as to specified concentrations of GTS-21 in the presence or absence of 10 M BAPTA-AM. 15 min later, cells were fractionated to membrane and soluble portions, each of which was assayed for phorbol-stimulated PKC activity/mg protein. Values are expressed as the ratios of membrane/soluble PKC activity; N = 6 plates/group from 3 separate experiments. *p < 0.05 compared to BAPTA-treated group, same GTS-21 concentration.

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49 Effects of 4OH-GTS-21 on PKC isozyme translocation were tested. Different concentrations of 4OH-GTS-21 were used in serum and NGF withdrawn differentiated PC12 cells. 3 M and 10 M 4OH-GTS-21 increased PKC alpha translocation (Figure 3-4). But 30 M 4OH-GTS-21 decrease the membrane/soluble ratio of PKC alpha. PKC delta membrane/soluble ratios were decreased at higher concentrations of 4OH-GTS-21. PKC gamma was unaffected. Figure 3-4. Effects of 4OH-GTS-21 on PKC isozyme translocation. Neuroprotective concentration of 4OH-GTS-21 increased the membrane/soluble ratio of PKC alpha, a measure of its activation. PKC delta membrane/soluble ratios were decreased, but over a higher concentration range. No effect was seen on translocation of PKC gamma. In order to determine the roles of different calcium channels in 7 receptor induced neuroprotection, some cells were treated either with L-type channel blocker nifedipine, ryanodine receptor blocker ryanodine, IP3 channel antagonist xestospongin C, phospholipase C inhibitor U-73122 with or without 10 M GTS-21. The voltage sensitive L-type calcium channel blocker nifedipine had no effect on cell viability when applied alone or when with GTS-21 (Figure 3-5). Ryanodine partially attenuated the GTS-21 induced protection, also without direct effect alone, indicating that only some

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50 cells appeared to depend on this channel activation for survival in this model. Xestospongin C, the IP3 channel antagonist, completely blocked 7 receptor mediated protection and had no effect on cell density when applied alone. Figure 3-5. Effects of calcium channel antagonists on GTS-21 induced cytoprotection in NGF-deprived PC12 cells. Cells differentiated as in Figure 1 were treated with 10 M GTS-21 immediately after NGF + serum removal, with or without 10 M ryanodine, 10 M xestospongin C, 10 M U-73122 or 500 nM nifedipine. Each value is the mean + SEM of 6-8 plates/group from 3 separate experiments. *p < 0.05 compared to same treatment without GTS-21;#p < 0.05 compared to GTS-21 only treatment group (one way ANOVA). Serum and NGF were withdrawn from differentiation PC12 cells and 10 M GTS-21 was added to cells. ERK1/2, p38 and JNK-phosphorylation were measured at 5 and 180-minute time interval. ERK1/2-phosphorylation was increased by 10 M of GTS-21 within 5 minutes, and this effect increased by 3 hours (Figure 3-6). Increased ERK1/2 phosphorylation was blocked by 500 nM MLA applied 5 minutes before GTS-21 addition (not shown). p38and JNK-phosphorylation were unaffected by GTS-21 at either time interval.

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51 Figure 3-6. Effects of GTS-21 on the phosphorylation of several MAP kinases in PC12 cells. Cells differentiated as in Figure 1 were treated with 10 M GTS-21 for the specified interval upon removal of the NGF + serum. Whole cell extracts were assayed for phospho-ERK1/2, phospho-JNK and phospho-p38 by western blotting and expressed as the mean + SEM of 4 samples/group, normalized to the 5 minute control value for that experiment. Each gel contained two lanes from the same 5-minute control sample that were averaged and used for normalization. *p < 0.05 compared to control from same time point (one way ANOVA). Four different concentrations of GTS-21 (0, 0.5,3 and 10 M) were used to test ERK1/2 phosphoration (180 minutes later). Some of groups pretreated with 100 nM MLA. The effect of GTS-21 on ERK1/2 phosphorylation was concentration dependent. This effect was blocked by MLA, demonstrating the role of 7 receptors (Figure 3-7). GTS-21 mediated neuroprotection was blocked by either pretreatment with the PKC blocker BIM or the ERK1/2 blocker U0126 and PD98059 (Figure 3-8). Various kinase inhibitors were tested on the effects of 4OH-GTS-21 on differentiation PC12 cells. 7 antagonist MLA (1 M), PKC antagonist BIM1 (100 nM), ERK1/2 inhibitor PD98059 (20 M) and PKA inhibitor H89 (1 M) blocked 4OH-GTS-21 provided cytoprotection (Figure 3-9).

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52 Figure 3-7. MLA blocks the ERK1/2 phosphorylation triggered by GTS-21. Cells were treated as in Figure 4, except with varying concentrations of GTS-21 for 180 minutes, plus or minus 100 nM MLA. Phospho-ERK was assayed and expressed as the mean + SEM of 4 samples/group, normalized to untreated control values on the same gel. *p < 0.05 compared to either the corresponding MLA treatment group.

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53 Figure 3-8. GTS-21 induced cytoprotection is dependent on ERK phosphorylation and PKC activation. Differentiated PC12 cells were treated with10 M GTS-21 for 3 days upon NGF + serum removal, with or without 10 M PD98059, 500 nM bis-indole maleimide (BIM) or 10 M U0126. Cell densities are expressed as the mean + SEM of 6 plates/group from 3 experiments; *p < 0.01 compared to untreated control (one way ANOVA). 0 25 50 75 100NGF-free + MLA + BIM1 + PD98059 + H89 03uM10uM30uM03uM10uM30uM03uM10uM30uM03uM10uM30uM03uM10uM30uM4OH-GTS-21Cell viability (%NGF-treated control) Figure 3-9. Effects of 4OH-GTS-21 and various kinase inhibotor on PC12 cells. NGF and serum were removed 1 hour before agonist treatment. 1 M 7 antagonist MLA, 100 nM PKC antagonist BIM1, 20 M ERK1/2 inhibitor and 1 M PKA inhibitor H89 were added 15 minutes prior to 4OH-GTS-21. Cell density was measured 3 days after NGF-removal. Values are means + SEM of 4 plates/group.

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54 Various kinase inhibitors were also tested on the protective effects of 4OH-GTS-21 against an amyloid peptide in human SK N SH cells. Abeta 25-35 (20 M) caused a significant reduction in cell density alone, which was partially blocked by 4OH-GTS-21 in a concentration dependent manner. At 30 M 4OH-GTS-21, additional toxicity was seen over that with Abeta peptide alone. The 7 antagonist MLA (1 M), PKC antagonist BIM1 (100 nM), ERK1/2 inhibitor PD98059 (20 M) and PKA inhibitor H89 (1M) all blocked this 4OH-GTS-21 provided cytoprotection (Figure 3-10). However, only MLA blocked the additional toxicity caused by 30 M 4OH-GTS-21. 0 50 100 150Abeta 25-35 + MLA + BIM 1 + PD98059 + H89 03uM10uM30uM03uM10uM30uM03uM10uM30uM03uM10uM30uM03uM10uM30uMCell viability(%untreated control) Figure 3-10. Effects of Abeta 25-35, 4OH-GTS-21 and various kinase inhibitor on SK N SH cells. All cells were treated with specified concentrations of 4OH-GTS-21 for 1 hour prior to adding 20 M Abeta 25-35. 1 M 7 antagonist MLA, 100 nM PKC antagonist BIM1, 20 M ERK1/2 inhibitor and 1 M PKA inhibitor H89 were added 15 minutes prior to 4OH-GTS-21. Cell density was measured 3 days after NGF-removal. Values are means + SEM of 4 plates/group. Discussion Previous studies have demonstrated GTS-21-induced calcium elevations (Gueorguiev et al., 2000; Li et al., 2002) and have suggested the importance of multiple calcium-activated processes for 7 receptor-induced neuroprotection (Li et al., 1999c;

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55 Kihara et al., 2001; Dajas-Bailador et al., 2002b; Bell et al., 2004; Salehi et al., 2004). The present results show for the first time that chelation of intracellular calcium ions blocks GTS-21-induced, 7 mediated neuroprotection, demonstrating a direct role for these divalent cations in the receptor-modulation of cell viability. This is consistent with previous studies that chelated or removed extracellular calcium ions to attenuate 7-mediated protection or kinase-activation (Donnelly-Roberts et al., 1996; Dajas-Bailador et al., 2002b). The concentration of BAPTA-AM used in our study was selected for its ability to prevent nicotine-induced elevations in both cytoplasmic calcium and tyrosine hydroxylase activity in PC12 cells (Gueorguiev et al., 1999). It therefore appears that the calcium-elevations triggered by 7 receptor activation have important roles both for the phenotypic properties of these cells as well as for their long-term viability in the presence of toxic insults. It is interesting to note that a careful study of intracellular calcium chelation in hippocampal neurons recently concluded that modest increases in intracellular calcium-concentrations were also associated with improved viability (Bickler and Fahlman, 2004). It remains to be determined to what extent BAPTA-treatment reduces calcium concentrations in mitochondria, smooth endoplasmic reticulum, and other storage sites under our experimental conditions. The effects of 7 receptor activation on cell viability and their blockade by BAPTA were seen without any acute change in 7 nicotinic receptor binding density and a modest increase in receptor density by 3 day. These observations are significant because 7 binding density was recently found to modulate the neuroprotection seen in PC12 cells (Jonnala and Buccafusco, 2001). The reduction in 7 receptor binding density over three days of NGF + serum removal is probably related to the observation that NGF

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56 increases this receptor subunit expression in PC12 cells(Takahashi et al., 1999). GTS-21 has not been reported to increase 7 receptor expression or lead to PC12 cell differentiation, which may account for its inability to prevent the loss of 7 receptor density over the 3-days of NGF-deprivation. The lack of effect of GTS-21 on 7 receptor binding density following the 3 days of NGF + serum deprivation was a surprising result since the less selective agonist nicotine was found to increase 7 receptor expression in these cells (Jonnala and Buccafusco 2001). MLA also increases 7 receptor density in PC12 cells (Jonnala and Buccafusco 2001), which might be expected with GTS-21 if it acted by desensitizing most 7 receptors. One possibility is that selective, low level activation of 7 receptors is insufficient to increase their density, which is consistent with the lack of effect of chronic GTS-21 administration on 7 receptor density in neocortex in vivo (Meyer et al., 1997). Alternatively, GTS-21 may preserve a population of PC12 cells that expresses fewer 7 receptors, while simultaneously increasing their 7 receptor expression, resulting in no net change in density under these conditions. Our results indicate that the cell viability effects of BAPTA are likely due to processes downstream from the receptor activation, since there was no decrease in binding density that would be expected to interfere with the actions of GTS-21. This is consistent with the observation that BAPTA exposure attenuated GTS-21 induced PKC activation. PKC-translocation and activation were previously found to be essential for 7 mediated protection in this apoptotic model (Li et al., 1999c). This increase in PKC activity occurs within 15 minutes; during the interval that neuroprotection is BAPTA-sensitive, suggesting that the kinase activation may be one of the relatively early steps in the protective process.

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57 The antiapoptotic effect of GTS-21 in PC12 cells requires extended activation of 7 receptors, since MLA blocks this cell survival when administered up to 1 hr post-agonist (Li et al., 1999c). The ability of BAPTA to block protection when applied 5 min after GTS-21 suggests that a protracted elevation in intracellular calcium ions is also essential for protection. Whether this is due to a slow or rapid increase in calcium is not clear. We previously demonstrated that neuroprotective concentrations of GTS-21-caused a long term, near steady state calcium influx through 7 receptors, without the desensitization of the overall receptor population seen at higher, non-protective agonist concentrations (Papke et al., 2000). However, while it is likely that this low-level receptor activation provides an early calcium-transient that is important for triggering the cytoprotective pathway, it appears that downstream calcium channel activation is important as well. The multichannel modulation of intracellular calcium by voltage sensitive L-type calcium channels, intracellular IP3 channels, and intracellular ryanodine channels provides a potentially complex mechanism for 7 receptors to affect cellular function and viability (Vijayaraghavan et al., 1992; Gueorguiev et al., 2000; Shoop et al., 2001; Dajas-Bailador et al., 2002a; Dajas-Bailador et al., 2002c). Activation of the intracellular IP3 calcium channel, and to a lesser extent the ryanodine receptor, are necessary for complete 7 mediated protection in this model, based on sensitivity to antagonists. This is consistent with the protective actions of metabotropic receptors such as bradykinin (Yamauchi et al., 2003) and mGluR4s (Maj et al., 2003) that also act on IP3 receptors. The concentration of xestospongin C used in the present study was found previously by our group to block IP3 receptors and reduce GTS-21 induced calcium accumulation in PC12 cells for as long as it was assayed (45 min)(Gueorguiev et al., 2000). This

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58 dependence on IP3 channels for most of the long term elevation in intracellular calcium triggered by 7 receptors is consistent with their involvement in cytoprotection, which similarly depends on long term receptor activation (Li et al., 1999c). Inhibition of phospholipase C with U-73122 (Kokoska et al., 1998), which blocks IP3 production, also attenuates GTS-21 induced protection, providing additional support for a role of IP3 receptors. Ryanodine channels were also involved in the protection of some NGF + serum deprived PC12 cells, though this effect was less dramatic than that seen with IP3 channel attenuation. Since ryanodine may only partially attenuate 7 mediated calcium elevations (Gueorguiev et al., 2000), it is not surprising that some cells remained sensitive to GTS-21 in the presence of this channel blocker. There are multipe types of ryanodine receptor that are differentially expressed in various tissues, however, so it is conceivable that this result may be difficult to extrapolate to other neuronal models (Berridge et al., 2000). In brain neurons, ryanodine receptors are primarily localized to the endoplasmic reticulum of postsynaptic entities, from which they release calcium in response to increased cytoplasmic calcium. Recently, however, they have been found presynaptically and may be involved in modulating transmitter release (Bouchard et al., 2003), another well characterized function of 7 receptors. The nifedipine-sensitive voltage sensitive L-type channel also underlies a significant amount of the calcium accumulation seen following 7 receptor activation. Although extracellular calcium is necessary for neuroprotection, as noted above, it appears that the channels other than L-type channels are involved in this action, probably the 7 receptors themselves. The observation that L-type channels are not essential for

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59 protection may be due to multiple factors. First, L-type channel openings may be for a shorter duration than is necessary for protection, e.g., if the voltage-dependent channel openings is attenuated over time through calcium-activated potassium channels. Alternatively, the calcium entry through these channels may be physically removed from the transduction processes essential for cytoprotection. A third possibility is that the intracellular calcium channels may provide sufficient calcium for protection even in the absence of voltage sensitive calcium channel activation. Our results indicate for the first time that the ERK1/2 MAP kinase pathway is required for 7 receptor mediated protection, based on its GTS-21 induced phosphorylation and attendant activation, as well as the ability of the ERK1/2 inhibitor PD98059 to block protection. ERK1/2 phosphorylation has been associated with cytoprotection in a variety of model systems e.g., (Hetman and Xia, 2000; Kyosseva, 2004), and with other anti-apoptotic processes found to be triggered by 7 receptors, including bcl2 elevations, increased mitochondrial membrane potential, and reduced cytochrome C release (Li et al., 1999c). Our results indicate therefore that calcium accumulation triggered by 7 receptor activation is necessary for this chain of kinase-mediated anti-apoptotic events. In contrast, neither the p38 nor the JNK pathway was apparently activated by a protective concentration of GTS-21. It is interesting to note that activation of ERK has been reported with very low concentrations amyloid peptides, suggesting that this may provide a dose-dependent protective role for the peptides and receptors under appropriate conditions, perhaps even in the sparing of 7 receptor expressing cells in Alzheimer's disease.

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60 In summary, 7 receptor activation provides cytoprotection against trophic factor deprivation and triggers PKC translocation through a mechanism that appears to involve intracellular calcium ion elevations (Figure 3-11). Among the several calcium channels triggered by 7 receptor activation, IP3and, to a lesser extent ryanodine-receptor calcium channels are likely mediators of these calcium-elevations and are essential for cytoprotection. Downstream ERK1/2 phosphorylation is also essential for protection, while other MAP kinases JNK and p38 are not. It now becomes important to determine the role of these various pathways in the protective actions of 7 receptors in brain in vivo. Figure 3-11. The potential mechanism of 7 nicotinic receptors mediated cytoprotection.

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CHAPTER 4 NEUROPRECTIVE AND ANTI-AMYLOIDOGENE EFFECTS OF THE 7 PARTIAL AGONIST 4OH-GTS-21 IN FIMBRIA FORNIX LESIONED MICE OF DIFFERENT GENOTYPES Introduction While 7 nicotinic receptors are neuroprotective in a variety of models, little is known about their protective properties in models of neuronal dysfunction related to AD in vivo. There is presently no animal model that mimics every aspect of AD, though various lesion and genetic manipulations have been used. Combinations of these approaches, however, are much less common. Investigations of genetic linkage and transgenic animal models have shown that no single genetic defect that accounts for all the features of AD. A certain number genes have been identified to be involved in the progressive neuron degeneration of this disease. Most AD cases have mutations in the genes encoding for APP, presenilin 1 (PS1) or PS2. All of these mutations have been shown to alter APP metabolism and increase the A peptide levels in the brain. Transgenic mice expressing mutated human genes that are associated with familial AD offer a powerful model to study A. The present study employed either single human mutated PS1-M146L transgenic mice or double transgenic mice expressing both human APP Swedish mutant K670N/M671L and PS1-M146L mutations (APP/PS1). Both types of transgenic APP/PS1 mice have elevated levels of fibrillogenic A 42 peptide in their brains, though only the APP/PS1 mice develop amyloid plaques starting 61

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62 around the age of 4 months of age. By 6 months of age, the amyloid plaque load is comparable to that of 12-month-old single APP K670N/M671L mice. This demonstrates that APP/PS1 mice develop amyloid plaques earlier than the APP-only transgenic mice. However, neither type of transgenic mice shows neurofibrillary tangles. These mice do have deficits in spatial memory in selected paradigms, notably the radial arm task in the Morris water paradigm, though not in the classic Morris water protocol. Mutations in PS1 may act synergistically with Swedish mutant APP to cause some pathology reminiscent of the AD brains. PS1 and PS2 play a critical role in mediating gamma secretase cleavage of the APP. Gamma secretase is one step in the formation of amyloidogenic A 1-42 or A 1-40. In addition, PS1 deficiency leads to alter intracellular Ca 2+ homeostasis involving endoplasmic reticulum Ca 2+ stores. PS1 transgenic mice that overexpress the mutant AD protein have been found to elevate levels of endogenous Abeta 1-42, but not Abeta1-40. Abeta1-42 levels are significantly elevated in the mutant PS1 mice, presumably by enhancing cleavage of APP at a gamma-secretase site. Evidence from in vitro studies also indicates that PS1 itself might either act as gamma-secretase or mediate the catalytic activity of the enzyme (De Strooper and Annaert, 2000). These PS1 transgenic mice do not form amyloid deposits upon aging, presumably because the levels of Abeta do not reach the level required to start the aggregation process in mice (McGowan et al., 1999). Mutated PS1 mice also do not show overt AD-like pathology or spatial learning deficits in the Morris water maze test. The densities of 7 nicotinic receptor-binding sites are unaltered in transgenic PS1 mice compare to non-transgenic controls.

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63 A recent study showed that no significant differences in size or number of cholinergic nerve terminals in the hippocampus or neocortical areas of mice overexpressing M146L PS1 (Wong et al., 1999). Conversely, studies from the double transgenic mice APP/PS1 mice have been found to display an extensive loss of cholinergic synapses in the frontal cortex and hippocampus (Vaucher et al., 2002). It was suggested that overexpression of human A peptide combined with a shift toward longer forms of A terminating at residues 42 or 43 due to mutation of PS1 is required to elicit cholinergic deficits in mice (Bronfman et al., 2000). Vaucher et al. showed that mutation of the human PS1 gene might alter sensorimotor activity and long-term retention of object recognition memory but not ChAT enzymatic activity or cholinergic receptor binding sites (Vaucher et al., 2002). Whether this cognitive deficit is due to an alteration in APP processing of the endogenous mouse APP holoprotein by the PS1 mutation or related to altered functions of other neurotransmitters is not known. A non-genetic approach to model AD involves lesions of neuronal pathways affected by the disease. One of the early effects of AD is the degeneration of basal forebrain cholinergic neurons, which results in loss of cholinergic function in the neocortex and hippocampus. The septo-hippocampal pathway has been the most thoroughly investigated basal forebrain cholinergic pathway. This pathway carries the acetylcholine and GABA as neurotransmitters in axons that project to the hippocampus. Fimbria fornix lesion (FFX-lesion) can result in partial or near-complete loss of cholinergic activity in the hippocampus and impairment in behavioral tasks, depending on the extent and type of lesion. For example, the aspirative lesions used in my studies affect from 50-90% of the cholinergic and GABA neurons projecting to the

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64 hippocampus. Following these lesions, animals become deficient in septal cholinergic neuronal density, memory related behavior and, as was recently reported, in hippocampal 7 nicotinic receptor functions. The cholinergical deficits in septum following FFX lesions may be due to the loss of retrograde axonal NGF transport from the hippocampus, based on the observations that: 1) NGF is typically transported in this manner by these cells retrograde; and 2) that NGF-administration can protect septal cholinergic neurons from the effects of the lesion. Since NGF-deprivation in differentiated PC12 cells is toxic in a manner that is protected by 7 nicotinic receptor agonists, my hypothesis was that the 7 nicotinic receptors partial agonist 4OH-GTS-21 would also be able to protect cholonergic neurons in vivo from axotomy. However, this protection was not expected in amyloid expressing mice such as the APP/PS1 double transgenic because of the 7 nicotinic receptor blockade by amyloid peptides. Mapping of cholinergic pathways based on choline acetyltransferase (ChAT) immunohistrochemistry could be considered to define the limits of nicotinic cholinergic signaling systems in the mammalian CNS. ChAT immunohistochemical maps identify major cholinergic projection from loosely-delimited nuclei of heterogeneous neurotransmitter phenotype in the medial septum (Mesulam, 1995). However, since 7 nicotinic receptors are also activated by choline, a natural byproduct of phosphatidylcholine metabolism and a circulating nutrient, it is possible that these cholinergic signals do not require and are not marked by ChAT staining. A small fraction (10-20%) of septal GABAergic neurons are intermingled with cholinergic neurons projecting to the hippocampus, although estimates about the number

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65 of GABAergic neurons in this region vary across different studies and species. Septal GABA neurons have morphologies similar to those of ChATpositive neurons, with a somewhat different topography. GABAergic neurons have been demonstrated a roughly similar number to the cholinergic neuron in rats, though a smaller fraction project to the hippocampus in at least some mouse stains (Sarter and Bruno, 2002). Interestingly, while these septohippocampal GABA neurons project alongside cholinergic neurons, they are not adversely affected by AD even as cholinergic neurons are lost. This may be due to the differential need of these cell types for NGF, since the GABA neurons do not posses NGF receptors. It has been reported that both septal GABA and cholinergic neurons possess 7 nicotinic receptors, but whether these receptors are protective for both populations is not known. One possible mechanism to account for 7 nicotinic receptor mediated neuroprotection is an increase in NGF expression. Nicotinic receptor activation was found to increase NGF levels and NGF-receptor levels, so this is a possibility. If so, then the 7 nicotinic receptor agonist treatment will likely be protective only for cholinerhic neurons and not GABA neurons. Alternatively, if 7 nicotinic receptors are protective more directly, then it would more likely that both types of neurons were protected by 7 nicotinic receptor agonists. I investigated these possibilities by comparing the neuroprotective actions of 4OH-GTS-21 in both populations of neurons following FFX lesions. While multiple studies have evaluated the effects of potential therapeutic agents in either transgenic mice or lesioned animals, no study has yet combined these two model systems. One of my project goals was therefore to test whether an 7 nicotinic receptor

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66 agonist could provide neuroprotection in a combination transgenetic and lesion model involving FFX lesions of wild type, APP/PS1 and PS1 transgenic mice. Another potential beneficial effect of 7 nicotinic receptor activations for AD may be reducing amyloid plaque density. Nicotine has been found recently to reduce amyloid plaque density in mutant APP transgenic mice over a period of several months of PO treatments. While it is not clear which of the nicotinic receptor subtypes cause this effect, my hypothesis was that 7 nicotinic receptors may be involved based on the observations, as noted in Chapter 1, that these receptors can stimulate APP -secretase activity, which would be expected to reduce substrate availability for Abeta amyloidogenic peptides. One method to determine the role of 7 nicotinic receptors in the nicotinic-induced reductions in amyloid density is to use selective agonists for the receptor. I addressed this possibility with the partial agonist 4OH-GTS-21 in double transgenic APP/PS1 mice. Results Mice receiving FFXlesions appeared to be no different from unlesioned animals with respect to body weight gain or visual assessments of health. In a preliminary study, AChE staining was used to verify the extent of the lesion relative to the loss of cholinergic innervation of the hippocampus. Aspirative FFX-lesions were performed unilaterally on male adults SD rat. The lesion resulted in a nearly complete loss of the hippocampal AChE-positive fibers on the lesion side. High levels of AChE staining were observed in the unlesioned side (Figure 4-1).

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67 Figure 4-1. Aspirative FFX-lesion of the septal hippocampal cholinergic pathway. The lesioned side hippocampus AChE staining is over 90% lost. Wild type C57/B16/J mice (9 month), PS1 mice (9 month) and APP/PS1 (9 month) also had significant, 55-60%, reductions in septal hippocampal cholinergic neuronal density following FFX-lesion as measured by septal ChAT staining two weeks post-lesion (Figure 4-2). There was no difference in the extent of this ChAT staining loss among the three groups when expressed as a percent of unlesioned, contralateral cell density. This normalization procedure of expressing neuronal density as a percent of the contralateral control side was used to control for inter-animal differences in perfusion, sectioning and staining. To what extent the loss of cholinergic neuron density was due to neuronal death versus phenotypic change is not known. There was also a significant reduction in the size of the septal cholinergic perikarya caused by this lesion. In order to evaluate the effect of chronic 7 nicotinic receptors activation on this septal cholinergic dysfunction, wild type, PS1 and APP/PS1 mice were injected IP twice/per day for two weeks (first injection 15 minutes prior to lesion) with either 0.9% saline vehicle or 1 mg/kg (salt weight) 4OH-GTS-21 (Figure 4-2 and 4-3). None of the strains appeared to be adversely affected by this dose of 4OH-GTS-21 based on weight

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68 gain, animal appearance, or gross behavior or physiology. For wild type and APP/PS1 mice, there was no apparent difference in septal ChAT staining between the 4OH-GTS-21 (1mg/kg) and 0.9% saline treatment groups. However, the PS1 mice treated with this 4OH-GTS-21 regimen retained more ChAT staining perikarya on the lesion side compared to the wild type and APP/PS1 mice. This stereological evaluation indicated that 4OH-GTS-21 could provide some protection of cholinergic neurons in PS1 mice, under conditions in which it did not affect APP/PS1 or wild type mice. Wild typePS1APP/PS1Wild type+GTS-21PS1+GTS-21APP/PS1+GTS-21 0 25 50 75 *Percentage of cellsurvive Figure 4-2. Septal ChAT neuron staining in 9 month old mice. 4OH-GTS-21 injected 2x daily (1 mg/kg IP) for 2 weeks increased septal ChAT staining neurons in unilaterally lesioned PS1 mice. 4OH-GTS-21 had no protective effect in 9 month old APP/PS1 and wild type mice. P<0.05 compared to other group (one way ANOVA).

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69 Saline 4OH-GTS-21 PS1 Wild type APP/PS1 Saline 4OH-GTS-21 PS1 Wild type APP/PS1 Figure 4-3. Septal ChAT-staining perilarya in 9 month old PS1, APP/PS1 and wild type C57/B16/J mice two weeks after unilateral aspirative FFX-lesions. Only the PS1 mice showed any neuroprotection with drug treatment.

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70 The septal GABAergic neuron quantification was conducted by using parvalbumin immunohistochemisty that accounts for a major fraction of the GABAergic innervation to hippocampus (Figure 4-4). The immunohistochemistry staining is shown in Figure 4-5. The cell-counting data showed that no change affected the GABAergic neuron density. Wild type+GTS-21Wild type+SalinePS1+GTS-21PS1+SalineAPP/PS1+GTS-21APP/PS1+Saline 0.8 1.0 1.2precentage of cellsurvive Figure 4-4. Septal GABAergic neuron staining in 9 month old mice. 4OH-GTS-21 was injected 2X daily (1 mg/kg IP) for 2 weeks. No change in septal GABAergic neurons staining in unilaterally lesioned mice. *p<0.05 compared to saline group (one way ANOVA).

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71 Saline 4OH-GTS-21 PS1 APP/PS1 Wild type Saline 4OH-GTS-21 PS1 APP/PS1 Wild type Figure 4-5. Septal GABAergic staining perilarya in 9 month old PS1, APP/PS1 and wild type C57/B16/J mice two weeks after unilateral aspirative FFX-lesions. No neuroprotection with drug treatment in these groups.

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72 6E10 and thioflavine S staining were conduced in sections of these same 9-month-old APP/PS1 mice (Figure 4-6). APP/PS1 mice were treated with 4OH-GTS-21 or 0.9% saline vehicle IP for two weeks. The 4OH-GTS-21 + lesion groups had few plaques compared to saline group (Figure 4-7). Figure 4-6. The thioflavine S and 6E10 staining in 9 month old APP/PS1 mice. Left pane: thioflavine S staining in hippocampus; Right panel: 6E10 staining in hippocampus and neocortex. 80 100 120No Drug +4OH-GTS-21 *TreatmentPlaque Density (% ofUnlesioned Side) Figure 4-7. APP/PS1 mice (9 months old; N=4-5/gp) had lower hippocampal amyloid density stained with 6E10 antibody after a combination of fimbrial lesion and 4OH-GTS-21 IP X2 for 2 wks than either treatment alone. *p<0.05 compared to unlesioned side; there was no difference between drug treatment groups in absence of lesion. Discussion

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73 This study investigated the 7 nicotinic receptor agonist effects on cholinergic and GABAergic neuron viability in wild type, APP/PS1 and PS1 mice receiving FFX-lesions. The aspirative FFX-lesions used in this study resulted in a substantial depletion of cholinergic markers in the mouse hippocampus though this depletion was less dramatic than reported previously, or than we saw based on AChE histochemistry, for rat. The extent of cholinergic depletion was the same for each group of saline-injected mice. Our data show that 4OH-GTS-21 could provide neuroprotection in PS1 lesioned mice, but no protection was observed in wild type or APP/PS1 lesioned mice treated with the drug. This difference may reflect additive or synergistic effects of PS1 and 7 nicotinic receptors. Kang et.al show that presenilins play important roles in Akt/GSK signaling and tau phosphorylation (Kang et al., 2005). The PS1 mutation was reported to enhance tau phosphorylation and reduces kinesin-based transport by increasing GSK-3 activity in primary neurons. However, the mechanism is not known for how the PS1 mutations could account for these effects. One factor may be the relative amount of PS1 vs. PS2 expression, since PS1 mutations led to a substantial reduction in PS2 fragment levels compared to wild type PS1. This could result in a corresponding decrease Akt and ERK activation by the PS1 mutation. Akbari et.al.(Akbari et al., 2004) reported that PS1 mutations lead to increased intracellular stores and an attenuation of capacity calcium entry. Thus, 7 nicotinic receptor activations may have additive effects on intracellular calcium levels with PS1, which, as noted in Chapter 3, is important for neuroprotection. They also demonstrated that in the presence of APP overexpression, an inverse relationship exists between gamma-secretase activities. Since PS1 overexpressing mice

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74 have increased levels of -Akt and ERK activation, both of which are important for 7 nicotinic receptor mediated neuroprotection, this may be another additive or synergistic interaction between these two systems. This may be the reason we saw the protection in cholinergic neurons in PS1 mice but not wild type mice. For the APP/PS1 mice, both the APP and PS1 are overexpressed. However, APP has a high affinity for blocking 7 nicotinic receptors. This could interfere with 4OH-GTS-21 binding to 7 nicotinic receptors, attenuating the drug-induced protection in APP/PS1 mice. If so, higher doses of this partial agonist or the use of a more efficacious agonist than 4OH-GTS-21 might still be effective. Alternatively, the pharmacokinetic properties of the 4OH-GTS-21 could be considered more carefully in designing the dosing in order to optimize the amount of drug at receptors over more extended intervals. As noted in Chapter 1, 4OH-GTS-21 is a relatively short-life drug. One interesting observation is that GABAergic neurons were not protected in the drug treatment group compared to the saline group. One factor is that the number of GABAergic neurons projecting to hippocampus is less than cholinergic neurons. Linke et al. showed that 38% of all retrograde labeled neurons were ChAT-positive whereas only 10% of all retrograde labeled cells were immunostained for parvalbumin (Linke et al., 1994). This is support our hypothesis. Another possible reason is that the proportion of GABAergic neurons might have been underestimated because immunostaining for parvalbumin only labels a subpopulation of GABAergic neurons. Mechanistically, however, these data suggest that some factor activated by 7 nicotinic receptors is more effective in cholinergic than GABAergic neurons. This factor may be NGF, which as

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75 noted above is protective for cholinergic but not GABA neurons in septum and which appears to be elevated by nicotine receptor activation. Given the toxicity of aggregated A to cells in vitro, it is perhaps surprising that even though A aggregates have been abundant throughout the brains of APP/PS1 mice for most of their lives, there is little neurotoxicity. One possible explanation for the apparent lacks of overt A toxicity in these mice involves the putative trophic effects of APP and PS1, which may counteract the degenerative effects of amyloid formation. Another explanation is that the effects of A aggregation in plaques may be very local. It is hard to discern local degeneration of neurons in close to plaques (Hernandez et al., 2001). 6E10 and thioflavine S staining were used to detect amyloid plaques in APP/PS1 mice with or without 4OH-GTS-21 treatments for two weeks. The density of plaques was slightly but significantly decreased in 4OH-GTS-21 treatment groups in the lesioned hemisphere compared to saline groups lesioned side, when both were normalized to the contralateral, unlesioned side. This normalization was necessary because of the wide inter-animal variability in plaque density, which was largely eliminated by this normalization. This result suggests that reducing septal input to the hippocampus combines with 7 nicotinic receptor activation to reduce plaque density. One hypothesis is that 4OH-GTS-21 treatments for 2 weeks may not be enough to decrease amyloid deposits as significantly as was reported for nicotine over a period of 3 months. We should also consider the alternative hypothesis that other nicotinic receptors are responsible for this action, either alone or in combination with 7 nicotinic receptors. A pharmacokinetic explanation is also possible; the short-term half-life of 4OH-GTS-21

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76 may not have permitted it to exert the same anti-plaque action when injected 2 X per day that was possible when nicotine was administered in the water, the route used previously to reduce amyloid plaques.

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CHAPTER 5 RAAV MEDIATED GENE TRANSFER IN VITRO AND IN VIVO Introduction Acetylcholine, acting through 7 nicotinic receptors, is an important modulator of electrical activity in the central nervous system and is involved in a variety of physiological processes and synaptic plasticity, including cognition and development (Volpicelli and Levey, 2004). The loss of cholinergic function has been implicated in AD. We already know that AChE inhibitors have in clinical studies shown palliative effects on symptoms and a trend to slow disease progression. But at the later stages of AD, the AChE inhibitors have little or no effect on AD. A consistent and significant loss of 7 nicotinic receptors has also been observed in neocortical autopsied brain tissue including hippocampus from AD patients compared to aged-matched healthy subjects. So the cholinergic receptors including 7 nicotinic receptors of the hippocampus are considered to be therapeutic targets for memory loss and dementia. 7 nicotinic receptors are highly expressed in the hippocampus and neocortex. Functional 7 nicotinic receptors in the hippocampus are mostly located at the cell bodies of the mossy cells and interneurons of the dentate gyrus. There is also evidence for the 7 nicotinic receptors being expressed on the synaptic terminals of interneurons and dendrites of pyramidal cells. This thesis tests whether increasing the density of 7 nicotinic receptors can be accomplished with gene delivery in a manner that is functional, long term, dose77

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78 dependent, and non-toxic. This would be a potential alternative method to the classic, pharmacological approach of increasing 7 nicotinic receptor activities with higher agonists concentrations. In our previous study, we showed that a high, 50 M concentration of the 7 nicotinic receptor selective agonist GTS-21 could cause cell toxicity in vitro (Li et al., 1999b). Although similar toxicity in vivo has not been seen and may be unlikely because of the very rapid desensitization of these receptors at such high concentrations, this desensitization would also be expected to interfere with efforts to increase 7 receptors function through high dose agonist regimens. Therefore, increasing the density of 7 nicotinic receptors is another promising target for therapy of AD, schizophrenia, and other conditions associated with dysfunctional 7 receptors. Despite pharmacological evidence that changes in 7-receptor activity affect behavior and cell survival, transgenic 7 knockout mice show few behavioral changes compared to wild type controls. One possible explanation of this observation involves compensatory processes to replace those normally mediated by the receptor. We have developed another approach to modify 7 receptors gene expression in adults, bypassing potential developmental compensatory processes, using the rAAV vector system. In this study, the rAAV is used as a gene delivery vector due to its apparent non-toxicity, ability to transduce postmitotic neuron and long-term expression gene (Robbins et al., 1998). A single injection into the hippocampus leads to widespread expression of transgene in interneurons and other neuronal types. The serotypes of rAAV2 and rAAV8/2 were compared in our study since little is known about the latter in brain and since it appears to be more effective with respect to spread of transgene in other tissues. The cells of the nervous system can be divided into two broad categories: neurons and a

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79 variety of supporting neuroglial cells. rAAV2 has been the most widely studied of the serotypes in brain and its tropism is neuronal predominantly. The possible mechanism of this tropism is either receptor-mediated or promoter-dependent selectivity. Early studies showed that rAAV2 mediated transgene expression occurred in hippocampal interneurons, and less strongly in hippocampal CA1 pyramidal neurons or dentate gyrus granule neurons. In this study, the rAAV8/2 vectors were also studied to determine the distribution and spread of the vectors in hippocampus. The rAAV2 and rAAV8/2 vectors used here both contained the chicken beta actin promoter combined with CMV enhancer. This is an extensively used promoter system for gene transfer in brain because of its high activity for extended intervals (Klein et al., 2002). We tested the rAAV mediated 7 nicotinic receptor gene deliveries in vitro and in vivo. For in vitro studies, rat pituitary tumor derived cells (GH4C1) were chosen. These do not express endogenous 7 receptors but can express functional 7 receptors after transfection because they synthesize the chaperone protein RIC-3. For the in vivo studies, wild type C57/Bl/J mice and Sprague Dawley rats, as well as 7 KO mice (strain B6.129S7-Chrna7 tm1Bay ) were used. The goal was to test the following hypotheses: 1) 7 nicotinic receptors could expressed in vitro and in vivo in a dose dependent and functional manner, with no greater toxicity observed than under normal conditions in the presence of an 7 agonist; 2) 7 nicotinic receptor gene transfer could restore function in KO mice; 3) different neuronal populations in the hippocampus are similarly able to express this transgenic receptor after transduction in vivo; and 4) 7 vector mediated gene transfer into hippocampus could improve memory related behavior analogous to agonist-treatment.

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80 Results The structures of the pUF12-rat 7-plasmid containing expression cassette are shown in Figure 5-1. The CBA promoter is the hybrid truncated CMV enhancer and chicken actin promoter. The size of the first plasmid from TR to TR was 5.1Kb that is slightly greater than the rAAV package size of 5 Kb. The pUF12-rat 7 plasmid without WPRE was used in subsequent parts of this study accordingly. The empty and GFP plasmids were controls as specified. TR CMV CBA Exon 1 Intron IRES GFP Poly A TR TR CMV CBA Exon 1 Intron Poly A TR TR CMV CBA Exon 1 Intron Rat alpha-7 WPRE Poly A TR TR CMV CBA Exon 1 Intron Rat alpha-7 Poly A TR Figure 5-1. Schematic diagram of the expression cassettes. To evaluate the ability of the plasmid constructs to code for rat 7 and GFP, GH4Cl cells were transfected with rAAV-rat 7 plasmid and pUF12 using a calcium phosphate method. GFP expression was observed 24 hours after transfection and around 15% of the cells expressed GFP at day 3. Rat 7 expressions was measured by ligand (MLA) binding assay at day 3 and found only in the 7 transfected cultures (Figure 5-2). In order to determine whether the 7 gene delivery had any effect on cell viability, either by itself or in the presence of a toxic agent, some of the transfected GH4C1 cells were also exposured to 20 M Abeta 25-35 for this 3 day interval. Cell viability was measured by cell counting using an NIH image system. GFP and 7 gene transfer alone had no effect on cell density, but 20 M Abeta 25-35 caused significant cell loss. Cells

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81 transfected with rat 7 receptors and then treated with Abeta 25-35 displayed no protection in total cell density, possibly because the transfection efficiency was too low and too few cells were protected overall in the culture. However, it appears that the few cells expressing these transgenic 7 receptors may have been protected. The 7 nicotinic receptor binding was 110 + 15 fmol/mg in the non-Abeta treated cultures and 217 + 20 fmol/mg in the Abeta 25-35 treated group (Figure 5-2). This increase in receptor density could reflect the relative sparing of cells expression 7 nicotinic receptors. Alternatively, the Abeta peptide may have acutely increased 7-receptor density by some other mechanism, though this has not been seen previously. GFPAlpha-7A-betaA-beta+GFPA-beta+Alpha7 -100 0 100 200 300 400MLA Binding( fmol/mg ) GFPAlpha-7A-betaA-beta+GFPA-beta+Alpha7 0 100000 200000 300000Cell Density (%) Figure 5-2. Transfection of GH4Cl cells with rat 7 nicotinic receptors:effect of Abeta 25-35 exposure on cell viability and receptor density. Cell viability was measured 72 hr later and expressed as the mean + SEM of 6-8 plates per group by cell counting using an NIH image system. The high affinity MLA binding to 7 nicotinic receptors was conducted in membranes from the same cultures. *p<0.05 compare to other group (one way ANOVA). The plasmids were packaged into rAAV2 and rAAV8/2 using the adenovirus-free method developed by Zolotukhin et al. (1999). The discontinuous iodixanol gradient was built (Figure 5-3A). After a two hour centrifugation, the rAAV distributed around the 40-60% density-interface (Figure 5-3B). A dot plot analysis was used to titer the number of

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82 total genomic particles (Figure 5-3B). This method quantified viral DNA by hybridizing with a biotin probe of pUF12 and by comparing the intensity of labeling against known quantities of plasmid pUF12-rat 7. The first and second lanes were standard curves. The highest band had 5 ng/l DNA with 1 ng = 4 x 1011 genomic particles/ml. The titer of rAAV2-rat 7 was 3 x 1012 genomic particles/ml. The titer of rAAV8/2-rat 7 was 4 x 1012 particles/ml. The titer of rAAV2-GFP was 1.5 x 1012 genomic particles/ml. The title of rAAV8/2 was 2x 1012 genomic particles/ml. AB AB Figure 5-3. Iodixanol gradient for the purification of rAAV and dot plots for titer. (A). Preformed gradients shown before (left tube) and after (right tube) the 2 hour centrifugation. (B). The first and second lanes were standard curves. The third and fourth lanes were samples. The different concentrations of rAAV2-rat 7 and rAAV2-GFP transductions of GH4C1 cells are shown in Figure 5-4. GFP expression was observed in culture within 24 hours. By day 5, over 90% of cell expressed GFP in the 30 x 10 9 genomic particles/ml treatment group. Functional rat 7 receptors expressions were detected by the [ 3 H] MLA binding assay. Increasing the concentration of vector resulted in more 7 nicotinic receptor binding.

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83 0 5 10 15 20 25 30 35 40 0 20 40 60 80 100transduction alpha 7 0 1000 2000 3000 4000 5000Genomic particles (x 109/ml)% GFP cellsMLA binding (fmol/kgprotein) Figure 5-4. The dose response of rAAV2-rat 7 receptor and rAAV-GFP. Binding assay was measured 5-day later and 6-8 plates per group. The expressions of 7 nicotinic receptors were compared among transduced GH4C1 cells and several other cell types using MLA binding. There were 960 fmol/mg protein expressed in rAAV2-rat 7 (5 x 10 9 genomic particles) transduced GH4C1 cells after 5 days (Figure 5-5). An 7 stable cell line (also GH4C1) had 750 fmol/mg protein expressed, while the PC12 cell line used for neuroprotection studies in Chapter 3 had 50 fmol/mg protein expression. There were approximately 100 fmol of receptor/mg protein expressed in pUF12-rat 7 plasmid transfected GH4C1 cells using the calcium phosphate method, although only a small fraction of these cells was transfected as noted above. This study demonstrated that the rAAV2-rat 7 was highly effective in increasing 7 nicotinic receptor expressions in GH4C1 cells.

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84 rAAV2-GFPrAAV-rat alpha7stable GH4C1PC12Alpha7 plasmid 0 250 500 750 1000TreatmentMLA Binding (fmol/mgprotein) Figure 5-5. 7 receptor expression was measured in different populations of transiently transfected, stably transfected, transduced, and normally expressing cells. 1.5 x10 12 genomic particles of rAAV2rat 7 and pUF12-rat 7 plasmid were used to introduce 7 receptors into GH4C1 cells. Binding assay were conducted 5-days after gene transfer. These receptor-binding values are compared to those of NGF-differentiated PC12 cells and stably transfected and selected GH4C1 cells. (N= 3 plates per group). After showing that rAAV2-rat 7 vectors were able to increase the rat 7 receptors expressing in vitro, the next step was to test whether this increase the rat 7-receptor density could cause toxicity in the absence or presence of increase the concentration of GTS-21 (Figure 5-6). GH4C1 cells were transduced with 2X109 genomic particles of rAAV2-rat a7 vectors and were exposed 5 days later to specified GTS-21 concentrations for another 3 days. PC12 cells were used as the positive control, though with much lower receptor density. GH4C1 cells transduced with rAAV2-GFP were used as the negative control. The binding assay was showed that PC12 had 50 fmol MLA binding/mg protein expressed. GH4C1 cells transduced with 2X109 genomic particles of rAAV2-rat a7 vectors had 750 fmol MLA binding/mg protein. The cell density was determined and normalized to drug-free control values (N = 3 plates/group). Despite much higher

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85 receptor binding density in transduced GH4C1 cells, there was no increase in agonist potency for toxicity. This suggests that toxicity depends on agonist concentration more than receptor density. It was previously established high agonist concentration were associated with much different receptor kinetics (i.e. peak + very rapid desensitization) than the low volume, steady state receptor activation caused by low agonist concentration. The present results indicate that the shape of this receptor response, which is agonist concentration dependent, is more important than the total number of receptors activated for affecting cell viability. Interestingly, GTS-21 appeared to increase cell density in GFP-transduced cells, though this effect did not reach significantly except at the lowest GTS-21 concentration. It may be that GTS-21 exerts an unknown action on these cells that is independent of 7 receptors. 0 10 20 30 40 50 60 70 0 100 200 300No Alpha7 Receptor + Alpha7 Receptor *PC12 Cells **[GTS-21], uMCell Viability (%untreated cells/field) Figure 5-6. Effects of GTS-21 on the viability of PC12 cells and 7 transduced GH4C1 cells. The cell viability was measure after exposure to GTS-21 for three days. It showed that 60 M GTS-21 could cause significant cell loss. The density of 7 receptor expression had no effect on cell viability. *p < 0.05 compared to same treatment with different concentration of GTS-21 (One-Way ANOVA). In order to investigate the in vivo effects of 7-receptor gene delivery in brain, a very low dose of 4 x 105 genomic particles of rAAV2-rat 7 with WPRE vector was injected unilaterally into male adult Sprague Dawley (SD) rats (250 g) hippocampus CA1

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86 region. Two weeks after injection, both sides of hippocampus were rapidly dissected from euthanized animals. The contralateral, uninjected side was the control. High affinity MLA binding increased in the hippocampus injected side compared to control (143 + 13% of contralateral control binding) (Figure 5-7). This result demonstrated that near physiological increases in receptor density were feasible with this gene delivery system, using a very low vector dose. UntreatedAlpha7 0 100 200HippocampusMLA binding ( fmol/mgprotein) Figure 5-7. Low vector dose: in vivo transduction with 7 vectors in hippocampus. 4 x 105 genomic particles of rAAV2-rat a7 were injected into hippocampus. High affinity MLA binding increased in the hippocampus 2 weeks after transduction with the rAAV2-rat 7. All values are mean + SEM of 4 animals. *P<0.05 compared to untreated group (t-test). Since one of my goals was to investigate the safety and function of 7 receptors in vivo, I next focused on the effects of high vector doses on this receptor expression. A higher dose of 5 x 109 genomic particles of rAAV2-rat 7 vectors without WPRE was injected unilaterally into male adult SD rat brain hippocampus CA1 region. Two weeks after injection, both sides of hippocampus were rapidly dissected from euthanized animals. The uninjected side was treated as the control for endogenous 7 receptors. MLA binding was dramatically increased in the injected side compared to controls

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87 (Figure 5-8A). An equal dose of rAAV2-GFP was also injected in rat hippocampus to determine the transduction efficiency with this type of vector (Figure 5-8B). Injected sideUninjected side 0 1000 2000 3000MLA binding (fmol/mgprotein) AB Injected sideUninjected side 0 1000 2000 3000MLA binding (fmol/mgprotein) AB Figure 5-8. Effects of a higher vector dose on 7 nicotinic receptor exprrssion in hippocampus. A. 5x109 genomic particles of rAAV2rat 7 were injected in the rat hippocampus. High affinity MLA binding increased in the injected side. B. rAAV2-GFP transduction was highly efficacious. *P<0.05 compared to untreated group (t-test). In order to identify and study exogenous 7 gene expression in the absence of endogenous receptors, 1.5 x 1010 genomic particles of rAAV2-rat 7 were injected unilaterally into the hippocampal CA1 region of 7 nicotinic receptor knockout mice. Two weeks after injection, each side of hippocampus and neocortex was assayed for MLA binding. Knockout mice on the injected side had high 7-receptor expression (500 fmol/mg protein), but no measurable binding on the uninjected side (Figure 5-9A). The 7 heterozygous mice had 1200 fmol MLA binding/mg protein expression in the injected hippocampus, but no nicotine displaceable MLA binding on the control side. The neocortex had 7-receptor expression in both knockout and 7 +/mice (Figure 5-9B). This study demonstrated that we could restore 7 receptors in knockout mice.

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88 0 500 1000 1500HippocampusMLA binding ( fmol/mgprotein) 0 250 500 750 1000KO mice injected side KO mice non-injected side alpha7 +/no treatment alpha7 +/injected side CortexMLA binding ( fmol/mgprotein)AB 0 500 1000 1500HippocampusMLA binding ( fmol/mgprotein) 0 250 500 750 1000KO mice injected side KO mice non-injected side alpha7 +/no treatment alpha7 +/injected side CortexMLA binding ( fmol/mgprotein) 0 500 1000 1500HippocampusMLA binding ( fmol/mgprotein) 0 250 500 750 1000KO mice injected side KO mice non-injected side alpha7 +/no treatment alpha7 +/injected side CortexMLA binding ( fmol/mgprotein)AB Figure 5-9. rAAV2-rat 7 vector gene transfer in 7 KO and 7 heterozygous (+/-) mice. 1.5 x 1010 genomic particles of rAAV2-rat 7 were injected into hippocampus of 7 KO or heterozygous mice. These results showed that the neuronal transgenic 7 receptor was expressed in hippocampus and, presumably through vector spread from the injection track through neocortex. and #p<0.05 compare to same genotype untreated side (t-test). Scatchard plots were used to calculate the Kd and Bmax values of MLA binding in the transduced hippocampus to determine whether there the binding properties of the transgenic receptors compared to endogenous receptors. 1.8 x10 10 or 3.6 x10 10 genomic particles were bilaterally injected into wild type mouse hippocampus. After two weeks, binding assays were conducted with varying concentrations of labeled MLA. The MLA Kd was found to range from 1.98~2.288 nM in these 3 treatments, indicating that the endogenous and transgenic receptors has similar MLA binding affinities (Table 5-1). The Bmax increased with the concentration of vector as expected.

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89 Table 5-1. Scatchard analyses of MLA Kd and Bmax in mouse with or without 7 knockout mice gene transfer Vector (genomic particles) Kd (nM) Bmax (fmol/mg protein) 0 2.228 23.24 1.8 2.062 855.5 3.6 1.98 926.4 Table 5-1. MLA Kd and Bmax. Each treatment had 5 mice/per group. The immunohistochemistry of wild type rats, wild type mice and 7 KO mice hippocampus is shown in Figure 5-10. The left panel shows the wild type rat CA1 region. The middle panel shows the wild type mice CA1 region. The right panel shows the knockout mice hippocampus. This study demonstrated that no 7 receptors could be detected in knockout mice, while there was endogenous 7 receptor expression in wild type rat and mice. Figure 5-10. mAB 306 immunohistochemical staining of rat, mice and 7 KO mice. 1.8 x10 10 genomic particle of rAAV2-rat 7 were unilaterally injected into knockout mice septum. Two weeks after injection, knockout mice were perfused and dissected and sections analyzed. Figure 5-11 shows the immunohistochemical staining of septum after injection.

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90 Figure 5-11. FITC-staining of knockout mouse septum after injection. The left panel shows that the injection side had 7 receptor expression. The middle panel is a high magnification of the injection side. The right panel is a cofocal image of injection side. In order to increase vector spread in brain in vivo, an rAAV8/2-rat 7 was generated. As noted above, this vector was reported to have greater spread than rAAV2 in brain and some other tissues. It should be noted that in preliminary studies, we found it less effective in vitro than rAAV2, which is the reason that we continued using rAAV2 for those studies. Since little is known about rAAV8/2 mediated gene transfer in brain, rAAV8/2-GFP was first evaluated for neuron specificity (Figure 5-12). No colocalization of GFP and astrocytes stained with GFAP was observed, indicating that rAAV8/2 transduced neurons primarily. This agrees with the morphological assessment of the GFP expressing cells, which was exclusively neuronal in hippocampus and neocortex. However, this study did not eliminate the possibility that other brain glia (microglia, oligodendrocytes) may have been transduced as well.

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91 Figure 5-12. rAAV8/2 transduction in rat brain. The left panel was stained with GFAP (red). The middle panel is GFP expression. The right panel shows merged astrocytes (red) and GFP expressing cells. In order to determine whether there was any toxicity caused by 7-receptor gene delivery into hippocampus, several measurements were used. First, fluorojade labeling of the hippocampus found no evidence of dying neurons two weeks following injection of a high dose of vector (3.6 x 1010 genomic particles of rAAV8/2-rat 7). Since 7 receptor binding density was very high at this 2 weeks interval, it is not likely that the receptor transduced neurons had already died. In another line of study, several animals transduced with this 1.8 or 3.6 x 1010 genomic particles of this vector (2 animals/dose) underwent Morris water task analyses and were found to be unaffected compared to control mice, indicating no apparent dysfunction in that complex spatial memory related behavior. In order to determine if the increase in 7 nicotinic receptor bindings after gene transfer in hippocampus was functional, electrophysiological responses were used. 1.8 x 10 10 genomic particles of rAAV8/2-rat 7 combined with 0.6 x 1010 genomic particles of rAAV8/2-GFP were injected together unilaterally into mouse hippocampus. The contralateral side received 0.6 x 1010 genomic particles of rAAV8/2-GFP only. Two weeks after injection, fresh hippocampus was rapidly removed and its single cell electrophysiological responses were measured. GFP-transduced cells were identified

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92 with a fluorescence microscope. The whole-cell current in response to 1 mM acetylcholine was near 5,000pA in transduced cells on the injected side (Figure 5-13 B). GFP-transduced neurons in the contralateral hippocampus had a smaller, conventional 7 type current (Figure 5-13 A). Figure 5-13. Electrophysiological responses in wild type mouse hippocampus following 7 gene delivery (A): 1 mM acetylcholine produces a typical 7 type current in a hippocampal stratum radiatum interneuron in control tissue. (B): A neuron located in the contralateral hippocampus, which received 7 gene delivery. The response is nearly 50 times greater in peak amplitude than that shown in A. The whole-cell current in response to acetylcholine is near 5000 pA. The wild type mouse and rat hippocampus dentate granule cells normally do not express 7 receptors and typically have no response to 1 mM acetylcholine in this region (Figure 5-14 Cell 1). When 1 mM acetylcholine was added to the injected hippocampus, there was a significant response in dentate granule cells (Figure 5-14 Cell 2), indicating that this gene delivery was effective even in typically non-7 receptor expressing neurons.

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93 Figure 5-14. Electrophysiological responses in wild type dentate granule rat cells. Response to 1 mM ACh (scale bars: vertical 100pA/horizontal 100 ms). Cell 1 is from GFP-transduced hippocampus. Cell 2 is in 7 receptor transduced side. In order to determine whether 7 gene delivery restored receptor function in 7 knockout mice, 2-month-old 7 knockout mice were injected with rAAV8/2-rat 7 (1.8 x10 10 genomic particles) combined with 0.6 x 1010 genomic particles of rAAV8/2-GFP in the hippocampus in order to detect likely transduced neurons. Rreceptor-transduced neurons from this region had evoked 7 nicotinic receptor responses in the presence of 1 mM acetylcholine, and this response was completely blocked with 50 nM MLA (Figure 5-15). Figure 5-15. Evoked 7 nicotinic receptor responses in 7 knockout mice transduced with rat 7 nicotinic receptors. Recording from stratum radiatum interneuron, which had a response in the presence of 1 mM acetylcholine. This response was completely blocked with 50 nm MLA (scale bars 1 nA and 100 ms).

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94 Dentate granule cells and pyramidal cells are populations of hippocampal neurons that do not normally produce functional 7 nicotinic receptors. Two-month-old mice were injected with 1.8 x 10 10 genomic particles of rAAV8/2-rat 7 receptor together with 0.9 x10 10 genomic particles of rAAV8/2-GFP, or with 0.9 x10 10 genomic particles of rAAV8/2-GFP alone, in hippocampus. One week after injection, dentate granule cells expressed GFP (Figure 5-16). Typical 7 response kinetics was produced by somatic application of 1 mM ACh in these GFP-labeled neurons (Figure 5-16 middle panel). This response was blocked by 50 nM MLA, demonstrating 7 selectivities (Figure 5-16 right panel). Figure 5-16. Histology following electrophysiological recording: 2 month old mice that received gene transfer with rAAV8/2 -rat 7 + rAAV8/2-GFP. 10X image of LA Simid with 1.8 x 10genomic particles of rAAVhowed s in Cell 1Cell 9 Cell 1Cell 9 dentate granule cells expressing GFP/rat 7. Middle panel: typical 7 response kinetics produced by somatic application of 1 mM acetylcholine. Right panel: acetylcholine evoked response was blocked with 50 nm M(scale bars: 50pA and 100ms). larly, three week old rats injecte10 8/2-rat 7 + 0.9 x1010 genomic particles rAAV8/2-GFP into hippocampus s7 functional responses in pyramidal cells. 300-micron sections were used for electrophysiological recordings. CA1 pyramidal layer cells expressing GFP were selected for recordings (Figure 5-17). 1 mM acetylcholine evoked somatic current

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95 CA1 pyramidal cells that were blocked by 50 nm MLA (Figure 5-17). CA1 Pyramidal cell, evok Figure 5-17. Electrophysiological recording of a CA1 pyramidal neuron in the ed somaticresponse hippocampus of a 3 week old 7 receptor-transduced mouse. Dark tracing: ) blocked The results of this study indicateime that 7 nicotinic receptor expreor 7 7 the functional vector-mediane fer current in the presence of 1 mM acetylcholine. MLA (lighter tracingthe current. Scale bars: 25 pA and 100 ms. Discussion for the first t ssion and function can be modulated in a dose-dependent, non-toxic manner fextended intervals by gene delivery. Further, this gene delivery approach can restore receptor function to brain neurons in receptor knockout animals deficient in their endogenous expression. It therefore appears that this approach may provide an alternative approach for treating conditions associated with dysfunction of the nicotinic receptor system, including AD and schizophrenia. There have been very few publications demonstrating ted gene delivery of any plasma membrane receptors in brain in vivo, and nousing rAAV. Bahi et al. (Bahi et al., 2004) used lentivirus vector mediated gene transto express D3 dopamine receptors into the nucleus accumbens, with resultant changes in cocaine-induced locomotion. This work demonstrated that vector mediated gene transfer could be functional even in the complex and highly regulated milieu of the brain, at least

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96 for some receptors. Our results indicate that this is likely to be true for 7 nicotinic receptors as well in multiple brain regions. One of the advantages of using rAAV2 vectors for brain has been their selectivity for neurons, permitting selective genetic modification of that cell type. Since 7 nicotinic receptors are primarily neuronal under most conditions, this vector is appropriate for attempting to restore 7 receptor functions without unwanted expression other cell types. However, little is known about cell type selectivity for rAAV8/2 vectors in brain. The present data indicate that this novel hybrid serotype also has neurotropism in brain since glias were not transduced with GFP. Further, the spread of transgene expression with rAAV8/2 was greater than that with rAAV2 based on qualitative evaluations following hippocampal injections, so rAAV8/2 became the system of choice in later functional studies involving ligand binding, electrophysiology, and behavior. One of the concerns about 7-receptor gene transfer into neurons has been the apparent requirement for the chaperone protein RIC-3 for functional expression of the receptor. RIC-3 is expressed by GH4C1 cells, unlike many other cell lines that do not normally synthesize 7 nicotinic receptors. This appears to account for the ability of these cells to express MLA-binding, plasma membrane associated, 7 receptors following gene transfer of the receptor, which is not seen in RIC-3 negative cells. Cell lines that intrinsically express 7 receptors (e.g., PC12 cells), also express RIC-3. Based on these observations, it was not clear whether 7 receptors would be expressed in neurons in which these receptors are not normally seen, such as hippocampal dentate gyrus or pyramidal neurons. Our observation that both of these cell types express

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97 functional 7 receptors in the rat only after 7 gene transfer indicates that the rate limiting step in their production is not normally RIC-3 but the receptor itself. Another concern about 7 gene transfer was whether it led to functional expression in adult knockout mice that had never expressed the receptor. Conceivably, these animals had developed compensatory processes that simultaneously interfered with ectopic receptor expression. The results of this study indicate that knockout mice could express ectopic 7 receptors at high levels after rAAV gene delivery. However, the level of this expression appeared to be lower than was seen in identically treated wild type mice. This reduction was seen in total hippocampal high affinity MLA binding levels, but was even more evident in single neuron electrophysiology, in which responses to acetylcholine by transduced KO mouse hippocampal interneurons were much less than those seen in wild type mice receiving the same 7 vector dose. While the mechanism underlying this differential level of functional 7 receptors is not clear, it may involve intrinsic modulatory factors recently found to affect these receptors. The levels of transgenic 7 receptors binding in hippocampus were dependent on vector dose over a wide range. At the highest rAAV 8/2 vector doses used, which required vector high titers, there were large, 30-50 fold increases in receptor binding throughout the entire hippocampus and in electrophysiological responses of individual neurons that express the receptor intrinsically. This comparable increase in 7-receptor expression in individual neurons and throughout the hippocampus is consistent with the widespread distribution of the rAAV8/2 vector in this region seen using GFP-expression. A lower, more physiological level (40-50% over control values) of transgenic 7 receptor expression could also be produced in rat hippocampus, though this required a much lower

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98 (10,000-fold) dose of vector. It should be noted that this lower expression of 7 receptors was seen using a different serotype (rAAV2) that also contained the WPRE insert to stabilize mRNA and potentially increase receptor expression. A comparable low, physiological level of 7 transgene expression has not been determined yet for rAAV8/2 because the focus of this project was to evaluate the efficacy and safety of the 7 receptor gene transfer under maximal expression conditions. This permits a more conservative determination of the safety than would be possible with much lower receptor expression, though both approaches will ultimately be important. The 7-receptor MLA binding characteristics in hippocampus were comparable between rAAV8/2 transgenic and wild type 7 receptors. Despite the much higher Bmax values for the vector treated mice, the MLA-binding affinities (Kds) were similar between these groups. This is important because it indicates: 1) that the processing of the plasma membrane transgenic receptor is comparable to that of the wild type; and 2) the binding affinities of drugs for wild type receptors are likely to be predictive of their activities at the transgenic receptors. Therefore, differences in the dose-response characteristics to agonists between wild type and transgenic receptors will more likely reflect the different densities of the receptors. Since, as noted in Chapter 3, 7-receptor density can affect neuroprotection, this will be an important area for investigation in vivo following gene delivery leading to very high levels of the receptor expression. The apparent non-toxicity of the gene transfer of 7 receptors was demonstrated in several manners. In vitro, overexpression of these receptors had no effect on the GTS-21 induced dose-response curve for acute toxicity. This result indicated that the dose-dependent agonist-receptor interactions were more important than the receptor density in

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99 determining cell survival. In vivo, there was no apparent loss of 7 receptor binding density over several weeks, suggesting that the cells expressing these receptors were intact. Further, there was no fluorojade labeling of dying neurons following the high dose 7 receptor gene delivery. These results suggest that restoration of neuroprotection will be possible using 7 gene transfer under neurodegenerative conditions in which 7 receptors are low, without the toxicity or desensitization associated with high agonist concentrations. The observation that receptor-mediated toxicity is agonist concentration dependent more than on receptor density is important for predicting the effects on cell survival of different approaches for increasing nicotinic receptor expression. For examples, it suggests that receptor modulator that are being developed commercially to increase 7 receptor function by increasing its opening time may be no more toxic when combined with an agonist than the agonist would be alone. Several agents have been found to increase 7-receptor function (e.g., 5-hydroxyindole) without being agonists themselves, and these may provide a future therapeutic approach without undue toxicity based on my results with gene transfer. With respect to 7 receptor mediated neuroprotection following gene transfer, the increase in high affinity MLA binding expressed per mg protein in cells transfected with 7 receptors and treated with ABeta peptide vs. those not treated with Abeta is consistent with the receptor-mediated sparing of transfected cells. Selective protection of the 7-receptor expressing cells from Abeta would result in a higher receptor/total cell protein ratio. While there was no accompanying increase in cell density in this 7-receptor transgene treatment group, this may reflect the low transfection efficiency for these cells

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100 (under 10%). An alternative explanation is that the 7-receptor density was elevated directly by Abeta peptide exposure. This possibility is supported by the observation that alpha7 receptor binding density is elevated in the brains of Abeta-overexpressing transgenic mice, at least for several months. Unfortunately, it is difficult to use behavioral analyses such as the Morris water task for most purposes other than demonstrating a lack of toxicity relative to memory related behavior. This is because of several reasons. First, the 7 knockout mouse does not show deficits in this behavior, so a gene delivery approach to improve the behavior would be difficult to interpret. Even more difficult to interpret would be an analysis of the effects of 7 gene transfer into wild type animals, since this gene transfer increases receptor expression in neurons that do not normally synthesize the receptor. Since 7 receptors are activated by choline, which is ubiquitous in brain, any of these ectopic receptors could have effects on neuronal circuitry that affect memory. Therefore, it would be difficult to determine whether increased levels of normal transmission or to this abnormal transmission caused any improvement in behavior. While these studies indicated that 7 nicotinic receptor genes could be functionally delivered into brain neurons using rAAV vectors, several important questions remain unanswered regarding their potential for therapeutic use. One issue is whether there may be toxicity over more extended intervals than those used here, up to 2 weeks posttransduction. Another is whether the 7-receptor gene transfer improves memory or is neuroprotective in impaired individuals. One way to do this study would be to investigate the effects of gene transfer into the APP/PS1 transgenic mice receiving fimbrial fornix lesions as described in Chapter 4. Since Dr. Meyer and his colleagues

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101 recently demonstrated that lesions reduce 7-receptor function in hippocampus, it would be important to determine whether 7-receptor gene delivery could counteract this deficit, even in mice that have significant amyloid loads. A third issue about the use of gene therapy targeting 7 nicotinic receptors is whether there may be behavioral or physiological side effects caused by these receptors being expressed in excessively high levels or in neurons that do not normally express them. This possibility would require careful analyses of many potential behaviors and physiological processes. While this could be very much work, it is also possible that it could lead to new ways to modulate these other behaviors. CHAPTER 6 CONCLUSIONS AND FUTURE STUDIES Conclusions The goal of this project was to improve our understanding of the mechanisms of the 7 nicotinic receptors mediated neuroprotection in vitro and in vivo and test whether the 7 nicotinic receptors gene delivery could become a potential approach to treat AD. This involved, testing several hypotheses: 1) that intracellular calcium, calcium channels, and several kinase-systems are necessary for 7 nicotinic receptormediated neuroprotection; 2) that an 7 nicotinic receptor agonist protected lesioned septal cholinergic neurons in

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102 vivo; 3) that rAAV mediated 7 nicotinic receptor gene transfer was functional in a non-toxic manner in vitro and in vivo. When 7 agonists bind to the 7 receptors, they trigger the calcium entry into cells. Calcium ion then can trigger PKC and PKA activation, as well as ERK1/2 phosphorylation. These processes were all found to be important for cytoprotection. PKC activation was necessary for protection in the NGF/serum-withdrawal model in PC12 cells but not in the Abeta 25-35 exposed SK N SH model. But the PKA activity was important in both models. ERK1/2 phosphorylation levels were increased when 7 receptors were activated. Neither p38 nor the JNK pathway was apparently activated by a protective concentration of 7 agonists. Intracellular calcium ions were necessary for 7 nicotinic receptor mediated protection based on data that showed that intracellular calcium chelation BAPTA blocked GTS-21 induced protection. Our data showed that BAPTA exposure attenuated GTS-21 induced PKC activation. PKC activation was found to be essential for 7 mediated protection in this apoptotic model. Multiple channels modulate intracellular calcium levels. Our data showed that activations of the intracellular IP3 calcium channel and to a lesser extent the ryanodine receptor were necessary for complete 7 mediated protection. But the L-type calcium channels were not essential for 7 receptor mediated protection. One possible reason is that L-type channels may not be open long enough for neuroprotection as they close upon repolarization of the cell. The effects of the 7 receptors selective agonist 4OH-GTS-21 on cholinergic and GABAergic neuron viability in wild type, APP/PS1 and PS1 mice receiving FFX-lesions were investigated. The data showed that 4OH-GTS-21 could provide cholinergic

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103 neuroprotection in PS1 lesioned mice, but no protection was observed in wild type or APP/PS1 lesioned mice treated with the drug. Our hypothesis is that there may be additive effects of mutant PS1 overexpression and 7 nicotinic receptor activations. There are reports showing that overexpression of this PS1 mutation leads to increased intracellular calcium stores and an attenuation of capacity calcium entry, as well as ERK1/2 phosphorylation. Thus, 7 nicotinic receptors activation may have additive effects on intracellular calcium levels and kinase systems with PS1. These intracellular calcium levels are important for cell survival as noted above. We did not observe neuroprotection in APP/PS1 mice, perhaps because the APP-derived peptides have high affinity for blocking 7 nicotinic receptors and may therefore interfere with drug-induced protection. GABAergic neurons were not protected in the drug treatment group compared to the saline group. This may be related to the small fractions of GABAergic neurons lost in the lesions, which in turn reflects the smaller percent of total septal GABAergic neurons projecting to hippocampus. One reason why activated 7 nicotinic receptors is more effective in cholinergic neurons than GABAergic neurons is that nicotinic receptor mediated increases in NGF release may be involved. As noted above, NGF is protective for cholinergic but not GABA neurons in septum and appears to be elevated by 7 nicotine receptor activation. For the amyloid plaques, our data suggests that reducing septal input to the hippocampus combines with 7 nicotinic receptor activation to reduce plaque density, while neither one alone has this effect. Based on previous studies with nicotine showing amyloid reductions, this was a surprising result. One hypothesis is that 4OH-GTS-21

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104 treatments for 2 weeks may not be long enough to decrease amyloid deposits in intact animals. An alternative hypothesis that other nicotinic receptors are responsible for this action, either alone or in combination with 7 nicotinic receptors. Why denervation of the hippocampus from septal projections would combine with 7 nicotinic receptors activation to reduce amyloid plaques is unclear. One possibility is that transmitters released from the septal projections (e.g., acetylcholine, GABA, neuropeptide transmitters) counteract the effects of the 7 nicotinic agonists, so that removal of this transmission permits the 7 agonists to be effective. If so, this observation would suggest that 7 nicotinic receptors activation would be particularly effective even in the early stages of AD when septal dysfunction is already observed. One potential pathway of 7 nicotinic receptors mediated neuroprotection is described in Figure 6-1 based on the results in aim #1 and aim #2, as well as other data in the literature. 7 agonists 7 nicotinic receptors APP Ca2+ IP3, Ryanodine receptorsPKCMEKERK 1/2 Cell Protection AKTPS1 -Ca2+ 7 agonists 7 nicotinic receptors APP Ca2+ IP3, Ryanodine receptorsPKCMEKERK 1/2 Cell Protection AKTPS1 -Ca2+ Figure 6-1. The potential pathway of 7 nicotinic receptors mediated neuroprotection.

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105 This thesis also demonstrated for the first time that rAAV mediated gene transfer is a feasible way to deliver a plasma membrane receptor transgene into rat or mouse brain for long-term expression that can be modulated in a dose-dependent manner without producing degenerative neurons. Functional receptors were restored into the 7-knockout mouse using this vector approach as well. Therefore, it appears that this method may provide a way to treat deficits in 7 receptors. In my study, rAAV8/2 had greater spread than rAAV2, though both were neuron selective. The levels of 7 receptors expressed in hippocampus were dependent on the vector dose. One of the interesting observations is that functional 7 nicotinic receptors were expressed after gene transfer in the hippocampal dentate gyrus or pyramidal neurons that normally do not have functional 7 nicotinic receptors. This result indicates that the rate-limiting step in 7 productions is not normally the RIC-3 chaperone or other potential factors necessary for receptor expression, but the receptor itself. This had been a matter of some speculation in the field. No apparent toxicity due to the gene transfer of 7 receptors was observed in vitro or in vivo. In vitro, overexpression of these receptors had no effect on the GTS-21 induced dose-response curves for acute toxicity compared to those seen at physiological receptor levels. This result indicated that the dose-dependent agonist-receptor interactions were more important than the receptor density. In vivo, there was no fluorojade labeling of dying neurons after gene transfer 3 months after gene transfer.

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106 Future Studies More studies will be need to determine the complex relationship between APP and 7 receptors, and the functions of 7 receptors expressed after gene transfer in the 7-knockout mice, e.g., with respect to neuroprotection in the septum after FFX-lesions. In chapter 4, we did not observe that 4OH-GTS-21 provided neuroprotection against FFX-lesioned APP/PS1 mice. Our hypothesis is that APP binding to the 7 receptors blocked the protection. In future studies, higher doses of the agonist could be injected to overcome this lack of protection or the rAAV-rat 7 vector could be injected to septum of APP/PS1 mice to increase drug-sensitivity relative to neuroprotection, receptor expression and behavior. The goals of this future study would be to test: 1) whether the septum cholinergic neurons are protected in this AD model; 2) whether the up-regulation of astrocyte 7 receptors in APP/PS1 mice is blocked by 4OH-GTS-21; 3) whether behavioral improvements are seen in the APP/PS1 mice following gene delivery; and 4) whether the amyloid plaques density could be reduced or prevented by the gene expression. A novel way to test 7 receptors function is restore the 7 receptors back into the 7-knockout mouse hippocampus and septum. One important study would be to test whether restoring functional 7 receptors in the knockout mouse also restores the neuroprotective effects of 4OH-GTS-21 and this drugs effects on septal cholinergic neuron viability, and second messenger systems such as the ERK1/2, PHA and PKC pathways.

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BIOGRAPHICAL SKETCH Ms. Ke Ren received her Doctor of Medicine at Zunyi Medical University in July 1994. And later Ke became a clinical doctor/researcher in ophthalmology in Guiyang Medical School Hospital for four years. Ke then joined the graduate program in the Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, Florida, in Jan. 2002. She finished her Ph.D. in pharmaceutics in Dec. 2004 under supervision of Dr. Jeffery Hughes. 116


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EFFECT OF ALPHA7 NICOTINIC RECEPTOR ACTIVATION AND OVER-
EXPRESSION IN BRAIN AND IN PC12 CELLS
















By

KE REN












A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


2005


































Copyright 2005

by

KE REN

































This document is dedicated to my parents: Xilin Ren and Aijuan Wang and my husband
Jiang Liu.















ACKNOWLEDGMENTS

I would like to express my sincere appreciation and grateful thanks to Dr. Jeffrey

A. Hughes for guiding me through the four years of Ph.D. study, giving me many

excellent suggestions and supporting me in every respect. I am also extremely grateful

for Dr. Edwin Meyer for providing a great environment in his laboratory and mentorship.

He opened the door for me to explore the opportunities in research areas of nicotinic

receptors and gene delivery. I would like to thank the members of my supervisory

committee, Dr. Sean Sullivan and Dr. Sihong Song, for their valuable and kind advice

throughout my doctoral research. I take this opportunity to express my gratitude to Dr.

Mike King, Aaron Hirko, Craig Meyers, Jeffrey Thinshmidt and Dr. Yan Gong for all

their help in conducting studies and in understanding them.

I also would like to extend my thanks to all the graduate students, exchange

students, and post-doctoral fellows in the Department of Pharmaceutics who were my

colleagues and friends for their support. Finally, I thank my husband Jiang, my parents

and my sister for supporting me in many, many ways.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

L IST O F T A B L E S ........ ............................................... .. ...... .. .. ...... ............. .. vii

LIST OF FIGURES ............. ...................... ................. .... .... ............. viii

ABSTRACT .............. .......................................... xi

CHAPTERS

1 IN TR OD U CTION ............................................... .. ......................... ..

A lzheim her's D disease ............................................... .... ..... .............. ..
a 7 N icotinic R eceptors ...................................................... ..... ........................ 5
c7 Nicotinic Receptors: Effects On Cell Viability in vitro.......................................7
a7 Nnicotinic Receptors: Neuroprotection Properties in vivo............... ............... 9
3-benzylidene- and 3-cinnamylidene-anabaseine Compounds ..................................9
a7 Nicotinic Receptors and Memory Related Behaviors...........................................11
Models Of a7 Nicotinic Receptor Dysfunction: a7 Receptor KO Mice And
Septohippocam pal Lesions ............. .................................................................. 12
a7 Nicotinic Receptor And Beta Amyloid......................................................15
M em ory R elated B ehavioral Tests ........................................ ......... ............... 16
rAAV Mediated Gene Transfer Into Brain......................................................17
Specific A im s........................................................................................ 20

2 M ATERIALS AND M ETHOD S ........................................ ......................... 21

Construction of rA A V Plasm ids .......................................................... ............... 21
S u b c lo n in g ......................................................................................................2 2
Plasm id Preparations ............................ ......... .. ..... ....... .... .............. 24
Packaging rA A V V sectors A nd Titration ........................................ .....................26
Cell Transfections .................................................................................... ........ ..... ........ 31
Stereotaxic Surgeries ......................... .................. ................... ........ 32
High Affinity [3 H] MLA Binding Assay ....................................... ............... 32
Im m unohistrochem istry ............................................................... ......... ................ 34
FluoroJade Staining .......................... .............. ................. .... ... ....35
W estern-blot ..................... ........... .. ............ .. .............35
M o rris W after T a sk ................................................................................................ 3 6









Electrophysiological Recordings ....................................... ................... .......... 37
Fimbria Fornix Lesions And 40H-GTS-21 Injections............... ........ ..............38
D ifferentiation O f PC 12 Cells ......................................................... ............... 39
Protein K inase C (PK C) A ssay ......................................................... ............... 40
Tail DN A Extraction And Genetype ........................................ ........................ 41
S statistical A n aly ses ................................................................. ...... .......... .. 4 2

3 MECHANISMS UNDERLYING ALPHA7 NICOTINIC RECEPTOR
NEUROPROTECTION IN PC12 CELLS ........................................ ............... 43

Introduction ....................................................................................... .. .. .... 43
R e su lts ...................................... .................................................... 4 5
D iscu ssio n ...................................... ................................................. 5 4

4 NEUROPRECTIVE AND ANTI-AMYLOIDOGENE EFFECTS OF THE
ALPHA7 PARTIAL AGONIST 40H-GTS-21 IN FIMBRIA FORNIX
LESIONED MICE OF DIFFERENT GENOTYPES .................................................61

In tro d u ctio n .......................................... ....... ................. ................ 6 1
R esu lts ......... ...... ........... ...................................... ............................6 6
D iscu ssion ......... .................. .................................... ............................73

5 RAAV MEDIATED GENE TRANSFER IN VITRO AND IN VIVO.......................77

In tro d u ctio n ............ .................................................. ................ 7 7
R results ............. ......... .. ............. ... ............................................80
D discussion ................ ...... ...................................................... ....... 95

6 CONCLUSIONS AND FUTURE STUDIES .................................. ...........101

C o n clu sio n s.................................................... ................ 10 1
Future Studies ....................... ....................... ................ ........ 106

LIST OF REFEREN CES .. ................................................................. ............... 107

BIOGRAPHICAL SKETCH ........................................................................... 116
















LIST OF TABLES


Table p

1-1. Mean SD Pharmacokinetic parameters for GTS-21 after oral administration of
25, 75 and 150 mg of GTS-21 for 5 Days......................................... 11

1-2. Mean SD Pharmacokinetic parameters for 40H-GTS-21 after oral
administration of 25, 75 and 150 mg of 40H-GTS-21 for 5 Days ........................11

3-1. Effects of BAPTA and GTS-21 on a7 receptor binding density in PC12 cells ........48

5-1. Scatchard analyses of MLA Kd and Bmax in mouse with or without a7 knockout
mice gene transfer .................................... .. .. ..... .. ............89
















LIST OF FIGURES


Figure p

1-1. Chemical structures of GTS-21 and 40H-GTS-21. ................................................10

2-1. T he 7 genotype of m ice .................................................................. .............. 42

3-1. GTS-21 induced protection of PC12 cells during trophic factor deprivation.............46

3-2. Effects of intracellular calcium chelation on GTS-21 induced protection of PC12
cells during trophic factor deprivation. ....................................... ............... 47

3-3. Effects of intracellular calcium chelation on PKC activation by GTS-21 in PC12
c e lls ........................................................................... 4 8

3-4. Effects of 40H-GTS-21 on PKC isozyme translocation............. .................49

3-5. Effects of calcium channel antagonists on GTS-21 induced cytoprotection in
N GF-deprived PC12 cells. .................................. .......................................50

3-6. Effects of GTS-21 on the phosphorylation of several MAP kinases in PC12 cells. ..51

3-7. MLA blocks the ERK1/2 phosphorylation triggered by GTS-21.............................. 52

3-8. GTS-21 induced cytoprotection is dependent on ERK phosphorylation and PKC
activation .............................................................................53

3-9. Effects of 40H-GTS-21 and various kinase inhibotor on PC12 cells...................53

3-10. Effects of Abeta 25-35, 40H-GTS-21 and various kinase inhibitor on SK N SH
c e lls ........................................................................... 5 4

3-11. The potential mechanism of a7 nicotinic receptors mediated cytoprotection..........60

4-1. Aspirative FFX-lesion of the septal hippocampal cholinergic pathway...................67

4-2. Septal ChAT neuron staining in 9 month old mice. ................................................68

4-3. Septal ChAT-staining perilarya in 9 month old PS1, APP/PS1 and wild type
C57/B 16/J mice two weeks after unilateral aspirative FFX-lesions ...................69

4-4. Septal GABAergic neuron staining in 9 month old mice ........................................70









4-5. Septal GABAergic staining perilarya in 9 month old PS1, APP/PS1 and wild type
C57/B 16/J mice two weeks after unilateral aspirative FFX-lesions .......................71

4-6. The thioflavine S and 6E10 staining in 9 month old APP/PS1 mice .......................72

4-7. APP/PS1 mice (9 months old; N=4-5/gp) had lower hippocampal amyloid density
stained with 6E10 antibody after a combination of fimbrial lesion and 40H-
GTS-21 IP X2 for 2 wks than either treatment alone ...........................................72

5-1. Schematic diagram of the expression cassettes. ........................ .. .. ............... 80

5-2. Transfection of GH4Cl cells with rat a7 nicotinic receptors:effect of Abeta 25-35
exposure on cell viability and receptor density ........................................... ........... 81

5-3. Iodixanol gradient for the purification of rAAV and dot plots for titer. ....................82

5-4. The dose response of rAAV2-rat a7 receptor and rAAV-GFP.............................83

5-5. a7 receptor expression was measured in different populations of transiently
transfected, stably transfected, transduced, and normally expressing cells ............84

5-6. Effects of GTS-21 on the viability of PC12 cells and a7 transduced GH4C1 cells. .85

5-7. Low vector dose: in vivo transduction with a7 vectors in hippocampus .................86

5-8. Effects of a higher vector dose on a7 nicotinic receptor expression in
hippocam pus........................................................................ ... ...... ....... 87

5-9. rAAV2-rat a7 vector gene transfer in a7 KO and a7 heterozygous (+/-) mice........88

5-10. mAB 306 immunohistochemical staining of rat, mice and a7 KO mice ...............89

5-11. FITC-staining of knockout mouse septum after injection ..................................90

5-12. rAAV 8/2 transduction in rat brain ................................................... ................91

5-13. Electrophysiological responses in wild type mouse hippocampus following a7
g en e deliv ery .......... ........................... ..........................................92

5-14. Electrophysiological responses in wild type dentate granule rat cells ...................93

5-15. Evoked a7 nicotinic receptor responses in a7 knockout mice transduced with rat
a7 nicotinic receptors. ...................... .. ...................... .. .... .... ............... 93

5-16. Histology following electrophysiological recording: 2 month old mice that
received gene transfer with rAAV8/2 -rat a7 + rAAV8/2-GFP.............................. 94









5-17. Electrophysiological recording of a CAl pyramidal neuron in the hippocampus
of a 3 week old a7 receptor-transduced mouse............................................ 95

6-1. The potential pathway of c7 nicotinic receptors mediated neuroprotection............104















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

EFFECT OF ALPHA7 NICOTINIC RECEPTOR ACTIVATION AND OVER-
EXPRESSION IN BRAIN AND IN PC12 CELLS

By

Ke Ren

December 2005

Chair: Jeffrey Hughes
Major Department: Pharmaceutics

Brain a7 nicotinic receptors are implicated in Alzheimer's disease through their

actions on memory related behaviors, binding to beta-amyloid. a7 nicotinic receptors are

calcium permeant and provide neuroprotection against many insults. The mechanism of

c7 nicotinic receptors, intracellular calcium ions and downstream calcium channels in the

protection were investigated. The a7 agonist GTS-21 prevented PC12 cell death induced

by NGF + serum deprivation over a 3 day interval. This effect was blocked by the

intracellular calcium chelator BAPTA-AM in a manner that did not appear to involve

changes in receptor-density. BAPTA-AM blocked GTS-21 induced PKC activation, a

necessary process for protection. The IP3 calcium channel blocker xestospongin C and

the phospholipase C inhibitor U-93122 blocked protection and ryanodine receptor

blocker ryanodine partially attenuated protection, but the L-type channel antagonist

nifedipine had no effect. ERK1/2 but not JNK and p38 were activated by GTS-21, and

the ERK phosphorylation inhibitors PD98059 and U0126 blocked protection. In vivo,









GTS-21 appeared to prevent cholinergic cell loss in septum following fimbria/fornix

lesions in PS1 mice, but no protection in APP/PS1 and wild type mice.

Another approach to investigate the effects of a7 receptors on Alzheimer's

disease-related processes involves elevating the levels of this receptor in the plasma

membrane. We therefore took a somatic gene transfer approach to modify the expression

of a7 nicotinic receptors in the brain. Recombinant adeno-associated virus (rAAV) was

used. Vector containing rat a7 nicotinic receptors driven by the hybrid chicken beta

actin/cytomegalovirus promoter was injected stereotaxically into wild type and a7

knockout mice hippocampus. Within three weeks, receptors were detected through

binding assay and western-blots. The functional receptors were observed through

electrophysiology response. rAAV 8/2-rat a7 nicotinic receptors gene transfer into wild

type mice may improve acquisition performance in a dose-dependend manner.

In conclusion 1) IP3 calcium channel and ryanodine receptor are important for a7

nicotinic receptors mediated neuroprotection, but the L-type channel is not necessary for

long term protection; 2) GTS-21 could prevent cholinergic cell loss in septum following

fimbria/fomix lesions in PS1 mice; 3) rAAV-rat a7 nicotinic receptor could express

functional receptors in rat, wild type and a7 knockout mice.














CHAPTER 1
INTRODUCTION

Alzheimer's Disease

Alzheimer's disease is an age related neuropathological disorder characterized by

the presence of large numbers of neuritic plaques, neurofibrillary tangles and the

progressive atrophy and loss of neurons. Aging is a major factor for this and many other

neurodegenerative disorders. The United Nations population projections estimate that

50% of people older than 85 years of age are afflicted with AD (Allsop et al., 2001). To

date, the cause and progression of AD have not been fully elucidated. The "cholinergic

hypotheses" was initially presented over 20 years age and suggests that a dysfunction of

acetylcholine containing neurons in the brain contributes to the cognitive decline in AD

(Terry and Buccafusco, 2003). Aging and AD have also been associated with

degeneration of neurons of the ascending cholinergic pathway. The cell bodies of these

neurons are located in the basal forebrain, and their axons innervate the amygdala,

hippocampus and neocortex. Brain neuronal cholinergic transmission has not only been

suggested to be affected in these pathologies, but also in human cognitive disorders

associated with the normal process of aging. Among these are the age-related deficits in

short- and long -term memory, impairment of attention, and delayed reaction time.

Among several other observations relevant to the cholinergic systems, nicotinic

acetylcholine receptors (AchR) binding sites have been reduced in number in the cerebral

cortex of patients with AD (London et al., 1989). The only FDA recognized therapies for









the disease are acetylcholinesterase (AChE) inhibitors such as aricept, tacrine, donepezil,

rivastigmine and galantamine, and the NMDA antagonist memantine.

The rationale for using AChE inhibitors is to enhance cholinergic transmission in

the brain by decreasing the metabolism of the neurotrasmitter acetylcholine. Basal

forebrain cholinergic neurons that are important for memory related behavior are

decreased in early AD, and this decrease becomes more evident as the disease progresses

(Nordberg and Svensson, 1998). These basal forebrain neurons have their perikarya

located primarily in the septum or the nucleus basalis. Septal cholinergic neurons send

axons and nerve terminals to the hippocampus, providing the only cholinergic innervation

of that region. Nucleus basalis cholinergic neurons send their projections throughout the

cerebral cortex. This cerebral cortical innervation from nucleus basalis accounts for over

90% of the cholinergic activity in rodents; in humans, it accounts for about 60% of the

cholinergic activity with the remainder due to intrinsic neurons in the neocortex. All of

the AChE inhibitor drugs for AD are most effective in the early stages of the disease,

presumably because there are more cholinergic neurons remaining for the drugs to act

upon. Since AChE inhibitors protect acetycholine in the synapse, they increase all types

of cholinergic transmission throughout the brain, including multiple muscarinic and

nicotinic receptors. Which of these receptor subtypes collectively or individually are

important for the beneficial effects of AChE inhibitors is an active area of study.

Several studies have shown that the expression and distribution of AChE in AD

patients has changed (Kasa et al., 1997; Talesa, 2001). AChE activity is lost in specific

regions of the AD brain, such as the neocortex and hippocampus, which are important for

memory-related behaviors. The relative proportions of different forms of AChE also









changed in this disease, suggesting changes in function. AChE has been found to co-

localize with Abeta peptides, which appear to be involved in the disease as discussed

below.

The rationale for using an NMDA antagonist in AD is to block the effects of

elevated levels of glutamate that may lead to neuronal dysfunction. Glutamate receptors

are already greatly reduced in this disease, and this is thought to be due to their

excitotoxic properties. According to this model, excessive glutamate receptor activity in

AD, especially that triggered by NMDA receptors, results in the excitotoxic loss of

glutamate receptor expressing neurons. Memantine would prevent this toxicity,

preventing further glutamate receptor loss and loss of neurons expressing these neurons.

Memantine is the only one of the FDA approved drugs found to have efficacy in later

stages of AD.

The "amyloid cascade" hypothesis is another of the central trends in AD research

community (Allsop et al., 2001). According to this hypothesis, neurodegeneration in AD

begins with abnormal processing of the amyloid precursor protein (APP) and results in

the production and aggregation of Abeta peptide-forming oligomers and amyloid fibrils

that form the senile plaques. Less clear is whether these Abeta species trigger the

formation of neurofibrillary tangles or eventually causes neuronal cell atrophy or death.

The amyloid cascade hypothesis has received considerable support from genetic

studies into the early-onset familial forms of AD, for which mutations in APP or

presenilin genes causing AD lead to an increase in Abeta production (Allsop et al., 2001).

Human APP mutations can cause amyloid plaques in transgenic in an age-related manner.

These plaques are typically not seen until about 10 months of age, and become more









pronounced thereafter, especially in hippocampus and neocortex. Interestingly,

transgenic mice with high levels of plaques exhibit only very modest decreases in

memory related behaviors and few neuronal deficits. This suggests that AD may require

more than just amyloid plaques.

Presenilins I and II were originally discovered and cloned through their

involvement in genetic forms of AD. Subsequently, it was found that both of these gene

products were involved in APP gamma secretase activity that leads to amyloidogenic

peptides such as Abeta 1-42 (W., 2001). While once thought to be gamma secretase itself,

presenilin was later shown to be a component of a multiprotein complex that has this

enzymatic activity. Overexpression of mutant, AD-causing, human presenilin I in

transgenic mice does not cause amyloid plaque formation. However, it does increase the

levels of APP peptides and also causes modest behavioral changes. Further, double

transgenic mice expressing mutant human APP and mutant presenilin I express amyloid

plaques much more quickly than do single transgenics making mutant human APP alone.

Presenilins also appear to have a variety of additional cellular effects that may not

be due to gamma secretase activity. These effects include the activation of multiple

protein kinase pathways such as AKT and ERK1/2 that are involved in cell viability. It is

possible that increased presenilin expression has complex effects on cell function, some

reducing it (e.g., by increasing amyloid expression) and other improving it (increased

kinase activity).

Important questions that remain are 1) how do mutations in presenilin or Abeta

cause any of the symptoms seen in A? And 2) how do these mutations affect our ability

to treat the disease pharmacologically? Recent studies suggest that both presenilin and









Abeta peptides affect pathways involved in cholinergic transmission, particularly

involving nicotinic receptor mediated transmission.

A consistent and significant loss of some subtypes of nAChRs has observed in

cortical autopsy brain tissue from AD patients compared to aged-matched healthy

subjects. The neocortical nAChRs deficits significantly correlate with cognitive

impairment in AD patients (Warpman and Nordberg, 1995). It was suggested that the

nAChRs deficits in AD brains probably represent an early phenomenon in the course of

this disease (Nordberg, 1994). Of the many nicotinic receptor subtypes found in the

brain, the most attention has been focused on a7 nicotinic receptors because of their

interactions with Abeta peptides, their importantance for memory related behaviors, their

ability to be neuroprotective, and their loss of expression in AD.

ca7 Nicotinic Receptors

AChRs exist as a variety of subtypes. Each of AChR subunits encoded by these

genes is thought to have an extensive N-terminal domain positioned extracellularly, four

transmembrane domains (MI-M4) that anchor these integral membrane proteins, and an

extracellular C-terminus. nAChRs are ligand-gated ion channels that can be divided into

two groups: muscle receptors and neuronal receptors. My research focuses on neuronal

receptors because of their behavioral importance. Neuronal nicotinic receptors form a

family of receptors that are differentially expressed in many regions of the CNS.

nAChRs have many functions, such as cognition, sleeping, arousal, feeding behavior,

neuronal development, and cell survival, but it remains to be determined which receptor

subtypes are involved with which functions. Dysfunctions of nAChRs have been linked

to a number of human diseases such as AD, Parkinson's disease and schizophrenia.









Nicotinic receptors consist of pentameric subunits that are either homemeric or

heteromeric. Heteromeric brain nicotinic receptors consist generally of multiple a

(mostly c2-4) and 3 (32-4) subunits. The a432 combination is the most common

combination of a and 3 subunits in the brain. a7 receptors, in contrast, are homomeric

receptors. Mapping of AchR distribution at low resolution based on radioligand binding

autoradiography is consistent with expression of some form of AchR in most of these

"major" or "minor" cholonergic targets. Anatomic analyses also suggest that a432 and

a7 (labeled using 3 H-labeled nicotinic agonists and 125 I-labeled Btx, respectively) have

largely unique but sometimes overlapping distribution. Btx sites predominate in the

septum, hippocampus, neocortex and hypothalamus, with lower density in the striatum.

It has been suggested that u7 nicotinic receptors may play an important role in cognitive

processes.

a7 receptors have a number of unique physiological and pharmacological

properties among nicotinic receptors, including high permeability to calcium, rapid and

reversible desensitization, and pronounced inward rectification. a7 receptors have a high

affinity for the antagonists alpha-bungarotoxin and methyllycaconitine (MLA). The c7-

receptor channel is highly permeable to calcium ions. Calcium acts as a second

messenger inside the neuron and not only stimulates neurotransmitter release, but also

stimulates signal transduction events through stimulation of protein kinases, calcineurins,

nitric oxide synthetases and other enzymes (Kem, 2000).

a7 nicotinic receptors are located both pre- and post-synaptically (Albuquerque et

al., 1997). Their presynaptic location and ability to promote the conductance of calcium

ions are consistent with an important role in the depolarization-dependent and calcium









ion-dependent release of neurotransmitters (Albuquerque et al., 1997). These receptors

have a very important postsynaptic location on gamma-aminobutyric acid-ergic

(GABAergic) inhibitory tone (Adler et al., 1998).

One of the difficulties in studying a7 receptor function is rapid desensitization

(Seguela et al., 1993). Selective agonists may act through receptor activation or

subsequent desensitization. A standard approach to distinguish these possibilities is to

use antagonists such as the non-selective mecamylamine, or the more selective MLA and

a-bungarotoxin. If receptor-activation is required, these antagonists block the action;

alternatively, their effects mimic antagonism if through desensitization. Nicotinic

receptor antagonists that block a7 receptors interfere with memory related behaviors and

block their neuroprotective properties. This indicates that agonist-activity is needed for

these receptor functions.

o7 Nicotinic Receptors: Effects On Cell Viability in vitro

The pattern of activation of a7 nicotinic receptors by agonists is concentration

dependent. Low concentrations of agonists induce a long term, low level and almost

steady state increase in net cationic influx (Papke et al., 2000). High concentrations of

agonists, in contrast, cause a much larger spike in conductance that is also much shorter

lived because of desensitization of the receptors. Only the longer term activation of the

receptor by low agonist concentrations is associated with the neuroprotective effects of

a7 receptor activation.

To date, a7 nicotinic receptor-mediated neuroprotection has been demonstrated in

the following models: NGF- and serum-withdrawal from differentiated PC12 cells (Li et

al., 1999b); glutamate induced excitotoxicity in primary rat brain neuron cultures









(Shimohama et al., 1998); Abeta amyloid exposure (Meyer et al., 1998a); ethanol toxicity

in primary neuronal cultures and PC12 cell (Li et al., 1999a). In all these models,

pretreatment with, and continued exposure to, agonist is necessary for neuroprotection.

An overview of this subject recently concluded that nicotinic receptor mediated

neuroprotection was attributable to a7 receptors (O'Neill et al., 2002). For a7 receptors,

the density of receptors and the binding affinity of the agonist could affect the extent of

neuroprotection (Jonnala and Buccafusco, 2001). Even indirect activation of c7 nicotinic

receptors by blocking cholinesterase activity is sufficient to be protective.

The mechanisms of a7 nicotinic receptor mediated neuroprotection in vitro have

been studied with respect to several intracellular processes. Protein kinase C (PKC)

activation, mitochondria membrane stabilization, reduced release of mitochondria

cytochrome oxidase and increased BCL-2 expression have all been found to be involved

in this neuroprotection (Li et al., 1999a; Dajas-Bailador et al., 2002a; Dajas-Bailador et

al., 2002c). a7 receptor mediated neuroprotection has also been found to require the Akt

antiapoptic pathway (Shimohama and Kihara, 2001). However, many steps in the

neuroprotection pathway are not known, starting with the role of calcium ions

themselves, various calcium channels, other intermediary kinase systems, and even the

level of a7-receptor density. It is also not known whether the pathways involved in

neuroprotection depend on the model system.

While low levels of agonist activation are neuroprotective, high concentrations of

agonists may be toxic to cells if applied quickly in vitro (Li et al., 1999b). There is no

evidence that this occurs in vivo, even at high doses of agonist. This may be due to the

relatively slow delivery of drug through the circulation vs. rapid receptor desensitization.









However, it is a potential concern that must be addressed when considering any new

approach that involves increased a7 receptor activity, since different approaches may

affect the receptors and their interactions with cellular function differently.

o7 Nnicotinic Receptors: Neuroprotection Properties in vivo

The potential neuroprotective effects of c7 nicotinic receptors in vivo are not well

understood. Pretreatment with the selective a7 receptor partial agonist GTS-21 was

found to reduce the penumbral damage caused by focal ischemia in the rat cerebral cortex

(Shimohama et al., 1998). Dr. Meyer's laboratory suggested that GTS-21 could protect

rat septal cholinergic neurons from partial fimbria-fornix lesions, though these lesions

were minimal (fewer than 15% of the cholinergic neurons were lost even in controls). In

a longer-term study of trans-synaptic cell loss, GTS-21 and nicotine were both found to

protect cerebral cortical neurons from trans-synaptic loss or atrophy following lesioning

of ascending nucleus baslis innervation (Meyer et al., 1998b). This result was also seen

in other nicotinic agonists (Socci and Arendash, 1996). However, no study has yet

evaluated the neuroprotective effects of nicotinic receptor agonists in genetic models of

AD such as the PS1 or APP/PS1 mouse.

3-benzylidene- and 3-cinnamylidene-anabaseine Compounds

Dr. Meyer's laboratory was the first to characterize families of 3-benzylidene- and

3-cinnamylidene anabaseine compounds as potential a7 receptor agonists for the

treatment of brain disorders. While the 3-benzylidene anabaseine compounds (e.g., GTS-

21) are typically selective partial agonists, the cinnamylidene anabaseines (e.g., 3-CA)

are full or at least more efficacious agonists. Both types of compounds have been used to

demonstrate neuroprotection through a7 nicotinic receptors. GTS-21 (also known as









DMXB) is 2,4- dimethoxybenzylidene anabaseine. It has been widely studied in

preclinical animal models and evaluated in a Phase I trial for Alzheimer's disease

(Kitagawa et al., 2003). This drug has much lower efficacy for primate or human a7

nicotinic receptors than for rodent receptors. GTS-21 rapidly enters the brain after oral

administration and improves memory related performance in nonhuman primates.

The pharmacokinetics properties of GTS-21 and 40H-GTS-21 have been studied to

a limited extent. Less than 1% of orally administered GTS-21 is recovered in the urine.

Three-hydroxy metabolites are generated in vitro by rat hepatic microsomal 0-

dealkylation of the two methoxy substituents on the benzylidene ring. These metabolites

are also found in plasma of rats after oral administration, but at significantly lower

concentrations relative to the parent compound. However, the principal metabolite, 3-(4-

hydroxy, 2-methoxy-benzylidene) anabaseine (40H-GTS-21), displays a higher efficacy

than GTS-21 on human as well as rat a7 nicotinic receptors (Figure 1-1). 40H-GTS-21

has a similar level of efficacy for both rat and human a7 nicotinic receptors. The

hydroxy metabolites are all more polar than GTS-21, derived from their octanol/water

partition coefficients, and they enter the brain less readily than GTS-21 (Kem, 2000).

However, they are behaviorally active when administered IP, indicating the ability to

enter the brain at therapeutic levels.


N H N H
SOCH, HCI N OH

SOGH3 N OCH3
GTS-21
4-OH-GTS-21


Figure 1-1. Chemical structures of GTS-21 and 40H-GTS-21.









GTS-21 and 40H-GTS-21 have been evaluated clinically. The pharmacokinetics of

GTS-21 and 40H-GTS-21 are shown in Table 1-1 and Table 1-2 (Kitagawa et al., 2003).

Table 1-1. Mean + SD Pharmacokinetic parameters for GTS-21 after oral administration
of 25, 75 and 150 mg of GTS-21 for 5 Days


Parameter

Cmax (ng/ml)

Tmax (h)

AUC

tl/2 (h)


25 mg

3.49+ 0.09

1.19+ 0.6

4.57 + 2.62

0.76+ 0.38


Table 1-1. Pharmacokinetic parameters of GTS-21.

Table 1-2. Mean + SD Pharmacokinetic parameters for 40H-GTS-21 after oral
administration of 25, 75 and 150 mg of 40H-GTS-21 for 5 Days


Parameter

Cmax (ng/ml)

Tmax (h)

AUC

tl/2 (h)


25 mg

2.71+ 1.12

1.3+ 0.58

7.49 + 4.73

0.81+0.30


75 mg

9.87+ 5.19

1.23+ 0.51

26.2+ 5.72

1.18+ 0.41


150 mg

32.9+ 18.3

1.11+ 0.48

71.3+ 24.9

1.15+ 0.37


Table 1-2. Pharmacokinetic parameters of 40H-GTS-21.

a7 Nicotinic Receptors and Memory Related Behaviors

a7 selective agonist such as GTS-21 improve several types of memory related

behaviors, including spatial memory in the Morris water task (Meyer et al., 1998a),

passive and active avoidance behaviors in rats (Meyer et al., 1997), radial arm maze in

aged rats (Arendash et al., 1995), delayed eye blink behavior in rabbits (Woodruff-Pak,

2003), hippocampal gating behavior in mice (Simosky et al., 2001), and delayed pair


75 mg

12.8+ 8.23

1.09+ 0.28

32.7+ 16.4

0.93+ 0.32


150 mg

47.8+ 28.4

1.02+ 0.38

85.1+ 32.9

0.93+ 0.19









matching and word recall (Briggs et al., 1997). In contrast, the selective antagonist MLA

reduces performance in radial arm maze when injected directly into the hippocampus at

low concentrations (Bettany and Levin, 2001). When injected peripherally, MLA also

interferes with several memory related behaviors, though this effect is more obvious in

ages, cognitively unimpaired rats than young adults rats or aged, impaired rats (personal

communication; Dr. Greg Rose, Memory Pharmaceuticals). The less selective antagonist

mecamylamine also interferes with memory related behaviors including the Morris water

task in FFX-lesioned rats when administered peripherally (Brown et al., 2001). As with

many drugs that improve memory related behaviors in rodents, a7 nicotinic receptor

agonists increase performance in lesioned or hypofunctional animals. Few reports show

improvement in intact animals. However, GTS-21 was found to improve performance in

delayed word match behavior in a small cohort of healthy adult humans (Kitagawa et al.,

2003). This observation suggests that these receptors may be effective for some

behaviors even in normal individuals.

Models Of a7 Nicotinic Receptor Dysfunction: a7 Receptor KO Mice And

Septohippocampal Lesions

a7 receptor knockout mice grow to normal size and show no obvious physical or

neurological deficits. These animals do exhibit expected deficits in MLA-sensitive

electrophysiological responses in hippocampus. This permits the knockout mouse to be

used for a7 receptor gene delivery studies to demonstrate restoration of function.

Behavioral responses in a7 knockout mice also appear normal, except for anxiety-

related behaviors in the open-field test. These observations may reflect developmental

compensatory differences between normal and knockout mice, since, as noted above,









modulating a7 receptors function in wild type adults causes behavioral changes.

Alternatively, it may be that some a7 receptors are modulatory but not necessary for

neuronal function and thus, when present, are able to affect behaviors that do not depend

on their presence. The a7 knockout mice were generated by introducing a 7 kb deletion

into embryonic stem cells followed by transmission to the germline. The mutation

deletes the last three exons (8-10) of the a7 locus. These exons encode the second

transmembrane domain, forming the putative ion channel, and the third and fourth

transmembrane domains and cytoplasmic loop (Orr-Urtreger et al., 1997).

Neuropathological examination of the brains of knockout mice revealed normal structure

and cell layering, including normal neocortical barrel fields. The histochemical

assessments of the hippocampus are also normal.

In contrast to the a7 knockout mice, mice expressing the a7 Leu250 point mutation

show extensive apoptosis throughout the neocortex and most of them die within 24 hours

of birth. This is probably due to the relative inability of this mutant to undergo

desensitization. As previously suggested, the fast desensitization kinetics of wild type a7

nicotinic receptors, which limits large acetylcholine-elicited Ca2+ influxes, might protect

against extensive neurodegeneration. The lack of additional neuroprotection in this a7

mutation relative to wild type mice suggests that a threshold Ca2+ level has to be reached

for toxicity.

Another model of a7-receptor dysfunction involves lesions of the

septohippocampal pathway, which reduce the cholinergic input to the hippocampus. The

hippocampus has been organized to play a fundamental role in some forms of learning

and memory, as shown since the early 1950s. The unusual shape of the human









hippocampus resembles that of a sea house, which is what led to its most common name

(in Greek, hippo means "horse" and "kampos means "sea monster") (Johnston et al.,

2003). It has been shown that damage to certain subregions of hippocampus can result in

an enduring amnesic syndrome. Affected patients are incapable of recording everyday

events and facts.

The hippocampus has been implicated in a number of neurological and psychiatric

disorders, including epilepsy, AD and schizophrenia. Because of the important role of

the hippocampus in learning and memory, it is not surprising that the hippocampus is

functionally disconnected from the rest of the brain in this disease during later stages.

Lesioning the septal input to the hippocampus by ablating the fimbria-fornix

pathway has recently been shown by Dr. Meyer and his colleagues to reduce a7 nicotinic

receptor function as measured electrophysiologically in hippocampus in a manner that is

overcome by chronic, twice-per-day injections of GTS-21 (Thinschmidt et al., 2005).

This reduction in function is paralleled by reductions in septal cholinergic density, but not

changes in receptor binding density in the hippocampus. The effects of these fimbrial-

fornix lesions on septal GABA neuronal survival are not known, but may be less than

seen in the cholinergic system because only a small fraction of GABA neurons in septum

project to the hippocampus. Selective lesions of either the cholinergic neurons or the

GABA neurons in the septum are not enough to cause these receptor-function deficits.

Instead, both types of neurons must be lesioned together such as by aspiration. Similarly,

both types of neuron deficits are necessary for memory related behavioral impairments,

with selective lesions in either type of nerve not causing much impairment (Yoder and

Pang, 2005).









a7 Nicotinic Receptor And Beta Amyloid

There are data showing that 1) a7 nicotinic receptors are blocked by low

concentrations of soluble Abeta 1-42 and other APP-derived peptides in a manner that is

likely to be competitive (Liu et al., 2001); 2) chronic oral treatment with nicotine reduces

amyloid plaque accumulation in transgenic Swedish mutation overexpressing mice

(Nordberg et al., 2002); and 3) nicotine increases a-secretase products (APP soluble)

(Efthimiopoulos et al., 1996) while decreasing gamma-secretase products (Utsuki et al.,

2002). GTS-21 was also found to increase soluble APP in isolated rat brain nerve

terminals, which would be expected to reduce amyloidogenic A3 expression. These data

suggested that a complex reciprocal interaction exists between nicotinic receptors and A3

1-42. Nicotinic receptor activation can be attenuated by the APP peptide but the levels of

AP 1-42 and A3 -containing plaques in turn can be reduced by nicotinic receptors

activation. In my project, a selective a7 nicotinic receptor agonist, 40H-GTS-21, was

evaluated for neuroprotection in fimbria fornix (FFX)-lesioned mice of several

genotypes: wild type, PSI only transgenic, and APP/PS1 double transgenic mice. The

aspirative lesion used for the study affected both cholinergic and GABAergic neurons

projecting to the hippocampus. This was the first time that an a7 nicotinic receptor

agonist was tested for neuroprotection in this species, as well as in any combination

model system consisting of both genetic changes and lesions.

I also investigated whether 40H-GTS-21 could decrease the amyloid load in these

mice. While it had been shown that nicotinic administered orally over a longer time

interval was effective in reducing amyloid plaque load, it was not clear which receptor

subtype or subtypes were mediated this effect. My study was designed to test the role of









a7 nicotinic receptors directly over a shorter interval (2 weeks), thereby testing the

hypothesis that a7 nicotinic receptors could be targets for these components of this

disease.

Memory Related Behavioral Tests

Morris water maze (MWM) is primarily designed to measure spatial learning and

recall, and has become quite useful for evaluating the effects of aging, experimental

lesions and drug effects (Jonasson et al., 2004). MWM is a challenging task that employs

a variety of sophisticated processes. These processes include the acquisition and spatial

localization of relevant visual cues that are subsequently processed, consolidated,

retained and retrieved. Several observations regarding the utility of the MWM are

notable: 1) The functional integrity of forebrain cholinergic systems that are critical for

efficient performance of that MWM appears to be consistently disrupted in patients who

suffer AD (Perry et al., 1999). 2) Neocortical and hippocampal projections from the

nucleus basalis magnocellularis and septum are reproducibly devastated in AD.

Accordingly, reductions in central cholinergic activity in rodents resulting from brain

lesions and age reproducibly impair spatial learning in the MWM (McNamara and

Skelton, 1993). 3) Other data implicated the hippocampus as an essential structure for

place learning which is commonly atrophic in patients with AD (Terry and Katzman,

1983; Mann, 1991). 4) Anticholinergic agents that are used routinely to impair

performance in the MWM also impair memory in humans and worsen the dementia in

those with AD (Ebert and Kirch, 1998). 5) Spatial orientation, navigation, learning and

recall are quite commonly disrupted in AD patients (Morris, 2003). However, the

transgenic mice carrying human APP Swedish mutation and that develop amyloid









plaques, with or without mutant PSI co-expression, do not show large deficits in spatial

learning and memory in MWM (Holcomb et al., 1998).

The MWM procedure has several advantages as a means of assessing cognitive

function in rodents when compared to other methods: 1) It requires no pre-training period

and can be accomplished in a short period of time with a relatively large number of

animals. 2) Through the use of training as well as probes, learning as well as retrieval

processes can be analyzed and compared among groups. 3) Through the use of video

tracking devices and the measures of swim speeds, behaviors can be delineated and

motoric or motivational deficits can be identified. 4) By changing the platform location,

both learning and re-learning experiments can be accomplished. This method is quite

useful in drug development studies for screening compounds for potential cognitive

enhancing effects.

rAAV Mediated Gene Transfer Into Brain

One of the approaches to increase receptor function without increasing agonist

concentration, which as noted above is not always desirable in the case of c7 nicotinic

receptors, is to increase receptor density by gene delivery. And one of the keys to

successful gene delivery is the selection of the appropriate therapeutic genes and their

molecular vehicle. A recombinant adeno-associated virus (rAAV) vector offers the

advantage of the ability to infect non-dividing cells, affording a non-pathogenic, long-

term transgene expression without a substantial inflammatory response when combined

with appropriate promoters. One goal of my project was to develop a7 nicotinic

receptors gene delivery systems for brain. This was hypothesized to provide safe,

effective and long-term therapy to counteract a7 deficits.









rAAV is one of the most promising viral vectors for gene therapy due to its wide

tropism and persistent transgene expression in vivo, and several clinical trials using

rAAV to treat genetic disease have been carried out. rAAV is also a safe and effective

means for studying transgene function in the brain.

AAV is a parvovirus with a diameter around 25 nm (Berns and Giraud, 1996). It is

a single-strand 4.7 kb DNA (ssDNA) genome packaged into three viral capsid proteins:

VP1 (87 kDa), VP2 (73 kDa) and VP3 (62 kDa). They form the 60-subunit viral particle

in a ratio of 1:1:20 (Muzyczka et al., 1984). The relatively high density of AAV particles

allows us to be easily separated by CsCl density centrifugation from adenovirus helper

particles whose density is approximately 1.35g/cm3. The linear ssDNA contains two

open reading frames flanked by two inverted terminal repeats of 145 nucleotides each

(Sperinde and Nugent, 1998). The upstream open reading frame (ORF) encodes four

overlapping nonstructural replication proteins (Rep), Rep78, Rep68, Rep52, and Rep40

(McLaughlin et al., 1998). The downstream ORF codes for the capsid proteins. After

entering host cells, the ssDNA genome of AAV is converted to the double-strand

template in cell nuclei and finally integrated into the host genome at chromosome

19q13.3 in humans (Leopold et al., 1998). This chromosome-selective integration is lost

in rAAV vectors in which the Rep coding sequences are removed.

AAV has demonstrated a broad tropism of infection, including lung, brain, eye,

liver, muscle, hematopoietic progenitors, joint synovium and endothelial cells (Miao et

al., 2000). The brain is a particularly good target for rAAV vector approaches because of

the topic maps of neuroanatomical organization. rAAV vectors retain much of this

tropism, with significant variations seen among serotypes depending on the tissue. These









serotypes differ in the composition of their capsid protein coat. rAAV serotype 2

(rAAV2) has been the most widely studied and best described among these. It binds to

both heparan sulfate proteoglycans and fibroblast growth factor receptors as an essential

step for cellular entry (Summerford et al., 1999). Recently, rAAV5 and rAAV8 have

also been investigated and found to bind to different cellular receptors. This probably

accounts for their different biodistribution properties when injected in brain and other

tissues. The mechanisms whereby other AAV serotypes enter host cells are actively

being studied. Early studies showed that rAAV2 mediated transgene expression occurred

in hippocampal interneurons, but not as strongly in hippocampal CA1 pyramidal neurons

or dentate gyms granul neurons. In my project, the rAAV8 vectors were also studied in

hippocampus.

rAAV mediated transgene expression has persisted for up to 2 years after in vivo

injection in rodent brains. rAAV-mediated expression is dose-dependent, but an accurate

dose dependence of expression has not yet been established. It also depends on the kind

of promoters chosen. Recently, we used the chick beta actin promoter/CMV enhancer,

which provided very high, stable, and long term transgene expression. Staining for

astrocytes or microglia following AAV-mediated transduction revealed no sign of gliosis

or infiltration relative to vehicle injection. Given the lack of inflammatory response or

pathogenicity, the reasonably high transduction efficiencies and the long persistence of

transgene expression, rAAV has become a leading candidate vector for somatic gene

transfer into brain in vivo.

AD is characterized by cholinergic deficits and degeneration of basal forebrain

cholinergic neurons. Cholinergic degeneration correlates with loss of memory function.









Therefore, rAAV mediated a7 nicotinic receptor gene transfer may become a potential

target for the treatment of AD.

Specific Aims

The goal of this project is to investigate the mechanism of c7 nicotinic receptors

mediated neuroprotection and the potential of these receptors as targets for developing

drugs for AD. I propose to evaluate a7 nicotinic receptors using selective a7 nicotinic

receptors agonist such as GTS-21 and 40H-GTS-21. Studies are focusing on three

models: 1) cell cultures expressing a7 nicotinic receptors; 2) wild type and a7 knockout

mice; 3) wild type, APP/PS1 and PS1 mice that have undergone FFX axotomy of the

septohippocampal pathway. The specific aims of these projects are as follows:

1. Determine the roles of calcium, calcium channels, and several kinase-

systems in a7 nicotinic receptor-mediated neuroprotection in vitro.

2. Test the hypothesis that the a7 nicotinic receptors agonist GTS-21 is

efficacious in protecting septal cholinergic neurons from axotomy in wild

type, PS1 overexpressing, and amyloid expressing mice.

3. Test the hypothesis that rAAV-rat a7 nicotinic receptor gene transfer

increases receptor expression in vitro and in vivo without toxicity.














CHAPTER 2
MATERIALS AND METHODS

Construction of rAAV Plasmids

Vector plasmids used for these studies are pUF 12-rat c7 with WPRE, pUF 12-GFP,

pUF 12-rat c7 without WPRE were constructed from pUF 12, which contains the chick P

actin promoter with truncated cytomegalovirus (CMV) enhancer (CBA promoter). All

plasmids had wild type AAV-2 terminal repeats (TR) and a poly (A) tail from bovine

growth hormone. The mRNA stabilizer woodchuck posttranscriptional regulatory

element (WPRE) was attached downstream to the u7 receptors in some rAAV2 vectors

preparations. Rat c7 cDNA was subcloned into this plasmid using Clal and verified

using EcoRI and HindIII. GFP was subcloned into this plasmid using HindIII and verified

with BamHI and BgIII. The DNA Core of the University of Florida analyzed both of the

DNA sequences. The biological activities of the u7 nicotinic receptor-expression vectors

were confirmed by western immunolabeling and binding assays following calcium

phosphate transfection of rat pituitary tumor derived (GH4C1) cells. GH4C1 cells were

incubated in 60 mm cell culture dishes in F-10 nutrient mixture (Ham) (Gibco BRL,

Grand Island, NY) supplement with 10% fetal calf serum (FBS, Gibco, Invitrogen

Corporation, CA) and 1% penicillin/streptomycin (Gibco, Invitrogen Corporation, CA) in

a 5% CO2 humidified atmosphere at 370C until 60% confluence. A mixture of 4 gg of

plasmid DNA, 12.4 [l of 2 M CaC12 was prepared in a total volume of 100 [l filtered

water. This mixture was added dropwise into equal volumes of 2 X HBS (280 mM NaC1,









10 mM KC1, 1.5 mM Na2HPO4, 12 mM dextrose, 50 mM HEPES, pH 7.1). There were

small precipitates at the bottom of dishes after two hours of transfection. The medium

was changed after 8 hours of transfection and the cultures were incubated for up to 72

hours. For control groups, GFP was monitored in cells after transfection. Rat a7

expression was detected by western blot and binding assays at day 3 post-transfection.

Subcloning

A backbone was prepared of rAAV2- rat a7 vector. 20 tg of pUF 12 was digested

with Hind III at 37 C for 1 hour. This digested reaction solution contained 6 [l Hind III,

6 tl NEB buffer 2, 20 [tg DNA in 60[l water. The digested reaction solution was mixed

with 12 [l 6 X DNA gel loading dye (30% glycerol, 6 mM EDTA, 0.06% bromophenol

blue and 0.06% xylene cyanol FF) and loaded on the 1% agarose gel for a hour

electrophorsis at 80 V. The larger size band was cut from the gel and extracted using an

agarose gel extraction kit (Qiagen). The backbone was dephosphorylated using calf

intestinal alkaline phosphatase (CIP, NEB). After dephosphorylation, the reaction was

extracted with phenol-chloroform, followed by chloroform. The upper layer was

collected. 2.5 volumes of 100% ethanol and one-tenth volume of 3 M NaAc pH 5.1 were

added into the sample and mixed and precipitated at -80 C for 2 hour. The sample was

centrifuged at 5,000 g for 15 min. The supernatant was discarded. One ml of 75%

ethanol was added to the pellet. The sample was centrifuged at 5,000 g for 15 min. The

supernatant was removed and the pellet was air dried for 15 min and dissolved in 20 [l

filtered water.

The second step was to add HindIII linker to rat a7 insert. Rat a7 insert was

flanked by Clal and blunt end ligated with T4 DNA polymerase. The blunt end reaction









contained 10 ul 10x Buffer T4 pol (NEB), 0.5 ul 25 mM dNTP (NEB), 3 ul T4 pol and

48 ul rat a7 insert and 38.5 ul water. The reaction was incubated at room temperature for

15 min. The whole reaction was mixed with 12 ul of 6-x DNA gel loading dye and

loaded on the 1% agarose gel for a 1-hour electrophoresis at 80 V. The rat a7 insert band

was cut from the gel and extracted using an agarose gel extraction kit (Qiagen). The

insert was extracted by phenol-chloroform, then chloroform. DNA was precipitated by

ethanol. The linker ligation reaction solution contained 17.5 ul rat a7 insert, 5 ul 5x

DNA dilution buffer, 2.5 ul linkers (2.5 [tg), 25 ul 2x ligation buffer and 2.5 ul ligase

(Boehringer). The reaction mixture was incubated at room temperature for 5 min. The

linker reaction was mixed with 6 ul of 6-x DNA gel loading dye and loaded on the 1%

agarose gel for 1-hour electrophoresis at 80 V. At the same time rat a7 insert was loaded

on the gel. The ligated linker shifted up a bit. The rat a7 insert linker band was cut from

the gel and extracted using an agarose gel extraction kit (Qiagen). The fragment was

extracted by phenol-chloroform, then chloroform. DNA was precipitated by ethanol. The

pellet was dissolved in 10 ul water.

Gel electrophoresis was run to compare the relative concentrations of vector and

insert. The third step was ligation. The ligation reaction contained approximately

equimoloar concentration of backbone and insert, 2 ul 10 x ligation buffer and 2 ul T4

DNA ligase (NEB) and water. The total volume was 20 ul which was added to a PCR

tube. The ligation mixed was incubated in a PTC-200 DNA Engine Thermal Cycles (MJ

Research) for overnight at 160 C. The next day, 180 ul water was added into the ligation

reaction. The ligation product was extracted by phenol-chloroform, then chloroform,









finally precipitated by ethanol. The pellet was dissolved in 10 [l H20. 2 [l obligation

product was used for transformation.

The next step was transformation. 2 [l of ligation product were transformed into

100 [l competent SURE cells (Stratagene, La Jolla, CA) using electroporation

transformation methods. The cuvette was placed in the BioRad Gene Pulser II (Bio-Rad

Laboratories). A single exponential decay pulse of 2.5 kV, 25 tF and 200 Q were

delivered. The whole reaction was added to 1 ml NZY (Fisher Scientific) without

antibiotics and incubated in the shaker for 1 hour at 370 C. 50 al, 100 al, 250 [l or 600 [l

were added to 10 cm NZY/AMP (100 [tg/ml) dishes and incubated at 370 C overnight.

Next day, six single colonies were selected and grown overnight. Minipreps of 6 single

colonies were performed using Miniprep kit (Qiagen). The colonies were selected using

restriction digestion and gel electrophoreses.

Plasmid Preparations

The plasmid was grown overnight in 5 ml of NZY/Amp (100 [tg/ml) at 370C on a

shaking platform by pick a single colony. Next day, the culture inoculated into 2 L

NZY/Amp (100 [tg/ml) and incubated for 16-18 hour. The overnight culture was Spin

down in 4 X 250 ml bottles at 5000 g for 15 min in a Sorvall RC-5B refrigerated

superspeed centrifuge (DuPont Instruments). The pellets were resupsend in 20 ml of

lysozyme buffer (25 ml Tris-HCl pH 8, 20 ml 0.5 M EDTA, 17.115 g of sucrose, dH20

for a total volume of 1 liter). 12mg/ml of lysozyme (Sigma) was added, mixed and put

on the ice for 5 min. 48 ml of solution II (20 ml 10% SDS, 4ml 10N NaOH, water for a

total volume of 200 ml) was added, mixed and stored the bottles at room temperature for

4 min. 36ml of3M NaAc (pH4.6-5.2) and 0.2 ml chloroform were added and mixed and









iced for 20 min. The mixtures were centrifuged at 5,000 g for 20 min. The clear

supernatant was transferred into new 250 ml bottle through gauze. 33 ml of 40% PEG

(Fishrs Scientific) was added, mixed and stored at -80 C overnight. The mixtures were

centrifuge at 5, 000g for 10min. The supernatant was removed and dissolved pellet in 10

ml of dH2O. 10 ml of 5.5 M LiCl (Fisher Scientific) was added, mixed and iced for 10

min followed by centrifugation at 5,000 g for 10 min. 21 ml of supernatant were

collected and transferred into 2 40 ml tubes. 6 ml isopropanol (Fisher Scientific) were

added, mixed and iced for 20 min. The mixtures were centrifuged at 5,000 g for 10 min

in small rotor. Each pellet was dissolved in 6.7 ml of TE buffer (10 mM Tris pH 8 and 1

mM EDTA). 8 g CsCl (Fisher Scientific) were added and mixed until dissolved. 0.44 ml

of ethidium bromide (Fisher Scientific, 10 mg/ml) was added to each bottle. 8.9 ml of the

solution were loaded into an Optiseal Beckman tube (Beckman Instruments). All tubes

were loaded into a 70.1 Ti rotor and ultracentrifuged at 100,000 g in a Beckman L8-70M

Ultracentrifuge for at least 19 hours at 200 C. A hand held long wave UV 366 nm was

used to estimate DNA density. There were two bands in the tube. The lower band was

collected with 16 G needle (Becton Dickinson) and transferred to a 15 ml tube. The

samples were extracted with equal volume of isoamyl alcohol (Fisher Scientific) until the

sample was clear, then mixed and centrifuged at 3000 g for 2 min. 3 ml of the extracted

sample were put into Corex tubes and 2.5 volume of H20 was added, followed by 2

combined volumes of ethanol. The mixture was iced for 30 min. The mixtures were

centrifuged at 5,000 g for 15 min. The supernatant was removed and the pellet dissolved

in 400 [tl of TE buffer in a 15 ml tube. It was washed with 400 [tl of TE, which was

pooled into the same tube. The sample was extracted with an equal volume of









phenol/chloroform (Amresco) until the interphase was clear, followed by chloroform

extraction. The extracted sample (400 [tl each) was transferred into 1.5 ml microfuge

tubes. 40 [tl of 3 M NaAc and 1 ml of ethanol were added, mixed and iced for 10 min,

followed by microfuge for 5 min at max speed in a 5414 Centrifuge (Hamburg, German).

The pellet was washed with 1 ml of 75 % ethanol, followed by 5 min at max speed. The

supernatant was removed. The pellets were air dried for 15 min. Each pellet was

dissolved in 200 [tl sterile TE buffer. The DNA concentration was detected at 260/280

nm in a Beckman DU 650 Spectrophotometer. The plasmid was confirmed by restriction

digestion and gel electrophoresis.

Packaging rAAV Vectors And Titration

The method used to package constructs and purify the rAAV was described by or

modified from Zolotukhin et al (Zolotukhin et al., 1999). HEK 293 cells at 70%

confluence were transfected with two plasmids by the calcium phosphate method. One

plasmid was pUF 12-rat a7 or pUF12. The other was helper plasmid. The rAAV2 helper

plasmid was pDG, which contained replication-deficient AAV genes for the rAAV

protein coat and replication-defective adenovirus genes for helper function in packaging.

The rAAV5/2 helper plasmid was pXYZ5. The rAAV8/2 helper plasmid was pXYZ8.

These two plasmids were used in equal molar ratios for transfection. 10 cell culture

dishes of 15 cm diameter were used. 1.25 ml of 2 M CaC12, 0.6 mg helper plasmid, 0.3

mg of pUF 12-rat a7 or pUF 12 and sterile water were mixed to the total volume of 10 ml

for ten dishes. This mixture was added dropwise into equal volumes of 2x HBA while

vortexing. This whole transfection mixture was added to 200 ml of warmed DMEM,

which contain 10% FBS and 1% penicillin/streptomycin. 22 ml of this medium mixture









were added to each dish of HEK 293 cells. After a 6-hour transfection, the medium was

removed and replaced with fresh DMEM. Seventy-two hours after the transfection, cells

were harvested using cell a cell scraper (Corning Incorporated) and centrifuged at 3000 g

at 4 C for 20 min. The cells were resuspended in 15 ml of lysis buffer (150 mM NaC1,

50 mM Tris, pH 8.5). The suspension underwent 3 cycles of freeze thawing with dry

ice/ethanol (10 min freeze, 15 min thaw, vortexing every 5 min). The samples were

treated with benzonase (endonuclease, Sigma) to digest unpackaged DNA. 6 [tl of 4.82 M

MgCl2 and 750 units of benzonase were added to 15 ml lysate and incubated at 370 C for

30 min. The lysate was centrifuged for 30 min at 3,000 g at 4 C. 15 ml of supernatant

were collected in a 39 ml Optiseal tube through 16G needle. A Pump Pro (Watson-

Marlow, UK) was set up as follows. 200 [tl glass pipettes were used for intake and 100 dtl

glass pipettes were used for output. Pump speed was set at 37 rpm counterclockwise.

The tube was rinsed with 40 ml water and 15 ml 15% iodixanol (IOD). The output

pipette was loaded into Optiseal tubes. Pumping was started with 15% IOD (1:47 min),

25% IOD (1:15 min), 40% IOD (1:47 min) and 60% IOD (1:50 min). (180 ml of 15%

IOD contained 45 ml of OptiPrep (Axis-Shield Poc AS, Norway), 36 ml of 5 M NaC1, 36

ml of 5x TD (5x PBS, 5 mM MgCl 2, 12.5 mM KC1) and 63 ml water. 120 ml of 25%

IOD contained 50 ml of OptiPrep, 24 ml of 5x TD, 46 ml of water and 300 [tl of 0.5 %

phenol red solution (Sigma). 100 ml of 40% IOD solution contained 68 ml of OptiPrep,

20 ml of 5x TD and 12 ml of water; 100 ml of 60% IOD contained 100 ml of OptiPrep

and 250 [tl of 0.5% phenol red solution). Tubes were sealed by heat and were

untracentrifuged at 100,000 g in a Beckman L8-70M Ultracentrifuge for 2 hour at 180

C. The rAAV band was located at the 1st interface (between 60% and 40%) from the









bottom of tube and up to 0.5 cm below the second interface; it was collected through a 16

G needle.

Heparin columns were used to purify AAV stereotype 2. Q sepharose was used for

AAV serotypes 5 and 8. The heparin column was made as follows: the bottom of the

Bio-Rad Econo-pac disposable chromatography column was snapped off and 6 ml of

well-mixed heparin immobilized on cross-linked 4% beaded agarose (Sigma) were added

to the column. The heparin column was equilibrated with 15 ml of 1xTD (lx PBS, 1 mM

MgCl2 and 2.5 mM KC1). The rAAV2 lysate was added onto the column, and then the

column was washed with 20 ml of lx TD. The rAAV2 was eluted with 15 ml of lx TD/1

M NaC1. The 15 ml of eluted solution was collected into a 50 ml conical tube. The Q

sepharose column was made as follows: the bottom of the Bio-Rad Econo-pac disposable

chromatography column was snapped off and 5 ml of well-mixed Q sepharose (Sigma)

were added into the column. The Q sepharose column was equilibrated with 20 ml of

solution A (20 mM Tris/15 mM NaC1, pH 8.5) and the column was washed with 20 ml

solution B (20 mM Tris/lM NaC1, pH 8.5) and again with 30 ml solution A. rAAV5/2 or

rAAV8/2 sample was diluted with two times solution A and loaded to the column. After

loading the sample, 50 ml of solution A were added to the column. The sample was

eluted with 20 ml of solution C (20 mM Tris/355 mM NaC1, pH 8.5) and collected into a

50 ml conical tube. The sample was added into the concentrator (Minipore: Biomax-

100K NMWL membrane) and centrifuged at 1,000 g for 5 min. The lysate was

concentrated to 1 ml and diluted with 9 ml of Ringer's solution twice. The virus was

concentrated to a final volume of 300 [Il. Virus was collected into siliconized tubes

(Fisher) and stored at -200 C.









A dot-blot assay was used to determine the titer of rAAV virus based on total

genomic particles. A 4 [tl aliquot of the virus was treated with DNAse I (Roche) mixture

for 1 hour at 370 C. This reaction contained 20 [tl of 10X DNAse buffer (50 mM Tris-

HC1, pH 7.5 and 10 mM MgC12), 2 [tl of DNAse, 174 [tl of dH20 and 4 [tl virus. After 1

hour, 22 [tl Proteinase buffer (10 mM Tris-HC1, pH 8.0, 10 mM EDTA and 10% sodium

dodecyl sulfate) were added and incubated for 1 hour at 370 C. An equal volume of

phenol-chloroform was added to the sample, vortexed for 5 min and microfuged for 5

min at 14,000 g. This was followed by an extraction with an equal volume of chloroform

and microfuging for 5 min at 14,000 g. The aqueous layer was transferred into a new

microfuge tube to which was added 1/10 volume of 3 M NaAc (pH 5.2) and 2.5 volumes

of ethanol. DNA was precipitated overnight at -80 C. Next day; the sample was

microfuged at 14,000 g for 20 min. The supernatant was discarded. 0.5 ml of 75%

ethanol was added to the pellet and microfuged at 14,000 g for 10 min. The pellet was air

dried for 15 min and dissolved in 40 [tl of water. The sample was quantified by a DNA

slot blot assay using 1.7 kb EcoRI segment ofpUF12 and a series of dilutions of pUF12

as standard curve. The dilution series of the pUF12 was started with 20-ng/dtl and

continued for 12 tubes. Each tube received 50 [tl of water. 50 [tl of DNA (20ng/[tl) were

pipetted from tube 1 to tube 2. Tube 2 was vortexed and 50 [tl of DNA removed from

tube 2 to tube 3. This dilution procedure was continued for all 12 tubes. A second set of

tubes labeled 1-12 were set up behind the DNA dilution set. 200 [tl of alkaline buffer (0.4

M NaOH, 10mM EDTA pH 8.0) were added to each tube. To each alkaline tube, 10 [tl

of DNA dilution set was added to each tube. For each virus sample, 10 [tl of 1:10

dilution and 1:1 dilution each were added to 200 [tl of alkaline buffer tube. The next step









was to set up the dot blotter (Bio-Rad). Two pieces of filter paper (Bio-Rad) were placed

in water and then put on the dot blotter. Membranes (Bio-Rad, Zeta-Probe Blotting

membranes) were put on the top of filter paper. The dot blotter was closed and 400 [tl of

water was added to each well. Vacuum was used to remove excess water. The standards

of all 12 tubes (in alkaline buffer) and samples were boiled for 10 minutes. The standard

curve tubes in alkaline buffer and samples were loaded to each well. The samples were

aspirated slowly with vacuum until all samples disappeared. The vacuum was

disconnected. 400 [tl alkaline buffer were added to each well and allowed to stand for 5

min, followed by vacuuming remaining solution. The membrane was removed from the

dot blotter and crosslinked by a UV crosslinker (UV Statalinker 1800, Strategene). The

membrane was placed in a small Biometra bottle. Prehybridyzation buffer (7.5 ml, 7%

SDS, 0.25 M NaHPO4 pH 7.2, 1 mM EDTA pH 8.0) was added into the bottle and

incubated at 650C for 1 hour before labeling probe was added. 6 [tl of biotinylated probe

was diluted with 54 [tl of 10 mM EDTA and denatured at 900C for 10 min. The total 60

[tl of denatured probe were added to the hybridization buffer and quickly mixed and

incubated overnight at 65 C in Biometra oven. A Brightstar Bio Detect kit (Ambion,

TX) was used for detection. The membrane was placed into a staining container, then

washed twice for 5 min in 1 X wash buffer (Ambion), followed by washing twice in

blocking buffer (Ambion) for 5 min each. The membrane was then incubated for 30 min

in blocking buffer. The membrane was incubated with 1 [tl strep-alkaline phosphatase

(Strep-AP) mixed with 10 ml blocking buffer for 30 min, followed by a 15-min

incubation in blocking buffer. The membrane was washed 3 times for 5 min each in 1 X

wash buffer and incubated with 1 X assay buffer for 2 min. The membrane was









incubated for 5 min in 5 ml of 1 X CDP-Star solution. The membrane was wrapped in a

single layer of plastic wrap and exposed to film at room temperature in a dark room. The

film exposure was for 15 mins. The blot was then scanned into computer and the

intensity of the bands analyzed with NIH Scion image software (Scion Corporation,

Maryland). The intensity of the bands for standard DNA was used to build a standard

curve. The titers was calculated using the coefficients: 1 ng DNA = 4 x 1011 particles/ml.

Vector stocks were ranged from 1012_1013 genomic particles per ml, except for that

containing the WPRE sequence genomicc lengths over 5000 kb) was 107 genomic

particles/ml.

Cell Transfections

GH4C 1 cells were obtained from American Type Culture Collection (ATCC). The

cells were grown in F-10 nutrient mixture containing 10% FBS and 1%

penicillin/streptomycin at 370 C in a 5% CO2 and 90-92% humidity. The cells were split

at a 1:3 ratio every 5 days, up to 10 passages. Cell confluence at the time of study was

approximately 60%. The cell culture medium was removed. The cells were exposed to a

0.05% trypsin/0.53 mM EDTA solution for 5 min. After 5 min, the trypsin/EDTA was

removed from the cells by transferring the cellular suspension to sterile conical tubes.

These tubes were centrifuged at 3,000 g for 5 min. The supernatants were removed and

the pellets were resuspended in 150 ptl fresh medium. Increasing concentrations of 1.25 x

109 /ml, 2.5 x 109 /ml, 5 x 109 /ml and 15 x 109 /ml genomic particles were added to the

medium and incubated for 30 min at 370 C. After incubation, the cells were plated at the

60 mm dishes and added 2 ml of fresh medium was added to the dishes. 3-5 days after

transfection, the cells were harvested for binding assays.











Stereotaxic Surgeries

Male Sprague-Dawley rats (-250g, 2 months old) were obtained from the Harlan

Sprague Dawley Farm (Indiana). a7 heterozygous mice were obtained from the Jackson

laboratory. They were housed and bred the in animal facility at the Health Science

Center of the University of Florida. Rats or mice were anesthetized with 4% isoflurane/

oxygen. rAAV2-rat a7, rAAV2-GFP, rAAV8-rat a7 and rAAV8-GFP vectors were

injected bilaterally into hippocampus or septum through a 27-gauge cannula connected

via 26 gauge I.D. polyethylene tubing to a 10 [l syringe mounted to a CMA/100

microinjection pump. The pump delivered 2 [tl virus (1010 genomic particles) at a rate of

0.15 tl /min. The injection coordinates for the hippocampus were -3.6 mm bregma, 2.2

mm medial-lateral, and 2.8 mm dorsal-ventral. For the medial septum, they were 0.7 mm

bregma, 0.2 mm medial-lateral and 7.0 mm dorsal-ventral. The cannula was removed

slowly removed after the injection. The skin was sutured. All animal care and procedures

were in accordance with institutional IACUC and NIH guidelines. Two weeks or 6

months after injections, the animals were euthanized and their brains evaluated for

receptor binding, immunohistochemistry, electrophysiology, or western blot.

High Affinity [3 H] MLA Binding Assay

Brain tissues or cell culture samples were prepared for nicotine-displaceable, high-

affinity [3H] methyllycaconitine (MLA) binding assay as follows. Septum or

hippocampus was rapidly dissected from animals euthanized while fewer than 4%

isoflurane/oxygen anesthesia and suspended in ice-cold Krebs Ringer buffer (KRH; in

mM 118 NaC1, 5 KC1, 10 glucose, 1 MgCl2, 2.5 CaCl2, 20 HEPES; pH 7.5). Ice-cold









KRH was also used to wash, and then harvest culture cells. Brain tissues or cell culture

samples were homogenized in ice-cold KRH buffer with a Polytron (setting 4 for 15 sec).

After 2 1 ml washes with KRH at 20,000 g, the membranes were incubated in 0.5 ml

Krebs Ringer with 2.3 nM [3 H] MLA (Tocris, Ellisville,MO), unless specified otherwise,

for 60 min at 4 C, plus or minus 5 mM nicotine. For determining the Kd and Bmax

values, a range of total MLA was used from 0.5 nM to 50 nM for Scatchard analyses.

Tissues were washed 3 times with 5 ml ice-cold KRH buffer by filtration through

Whatman GF/C filters that were preincubated for 30 min with 0.5% polyethylenimine

(Sigma). Liquid scintillation (EcoLite) counting of radioactivity was conducted in a

Beckman LS1800. Nicotine-displaceable binding was calculated for each sample in

triplicate in each experiment. Scatchard analyses are evaluated with the Statview

program. ANOVA was performed for treatment effects.

In Bradford protein assay, 100 ptleach of 0.1, 0.2, 0.4, 0.6 and 0.8 [tg/[tl of bovine

serum albumin (BSA, Sigma) were used as standards to measure the protein

concentration of the medium, brain samples and cell lysate samples. 50 [tl of each

samples were added into 5ml tubes containing 200 [tl of 1 x KRH. Five ml of diluted (1:4

with water) BioRad protein assay dye reagent (Bio-Rad) was added to each tube,

vortexed and sit for 30 min at room temperature. The O.D. of each tube was detected at

595 nm in a Beckman DU 650 Spectrophotometer (Beckman). A standard curve was

prepared according to the concentrations and absorbance of the BSA standards at 595 nm

and the protein concentration of each sample was determined using Prism software

(GraphPad Software, Inc.CA).









Immunohistrochemistry

Animals anesthetized with 4% isoflurane/oxygen were perfused with 100 ml of

cold phosphate-buffered saline (PBS), followed by 400 ml of cold 4% paraformaldehyde

(Sigma) in PBS. The brain was removed and equilibrated in a cryoprotectant solution of

30% sucrose/PBS and stored at 4 C. Coronal sections (50 |tm thick) were cut on a

sliding microtome with freezing stage. Antigen detection was conducted on floating

sections by incubation in a blocking solution (2% goat serum/ 0.3% Triton X-100/ PBS)

for 30 min at room temperature, followed by primary antibody incubation overnight at

4C. Primary antibodies used were: anti-a7-antibody mAb 306 (1:500, Sigma), 6E10

(1:1000, Signet, Dedham MA), glial fibrillary acidic protein (GFAP) (1:2000, Chemicon,

Temecula, CA), NeuN (1:1000, Chemicon), anti-Chat (1:1000, Chemicon) and anti-

parvalbumin (1:1000, Chemicon). To optimize a7 immunohistochemistry, 250 [l of

50% ethanol were added to each section and incubated for 30 min before added anti-a7

antibodies. The sections were washed in PBS 3 times for 5 min each and then incubated

with biotinylated anti mouse IgG or anti rabbit IgG (1:1000, Dako, Carpinteria, CA) for 1 hour

at room temperature. The sections were then washed with PBS three times for 5 min

each and labeled with ExtrAvidin peroxidase (HRP) conjugate (1:1000, Sigma) for 30

min at room temperature. Development of color was conducted with a solution of 0.67

mg/ml diaminobenzidine (DAB, Sigma)/0.1 M sodium acetate/8 mM imidazole/2%

nickel sulfate/0.003% H202. The sections were mounted on Fisher Superfrost Plus slides

and air dried for 30 min. They were passed through water, 70% ethanol, 95% ethanol

and 100% ethanol, and then the slides were xylene dehydrated. The slides were

coverslipped with Eukitt (Calibrated Instruments,NY).









Some sections were used with fluorescent secondary antibody. After incubating

with primary antibody, sections were washed in PBS 3 times and incubated with

fluorescein (FITC)-conjugated anti-mouse (Jackson Immunoresearch, 1:1000) secondary

antibody overnight at 4 C. Sections were washed in PBS and then mounted on Fisher

Superfrost Plus slides, air-dried and coverslipped with glycerol gelatin (Sigma).

FluoroJade Staining

FluoroJade staining is a method to stain degenerating neurons. Floating brain

sections prepared as describe above were treated with 100% ethanol for 3 min, followed

by 70% ethanol and dH20 for 1 min each, 0.06% potassium permanganate for 15 min, and

a wash with dH20 for 1 min. The sections were treated in the dark with 0.001% Fluoro-

Jade for 30 min and then mounted on slides, air-dried and coverslipped with glycerol

gelatin.

Western-blot

Septum or hippocampus was rapidly dissected from animal's euthanized under 4%

isoflurane/oxygen anesthesia. Ice-cold sodium phosphate buffer (50 mM sodium

phosphate, 50 mM NaC1, 2 mM EDTA, 2 mM EGTA, and 1 mM phenylmethysulfonyl

fluoride, pH 7.4) was used to harvest cells. Fresh brain tissues or cell culture samples

were homogenized with ice-cold 50 mM sodium phosphate buffer and centrifuged at

60,000 g for 60 min at 4 o C. The supernatant was discarded. The pellets were

resuspended in an ice-cold buffer containing 2% Triton X-100 and protease inhibitor

cocktail (Sigma). The suspension was mixed for 2 hour at 40C and then centrifuged at

100,000 g for 60 min at 4C. The supernatants were used for western blot analysis.

Protein contents in the fraction were assayed with the Bio-Rad protein assay kit. 25 itg

each sample protein were mixed with 2x Laemmli sample buffer (Bio-Rad) and fresh 5%









2-mercaptoethanol. Samples were boiled for 5 min. Each sample was separated on 10%

sodium dodecyl sulfate-polyacrylamide (SDS) gels by electrophoresis (150 V for 60

min). The proteins in the gel were blotted onto polyvinylidene difluoride membranes

using a transfer unit (Bio-Rad) (120 V for 2 hour) at 40C. The membrane was blocked

with 5% nonfat dry milk/ PBS buffer containing 0.05% Tween 20 for 60 min at room

temperature. The membrane was incubated with monoclonal anti-a7 antibody (Sigma,

mAb 306, 1:500) overnight at 40C, then washed three times with PBS/ 0.05% Tween 20

for 5 min each. The membrane was incubated with secondary antibody horseradish

peroxidase-conjugated anti-mouse IgG (Amersham, 1:10,000 dilution) for 60 min at

room temperature. The membrane was washed 3 times with PBS/ 0.05% Tween 20 for 5

min each. The membrane was exposed to ECL PLUS reagent (Amersham) for 1 min and

the membrane was exposed to Hyper Performance Chemiluminescence film (Amersham).

c7 nicotinic receptor band (MW 38,000) was quantified with the NIH Image program.

Morris Water Task

Morris water maze tests were performed to test the spatial learning and memory of

mice injected rAAV2-rat a7 vectors. These tests were conducted using a specially

designed water tanks (1 m) for mice with a fixed platform hidden just below (- 1.0 cm)

the surface of the water. The platform was rendered invisible by adding powdered milk

to render the water opaque. Various geometric images (e.g., circles, squares, triangles)

were placed in the testing room or hung on the wall in order that mice can use these

visible objects as a means of navigating in the maze. Mice received 2 blocks of training

trials (4 trials/block) daily for 8 days. With each subsequent entry into the maze the mice

progressively become more efficient at locating the platform. The ninth day was for









probe trials. The mice had 60 seconds to search for the platform and they were hand

guided to the platform if they did not reach it during that interval. The mouse was

allowed 30 seconds on the platform. For each trial, latency to find the platform, path

length to the platform, and swim speed were recorded by a video-tracking/computer-

digitizing system (HVS Image, Hampton, UK). On day 9, animals were given a probe

test in which the platform was removed and the rats had 60 seconds to search for the

platform. The swim distance and percentage of time spent in each quadrant were

recorded. The total time required for each animal to reach the platform was determined

for the 8 days training period, and these values were compared among the treatment

group by ANOVA. ANOVA was also used to compare probe (retention and recall)

performance among the treatment groups.

Electrophysiological Recordings

Fresh horizontal slices (300 gim) were prepared using a vibratome (Pelco, Redding,

CA). Slices were incubated at 300C for 30 min and then maintained submerged at room

temperature. The artificial cerebral spinal fluid (ACSF) used for cutting and incubating

slices contained in mM: 124 NaC1, 2.5 KC1, 1.2 NaH2PO4, 2.5 MgSO4, 10 D-glucose, 1

CaC12, and 25.9 NaHCO3, saturated with 95% 02 / 5% CO2. Following incubation, slices

were transferred to a recording chamber where they were superfused at a rate of 2 ml/min

with ACSF at 300C containing in mM: 126 NaC1, 3 KC1, 1.2 NaH2PO4, 1.5 MgSO4, 11

D-glucose, 2.4 CaC12, and 25.9 NaHCO3, saturated with 95% 02 / 5% CO2. Individual

neurons were identified with infrared differential interference contrast microscopy (IR

DIC) using a Nikon E600FN microscope (Nikon, Inc.). Whole-cell patch-clamp

recordings were made with pipettes pulled on a Flaming/Brown electrode puller (Sutter









Instruments, Novato, CA). Pipettes were typically 3-5 MQ when filled with an internal

solution that contained in mM: 140 Cs-MeSO3, 8 NaC1, 1 MgC12, 0.2 EGTA, 10 HEPES,

2 mg ATP, 0.3 Na3GTP, and 5 QX-314. This solution blocked all action potentials and

allowed stable voltage-clamp recording at depolarized membrane potentials. All internal

solutions were pH adjusted to 7.3 using additional CsOH or KOH and volume adjusted to

-285 mOsm.

For experiments involving fluorescence microscopy, GFP-expressing neurons were

visualized using light from a mercury lamp filtered at 510-560 nM. For all local

application experiments, a picospritzer (General Valve, Fairfield, NJ) was used to apply

ACh (1 mM) from pipettes identical to those used for whole-cell recording or from

double-barreled pipettes made using theta tubing (Sutter Instruments, Novato, CA). An

Axon Multiclamp 700A amplifier (Axon Instruments, Union City, CA) was used to

amplify voltage and current records. The data were sampled at 20 kHz, filtered at 2 kHz,

and recorded on a computer via a Digidata 1200A or 1321 analogue-to-digital converter

using Clampex version 8-8.2 (Axon Instruments). Data were analyzed using Clampfit

version 8-8.2 (Axon Instruments), OriginPro v. 7.0 (OriginLab, Boston, MA) and

Graphpad Prism v. 3.0 (Graphpad Software, San Diego, CA).

Fimbria Fornix Lesions And 40H-GTS-21 Injections

Wild type (Swiss Webster) and APP/PS1 (B6/D2 x Swiss Webster; a gift from

Dr.Karen Duff) or PS1 (Taconic Swiss Webster x B6D2F 1 crosses) mice are anesthetized

with sodium 2-6% isoflurane/oxygen gas. Depth of anesthesia was determined by toe

pinch and corneal reflex. Body temperature was maintained at 370 C with an isothermal

pad. The skull was exposed, and the bone from the region overlaying the septal area was









removed. A modified Pasteur pipette (pulled to a tip-diameter of 0.4mm) was lowered

under visual control with a surgical microscope and mild vacuum applied to remove the

fomix and small amounts of surrounding neocortical tissue. Stereotaxic coordinates for

the lesion sites in mice were A.P. 0.1mm, M.L. + 0.5mm, D.V.-2-3.8mm. Following the

lesioning procedures, a piece of gel foam was placed in the skull hole, antibiotic powder

was sprinkled over the skull, and the scalp was closed with tissue clamps. Animals were

returned to their cages after fully reaching consciousness. 40H-GTS-21 (1mg/kg) or

0.9% saline diluents was injected IP 2x daily (8AM or 6 PM) for two weeks post-lesion,

with the first injection 30 min prior to the lesion.

Differentiation Of PC12 Cells

PC12 cells were obtained from American Type Culture Collection (ATCC). The

cells were grown in RPMI Medium 1640 containing 10% heat-inactivated horse serum,

5% FBS and 1% penicillin/streptomycin at 37 C in a 5% CO2 and 90-92% humidity.

The cells were plated in culture plates that had been pre-coated with collagen (BD

Biosciences) for 3 hours. The cells were split at a 1:4 ratio every 3 days, up to 10

passages. Cell confluence at the time of study was approximately 60%. Cultures were

maintained for 7 days in serum-supplemented media with 100-ng/ml nerve growth factor

(NGF, BD Biosciences), which had been added at day 1 and day 3. On the day 7 of

differentiation, the medium was replaced with one of the following conditions: serum

medium with 100 ng/ml NGF, serum-free medium with or without NGF or serum-free

medium containing specified concentrations of various drugs. 10 ptM of 1,2-Bis (2-

aminophenoxy) ethane-N,N,N,N tetraacetic acid (BAPTA-AM, Molecular Probles

Eugene), 500 nM of nifedipine (Sigma), 10 ptM of xestospongin C (Calbiochem), 10 tpM









of ryanodine (Sigma), 10 [tM of PD98059 (Calbiochem), 10 [tM of U0126 (Cell

Signaling), 10 [iM of U-73122 (Calbiochem), 100 nM of MLA (RBI, South Natick,MA)

were added at specified time point in 100 [tl sterile water immediately after medium

replacement. 10 [LM GTS-21 was added in 50 [tl sterile water. Three days after NGF+

serum removal, five random photographs were taken in each plate with a Nikon inverted

microscope. Images were analyzed in a blinded manner for cell density using the NIH

Immage 1.55 program. There were 6 plates per treatment group. Cell counts are

expressed as a mean+ SEM. Only the cells attached to the bottom of the dishes were

analyzed.

Protein Kinase C (PKC) Assay

The total PKC activity was measured in membrane and soluble fractions of cell

lysates obtained by probe sonication using a kit purchased from Amersham. The medium

was removed from each well and replaced with HEPES-PO4 buffer (137 mM NaC1, 5.4

mM KC1, 0.3 mM Na2HPO4, 0.4 mM KH2PO4, 1 mg/ml dextrose, 20 mM HEPES, ImM

CaCl2 ,pH 7.2) and incubated for 10 min. The HEPES-PO4 buffer was removed and 40 [tl

of kinase assay buffer (KAB) was added. 50 [tg/ml digitonin, 200 [LM MBP [4-14]

substrate peptide, 100 [LM NaATP and 30 [tCi/ml (32 P) ATP were added immediately.

The enzyme activity was measured for 10 min. The reaction was terminated with 10 [tl

of 25% trichloracetic acid. 45 [tl of the acidified assay mixture from each well were

spotted onto 2 x 2 cm Whatman P-81 phosphocellulose filter paper. Phosphorylated

MBP [4-14] was quantified by liquid scintillation spectrophotometry in a Beckman

LS1800 counter.









Tail DNA Extraction And Genetype

Mice homozygous for the Chrna7t1mlBa mutation are viable and fertile, but are

inconsistent breeders, producing small litters. The heterozygous Chrna7tmlBay mutation

breeder mice were obtained from Jackson laboratory. Two-week-old mouse-tails were

cut off at the tip (2-5 mm) and were transferred to a pre-labeled tube. 700 [l of lysis

buffer (50 mM Tris, 100 mM EDTA, 0.05% SDS and 20 tg/ml proteinase K) were added

to the tube. The tubes were incubated at 55 C overnight. The tubes were removed from

55 C and 150 [tl 5 M NaCl were added. An equal volume of phenol-chloroform was

added to the sample, vortexed for 5 min and microfuged for 5 min at 14,000 g. This was

followed by an extraction with an equal volume of chloroform and microfuging for 5 min

at 14,000 g. The aqueous layer was transferred into a new microfuge tube to which was

added 2.5 volumes of ethanol. DNA was precipitated at -80 C for 30min. The sample

was microfuged at 14,000 g for 20 min. The supernatant was discarded. 0.5 ml of 75%

ethanol was added to the pellet and microfuged at 14,000 g for 10 min. The pellet was air

dried for 15 min and dissolved in 50 [tl of water. 4 [tl tails DNA were added to the 46 [tl

mix of the components for PCR amplification (9 [tl 10X PCR buffer, 4 [tl 2.5 mM dNTP,

2.5 [tl 20 [M 1002 primer, 1.25 tl 20 [LM 1003 primer, 1.875 [tl 20 [tM 1004 primer, 1

[tl Taq DAN polymerase and 31.5 [tl ddH20). The PCR reaction product was loaded to

1.5% agrose gel (Figure 2-1). The size of the u7 KO mouse band is 750 bp. The sizes of

u7 bands for heterozygous is 440 bp and 750 bp. The size of wild type mice is 440 bp.

















1 2 3 4 5

Figure 2-1. The a7 genotype of mice. 1: wild type mouse; 2: a7 knockout mouse; 3: a7
heterozygous mouse; 4: negative control; 5: 100 bp marker.

Statistical Analyses

Statistical analyses involved one way or multiple ANOVA for comparisons of

parametric populations, using the Statview program. For non-parametric analysis, such

as groups that undergo multiple treatments that may be interactive, resulting in a non-

normal population, using a rank order test on the Statview program (acquisition behavior

Morris water task).














CHAPTER 3
MECHANISMS UNDERLYING a7 NICOTINIC RECEPTOR NEUROPROTECTION
IN PC12 CELLS

Introduction

PC12 cells and SK N SH cells were being chosen in this aim study. Both of them

express endogenous a7 nicotinic receptors. It had shown that NGF-differentiated PC12

cells would undergo apoptosis following removal of both NGF and serum. NGF-

deprivation is hypothesized to occur in AD in septal cholinergic neurons. SK N SH cells

line is very sensitivity to exposure Abeta. We therefore used these two models to test the

cytoprotective action of u7 nicotinic receptors.

Several groups including ours have demonstrated that GTS-21 and 40H-GTS-21

a7 agonists increase intracellular calcium concentrations (Gueorguiev et al., 2000; Li et

al., 2002). They also activate the calcium-sensitive transduction processes such as

protein kinases A (PKA)(Dajas-Bailador et al., 2002b) and C (PKC), inositol

triphosphate (IP-3) kinase (Kihara et al., 2001), ERK (Dajas-Bailador et al., 2002b; Bell

et al., 2004), and janus kinase (Salehi et al., 2004). PKC (Li et al., 1999c), IP-3 kinase

(Kihara et al., 2001) and janus kinase (Salehi et al., 2004) are each essential for a7

mediated protection against one or more apoptotic insults, while the roles of the other

kinase pathways have not been studied. This protection through calcium-sensitive

kinases suggests a protective role for the increased intracellular calcium ion

concentrations observed following treatment with a7 agonists. However, there remains

no direct demonstration that a7 receptor mediated protection or kinase activation depends









on intracellular calcium ions. We tested this hypothesis by investigating the effects of

intracellular calcium ion chelation on both protection and PKC activation in NGF-

differentiated rat pheochromocytoma (PC12) cells. These cells undergo apoptosis

following NGF- removal and may provide a model for the dysfunction of ascending basal

forebrain neurons associated with decreased NGF-transport seen in Down's syndrome

and Alzheimer's disease (Kerwin et al., 1992; Scott et al., 1995; Cooper et al., 2001;

Salehi et al., 2004). They also express a7 nicotinic receptors and are protected from

apoptosis by prolonged treatment with a7 receptor agonists

Activation of a7 receptors can increase calcium accumulation both directly as well

as through activation of downstream L-type voltage sensitive channels, IP-3 channels,

and ryanodine channels (Vijayaraghavan et al., 1992; Gueorguiev et al., 2000; Shoop et

al., 2001; Dajas-Bailador et al., 2002a), analogous to what is seen with metabotropic

glutamate receptors (Fagni et al., 2000). L-type channels are activated by depolarization

triggered by the influx of sodium ions and calcium ions through these receptors, while the

intracellular calcium channels are likely activated through calcium-influx through both

a7 receptors and L-type channels. Blocking IP-3 channels with xestospongin C

attenuates the long term increase in calcium accumulation following a7 receptor

activation almost completely in PC12 cells, implicating these channels in neuroprotection

(Gueorguiev et al., 2000). An analysis of the calcium elevations triggered by nicotine in

SH-SY5Y cells indicated that xestospongin C-sensitive IP-3 channel activation appeared

to be more important than nifedipine-sensitive L-type channels or ryanodine-sensitive

channels for the long term effects of a7 receptors (Dajas-Bailador et al., 2002a). We

therefore hypothesized that these three channels may be differentially important for the









long term cytoprotective actions of a7 receptors, which we tested in PC12 cells that

express each of these calcium channels (Gafni et al., 1997; Tully and Treistman, 2004)).

A third goal of this study was to evaluated the role of the calcium-sensitive MAP

kinases ERK1/2, p38, and JNK in the a7 receptor mediated neuroprotection, since they

are also differentially involved in the cytoprotective effects of other anti-apoptotic agents

(Hetman and Xia, 2000; Hsu et al., 2004; Kyosseva, 2004). ERK1/2 phosphorylation has

been reported to be essential for the antiapoptotic effects of a wide variety of drugs

(Hetman and Xia, 2000; Kyosseva, 2004). It was therefore a likely candidate for

involvement in a7 receptor mediated protection, along with the other kinases mentioned

above. Two other MAP kinase pathways, p38 and JNK, which are more frequently

involved in stress responses than cytoprotection (Hsu et al., 2004; Kyosseva, 2004), were

also evaluated following a7 receptor activation to determine their potential involvement

in the protective effects of these receptors.

Results

PC12 cells were plated in 60 mm culture dishes and differentiated for 7 days in the

presence of 100 ng/ml NGF (Figure 3-1). NGF and serum were withdrawn from the

medium at that time. The cells were treated with different concentrations of GTS-21 (0,

3 ptM, 10 JIM and 30 pIM) for 72 hours. Cell density was measured after 3 days by the

NIH Image system program (Figure 3-1). This figure demonstrates that NGF and serum

withdrawn caused approximately a 50% cell loss. 3 JIM and 10 JIM GTS-21 induced

protection and provided 80-90% cell survival. But 30 JIM GTS-21 cause even more

extensive cell loss.













0. 75-

S50-
0I-
4) 25-


0 3uM 10uM 30uM
GTS-21


Figure 3-1. GTS-21 induced protection ofPC12 cells during trophic factor deprivation.
PC12 cells were plated in the 60 mm culture dishes and differentiated with
NGF for 7 days. Cells were rinsed and the media was replaced with fresh
media containing the treatment indicated above. Cell density was measured 3
days later and expressed as the mean + SEM of 6-8 plates/group from 3
experiments. Three random areas were counted per plate and these 3 values
were averaged to yield one value per plate. *p < 0.05 compared to untreated
group (one way ANOVA).

In order to evaluate the role of intracellular calcium ions and a7 receptors in this

cytoprotection. 10 [tM BAPTA-AM and 100 nM MLA were added either 30 min before

(pretreatment) or 30 min after (posttreatment) GTS-21. Treatment with BAPTA alone

had no effect on cell survival during NGF + serum removal or in the presence of NGF,

though it blocked the cytoprotective action of 10 [tM GTS-21 when added 30 min prior to

or 30 min after the receptor agonist. MLA similarly blocked the GTS-21 induced

protection when added at both time points (Figure 3-2).

None of the treatments affected the Kd for high affinity MLA binding to a7

receptors (range: 1.6-2.3 nM). Removal of NGF + serum reduced the density of a7

receptors over the 3 day but not 30 min interval (Table 3-1); this reduction was not seen

with addition of 100 ng/ml of NGF. Addition of BAPTA to the NGF + serum deprived








medium had no acute effect on a7 nicotinic receptor binding density, but also modestly

increased density over the 3 day interval compared to cells without the chelator. Neither

GTS-21 nor GTS-21 + BAPTA preserved receptor density in this manner.


Pretreatments


BAPTA


MLA


mr'lrfl


0 10


0 10
[GTS-21], gM


Posttreatments


BAPTA


MLA


Kflr nf


0 10


0 10


Figure 3-2. Effects of intracellular calcium chelation on GTS-21 induced protection of
PC12 cells during trophic factor deprivation. Cells were differentiated for 7
days with 100 ng/ml mouse NGF and then exposed to serum-free medium
containing either BAPTA-AM (10 [tM), GTS-21, or MLA (100 nM).
BAPTA-AM and MLA were added either 30 min before (pretreatment) or 30
min after (posttreatment) the GTS-21. Cell density was measured 3 days later
and expressed as the mean + SEM of 6-8 plates/group from 3 experiments,
normalized to the NGF-treated values for each experiment. *p < 0.05
compared to untreated group (one way ANOVA).
10 [tM of BAPTA also blocked the concentration-dependent GTS-21 induced

elevation in PKC membrane translocation seen in these NGF and serum withdrawn


differentiated PC12 cells (Figure 3-3).


UL
Z
- 100-


50-


0-


0 10










Table 3-1. Effects of BAPTA and GTS-21 on a7 receptor binding density in PC12 cells
Treatment High affinity MLA binding (Bmax in fmol/mg protein)

30 min 3 days
NGF+serum removal 152 + 11 107 + 10^

+ NGF (100 ng/ml) 165+ 18 172 + 15*

+ BAPTA (10 MM) 167 + 13 144 + 13*

+ GTS-21 (10 tM) 155+ 17 101 + 14A

+ BAPTA + GTS-21 172 + 16 98 + 12A

Table 3-1. PC12 cells were differentiated for 7 days with 100 ng/ml NGF and then
exposed to NGF + serum withdrawal. Specified concentrations ofNGF, BAPTA, or
GTS-21 were added and high affinity [3H] MLA was measured 30 min or 3 days later.
Each value is the mean + SEM of three samples, each assayed in tripicate. *p < 0.05
compared to same time point, no drug-treatment (two way ANOVA); Ap < 0.05 compared
to 30 min interval, same treatment.




1. -- GTS-21
-- GTS-21 + BAPTA
o
0 o 1.0-


S0.5-


0.0
0 2 4 6 8 10
[GTS-21], LiM


Figure 3-3. Effects of intracellular calcium chelation on PKC activation by GTS-21 in
PC12 cells. Cells differentiated as in Figure 3-1 were simultaneously exposed
to serum and NGF removal, as well as to specified concentrations of GTS-21
in the presence or absence of 10 [tM BAPTA-AM. 15 min later, cells were
fractionated to membrane and soluble portions, each of which was assayed for
phorbol-stimulated PKC activity/mg protein. Values are expressed as the
ratios of membrane/soluble PKC activity; N = 6 plates/group from 3 separate
experiments. *p < 0.05 compared to BAPTA-treated group, same GTS-21
concentration.









Effects of 40H-GTS-21 on PKC isozyme translocation were tested. Different

concentrations of 40H-GTS-21 were used in serum and NGF withdrawn differentiated

PC12 cells. 3 [tM and 10 [tM 40H-GTS-21 increased PKC alpha translocation (Figure

3-4). But 30 [tM 40H-GTS-21 decrease the membrane/soluble ratio of PKC alpha. PKC

delta membrane/soluble ratios were decreased at higher concentrations of 40H-GTS-21.

PKC gamma was unaffected.


S4-
ST -- Alpha
o. T Delta
0 =0 Gamma

EE2
(iU
oEs



c 0S 0



Figure 3-4. Effects of 40H-GTS-21 on PKC isozyme translocation. Neuroprotective
concentration of 40H-GTS-21 increased the membrane/soluble ratio of PKC
alpha, a measure of its activation. PKC delta membrane/soluble ratios were
decreased, but over a higher concentration range. No effect was seen on
translocation of PKC gamma.

In order to determine the roles of different calcium channels in a7 receptor induced

neuroprotection, some cells were treated either with L-type channel blocker nifedipine,

ryanodine receptor blocker ryanodine, IP3 channel antagonist xestospongin C,

phospholipase C inhibitor U-73122 with or without 10 [tM GTS-21. The voltage

sensitive L-type calcium channel blocker nifedipine had no effect on cell viability when

applied alone or when with GTS-21 (Figure 3-5). Ryanodine partially attenuated the

GTS-21 induced protection, also without direct effect alone, indicating that only some









cells appeared to depend on this channel activation for survival in this model.

Xestospongin C, the IP3 channel antagonist, completely blocked a7 receptor mediated

protection and had no effect on cell density when applied alone.

100- *


7 75
o T
S50

S25
00
O Nv










Figure 3-5. Effects of calcium channel antagonists on GTS-21 induced cytoprotection in
NGF-deprived PC12 cells. Cells differentiated as in Figure 1 were treated
with 10 [tM GTS-21 immediately after NGF + serum removal, with or without
10 [tM ryanodine, 10 [tM xestospongin C, 10 [tM U-73122 or 500 nM
nifedipine. Each value is the mean + SEM of 6-8 plates/group from 3 separate
experiments. *p < 0.05 compared to same treatment without GTS-21;#p <
0.05 compared to GTS-21 only treatment group (one way ANOVA).

Serum and NGF were withdrawn from differentiation PC12 cells and 10 [tM GTS-

21 was added to cells. ERK1/2, p38 and JNK-phosphorylation were measured at 5 and

180-minute time interval. ERK1/2-phosphorylation was increased by 10 [tM of GTS-21

within 5 minutes, and this effect increased by 3 hours (Figure 3-6). Increased ERK1/2

phosphorylation was blocked by 500 nM MILA applied 5 minutes before GTS-21 addition

(not shown). p38- and JNK-phosphorylation were unaffected by GTS-21 at either time

interval.











1 n5 Min
gg *
SO 200-
2o 180 Min


100-
II I
0'-="=- O--I



Control ERK1/2 JNK p38
MAP Kinase

Figure 3-6. Effects of GTS-21 on the phosphorylation of several MAP kinases in PC12
cells. Cells differentiated as in Figure 1 were treated with 10 pM GTS-21 for
the specified interval upon removal of the NGF + serum. Whole cell extracts
were assayed for phospho-ERK1/2, phospho-JNK and phospho-p38 by
western blotting and expressed as the mean + SEM of 4 samples/group,
normalized to the 5 minute control value for that experiment. Each gel
contained two lanes from the same 5-minute control sample that were
averaged and used for normalization. *p < 0.05 compared to control from
same time point (one way ANOVA).

Four different concentrations of GTS-21 (0, 0.5,3 and 10 pM) were used to test

ERK1/2 phosphoration (180 minutes later). Some of groups pretreated with 100 nM

MLA. The effect of GTS-21 on ERK1/2 phosphorylation was concentration dependent.

This effect was blocked by MLA, demonstrating the role of a7 receptors (Figure 3-7).

GTS-21 mediated neuroprotection was blocked by either pretreatment with the

PKC blocker BIM or the ERK1/2 blocker U0126 and PD98059 (Figure 3-8).

Various kinase inhibitors were tested on the effects of 40H-GTS-21 on

differentiation PC12 cells. a7 antagonist MLA (1 [ M), PKC antagonist BIM1 (100 nM),

ERK1/2 inhibitor PD98059 (20 [LM) and PKA inhibitor H89 (1 [tM) blocked 40H-GTS-

21 provided cytoprotection (Figure 3-9).


















300-
--- GTS-21
S* --- GTS-21+MLA










0 2 4 6 8 10

[GTS-21], iM


Figure 3-7. MLA blocks the ERK1/2 phosphorylation triggered by GTS-21. Cells were
treated as in Figure 4, except with varying concentrations of GTS-21 for 180
minutes, plus or minus 100 nM MLA. Phospho-ERK was assayed and
expressed as the mean + SEM of 4 samples/group, normalized to untreated
control values on the same gel. *p < 0.05 compared to either the
corresponding MLA treatment group.
corresponding MLA treatment group.









-100-

S75-



4 25-


-


Tm r


n


40 e
cf> % g
.^O / 0"^


Figure 3-8. GTS-21 induced cytoprotection is dependent on ERK phosphorylation and
PKC activation. Differentiated PC12 cells were treated with l0 [M GTS-21
for 3 days upon NGF + serum removal, with or without 10 [tM PD98059, 500
nM bis-indole maleimide (BIM) or 10 [tM U0126. Cell densities are
expressed as the mean + SEM of 6 plates/group from 3 experiments; *p < 0.01
compared to untreated control (one way ANOVA).


M
x \ \
\ \ v
V Y 'Y
\ ^ \
^ ^
^^
^^
N ^ \
v \ v
X \ \
\ \ V
V Y 'Y
V ^ \
^ ^ ^


|I|


= NGF-free
-+ MLA
8+ BIM1
S+ PD98059
=+ H89


40H-GTS-21
Figure 3-9. Effects of 40H-GTS-21 and various kinase inhibotor on PC12 cells. NGF and
serum were removed 1 hour before agonist treatment. 1 [ M a7 antagonist
MLA, 100 nM PKC antagonist BIM1, 20 [tM ERK1/2 inhibitor and 1 [ M
PKA inhibitor H89 were added 15 minutes prior to 40H-GTS-21. Cell density
was measured 3 days after NGF-removal. Values are means + SEM of 4
plates/group.


o
f- C

4-
n o


= u-
0
z3


II~YYYLYLY









Various kinase inhibitors were also tested on the protective effects of 40H-GTS-21

against an amyloid peptide in human SK N SH cells. Abeta 25-35 (20 pM) caused a

significant reduction in cell density alone, which was partially blocked by 40H-GTS-21

in a concentration dependent manner. At 30 [tM 40H-GTS-21, additional toxicity was

seen over that with Abeta peptide alone. The a7 antagonist MLA (1 pM), PKC

antagonist BIM1 (100 nM), ERK1/2 inhibitor PD98059 (20 [tM) and PKA inhibitor H89

(1pM) all blocked this 40H-GTS-21 provided cytoprotection (Figure 3-10). However,

only MLA blocked the additional toxicity caused by 30 [tM 40H-GTS-21.


150-
m Abeta 25-35
o EM + MLA
S+ BIM 1
0o 100- 1
T ES + PD98059
> E T | + H89
(4 50-






Figure 3-10. Effects of Abeta 25-35, 40H-GTS-21 and various kinase inhibitor on SK N
SH cells. All cells were treated with specified concentrations of 40H-GTS-21
for 1 hour prior to adding 20 [tM Abeta 25-35. 1 [tM a7 antagonist MLA, 100
nM PKC antagonist BIM1, 20 [iM ERK1/2 inhibitor and 1 [M PKA inhibitor
H89 were added 15 minutes prior to 40H-GTS-21. Cell density was measured
3 days after NGF-removal. Values are means + SEM of 4 plates/group.

Discussion

Previous studies have demonstrated GTS-21-induced calcium elevations

(Gueorguiev et al., 2000; Li et al., 2002) and have suggested the importance of multiple

calcium-activated processes for a7 receptor-induced neuroprotection (Li et al., 1999c;









Kihara et al., 2001; Dajas-Bailador et al., 2002b; Bell et al., 2004; Salehi et al., 2004).

The present results show for the first time that chelation of intracellular calcium ions

blocks GTS-21-induced, a7 mediated neuroprotection, demonstrating a direct role for

these divalent cations in the receptor-modulation of cell viability. This is consistent with

previous studies that chelated or removed extracellular calcium ions to attenuate a7-

mediated protection or kinase-activation (Donnelly-Roberts et al., 1996; Dajas-Bailador

et al., 2002b). The concentration of BAPTA-AM used in our study was selected for its

ability to prevent nicotine-induced elevations in both cytoplasmic calcium and tyrosine

hydroxylase activity in PC12 cells (Gueorguiev et al., 1999). It therefore appears that the

calcium-elevations triggered by a7 receptor activation have important roles both for the

phenotypic properties of these cells as well as for their long-term viability in the presence

of toxic insults. It is interesting to note that a careful study of intracellular calcium

chelation in hippocampal neurons recently concluded that modest increases in

intracellular calcium-concentrations were also associated with improved viability

(Bickler and Fahlman, 2004). It remains to be determined to what extent BAPTA-

treatment reduces calcium concentrations in mitochondria, smooth endoplasmic

reticulum, and other storage sites under our experimental conditions.

The effects of a7 receptor activation on cell viability and their blockade by

BAPTA were seen without any acute change in a7 nicotinic receptor binding density and

a modest increase in receptor density by 3 day. These observations are significant

because a7 binding density was recently found to modulate the neuroprotection seen in

PC12 cells (Jonnala and Buccafusco, 2001). The reduction in a7 receptor binding density

over three days of NGF + serum removal is probably related to the observation that NGF









increases this receptor subunit expression in PC12 cells(Takahashi et al., 1999). GTS-21

has not been reported to increase a7 receptor expression or lead to PC12 cell

differentiation, which may account for its inability to prevent the loss of a7 receptor

density over the 3-days of NGF-deprivation. The lack of effect of GTS-21 on a7 receptor

binding density following the 3 days of NGF + serum deprivation was a surprising result

since the less selective agonist nicotine was found to increase a7 receptor expression in

these cells (Jonnala and Buccafusco 2001). MLA also increases a7 receptor density in

PC12 cells (Jonnala and Buccafusco 2001), which might be expected with GTS-21 if it

acted by desensitizing most a7 receptors. One possibility is that selective, low level

activation of a7 receptors is insufficient to increase their density, which is consistent with

the lack of effect of chronic GTS-21 administration on a7 receptor density in neocortex

in vivo (Meyer et al., 1997). Alternatively, GTS-21 may preserve a population of PC12

cells that expresses fewer a7 receptors, while simultaneously increasing their a7 receptor

expression, resulting in no net change in density under these conditions.

Our results indicate that the cell viability effects of BAPTA are likely due to

processes downstream from the receptor activation, since there was no decrease in

binding density that would be expected to interfere with the actions of GTS-21. This is

consistent with the observation that BAPTA exposure attenuated GTS-21 induced PKC

activation. PKC-translocation and activation were previously found to be essential for a7

mediated protection in this apoptotic model (Li et al., 1999c). This increase in PKC

activity occurs within 15 minutes; during the interval that neuroprotection is BAPTA-

sensitive, suggesting that the kinase activation may be one of the relatively early steps in

the protective process.









The antiapoptotic effect of GTS-21 in PC12 cells requires extended activation of a7

receptors, since MLA blocks this cell survival when administered up to 1 hr post-agonist

(Li et al., 1999c). The ability of BAPTA to block protection when applied 5 min after

GTS-21 suggests that a protracted elevation in intracellular calcium ions is also essential

for protection. Whether this is due to a slow or rapid increase in calcium is not clear. We

previously demonstrated that neuroprotective concentrations of GTS-21-caused a long

term, near steady state calcium influx through a7 receptors, without the desensitization of

the overall receptor population seen at higher, non-protective agonist concentrations

(Papke et al., 2000). However, while it is likely that this low-level receptor activation

provides an early calcium-transient that is important for triggering the cytoprotective

pathway, it appears that downstream calcium channel activation is important as well.

The multichannel modulation of intracellular calcium by voltage sensitive L-type

calcium channels, intracellular IP3 channels, and intracellular ryanodine channels

provides a potentially complex mechanism for a7 receptors to affect cellular function and

viability (Vijayaraghavan et al., 1992; Gueorguiev et al., 2000; Shoop et al., 2001; Dajas-

Bailador et al., 2002a; Dajas-Bailador et al., 2002c). Activation of the intracellular IP3

calcium channel, and to a lesser extent the ryanodine receptor, are necessary for complete

a7 mediated protection in this model, based on sensitivity to antagonists. This is

consistent with the protective actions of metabotropic receptors such as bradykinin

(Yamauchi et al., 2003) and mGluR4s (Maj et al., 2003) that also act on IP3 receptors.

The concentration of xestospongin C used in the present study was found previously by

our group to block IP3 receptors and reduce GTS-21 induced calcium accumulation in

PC12 cells for as long as it was assayed (45 min)(Gueorguiev et al., 2000). This









dependence on IP3 channels for most of the long term elevation in intracellular calcium

triggered by a7 receptors is consistent with their involvement in cytoprotection, which

similarly depends on long term receptor activation (Li et al., 1999c). Inhibition of

phospholipase C with U-73122 (Kokoska et al., 1998), which blocks IP3 production, also

attenuates GTS-21 induced protection, providing additional support for a role of IP3

receptors.

Ryanodine channels were also involved in the protection of some NGF + serum

deprived PC12 cells, though this effect was less dramatic than that seen with IP3 channel

attenuation. Since ryanodine may only partially attenuate a7 mediated calcium

elevations (Gueorguiev et al., 2000), it is not surprising that some cells remained

sensitive to GTS-21 in the presence of this channel blocker. There are multiple types of

ryanodine receptor that are differentially expressed in various tissues, however, so it is

conceivable that this result may be difficult to extrapolate to other neuronal models

(Berridge et al., 2000). In brain neurons, ryanodine receptors are primarily localized to

the endoplasmic reticulum of postsynaptic entities, from which they release calcium in

response to increased cytoplasmic calcium. Recently, however, they have been found

presynaptically and may be involved in modulating transmitter release (Bouchard et al.,

2003), another well characterized function of a7 receptors.

The nifedipine-sensitive voltage sensitive L-type channel also underlies a

significant amount of the calcium accumulation seen following a7 receptor activation.

Although extracellular calcium is necessary for neuroprotection, as noted above, it

appears that the channels other than L-type channels are involved in this action, probably

the a7 receptors themselves. The observation that L-type channels are not essential for









protection may be due to multiple factors. First, L-type channel openings may be for a

shorter duration than is necessary for protection, e.g., if the voltage-dependent channel

openings is attenuated over time through calcium-activated potassium channels.

Alternatively, the calcium entry through these channels may be physically removed from

the transduction processes essential for cytoprotection. A third possibility is that the

intracellular calcium channels may provide sufficient calcium for protection even in the

absence of voltage sensitive calcium channel activation.

Our results indicate for the first time that the ERK1/2 MAP kinase pathway is

required for a7 receptor mediated protection, based on its GTS-21 induced

phosphorylation and attendant activation, as well as the ability of the ERK1/2 inhibitor

PD98059 to block protection. ERK1/2 phosphorylation has been associated with

cytoprotection in a variety of model systems e.g., (Hetman and Xia, 2000; Kyosseva,

2004), and with other anti-apoptotic processes found to be triggered by a7 receptors,

including bcl2 elevations, increased mitochondrial membrane potential, and reduced

cytochrome C release (Li et al., 1999c). Our results indicate therefore that calcium

accumulation triggered by a7 receptor activation is necessary for this chain of kinase-

mediated anti-apoptotic events. In contrast, neither the p38 nor the JNK pathway was

apparently activated by a protective concentration of GTS-21. It is interesting to note

that activation of ERK has been reported with very low concentrations amyloid peptides,

suggesting that this may provide a dose-dependent protective role for the peptides and

receptors under appropriate conditions, perhaps even in the sparing of a7 receptor

expressing cells in Alzheimer's disease.









In summary, a7 receptor activation provides cytoprotection against trophic factor

deprivation and triggers PKC translocation through a mechanism that appears to involve

intracellular calcium ion elevations (Figure 3-11). Among the several calcium channels

triggered by a7 receptor activation, IP3- and, to a lesser extent ryanodine-receptor

calcium channels are likely mediators of these calcium-elevations and are essential for

cytoprotection. Downstream ERK1/2 phosphorylation is also essential for protection,

while other MAP kinases JNK and p38 are not. It now becomes important to determine

the role of these various pathways in the protective actions of a7 receptors in brain in

vivo.


Figure 3-11. The potential mechanism of a7 nicotinic receptors mediated cytoprotection.














CHAPTER 4
NEUROPRECTIVE AND ANTI-AMYLOIDOGENE EFFECTS OF THE a7 PARTIAL
AGONIST 40H-GTS-21 IN FIMBRIA FORNIX LESIONED MICE OF DIFFERENT
GENOTYPES

Introduction

While a7 nicotinic receptors are neuroprotective in a variety of models, little is

known about their protective properties in models of neuronal dysfunction related to AD

in vivo. There is presently no animal model that mimics every aspect of AD, though

various lesion and genetic manipulations have been used. Combinations of these

approaches, however, are much less common.

Investigations of genetic linkage and transgenic animal models have shown that no

single genetic defect that accounts for all the features of AD. A certain number genes

have been identified to be involved in the progressive neuron degeneration of this

disease. Most AD cases have mutations in the genes encoding for APP, presenilin 1

(PS1) or PS2. All of these mutations have been shown to alter APP metabolism and

increase the A3 peptide levels in the brain.

Transgenic mice expressing mutated human genes that are associated with familial

AD offer a powerful model to study A3. The present study employed either single

human mutated PS1-M146L transgenic mice or double transgenic mice expressing both

human APP Swedish mutant K670N/M671L and PS1-M146L mutations (APP/PS1).

Both types of transgenic APP/PS 1 mice have elevated levels of fibrillogenic A3 42

peptide in their brains, though only the APP/PS1 mice develop amyloid plaques starting









around the age of 4 months of age. By 6 months of age, the amyloid plaque load is

comparable to that of 12-month-old single APP K670N/M671L mice. This demonstrates

that APP/PS1 mice develop amyloid plaques earlier than the APP-only transgenic mice.

However, neither type of transgenic mice shows neurofibrillary tangles. These mice do

have deficits in spatial memory in selected paradigms, notably the radial arm task in the

Morris water paradigm, though not in the classic Morris water protocol. Mutations in

PS1 may act synergistically with Swedish mutant APP to cause some pathology

reminiscent of the AD brains.

PS1 and PS2 play a critical role in mediating gamma secretase cleavage of the

APP. Gamma secretase is one step in the formation of amyloidogenic A3 1-42 or A3 1-

40. In addition, PS1 deficiency leads to alter intracellular Ca2+ homeostasis involving

endoplasmic reticulum Ca2 stores. PS1 transgenic mice that overexpress the mutant AD

protein have been found to elevate levels of endogenous Abeta 1-42, but not Abetal-40.

Abetal-42 levels are significantly elevated in the mutant PS1 mice, presumably by

enhancing cleavage of APP at a gamma-secretase site. Evidence from in vitro studies

also indicates that PS1 itself might either act as gamma-secretase or mediate the catalytic

activity of the enzyme (De Strooper and Annaert, 2000). These PS1 transgenic mice do

not form amyloid deposits upon aging, presumably because the levels of Abeta do not

reach the level required to start the aggregation process in mice (McGowan et al., 1999).

Mutated PS1 mice also do not show overt AD-like pathology or spatial learning deficits

in the Morris water maze test. The densities of c7 nicotinic receptor-binding sites are

unaltered in transgenic PS1 mice compare to non-transgenic controls.









A recent study showed that no significant differences in size or number of

cholinergic nerve terminals in the hippocampus or neocortical areas of mice

overexpressing M146L PS1 (Wong et al., 1999). Conversely, studies from the double

transgenic mice APP/PS1 mice have been found to display an extensive loss of

cholinergic synapses in the frontal cortex and hippocampus (Vaucher et al., 2002). It was

suggested that overexpression of human A3 peptide combined with a shift toward longer

forms of A3 terminating at residues 42 or 43 due to mutation of PS1 is required to elicit

cholinergic deficits in mice (Bronfman et al., 2000). Vaucher et al. showed that mutation

of the human PS1 gene might alter sensorimotor activity and long-term retention of

object recognition memory but not ChAT enzymatic activity or cholinergic receptor

binding sites (Vaucher et al., 2002). Whether this cognitive deficit is due to an alteration

in APP processing of the endogenous mouse APP holoprotein by the PS1 mutation or

related to altered functions of other neurotransmitters is not known.

A non-genetic approach to model AD involves lesions of neuronal pathways

affected by the disease. One of the early effects of AD is the degeneration of basal

forebrain cholinergic neurons, which results in loss of cholinergic function in the

neocortex and hippocampus. The septo-hippocampal pathway has been the most

thoroughly investigated basal forebrain cholinergic pathway. This pathway carries the

acetylcholine and GABA as neurotransmitters in axons that project to the hippocampus.

Fimbria fornix lesion (FFX-lesion) can result in partial or near-complete loss of

cholinergic activity in the hippocampus and impairment in behavioral tasks, depending

on the extent and type of lesion. For example, the aspirative lesions used in my studies

affect from 50-90% of the cholinergic and GABA neurons projecting to the









hippocampus. Following these lesions, animals become deficient in septal cholinergic

neuronal density, memory related behavior and, as was recently reported, in hippocampal

a7 nicotinic receptor functions.

The cholinergical deficits in septum following FFX lesions may be due to the loss

of retrograde axonal NGF transport from the hippocampus, based on the observations

that: 1) NGF is typically transported in this manner by these cells retrograde; and 2) that

NGF-administration can protect septal cholinergic neurons from the effects of the lesion.

Since NGF-deprivation in differentiated PC12 cells is toxic in a manner that is protected

by a7 nicotinic receptor agonists, my hypothesis was that the a7 nicotinic receptors

partial agonist 40H-GTS-21 would also be able to protect cholonergic neurons in vivo

from axotomy. However, this protection was not expected in amyloid expressing mice

such as the APP/PS1 double transgenic because of the a7 nicotinic receptor blockade by

amyloid peptides.

Mapping of cholinergic pathways based on choline acetyltransferase (ChAT)

immunohistrochemistry could be considered to define the limits of nicotinic cholinergic

signaling systems in the mammalian CNS. ChAT immunohistochemical maps identify

major cholinergic projection from loosely-delimited nuclei of heterogeneous

neurotransmitter phenotype in the medial septum (Mesulam, 1995). However, since a7

nicotinic receptors are also activated by choline, a natural byproduct of

phosphatidylcholine metabolism and a circulating nutrient, it is possible that these

cholinergic signals do not require and are not marked by ChAT staining.

A small fraction (10-20%) of septal GABAergic neurons are intermingled with

cholinergic neurons projecting to the hippocampus, although estimates about the number









of GABAergic neurons in this region vary across different studies and species. Septal

GABA neurons have morphologies similar to those of ChAT- positive neurons, with a

somewhat different topography. GABAergic neurons have been demonstrated a roughly

similar number to the cholinergic neuron in rats, though a smaller fraction project to the

hippocampus in at least some mouse stains (Sarter and Bruno, 2002). Interestingly, while

these septohippocampal GABA neurons project alongside cholinergic neurons, they are

not adversely affected by AD even as cholinergic neurons are lost. This may be due to

the differential need of these cell types for NGF, since the GABA neurons do not posses

NGF receptors.

It has been reported that both septal GABA and cholinergic neurons possess a7

nicotinic receptors, but whether these receptors are protective for both populations is not

known. One possible mechanism to account for a7 nicotinic receptor mediated

neuroprotection is an increase in NGF expression. Nicotinic receptor activation was

found to increase NGF levels and NGF-receptor levels, so this is a possibility. If so, then

the a7 nicotinic receptor agonist treatment will likely be protective only for cholinerhic

neurons and not GABA neurons. Alternatively, if c7 nicotinic receptors are protective

more directly, then it would more likely that both types of neurons were protected by a7

nicotinic receptor agonists. I investigated these possibilities by comparing the

neuroprotective actions of 40H-GTS-21 in both populations of neurons following FFX

lesions.

While multiple studies have evaluated the effects of potential therapeutic agents in

either transgenic mice or lesioned animals, no study has yet combined these two model

systems. One of my project goals was therefore to test whether an a7 nicotinic receptor









agonist could provide neuroprotection in a combination transgenetic and lesion model

involving FFX lesions of wild type, APP/PS 1 and PS1 transgenic mice.

Another potential beneficial effect of c7 nicotinic receptor activations for AD may

be reducing amyloid plaque density. Nicotine has been found recently to reduce amyloid

plaque density in mutant APP transgenic mice over a period of several months of PO

treatments. While it is not clear which of the nicotinic receptor subtypes cause this

effect, my hypothesis was that a7 nicotinic receptors may be involved based on the

observations, as noted in Chapter 1, that these receptors can stimulate APP a-secretase

activity, which would be expected to reduce substrate availability for Abeta

amyloidogenic peptides.

One method to determine the role of c7 nicotinic receptors in the nicotinic-induced

reductions in amyloid density is to use selective agonists for the receptor. I addressed

this possibility with the partial agonist 40H-GTS-21 in double transgenic APP/PS 1 mice.

Results

Mice receiving FFX- lesions appeared to be no different from unlesioned animals

with respect to body weight gain or visual assessments of health. In a preliminary study,

AChE staining was used to verify the extent of the lesion relative to the loss of

cholinergic innervation of the hippocampus. Aspirative FFX-lesions were performed

unilaterally on male adults SD rat. The lesion resulted in a nearly complete loss of the

hippocampal AChE-positive fibers on the lesion side. High levels of AChE staining were

observed in the unlesioned side (Figure 4-1).
















-V

ah


Figure 4-1. Aspirative FFX-lesion of the septal hippocampal cholinergic pathway. The
lesioned side hippocampus AChE staining is over 90% lost.

Wild type C57/B16/J mice (9 month), PS1 mice (9 month) and APP/PS1 (9 month)

also had significant, 55-60%, reductions in septal hippocampal cholinergic neuronal

density following FFX-lesion as measured by septal ChAT staining two weeks post-

lesion (Figure 4-2). There was no difference in the extent of this ChAT staining loss

among the three groups when expressed as a percent of unlesioned, contralateral cell

density. This normalization procedure of expressing neuronal density as a percent of the

contralateral control side was used to control for inter-animal differences in perfusion,

sectioning and staining. To what extent the loss of cholinergic neuron density was due to

neuronal death versus phenotypic change is not known. There was also a significant

reduction in the size of the septal cholinergic perikarya caused by this lesion.

In order to evaluate the effect of chronic a7 nicotinic receptors activation on this

septal cholinergic dysfunction, wild type, PS1 and APP/PS 1 mice were injected IP

twice/per day for two weeks (first injection 15 minutes prior to lesion) with either 0.9%

saline vehicle or 1 mg/kg (salt weight) 40H-GTS-21 (Figure 4-2 and 4-3). None of the

strains appeared to be adversely affected by this dose of 40H-GTS-21 based on weight







68


gain, animal appearance, or gross behavior or physiology. For wild type and APP/PS1

mice, there was no apparent difference in septal ChAT staining between the 40H-GTS-

21 (Img/kg) and 0.9% saline treatment groups. However, the PS1 mice treated with this

40H-GTS-21 regimen retained more ChAT staining perikarya on the lesion side

compared to the wild type and APP/PS1 mice. This stereological evaluation indicated

that 40H-GTS-21 could provide some protection of cholinergic neurons in PS1 mice,

under conditions in which it did not affect APP/PS1 or wild type mice.


ow

0)
w

C (,

a-


Figure 4-2. Septal ChAT neuron staining in 9 month old mice. 40H-GTS-21 injected 2x
daily (1 mg/kg IP) for 2 weeks increased septal ChAT staining neurons in
unilaterally lesioned PS1 mice. 40H-GTS-21 had no protective effect in 9
month old APP/PS1 and wild type mice. P<0.05 compared to other group
(one way ANOVA).







69









Saline 40H-GTS-21
IF-
,' : ..

P.* 4.
*S ? Ti. ,.. '



.















APP/PSFigure 4-3. Septal ChAT-staining perilarya in 9 month old PS, APP/PS and wild type

PS mice showed any neuroprotection with drug treatment.






Figure 4-3. Septal ChAT-staining perilarya in 9 month old PSI, APP/PS 1 and wild type

PSI mice showed any neuroprotection with drug treatment.






70


The septal GABAergic neuron quantification was conducted by using

parvalbumin immunohistochemisty that accounts for a major fraction of the GABAergic

innervation to hippocampus (Figure 4-4). The immunohistochemistry staining is shown

in Figure 4-5. The cell-counting data showed that no change affected the GABAergic

neuron density.


1.2-
o


E 1.0-
I-
CL


0.8-'


Figure 4-4. Septal GABAergic neuron staining in 9 month old mice. 40H-GTS-21 was
injected 2X daily (1 mg/kg IP) for 2 weeks. No change in septal GABAergic
neurons staining in unilaterally lesioned mice. *p<0.05 compared to saline
group (one way ANOVA).














40H-GTS-21


PS1











APP/PS1












Wild type


Figure 4-5. Septal GABAergic staining perilarya in 9 month old PS1, APP/PS1 and wild
type C57/B16/J mice two weeks after unilateral aspirative FFX-lesions. No
neuroprotection with drug treatment in these groups.


Saline






72


6E10 and thioflavine S staining were conduced in sections of these same 9-month-

old APP/PS1 mice (Figure 4-6). APP/PS1 mice were treated with 40H-GTS-21 or 0.9%

saline vehicle IP for two weeks. The 40H-GTS-21 + lesion groups had few plaques

compared to saline group (Figure 4-7).


a~-


-V
,I


A 6.


Figure 4-6. The thioflavine S and 6E10 staining in 9 month old APP/PS1 mice. Left pane:
thioflavine S staining in hippocampus; Right panel: 6E10 staining in
hippocampus and neocortex.


120-




II
0



80-
Fa


I- No Drug

m +40H-GTS-21


-1T


Treatment

Figure 4-7. APP/PS1 mice (9 months old; N=4-5/gp) had lower hippocampal amyloid
density stained with 6E10 antibody after a combination of fimbrial lesion and
40H-GTS-21 IP X2 for 2 wks than either treatment alone. *p<0.05 compared
to unlesioned side; there was no difference between drug treatment groups in
absence of lesion.Discussion


-F









This study investigated the a7 nicotinic receptor agonist effects on cholinergic and

GABAergic neuron viability in wild type, APP/PS1 and PS1 mice receiving FFX-lesions.

The aspirative FFX-lesions used in this study resulted in a substantial depletion of

cholinergic markers in the mouse hippocampus though this depletion was less dramatic

than reported previously, or than we saw based on AChE histochemistry, for rat. The

extent of cholinergic depletion was the same for each group of saline-injected mice. Our

data show that 40H-GTS-21 could provide neuroprotection in PS1 lesioned mice, but no

protection was observed in wild type or APP/PS1 lesioned mice treated with the drug.

This difference may reflect additive or synergistic effects of PS1 and a7 nicotinic

receptors.

Kang et.al show that presenilins play important roles in Akt/GSK signaling and tau

phosphorylation (Kang et al., 2005). The PS1 mutation was reported to enhance tau

phosphorylation and reduces kinesin-based transport by increasing GSK-3 activity in

primary neurons. However, the mechanism is not known for how the PS1 mutations

could account for these effects. One factor may be the relative amount of PS1 vs. PS2

expression, since PS1 mutations led to a substantial reduction in PS2 fragment levels

compared to wild type PS1. This could result in a corresponding decrease Akt and ERK

activation by the PS1 mutation. Akbari et.al.(Akbari et al., 2004) reported that PS1

mutations lead to increased intracellular stores and an attenuation of capacity calcium

entry. Thus, a7 nicotinic receptor activations may have additive effects on intracellular

calcium levels with PS1, which, as noted in Chapter 3, is important for neuroprotection.

They also demonstrated that in the presence of APP overexpression, an inverse

relationship exists between gamma-secretase activities. Since PS1 overexpressing mice









have increased levels of -Akt and ERK activation, both of which are important for a7

nicotinic receptor mediated neuroprotection, this may be another additive or synergistic

interaction between these two systems. This may be the reason we saw the protection in

cholinergic neurons in PS1 mice but not wild type mice.

For the APP/PS1 mice, both the APP and PS1 are overexpressed. However, APP

has a high affinity for blocking a7 nicotinic receptors. This could interfere with 40H-

GTS-21 binding to a7 nicotinic receptors, attenuating the drug-induced protection in

APP/PS1 mice. If so, higher doses of this partial agonist or the use of a more efficacious

agonist than 40H-GTS-21 might still be effective. Alternatively, the pharmacokinetic

properties of the 40H-GTS-21 could be considered more carefully in designing the

dosing in order to optimize the amount of drug at receptors over more extended intervals.

As noted in Chapter 1, 40H-GTS-21 is a relatively short-life drug.

One interesting observation is that GABAergic neurons were not protected in the

drug treatment group compared to the saline group. One factor is that the number of

GABAergic neurons projecting to hippocampus is less than cholinergic neurons. Linke

et al. showed that 38% of all retrograde labeled neurons were ChAT-positive whereas

only 10% of all retrograde labeled cells were immunostained for parvalbumin (Linke et

al., 1994). This is support our hypothesis. Another possible reason is that the proportion

of GABAergic neurons might have been underestimated because immunostaining for

parvalbumin only labels a subpopulation of GABAergic neurons. Mechanistically,

however, these data suggest that some factor activated by a7 nicotinic receptors is more

effective in cholinergic than GABAergic neurons. This factor may be NGF, which as









noted above is protective for cholinergic but not GABA neurons in septum and which

appears to be elevated by nicotine receptor activation.

Given the toxicity of aggregated A3 to cells in vitro, it is perhaps surprising that

even though A3 aggregates have been abundant throughout the brains of APP/PS1 mice

for most of their lives, there is little neurotoxicity. One possible explanation for the

apparent lacks of overt A3 toxicity in these mice involves the putative trophic effects of

APP and PS1, which may counteract the degenerative effects of amyloid formation.

Another explanation is that the effects of A3 aggregation in plaques may be very local. It

is hard to discern local degeneration of neurons in close to plaques (Hernandez et al.,

2001).

6E10 and thioflavine S staining were used to detect amyloid plaques in APP/PS1

mice with or without 40H-GTS-21 treatments for two weeks. The density of plaques

was slightly but significantly decreased in 40H-GTS-21 treatment groups in the lesioned

hemisphere compared to saline group's lesioned side, when both were normalized to the

contralateral, unlesioned side. This normalization was necessary because of the wide

inter-animal variability in plaque density, which was largely eliminated by this

normalization. This result suggests that reducing septal input to the hippocampus

combines with a7 nicotinic receptor activation to reduce plaque density. One hypothesis

is that 40H-GTS-21 treatments for 2 weeks may not be enough to decrease amyloid

deposits as significantly as was reported for nicotine over a period of 3 months. We

should also consider the alternative hypothesis that other nicotinic receptors are

responsible for this action, either alone or in combination with a7 nicotinic receptors. A

pharmacokinetic explanation is also possible; the short-term half-life of 40H-GTS-21






76


may not have permitted it to exert the same anti-plaque action when injected 2 X per day

that was possible when nicotine was administered in the water, the route used previously

to reduce amyloid plaques.














CHAPTER 5
RAAV MEDIATED GENE TRANSFER IN VITRO AND IN VIVO

Introduction

Acetylcholine, acting through a7 nicotinic receptors, is an important modulator of

electrical activity in the central nervous system and is involved in a variety of

physiological processes and synaptic plasticity, including cognition and development

(Volpicelli and Levey, 2004). The loss of cholinergic function has been implicated in

AD. We already know that AChE inhibitors have in clinical studies shown palliative

effects on symptoms and a trend to slow disease progression. But at the later stages of

AD, the AChE inhibitors have little or no effect on AD. A consistent and significant loss

of a7 nicotinic receptors has also been observed in neocortical autopsied brain tissue

including hippocampus from AD patients compared to aged-matched healthy subjects.

So the cholinergic receptors including a7 nicotinic receptors of the hippocampus are

considered to be therapeutic targets for memory loss and dementia.

a7 nicotinic receptors are highly expressed in the hippocampus and neocortex.

Functional a7 nicotinic receptors in the hippocampus are mostly located at the cell bodies

of the mossy cells and interneurons of the dentate gyms. There is also evidence for the

a7 nicotinic receptors being expressed on the synaptic terminals of interneurons and

dendrites of pyramidal cells.

This thesis tests whether increasing the density of u7 nicotinic receptors can be

accomplished with gene delivery in a manner that is functional, long term, dose-









dependent, and non-toxic. This would be a potential alternative method to the classic,

pharmacological approach of increasing a7 nicotinic receptor activities with higher

agonists concentrations. In our previous study, we showed that a high, 50 [tM

concentration of the a7 nicotinic receptor selective agonist GTS-21 could cause cell

toxicity in vitro (Li et al., 1999b). Although similar toxicity in vivo has not been seen

and may be unlikely because of the very rapid desensitization of these receptors at such

high concentrations, this desensitization would also be expected to interfere with efforts

to increase a7 receptors function through high dose agonist regimens. Therefore,

increasing the density of c7 nicotinic receptors is another promising target for therapy of

AD, schizophrenia, and other conditions associated with dysfunctional a7 receptors.

Despite pharmacological evidence that changes in a7-receptor activity affect

behavior and cell survival, transgenic a7 knockout mice show few behavioral changes

compared to wild type controls. One possible explanation of this observation involves

compensatory processes to replace those normally mediated by the receptor. We have

developed another approach to modify a7 receptors gene expression in adults, bypassing

potential developmental compensatory processes, using the rAAV vector system.

In this study, the rAAV is used as a gene delivery vector due to its apparent non-

toxicity, ability to transduce postmitotic neuron and long-term expression gene (Robbins

et al., 1998). A single injection into the hippocampus leads to widespread expression of

transgene in interneurons and other neuronal types. The serotypes of rAAV2 and

rAAV8/2 were compared in our study since little is known about the latter in brain and

since it appears to be more effective with respect to spread of transgene in other tissues.

The cells of the nervous system can be divided into two broad categories: neurons and a









variety of supporting neuroglial cells. rAAV2 has been the most widely studied of the

serotypes in brain and its tropism is neuronal predominantly. The possible mechanism of

this tropism is either receptor-mediated or promoter-dependent selectivity. Early studies

showed that rAAV2 mediated transgene expression occurred in hippocampal

interneurons, and less strongly in hippocampal CA1 pyramidal neurons or dentate gyms

granule neurons. In this study, the rAAV8/2 vectors were also studied to determine the

distribution and spread of the vectors in hippocampus. The rAAV2 and rAAV8/2 vectors

used here both contained the chicken beta actin promoter combined with CMV enhancer.

This is an extensively used promoter system for gene transfer in brain because of its high

activity for extended intervals (Klein et al., 2002).

We tested the rAAV mediated a7 nicotinic receptor gene deliveries in vitro and in

vivo. For in vitro studies, rat pituitary tumor derived cells (GH4C 1) were chosen. These

do not express endogenous a7 receptors but can express functional a7 receptors after

transfection because they synthesize the chaperone protein RIC-3. For the in vivo

studies, wild type C57/B1/J mice and Sprague Dawley rats, as well as a7 KO mice (strain

B6.129S7-Chra7tmlBay) were used. The goal was to test the following hypotheses: 1) a7

nicotinic receptors could expressed in vitro and in vivo in a dose dependent and

functional manner, with no greater toxicity observed than under normal conditions in the

presence of an a7 agonist; 2) a7 nicotinic receptor gene transfer could restore function in

KO mice; 3) different neuronal populations in the hippocampus are similarly able to

express this transgenic receptor after transduction in vivo; and 4) a7 vector mediated

gene transfer into hippocampus could improve memory related behavior analogous to

agonist-treatment.









Results

The structures of the pUF12-rat uc7-plasmid containing expression cassette are

shown in Figure 5-1. The CBA promoter is the hybrid truncated CMV enhancer and

chicken actin promoter. The size of the first plasmid from TR to TR was 5.1Kb that is

slightly greater than the rAAV package size of 5 Kb. The pUF 12-rat a7 plasmid without

WPRE was used in subsequent parts of this study accordingly. The empty and GFP

plasmids were controls as specified.

TR CMV CBA Exon 1 Intron WPRE Poly A TR

TR CMV CBA Exon 1 Intron Poly A TR

TR CMV CBA Exon 1 Intron Poly A TR

TR CMV CBA Exon 1 Intron IRES GFP Poly A TR

Figure 5-1. Schematic diagram of the expression cassettes.

To evaluate the ability of the plasmid constructs to code for rat a7 and GFP,

GH4C1 cells were transfected with rAAV-rat a7 plasmid and pUF 12 using a calcium

phosphate method. GFP expression was observed 24 hours after transfection and around

15% of the cells expressed GFP at day 3. Rat a7 expressions was measured by ligand

(MLA) binding assay at day 3 and found only in the a7 transfected cultures (Figure 5-2).

In order to determine whether the a7 gene delivery had any effect on cell viability,

either by itself or in the presence of a toxic agent, some of the transfected GH4C1 cells

were also exposure to 20 [tM Abeta 25-35 for this 3 day interval. Cell viability was

measured by cell counting using an NIH image system. GFP and a7 gene transfer alone

had no effect on cell density, but 20 [tM Abeta 25-35 caused significant cell loss. Cells










transfected with rat a7 receptors and then treated with Abeta 25-35 displayed no

protection in total cell density, possibly because the transfection efficiency was too low

and too few cells were protected overall in the culture. However, it appears that the few

cells expressing these transgenic a7 receptors may have been protected. The a7 nicotinic

receptor binding was 110+ 15 fmol/mg in the non-Abeta treated cultures and 217+ 20

fmol/mg in the Abeta 25-35 treated group (Figure 5-2). This increase in receptor density

could reflect the relative sparing of cells expression a7 nicotinic receptors. Alternatively,

the Abeta peptide may have acutely increased a7-receptor density by some other

mechanism, though this has not been seen previously.


3=i -T 4Dwi


.4-
IL

0- 1 0:









25-35 exposure on cell viability and receptor density. Cell viability was

group by cell counting using an NUT image system. The high affinity MLA

cultures. *p<0.05 compare to other group (one way ANOVA).
-LL LL







method developed by Zolotukhin et al. (1999). The discontinuous iodixanol gradient was
< 0 0 3



Figure 5-2. Transfection of GH4Cl cells with rat io7 nicotinic receptors:effect of Abeta
25-35 exposure on cell viability and receptor density. Cell viability was
measured 72 hr later and expressed as the mean + SEM of 6-8 plates per
group by cell counting using an NIH image system. The high affinity MLA
binding to u.7 nicotinic receptors was conducted in membranes from the same
cultures. *p<0.05 compare to other group (one way ANOVA).

The plasmids were packaged into rAAV2 and rAAV8/2 using the adenovirus-free

method developed by Zolotukhin et al. (1999). The discontinuous iodixanol gradient was

built (Figure 5-3A). After a two hour centrifugation, the rAAV distributed around the 40-

60% density-interface (Figure 5-3B). A dot plot analysis was used to titer the number of









total genomic particles (Figure 5-3B). This method quantified viral DNA by hybridizing

with a biotin probe of pUF 12 and by comparing the intensity of labeling against known

quantities of plasmid pUF 12-rat a7. The first and second lanes were standard curves.

The highest band had 5 ng/[il DNA with 1 ng = 4 x 1011 genomic particles/ml. The titer

of rAAV2-rat a7 was 3 x 1012 genomic particles/ml. The titer of rAAV8/2-rat a7 was 4

x 1012 particles/ml. The titer of rAAV2-GFP was 1.5 x 1012 genomic particles/ml. The

title of rAAV8/2 was 2x 1012 genomic particles/ml.


A B













Figure 5-3. Iodixanol gradient for the purification of rAAV and dot plots for titer. (A).
Preformed gradients shown before (left tube) and after (right tube) the 2 hour
centrifugation. (B). The first and second lanes were standard curves. The third
and fourth lanes were samples.

The different concentrations of rAAV2-rat a7 and rAAV2-GFP transductions of

GH4C1 cells are shown in Figure 5-4. GFP expression was observed in culture within 24

hours. By day 5, over 90% of cell expressed GFP in the 30 x 109 genomic particles/ml

treatment group. Functional rat a7 receptors expressions were detected by the [3 H]

MLA binding assay. Increasing the concentration of vector resulted in more a7 nicotinic

receptor binding.











-5000
100-
-4000
80 80-
/ -3000 Q-
60 -
LL _D.
40- alpha 7 -2000 h -
S 40

20- -transduction -1000


0 5 10 15 20 25 30 35 40

Genomic particles (x 10 9/ml)

Figure 5-4. The dose response of rAAV2-rat a7 receptor and rAAV-GFP. Binding assay
was measured 5-day later and 6-8 plates per group.

The expressions of c7 nicotinic receptors were compared among transduced

GH4C1 cells and several other cell types using MLA binding. There were 960 fmol/mg

protein expressed in rAAV2-rat a7 (5 x 109 genomic particles) transduced GH4C1 cells

after 5 days (Figure 5-5). An a7 stable cell line (also GH4C1) had 750 fmol/mg protein

expressed, while the PC12 cell line used for neuroprotection studies in Chapter 3 had 50

fmol/mg protein expression. There were approximately 100 fmol of receptor/mg protein

expressed in pUF 12-rat a7 plasmid transfected GH4C 1 cells using the calcium phosphate

method, although only a small fraction of these cells was transfected as noted above.

This study demonstrated that the rAAV2-rat a7 was highly effective in increasing a7

nicotinic receptor expressions in GH4C1 cells.










1000-

o 750

500


250







Treatment

Figure 5-5. a7 receptor expression was measured in different populations of transiently
transfected, stably transfected, transduced, and normally expressing cells. 1.5
x10 12 genomic particles of rAAV2- rat a7 and pUF12-rat a7 plasmid were
used to introduce a7 receptors into GH4C1 cells. Binding assay were
conducted 5-days after gene transfer. These receptor-binding values are
compared to those of NGF-differentiated PC 12 cells and stably transfected
and selected GH4C1 cells. (N= 3 plates per group).

After showing that rAAV2-rat a7 vectors were able to increase the rat a7 receptors

expressing in vitro, the next step was to test whether this increase the rat a7-receptor

density could cause toxicity in the absence or presence of increase the concentration of

GTS-21 (Figure 5-6). GH4C1 cells were transduced with 2X109 genomic particles of

rAAV2-rat a7 vectors and were exposed 5 days later to specified GTS-21 concentrations

for another 3 days. PC12 cells were used as the positive control, though with much lower

receptor density. GH4C1 cells transduced with rAAV2-GFP were used as the negative

control. The binding assay was showed that PC12 had 50 fmol MLA binding/mg protein

expressed. GH4C1 cells transduced with 2X109 genomic particles of rAAV2-rat a7

vectors had 750 fmol MLA binding/mg protein. The cell density was determined and

normalized to drug-free control values (N = 3 plates/group). Despite much higher










receptor binding density in transduced GH4C1 cells, there was no increase in agonist

potency for toxicity. This suggests that toxicity depends on agonist concentration more

than receptor density. It was previously established high agonist concentration were

associated with much different receptor kinetics (i.e. peak + very rapid desensitization)

than the low volume, steady state receptor activation caused by low agonist

concentration. The present results indicate that the shape of this receptor response, which

is agonist concentration dependent, is more important than the total number of receptors

activated for affecting cell viability. Interestingly, GTS-21 appeared to increase cell

density in GFP-transduced cells, though this effect did not reach significantly except at

the lowest GTS-21 concentration. It may be that GTS-21 exerts an unknown action on

these cells that is independent of c7 receptors.


300-
-- No Alpha7 Receptor
A --+ Alpha7 Receptor
>' 2-0 ----- PC12 Cells

2 100-



0
0 10 20 30 40 50 60 70
[GTS-21], uM


Figure 5-6. Effects of GTS-21 on the viability of PC12 cells and a7 transduced GH4C1
cells. The cell viability was measure after exposure to GTS-21 for three days.
It showed that 60 [tM GTS-21 could cause significant cell loss. The density of
a7 receptor expression had no effect on cell viability. *p < 0.05 compared to
same treatment with different concentration of GTS-21 (One-Way ANOVA).

In order to investigate the in vivo effects of u7-receptor gene delivery in brain, a

very low dose of 4 x 105 genomic particles of rAAV2-rat a7 with WPRE vector was

injected unilaterally into male adult Sprague Dawley (SD) rats (250 g) hippocampus CA1









region. Two weeks after injection, both sides of hippocampus were rapidly dissected

from euthanized animals. The contralateral, uninjected side was the control. High

affinity MLA binding increased in the hippocampus injected side compared to control

(143 + 13% of contralateral control binding) (Figure 5-7). This result demonstrated that

near physiological increases in receptor density were feasible with this gene delivery

system, using a very low vector dose.


200-




0 00-





Untreated Alpha7
Hippocampus

Figure 5-7. Low vector dose: in vivo transduction with a7 vectors in hippocampus. 4 x
105 genomic particles of rAAV2-rat a7 were injected into hippocampus. High
affinity MLA binding increased in the hippocampus 2 weeks after
transduction with the rAAV2-rat a7. All values are mean + SEM of 4 animals.
*P<0.05 compared to untreated group (t-test).

Since one of my goals was to investigate the safety and function of c7 receptors in

vivo, I next focused on the effects of high vector doses on this receptor expression. A

higher dose of 5 x 109 genomic particles of rAAV2-rat a7 vectors without WPRE was

injected unilaterally into male adult SD rat brain hippocampus CA1 region. Two weeks

after injection, both sides of hippocampus were rapidly dissected from euthanized

animals. The uninjected side was treated as the control for endogenous a7 receptors.

MLA binding was dramatically increased in the injected side compared to controls









(Figure 5-8A). An equal dose of rAAV2-GFP was also injected in rat hippocampus to

determine the transduction efficiency with this type of vector (Figure 5-8B).


A B
3000-
E

-E -2000-


,- 1000



6 4


Figure 5-8. Effects of a higher vector dose on a7 nicotinic receptor expression in
hippocampus. A. 5x109 genomic particles of rAAV2- rat a7 were injected in
the rat hippocampus. High affinity MLA binding increased in the injected
side. B. rAAV2-GFP transduction was highly efficacious. *P<0.05 compared
to untreated group (t-test).

In order to identify and study exogenous a7 gene expression in the absence of

endogenous receptors, 1.5 x 1010 genomic particles of rAAV2-rat a7 were injected

unilaterally into the hippocampal CA1 region of c7 nicotinic receptor knockout mice.

Two weeks after injection, each side of hippocampus and neocortex was assayed for

MLA binding. Knockout mice on the injected side had high a7-receptor expression (500

fmol/mg protein), but no measurable binding on the uninjected side (Figure 5-9A). The

a7 heterozygous mice had 1200 fmol MLA binding/mg protein expression in the injected

hippocampus, but no nicotine displaceable MLA binding on the control side. The

neocortex had a7-receptor expression in both knockout and a7 +/- mice (Figure 5-9B).

This study demonstrated that we could restore a7 receptors in knockout mice.











A B

1500- 1000 KO nice injected side
SKO mice non-injected side
S100- 750- alpha7 +- no treatment
F' 500 1 lialpha7 +- injected side
a O 500- ,5
0250-

Hippocanpus Cortex

Figure 5-9. rAAV2-rat a7 vector gene transfer in a7 KO and a7 heterozygous (+/-) mice.
1.5 x 1010 genomic particles of rAAV2-rat a7 were injected into
hippocampus of c7 KO or heterozygous mice. These results showed that the
neuronal transgenic a7 receptor was expressed in hippocampus and,
presumably through vector spread from the injection track through neocortex.
and #p<0.05 compare to same genotype untreated side (t-test).

Scatchard plots were used to calculate the Kd and Bmax values of MLA binding in

the transduced hippocampus to determine whether there the binding properties of the

transgenic receptors compared to endogenous receptors. 1.8 x1010 or 3.6 xl1010 genomic

particles were bilaterally injected into wild type mouse hippocampus. After two weeks,

binding assays were conducted with varying concentrations of labeled MLA. The MLA

Kd was found to range from 1.98-2.288 nM in these 3 treatments, indicating that the

endogenous and transgenic receptors has similar MLA binding affinities (Table 5-1). The

Bmax increased with the concentration of vector as expected.