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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2012-12-31.

Permanent Link: http://ufdc.ufl.edu/UFE0042660/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2012-12-31.
Physical Description: Book
Language: english
Creator: Wang, Zhaohua
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: Pharmaceutics -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, M.S.P.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Zhaohua Wang.
Thesis: Thesis (M.S.P.)--University of Florida, 2010.
Local: Adviser: Derendorf, Hartmut C.
Local: Co-adviser: Palmieri, Anthony.
Electronic Access: INACCESSIBLE UNTIL 2012-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042660:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042660/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2012-12-31.
Physical Description: Book
Language: english
Creator: Wang, Zhaohua
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: Pharmaceutics -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, M.S.P.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Zhaohua Wang.
Thesis: Thesis (M.S.P.)--University of Florida, 2010.
Local: Adviser: Derendorf, Hartmut C.
Local: Co-adviser: Palmieri, Anthony.
Electronic Access: INACCESSIBLE UNTIL 2012-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042660:00001


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1 HUMAN PLASMA GELSOLIN GENE DELIVERED VIA AAV8 RETARDS MEMORY LOSS IN APP/PS1 MICE BY ZHAOHUA WANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Zhaohua Wang

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3 To my son, Alex Chen Wang

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4 ACKNOWLEDGMENTS I would like to thank my parents, Zhongxin and Shuyun Wang, for all of their love and support. I also need to thank my wife, Xiuhua Chen; and my son Alex for their inspiration, love, and support.

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5 TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................................. 4 page LIST OF FIGURES .......................................................................................................... 7 LIST OF ABBREVIATIONS ............................................................................................. 9 ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUCTION .................................................................................................... 12 Dementia ................................................................................................................ 12 Discovery of Alzheimers Disease ........................................................................... 12 Current Therapies ................................................................................................... 13 Cause of Alzheimers Disease ................................................................................ 13 Amyl oid Precursor Protein ...................................................................................... 14 Amyloid Beta ........................................................................................................... 15 Amyloid Cascade Hypothesis ................................................................................. 15 Sink Hypothesis ...................................................................................................... 17 Gelsolin ................................................................................................................... 17 Gene Therapy ......................................................................................................... 19 2 MATERIALS AND METHODS ................................................................................ 22 Reagents ................................................................................................................ 22 Subcloning Vectors ................................................................................................. 22 Large Scale Plasmid Preparation ........................................................................... 22 Cell Culture ............................................................................................................. 24 Animals and Procedures ......................................................................................... 24 Western Blot ........................................................................................................... 26 Enzyme Linked Immuno ........................................................ 26 Sandwich ELISA for Gelsolin .................................................................................. 27 New Method of ELISA for Gelsolin ......................................................................... 28 Histochemistry ........................................................................................................ 29 Thiazine Red Staining ............................................................................................. 29 Image Analysis ....................................................................................................... 29 3 AAV8 VECTOR WITH HUMAN PLASMA GELSOLIN DELIVERY AND EFFECT IN APP/PS1 MALE MICE BEHAVIOR ..................................................... 31 Gene Therapy and Behavior Training ..................................................................... 31 Results and Discussion ........................................................................................... 33 Behavior Training of Male Mice .............................................................................. 33

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6 Male Mice Results Analysis .................................................................................... 34 4 AAV8 VECTOR WITH HUMAN PLASMA GELSOLIN DELIVERY AND EFFECT IN APP/PS1 FEMALE MICE BEHAVIOR ................................................. 42 Behavior Training of Female Mice .......................................................................... 42 Results and Discussion ........................................................................................... 42 Female Mice Results Analysis ................................................................................ 43 5 AAV8 VECTOR WITH HUMAN PLASMA GELSOLIN DELIVERY AND ................................. 48 A myloid Burden in Brain ......................................................................................... 48 Results .................................................................................................................... 49 Dense Cored Amyloid Deposits .............................................................................. 49 ......................................................................................... 50 A myloid Burden Results Analysis ........................................................................... 50 6 NEW ELISA METHOD FOR HUMAN PLASMA GELSOLIN QUANTIFICATION STYELS .................................................................................................................. 61 Methods of Protein Concentration Quantification .................................................... 61 New ELISA Method for Gelsolin ............................................................................. 63 LOD & LOQ ............................................................................................................ 64 The Results of Gelsolin Spiked with Mouse Plasma Compared with Those in PBS ..................................................................................................................... 64 ELISA Results Analysis .......................................................................................... 64 7 DISCUSSI ON AND FUTURE DIRECTIONS .......................................................... 70 LIST OF REFERENCES ............................................................................................... 75 BIOGRAPHICAL SKETCH ............................................................................................ 87

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7 LIST OF FIGURES Figure page 1 1 Mechanism of APP cleavage to amyloid beta. .................................................... 20 1 2 Hypothesis of amyloid cascade theory. .............................................................. 21 2 1 HindIII and BglII digest plasmids.. ...................................................................... 30 3 1 Morris Water Maze learning curve. .................................................................... 35 3 2 Morris Water Maze test. ..................................................................................... 36 3 3 Radial Arm Water Maze test. ............................................................................. 37 3 4 Eleven month old male mice radial arm water maze.. ........................................ 38 3 5 Fourteen month old male mice radial arm water maze. ...................................... 39 3 6 Morris water maze system. ................................................................................. 40 3 7 Radial arm water maze system. ......................................................................... 41 4 1 Seven month old female mice radial arm water maze. ....................................... 44 4 2 Eleven month old female mice radial arm water maze. ...................................... 45 4 3 Latency time before female mice find target platform. ........................................ 46 4 4 Errors made before female mice find target platform. ......................................... 47 5 1 Dense core amyloid deposits in APP/PS1 male mice.. ...................................... 51 5 2 Analysis of dense core amyloid deposits in APP/PS1 male mice. ...................... 52 5 3 Analysis of dense core amyloid deposits in APP/PS1 male mice hippocampus area.. ............................................................................................ 53 5 4 Analysis of dense core amyloid deposits in APP/PS1 male mice cortex area... ................................................................................................................. 54 5 5 ........ 55 5 6 Dense core amyloid deposits in APP/PS1 female mice. .................................... 56 5 7 Analysis of dense core amyloid deposits in APP/PS1 female mice.. .................. 57

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8 5 8 Analysis of dense core amyloid deposits in APP/PS1 female mice hippocampus area. ............................................................................................. 58 5 9 Analysis of dense core amyloid deposits in APP/PS1 female mice cortex area.. .................................................................................................................. 59 5 1 0 section.. .............................................................................................................. 60 6 1 Gelsolin function assay. ...................................................................................... 65 6 2 Western blot of gelsolin. ..................................................................................... 66 6 3 Showed that O.D. value not changed when gelsolin amount increased. ............ 67 6 4 Gelsolin standard curve. ..................................................................................... 68 6 5 Standard curve of gelsolin in PBS and spike with mouse plasma. ..................... 69

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9 LIST OF ABBREVIATION S AAV Associate Adeno Virus amyloid beta Ach acetylcholine AD Alzheimers disease APP Amyloid Precursor Protein BBB blood brain barrier CNS central nervous system CSF cerebrospinal fluid ELISA Enzyme Linked Immunosorbent Assay FAD familial Alzheimers disease HRP Horseradish peroxidase IDE i nsulin degrading enzyme LOD limit of detection L OQ limit of quantitation NFTs neurofibrillary tangles NMDA N methyl D aspartate PS presenilin RAWM radial arm water maze MWM morris water maze RAGE receptor for advanced glycation end

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science HUMAN PLASMA GELSOLIN GENE DELIVERED VIA AAV8 RETARDS MEMORY LOSS IN APP/PS1 MICE By Zhaohua Wang December 2010 Chair: Hartmut Derendorf Cochair: Anthony Palmieri Major: Pharmaceutic al Sciences Pharmacy Alzheimers disease (AD) is the most common form of dementia. It is progressive and unrecovered. Memory loss is the earliest and important syndrome. AD is characterized by a progressive accumulation of extracellular amyloid plaques, and intracellular neurofibrillary tangles (NFTs) in the brain. Evidence suggests that the increased production or decreased clearance of a myloid (A ) which is the main constituent of amyloid plaques triggers a cascade that ultimately leads to Alzheimers pathology, making it a promising target for the treatment of AD. One strategy targeting A that has shown promise is increasing its clearance by binding A peripherally, either by immunization or treatment with agents that bind A creating a peripheral sink that shifts the equilibrium of One such agent is a protein named plasma gelsolin, which has 6 subunits. which can be expressed peripherally, in a mouse model of Alzheimers disease can

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11 ome questions around the treatments of AD. For example, in 2008, more than 100 clinical trials of AD failed, dosing because the traditional medicines are not reproducible by themselves. In order to f ou nd an ideal treatment method for one dose life long term treatment, We packaged the coding sequence for human plasma gelsolin into an AAV 8 (Associate Adeno Virus) vector. Then we peripherally delivered, via the tail vein, this vector into APP/PS1 mice, a kind of AD mouse model. After behavior training, we find that peripheral expression of plasma gelsolin retards the memory loss in treated mice compare with control mice. W e f ound that results show that we may have developed a new method for one dose life long t erm

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12 CHAPTER 1 INTRODUCTION Alzheimers disease (AD) is the most common f orm of dementia. It is estimated that there are 5.3 million Americans have AD in 2010. 8 And this number is still increasing. T he number of Americans impacted by AD is e stimated more than 10 million in 2050. This increased numbers of AD patients can be att ributed to the aging of the population. 1, 2 A lzheimers disease would impact a lot on a persons life quality. 3 Besides, as the disease progresses, independence decreases, this would plac e a huge financial problem on social and family caregivers. Total payments for AD are expected to be $172 billion in 2010 based on 2004 data. 4, 5 6 7 So it is important to find an efficacious treatment for AD to ease the social and family burden of the disease. Dementia Dementia is taken from Latin meaning "madness" 9 It is characterized by the loss in memory and cognitive abilities which is caused by many different diseases that would damage brain cells. The syndrome of dementia is the loss of abilit ies of language, perception, judgment and memory. Discovery of Alzheimers D isease In 1901, German psychiatrist Alois Alzheimer met a patient named Mrs. Auguste Deter who had behavioral problems including a loss of short term memory. Alzheimer followed her case until she died and he was the first scientist who reported the case publicly. 10 The disease was first described as a distinctive disease by Emil Kraepelin 11,12 Since then Alzheimers disease has been recognized as the most common form of dementia in the world.

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13 Current Therapies Currently only four drugs in two groups have been approved by the FDA for the treatment of AD. Three are acetylcholinesterase inhibitors (donepezi l, rivastigmine, and galantamine) and the other one is an NMDA receptor antagonist ( memant ine ). The activity of the cholinergic neurons is reduced in AD. 13 Acetylc holinesterase inhibitors can be used to decrease the catabolism of acetylcholine (ACh), which is lost in AD due to the death of cholinergic neurons. 14 Increasing the level of acetylcholine can improve the memory and learning abilities 15 Memantine is a noncompetitive NMDA receptor antagonist which is used to block NMDA receptors and inhibit their overstimulation by glutamate whose elevated levels may lead to neuronal dysfunction. 16 Both treatment have just shown modest improvements in Alzheimer's disease patients with behavioural problems, but they are far from an ideal treatment. They only delay the progression of AD and are not a cure. 17, 18 Cause of Alzheimers Disease Senile plaques are a characterization of AD. In 1983, Allsop identified that a 40 42 amino ac id peptide named amyloid beta A is the main element 19 This peptide come from a larger protein named amyloid precursor protein (APP) after APP had been cleaved by secretases. 20 The discovery that all of the autosomal dominant familial AD can be attributed to mutations in one of three genes: amyloid precursor protein (APP) and presenilins 1 and 2. 21, 22, 23, 24 Most mutations increase the production of 42. Some of the mutations only alter the ratio betw een 25, 26 This suggests that presenilin mutations can cause cascade hypothesis proposed by Hardy and Allsop, which suggested that the

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14 mismetabolism of APP triggers AD pathogenesis. And formation of neuritic plaques would induce a reduction of neurotransmitters, and then intrigue the death of neurons and dementia, it means that increased concentration and may be a cause of AD 27 Although autosomal dominant familial AD is less than 0.1% of all Alzheimers cases, every autosomal dominant familial AD involves the oligomerization and precipitation. 28 Since 1991, the amyloid cascade hypothesis turn to be the fundamental cause of the disease. 29,30 Further support evidence that transgenic mice that express a mutant form of the human APP gene develop amyloid plaques and Alzheimer's like brain pathology with spatial learning de ficits. 31,32,33,34 Amyloid Precursor Protein Human Amyloid Precursor Protein (APP) is a transmembrane protein. It is expressed in many different types of cells. 35 Scientists have identified ten isoforms of APP, which are created by alternative splicing. 36 There are three major isoforms, C, B, and A which have 695, 751, and 770 amino acids, seperately. The C isoform is primarily present in neurons. 35 The primary function of APP is not known, though evidence suggests it may play roles as regulator of synapse formation and neural plasticity 37, 38 Besides, the roles of APP in cell signaling long term potentiation, and cell adhesion have been proposed and supported by limited research. 39 Although the function of APP is not clear, APP knockout mice showed relatively minor phenotypic effects of impaired long term potentiation and memory loss comparing with transgenic mice of upregulated APP expression. 40, 41

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15 Amyloid Beta cleavage of the amyloid precursor protein (APP). There are three cleavage sites identified on APP. The proteases for the cleavage are known as and secretases secretase cut APP on the extracell ular site, and then secretase cut the remaining part on the transmembrane site, the fragment between the and se cretases cutting sites important to oligomerzation and fibril formation and associated with the disease. 42 Evidence showed that autosomal dominant 43,44,45,46 helical secondary structure, then structure sheet structure as soluble oligomers. The soluble oligom ers aggregate to protofibrils and fibrils, finally come together to make up the plaques. 29 Amyloid Cascade Hypothesis One of the major questions is central to the pathogenesis of AD is known as the amyloid cascade hypothesis which was proposed in 1991 by John Hardy and David Allsop. 29 This hypothesis suggests that the mismetabolism of APP would induce AD due to increased level increased polymerized to oligomers, and then oligomers aggregate to fibrils and deposit as plaques. The plaques would active of microglia and astrocytes, make them release proinflammatory cytokines, finally induce neuron injury and lead to dementia. 47, 48, 49

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16 The evidence support the amyloid cascade hypothesis is that every form of familial Alzheimers disease (FAD) involves mutations of either APP or the enzymes that cleave APP, make an increase level of A 42. 50 And transgenic mice expressing mutations of APP and PS1 would increase levels and amyloid plaques. 51 have been shown to be neurotoxic to cells via a variety of ways. 52, 53, 54, 55, 56, 57, 58 In enzymes, which include the insu lin exported via binding of A2M and APOE to LRP, and degrade through the lysosomal pathway. mediated endocytosis, and then degraded peripheral ly Endosomal dysfunction is one of pathologies of sporadic AD and abnormally enlarged endosomes may induce deposition. 59 These data suggest that increased level But there are some research not supp ort amyloid cascade hypothesis 60, 61 They but it did not show in animal models, the in vitro toxicity may due to artifact cause They showed that neurofibrillary tangles (NFT) and neuron number s may be better para meter s than amyloid beta. 62 And there are some evidences that soluble oligomer had higher correlation with memory loss s as an antioxidant or a neurotrophin. 63, 64, 65, 66, 67, 68, 69, 70 Although there are some evidence s argue the a myloid cascade hypothesis, the majority Alzheimers disease.

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17 Sink Hypothesis could reduce burden in mice and pr event memory loss promising target to treat AD. 71, 72, 73 Although the clinical trial has failed due to the reason that the 6% patients developed meningoencephalitis, the following studies showed that antibody produc ers get sig nificantly improved memory ability 74, 75 Sink theory had been proposed to explain how immunization works, the CNS to the periphery supported by the finding that less than 0.1% of antibodies in the serum across the blood brain barrier (BBB). 76 And the support evidence for the sink theory come from the studies of many were administered peripherally. Soluble truncated RAGE protein (receptor for advanced glycation end) w hAPP mice show ed el along with a decrease level in brain. 77 binding agents GM1 and plasma gelsolin. 78, 79 Our study f ocused on the effects on the relationship of the memory loss and amyloid deposition by plasma gelsolin gene expression. Gelsolin Gelsolin is an 82 kD, with six sub units actin binding protein which play a key r ol e on actin polymerization and actin filament depolymerization. 80, 81 Gelsolin can be divided into two groups by location, which are intracellular (cytoplasmic gelsolin) and extracellular (plasma gelsolin). 82 Gelsolins major function is that work as a n actin binding protein for its polymerization and actin filaments depolymerization. 83 The gelsolin function is regulated by Ca2+ and phosphoinositides. 84 Intracellular g elsolin is in

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18 the cell whereas the plasma gelsolin is a secreted protein with a signal peptide. 85 Both kinds of gelsolin come from same gene but splicing on different sites also, their structure are different and plasma gelsolin has 25 amino acids residue on aminoterminus compare with the intracellular gelsolin. 86 An d t he concentration of gelsolin in the plasma is 179 87 Several studies suggest that gelsolin involved in other event s besides regulate actin polymerization which include calcium channels and N methyl D aspartate (NMDA) receptors, apoptosis modulation, tumor suppression, immune re actions and cell motility and endocytosis. 88, 89, 90, 91,92,93, 94, 95, 96, 97, 98, 99, 103 An important result is a gelsolin mutation would induce an aberrant cleav age of it and produce a 68kD anomal gelsolin fragm ent to be secreted and then deposited in the Finish type of amyloidosis. 100, 101, 102 But non mutated gelsolin plays a different role in AD, which is emphasized below. Plasma gelsolin could against inflammatory reactions which is induced by injury. 104, 105 And plasma gelsolin can reduce the viscosity of cystic fibrosis sputum. 106 Some e vidence s showed that human plasma gel fibrillization, and even to of gelsolin in the amyl oid beta clearance. 107, 108 A fact that gelsolin level in AD patients was significantly lower than controls suggests that decreased gelsolin concentration may be a cause in the increased amyloid beta level in AD. 109 Matsuoka reported that inject bovine plas ma gelsolin can prevent deposit in the brain in younger huAPP K670N,M671L/ PS 1 M146L mouse, but they did not see the gelsolins effects in older mice and on memory related behavior. 110 Furthermore, Hirko showed that peripheral deliver plasmid of

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19 plasma gelsolin can decrease A burden in the brain in APP/PS1 mice, but they did not find correlation between the reductions and memory related behavior 112 Gene T herapy Gene therapy is a method through transfer genes into an patients to treat disease There are three ways to deliver genes into patient s, directly delivery, nonvirus vector and virus vector delivery. Since most traditio nal treatments, directly delivery and nonvirus vector delivery are short term, multiple dosing treatments, virus vector delivery show certain advantage that it provide a long term, one dose treatment which will be cheaper, more convenience to use in the f uture. There are several virus vectors that can be used for gene delivery The recombinant Adenoassociated viruses (rAAV) would be a good choice. rAAV does not contain any virus genes but only the therapeutic using gene so that it could not integrate into the patients genome and this character would make it safe r than others. There are a few disadvantages to using rAAV, including the low DNA capacity and the difficult to produce. But the production problem has recently been solved by Amsterdam Molecular Therapeutics 111 Compare to most other virus, most patients who had been treated by rAAV would not be induced an immune reaction to block and clearance the virus so that the treatments would be successful for the disease The focus of our study is on the effects of peripherally deliver AAV 8 packaged human plasma gelsolin gene expression and memory related behavior on AD animal model APP/PS1 mice.

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20 Figure 11 Mechanism of APP cleavage to amyloid beta.

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21 Figure 1 2 Hypothesis of amyloid cascade theory.

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22 CHAPTER 2 MATERIALS AND METHOD S Reagents Except noted, all chemicals were purchased from Fisher colorimetric ELISA kits were purchased from Invitrogen (catalog #: KHB3441). Molecular biology reagents and enzymes were purchased from New England Biolabs. The PCR primers were ordered from Sigma Genosys. PVDF membranes were purchas ed from BioRad Laboratories. His tagged Human Plasma Gelsolin was purchased from Cytoskeleton. Phosphate Buffered Saline (PBS) was purchased from Mediatech. Gelsolin Antibody (C 18) was purchased from Santa Cruz Biotechnology. His Tag Polyclonal Antibody was purchased from Cell Signaling Technology. Donkey Anti Goat IgG (H&L) Horseradish peroxidase (HRP) Polyclonal Antibody was purchased from GenScript. Chitosan and O Phenylenediamine dihydrochloride tablet set were purchased from Sigma Aldrich. Subcloning Vectors Plasma gelsolin plasmid, pPGLE, was kindly provided by Dr. Hisakazu Fujita. 113 The sequence of plasma gelsolin was removed and then inserted into a pGFP plasmid backbone which is described by Dr. Hirko. 112 Plasmids were propagated in SURE cells and purified. AAV 8 vector packaging purified plasmid is provided by Dr. Zolotukhin. 114 Large Scale Plasmid Preparation SURE cells were transformed with the human plasma gelsolin plasmid, using a Bio Rad electroporator. Transformed bacteria were grown for an hour in 1mL of LB medium at 37C, followed by plating on LB agar plates containing ampicillin (50mg/L), and then grown overnight at 37C. Several colonies were selected for screening, and

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23 each was grown overnight in 5 mL of ampicillincontaining LB medium. Plasmids were purified from the cultures, using Qiagen mini plasmid prep kits. Plasmids were then subjected to a BglII digest to confirm. (Figure 21) After we collected the sample of plasmids, we had to pick up the correct clone for large scale preps since recombined plasmids may lose insert DNA sequence. In order to find the correct clone which still keeps the human plasma gelsolin sequence, we used BglII digest plasmid then run agarose g el to separate the DNA strend, from the results we can pick up the correct plasmids for use. First we placed a comb in the agarose apparatus. Then we prepared a 1% agarose solution in TBE buffer and poured it into the tray. After the gel was set, we added TBE buffer and removed the comb. Mixed the samples with loading dye then loaded samples into the wells 30 ul per well Also, we loaded lader as a marker. Closed the lid and t urned on the power, increased the voltage to 80 V, bubbles should be seen at the electrodes and the bromophenol blue tracking dye should enter th e gel. Run the gel until the fastest moving dye has run 3/4 of the gel. Turn off the power R emoved the gel and stain it with ethidium bromide solution (1ug/ml) for 10 min. Drained off the ethidium bromide solution. We rinsed the gel. Finally placed the gel on a UV transillum inator and took a picture. (Figure. 2 1 ) From picture, we can see sample 3, 4 have two more DNA bands than sample 1, 2 because plasmids sample 3, 4 still keep the human plasma gelsolin DNA sequence. For large scale preparations, picked a single colony and inoculate a starter culture of 10 ml ampicillin containing LB medium for 8 h at 37C with 300 rpm shaking. This was used to inoculate 2 L of ampicillin containing LB medium. We grew the bacteria at 37C for 12h with 300 rpm sh aking again. Harvest the bacterial cells by centrifugation at

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24 6000 x g for 15 min at 4C. Then we used Qiagen Plasmid Mega Kits to purify the plasmid DNA. Resuspend the bacterial pellet in 125 ml of Buffer P1. Added 125 ml of Buffer P2, mixed thoroughly by vigorously inverting 4 6 times, and incubated at room temperature for 5 min. Added 125 ml of chilled Buffer P3, mixed immediately and thoroughly by vigorously inverting 4 6 times, and incubated on ice for 30 min. Centrifuged at 20,000 x g for 30 min at 4 C. We collected supernatant. Centrifuge the supernatant again at 20,000 x g for 15 min at 4C. We collected supernatant. Equilibrate a QIAGEN tip 10000 by applying 75 ml Buffer QBT, and allowed the column to empty by gravity. Added the supernatant into the QIAGEN tip and allowed it to enter the resin by gravity. We washed the QIAGEN tip with 600 ml Buffer QC. Eluted DNA with 100 ml Buffer QF. Precipitate DNA by adding 70 ml isopropanol to the eluted DNA. Mixed and centrifuge immediately at 15,000 x g for 30 min at 4C. We decanted the supernatant. Washed DNA pellet with 10 ml 70% ethanol, and centrifuge at 15,000 x g for 10 min. Decanted the supernatant. Air dry the pellet for 10 20 min, and redissolve the DNA in 100ul water. Concentration and purity of the samples were determined by UV absorbance at 260/280 nm. Cell Culture Human embryonic kidney (HEK) 293 cells were cultured in DMEM with 10% fetal Invitrogen, CA) in a 5% CO2 inc ubator at 37C. Animals and Procedures All procedures were performed with approval of the University of Florida's Institutional Animal Use and Care Committee. Doubletransgenic mice expressing both of mutant APP695K594N,M595L and mutant presenilin1E9 (AP P/PS1E9 mice) were

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25 supplied by the Jackson Laboratories. 115 Gelsolin transgene expression was accomplished by injecting 1011 rAAV8 viral genomes per mouse via the tail vein. Because the A deposit will be formed starting around 3 months old for the APP/P S1 mice, the n=16 APP/PS1 male mice were divided into 3 groups: control group: no treatment; young age treatment group: treatment at 4 months old; older age treatment group: treatment at 6 months old. The Morris Water Maze test was used to evaluate memory. The mice were trained 4 one minute trials per day for 9 days, then a probe test for memory evaluation was performed. The Radial Arm Water Maze test was used to train the mice in the same way as the Morris Water Maze, except probe evaluation was done daily And the n=24 APP/PS1 female mice were divided into 2 groups: control group: no treatment; treatment group: treatment at 4 months old. The Radial Arm Water Maze test was used to train the mice. During the period, all blood samples were collected from mice arteria caudilis by using plasma collect tube and kept in ice. Then the tubes were centrifuged at 1000*g for 5 minutes, the blood cell and plasma were separated by gel in the tube, blood cells below the gel, plasma above the gel. Finally, animals were anesthetized with isoflourane and perfused with phosphatebuffered saline (PBS). The brains were excised and divided into two halves. One hemi brain was frozen in liquid nitrogen and stored at 80 C for analysis by ELISA. The whole hemi brains from male mice were analysis by ELISA, but we just picked up hippocampi of the hemi brains to do ELISA analysis from female mice. The remaining hemi brain was fixed for 48 hours in a 4% paraformaldehyde in PBS solution and then cryoprotected in 30% sucrose PBS solution We then performed brain slicing and Thioflavin S staining to obtain A deposits.

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26 Western Blot mercaptoethanol, boiled for 5 minutes, and then loaded onto a precast SDS 10% PAGE Tris HCl gel. Gels were run using a Biorad power supply set at 100 V for one hour. Separated proteins were then transferred to a PVDF membrane at 75V for 2 hours on ice. Membranes were incubated overnight in a blocking solution (5% nonfat dry milk and 0.05% Tween 20 in PBS) at 4C. Primary antibodies were then added monoclonal anti gelso lin antibody from CDBR lab in UF was used at 1:1000 dilution and incubated at room temperature for 2 hours. Membranes were washed three times in PBS with 0.05% Tween 20, and incubated with horseradish peroxidase (HRP) conjugated anti mouse antibody [Amersh am (Piscataway, NJ) at 1:5000] in blocking solution for 1 hr at room temperature. Following three more washes, they were incubated with substrate [electrochemiluminescence (ECL), Amersham(Piscataway, NJ)] for 1 min and exposed (Kodak, Rochester, NY). Enzym For enzyme linked immunosorbent assay (ELISA) Biosource colorimetric amyloids 1 40 and 142. Frozen hippocampi 0mM NaCl, protease inhibitors, pH 11.5) and centrifuged at 20,000 g for 25min by using a Polytron homogenizer (Brinkmann Instruments). The carbonatesoluble supernatant was collected as soluble and the pellet was rebuffer (5 M guanidine, 50mM Tris, protease inhibitors, pH 8.0). The homogenate was centrifuged at 20,000 g for 25 min, and the guanidine extract was collected as

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27 unsoluble 116 ion was performed by Invitrogen Inc. produced Human room temperature for two hours in the wells provided with the Biosource kit with an equal volume of primary antibody solut ion. Samples were then washed four times, and incubated in HRP solution for one half hour. Samples were washed four times again and then incubated for a half hour with HRP substrate, in a box to protect the samples from light. Stop solution was then added and absorbance at 450 nm was measured determined from standards provided with the kit. Sandwich ELISA for Gelsolin Centrifuge the blood sample (5min). Coated ELISA plate (96 well plate) with his tag Polyclonal antibody at 4 C. Deposed samples and then washed plate with PBS T 3 times. We blocked plate with BSA (100 /well) at room temperature for 1 hour. Disposed solution and then washed plate with PBS T 3 times. We added PBS as control groups and the standard preparation of 5 ng 10 ng 20ng, 40ng and 80ng, and waited at room temperature for 1 hour. Disposed solution and then washed plate with PBS T 3 times. We add ed gelsolin antibody (1 / ; 100 /well),and waited at room temperature for 1 hour, then prepared substrate solution (20mL) of phosphatecitrate buffer with urea hydrogen peroxide tablets. Disposed solution samples and then washed plate with PBS T 3 times Donkey anti goat IgG (H&L) horseradish peroxidase (HRP) polyclonal antibody (0.2 / ; substrate of 1, 2 phenylenediamine dihydrochoride into the substrate solution of

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28 phosphatecitrate buffer with urea hydrogen peroxide tablets (step 5) and mixed them. room temperature for 15Finally we recorded the absorbance at 490 nm on a plate reader within 30 minutes of stopping the reaction. New Method of ELISA for Gelsolin Centrifuge the blood sample (5min). Coated ELISA plate (96 well plate) with the standard preparation of 1ng, 2ng, overnight at 4C. Disposed of samples and then washed plate with PBS T 3 times. for 1 hour. Disposed solution and then washed plate with PBS phosphatecitrate buffer with urea hydrogen peroxide tablets w as prepared. Disposed solution samples and then washed plate with PBS T 3 times. Donkey anti goat IgG added, then wait at room temperature for 1 hour. Then we add substrate o f 1, 2 phenylenediamine dihydrochoride into the substrate solution of phosphatecitrate buffer with urea hydrogen peroxide tablets (step 5) and mixed them. Developed color using re for 1530 min. Stoppped reaction by addition of 4N H2S04 490 nm on a plate reader within 30 minutes of stopping the reaction was recorded.

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29 Histochemistry brains on a sl iding microtome with a freezing stage. Four sections, six sections apart each, were mounted on slides for thioflavine S staining. Sections were allowed to dry on the slides for 15 minutes. The slides were then placed in deionized water for five minutes. They were then placed in filtered Mayers Hematoxylin for five minutes. Next, the slides were rinsed under running water for five minutes, followed by a five minute rinse in deionized water. The slides were then placed in a 1% thioflavine S solution (in dH2O filtered, Sigma) for five minutes. The slides were differentiated in 70% ethanol for five minutes, given short rinses in deionized water followed by PBS, and cover slipped with glycerol gelatin (Sigma). Thiazine R ed S taining Free floating sections were s tained for 30 minutes at room temperature in 0.01% thiazine red in PBS solution. Sections were washed 3 times in PBS for 5 minutes each time, then mounted and cover slipped using glycerol gelatin. Image Analysis For percent amyloid burden measurements (bot h dense cored and diffuse) sections were analyzed in a blinded manner using the NIH Image J software. Regions of interest (ROI) were created encompassing both the hippocampus and neocortex of digital micrographs of each stained section. The ROIs area was measured in pixels2. The number of plaques stained, plaque sizes (in pixels2), and total stained areas in the hippocampus and cortex (in pixels2) were determined by thresholding segmentation. Total stained areas were divided by total area, and then multipl ied by 100% to give the percent amyloid burden.

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30 L 1 2 3 4 Figure 21 HindIII and BglII digest plasmids. L (Laddar), 1, 2 (sample 1, 2), 3, 4 (sample 3, 4). From picture, we can see sample 3, 4 have two more DNA bands than sample 1, 2 because plasmids sample 3, 4 still keep the human plasma gelsolin DNA sequence.

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31 CHAPTER 3 AAV8 VECTOR WITH HUMAN PLASMA GELSOLIN DELI VERY AND EFFECT IN APP/PS1 MALE MI CE BEHAVIOR Gene Therapy and Behavior Training Gene therapy is a new method to use specific sequences of DNA or RNA to treat, cure, or ultimately prevent disease. There are still many hurdles to overcome before it could be used in the future as a mature technic. T he major hurdle is degradation of DNA or RNA due to endogenous nucleases. The other challenge is the ability of the cells to transcript and translates genes. Another one to overcome is the period that the cells can keep the gene expression on a rea sonable level for cure that accumulates and deposit s in the brain of patients who have Alzheimers disease. it fibrillization, and even disassembles amyl giving a possible role for gelsolin of clearence 93, 108 Matsuoka showed injections with bovine plasma gelsolin 1 M146L, but they failed of the testing gelsolins effects in older mice and on memory related behavior 110 And Hirko showed peripheral deliver plasmid of plasma gelsolin can decrease A burden in the brain in APP/PS1 mice, however they did not correlate these reductions with retard memory los s. 112 Since gene therapy can be used as a treatment all these results triggered us to ask a question whether we can use rAAV 8 to deliver the human plasma gelsolin gene and test it in an AD mouse model as a lifelong, one dose ideal treatment. In this chapter we characterize the relationship between the rAAV8 with human plasma gelsolin gene treatment and AD model male mice memory ability.

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32 The Morris water maze is a behavioral test used to study spatial learning and memory It was developed by neuroscientist Richard G. Morris in 1981. 118, 119 In a standard protocol, a mouse is putting into a pool of water with an platform below the water surface. Some signs are placed around the pool. After released, the mouse swims and tries to escape from the water. The whole experiments are tap ed. During the experiment, the mouse would learn to find the platform, and the mouse would find the platform more rapidly than beginning of the experiment This imp rovement of performance due to mouse learned and kept the memory where the hidden platform is. After training a capable mouse would spent less time before it find the platform. Therefore we used Morris water maze to compare the difference of the memory ability between the treated mice and untreated control mice by probe trial after all mice learned where the platform is located in the pool through the learning trial. Since standard Morris water maze just can provide one group data to be used for the learning and memory ability analysis in ten days so that the chance of random results are increased, we use the radial arm water maze instead of Morris water maze laterly. Radial arm water maze provide data which can be used for learning ability analysis and we can have one group of data for memory ability analysis each day in ten days so that the chance of random results are decreased. In a typical radial arm water maze system six arms were placed in a pool of water which would make six swim channels And si gns were put on the walls in each arm The standard r adial arm water maze testing procedure would have four learning trials and one memory trial each day for 10 days. During the experiment the re is a submerged escape platform was p ut in the target arm; th e target arm was randomly changed for each day experiment A nd four

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33 starting arms were randomly selected for learning trials, the number of errors during the fourth trial would be an index of learning. Each trial would be 1min, and the number of errors of going into wrong arms before mouse found platform was recorded. A memory ability trial was then done by using the fourth arm as the starting arm Over 1 min trial, the number of errors was recorded. Finally, we will have more data than Morris water maze t o analysis the learning and memory ability. 120 Results and Discussion Behavior T raining of Male Mice Doubletransgenic mice expressing both of mutant APP695K594N,M595L and mutant presenilin1E9 (APP/PS1E9 mice) were supplied by the Jackson Laboratories Gelsolin transgene expression was accomplished by injecting 1011 rAAV8 viral genomes per mouse via the tail vein. Because A deposits will be formed starting around 6 months old for the APP/PS1 mice, the n=16 APP/PS1 male mice were divided into 3 groups: control group: no treatment; young age treatment group: treatment at 4 months old; older age treatment group: treatment at 6 months old. When mice were at 8 months old, Morris Water Maze test was used to evaluate memory. The mice were trained for 4 times with one minute trials each time per day for 9 days, and then a probe test for memory evaluation was performed. Morris water maze learning curve shows that all male mice shorten the latency to the target platform from around 30 seconds to around 20 seconds after nine days training, indicating that all male mice learned the target platform position. The following probe test results would not be random results. (Figure 3 1) After statistics tests were performed using SAS software based on a oneway ANOVA tes t, take p value: 0.05. results showed that s patial memory retention of the 4 month old treated group was significantly different compared to the control group

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34 because the 4 monthold treated group spend more time in target quadrant area compare with the control group. (Figure 32) 6 monthold treated group show similar results but not show significant difference compare with control group. At the age of 11 and 14 months old, the radial arm water maze test was used to test memory. Training the mice occu rred in the same way as the Morris water maze, except probe evaluation was done daily. (Fig.33) The results showed that the treated groups make less mistakes before they selected the target arm and found target compared with the control group, but failed to show significant difference base on one way ANOVA test, take p value: 0.05. Male Mice Results Analysis In this chapter we demonstrate that human plasma gelsolin gene delivery via AAV 8 vector in APP/PS1 male mice will retard memory. Treated mice stayed for longer in target area than control mice in the Morris water maze probe test and also made fewer mistakes before they found the platform than control mice during the radial arm maze. We also showed that the earlier age treatment showed more efficacies at preventing memory deficits than the older age treatment in mice. At the same time, we observed that plasma gelsolin gene delivery via AAV 8 did not affect the learning ability of mice.

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35 Figure 3 1 Morris Water Maze learning curve. Morris water maze learning curve showed that all male mice shorten spent time to find target platform after nine days training from around 30 seconds to around 20 seconds. 0 5 10 15 20 25 30 35 40 0 1 2 3 4 5 6 7 8 9 10Time (sec)Time (day)Training curve control 6 month old trt group

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36 Figure 3 2 Morris Water Maze test. Morris water maze probe test of percentile of time spent in target quadrant area by trained mice. Graph showed significant difference (p value < 0.05) by comparing young age treated group and control group. The treated group spent more time on target area than control group. Control 4 Month-old treated 6 Month-old treated 0 5 10 15 20 25 30 35* p=0.0118% time in target quadrant

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37 Figure 33 Radial Arm Water Maze test. Radial arm water maze probe test of mistake times made by trained mice. Graph showed treated group make less mistake by comparing with control group. Control 4 Month-old treated 6 Month-old treated 0 1 2 3 411 month oldNumber of Errors Control 4 Month-old treated 6 Month-old treated 0 1 2 3 4 14 month oldNumber of Errors

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38 Figure 34 Eleven month old male mice radial arm water maze. Treated group made less mistake than control group during the probe test of the memory ability, but the learning ability did not show a difference among three group. 0 1 2 3 4 0 1 2 3 4time (mistake)trialtrial and probe (total) control 6 mon old trt group 3 mon old trt group

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39 Figure 35 Fourteen month old male mice radial arm water maze. Three month old treated group show made less mistakes than control group and six month old treated group during the probe test of the memory ability, but the learning ability did not show a difference among three group. 0 1 2 3 4 0 1 2 3 4miataketrial trial and test (paper)

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40 Figure 36 Morris water maze system.

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41 Figure 37 Radial arm water maze system.

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42 CHAPTER 4 AAV8 VECTOR WITH HUMAN PLASMA GELSOLIN DELI VERY AND EFFECT IN APP/PS1 FEMALE MICE BEHAVIOR Behavior Training of Female Mice From the previous chapters results, we noticed that the sample size of each group of male mice was too small to have enough power to do statistical analysis and the results of the younger age treatment group had a better effect of treatment than the older age treat ment group and control group. Twenty four APP/PS1 female mice were purchased and divided into 2 groups, each group having 12 female mice: control group: no treatment; treatment group: treatment at 4 months old. As a positive control w e included 12 same strain wild type female mice. The Radial Arm Water Maze test was used to train the mice instead of the Morris water maze. During the period, all blood samples were collected from mice arteria caudilis by using plasma collect tube and kep t in ice. Then the tubes were centrifuged at 1000*g for 5 minutes, the blood cell and plasma were separated by gel in the tube, blood cells below the gel, plasma above the gel. Results and Discussion Same strain female APP/PS1 mice were purchased and the s ame treatment and procedure were used except using Radial arm water maze instead of Morris water maze in order to compare the results with male mice. When female mice aged to seven months old, eleven months old and sixteen months old, we performed the redi al arm water maze to match the male mice behavior training time points. After analysis of the data generated from seven month and eleven month radial arm water maze experiment, we found no differences of learning and memory ability. (Figure 4 1, 4 2) We an alyzed the latency to the target and the number

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43 of errors made before they found the target platform. No differences of learning ability and memory ability between the treated and control female mice were observed even when they are sixteen months old. (Fi gure 43, 4 4) Female Mice Results Analysis In this chapter we show that hu man plasma gelsolin gene delivery via AAV 8 vector in APP/PS1 female mice has no effect on the learning and memory ability of female APP/PS1 mice.

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44 Figure 41 Seven month old female mice radial arm water maze. 0 1 2 3 4 5 6 0 1 2 3 4 control trt

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45 Figure 42 Eleven month old female mice radial arm water maze. 0 1 2 3 4 5 6 7 0 1 2 3 4timestrial trt cont

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46 Figure 43 Latency time before female mice find target platform. 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60Gelsolin Control Wild-Type DayLatency (sec)

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47 Figure 44 Errors made before female mice find target platform. 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8Gelsolin Control Wild-Type DayErrors

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48 CHAPTER 5 AAV8 VECTOR WITH HUMAN PLASMA GELSOLIN DELI VERY AND EFFECT OF A BURDEN IN APP/PS1 MI CE BRAIN AREA A myloid Burden in Brain As shown in the previous promising target to treat AD and APP/PS1 mice would be a good animal model for research of AD since the mutation of APP gene and the mism etabolism of APP would intriguer AD pathogenesis due to increase the le vel finally induce the aggregation 21, 22, 23, 24 and transgenic mice that express a mutant form of the human APP gene would develop amyloid plaques and then intriguer Alzheimer's pathology with spatial learning deficits. 31,32,33,34 prevents fibrillization, 93, 108 Matsuoka showed injections with bovine plasma gelsolin can 1 M146L, 110 and the brain in APP/PS1 mice, 112 all these results give the supports that gene therapy by using human plasma gelsolin gene may be an ideal treatment for AD. We have shown that AAV 8 with human plasma gelsolin gene delivery can retard memory loss in APP/PS1 male mice, but there still have a question of co rrelation explain how it works after gene expressing.

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49 Results Dense Cored Amylo id Deposits Thioflavin S staining was used to examine the amyloid plaques in male mice brain 121 were sliced and stained (Fig.5 1, 5 6), and digital micrographs were taken as described previously. Images were analyzed in a blinded manner using Image J software. The area of the hippocampus and cortex, total stained area, and the number of plaques was determined. The amyloid burden was determined by dividing the total area st ained by the total area of the hippocampus and cortex. A oneway ANOVA was performed for statistical analysis. The images of the male mice brain slices shown less amyloid deposit in the brain in treated group mice, (Figure 51) but female mice show different results. (Figure 5 6). Then we quantify the amyloid burden level by comparing percentage area stained, amyloid plaque numbers, amyloid plaque average size and amyloid beta concentration between treated and control mice. Figure 52 shows the male mice am yloid plaques deposit percentage area results, we noticed that 4 month old group has less than the control group in both the hippocampus and cortex area. We then count amyloid plaque numbers and measure plaque size, we observed that the mice in 4 month old age treated group show less numbers and smaller size of plaques compared to the control group, especially in memory related hippocampus area. The mice in 6 month old treated group did not show very close level to control group. (Figure 53, 5 4) After analysised of the female mice results, we find similar results except the plaques numbers for the treated group are increased compared to the control group. (Fig 5 7) Since we increased the sample size, we have more power for data analysis,

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50 and we observe that there are significant differences of the plaques size between the treated group and control group. Also, the hippocampus, an important structure for memory and learning, shows a significant difference of percentage area between these two groups. (Fig 58 5 9) Brain A oncentrations The results from the image analysis only show a 2D level of the amyloid burden. We use an ELISA method to quantify amyloid beta 42 concentration by using frozen half ELISA kits were used to measure the 42) in the hemi brains from the mice. By comparing the analysis results, we find that the unsoluble centration increased in gelsolin gene delivery mice compare with control mice. Especially in significant difference by comparing the gelsolin gene delivery mice compare wit h control mice. (Figure. 55, 5 10) A myloid Burden Results Analysis In this study, we examined the effects of peripheral human plasma gelsolin gene delivery via AAV densecored amyloid plaque results, w e observe that the treatment reduced especially in the hippocam pus area.

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51 Figure 51 Densecore amyloid deposits in APP/PS1 male mice. Deposits visualized by thioflavin S Staining. Negative digital micrographs of control mouse (left), 6 month old treated mouse (middle) and 3 monthold treated mouse (right).

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52 a. b. Figure 52 Analysis of densecore amyloid deposits in APP/PS1 male mice. Deposits visualized by thioflavin S staining. a) Percent amyloid burden in cortex area. b) Percent amyloid burden in hippocampus area. Bars represent group means the standard error. Thioflavin-S % area in cortex control 3 month old treated 6 month old treated 0.0 0.5 1.0 1.5 2.0 2.5 Thioflavin-S % area in hippocampus control 6 month old treated 3 month old treated 0.0 0.5 1.0 1.5 2.0

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53 a. b. Fig ure 5 3 Analysis of densecore amyloid deposits in APP/PS1 male mice hippocampus area. Deposits visualized by thioflavin S staining. a) Amyloid plaques total numbers. b) Average size of amyloid plaques. Bars represent group means the standard error. amloid beta numbers in hippocampus area Control 6 month old treated 4 month old treated 0 50 100 150 200 250 amyloid plaques average size in hippocampus control 6 month old treated 3 month old treated 0 10 20 30 40

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54 a. b. Fig ure 5 4 Analysis of densecore amyloid deposits in APP/PS1 male mice cortex area. Deposits visualized by thioflavin S staining. a) Amyloid plaques total numbers. b) Average size of amyloid plaques. Bars represent group means the standard error. amyloid plaques numbers in cortex control 6 month old treated 3 month old treated 0 100 200 300 400 Amyloid plaqus average size in cortex Control 6 month old treated 3 month old treated 0 10 20 30 40

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55 a. b. Fig ure 5 5 higher in the mice which were treated by gelsolin gene delivery than control mice. Unsoluble Amyloid 42 Control 4 Month-old treated 6 Month-old treated 0 25 50 75 100 125pg/mL Soluble Amyloid 42 Control 4 Month-old treated 6 Month-old treated 0 25 50 75pg/mL

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56 Figure 56 Densecore amyloid deposits in APP/PS1 female mice. Deposits visualized by Thi azine red Staining. Digital micrographs of control mouse (left), treated mouse (right).

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57 a. b. Figure 57 Analysis of densecore amyloid deposits in APP/PS1 female mice. Deposits visualized by Thiazine red staining. a) Percent amyloid burden in cortex area. b) Percent amyloid burden in hippocampus area. Bars represent group means the standard error. amyloid plaques % area in cortex treated control 0 1 2 3 4 5 6 7 8 9 amyloid plaques % area in hippocampus treated control 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

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58 a. b. Fig ure 5 8 Analysis of densecore amyloid deposits in APP/PS1 female mice hippocampus area. Deposits visualized by thiazine red stai ning. a) Amyloid plaques total numbers. b) Average size of amyloid plaques. Bars represent group means the standard error. amyloid plaques numbers in hippocampus treated control 0 50 100 150 200 250 300 350 400 450 500 550 amyloid plaques average size in hippocampus treated control 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0035 0.0040 0.0045 0.0050 0.0055 0.0060 0.0065

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59 a. b. Fig ure 5 9 Analysis of densecore amyloid deposits in APP/PS1 female mice cortex area. Deposits visualized by thiazine red staining. a) Amyloid plaques total numbers. b) Average size of amyloid plaques. Bars represent group means the standard error. amyloid plaques numbers in cortex treated control 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 amyloid plaques average size in cortex treated control 0.000 0.001 0.002 0.003 0.004 0.005 0.006 0.007

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60 Fig ure 5 10. ELISA analysis of t group and control group, but the illustration shows that there are significant etween treated group and control group. Amyloid Beta 42 ELISA Control Soluble A Gelsolin Soluble A Control Unsoluble A Gelsolin Unsoluble A 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 *p = 0.05 pg/g of brain

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61 CHAPTER 6 NEW ELISA METHOD FOR HUMAN PLASMA GELSOLI N QUANTIFICATION S TYELS Methods of Protein Concentration Quantification We have shown that peripheral human plasma gelsolin gene delivery via AAV 8 can retard the memory loss observed in APP/PS1 male mice and this effect may be due results encourage us to try to develop the PK/PD model of this treatment in order to help us understand how this treatment works. Low level of plasma gelsolin correlated with severe trauma, sepsis, myonecrosis, conditioning regimens for stem cell transplantation, acute r espiratory distress syndrome (ARDS), and liver necrosis, 122, 123, 124, 125, 126, 127. Increase plasma gelsolin level could improve pulmonary injury in rodents which was ex perienced to hyperoxia or burns, 124, 128. Due to these observations and the fact that a biotechnology company has begun to produce gelsolin for future clinical use, knowledge of human plasma gelsolin metabolism appears worthy of consideration, and development of PK model is necessary. Currently t here is no published data for the PK/PD parameter of the plasma gelsolin. Human plasma gelsolin has high homology with mouse plasma gelsolin. In order to quantify human plasma gelsolin concentration in the mice we had to develop a method which can be used to differentiate the signal of the human and mouse plasma gelsolin. We tried several methods to quantify plasma gelsolin concentration. First, we tried a gelsolin function assay to quantify gelsolin concentration. Since enzyme like properties of gelsolin could p olymerize actin monomer and cleave actin filaments under different pH ion strength. The time of reactions is dependent on the gelsolin concentration, so we can measure the reaction signal to quantify the gelsolin

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62 concentration. There are two stand ard gelsolin functional assays. One assay is to cleave rhodamine phalloidin labeled actin filaments; the other assay is to p olymerize pyrine labeled actin monomer. Fluorescence intensity was measured to quantify gelsolin concentration. 129, 130 We used rh odamine phalloidin method to quantify gelsolin concentration. Rabbit muscle actin was dissolved in a solution (TRIS 10 mM, CaCl2 0.2 polymerising buffer (KCl 150 mM, MgC l2 2 mM, TRIS 10 mM, CaCl2 0.2 mM, ATP 0.5 mM and dithiothreitol 0.2 mM), incubated at room temperature for 2 hours and stored at 20C. Human plasma gelsolin was dissolved in a solution ( KCl 75 mM, EGTA 0.2 mM, and HEPES 10 mM ) at 400 nM and stored at 80C. The solution used throughout the experiments. The assay is based on the fluorescence increase of rhodaminelabelled phalloidin after binding to polymerized actin. 131, 132 Flu orescence intensity was measured by a fluorescence microplate reader, using 96well black clear bottom plates. We used transmittance at 540 and 590 nm for excitation and emission. Actin was incubated with phalloidin for at least 8 minutes before test Add i ng of gelsolin would reduce the fluorescence. The gelsolin concentration was normalised by using phalloidin plus actin and monomer actin alone as 100% and 0% (Fig 6 1) These methods cannot be used because the physiological concentration of gelsolin in the and the background signal of gelsoin is too strong to eliminate. Then we attempted to use the monoclonal antibodies and develop western blot methods to quantify human plasma gelsolin concentration in the mouse plasma. Antibodies were developed by us with help from the CDBR lab. The western blot is used

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63 to detect gelsolin concentration. It uses electrophoresis to separate denatured proteins. The n the proteins are transferred to a PVDF membrane, where they are tested by antibodies 133, 134 But this method still cannot be used because this method requires denaturation of the protein res ulting in the lose their three dimensional structure and turn to linear structure exposing all the antibody binding sites making it difficult to differentiate the signal of human and mouse plasma gelsolin. Since western blot cannot differentiate signals between mouse and human plasma gelsolin, we attempted to develop an ELISA method using commercial antibodies. First we tried to develop Sandwich ELISA. The his tag polyclonal antibody in the Sandwich ELISA was used to selectively bind to the commercial hi stagged human plasma gelsolin, which should to eliminate the influence of mouse plasma gelsolin and help to quantify the amount of human plasma gelsolin more precisely. Figure 6 3 shows that there were not enough differences by comparing with the control groups. So the Sandwich ELISA can not being used to quantify human plasma gelsolin either New ELISA Method for Gelsolin We concluded that the his tag antibody might not bind the human plasma gelsolin as a reason why this method did not worked. The new EL ISA method was developed by coating plasma gelsolin sample on plates directly Figure 64 showed the standard curve looks very well on linear relationship between O.D. value and the gelsolin concentration from 0 ng to 20 ng range. As gelsolin concentration increased, antibodies became more and more saturated so that curve t u r ned to flat

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64 LOD & LOQ The limit of detection (LOD) and the limit of quantitation (LOQ) were used to verify the effecacy of our experiments. LOD was around 0.18ng and LOQ was around 2.5ng, both of them were lower than 5ng.This result means that the standard curve range which covers from 5ng to 20ng mig ht be effective to quantify the amount of human plasma gelsolin. The R esults of G elsolin S piked with Mouse P lasma C ompared w ith T hose in PBS The further experiment was done to compare the results between gelsolin spiked with mouse plasma and in PBS (Figure 6 5) We noticed that both of curves show same trend and almost parallel. But the O.D. value in spike sample is lower than PBS sample. We hypothesized that this may due to proteases in the mouse plasma that degrade human plasma gelsolin protein which gives us a warning that for further experiments we might have to use protease inhibitors. ELISA Results Analysis In this study, we developed a new method to quantify the human plasma gelsolin, the beauty of this method is that it can differentiate human and mouse plasma gelsolin signal which provide a powerful tool for the future research.

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65 Fig ure 6 1 Gelsolin function assay. Assay shows that it works for quantify gelsolin concentration because monomer actin solution show lowest signal strength, polymerized actin binding by rhodanmine phalloidin show strongest signal strength and addin gelsolin polymerized acti n solutions signal strength between monomer and polymerized actin. 0 2 4 6 8 10 12 14 16 18 20 0 5 10 15fluorencencegelsolin assay mono actin poly actin poly actin +1ul gel

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66 a. 10 20 50 100 ng b. S 1 2 3 6 10 11 12 13 4 5 7 9 L S 14 15 S4 S15 Figure 6 2 Western blot of gelsolin. Western blot results illustrated it could be use to quantify human plasma gelsolin concentration alo ne, but it failed when it is used to try to quantify human plasma gelsolin in mouse plasma. a) western blot signal is stronger when gelsolin amount increase. b) western blot signal show no difference among different groups of mice. S, standard gelsolin; L, ladder; black numbers: 1, 2, 3, 4, 5 are mice number in control group; green numbers: 6, 7, 9 are mice number in 6 month old treated group; red numbers: 10, 11, 12, 13, 14, 15 are mice number in control group; S4, standard gelsolin spike with NO. 4 mouse; S15, standard gelsolin spike with NO. 15 mouse.

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67 Figure 6 3 S howed that O.D. value not changed when gelsolin amount increased.

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68 Figure 6 4 Gelsolin standard curve.

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69 Fig ure 6 5 Standard curve of gelsolin in PBS and spike with mouse plasma.

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70 CHAPTER 7 DISCUSSION AND FUTURE DIRECTIONS Previous studies have demonstrated that human plasma gelsolin can bind to 107, 108 Matsouka showed that administration of bovine plasma gelsolin could reduce amyloid 110 Hirko et al. showed that peripheral delivery of human plasma gelsolin plasmid DNA could reduce amyloid levels in APP/PS1 mice. 112 Based on these results, we hypothesized that with human plasma gelsolin gene delivery via AAV8 peripherally would be a single dose, long term ideal treatment for AD. To test this hypothesis, first we prepared large amount of human plasma gelsolin plasmi d for AAV 8 packaging. Then we injected AAV 8 coding for human plasma gelsolin into the AD mouse model APP/PS1 mice through the tail vein. To test the effect of the treatment, we performed behavior training and finally sacrificed the mice and examined their brains for amyloid pathology. Through comparing the results of behavior training from the Morris Water Maze test and radial arm water maze test, we determined that memory loss associated with the APP/PS1 line of mice used had been retarded in the male mice that received gelsolin gene delivery compared to the control males. We did not oberve any effect on memory in female mice through behavior training. The learning ability showed no difference for all mice. Arendash reported similar radial arm water maze behavior training results comparing APP/PS1 and wild type male mice. 117 Their results showed that wild type male mice slow down memory loss by comparing with APP/PS1 male mice and learning ability showed no difference between those two groups of male m ice. That means our gelsolin gene delivery via AAV 8 treatment in male mice behavior

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71 training results is matching the wild type mice. The reason that the female mice did not retard memory loss may due to gender behavior differences. Although memory loss had been retarded after treatment, we did not find any significant difference of brain slice staining results between treated male mice and control mice. A larger sample group size to show the statistical significant difference may be needed. From the ELISA analysis we find that after treatment, comparing the treated mice and control mice. At the same time, thioflavinS and thiazine red staining of brain slices sh ow that amyloid deposits in the brain decreased in the treated mice compared to the control mice, especially in the hippocampus area which is a critical structure for learning and memory. These results may support the sink theory. Gelsolin gene expression in the body after gene delivery via AAV 8, results in the would shift the equilibr APP/PS1 male mice. Therefore, we believe that the treatment strongly showed and sink theory may be used to explain how peripherally expressed gelsolin acted as a Nearly all cl inical trials for Alzheimers disease have failed the past several years. We suspect that this may be due to poor volunteer selection. First, we recognized that

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72 one chemical for the treatment may focus on one biomarker. We understand there are many hypotheses to explain which one is the critical reason for Alzheimers disease progression, such as amyloid beta, tau, neuron cell signal receptors, etc. It may be true that there is more than one factor for Alzheimers disease progression that may require the need to differentiate Alzheimers disease into several subtypes. This may help to make more clear how to explain that Alzheimers disease can be induced and explore more chemicals to focus on different biomarkers due to different hypothesis. Before we do c linical trials, we need to screen the volunteers by using different biomarkers in order to target therapies via the specific chemicals, then we have a higher chance to find a fitting treatment chemical for a specific subtype of Alzheimers disease patient. It may be necessary to go back and check all chemicals we have to find useful chemicals. Second, we always choose aged volunteers for the clinical trials. It may be another reason for the trial failure because we know if a volunteer shows symptoms, that m eans neurons have already been injured and lost, and no method can be used recovery them, then memory will be lost forever due to neurons dying. This may be the reason clinical trials fail on not show significant results at halting the progression of memor y loss by comparing the treatment groups with control groups. In the future, we can choose volunteers through biomarker screens in order to find high risk group base on the specific biomarkers for the clinical trials and perform clinical trials earlier bef ore symptoms have already been shown. For the methods to quantify human plasma gelsolin concentration, by comparing the three methods of the western blot, sandwich ELISA, the new ELISA method, the new ELISA method showed to be a better method. The standar d curve showed a good

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73 linear relationship in a measurable range. This advantage made it possible to quantify human plasma gelsolin. Although the data in vitro appeared positive, it did not work very well in vivo There were no significant differences between administration groups and the background. We hypothesis that i.v. injection of human plasma gelsolin may bind to blood cells or tissue, so when we harvest plasma samples there was no human plasma gelsolin signal showing in the plasma fraction. We need to perform more experiments to confirm our hypothesis. The vast majority of gene therapy approaches for AD involve delivery of vectors coding for amyloid degrading enzymes (neprilysin or insulin degrading enzyme) (Eckman an d Eckman, 2005; Marr et al., 2003). the rationale behind using amyloid degrading enzymes is to increase the clearance of amyloid. Future directions for study should include the development of a PK /PD model of AAV8 with human plasma gelsolin gene delivered peripherally, determination which secretase, human plasma gelsolin and amyloid degrading enzymes. The first two studies will be im portant for understanding how the treatment works, and the cocktail treatment will provide an ideal treatment finally increase clearance. In conclusion, our study demonstrat es that peripheral delivery of AAV 8 coding for human plasma gelsolin may be a potential treatment for AD because it results in

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74 advantage of being a one dose, long term treatm ent compared with the traditional therapeutic approaches for AD.

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87 BIOGRAPHICAL SKETCH The author was born in China in 1973. In 1994, he received his B.S (Bachelor of Science) degree in Nanjing University. In 2006, he joined in Department of P harmaceutics.