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Up-Regulation and Activation of Caspase-12 and Caspase-7 Following Traumatic Brain Injury in Rats

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Title:
Up-Regulation and Activation of Caspase-12 and Caspase-7 Following Traumatic Brain Injury in Rats
Creator:
LARNER, STEPHEN FRANK ( Author, Primary )
Copyright Date:
2008

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Antibodies ( jstor )
Apoptosis ( jstor )
Cell death ( jstor )
Complementary DNA ( jstor )
Hippocampus ( jstor )
Messenger RNA ( jstor )
Physical trauma ( jstor )
Protein isoforms ( jstor )
Rats ( jstor )
Traumatic brain injury ( jstor )

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University of Florida
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University of Florida
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Copyright Stephen Frank Larner. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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8/31/2005
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73333923 ( OCLC )

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UP-REGULATION AND ACTIVATION OF CASPASE-12 AND CASPASE-7 FOLLOWING TRAUMATIC BRAIN INJURY IN RATS By STEPHEN FRANK LARNER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Stephen Frank Larner

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This document is dedicated to my parents Fra nk H. and Verlee B. Larner in honor of their curiosity, interest and loving support during this time of academic challenge.

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ACKNOWLEDGMENTS First and foremost I would like to thank my parents, Frank H. and Verlee B. Larner, for their love and support as I travailed over the years as I changed careers and returned to the academic life to achieve this doctoral degree. Their constant encouragement even as they struggled, and still do to understand just what it is I am studying has been appreciated. I would like to thank my mentor, Ronald Hayes, for the opportunity to pursue research in this novel area that allowed me to push back the frontiers of knowledge in ways that neither us could have envisioned when I began this trek. I would also like to thank my past and present committee members, Brian Pike, Gerry Shaw, Kevin Wang, and Nancy Denslow, for providing their expertise and input through my research endeavors. Their help in overcoming the bumps that normally occur in any research project is very much valued. I thank all of the members of the Ronald L. Hayes and Kevin K.W. Wang laboratories for their great friendships and invaluable assistance over the years. The completion of this research as preparation for this dissertation would not have been possible without their support. I am especially grateful to Deborah M. McKinsey for support during the early years as I worked on cloning and sequencing the CASPASE-12 gene and Barbara O’Steen and Erik Johnson for their assistance in working with the rats and immunohistochemistry protocols, respectively. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES.........................................................................................................viii ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION AND BACKGROUND.................................................................1 Introduction...................................................................................................................1 Rationale: Traumatic Brain Injury................................................................................2 Apoptosis: Definition and Pathways............................................................................3 Caspases........................................................................................................................4 Endoplasmic Reticulum and ER Stress........................................................................6 Unfolded Protein Response Following BiP (GRP78) Activation.................................7 PERK...................................................................................................................10 ATF6....................................................................................................................10 IRE1and Caspase-12........................................................................................12 Caspase-12..................................................................................................................13 Caspase-7....................................................................................................................16 Calpains......................................................................................................................18 Activation of Caspase-12 by Caspase-7 and Calpains...............................................19 2 INCREASED EXPRESSION AND PROCESSING OF CASPASE-12 AFTER TRAUMATIC BRAIN INJURY IN RATS...............................................................22 Introduction.................................................................................................................22 Results.........................................................................................................................25 Immunoblot Analysis of Caspase-12 Expression after Traumatic Injury:..........25 Immunoblot analysis of caspase-12 expression in cortex after traumatic injury.......................................................................................................28 Immunoblot analysis of caspase-12 expression in hippocampus after traumatic injury.......................................................................................28 Standard Curve Generation for Semi-Quantitative RT-PCR Using Serially Diluted cDNA..................................................................................................29 Semi-quantitative PCR Analysis of Experimental Samples................................30 v

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Immunohistochemical Analysis of Caspase-12 Expression after Traumatic Injury................................................................................................................32 Immunohistochemical analysis of cortical caspase-12 expression after trauma.....................................................................................................32 Immunohistochemical analysis of hippocampal caspase-12 expression after trauma.....................................................................................................32 Discussion...................................................................................................................35 3 CASPASE-7: INCREASED EXPRESSION AND ACTIVATION AFTER TRAUMATIC BRAIN INJURY IN RATS...............................................................44 Introduction.................................................................................................................44 Results.........................................................................................................................46 In Vitro Model: Confirmed Presence of Caspase-7 Protein in PC12 Cells.........46 Immunoblot Analysis of Caspase-7 Activation Following Traumatic Brain Injury................................................................................................................48 Semi-quantitative RT-PCR Analysis of Caspase-7 mRNA levels Following TBI52 Semi-quantitative PCR Analysis of Experimental Samples................................54 Immunohistochemical Analysis of Caspase-7 Up-regulation Following TBI....54 Discussion...................................................................................................................58 4 RAT CASPASE-12 ISOFORM..................................................................................64 Introduction.................................................................................................................64 Results.........................................................................................................................66 Discussion...................................................................................................................72 5 EXPERIMENTAL PROTOCOLS.............................................................................77 Rat Pheochromocytoma (PC12) Cell Culture, Collection and Preparation................77 Surgical Preparation and Controlled Cortical Impact Traumatic Brain Injury...........78 Tissue Lysis and Protein Purification.........................................................................79 Semi-Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR)...80 RNA Purification.................................................................................................80 Reverse Transcription..........................................................................................80 Primer Selection..................................................................................................80 Standard PCR......................................................................................................81 Semi-quantitative/LightCycler PCR....................................................................81 Standard Curve Preparation and Semi-Quantitative PCR Analysis....................82 Immunoblot Analysis..................................................................................................83 Test for Anti-caspase-7 Antibody Specificity............................................................84 Immunohistochemistry...............................................................................................85 Immunohistochemistry Preparation.....................................................................85 Analysis...............................................................................................................85 Statistical Analyses.....................................................................................................86 Preparation of Novel Fragment-Specific Antibodies.................................................87 Clone and sequence Caspase-12 cDNA in rat............................................................88 vi

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Primer Selection..................................................................................................88 Total RNA Isolation from Tissue........................................................................88 Reverse Transcription-PCR and Cloning of PCR Product..................................89 TOPO TA Cloning..............................................................................................89 Sequenced Results on CEQ 2000 DNA Analysis System...................................90 Extending Sequence Using the 3’-Rapid Amplification of cDNA Ends (RACE)...................................................................................................90 6 CONCLUSION AND FUTURE DIRECTIONS........................................................92 APPENDIX A RAT CASPASE-12 ISOFORM GENETIC cDNA SEQUENCE..............................97 B RAT CASPASE-12 ISOFORM PROTEIN SEQUENCE..........................................99 Caspase-12 Published and “In House” Isoform Protein Sequence Alignment...........99 Caspase-12 Isoform Gene to Protein Sequence Translation.....................................100 C THE COMPLETE RAT CASPASE-12 GENE........................................................102 LIST OF REFERENCES.................................................................................................110 BIOGRAPHICAL SKETCH...........................................................................................127 vii

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LIST OF FIGURES Figure page 1-1 Apoptotic pathways are induced by a variety of pathological conditions..................6 1-2 Traumatic injury induces the unfolded protein response...........................................8 1-3 Effector caspase-7 with a short prodomain..............................................................18 1-4 Traumatic injury induces specific cleavage of pro-caspase-12................................20 2-1 Immunoblot analysis of proand cleaved caspase-12 expression in ipsilateral cortex........................................................................................................................26 2-2 Immunoblot analysis of proand cleaved caspase-12 expression in ipsilateral hippocampus.............................................................................................................27 2-3 Standard curve generation for caspase-12 and GAPDH semi-quantitative real-time PCR...........................................................................................................30 2-4 Semi-quantitative real-time PCR analysis of caspase-12 mRNA expression................31 2-5 Immunohistochemical analysis of the ipsilateral cortex..........................................33 2-6 Immunohistochemical analysis of the ipsilateral hippocampus...............................34 3-1 Confirmation of anti-caspase-7 antibody specificity...............................................47 3-2 Thapsigargin mediated caspase-7 activation in PC12 cells.....................................48 3-3 Z-D-DCB inhibits thapsigargin mediated caspase-7 activation in PC12 cells.........49 3-4 Traumatic brain injury mediated caspase-7 activation in the ipsilateral cortex.......51 3-5 Traumatic brain injury mediated caspase-7 activation in the ipsilateral hippocampus.............................................................................................................52 3-6 Standard curve generation for caspase-7 and GAPDH semi-quantitative real-time PCR..........................................................................................................................53 3-7 Semi-quantitative real-time PCR analysis of caspase-7 mRNA expression............55 viii

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3-8 TBI up-regulation of caspase-7 in neurons in the cortex and hippocampus............56 3-9 TBI up-regulation of caspase-7 in astrocytes in the cortex and hippocampus.........57 4-1 RT-PCR analysis of rat caspase-12 in selected adult tissues using primer pair P1/P2........................................................................................................................66 4-2 A schematic of the caspase-12 cloned and sequenced segments.............................67 4-3 The rat caspase-12 isoform’s additional exon’s genetic sequence...........................69 4-4 The rat caspase-12 isoform’s additional exon’s protein sequence...........................69 4-5 Caspase-12 rat isoform sequence compared to the full gene sequence....................70 4-6 Caspase-12 isoform intron and exon boundaries.....................................................70 4-7 Immunoblot of the anti-caspase-12 isoform-specific antibody................................71 5-1 Summary of the 3’ RACE system procedure...........................................................91 ix

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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 UP-REGULATION AND ACTIVATION OF CASPASE-12 AND CASPASE-7 FOLLOWING TRAUMATIC BRAIN INJURY IN RATS By Stephen Frank Larner August, 2004 Chair: Ronald L. Hayes Major Department: Neuroscience Apoptosis, a programmed sequence of events leading to the elimination of cells, is a genetically conserved cellular process that plays an important physiological role during development and homeostasis of tissues. Traumatic brain injury (TBI) causes disruption of tissue homeostasis that may result in pathological activation of apoptosis. Apoptosis is mediated, in part, by caspases, a 14-member family of aspartate-specific cysteine proteases. The recently characterized caspase-12 was found to be both induced and activated during the unfolded protein response by caspase-7 and calpain following excess endoplasmic reticulum (ER) stress. In this study, we examined expression and activation levels of caspase-12 and caspase-7 using the lateral controlled cortical impact model of TBI in rats. Using immunoblots, caspase-12’s proform (60 kDa) and active form (12 kDa) were found to be elevated within six hours of injury. In the ipsilateral cortex, caspase-12 reached peak induction for both the proform and active form within 24 hours x

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post-injury and remained elevated for the proform up to three days after injury. In the ipsilateral hippocampus, caspase-12 induction also peaked at 24 hours post injury for the proform and remained elevated up to five days after injury, but earlier, at six hours post injury, for the active form. For caspase-7, immunoblots revealed that the pre-active (32 kDa) and the active (18 kDa) forms were elevated at 5 days post-injury and remained elevated to 7 days. Semi-quantitative PCR analysis confirmed that caspase-12 and caspase-7 mRNA levels were elevated in injured rat brains. Increasing levels of injury severity (1.0, 1.2, or 1.6mm compression injury) associates with increased mRNA expression levels, peaking at day 5 in the ipsilateral cortex for both caspases and earlier, six hours post-injury, in the ipsilateral hippocampus, for caspase-12, and 6 to 24 hours for caspase-7. Immunohistochemical studies show that both neurons and astrocytes are affected. These studies are the first to document that the caspase-12 and caspase-7 play a role in apoptotic cell death following TBI in rats and that caspase-7 is found in the brain and activated by TBI. xi

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CHAPTER 1 INTRODUCTION AND BACKGROUND Introduction Traumatic brain injuries (TBI) in the U.S.A. afflict each year approximately 1.5 million people of whom 50,000 people will die and another 80,000 plus who survive will experience the onset of disabilities, all at the estimated cost of over $56 billion annually. Yet, currently, there is little to offer in the way of pharmacological treatment as a result of these injuries. Apoptotic cell death has been suggested to play an important role in the cascade of neuronal degeneration that follows TBI (Tymianski and Tator, 1996; Weber et al., 2001), but the biochemical and molecular mechanisms involved are still in the early stages of elucidation. Recent findings suggest the endoplasmic reticulum (ER) when stressed may play a more important role than previously recognized in modulating apoptosis. Recently, a newly identified and novel member within the cysteine protease family of apoptosis mediators, caspase-12, has been cloned and found similar to the other family members (Van de Craen et al., 1997). This caspase was shown to be involved in apoptosis as an upstream activator, and the novel finding was that caspase-12 is localized to the ER (Nakagawa et al., 2000). This protease is believed to be explicitly involved in programmed cell death that results from ER stress such as the disruption of Ca 2+ homeostasis. It has, also, been previously reported that cytosolic Ca 2+ concentrations increase after TBI resulting in the activation of calpain, a reported activator of caspase-12 (Nakagawa and Yuan, 2000). Similarly, caspase-7, a more well characterized caspase normally considered an effector or downstream caspase, was discovered to activate 1

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2 caspase-12 as well (Rao et al., 2001). Thus, the general hypothesis of this dissertation is that traumatic brain injury causes increased expression of caspase-12 and caspase-7 mRNA and protein and induces their activation. The results of this research will advance our understanding of the apoptotic process, specifically the ER stress induced response and the role caspase-12 and caspase-7 play in the proteolytic cascade. If caspase-12 is confirmed to be restricted to stress responses in the ER, it will provide a novel target for therapeutic treatment of neurodegenerative diseases (e.g., TBI and AD) of the central nervous system. This dissertation is the first to characterize caspase-12 and caspase-7 in rat after TBI providing new insights into the mechanisms of cell death following injury. Rationale: Traumatic Brain Injury Traumatic brain injury (TBI) is a serious health issue in the United States as well as other nations. According to the Centers for Disease Control and Prevention, U.S.A., traumatic brain injury is frequently referred to as the silent epidemic because the problems that result from it (e.g., impaired memory, change in character traits, difficulty concentrating) often are not visible (Gerberding, 2003). Any blow or jolt to the head that results in the disruption of the normal functions of the brain is defined as TBI. The severity of the injury may range from mild, a brief change in mental status or consciousness, to severe, an extended period of unconsciousness (30 minutes or more), prolonged amnesia after the injury, or a penetrating skull injury. Any TBI can result in shortand long-term disabilities (Gerberding, 2003). Brain injuries are among the most likely types of injury to cause death or permanent disability. Each year in the United States, an estimated 1.5 million people sustain a TBI, which is 8 times the number of people diagnosed with breast cancer

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3 and 34 times the number of new cases of HIV/AIDS. Of these, 50,000 people die and the 80,000 to 90,000 who survive will experience the onset of disabilities. TBI may cause problems with cognition – concentration, memory, judgment, and mood; movement abilities – strength, coordination, and balance; sensation – tactile sensation and special senses such as vision; and emotion – instability and impulsivity (Thurman et al., 1999). At least 5.3 million Americans, about 2% of the U.S. population, currently live with disabilities resulting from TBI (Thurman et al., 1999). This estimate is based on the number of people hospitalized with TBI each year and does not include people seen in emergency departments who were not admitted to the hospital, those seen in private doctor's offices, and those who do not receive medical care. An estimated 15% of persons who sustain a mild brain injury continue to experience negative consequences including chronic untreatable pain one year after injury (Guerrero et al., 2000). In the U.S., direct and indirect annual costs of TBI are estimated to totaled $56.3 billion (NIH, 1998). The tragedy is that currently there are no known pharmacological treatments available. Apoptosis: Definition and Pathways Programmed cell death is a conserved dedicated molecular program that eradicates excess or potentially dangerous cells. This genetically regulated program allows multicellular organisms to tightly control cell numbers, tissue size and to protect itself from rogue cells that threaten homeostasis. The term for this phenomenon adopted in 1972 by Currie and colleagues is apoptosis (cited in Kerr et al., 1972). Interest in the process was modest until the early 1990’s when a number of observations noted that cell death was not incidental but a critical factor in the pathobiology of many acute and chronic neurodegenerative disorders. Apoptosis refers to a particular physiological change the cell undergoes as it is dying. Morphologically chromatin condenses to

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4 heterochromatin at the periphery of the still intact nuclear membrane often referred to as margination of the chromatin. The cells also shrink and become denser as determined by staining or flow cytometry, and the plasma membrane blebs and often fragments into multiple membrane-enclosed vesicles (Bredesen, 2000). This morphology derived from the activation of proteases, such as the caspase family, is common to many but not all cell deaths since caspase-independent cell death has been reported (Hengartner, 2000). Programmed cell death, therefore, is an active molecular process that often requires active transcription and translation of proteins for initiation (Zipfel et al., 2000) as opposed to necrosis or oncosis, which are accidental and in which the cell normally has no active role. This manner of regulating cell death calls attention to the fact that there are different signals and different evoked pathways all of which are genetically controlled. Necrotic cells most typically lyse, provoking a substantial inflammatory response (Lockshin and Zakeri, 2001). It is now generally understood that even in necrosis the genetic machinery can be evoked so that the necrosis versus apoptosis process is more of a continuum than sharply delineated patterns of cell death. It should be noted that apoptosis can occur either with or without caspases, such as might occur with AIF and endoG, though in the latter cases the results often bear little morphological resemblance to its classical definition (Hengartner, 2000). Caspases The family of cysteine-dependent aspartate-specific proteases, or caspases, has been found to be critical mediators of apoptosis. To date 14 members have been identified, 12 in humans. Nearly all studies on programmed cell death have focused on two canonical pathways (Kaufmann and Hengartner, 2001). The extrinsic pathway (Figure 1-1) initiates with the ligation of a specialized family of plasma membrane

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5 receptors termed death receptors (DR) by the FAS-L or TNFrelated apoptosis-inducing ligand (TRAIL) death factors. Ligand-induced sequential binding and assembly of adaptor proteins with Fas-associated death domains (FADDs) with the death effector domains (DEDs) of procaspase-8 or procaspase-19 ultimately lead to the caspases proteolytic activation. Depending on the context, caspases-8 and -10 can proteolytically activate the effector or executioner caspases-3, -6, and -7 which are responsible for dismantling cellular proteins or initiate a series of events through Bid that will activate the mitochondrial or intrinsic pathway, caspase-9, and then, eventually, the executioner caspases (Hengartner, 2000). The intrinsic pathway (Figure 1-1) involves cytochrome c release from the mitochondria into the cytosol as well as other mitochondrial polypeptides. Once released cytochrome c promotes the assembly of the apoptosome macromolecule whose members include Apaf-1, cytochrome c, and procaspase-9 along with ATP. This assemblage then activates caspase-9, which then is able to activate the executioner caspases (Hengartner, 2000). A novel caspase activation pathway has emerged with the discovery that caspase-7 (normally downstream of the initiator caspases) activates caspase-12 (Rao et al., 2001). This unusual sequence suggests that there may be a feedback loop involving these two caspases. This ER apoptotic pathway (Figure 1-1) has come under intense scrutiny once it was discovered that it can be activated independent of the intrinsic and extrinsic pathways (Nakagawa et al., 2000) and appears to be involved in a number of neuropathologies.

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6 Figure 1-1: Apoptotic pathways are induced by a variety of pathological conditions Endoplasmic Reticulum and ER Stress The ER plays a critical role in a number of processes including lipid synthesis, maintenance of intracellular Ca 2+ homeostasis, and the synthesis, initial post-translational modification and proper folding (all calcium dependent) of one-third of all cellular proteins, mostly secretory and transmembrane, as well as their sorting and export for delivery to appropriate cellular destinations (Lodish et al., 2000). Most proteins depend upon their precise tertiary structures to perform their intended functions. Misfolding of these proteins detrimentally affect the cell’s function. The protein folding process is prone to occasional errors and normally is handled by either degrading or refolding the miscreant. If the cell’s ability to degrade or refold abnormal polypeptides is exceeded, the

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7 denatured or partially unfolded molecules accumulate and tend to aggregate. A recent study by Bucciantini and co-workers (2002) demonstrated that when two normally harmless proteins, SH3 domain of bovine phosphatidyl-inositol-3’-kinase and the amino-terminal domain of the E. coli HypF protein, are allowed to aggregate into fibrils, the species that form early in the aggregation process are highly toxic to the cell, while the end product, the fibrils themselves are relatively non-toxic. The high concentrations of these macromolecules in the ER, generally kept under control by molecular chaperones such as BiP (GRP78) which shield the exposed surfaces, can aggravate the problem when the chaperones are unable to handle the excess due to environmental disruptions. Unfolded Protein Response Following BiP (GRP78) Activation There are a number of factors that can cause ER stress and the accumulation of misfolded proteins. It is becoming increasingly evident that the ER function is disturbed in many acute and chronic diseases of the brain (Yuan and Yankner, 2000) such as global and focal ischemia, including the ischemia-reperfusion type (DeGracia et al., 2002), which occurs in stroke and cardiac arrest, and epileptic seizures, Alzheimer’s (AD) (Imaizumi et al., 2001) and Parkinson’s disease, and TBI (Tymianski and Tator, 1996; Weber et al., 2001). ER dysfunction necessitates the activation of the pathway responsible for communicating changes required to modulate the behavior of the cell to allow it to adapt and survive. This pathway is called the unfolded protein response (UPR) (Figure 1-2). UPR stimuli may include glucose or oxygen deprivation, inhibition of protein glycosylation, shift of the lumen from an oxidizing to a reducing environment, increased NO and free radical formation (Liu et al., 2001), acidic and alkaline pH-shifts, disruption of ER-associated protein degradation, and, most notably, the disruption of ER Ca2+ homeostasis leading to ER Ca2+ depletion which is toxic to cells (Nguyen et al.,

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8 2002; Paschen and Doutheil, 1999; Paschen and Frandsen, 2001). Almost every activity of the cell is mediated by Ca2+, an intracellular messenger. To coordinate these functions Figure 1-2: Traumatic injury induces the unfolded protein response. This leads to the cleavage and activation of procaspase-12 by caspase-7 and calpain. Ca 2+ signals need to be flexible yet precisely regulated (Berridge et al., 1998; Berridge et al., 2000). To this end recent evidence suggests that the ER Ca 2+ stores consist of spatially-distinct compartments that can be individually controlled (Blaustein and Golovina, 2001). While high concentrations of cytosolic Ca 2+ can lead to necrosis via the activity of Ca 2+ -sensitive proteases (e.g., calpains) they has also been implicated in apoptosis (Raghupathi et al., 2000). If Ca 2+ stored in the ER is depleted, the mitochondria can play a backup role by absorbing excess calcium from the cytosol and storing it, but it can become overloaded. Excess Ca 2+ in the mitochondria is normally shuttled back to the

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9 ER but becomes toxic, if excessive, initiating the mitochondrial apoptotic pathway (Duchen, 2000; Paschen and Doutheil, 1999; Siesjo et al., 1999). The objective of UPR is to quickly reduce the requirement for ER protein processing and to eliminate the misfolded proteins as rapidly as possible. To achieve this eukaryotic cells have three required compensatory and coordinated responses: 1) degradation, 2) translational attenuation, and 3) transcriptional induction (Mori, 2000). The protein degradation mechanism arranges for the misfolded proteins to be transported out of the ER through a translocon to the cytoplasm where they are ubiquitinated and degraded by the action of the 26s proteasome (Lodish et al., 2000). When the capacity of the ER to cope is saturated, it necessitates the UPR-mediated induction of molecular chaperones and other components required for degradation to remove the polypeptides that fail to fold properly (Travers et al., 2000). This improves the folding and processing efficiency of the system at the same time reducing the flow of proteins into the ER compartment. If the adaptive capacity of the UPR to handle the problem is exceeded the UPR response includes the induction of pro-apoptotic events including the up-regulation of a number of cell death genes and their eventual activation, including caspase-12. The three primary mammalian UPR ER-transmembrane proteins controlling transcription, translation, and apoptosis are PERK (PKR-like ER kinase), IRE1, and ATF6 (Activating Transcription Factor). All three proteins are required for cell survival. Activation of PERK and IRE1 is mediated by their homologous ER-luminal N-terminal sequences. Normally, the luminal domains of each are kept in an inactive state by forming 1 to 1 complexes with the molecular chaperone BiP (grp78) (Bertolotti et al., 2000). When ER-luminal unfolded proteins reach critical levels, BiP disassociates from

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10 PERK and IRE1 to bind the offenders to prevent their aggregation (Bertolotti et al., 2000; Laitusis et al., 1999; Yu et al., 1999). PERK Once PERK is released from BiP it oligomerizes, transautophosphorylates, and once activated phosphorylates the eukaryotic translation initiation factor 2 subunit (eIF2) (Harding et al., 2000; Scheuner et al., 2001). Phosphorylation of eIF2 prevents the assembly of the ribosomal 60S and 40S subunits by interfering with the formation of a 43S initiation complex inhibiting protein translation. Also, by acting through ATF4, PERK induces the transcription factor CHOP (GADD153) which promotes apoptosis (Wang et al., 1998; Zinszner et al., 1998) by down-regulating the expression of the anti-apoptotic protein Bcl-2 and drastically depleting cells of glutathione, the primary intracellular scavenger of reactive oxygen species (McCullough et al., 2001). While the phosphorylation of eIF2 inhibits protein translation during ER stress, there are those mRNAs up-regulated in response to ER-stress conditions that have found other translation routes. Two mechanisms are currently known to be involved. The first is mediated by ribosome binding to an internal ribosome entry site (IRES) element located in the 5 untranslated region bypassing the normally used 5 m 7 G-cap structure. The second entails the assembly of those initiation factors that favor stress related translation (Holcik et al., 2000) initiated through ATF6. ATF6 The second important protein, ATF6, a key UPR transcription factor, has no known pro-apoptotic proclivity (DeGracia et al., 2002). ATF6 is normally synthesized as a 90 kDa ER-resident type II transmembrane protein with its NH 2 -terminal DNA binding

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11 domain facing the cytosol and its COOH terminal in the ER lumen (Haze et al., 1999). ATF6 is also kept in an inactive state by forming at least 1 to 3 complexes with the molecular chaperone BiP. UPR induces the release of BiP allowing ATF6 to transmigrate to the Golgi (Shen et al., 2002) where the Golgi-localized site-1 protease (S1P) and site-2 protease (S2P), the enzymes that process SREBPs in response to cholesterol deprivation, proteolyze ATF6 into its active 50 kDa form (Shen et al., 2002; Ye et al., 2000). This cleavage liberates the N-terminal DNA-binding portion allowing it to migrate to the nucleus to induce expression, in collaboration with other transcription factors (e.g., NF-Y and YY1) (Li et al., 2000), of genes containing the mammalian ER stress-response element (ERSE) (Roy and Lee, 1999). The unique sequence of ERSE consists of 19 nucleotides (CCAAT-N 9 -CCACG) (Roy and Lee, 1999; Yoshida et al., 1998), is commonly present in the promoter regions of UPR target genes and requires two transcription factors. When the general transcription factor NY-Y binds the CCAAT part and ATF6 binds the downstream CCACG element this provides UPR activation specificity (Yoshida et al., 2000). One inducible target gene of ATF6 is the transcription factor X-box protein 1 (XBP-1). XBP-1 protein is processed into a new “chimeric” protein when the activated IRE1 acts as the site-specific endonuclease cleaving a sequence of 26 bases out of the coding region of the mRNA leading to a shift of the open reading frame of the message (Calfon et al., 2002; Lee et al., 2002; Shen et al., 2001). This modified XBP-1 protein then enacts transcription of ERSE target genes including the molecular chaperones such as BiP (GRP78) and PDI.

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12 IRE1and Caspase-12 The functions of the third protein, IRE1 which also oligomerizes and transautophosphorylates when released from BiP, are more complex. Activated IRE1 triggers the apoptosis-associated stress-induced protein Ser/Thr kinases known as SAPKs (stress-activated protein kinases) or JNKs. This is accomplished by recruiting, via its cytoplasmic domain, TRAF2 (tumor necrosis factor receptor-associated factor-2) adaptor (Urano et al., 2000) and JIK (Jun inhibitor kinase) (Yoneda et al., 2001) into a protein complex. JNK activates apoptosis by multiple mechanisms. It phosphorylates p53 which reduces p53 degradation and, since this form is less susceptible to ubiquitination, the resulting accumulation can trigger apoptosis (Fuchs et al., 1998). JNK also induces the Fas ligand and the caspase-8 pathway (Faris et al., 1998). In addition, JNK phosphorylates the major anti-apoptotic proteins Bcl-2 and Bcl-xL, reducing their anti-apoptotic activity (Kharbanda et al., 2000; Srivastava et al., 1999). Finally, by promoting the release of cytochrome c from the mitochondria JNK can activate the intrinsic pathway (Tournier et al., 2000). TRAF2 involvement in the pro-survival pathway is dependent on protein synthesis mediated by activation of NF-B via the interaction with NF-B-inducing kinase (NIK) (Bradley and Pober, 2001; Tada et al., 2001). Activated NF-B has, also, been shown to repress the transcription of the CHOP gene as part of the cellular defense against ER stress-induced apoptosis (Nozaki et al., 2001). TRAF2 also has an important role in the ER stress-induced apoptosis (Yoneda et al., 2001). In unstressed cells TRAF2 forms a stable complex with procaspase-12. The stimuli that induce ER stress and the recruitment

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13 of TRAF2 to IRE1 lead to its disassociation from procaspase-12 promoting the latter’s oligomerization, positioning it for activation. The signaling pathway that initiates ER stress-induced apoptosis appears to be dependent on the ER-associated caspase-12 (Nakagawa et al., 2000). Caspase-12 Though the mouse cDNA sequence for caspase-12 was reported in 1997 (Van de Craen et al., 1997) it was not until early 2000 that Yuan and colleagues (Nakagawa et al., 2000) were able to detail the association of caspase-12 with the ER. Caspase-12 is ubiquitously expressed in mouse tissues including moderate levels in the brain where it is found in cortical neurons, Purkinje cells, and brainstem neurons. They showed that treatment with tunicamycin (inhibitor of ER N-glycosylation), thapsigargin (intracellular Ca 2+ homeostasis disruptor), and A23187 (a Ca 2+ ionophore) triggers apoptosis exclusively through the ER via caspase-12 and that membrane-targeted (Fas activation) or mitochondrial-targeted apoptotic signals (serum deprivation) do not. Caspase-12 has been found to have several roles since it was first discovered to be an ER resident protein. For example the phylogenic clustering, do to sequence homology, of caspase-12 with -1, -4, -5, and -11 suggests that it has a role in the immune or inflammatory responses. Caspase-12 has been implicated in viral infection (Bitko and Barik, 2001) and later was found to be stimulated by IFNbut not by IFNor IFNin fibrosarcoma and melanoma cells. It is interesting that it is IFNin synergy with dsRNA that increased the expression of caspase-12, because normally IFNis considered the antiviral IFN and IFNwas thought to be restricted to immune system cells (Kalai et al., 2003). In the study on increased apoptotic potential in aged skeletal muscle showed that

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14 with a lifelong reduction in calorie intake that the levels of procaspase-12 and cleaved caspase-12 were significantly reduced in rats suggesting improved calcium homeostasis (Dirks and Leeuwenburgh, 2004). Also, in an examination of methamphetamine (METH) it was observed that METH injection was followed by an almost immediate activation in the brain of the proteases calpain and caspase-12. The data suggest that neuronal apoptosis is caused in part by interactions between the ER stress and mitochondrial death pathways and by the activation and cross-talk between caspase-dependent and independent pathways (Jayanthi et al., 2004). It was originally reported that caspase-12 was not functional in humans due to 2 deleterious mutations (Fischer et al., 2002). However new data indicate that 20% of African-Americans have a polymorphism that allows production of the full-length caspase-12 (Saleh et al., 2004). Although these data need to be expanded and clarified, the presence of a functional CASPASE-12 gene in this population subset could predispose this group to an increased pathophysiological response to TBI given that caspase-12-deficient cortical neurons have been shown to be defective in apoptosis induced by amyloidprotein (Nakagawa et al., 2000). TBI is a potent environmental risk factor for development of AD; however, the reasons for this relationship are not clear although one potential explanation involves the amyloid(A) peptide. A accumulation has been reported to be neurotoxic (Yankner et al., 1990) in AD (Yan et al., 1997) and is associated with TBI (Graham et al., 1996; Graham et al., 1999) where damaged axons may serve as a large reservoir and may contribute to A plaque formation following TBI in humans (Smith et al., 2003). A accumulation elicits ER stress and the activation of the UPR pathway leading to caspase-12 activation and apoptotic cell death

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15 (Nakagawa et al., 2000). These findings suggest, similar to apolipoprotein E (APOE4) (Guo et al., 2000; Mayeux et al., 1995; Mayeux et al., 1993; Strittmatter et al., 1993), a CASPASE-12 genetic variant could result in subsequent increased neurodegenerative cascades following TBI in the subset of the population encoding for a full length CASPASE-12 gene. More related to TBI, two recent studies of focal cerebral ischemia in mouse and rat, produced by middle cerebral artery occlusion and the resultant reperfusion injury, offered evidence that stress to the ER leads to the increase in the protein expression of caspase-12 and that it is an important component in the neuronal death following ischemia/reperfusion (Mouw et al., 2003; Shibata et al., 2003). The elements involved in the tissue damage induced by reperfusion include excess free radical production and perturbation of intracellular calcium homeostasis (Paschen and Doutheil, 1999), the factors known to trigger ER stress and UPR. The finding by Shibata and colleagues (2003) that there is increased expression of BiP/grp78 with the same temporal profile as the increased protein expression of the activated caspase-12 strongly suggests that the ER was stressed. These findings complement our study (see Chapter 2 and Larner et al., 2004) suggesting there is a mechanistic link between the ischemic-like conditions that occur following TBI, the increase in caspase-12 expression, and neuronal death in the cortex and hippocampus. Our recently published results showed caspase-12 is up-regulated and processed in rat brains after TBI (see Chapter 2 and Larner et al., 2004). This study examined rat caspase-12 expression using the controlled cortical impact TBI model. Immunoblots of fractionated cell lysates found elevated caspase-12 proform and processed form with

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16 peak induction observed within 24 hours post-injury in the cortex. Hippocampus caspase-12 proform induction peaked at 24 hours post-injury while processed form induction peaked at 6 hours. Semi-quantitative RT-PCR analysis confirmed elevated caspase-12 mRNA levels after TBI. Injury severity of 1.6 mm compression was associated with increased caspase-12 mRNA expression, peaking at 5 days in the cortex and at 6 hours in the hippocampus. Immunohistochemical analysis revealed caspase-12 induction in neurons in both the cortex and hippocampus, as well as in astrocytes at the cortical contusion site. This was the first report of increased expression of caspase-12 following TBI. The time series for our study of cortical mRNA levels ended after 5 days at their highest point. Additional research is required to understand the reason for the continual elevated levels when the protein levels have ostensibly returned to normal and how far into the future it continues to remain elevated and the consequences of these elevated levels. Caspase-7 The three caspase-7 isoforms that have been cloned (Juan et al., 1997) are produced as a catalytically inactive zymogen which must be proteolytically processed to become an active protease. The contribution of caspase-7 to apoptosis remains controversial, but one study did implicate it in the early stages of apoptosis (Korfali et al., 2004). Until very recently, there was a prevailing belief that caspase-7 was not present in the brain (Juan et al., 1997; Ray and Cardone, 2002), not activated if present (Henshall et al., 2002), or activated, but ineffective in neurons and astrocytes (Zhang et al., 2000). The structure of caspase-7 exhibits a high degree of similarity with caspase-3 (Riedl et al., 2001; Wei et al., 2000) though the two share only 54% sequence identity (Juan et al., 1997). Because of this similarity, it was also believed that caspase-7 was redundant in terms of caspase-3,

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17 thus minimizing the role of caspase-7 in the apoptotic cascade. However, the presence of a unique negative electrostatic potential at the S4 region of the catalytic site of caspase-7 versus caspase-3s neutral electrostatic potential may allow this seemingly redundant caspase to act on different substrates than caspase-3, in different cell types, or different cellular compartments. There are at least three known caspase-7 targets that are not shared by caspase-3: caspase-12 (Rao et al., 2001), kinectin (Machleidt et al., 1998), and tumor necrosis factor receptor-I (TNFRI) (Ethell et al., (2001). Recent studies strongly suggest that caspase-7 has an important, non-redundant role in normal physiology and in apoptotic cell death. One study examining caspase-3 deficient mice found no evidence of any compensatory activation of caspase-7 in the CNS following in vivo cerebral ischemia or after in vitro oxygen glucose deprivation (Le et al., 2002). Immunohistochemical data on rat C6 glioma cells also suggests that there are marked differences in the subcellular distribution of caspase-3 and caspase-7 during apoptosis (Meller et al., 2002). In addition, caspase-3 -/mice exhibit neurodegenerative disorders (Kuida et al., 1996), while mice that are caspase-7 -/do not survive in utero (Slee et al., 2001). Given that studies of caspase-7 have shown that it is not redundant (Nicolini et al., 2001; Pompl et al., 2003; Repici et al., 2003; Soung et al., 2003; Suen et al., 2003), the contribution this caspase makes to apoptosis following TBI and to ER stress-induced UPR requires evaluation. Importantly, Rao and colleagues (2001) showed evidence of caspase-12 cleavage and activation by caspase-7 under conditions of ER stress. Our results showed caspase-7 is up-regulated and activated in rat brains after TBI (see Chapter 3). This study examined rat caspase-7 expression using the controlled

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18 cortical impact TBI model. Immunoblots of fractionalized cell lysates found elevated caspase-7 proform, pre-active, and active forms with peak induction observed at 3, 7, and 5 days post-injury, respectively, in the cortex. Hippocampus caspase-7 pre-active and active forms peaked at 5 days post-injury while proform showed no induction. Semi-quantitative RT-PCR analysis confirmed elevated caspase-7 mRNA levels after TBI. Injury severity of 1.6 mm compression was associated with increased caspase-7 mRNA expression, peaking at 5 days in the cortex and day 1 in the hippocampus. Immunohistochemical analysis revealed caspase-7 induction in neurons and astrocytes in both the cortex and hippocampus. This study will be the first report of caspase-7 expression in the brain and activation following TBI. The time series for our study of cortical mRNA levels ended after 5 days at their highest point, additional research is required to understand the reason for the continual elevated levels when the protein levels have ostensibly returned to normal and how far into the future it continues to remain elevated and the consequences of these elevated levels Figure 1-3: Effector caspase-7 with a short prodomain Calpains The other family of cysteine proteases, the cytosolic calcium-activated neutral cysteine endopeptidase referred to as calpains, is found ubiquitously in mammalian cells and has been implicated in both necrosis and apoptosis. The two isoenzymes of calpain (calpain-1 and calpain-2) exist as a pro-enzyme heterodimer (80 kDa-29 kDa) in resting cells but are activated by calcium and autolytic processing to produce a heterodimer of 78

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19 kDa-18 kDa (Wang, 2000). Calpain activation is reliably detected and activated in a variety of CNS injuries including ischemia (Bartus et al., 1994; Rami et al., 2000; Roberts-Lewis et al., 1994; Yokota et al., 1995) and TBI (Kampfl et al., 1996; Newcomb et al., 1997; Pike et al., 1998a; Ringger et al., 2004; Saatman et al., 1996). While early work suggested that calpain was involved solely in necrotic/oncotic cell death, calpain activation has also been implicated in various in vitro models of apoptosis (Nath et al., 1996; Pike et al., 1998b; Squier et al., 1994; Vanags et al., 1996). Moreover, calpain activation is directly associated with apoptotic cell death detected following glucose oxygen deprivation (Newcomb-Fernandez et al., 2001). Following disruption of the ER Ca 2+ homeostasis, calpain is activated and has been shown in one in vitro study to cleave caspase-12 in the presence of millimolar but not micromolar calcium, thus implicating calpain-2 into potentially active fragments (Nakagawa and Yuan, 2000). Previous studies conducted in Dr. R.L. Hayes’ and Dr. K.K.W. Wang’s laboratories have detected both calpain-1 and calpain-2 activity following in vivo TBI (Rink et al., 1995; Zhao et al., 1998). Activation of Caspase-12 by Caspase-7 and Calpains Caspase zymogens require a minimum of two cleavages to be converted to a mature enzyme, one separating the prodomain from the large subunit and small subunit and a second to separate the two subunits. The Yuan laboratory (Nakagawa and Yuan, 2000) showed that milli-calpain (calpain-2) activated by the disruption of the ER Ca 2+ homeostasis, translocates from the cytosol to the ER membrane where is cleaves the ~60-kDa caspase-12 precursor. The two major calpain-2 cleavage sites are T132 and K158 which are between the prodomain and the large subunit (Figure 1-4). The data suggest that the calpain-2-cleaved ~35-kDA caspase-12 fragments are active and may

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20 spontaneously cleave themselves between the large and small subunits at the D318E site to generate the mature caspase-12. In this study the authors, also, suggest that calpain-2 activation by itself is insufficient to induce apoptosis and that caspase-12 plays a key role. This result is particularly interesting since Chua and colleagues (2000) show, even after it had been suggested that calpain was involved in apoptosis, that calpain cleaves caspases-7, -8, and -9 generating proteolytically inactivate fragments preventing the activation of caspase-3 via these routes. Rao and colleagues (2001)demonstrated that caspase-12 could be activated by caspase-7. They showed that ER stress affects the translocation of active cytosolic caspase-7 to the ER surface where it cleaved procaspase-12 at the D94 site generating a 42 kDa fragment (Figure 1-4). A 35 kDa fragment was also discovered suggesting the 42 kDa fragment may represent an active form of caspase-12 and that it may automatically self-cleave generating the 35 kDa fragment. A second cleavage site was also uncovered lying between the large and small subunit at D341 and is presumed to be a second cleavage site for generating the two subunits. The resulting two subunits associate to form an (alpha)2(beta)2-tetramer, two large subunits and two small subunits, which is the active enzyme. Figure 1-4: Traumatic injury induces specific cleavage of pro-caspase-12. Cleavage is accomplished by caspase-7 and calpain at specific cleavage sites. Though little is known about how the activation of caspase-12 leads to the execution of apoptosis a study evaluating the results of thapsigargin treatment to induce

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21 prolonged ER stress demonstrated that caspase-9 co-immunoprecipitated with caspase-12 (Rao et al., 2001). Later work by the same laboratory suggested this ER pathway is mitochondrial and Apaf-1-independent (Rao et al., 2002a). Another investigation reported that caspase-12, in aggregation with Apaf-1 and caspase-9 may result in activation of caspase-7 (Rao et al., 2002b). This report raises the possibility that, under some circumstances caspase-12 could provide feedback activation of caspase-7. A recent work showed that BiP forms a complex with caspase-7 and caspase-12 and prevents release of caspase-12 from the ER. The addition of (d)ATP dissociates this complex and may facilitate movement of caspase-12 into the cytoplasm setting in motion the cytosolic component of the ER stress-induced apoptotic cascade. These results define a novel protective role for BiP in preventing ER stress-induced cell death (Rao et al., 2002b). Calpain and caspase-7 are important activators of caspase-12 and therefore initiate downstream caspase processing, activation, and cell death. It is important if therapeutic interventions are to be considered to understand how the caspase-7/caspase-12 pathway differs from the calpain/caspase-12 pathway as well as the relevance of each of these pathways in ER stress-induced cell death. Studies employing calpain and caspase site-specific antibodies to caspase-12 should prove useful in elucidating these specific pathways.

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CHAPTER 2 INCREASED EXPRESSION AND PROCESSING OF CASPASE-12 AFTER TRAUMATIC BRAIN INJURY IN RATS Note: The work presented in this chapter was published in Journal of Neurochemistry 88, 78-90 (2004). Deborah M. McKinsey assisted with the semi-quantitative RT-PCR analysis. 1 Introduction Traumatic brain injury (TBI) causes progressive neuronal degeneration resulting from acute and delayed cell death that is mediated in part by caspases (Clark et al., 1999; Clark et al., 2000; Yakovlev et al., 1997). Previously, necrosis was thought to be the primary mode of cell death after TBI, but current reports have implicated apoptosis in the neuropathology of TBI (Clark et al., 2000; Colicos et al., 1996; Conti et al., 1998; Newcomb et al., 1999; Rink et al., 1995; Yakovlev et al., 1997). Apoptosis is critical to the sculpting and pruning of the CNS during development (Oppenheim, 1991; Raff et al., 1993; Vaux and Korsmeyer, 1999) and for homeostasis of tissues that require the elimination of aged and abnormal cells (Johnson et al., 1999). However, while apoptosis is normally under strict control, during acute and chronic pathological conditions such as after stroke or TBI, apoptosis can contribute to neuronal death. To date, studies of apoptosis in TBI have concentrated on two caspase-mediated apoptotic pathways, termed the extrinsic and intrinsic pathways (Daniel, 2000; Yakovlev and Faden, 2001). The 1 Larner, S.F., Hayes, R.L., McKinsey, D.M., Pike, B.R., and Wang, K.W.W. (2004). Increased expression and processing of caspase-12 after traumatic brain injury in rats. J. Neurochem. 88, 78-90. Used with permission of the International Society for Neurochemistry and Blackwell Publishing. 22

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23 extrinsic pathway is initiated by the binding of specialized ligands to a family of plasma membrane receptors termed death receptors that results in the activation of caspases-8/10. In contrast, the intrinsic pathway involves the release of cytochrome c from the mitochondria, which in turn activates caspase-9. Both pathways ultimately lead to the activation of caspase-3. Recently, a novel caspase-12-mediated apoptotic pathway has been described that involves endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) (Nakagawa et al., 2000). The protease family of caspases plays a key role in the implementation of apoptosis in vertebrates (Jacobson et al., 1997). They are constitutively expressed as precursor proteins (pro-caspases) and are believed to have little or no enzymatic activity. Once activated, they are processed into large subdomains of approximately 20 kDa (p20) and small subdomains of approximately 10 kDa (p10) (Cohen, 1997), that form heterotetramers, which possess enzymatic activity. The caspase family has been divided into two broad functional categories: initiator caspases (caspase-8, -9, -10, and -12) and effector caspases (caspase-3, -6, and -7). The initiator caspases are upstream in the apoptotic pathway and respond to apoptotic stimuli by undergoing autoproteolytic activation. The downstream effector caspases are processed by the active initiator caspases and are responsible for dismantling cellular structure. Recent studies have shown that caspase-12 functions as an initiator caspase and has been implicated in ER stress-induced apoptosis (Nakagawa et al., 2000; Rao et al., 2001; Rao et al., 2002b; Yoneda et al., 2001). The ER plays a critical role in a variety of processes including protein synthesis and folding, and the maintenance of Ca 2+ homeostasis. A number of recent studies

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24 demonstrate that ER stress causes the disruption of these and other normal functions, thus the ER has garnered increased interest for its putative role in cellular pathology. In addition, the disruption of ER homeostasis is considered a causal factor in pathologically relevant apoptosis and has been implicated in several neurodegenerative disorders (Aridor and Balch, 1999; Soto, 2003). For example, disruption of Ca 2+ homeostasis leads to increased levels of cytosolic Ca 2+ , which induce pathological activation of calpains after TBI (Pike et al., 1998a), and the translocation of calpains to the ER surface where they can activate caspase-12 (Nakagawa and Yuan, 2000). Recent attention has centered on the role of caspase-12 and its unique functional characteristic of being specifically activated by ER stress. For example, Nakagawa and colleagues (Nakagawa et al., 2000) have shown that caspase-12 deficient cells are resistant to inducers of ER stress-induced apoptosis such as brefeldin-A, tunicamycin, and thapsigargin. The molecular mechanisms by which caspase-12 mediates apoptosis are still under investigation, but recent work suggests that it functions by cleaving pro-caspase-9 without the involvement of cytochrome c and the apoptosome (Morishima et al., 2002; Rao et al., 2001; Rao et al., 2002a). The hypothesized pathway of caspase activation in response to ER stress is active caspase-12 activates caspase-9, which in turn, activates caspase-3 (Morishima et al., 2002; Rao et al., 2001; Rao et al., 2002a). Importantly, this hypothesis suggests a putative therapeutic target that is temporally upstream of those focused on attenuating the mitochondrial-mediated (intrinsic) or extrinsic pathway. This study tests the hypothesis that TBI induces increased expression of caspase-12 mRNA and protein levels that are related to injury severity in the cortex and hippocampus of rats and that this expression is found primarily in neurons. Using a

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25 rodent model of lateral controlled cortical impact injury, this investigation characterized the time course for induction of caspase-12 mRNA, upregulation of the zymogen, and cleavage of caspase-12 into its processed and alleged active form. Induction of caspase-12 mRNA was observed with semi-quantitative real-time PCR analysis within 6 hours of TBI in the hippocampus and within 3 days in the cortex. Immunoblot analyses of the caspase-12 proform (~60 kDa) and processed form (~12 kDa) showed increased protein expression within 6 hours as well. Immunochemical analyses revealed that caspase-12 was induced in neurons in both the ipsilateral cortex and hippocampus. There was also evidence that caspase-12 was upregulated in astrocytes at the contusion site. Our findings demonstrate that caspase-12 is rapidly induced and processed after TBI suggesting that caspase-12 may be an important upstream mediator and part of a newly discovered pathway leading to apoptosis following TBI. Results Immunoblot Analysis of Caspase-12 Expression after Traumatic Injury: Total cellular fractions, less nuclear and mitochondrial components as described above for the 1.6mm cortical impact injury, were prepared from rat ipsilateral cortex and hippocampus to test the results of 1 of the 3 injury levels examined in the mRNA expression experiments. Caspase-12 expression was examined using the anti-caspase-12-specific antibody (kind gift from Drs. Michael Kalai and Peter Vandenabeele, Flanders Interuniversity Institute for Biotechnology, University of Ghent, Ghent, Belgium), quantified by densitometric analysis, and expressed as percent of naive control levels adjusted for the sham effect (Figures 2-1 and 2-2). Figures 2-1A and 2-2A show immunoblot analyses for caspase-12 expression in rat ipsilateral cortex and hippocampus, respectively, for nave and sham-injured control animals, and for TBI (1.6 mm controlled

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26 cortical impact) animals from 6 hours to 14 days post-injury (n = 4 for each time point). Figure 2-1: Immunoblot analysis of proand cleaved caspase-12 expression in ipsilateral cortex. (A) Protein samples collected from nave (N) rats to serve as injury controls, sham-operated rats 1 and 7 days after surgery (S1, S7), and injured rats 6 hours to 14 days after injury (6h, 1d, 3d, 5d, 7d, and 14d). -actin was directly assessed as an internal methods control. (B) Quantitative analysis of immunoblots were quantified by densitometry and the caspase-12 (proand cleaved) expression levels in the ipsilateral cortex of injured animals after adjustment for the sham effect were calculated as a percentage of nave control caspase-12 expression and were statistically significant for pro-casp12 (## p< 0.01) and cleaved casp12 (** p< 0.01). Using the anti-caspase-12 antibody (kind gift from Drs. Michael Kalai and Peter Vandenabeele, Flanders Interuniversity Institute for Biotechnology, University of Ghent, Ghent, Belgium), 2 distinct protein bands were identified: an approximately 60 kDa band corresponding to the zymogen (Pro-Casp12) and an approximately 12 kDa band that

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27 represents the small subunit of the active form of caspase-12 (Cleaved Casp12) (Rao et al., 2001). Immunoblots were also run with equivalent protein amounts and probed with the anti--actin antibody, which served as an internal control for protein loading and transfer. Loading and transfer were essentially equivalent in all wells, as shown by comparable 42-kDa signal intensities. Figure 2-2: Immunoblot analysis of proand cleaved caspase-12 expression in ipsilateral hippocampus. (A) Protein samples were collected from nave (N) rats to serve as injury controls, sham-operated rats 1 and 7 days after surgery (S1, S7), and injured rats 6 hours to 14 days after injury (6h, 1d, 3d, 5d, 7d, and 14d). -actin was directly assessed as an internal methods control. (B) Quantitative analysis of immunoblots were quantified by densitometry and the caspase-12 (proand cleaved) expression levels in the ipsilateral hippocampus of injured animals were calculated after adjustment for the sham effect as a percentage of nave control caspase-12 expression and were statistically significant for pro-casp12 (# p< 0.05, ## p< 0.01) and for cleaved casp12 (** p< 0.01).

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28 Immunoblot analysis of caspase-12 expression in cortex after traumatic injury Tissue from sham-operated animals showed a modest increase in caspase-12 proform expression 1 day after craniotomy that was not statistically significant and declined to nave levels by 7 days (Figure 2-1A). After adjustment for sham effect, significant induction of the caspase-12 proform was observed within 6 hours of cortical injury (p<0.01) and peaked on day 1 (418% 30%, p<0.01). Elevated expression was also detected 3 days post-injury before declining to nave levels by 7 days (Figure 2-1B, p<0.01). Increased levels of processed caspase-12 were also observed within 6 hours after cortical injury, with peak expression 1 day after injury (503% 40%, p<0.01). Elevated expression levels continued to be statistically significant up to 3 days following injury before declining to near nave levels (Figure 2-1B). One-way ANOVA with Dunnett’s multiple comparison tests was performed to evaluate statistical significance. Immunoblot analysis of caspase-12 expression in hippocampus after traumatic injury Hippocampi from sham-injured animals showed a slight increase in proform and processed caspase-12 expression 1 day after craniotomy that was not statistically significant and declined to nave levels by 7 days (Figure 2-2A). After adjustment for the sham effect, elevated levels of caspase-12 induction for both the proform (p<0.01) and processed form (p<0.01) were observed within 6 hours of TBI. In fact, levels of processed caspase-12 expression peaked at this early time point (620% 46%) and remained significantly elevated 1-day post injury (Figure 2-2B). Pro-caspase-12 induction peaked later at 1 day after injury (641% 124%) and expression remained significantly elevated at 3 and 5 days following injury (Figure 2-2B). Compared to the ipsilateral cortex, the ipsilateral hippocampus possessed greater caspase-12 protein

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29 induction over nave controls for both the proform and the processed form. In addition, peak induction of the processed form occurred earlier in the hippocampus compared to the cortex (6 hours versus 1 day). One-way ANOVA with Dunnett’s multiple comparison tests was performed to evaluate statistical significance. Standard Curve Generation for Semi-Quantitative RT-PCR Using Serially Diluted cDNA The highest levels of caspase-12 transcript expression were observed in ipsilateral cortex and hippocampus from 6 hours to 1 day after injury (Figure 2-3). Therefore, total RNA was collected 6 hours and 1 day after injury from the ipsilateral cortex and hippocampus, respectively. Total RNA was extracted from rat ipsilateral hippocampal (6 hours post-injury) and cortical (1 day post-injury) tissue for caspase-12 expression and from ipsilateral cortical tissue (3 days post-injury) for GAPDH expression. RNA samples were reverse transcribed and the cDNAs were serially diluted. Aliquots sample (100%, 33.3%, 11.1%, and 3.7%) of the cDNA underwent real-time PCR using primer pairs for caspase-12 mRNA or GAPDH mRNA. For each dilution and each primer set, the cycle number at which the PCR amplification entered the log-linear region was identified (crossing point cycle number). Standard curves were generated by plotting the log concentration of total mRNA versus the crossing point cycle number. A linear regression analysis was performed, the r 2 ranged from 0.9895 to 1.000. For quantitation, mRNA samples from nave, sham, and injured rats underwent real-time PCR, generating a crossing point cycle number for each primer set. Using the standard curves, the cycle number was converted to an amount of mRNA. These amounts are expressed as a percentage of sham control mRNA (=100%).

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30 Figure 2-3 shows the linear regression analysis of each primer set’s crossing point cycle number for each brain region versus the logarithm of the dilution factor. For each primer set, the range of crossing point cycle numbers required to cover the serially diluted standard curve varied: 14-21 cycles for GAPDH, 25-29 cycles for caspase-12 (cortex), and 27-31 cycles for caspase-12 (hippocampus). These differences primarily reflect the abundance of the transcripts. GAPDH mRNA was the most abundant transcript requiring the fewest cycles, whereas caspase-12 hippocampal mRNA was the least abundant transcript, and therefore required the most cycles. Figure 2-3: Standard curve generation for caspase-12 and GAPDH semi-quantitative real-time PCR. The standard curve generation was performed using serially-diluted cDNA. For each dilution and each primer set, the cycle number at which the PCR amplification entered the log-linear region was identified (crossing point cycle number). Standard curves were generated by plotting the log concentration of total RNA versus the crossing point cycle number. Semi-quantitative PCR Analysis of Experimental Samples Using the standard curves generated as described above, the crossing point cycle numbers were converted to relative amounts of mRNA. These relative amounts were then expressed as percent of naive control adjusted for the sham effect. Figure 2-4 shows the

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31 time course of caspase-12 mRNA expression in ipsilateral cortex and hippocampus after cortical injury for 3 magnitudes of injury. The data noticeably convey the similarities in the upregulation of mRNA levels following injury and illustrate the effect of injury severity on caspase-12 mRNA expression. Figure 2-4: Semi-quantitative real-time PCR analysis of caspase-12 mRNA expression. mRNA in (A) cortex and (B) hippocampus levels after adjustment for sham effect are expressed as a percentage of nave control Values are mean and SEM and one-way ANOVA with Dunnet’s multiple comparison test was performed to evaluate statistical significance (n = 3; *p<0.05, **p<0.01). In the cortex, maximal and statistically significant caspase-12 mRNA expression was observed for all 3 injury magnitudes 5 days post-injury: 1.0 mm 657% 119% (n=3, p<0.01); 1.2 mm 651% 65% (n=3, p<0.05); and 1.6 mm – 1,259% 465% (n=3, p<0.01). The 1.2 mm and 1.6 mm injury magnitudes reached significant increases in mRNA expression (p<0.05 and p<0.01, respectively) three days after injury. However, in the hippocampus, maximal and significant caspase-12 mRNA levels were observed at 6 hours for all 3 injury magnitudes: 1.0 mm 435% 98% (n=3, p<0.01); 1.2 mm 451% 10% (n=3, p<0.05); and 1.6 mm 460% 36% (n=3, p<0.01). Significantly elevated expression was detected at the 1-day (1.0 and 1.6 mm injury, p<0.05 and p<0.01, respectively) and 3-day (1.6 mm injury, p<0.05) time points.

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32 Immunohistochemical Analysis of Caspase-12 Expression after Traumatic Injury Ipsilateral and contralateral cortical and hippocampal tissues were examined for caspase-12 induction 1 day following TBI (1.6 mm controlled cortical impact injury). High magnification photomicrographs of the Alexa Fluor stains of uninjured, nave, animals revealed healthy cell bodies and little detectable caspase-12 expression (Figure 2-5A, panel A and Figure 2-6A, panel A). In contrast, one day following injury, caspase-12 expression was readily observed in both the ipsilateral cortex and hippocampus with significant levels in the former immediately below the impact site (Figure 2-5, panels B and F; Figure 2-6, panels B and F). Immunohistochemical analysis of cortical caspase-12 expression after trauma The ipsilateral cortex at the site of the contusion revealed a considerable increase in caspase-12 expression with decreasing levels distal to the site of impact. The morphology of the injury site where the highest levels of caspase-12 induction was located had a decidedly disorganized, almost chaotic appearance when compared to the contralateral and nave tissue. Caspase-12-immunopositive neurons (Figure 2-5A, panel E) included those cells with apoptotic bodies (Figure 2-5A, panel D). It was also clear that caspase-12 was induced in astrocytes at the site of the contusion (Figure 2-5B, panel I) but could not be specifically identified in astrocytes distal to this location. Immunohistochemical analysis of hippocampal caspase-12 expression after trauma The ipsilateral hippocampus revealed induction of caspase-12 with caspase-12-immunopositive cells co-localizing with neuronal cell-specific marker NeuN (Figure 2-6A, panel E) but not with the astrocytic marker GFAP (Figure 2-6B, panel I). NeuN stained neuronal cells (Figure 2-6A, Panel C) shows no evidence of morphopathology in the hippocampus as compared to the cortex.

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33 Figure 2-5: Immunohistochemical analysis of the ipsilateral cortex. (A) Nave brain tissue shows no caspase-12-immunoreactivity (Panel A). Apoptotic bodies revealed via DAPI staining (arrows, Panel D, see insert) co-localize with caspase-12 and the neuronal marker NeuN (arrows, Panel E). Caspase-12 also co-localizes with NeuN without apoptotic bodies (curved arrows, Panel E). (B) Caspase-12 is found in cells labeled with the astrocytic marker GFAP (arrowheads, Panel I) and without (arrows, Panel I). Cells showing apoptotic bodies (arrow, Panel H, see insert). There were astrocytes examined (curved arrow, Panel I) that did not show caspase-12. Photomicrographs 400x; scale bar 20 m; caspase-12 – panels A, B, and F; NeuN – panel C; GFAP – panel G; DAPI – panels D and H; co-localized images – panels E and I.

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34 Figure 2-6: Immunohistochemical analysis of the ipsilateral hippocampus. (A) Nave tissue shows no caspase-12 immunoreactivity (Panel A) while injured brain tissue revealed caspase-12 positive cells (Panel B). Caspase-12 co-localizes with the neuronal cell marker NeuN (arrows, Panel E). (B) Caspase-12 does not appear to co-localize with the astrocytic marker GFAP (arrows, Panel I). Photomicrographs 400x; scale bar 20 m; caspase-12 – panels A, B, and F; NeuN – panel C; GFAP – panel G; DAPI – panels D and H; co-localized images – panels E and I.

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35 Discussion This is the first study to show increased expression of caspase-12 mRNA in injured cortex and hippocampus after mild to severe traumatic brain injury in rats and increased caspase-12 protein expression and processing after severe injury. The study also shows evidence that the increased caspase-12 expression after TBI is found, predominantly, in neurons in the ipsilateral cortex and hippocampus but also in cortical astrocytes located at the site of the contusion. In the ipsilateral cortex, immunoblot analysis revealed that levels of both the zymogen and the processed form of caspase-12 protein rapidly increased within 6 hours of injury, peaking by about 1 day after injury when compared to nave control. This was followed by a slow decline over the next 2 weeks with protein levels returning to nave levels by day 14. In the ipsilateral hippocampus, caspase-12 protein expression increased rapidly within the first 6 hours where expression peaked for the processed form. Levels of the proform peaked later, at 1-day post-injury when compared to nave control. The increase in protein expression was more robust in the hippocampus than in the cortex when compared to naive. Immunohistochemical analysis demonstrated that caspase-12 immunoreactivity was considerably increased 24 hours following injury, when compared to nave animals, and was found primarily in neurons at the site of the contusion and, to a lesser extent, in areas distal to the site of impact, including the hippocampus (Figures 2-5 and 2-6). In addition, GFAP labeled astrocytes expressing caspase-12 could be found at the contusion site as well (Figure 2-5B). However, no astrocytes located distal to this area appeared to show evidence of caspase-12 immunoreactivity. Examination of the NeuN stained neuronal brain tissue at the contusion location, in cells displaying caspase-12 immunoreactivity, showed evidence of pronounced morphological changes, when

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36 compared to the contralateral side or in nave animal brain tissue (results not shown). These observations further confirm the previous findings by our laboratory that morphopathological changes occur within 24 hours following TBI (Newcomb et al., 1997; Newcomb et al., 1999). Previous studies of TBI have concentrated on the well-characterized extrinsic and intrinsic pathways that were found to be involved in neuronal cell death after TBI (Keane et al., 2001). The extrinsic apoptotic pathway is triggered by binding of specialized ligands to death domain-containing members of the tumor necrosis factor receptor (TNFR) superfamily, such as Fas and tumor necrosis factor receptor-1 (TNFR-1), allowing for the formation of a death-inducing signaling complex (DISC). Increased expression and interaction of the Fas receptors with FasL ligands (Beer et al., 2000; Qiu et al., 2002) and TNFR-1 with TNF (Beer et al., 2000; Taupin et al., 1993) have been demonstrated in TBI. The Fas DISC containing the adaptor protein Fas-associated death domain protein (FADD), and the TNFR-1 DISC containing the TNF-associated receptor with death domain (TRADD), and their respective procaspases-8 and -10, leads to the autoproteolytic processing of these zymogens, initiating the subsequent activation of procaspase-3. The intrinsic pathway involves alterations in mitochondria homeostasis and plays a key role in TBI-associated cell death (Raghupathi et al., 2000; Xiong et al., 1997; Yang et al., 1985). These disruptions generally involve the release of cytochrome c and the formation of the apoptosome consisting of Apaf-1, dATP, cytochrome c, and the recruitment and activation of caspase-9. Procaspase-9, generally believed to reside in and released from the mitochondria, is activated during apoptosis, and then activates the executioner procaspase-3.

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37 Recent studies have challenged the exclusivity of these two pathways with the discovery that apoptosis is independently induced by an ER stress pathway (Nakagawa et al., 2000; Rao et al., 2001; Rao et al., 2002b; Yoneda et al., 2001). The ER is the site for a number of critical processes including lipid synthesis, maintenance of intracellular Ca 2+ homeostasis, and the synthesis, initial post-translation modification, and proper Ca 2+ dependent folding of most secretory and transmembrane proteins. This includes their sorting and export for delivery to appropriate cellular destinations (Aridor and Balch, 1999; Kaufman, 1999; Paschen and Doutheil, 1999). ER functions are disturbed in many acute and chronic diseases of the brain (Yuan and Yankner, 2000) such as global and focal ischemia, ischemia-reperfusion injury that occurs during stroke and cardiac arrest (DeGracia et al., 2002), epileptic seizures (Henshall et al., 2000; Pelletier et al., 1999), Alzheimer’s disease (Imaizumi et al., 2001), and TBI (Weber et al., 2001). A variety of stimuli including changes in the luminal environment (Nakamura et al., 2000), glucose deprivation, oxidative stress, and the disruption of homeostasis and release of Ca 2+ from the ER, may induce ER stress and subsequently induce apoptosis (Mattson et al., 2000; Nakagawa et al., 2000; Weber et al., 2001; Yu et al., 1999). Any one of these conditions will result in an increase of unfolded or malfolded proteins and the induction of the ER molecular chaperone BiP/GRP78 (Bertolotti et al., 2000; Yu et al., 1999). The induction of BiP/GRP78 triggers the unfolded protein response. Once activated, the UPR pathway is responsible for communicating changes required to modulate the behavior of the cell allowing it to adapt and survive or to commit apoptosis. The 3 stressor proteins found in the ER membrane that generate the response include PERK (also known as PEK), ATF6,

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38 and IRE1. Caspase-12 appears to act within the UPR pathway as a key component of the reaction of IRE1 to trauma and the increase of unfolded or malfolded proteins. The functions of activated IRE1, which oligomerizes and transautophosphorylates when released from BiP/GRP78, are complex. Activated IRE1 recruits, via its cytoplasmic domain, JIK (Jun inhibitor kinase) (Yoneda et al., 2001) into a protein complex with cytosolic adaptor protein TRAF2 (tumor necrosis factor receptor-associated factor-2). TRAF2 is involved in a pro-survival pathway that is dependent on protein synthesis, mediated by activation of NF-B (Natoli et al., 1997) through the interaction with NF-B-inducing kinase (NIK) (Bradley and Pober, 2001; Tada et al., 2001). The activation of IRE1 stimulates the release of TRAF2 from its stable complex with procaspase-12 (Yoneda et al., 2001). Caspase zymogens require a minimum of 2 cleavages to be converted to a mature enzyme, one separating the prodomain from the large subunit and small subunit and a second to separate the 2 subunits. Once released from TRAF2, procaspase-12, which is found on the cytoplasmic side of the ER, dimerizes in preparation for the initial cleavage by the proteases, calpain (Nakagawa and Yuan, 2000) and/or caspase-7 (Rao et al., 2001). In a previous in vitro cell free (caspase buffer only) study (Van de Craen et al., 1999) using a purified and truncated form of procaspase-12 (~ 30 kDa), caspase-12 was found to be cleaved by both caspase-3 and caspase-7. Bredesen’s laboratory (Rao et al., 2001) found that the full length, intact procaspase-12, used in an in vitro cell culture model, was cleaved only by caspase-7 and not caspase-3. Calpain has been shown to be activated early in TBI (Pike et al., 1998a). When activated, both proteases are attracted to the ER membrane by some yet unknown mechanism. The processed form of caspase-12 then

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39 autoprocesses into the p20 and p10 subdomains and the resulting 2 subunits associate to form an (alpha)2(beta)2-tetramer which is the active enzyme. The activation of caspase-12 appears to lead to cellular apoptosis mediated by caspase-9 (Morishima et al., 2002; Rao et al., 2001; Rao et al., 2002a) and caspase-3. In contrast to rodent studies, a recent report suggests the human caspase-12 sequence has acquired deleterious mutations rendering it nonfunctional (Fischer et al., 2002). The study revealed that all splice variants examined had a frame shift mutation and a premature stop codon which would preclude expression of a full length protein. The evidence also points to a loss-of-function mutation within the SHG box, a critical catalytic site in caspases. On the other hand, the authors mentioned a caspase-12 allele residing in the SNP database (dbSNP Acc rs#648264) that restores the open reading frame, suggesting that a full length human caspase-12 allele may exist. The study was limited to genetic expression; as a result no protein expression analysis was done to confirm their conclusions. This analysis is important because currently there are at least 8 human cell lines with evidence for the presence of caspase-12 protein. These cell lines include HeLa cells (Nakagawa et al., 2000), HEK293T human embryonic kidney cells (Rao et al., 2001; Yoneda et al., 2001), A549 human lung epithelial cells (Bitko and Barik, 2001), T-ALL CCRF-CEM (subclone CEM-C7H2 vector control cell line – C7H2-VC) human T-cell acute lymphoblastic leukemia cells (Tinhofer et al., 2002), human fas-expressing L929sA cells (L929sAhFas) (Kalai et al., 2003), Huh7 human liver-derived cells (Xie et al., 2002), normal human mammary epithelial cells (HMEC) (Sergeev, 2004), and human breast carcinoma cells (MCF-7) (Sergeev, 2004). Caspase-12 was also observed in humans afflicted with sporadic and variant Creutzfeldt-Jakob

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40 disease, a misfolded prion protein pathology (Hetz et al., 2003). A second study recently reported that a full-length CASPASE-12 gene was present in humans but only in approximately 20% of Americans of African descent (Saleh et al., 2004). These differences will need to be clarified by additional research before human TBI can be interpreted from rodent TBI. Two recent studies of focal cerebral ischemia in mouse and rat, produced by middle cerebral artery occlusion and the resultant reperfusion injury, offered new evidence that stress to the ER leads to the increase in the protein expression of caspase-12 and that it is an important component in the neuronal death following ischemia/reperfusion (Mouw et al., 2003; Shibata et al., 2003). The elements involved in the tissue damage induced by reperfusion include excess free radical production and perturbation of intracellular calcium homeostasis (Paschen and Doutheil, 1999), the factors known to trigger ER stress and UPR. The finding by Shibata and colleagues (2003) that there is increased expression of BiP/GRP78 with the same temporal profile as the increased protein expression of the activated caspase-12 strongly suggests that the ER has been stressed. In addition they detected DNA fragmentation by TUNEL, a characteristic indication of the occurrence of apoptosis, in many of the caspase-12 positive cells. By utilization of semi-quantitative RT-PCR Mouw and colleagues (2003) observed a significant increase in caspase-12 mRNA in the ipsilateral side of the striatum following ischemia. Both studies found that the increase in caspase-12 protein expression was localized mainly in the ipsilateral striatal neurons. These findings complement our study suggesting there is a mechanistic link between the ischemic-like conditions that occur following TBI, the increase in caspase-12 expression, and neuronal death in the cortex and hippocampus.

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41 The Nakagawa and colleagues (2000) study showed that caspase-12 is an important component of apoptosis induced by the amyloidprotein as well as other ER stress inducing signals. TBI has been implicated in the pathogenesis of Alzheimer’s disease (AD) in aging suggesting that the induction by amyloidprotein of caspase-12 may contribute to the epidemiological observation that a subset of patients who experience a TBI are at greater risk for developing AD. Previous studies also found that patients with the apolipoprotein E-4 (APOE4) allele are at higher risk for developing AD than other genotypes (Saunders et al., 1993). When an elderly population was examined for the risks of AD it was discovered that those with the APOE4 allele had a two-fold increase in risk of developing AD. But the combination of the allele with a history of TBI increased the associated risk ten-fold (Mayeux et al., 1995). Follow up studies showed that the APOE4 frequency expression is higher in patients with amyloid -protein deposition after head injury than in head-injured patients without amyloid -protein deposition (Graham et al., 1999; Nicoll et al., 1995). Although the pathophysiological mechanism linking head trauma and AD has yet to be elucidated, a recent study using the PDAPP mouse model for AD (transgenic mice that develop AD-like pathology) suggested that APOE4 influences the amount of amyloid deposition by reducing the amount of amyloid clearance after TBI (Hartman et al., 2002). This finding complements the study done by Smith and colleagues (2003) that suggested a mechanistic link between TBI and amyloidlevels and neuronal death selectively in the hippocampus. Furthermore caspase-12 deficient cortical neurons are partially protected from apoptosis induced by amyloid-(Nakagawa et al., 2000).

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42 Caspase-12 protein induction, in our study, was further supported by semi-quantitative PCR transcript analysis. In the cortex mRNA transcripts showed statistically significant elevation within 3 days of injury and remained elevated for at least 5 days. The delayed and sustained induction of caspase-12 mRNA suggests the impairment of cellular mechanisms. The cortex may be most vulnerable to this disruption due to its proximity to the injury site. There are several possibilities that are not mutually exclusive. First, it suggests that caspase-12 is involved in protracted apoptosis as well as acute cell death. This is in keeping with proposal by Rao and co-workers (2001) that prolonged ER stress may induce further caspase-12 activation via caspase-7, contributing to a delayed but continuing apoptosis through its interaction with caspase-9 (Morishima et al., 2002; Rao et al., 2001; Rao et al., 2002a). Second, the normal cellular protein metabolism is most likely impaired. One of the responses of UPR, in response to ER stress, is for there to be a general global inhibition of protein synthesis following the activation of PERK (DeGracia et al., 2002; Harding et al., 2000; Harding et al., 1999) with exceptions for proteins that will either return the cell to normal activity or commit the cell to apoptosis (Kohno et al., 1993; Mori et al., 1992; Natoli et al., 1997; Yoshida et al., 1998). There is no evidence to suggest that UPR interferes with mRNA transcription. In addition, there may also be either a decrease in the normal degradation of proteins including transcription factors, due to the lack of sufficient amounts of the protein ubiquitin because of the inhibition of protein synthesis, or the process may be overloaded by the UPR as the cell attempts to reduce its backlog of unfolded and malfolded proteins engendered by the ER stress initiated by TBI. A third possibility is that while the transcription of the mRNA continues in response to UPR the proform is not being

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43 transcribed nor is it being processed to its active form, as the cells regain control over their cellular metabolism. Our study shows the proform returning to normal levels within 7 days, caspase-12 mRNA may follow later. This remains to be determined by future investigations. The early responses provide compelling evidence that caspase-12 mRNA induction and increased protein expression occur in response to TBI and that caspase-12 may be a major contributor to the more acute pathophysiological events of TBI. In previous studies, caspase-12 has been characterized as an initiator, or upstream, caspase (Rao et al., 2001; Rao et al., 2002a). The actual mechanisms and consequences of caspase-12 induction and activation in TBI remain to be examined. In summary this study provides the first characterization of caspase-12 mRNA and protein expression and processing in a model of TBI. These findings provide insight into a novel mechanism of cell death following injury. These data also provide clues into the potential ways TBI might contribute to the pathogenesis of a number of neuropathologies through the caspase-12 apoptotic pathway.

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CHAPTER 3 CASPASE-7: INCREASED EXPRESSION AND ACTIVATION AFTER TRAUMATIC BRAIN INJURY IN RATS Note: The work presented in this chapter is being prepared for publication. Deborah M. McKinsey assisted with the semi-quantitative RT-PCR analysis. Introduction Traumatic brain injury (TBI) is a serious health issue in the United States as well as other nations. According to the Centers for Disease Control and Prevention, USA, traumatic brain injury is frequently referred to as the silent epidemic because the pathologies that result tend to be emotional and cognitive in nature (e.g., impaired memory, change in character traits, difficulty in concentrating) rather than physical (Gerberding, 2003). The tragedy is that despite about 2% of the United States population suffering from some form of TBI related disability there is currently no known pharmacological treatment available (NIH, 1998). TBI causes progressive neuronal degeneration resulting from acute and delayed cell death that our laboratory as well as others have determined is mediated in part by calpains (Kampfl et al., 1996) and in part by apoptotic inducing caspases (Clark et al., 2000; Colicos et al., 1996; Conti et al., 1998; Newcomb et al., 1999; Rink et al., 1995; Yakovlev et al., 1997). Programmed cell death, apoptosis, a conserved active molecular process, often requires active transcription and translation of proteins for initiation of the molecular program (Rink et al., 1995). Apoptosis as a genetically controlled program is characterized by proteolysis of cellular components and is critical to the pruning of the 44

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45 CNS during development (Oppenheim, 1991; Raff et al., 1993; Vaux and Korsmeyer, 1999) and for homeostasis of tissues that require the elimination of aged and abnormal cells (Johnson et al., 1999). This regulated program allows multicellular organisms to tightly control cell numbers and tissue size, and protect against rogue cells that threaten homeostasis. Though apoptosis is under strict control under normal conditions, alterations in the apoptotic pathways have been implicated in many diseases, such as cancer and neurodegenerative disorders (Thompson, 1995; Yuan and Yankner, 2000) like the pathological conditions that occur after stroke or TBI where apoptosis contributes to neuronal cell death. The apoptotic mechanism is a conserved process found across species and is associated with the sequential activating cascade of a family of cysteine-dependent aspartate-specific proteinases known as caspases. All caspases are translated as inactive zymogens that must be proteolytically processed to become active (Cohen, 1997) with perhaps the exception of caspase-9 (Rodriguez and Lazebnik, 1999; Stennicke et al., 1999). Caspases can be divided into two broad groups. The upstream initiator caspases (including 2, 8, 9, 10, and 12) are triggered by cofactor-mediated transactivation that activate the downstream effector or executioner caspases (3, 6, and 7). The three executioners cleave distinct intracellular substrates that promote the characteristic apoptotic morphology. Caspase-7, along with caspase-3 and -6, contain short prodomains. Caspase-7 is structurally and functionally most similar to caspase-3 (Juan et al., 1997). The activation of the effector caspase-7, upon induction of apoptosis, is performed by the proteolytical cleavage of the proform (35 kDa) by the initiator caspase-9 by first converting the proform into a 32 kDa intermediate or pre-active form, which is then further processed

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46 into the two active subunits consisting of the p20 or large (18 kDa) subunit and the p10, or small (11 kDa) subunit (Swiss-Prot P55210; (Duan et al., 1996; Wolf and Green, 1999). Active caspase-7 has been shown to cleave the nuclear substrate PARP (Germain et al., 1999), kinectin (Machleidt et al., 1998), and caspase-12 (Rao et al., 2001). The current paradigm is that caspase-7, mRNA and protein, is either not present in the brain (Juan et al., 1997; Ray, 2002) or it has little impact (Henshall et al., 2002; Le et al., 2002; Slee et al., 2001; Zhang et al., 2000). It is our hypothesis that caspase-7 is present in the brain and is up-regulated and activated following traumatic injury. Results In Vitro Model: Confirmed Presence of Caspase-7 Protein in PC12 Cells Caspase-7 has frequently been characterized as redundant to the more studied caspase-3. It has been suggested that caspase-3 is the primary executioner of the three effector caspases while caspase-7 plays a minor if undetectable role especially in the brain. Although the two caspases only share a 53% homology (67% similarity) (Fernandes-Alnemri et al., 1995; Juan et al., 1997), to confirm that anti-caspase-7 antibody selectively binds to the caspase-7 and not to caspase-3, anti-caspase-7 and anti-caspase-3 antibodies were tested against human caspase-7 and caspase-3 recombinant proteins. As Figure 3-1 illustrates the anti-caspase-7 antibody is discriminatory in that there is no crossover effect between the two recombinant proteins. This suggests that the antibody for caspase-7 selectively distinguishes caspase-7 from caspase-3, the protein it is most closely related. Previous studies have established that the rat neuronal pheochromocytoma cell line (PC12) is a convenient cell model of sympathetic neurons and has proven useful in studies of apoptotic signaling pathways (Edsall et al., 2001; Haviv et al., 1998). In

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47 Figure 3-1. Confirmation of anti-caspase-7 antibody specificity. Antibodies to caspase-7 and caspase-3 were tested against caspase-7 and caspase-3 human recombinant proteins. (A) Anti-caspase-7 reacts solely with caspase-7 protein and not with caspase-3. (B) Anti-caspase-3 reacts solely with caspase-3 protein and not with caspase-7. Protein loading is in nanograms (ng). response to thapsigargin, an ER-associated Ca 2+ -ATPase inhibitor, challenge (1 M) fractionated PC12 cell lysates were examined 6, 12, and 24 hours post-treatment for caspase-7 activation. The endoplasmic reticulum/cytosolic (ER/cytosol) fraction and the nuclear fraction both demonstrated increasing levels and activation of the large p20 subunit (18 kDa) of caspase-7 peaking at 24 hours (Figure 3-2) at over 450%. To determine whether the data truly represented caspase-7, the cells were pre-treated for 1 hour with carbobenzoxy-Asp-CH 2 OC(O)-2,6-dichlorobenzena (Z-D-DCB; 100 M), a pan-caspase inhibitor, before thapsigargin treatment. The inhibitor provided significant protection from active caspase-7 reducing activation of the large subunit to near control levels (Figure 3-3).

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48 Figure 3-2. Thapsigargin mediated caspase-7 activation in PC12 cells. (A) Representative immunoblot showing caspase-7 levels for fractionated thapsigargin treated (1 M) PC12 cells after 6, 12, and 24 hours. The cell lysates were fractionated as described in the Experimental Procedures. The positive control (+ Cntrl) was camptothecin treated Jurket cells. (B) Quantification by densitometry of immunoblots showed caspase-7 levels were statistically significant for fractionated thapsigargin treated PC12 cells. The values are the mean and SEM; statistical analysis was done by one-way ANOVA with Dunnet’s multiple comparison test using Graphpad Prism: n = 5 samples; ** p < 0.01. Immunoblot Analysis of Caspase-7 Activation Following Traumatic Brain Injury To test the hypothesis that caspase-7 is present and could be up-regulated and activated in the brain we examined caspase-7 activation in our model organism, the rat, following traumatic brain injury. Since caspase-7 has been characterized pre-apoptosis as a protein that has been known to associate with the ER (Chandler et al., 1998; Meller et al., 2002), the ER/cytosolic fraction, as described in the Experimental Procedures for the 1.6 mm controlled cortical impact injury, was prepared from the ipsilateral cortex and

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49 Figure 3-3. Z-D-DCB inhibits thapsigargin mediated caspase-7 activation in PC12 cells. (A) With the pan-caspase inhibitor Z-D-DCB (100 M), fractionated thapsigargin treated PC12 cells showed inhibition of caspase-7 activation after 24 hours. (B) Quantification by densitometry of immunoblots showed caspase-7 levels were statistically significant for fractionated thapsigargin treated PC12 cells over PC12 cells treated with Z-D-DCB the pan-caspase inhibitor and control. The values are the mean and SEM; statistical analysis was done by one-way ANOVA with Dunnet’s multiple comparison test using Graphpad Prism: n = 4 samples; ** p < 0.01. hippocampus to test the results of one of the three injury levels examined in the mRNA expression experiments (Figure 3-7). Utilizing the 1.6 mm compression injury which represents a moderate-to-severe injury, caspase-7 expression was examined using an antibody that differentiates the proform, the pre-active form, and the large, or p20, active subunit of caspase-7. The results were quantified by densitometric analysis and expressed as a percentage of nave control levels adjusted for the craniotomy effect (Figure 3-4 and Figure 3-5). Figures 3-4A and 3-5A are representative immunoblots for caspase-7

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50 expression in rat ipsilateral cortex and hippocampus, respectively, for nave, craniotomized control animals for 1 and 7 days post-injury, and for TBI animals from 6 hours and 1, 3, 5, 7 and 14 days post-injury. Immunoblots were also run with equivalent amounts of protein and probed with the anti--actin antibody, which served as an internal protein loading and transfer control. Loading and transfer were essentially equivalent in all wells, as shown by comparable 42 kDa signal intensities. Tissue samples from the ipsilateral cortex are taken from the penumbra and lesion site where the damage was extensive with the brain structure in this vicinity in disarray. It is normal also to find, following TBI, blood clots in the samples as well which tend to disappear by day 14 post-injury along with necrotic and apoptotic tissue. The lesion site that remains displays glial scaring and cellular loss. The craniotomy-operated animals, while not displaying the hallmarks of TBI, the tissue samples did show a modest decrease in caspase-7 proform expression on day 1 after the craniotomy but that was not statistically significant and returned to nave levels by day 7 (Figure 3-4B). There was no increase in the active form for the craniotomy only animals. After adjustment for the craniotomy effect, a statistically significant induction of the caspase-7 pre-active (32 kDa) when compared to nave control was observed within 3 days of cortical injury (p<0.01). Both the pre-active (32 kDa) and the active (18 kDa) forms of caspase-7 peaked around day 5 around 400% (p<0.01) and remained significantly elevated at 7 days post-injury (p<0.01) before returning to near nave levels (Figure 3-4B) on day 14. Tissue samples from the ipsilateral hippocampus for the craniotomy-operated only animals, on the other hand, showed a modest increase in caspase-7 proform expression that was not statistically significant (Figure 3-5B) and, also, no detectable change in the

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51 active form. After adjustment for the craniotomy effect, a statistically significant induction of the caspase-7 pre-active and the active forms of caspase-7, when compared to nave, peaked around 5 days post-injury before returning to near nave levels (Figure 3-5B) by day 14. Neither the cortex nor the hippocampus showed any statistical significant changes for the proform of caspase-7 (Figures 3-4 and 3-5). Figure 3-4. Traumatic brain injury mediated caspase-7 activation in the ipsilateral cortex. (A) Representative immunoblot of the proform (35 kDa), pre-active (32 kDa), and the active (18 kDa) form of caspase-7 in ilsilateral cortical protein samples (n = 6 animals). -actin was directly assessed as an internal methods control. (B) Immunoblot analyses using the anti-caspase-7 antibody were quantified by densitometry and the levels of caspase-7 (proform, pre-active, and active form) expression of injured animals after adjustment for the craniotomy effect (C1, C7) were calculated as a percentage of nave (N) control. One-way ANOVA with Dunnett’s multiple comparison tests was performed to evaluated statistical significance (**p<0.01).

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52 Figure 3-5. Traumatic brain injury mediated caspase-7 activation in the ipsilateral hippocampus. (A) Representative immunoblot of the proform (35 kDa), pre-active (32 kDa), and the active (18 kDa) form of caspase-7 in ilsilateral hippocampal protein samples (n = 4 animals). -actin was directly assessed as an internal methods control. (B) Immunoblot analyses using the anti-caspase-7 antibody were quantified by densitometry and the levels of caspase-7 (proform, pre-active, and active form) expression of injured animals after adjustment for the craniotomy effect (C1, C7) were calculated as a percentage of nave (N) control. One-way ANOVA with Dunnett’s multiple comparison tests was performed to evaluated statistical significance (*p< 0.05). Semi-quantitative RT-PCR Analysis of Caspase-7 mRNA levels Following TBI Standard curves were prepared to determine the relative amounts of caspase-7 or GAPDH mRNA present in the tissue following injury or craniotomy as well as from

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53 uninjured tissue. After the standard curves were generated as discussed in the Experimental Procedures (see Chapter 5) by plotting the log concentration of total RNA versus the crossing point cycle number, a linear regression analysis was performed. The r 2 ranged from 0.9570 to 0.9992. Figure 3-6 shows the linear regression analysis of each primer set’s crossing point cycle number for each brain region versus the logarithm of the dilution factor. For each primer set, the range of crossing point cycle numbers required to cover the serially-diluted standard curve varied: 14-21 cycles for cortical GAPDH, 24-28 cycles for caspase-7 (hippocampus), and 24-29 cycles for caspase-7 (cortex). These differences primarily reflect the abundance of the transcripts. GAPDH mRNA was the most abundant transcript requiring the fewest cycles, whereas caspase-7 cortical mRNA was the least abundant transcript and therefore required the most cycles. Figure 3-6. Standard curve generation for caspase-7 and GAPDH semi-quantitative real-time PCR. The standard curve generation for semi-quantitative RT-PCR was performed using serially-diluted cDNA. For each dilution and each primer set, the cycle number at which the PCR amplification entered the log-linear region was identified (crossing point cycle number). Standard curves were generated by plotting the log concentration of total RNA versus the crossing point cycle number.

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54 Semi-quantitative PCR Analysis of Experimental Samples Using the standard curves generated as described, the crossing point cycle numbers of the experimental samples were converted to relative amounts of mRNA. These relative amounts were then expressed as percentage of nave control adjusted for the craniotomy effect (Figure 3-7). In order to evaluate the magnitude of injury on mRNA expression three levels of injury severity (1.0, 1.2, and 1.6 mm) were examined for the ipsilateral cortex and ipsilateral hippocampus after controlled cortical impact injury. The data convey the similarities in the up-regulation of mRNA levels following injury and illustrate the effect of injury severity on caspase-7 mRNA expression. In the ipsilateral cortex, maximal and statistically significant caspase-7 mRNA expression was observed for all three injury magnitudes 5 days after injury (Figure 3-7A). All three showed an upward trend earlier with the 1.2 and 1.6 mm injury magnitudes producing significant increases in mRNA expression on day 3. In the hippocampus, however, maximal and significant caspase-7 mRNA levels were observed (Figure 3-7B) for the 1.0 and 1.2 mm injury magnitudes as early as 6 hours post-trauma before declining and returning to near nave levels by day 5. The 1.6 mm injury magnitude peaked for this study at day 1 post-injury then it also declined to near nave levels by day 5. The difference in pattern of expression between the cortex and the hippocampus can be understood when the distance from the impact site and the different levels of damage each brain region sustains following impact is taken into consideration. Immunohistochemical Analysis of Caspase-7 Up-regulation Following TBI To determine in which cell type TBI mediated up-regulation of caspase-7 occurs, ipsilateral and contralateral cortical and hippocampal brain sections 5 days post-injury as

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55 Figure 3-7. Semi-quantitative real-time PCR analysis of caspase-7 mRNA expression. mRNA in (A) cortex and (B) hippocampus levels after adjustment for sham effect are expressed as a percentage of nave control. Values are the mean and the SEM and one-way ANOVA with Dunnet’s multiple comparison test was performed to evaluate statistical significance (n = 3; *p<0.05, **p<0.01). well as nave and craniotomy-operated brain sections were examined for caspase-7 induction. High magnification photomicrographs of the Alexa Fluor stains of uninjured and craniotomy-operated animals revealed healthy cell bodies and little detectable caspase-7 expression. In contrast, in 5 day post-injury tissue caspase-7 expression was readily observed in both the ipsilateral cortex and hippocampus (Figures 3-8 and 3-9), with significant levels in the cortex in the immediate vicinity of the impact site. The ipsilateral cortex at the site of the contusion revealed a considerable increase in caspase-7 expression with decreasing levels distal to the site of impact. The morphology of the injury site where elevated levels of caspase-7 induction were located had a decidedly disorganized, almost chaotic appearance when compared to the contralateral, nave, and craniotomy tissue. In the ipsilateral cortex caspase-7 co-localized with immunopositive neurons as marked by anti-NeuN, a neuronal cell-specific antibody,

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56 Figure 3-8. TBI up-regulation of caspase-7 in neurons in the cortex and hippocampus. Expression of caspase-7 was induced in (A) cortical and (B) hippocampal neurons following TBI using immunohistochemical techniques on 40 m brain tissue sections. Chromatin was visualized with DAPI, neurons with the neuron specific antibody NeuN, casp7 protein with anti-caspase-7 antibody with co-localization resulting in yellow and orange (see arrows). Photomicrographs 400x; scale bars = 20 m.

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57 Figure 3-9. TBI up-regulation of caspase-7 in astrocytes in the cortex and hippocampus. Expression of caspase-7 was induced in (A) cortical and (B) hippocampal astrocytes following TBI using immunohistochemical techniques on 40 m brain tissue sections. Chromatin was visualized with DAPI, astrocytes with the astrocytic specific antibody GFAP, casp7 protein with anti-caspase-7 antibody with co-localization resulting in yellow and orange (see arrows) including in cells showing apoptotic bodies (see insert and arrowhead). Photomicrographs 400x; scale bars = 20 m. (Figure 3-8A) and appeared to include those cells with evidence of morphopathology. The ipsilateral hippocampus also revealed that induced caspase-7 co-localized with the

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58 neuronal cell-specific marker NeuN (Figure 3-8B) but the hippocampal tissue did not display the same disorganized appearance such as that viewed in the cortex. Caspase-7 in the ipsilateral cortex was also found to co-localize with immunopositive astrocytes as marked by anti-GFAP, an astrocytic cell-specific antibody, (Figure 3-9A) and also appeared to include those cells with apoptotic bodies. The ipsilateral hippocampus likewise revealed that induced caspase-7 co-localized with the astrocytic cell-specific marker GFAP (Figure 3-9B). Discussion Our findings establish that caspase-7 is present in the brain and appears to play a role in the apoptotic cell death response. To our knowledge, this study is the first to demonstrate that caspase-7 is present both at the mRNA and protein levels and that it is quite abundant. I have also demonstrated that it is activated in both neurons and astrocytes in the cortex and hippocampus following TBI. Caspase-7 appears to be emerging as an important apoptotic protease in its own right suggesting that it is more than a redundant clone of caspase-3 Three caspase-7 isoforms have been cloned (Juan et al., 1997) and like all caspases, it is produced as a catalytically inactive zymogen which must be proteolytically processed to become an active protease. The main isoform of procaspase-7 contains 303 amino acids and has been crystallized and been found to exist as two monomers arranged side by side but with opposite orientations (Riedl et al., 2001). The structure of caspase-7 exhibits a high degree of similarity (Riedl et al., 2001; Wei et al., 2000) with caspase-3 even though the two only share 53% sequence identity (Juan et al., 1997). The prodomain of caspase-7 inhibits both its apoptosis-inducing activity and its nuclear localization (Yaoita, 2002). The single contiguous polypeptide chain of procaspase-7 has a L2 loop

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59 linking the large and small subunits. The cleavage site after the sequence IQAD 198 in this interdomain loop must be cleaved to allow rearrangement of the essential loops in the active site (Zhao et al., 1998). The surface potential at the enzymatic site of caspase-7 differs from caspase-3 in that it has an unpaired basic residue (Arg237) near the P4 aspartic acid giving it a negative electrostatic potential while caspase-3 is neutral in this region. This unique hydrophilic residue in caspase-7 may allow this seemingly redundant caspase to act on different substrates from caspase-3, in different cell types, or different cellular compartments. There are at least two known caspase-7 targets that are not shared by caspase-3: caspase-12 (Rao et al., 2001) and kinectin (Machleidt et al., 1998). The contribution of caspase-7 to apoptosis remains controversial. Up until very recently it was still the prevailing belief that either there was no caspase-7, mRNA and protein, present in the brain (Juan et al., 1997; Ray, 2002), it was not activated if present (Henshall et al., 2002), or if activated it was ineffectual in at least neurons and astrocytes (Zhang et al., 2000). This view was general held even though it was reported in one of the earliest studies that caspase-7 (Mch3) mRNA was found in the brain though at very low levels (Fernandes-Alnemri et al., 1995). In one study in caspase-3 deficient mice there was no evidence of compensatory activation in the nervous system of caspase-6 or -7 following in vivo cerebral ischemia or after in vitro oxygen glucose deprivation (Le et al., 2002), unlike that found in the liver. The authors suggested that stimulus-specific or organotypic differences may exist in caspase regulation and that caspase-7 may not play a major role in mediating PARP cleavage in ischemic mutant neurons despite caspase-7 constitutive expression (Le et al., 2002). In an immunohistochemical study on rat C6

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60 glioma cells data suggest that there may exist marked differences in the subcellular distribution of caspase-3 and caspase-7 during apoptosis (Meller et al., 2002). Of the three apoptotic pathways, the intrinsic pathway mediated through the mitochondria by caspase-9 and the extrinsic pathway mediated through the plasma membrane by caspases-8 and -10 have been the most studied. The third pathway initiated under ER stress is mediated through the ER membrane and involves caspase-12. It has been reported that the ER chaperone GRP78 (BiP) constitutively associates with pro-caspase-7 (Reddy et al., 2003) forming a complex with caspases-7 and -12 (Rao et al., 2002). Under ER stress BiP is released and caspase-7 cleaves caspase-12 (Rao et al., 2001) activating the caspase apoptotic cascade thus coupling ER stress to the cell death program. The data in this study on PC12 cells confirm that caspase-7 is activated by ER stress when initiated by thapsigargin treatment further verifying that caspase-7 has a strong ER associated activation component. The differential subcellular distribution of specific caspases during the induction of apoptosis in vivo was demonstrated by Chandler and colleagues (Chandler et al., 1998). Following Fas-induced apoptosis in vivo, active caspase-3 was found primarily in the cytosol, whereas active caspase-7 was associated almost exclusively with the mitochondrial and microsomal (endoplasmic reticulum) fractions. Both the activation of caspase-7 in the endoplasmic reticulum and the cleavage of the endoplasmic reticular-specific substrate, sterol regulatory element-binding protein 1 (SREBP-1), were blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-fmk). Kinectin, a 156 kDa protein, an exclusive target of caspase-7 (Machleidt et al., 1998), is found on the cytoplasmic face in the ER membranes (Toyoshima et al., 1992) and has been shown to interact with the cargo binding site of

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61 conventional kinesin and to activate its microtubule-stimulated ATPase activity. An interesting finding was that kinesin was found to be most abundant in the brain (Hollenbeck, 1989) suggesting that kinectin is as well. A previous study by our laboratory shows that caspase-12, another ER bound protein (Nakagawa et al., 2000), is activated following TBI (Larner et al., 2004). One of caspase-12’s two activators is caspase-7 (Rao et al., 2001), the other, calpain-2 (Nakagawa and Yuan, 2000). Based on our previous data which showed that caspase-12 peaks on day 1 post-injury (Larner et al., 2004) and with this study in which the data show that caspase-7 peaks on day 5 post-injury suggests that either calpain is an early activator of caspase-12 and that caspase-7 plays a more prominent role later or caspase-7 and caspase-12 are engaged in a feedback loop. This latter is suggested by the finding in DT40 cells that caspase-7 functions early in the apoptotic pathway induced by drugs such as etoposide and staurosporine (Korfali et al., 2004) and from our data that caspase-7 peaks after caspase-12. Recent studies clearly imply that caspase-7 has an important, non-redundant role in normal physiology and in apoptotic cell death. While caspase-3 -/mice exhibit neurodegenerative disorders (Kuida et al., 1996) mice that are CASPASE-7 -/have an early embryonic lethal phenotype (Slee et al., 2001). In a study of the CASPASE-7 gene in human solid cancers it was found that between 2% to 3% of the tumors examined had inactivating mutations in the CASPASE-7 gene, as determined by genomic DNA analysis, that appeared to lead to the loss of apoptotic function contributing to their pathogenesis (Soung et al., 2003). In a related study of the human neuroblastoma cell line SH-SY5Y exposed to the anticancer apoptotic inducing drug, paclitaxel, the addition of trans-resveratrol, a natural antioxidant present in grapes and red wine, was able to inhibit

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62 the activation of caspase-7 and the degradation of PARP thereby modulating the signals that committed these neuronal-like cells to apoptosis (Nicolini et al., 2001). An investigation of the molecular mechanisms of neuronal death in the dorsal lateral geniculate nucleus following visual cortical lesions found that caspase-7 was activated though its expression was mainly limited to the pretectal nuclei (Repici et al., 2003). Focusing more neuropathologically, in a clinical study of Alzheimer’s diseased patients elevated mRNA expression for caspase-7 was revealed in the entorhinal cortex. Caspase mRNA expression was closely associated with neurofibrillary tangles and, to a lesser extent, neuritic plaque density (Pompl et al., 2003). And in a related study the beta-amyloid (A) peptide, a peptide believed to play an important role in Alzheimer’s pathogenesis, was found to trigger ER calcium release in primary cortical neurons in culture. While the addition of Xestospongin C or FK506 partly attenuated A neurotoxicity shown by the reduced ER calcium release and up-regulation of BiP, other signaling machinery such as the activation of caspase-7 death signal transmissions from the ER to other organelles could not be altered (Suen et al., 2003). Finally, in a rat model of focal cerebral ischemia, a model that has previously been shown to have analogous bio-molecular responses to TBI, permanent middle cerebral artery occlusion (MCAO) affected caspase-7 mRNA expression. mRNA increased 1.5 fold 6 hours after MCAO in the ipsilateral cortex and better than 2 fold by 24 hours. In the contralateral cortex there was about 1.5 fold increase by 24 hours post-injury (Harrison et al., 2001). Given that caspase-7 studies have shown that it has less redundancy than previously supposed the contribution that it makes to apoptosis following traumatic brain injury and to other neurodegenerative conditions needs further evaluation. It has been

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63 successfully been shown in this study that caspase-7 is significantly up-regulated and activated in both neurons and astrocytes after traumatic brain injury.

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CHAPTER 4 RAT CASPASE-12 ISOFORM Introduction The gene is the functional unit of heredity. It occupies a specific locus on a chromosome and is reproduced exactly at each cell division. It is the source of information for the code that will translate a sequence of amino acids into a specific functional polypeptide (Stedman, 1997). A gene includes more than the nucleotides encoding the amino acid sequence, referred to as the coding region (exons), the gene also includes all the DNA sequences required for synthesis of a particular RNA transcript including the promoter region, the introns, and the poly(A) tail. Studies of mRNAs of specific genes have revealed that the inclusion or exclusion of exons in a certain cell type appears to be the result of the effective combination of several splicing repressors and enhancers (Grabowski and Black, 2001). Alternative exon splicing is particularly common in the vertebrate and invertebrate nervous systems where multiple isoforms of many proteins are required for neuronal development and function. The primary transcripts from these genes frequently demonstrate complex splicing patterns with different spliced forms expressed in different anatomical locations within the central nervous system (Lodish et al., 2000). One example of this regulated splicing can be found in the “hair cells” (ciliated neurons) gradient within the inner ear that detects low to high frequencies. Each hair cell responds to a particular frequency and one of its components is an ion channel that opens at particular Ca 2+ concentrations depending on the channel’s response to that frequency 64

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65 of sound for which it is attuned. The gene encoding this channel protein, slo, is expressed as multiple alternatively spiced mRNA isoforms with a different version being expressed depending on its position along the length of the cochlea. While there are 576 possible isoforms of slo, it is not known, however, if all possible isoforms are expressed (Black, 1998; Ramanathan et al., 1999). An extreme example of regulated alternative RNA processing occurs when the Dscam gene in Drosophila is expressed. During fly development this axon guidance receptor gene is responsible for the connections made by the axons of retinal neurons with neurons in a specific region of the brain. Analysis of the Dscam gene shows that the 95 exons that make up the gene could be alternatively spliced to generate over 38,000 possible isoforms. These results raise the possibility that the expression of different Dscam isoforms through regulated RNA splicing helps to specify the tens of thousands of different specific synaptic connections made between retinal and brain neurons. This suggests that the correct neuronal wiring of the brain may depend on regulated RNA splicing (Schmucker et al., 2000). During the cloning and sequencing of the rat caspase-12 mRNA, a sequence that was 78 nucleotides (26 amino acids) longer than the eventually published sequence was uncovered suggesting that there are at least two isoforms of caspase-12 in the rat. This would not be an unusual finding since, as was reported in Chapter 3, caspase-7 has at least 3 isoforms, and in the study of caspase-12 genetic sequence in humans (Fischer et al., 2002) the authors cited that there may be as many as 9 isoforms. Why are these different isoforms present? Where are they expressed and under what conditions? Why are there more isoforms expressed in the brain than in other tissues? These questions are

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66 interesting subjects for future studies as they may help us to better understand the complexity of the cells’ response to pathological trauma. Results This work is still in progress but the results are encouraging and are suggestive that there is a second functional caspase-12 isoform in the rat. The initial report of caspase-12 (Van de Craen et al., 1997) from the mouse provided a putative full length cDNA as well as protein sequence. The reported length of the mouse cDNA (GenBank Y13090) is approximately 2,262 base pairs (bp) with an open reading frame (ORF) of 1,260 bp. It was found to be most abundantly expressed in lung and skeletal muscle with lower amounts in the brain and other tissues. The protein product of an in vitro translation of the murine cDNA clone for the caspase-12 precursor revealed a molecular weight of about 47.5 kDa roughly what was expected from the gene sequence. To analyze the mRNA expression in rat tissues a panel of cDNAs from various tissues was tested by PCR with a primer pair specific for caspase-12 (P1/P2). As in the mouse (Van de Craen et al., 1997) and human (Fischer et al., 2002) the results appeared to confirm that the highest expression is also in the lung. For the rat this was followed by the skeletal muscle with still lesser amounts found in the liver, the brain, and the spleen (Figure 4-1). Figure 4-1: RT-PCR analysis of rat caspase-12 in selected adult tissues using primer pair P1/P2.

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67 Since the results appeared to confirm the findings for the mouse no additional work was attempted and no controls were run to confirm the normalization of the cDNA used for PCR. Cloning and sequencing the rat caspase-12 mRNA was accomplished in three segments (Figure 4-2). Thus far, the nearly complete rat cDNA sequence that has been generated is about 14 bases short of being a complete ORF, the first 14 bases at the 5’ end. Although the sequence is incomplete it was enough to isolate and identify a new isoform. Reverse transcriptase (RT) PCR was used to synthesize cDNA from total RNA from rat brain and lung tissue. The gene specific primer (GSP) pair, P1/P2, were initially selected using the published mouse caspase-12 cDNA sequence (Van de Craen et al., 1997) (GenBank Y13090) using Primer3 software (Rozen, 1998). PCR was used to amplify caspase-12 cDNA and the resulting PCR products were TOPO TA cloned. The PCR products were sequenced as described in the Experimental Protocol (Chapter 5). Figure 4-2: A schematic of the caspase-12 cloned and sequenced segments. The segments are aligned to a representative caspase-12 cDNA. The red boxes represent, in order, the SHG and QACRG enzymatic sites. The first segment cloned and sequenced was 672 bp (base pairs) in length and straddled the SHG and QAC(R/Q/G) G motifs which represent the enzymatic sites and appear to be conserved in all caspases. These are seen in Figure 4-2 as open red boxes

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68 and underlined sequences in Appendix A. These conserved sequences are important in the binding and catalytic activity of all caspases. Segment 3, 422 bp in length, was cloned and sequenced by primer pairs that represent the reverse of primer P2 and a universal amplification primer provided by the Rapid Amplification of cDNA Ends (RACE) 3 End kit from Clontech. RACE is a procedure for amplification of nucleic acid sequences from a messenger RNA template between a selected internal site of known sequences and the 3 end of the mRNA. Since PCR requires two sequence-specific primers that flank the unknown region to be amplified, to amplify and characterize regions of unknown sequences presents a challenge. 3 RACE takes advantage of the natural poly(A) tail found in most mRNAs as a generic priming site for PCR. In this procedure, mRNAs were converted into cDNA using reverse transcriptase and an oligo-dT adapter primer. The targeted cDNA was then amplified by PCR using a GSP that anneals to known sequences and a universal adapter primer (UAP) that targets the poly(A) tail region. Anti-P2 was the GSP that worked in this specific case. This permits the capture of unknown 3-mRNA sequences that lie between the known target site and the poly(A) tail. (See Experimental Protocols – Chapter 5, Figure 5-1 for illustration of the procedure.) Segment 2, 893 bp in length, was cloned and sequenced from the primer pairs P3/P4. P3 is located near the 5 end of the coding region was paired with P4, a known sequence that nested within segment 1. This segment contains the putative additional exon that would render this mRNA an isoform. The sequence for the addition is 78 bp in length and occupies a position between the reported second and third exon (Figure 4-3 and Appendix C). This is within the prodomain. The 78 bp sequence is exactly divisible

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69 by 3, the genetic triplet code that represents the codon for an amino acid, which corresponds to 26 additional amino acids (Figure 4-4). This new exon can be found in that part of the complete gene sequence just as the sequence goes into a series of Ns representing bases where the sequencers could not distinguish one base from another (Figure 4-5 and Appendix 3). This continues for what appears to be over 450 bp with few exceptions and only becomes resolved about 33 bp before the reported third exon. NM 130422 Rattus_norvegicus .CTTCAGAAAATGTTTACACCATCTTCTGCATCAG-----------”In House” Casp12 Isoform .CTTCAGAAAATGTTTACACCATCTTCTGCATCAGATTCTGAAAAAG ********************************** NM 130422 Rattus_norvegicus -------------------------------------------------”In House” Casp12 Isoform AAATTAAAGTCAACAAAAATGAGGGACAAGATTTATTGAAGCAGATAATG NM 130422 Rattus norvegicus ----------------AATCCAGGGGAAAAGTAGAAGATGAAGAAAT. ”In House” Casp12 Isoform CCTTTTTCTCTGATGGAACCCAGGGGAAAAGTAGAAGATGAAGAAAT. ** **************************** Figure 4-3: The rat caspase-12 isoform’s additional exon’s genetic sequence. The additional exon is flanked by the sequences common between the two. The C in the lower strand appears to be a difference in the two isoforms or between the sources of the DNA. (* = single, fully conserved residue.) NM 130422 Rattus_norvegicus .EKTEMAGKIFAGHIANSDKQLSLQFPSDDEEDELQKMFTPSSAS----------“In House” Casp12 Isoform .EKTEMAGKIFAGHIANSDKQLSLQFPSDDEEDELQKMFTPSSAS DSEKEIKVNKNEG ******************************************** NM 130422 Rattus_norvegicus -------------ESRGKVEDEEMEVNVGVAHASHLMLTVPQGIQSTEVQDSLKLCSRDW “In House” Casp12 Isoform QDLLKQ IMPFSLMEPRGKVEDEEMEVSVGVAHASHLMLTVPQGIQSTEVQDSLKLCSRDW * ***********.********************************* NM 130422 Rattus_norvegicus FCTMKTERAEEIYPVMEKEGRTRLALIICNKKFDYLFDRDDAETDILNMKELLQNLG “In House” Casp12 Isoform FCTMKTERAEEIYPVMEKEGRTRLALIICNKKFDYLFDRDDAETDILNMKELLQNLG ********************************************************* Figure 4-4: The rat caspase-12 isoform’s additional exon’s protein sequence. The exon flanked by the amino acids common between the two. The amino acids shown in blue appear to be differences in the two isoforms or between the sources of the DNA. (* = single, fully conserved residue; : = conservation of strong groups; . = conservation of weak groups) To determine if the inclusion actually represents an additional exon and not a read through of an intron segment an analysis was made of the exon-intron junctions or splice sites (Figure 4-6). There are moderately conserved short consensus sequences at intron-exon boundaries in eukaryotic pre-mRNAs. A pyrimidine-rich region (Cs and Ts) just

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70 upstream of the exon 5 splice site is also common in higher eukaryotes. The nucleotides that are most conserved, nearly 100% of the time, are GU (or GT for cDNA) found at the 5 end and AG found at the 3 end of introns and the C base just before the AG (for a CAG sequence) about 80% (Lodish et al., 2000). Also exons tend to end with the G base about 80% of the time. This new exon is found within intron 2 and follows all the above rules. The intron sequence immediately before the “New Exon” has the signature CAG at its 3 end and the “New Exon” ends with the G base (Figure 4-6). Currently there is no way to determine from the NIH data base the 5 end of the next intervening intron due to the series of Ns representing bases that could not be distinguished by the sequencer as discussed above. The region within intron 2 immediately before the new exon is pyrimidine-rich, another telltale sign that this is an authentic exon. NW 047797 R. norvegicus gene ATTCTGAAAAAGAATTAAAGTNAAAAAAATGANGGANNAGATTTATTGAAGNANATA “In House” Casp12 Isof. cDNA ATTCTGAAAAAGAAATTAAGTCAACAAAATGAGGGACAAGATTTATTGAAGCAGATA ************** * **** ** ******* *** ************* * *** NW 047797 R. norvegicus gene ATNNNTTTTTCTCTGATNNNNNN “In House” Casp12 Isof. cDNA ATGCCTTTTTCTCTGATGG ** ************ Figure 4-5: Caspase-12 rat isoform sequence compared to the full gene sequence. The Ns represent bases where the sequencers were unable to distinguish one base from another. (* = single, fully conserved residue) -Intron 1---Exon 2----------------------------Intron 2------------------------NW 047797 R. norvegicus gene GTTT CAG AATTTCNTTCTTGCATCAG GT NATTANN “In House” Casp 12 Isoform ~~~~~~~~AATTTCCTTCTTGCATCAG~~~~~~~~~~~~~~~~~~~~~ -(pyrimidine rich)-----------------New Exon---------------------Intron 2 cont. NW 047797 R. norvegicus gene TTCTCCTTTTCTTTGTNTACTC CAG ATTCTGAACTCTGATNN NN NNNN “In House” Casp 12 Isoform ~~~~~~~~~~~~~~~~~~~~~~~ATTCTGAACTCTGATGG~~~~~~~~~ -------------------Exon 3---------------------------------------------------------> NW 047797 R. norvegicus gene AATCCAGGGGAAA CTCTCTTTCAG “In House” Casp 12 Isoform ~~~~~~~~~~~AACCCAGGGGAAA Figure 4-6: Caspase-12 isoform intron and exon boundaries. The “New Exon” has the indicative signs that indicate this is a legitimate exon including the CAG consensus sequence and pyrimidine rich region before the exon and the base G that ends the exon.

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71 To determine if the mRNA sequence translates into an operational protein a rat anti-caspase-12 isoform-specific antibody was designed using the protein sequenced underlined in Figure 4-4. The antibody generated in rabbits was ELISA tested and affinity-purified. The reactivity and specificity of the antibody was tested on 2-day post-injury TBI cortical rat tissue with a 1.6 mm compression injury. The preliminary results (Figure 4-7) suggest that the isoform-specific antibody is able to detect the expected approximately 55 kDa proform and the cleaved 20 kDa prodomain (~110 Da per amino acid x approximately 185 amino acids for the isoform prodomain = 20.35 kDa) that would remain following activation of the proform. More work will be required to confirm these encouraging preliminary results including comparing the antibody head-to-head to one of the commercially available anti-caspase-12 antibodies. I hypothesize that the isoform-specific antibody will be able to detect a slightly larger (3 to 5 kDa) caspase-12 proform and a slightly larger prodomain (3 to 5 kDa) following activation of the protein. Figure 4-7: Immunoblot of the anti-caspase-12 isoform-specific antibody. The antibody appears to be able to selectively detect the proform (55 kDa) and the isolated prodomain after activation of caspase-12 (20 kDa). The remaining bands are non-specific. The antibody appears to need to be further purified. (N = Nave rat cortex; TBI = 2-day post-injury rat cortex after 1.6 mm compression injury)

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72 Analysis of the procaspase-12 rat protein, both the published and isoform-in-progress, using SMART (Simple Modular Architecture Research Tool) (Letunic et al., 2002) suggests there are two principle domains and, as was confirmed by a Kyte-Doolittle graphical plot (Kyte and Doolittle, 1982), no transmembrane elements. The first domain found in the N-terminus prodomain, about 85 amino acids in length, predicts a CARD (Caspase Recruitment Domain) motif. The second, extending from about amino acid 166, is the CASc (Caspase, interleukin-1 beta converting enzyme (ICE) homologue) motif representing the active domain in which the large and small subunit reside. The CARD domain is found in other caspases of this subfamily including caspases-1, -4, -5, and -11, as well as in caspase-9 which may belong to the CASP-2 or CASP-3 subfamily and caspase-2 of the subfamily of the same name. The CARD motif appears to be important in promoting interactions of these caspases with one another and with a range of other regulatory and adapter proteins such as Apaf-1, a protein required to create the apoptosome necessary to activate caspase-9 . The CARD domain typically associates with other CARD-containing proteins, forming either dimers or trimers. It has been predicted that the CARD is related in structure and sequence to both DD and DED domains, which work in similar pathways and show similar interaction properties (Earnshaw et al., 1999). Discussion Tissue-specific alternative splicing affects protein function in a variety of ways and this appears to be most evident in the nervous system. In the nervous system, mRNA isoforms play differing roles in learning and memory, neuronal cell recognition, neurotransmission, ion channel function, and receptor specificity. And now we can add apoptotic regulation. The different combinations of exons from the pre-mRNAs that are

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73 possible are a form of genetic control offering an essential mean of creating versatility. Yet the control mechanisms, the complex arrays of positive and negative RNA elements that allow for subtle responses to spatial and temporal cues that specify tissue, cell and developmental changes in alternative splicing, remain poorly understood despite progress in identifying regulatory elements and their RNA binding proteins (Black, 1998; Grabowski and Black, 2001). The human pathology that occurs when the cell loses it ability to regulate this function highlights its critical nature. Thus, alternative splicing appears to be a major means of generating proteomic diversity and in modulating protein activities in a temporal and spatial manner (Black, 1998; Grabowski and Black, 2001). The really interesting question when we focus on the biological role of alternative splicing is how does alternative spliced proteins, which increase proteome diversity, help distinct cell types and cellular responses carry out their particular functions? Alternative spliced caspases have been reported for some time but only recently has the different isoforms been studied for their different functions. The results reported for caspases-2, -3, -8, and -9 show that their isoforms are involved in regulating apoptotic events in a temporal and spatial manner. For example caspase-2 long form, caspase-2L, has been found to play a role in the Fas-mediated pathway that leads to cell death by contributing to the activation of caspase-8 at the DISC (death-inducing signaling complex) level (Droin et al., 2001a). Caspase-2L has also been found involved in the cleavage of II-spectrin. The long form binds to II-spectrin while the short form (2S) does not and like caspase-3 cleaves the protein at residue Asp1185 in vitro (Rotter et al., 2004). Caspase-2S on the other hand was reported to negatively interfere with the major features of apoptosis. It partially prevents the nuclear changes associated with cell death,

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74 prevented maturation of apoptotic bodies, delayed phophatidylserine externalization, and prevented cleavage and activation of caspase-2L (Droin et al., 2001b). It has also been reported that there are differences in the two caspase-2 isoforms’ promoter strength. It appears they have alternative transcriptional initiation sites and the 5-splicing events that regulate the expression of caspase-2L and 2S isoforms may be translated from alternative translation initiation codons (Logette et al., 2003). The other caspases have not as yet been studied with as much detail but reports have begun to present some interesting findings. It is now known that caspase-3 has two isoforms that are differentially regulated during the development of the rat brain. In naturally occurring neuronal death that takes place during brain sculpting, the inactive 32 kDa proform and an active 20 kDa form are both up-regulated during the late embryonic and early post-partum developmental stages before returning to base levels (Mooney and Miller, 2000). It has been reported that caspase-8 has 8 isoforms but that only 2 predominate (Scaffidi et al., 1997). Caspase-8L, an isoform that is missing the catalytic site, acts an inhibitor to the other isoforms by binding to FADD, thereby interfering with Fas-mediated apoptosis (Himeji et al., 2002). On the other hand, the extra (NEX) domain of caspase-8L allows it to be preferentially recruited to the BAP complex in the ER in response to apoptotic signaling by the oncogenic E1A protein. BAP contributes to the pro-caspase-8L processing thereby triggering several apoptotic pathways leading to the activation of downstream caspases and cell death (Breckenridge et al., 2002). Caspase-9S, found in the cytoplasm of multiple tissues is not cleaved in the apoptotic process thus appears to act as a dominant negative variant whose physiological role is to inhibit cell death (Angelastro et al., 2001).

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75 In a study to find the proteases that activate caspase-12 Nakagawa and Yuan (2000) reported protein expression in one of their immunoblots, of murine cultured glial cells challenged by oxygen and glucose deprivation (OGD), of a band that was “slightly bigger” than the full length caspase-12. They noted that this protein is expressed at very low levels in control conditions but showed increased expression when the cells were challenged. They made no attempt to follow up this finding noting that because the anti-caspase-12 antibody recognize it that it “may be related to caspase-12”. The band appears to be about 3 to 5 kDa larger that the established procaspase-12 band. This is about the expected size increase the new rat isoform would generate that has been documented in this report. This finding suggests that caspase-12 in mice may have two isoforms as well and this would appear to confirm the conclusion that caspase-12 in rat has two isoforms and based upon the OCD activated mouse glial cellular response that it is highly responsive to apoptotic challenges. The finding by Fischer and colleagues (2002) that there may be as many as 9 isoforms of caspase-12 in humans with product sizes that are approximately 1300 (3), 1200 (2), 1070, 960, 820, and 700 nucleotides in length suggests that this caspase may play an interesting role in both apoptotic and inflammatory responses. A great deal of additional research will be necessary to determine what those roles are. It is too early in the experimental process to define the role this newly cloned and sequenced rat caspase-12 isoform may play in the overall regulation of apoptotic cell death. With the sequence known, with the anti-caspase-12 isoform-specific antibody as an available tool, and through additional work it should be feasible to examine both isoforms’ roles during pathological trauma. Since the preliminary data show the protein is

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76 upregulated and activated following TBI (Figure 4-7), this suggests that this new isoform of the rat caspase-12 plays an important role in the apoptotic process within the brain. What that process is and its effect on TBI pathology will have to wait until the completion of the study at a future date.

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CHAPTER 5 EXPERIMENTAL PROTOCOLS Rat Pheochromocytoma (PC12) Cell Culture, Collection and Preparation Rat PC12 cells were grown on polystyrene tissue culture dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 5% horse serum, 1% fungizone, 100 units/ml penicillin, and 100 g/ml streptomycin and kept at 37 o C in humidified 5% CO 2 incubator from 12 to 24 hours prior to treatment. Cell cultures were pretreated 1 hour before thapsigargin (Research Biochemical International) challenge (1 M), when necessary with the pan-caspase (100 M) inhibitor carbobenzoxy-Asp-CH 2 OC(O)-2,6-diclorobenzena (Z-D-DCB) (Bachem). The cell cultures were suspended in ice-cold detergent-free buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 2 mM EGTA, 0.33 M sucrose, 1 mM DTT) containing a broad-range protease inhibitor cocktail (Roche Molecular Biochemicals) and then passed 3-5 times through a 27 guage needle. The samples were fractionated by centrifuging at 600g for 10 minutes at 4C to isolate the nuclei and at 10,000g at 4C for 10 minutes to remove the mitochondria. The nuclei pellet was resuspended in a lysis buffer (20 mM HEPES, 1 mM EDTA, 2 mM EGTA, 150 mM NaCl, 0.1% SDS, 1% IGEPAL, and 0.5% deoxycholic acid at pH 7.5) and sonicated in preparation for immunoblot analysis. The subcellular supernatant, containing the lysosomal/ER and cytosolic proteins, was collected. 77

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78 Surgical Preparation and Controlled Cortical Impact Traumatic Brain Injury A previously described cortical impact injury device was used to produce traumatic brain injury (TBI) in adult rats (Dixon et al., 1991; Pike et al., 1998a). Cortical impact TBI results in cortical deformation within the vicinity of the impactor tip associated with contusion, and neuronal and axonal damage that is confined to the hemisphere ipsilateral to the site of injury. Adult male (280-300 g) Sprague-Dawley rats (Harlan) were anesthetized with 4% isoflurane in a carrier gas of 1:1 O 2 /N 2 O (4 min.) followed by maintenance anesthesia of 2.5% isoflurane in the same carrier gas. Core body temperature was monitored continuously by a rectal thermistor probe and maintained at 37o 1 o C by placing an adjustable temperature controlled heating pad beneath the rats. Animals were mounted in a stereotactic frame in a prone position and secured by ear and incisor bars. A midline cranial incision was made, the soft tissues reflected, and a unilateral (ipsilateral to site of impact) craniotomy (7 mm diameter) was performed adjacent to the central suture, midway between bregma and lambda. The dura mater was kept intact over the cortex. Brain trauma was produced by impacting the right cortex (ipsilateral cortex) with a 5 mm diameter aluminum impactor tip (housed in a pneumatic cylinder) at a velocity of 3.5 m/s with a 1.0 mm, 1.2 mm, or 1.6 mm compression and 150 ms dwell time (compression duration). Velocity was controlled by adjusting the pressure (compressed N2) supplied to the pneumatic cylinder. Velocity and dwell time were measured by a linear velocity displacement transducer (Lucas Shaevitz model 500 HR) that produces an analogue signal recorded by a storage-trace oscilloscope (BK Precision, model 2522B). Sham-injured (craniotomy-injured) animals undergo identical surgical procedures, but did not receive an impact injury. Nave animals received no surgery or

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79 injury. Appropriate preand post-injury management was maintained, and these measures complied with all guidelines set forth by the University of Florida Institutional Animal Care and Use Committee and the National Institutes of Health guidelines detailed in the Guide for the Care and Use of Laboratory Animals. Tissue Lysis and Protein Purification Cortical and hippocampal tissues were collected from nave animals or at 6 hours to 14 days after sham-injury (craniotomy-injured) or TBI. At the appropriate post-injury time-points, the animals were anesthetized with 4% isoflurane in a carrier gas of 1:1 O 2 /N 2 O (4 min.) and subsequently sacrificed by decapitation. For immunoblot studies ipsilateral and contralateral (to the impact site) cortices and hippocampi were rapidly dissected and snap-frozen in liquid nitrogen. Tissue samples were stored at -20C. The samples were homogenized in a glass tube with a Teflon dounce pestle in 15 volumes of ice-cold detergent-free buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 2 mM EGTA, 0.33 M sucrose, 1 mM DTT) containing a broad-range protease inhibitor cocktail (Roche Molecular Biochemicals). Samples were fractionated by centrifuging at 600g for 10 minutes at 4C to isolate the nuclei then at 10,000g at 4C for 10 minutes to remove the mitochondria. The nuclei pellet were resuspended in a lysis buffer (20 mM HEPES, 1 mM EDTA, 2 mM EGTA, 150 mM NaCl, 0.1% SDS, 1% IGEPAL, and 0.5% deoxycholic acid at pH 7.5) and sonicated in preparation for immunoblot analysis. The subcellular supernatant containing the lysosomal/ER and cytosolic proteins were collected.

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80 Semi-Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) RNA Purification Total RNA was isolated from control and injured samples of cortical or hippocampal tissue using TRIzol reagent (Gibco BRL). Isopropanol precipitation and ethanol washes were performed according to the manufacturer’s instructions. Samples were resuspended in 50-100 L DEPC-treated water. Reverse Transcription Three g of total RNA were incubated with 1 L oligo(dT) (0.5 mg/mL, GibcoBRL) at 70C for 10 minutes, then at 4C for five minutes. A reverse transcription reaction mixture was added to the RNA-oligo(dT) sample for a final volume of 20 L, containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 5 mM MgCl 2 , 500 M dNTPs, 10 mM DTT, 50 units SuperScript II reverse transcriptase (GibcoBRL), and 40 units RNaseOUT recombinant ribonuclease inhibitor (GibcoBRL). The sample was incubated at 42C for 55 minutes, 70C for 15 minutes for enzyme denaturation, and then transferred to 4C. Each sample was diluted to a final volume of 100 L with DEPC-treated water. Primer Selection Caspase-12 specific primers: For all base pair designations refer to GeneBank locus AF317633, Rattus norvegicus caspase-12. 5’C12 (gcacattcctggtctttatgtccc, bp 743-766) recognizes an upstream mRNA-specific sequence in caspase-12 transcripts. 3’C12 (gccactgctgatacagatgaggaa, bp 961-984) recognizes a downstream caspase-12 mRNA-specific sequence. Caspase-7 specific primers: refer to GeneBank locus AF072124, Rattus norvegicus caspase-7. 5’C7 (tgagccacggagaagagaat, bp 427-446) recognizes an upstream mRNA-specific sequence in caspase-7 transcripts. 3’C7 (tttgcttactccacggttcc, bp 663

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81 682) recognizes a downstream caspase-7 mRNA-specific sequence. GAPDH-specific primers: refer to GeneBank locus AF106860. The upstream primer is designated 5’GPD (ggctgccttctcttgtgac, bp 903-921) and the downstream primer is designated 3’GPD (ggccgcctgcttcaccac, bp 1624-1641). Primer sets were identified using the Primer3 software from the Whitehead Institute for Biomedical Research (Rozen, 1998). Standard PCR A PCR reaction buffer was added to 2 L of reverse transcription product for a final volume of 25 L containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl 2 , 200 M dNTPs, 0.5% DMSO, and 1.25 units Taq DNA polymerase (GibcoBRL). Each denaturation, annealing, and extension step was held for 30 seconds (two cycles of 95, 65C, and 72C; then two cycles of 95C, 62.5C, and 72C; then 32 cycles of 95C, 60C, and 72C). Aliquots of PCR products were loaded onto 1.5% agarose gels and separated by electrophoresis in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 7.5) containing 5 g/mL ethidium bromide. To assay for genomic DNA contamination, RNA samples underwent PCR amplification without prior reverse transcription. Any samples showing genomic contamination underwent repurification and repeat assay for genomic contamination prior to PCR analysis for transcript expression. Semi-quantitative/LightCycler PCR Real-time PCR was performed using a LightCycler rapid thermal cycler system (Roche Diagnostics) according to the manufacturer’s instructions. Reactions were performed in a 10 L volume with 0.5 M primers and 2.5 mM MgCl2. Other reagents including nucleotides, FastStart Taq DNA polymerase, and buffer will be used as provided in the LightCycler-FastStart DNA Master SYBR Green I reaction mix (Roche Diagnostics). The amplification protocol included a 5-minute 95C denaturation; one

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82 cycle with 95C denaturation for 5 seconds, 65C annealing for 10 seconds, and 72C extension for 35 seconds; one cycle with 95C denaturation for 5 seconds, 62.5C annealing for 10 seconds, and 72C extension for 35 seconds; then 30-40 cycles of 95C denaturation for 5 seconds, 60C annealing for 10 seconds, and 72C extension for 35 seconds. Detection of the fluorescent product occurs at the end of the 72C extension periods. Specificity of the amplification product from each primer pair was confirmed by melting curve analysis of the PCR product and subsequent gel electrophoresis. Quantification was performed by online monitoring for identification of the exact time point at which the logarithmic linear phase can be distinguished from the background (crossing point). The crossing point was expressed as a cycle number. Standard Curve Preparation and Semi-Quantitative PCR Analysis Total RNA from ipsilateral cortex and hippocampus was collected 6 hours and 1 day after injury, respectively, and then reversed transcribed into cDNA and serially diluted to generate a standard curve of relative amounts of RNA. Samples underwent analysis using the LightCycler PCR with primer sets for caspase-12, caspase-7 and GAPDH mRNA. Aliquots of each cDNA dilution sample (100%, 33.3%, 11.1%, and 3.7%) underwent real time PCR for each primer pair (caspase-12, caspase-7 or GAPDH-specific). PCR analysis yielded a crossing point cycle number for each dilution for each transcript-specific primer set at which the PCR amplification entered the log-linear region. After the standard curves were generated by plotting the log concentration of total RNA versus the crossing point cycle number, a linear regression analysis was performed. Using the standard curve for each primer set, the amount of caspase-12, caspase-7, or GAPDH mRNA was determined. The amount of each transcript in craniotomy-injured

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83 animals was set at 100%, and the level of expression in an impact injured sample was calculated as a percent of craniotomy-injured expression. Immunoblot Analysis Protein concentrations of the subcellular cell lysate homogenate fractions were determined by the Detergent Compatible (DC) Assay for Protein (Bio-Rad Laboratories) with albumin standards. Aliquots (100 200 g) of each sample was prepared for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) by addition of 8X loading buffer (1X loading buffer contains 125 mM Tris-HCl (pH 6.8), 100 mM 2-mercapto-ethanol, 4% SDS, 0.01% bromophenol blue, and 10% glycerol). Samples with loading buffer were heated for 10 minutes at 96C, centrifuged for one minute at 10,000g, and then resolved by SDS-PAGE on 4-20% Tris/glycine gels (Invitrogen Life Technologies) at 150 V for 90 minutes at room temperature. Following electrophoresis, the fractionated proteins were transferred to Immobilon-P polyvinylidene fluoride (PVDF) membrane (Millipore) by the semi-dry method in a transfer buffer containing 39 mM glycine, 48 mM Tris, and 5% methanol at 20 V for 2 hours at room temperature. Ponceau Red (Sigma-Aldrich) was used to stain membranes to confirm successful transfer of protein and to insure that an equal amount of protein was loaded in each lane. Blots were blocked for 1 hour at room temperature in 5% nonfat dry milk in TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.003% Tween-20, pH 7.5). Immunoblots were probed with either anti--actin monoclonal (Sigma-Aldrich Co.) to confirm equal amounts of protein loading, anti-caspase-12-specific antibody (a kind gift from Drs. Michael Kalai and Peter Vandenabeele at the Flanders Interuniversity Institute for Biotechnology, University of Ghent, Ghent, Belgium) or anti-caspase-7 antibody (BD Biosciences Pharmingen).

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84 Following overnight incubation at 4C (at 1:1000 for anti--actin and anti-caspase-7, and 1:3000 for anti-caspase-12 primary antibodies) in 5% blocking solution (nonfat dry milk/TBST) for anti-caspase-7 and anti--actin and in 3% blocking solution plus 1% bovine serum albumin for caspase-12 and three washes, blots were incubated for 1 hour at room temperature in 5% blocking solution containing a biotinylated-conjugated goat-anti-rabbit IgG (1:1000; Amersham Life Science, Inc.) for anti-caspase-7 and anti--actin and in 3% blocking solution containing a biotinylated-conjugated goat-anti-rabbit IgG (1:5000, Amersham Life Science, Inc., Arlington Heights, IL, USA) for anti-caspase-12. Blots were then incubated for 30 minutes at room temperature in blocking solution containing a streptavidin alkaline phosphatase conjugate (1:3000 for anti-caspase-7 and anti--actin and 1:5000 for anti-caspase-12, Amersham Life Science, Inc.). Bound antibodies were visualized at room temperature by color development with BCIP/NBT Phosphatase Substrate (Kirkegaard & Perry Laboratories). Positive control, in the PC12 cells/caspase-7 analysis was camptothecin treated Jurkat cell lysates (20 L; BD Biosciences Pharmingen). -actin was directly assessed as an internal methods control. Since -actin may itself be cleaved by caspases, blots were examined for cleaved fragments, but no fragments were detected. Test for Anti-caspase-7 Antibody Specificity Aliquots of 1, 10, 25, and 50 ng of caspase-7 and caspase-3 human recombinant (Chemicon) were prepared and resolved by SDS-PAGE on 4-20% Tris/glycine gels before being transferred to PVDF blot. The blots were probed by anti-caspase-7 antibody (1:1000; BD Biosciences Pharmingen) and anti-caspase-3 antibody (1:1000; Cell Signaling). For details see Immunoblot Analysis above.

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85 Immunohistochemistry Immunohistochemistry Preparation Brain tissues were collected from animals that were uninjured, with craniotomy or with 1 day (caspase-12) and 5 day (caspase-7) TBI. At the appropriate time-point, the animals were anesthetized using 4% isoflurane in a carrier gas of 1:1 O 2 /N 2 O (4 min.). The animals were transcardially perfused with 200ml 2% heparin (Elkins-Sinn, Inc.; Cherry Hill, NJ) in 0.9% saline (pH 7.4) followed by 400ml 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4) and then subsequently sacrificed by decapitation and the brains were removed. A total of 2 hours in fix was followed by storage in either PBS or cryoprotection buffer. Vibratome cut 40-micron sections were fluorescent immunolabeled with cell type specific monoclonal antibodies, the anti-caspase-12 antibody or the anti-caspase-7 antibody, and the nuclear counterstain DAPI. Analysis Briefly, tissue sections or cell cultures were rinsed in PBS then incubated for 1 hour at room temperature in 2% (caspase-12) or 10% (caspase-7) goat serum/2% (caspase-12) or 10% (caspase-7) horse serum/0.2% Triton-X 100 in TBS (block) to decrease non-specific labeling. The sections were then incubated with the primary antibody: the anti-caspase-12 (IN) antibody (Cell Signaling, Technologies, Beverly, MA) or the anti-caspase-7 antibody (BD Biosciences Pharmingen), at a concentration of 1:500, and either the mouse anti-neuron-specific nuclear protein antibody (neuronal nuclei – anti-NeuN) (Chemicon) at a concentration of 1:1000 or the mouse astrocyte-specific anti-glial fibrillary acidic protein antibody (anti-GFAP) (Roche Molecular Biochemicals, Mannheim, Germany) at a concentration of 1:1000 for 4 days in block at 4C. After being rinsed in TBST, the tissue sections were incubated with species-specific Alexa Fluor (Molecular Probes, Inc.,

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86 Eugene, OR) secondary antibodies at a concentration of 1:3000 in block for 1 hour at room temperature. The sections were then washed in PBS, cover slipped in Vectashield with DAPI (Vector Laboratories), viewed and digitally captured with a Zeiss Axioplan 2 microscope equipped with a Spot Real Time (RT) Slider high resolution color CCD digital camera (Diagnostic Instruments, Inc.). Tissue sections without primary antibodies were similarly processed to control for binding of the secondary antibodies. Appropriate control sections were performed and no specific immunoreactivity was been detected. Statistical Analyses For the immunoblots, semi-quantitative analysis was performed by computer-assisted densitometric scanning (ImageJ, version 1.29x, NIH, USA). Data were acquired as integrated densitometric values and transformed to percentages of the densitometric levels obtained from nave animals visualized on the same blot. One-way ANOVA with Dunnett’s multiple comparison tests was performed on the data using GraphPad Prism Version 3.03 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). The one-day and seven-day densitometric values for sham-injured animals were pooled after analysis by one-way ANOVA with Dunnett’s multiple comparison tests for each specific day revealed no statistical differences. All values are given as mean SEM. Differences were considered significant if p<0.05. Verification that the semi-quantitative PCR method yielded results similar to Northern blot analysis was shown in a previous paper by our laboratory (Tolentino et al., 2002). In summary, the Northern blot analysis demonstrated a temporal profile of transcript induction in response to cortical injury. A linear regression analysis was performed comparing mRNA expression determined by PCR and Northern blot and the results from Northern blot and PCR analyses fit a linear correlation with a slope = 1.01, r 2

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87 = 0.95. A semi-quantitative PCR strategy that independently measures mRNA transcript levels has two major advantages over Northern blot analyses: 1) PCR amplification allows for detection of much lower levels of transcript expression, which is important for the analysis of caspase-12 mRNA expression in the brain; and 2) a PCR-based approach decreases the amount of total RNA required for analysis, thereby facilitating analysis of smaller tissue samples and obviating the need to pool RNA samples prior to analysis. One-way ANOVA with Dunnett’s multiple comparison tests was performed on the semi-quantitative PCR data using GraphPad Prism Version 3.03 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). The data were normalized using logarithmic transformation. All values are given as mean SEM. Differences were considered significant if p<0.05. Preparation of Novel Fragment-Specific Antibodies Rat caspase-12 isoform-specific antibody was designed and tested. The peptide was selected from the amino acid sequence that was determined from the cloned and sequenced additional exon bases (Figure 4-5). Based on this knowledge synthetic custom peptides representing the amino acid sequences that were caspase-12 isoform-specific (D-S-E-K-E-I-V-N-K-N-E-G-Q-D-L-L-K-Q-C 12 -Cys-OH) were generated (Califormia Peptide). A 12-carbon linker (C 12 = 11-aminoundecanoic acid) and a C-terminal cysteine residue were introduced for the subsequent coupling to the KLH (keyhole limpet hemocyanin) carrier protein using a sulfo-link crosslinking reagent (Pierce). Following coupling efficiency determinations, peptides were dialyzed and concentrated, and 2 mg of conjugated protein was used for multiple antigen injections into 2 rabbits. After 3 months, serum samples were collected and subjected to affinity purification using the custom peptide coupled to sulfo-linked resins (Pierce). The affinity-purified antibody was

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88 dialyzed against TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl), concentrated, and stored in 50% glycerol at -20 o C. Confirmation of the specificity of the antibodies employed Western blots comparing tissue lysates from rats subjected to TBI (2 days post-TBI) with tissue lysates from nave rats. Clone and sequence Caspase-12 cDNA in rat. Primer Selection Caspase-12 isoform-specific primers: For all base pair designations refer to GenBank Y13090, mus . 5’ P1 (ggctctcatcatctgcaaca) 3’ P2 (ccaggtgaactgacccagat); 5’ anti-P1 (ccaggtgaactgacccagat) 3’ UAP: provided by Clontech from their kit (oligo-dT); 5’ P3 (gacacatgaaagagatccaatctacaag) 3’ P4 (ccagatgttcttcatgatgacactatcttc). Primer sets were identified using the Primer3 software from Whitehead Institute for Biomedical Research (Rozen, 1998). Total RNA Isolation from Tissue Tissue was homogenized in 2 mL TRIzol (50-100 mg tissue); transferred to 15 mL conical tube and brought to total volume of 5 mL with TRIzol. Tubes were spun at 6,000g for 10 minutes at 4 o C and supernatant transferred to new 15 mL tube. 1 mL chloroform (0.2 mL CHCl 3 /1 mL TRIzol used for homogenization) was added, shaken by inverting for 30 seconds, incubated at room temperature 5 minutes, then spun at 6,000g for 15 minutes at 4 o C. Aqueous phase was transferred to new tube, 2.5 mL isopropanol (0.5 mL isopropanol/1 mL TRIzol use for homogenization) was added, and then incubated for 30-60 minutes at o C. The samples were transferred from 15 mL to 1.5 mL microcentrifuge tubes and centrifuged at 14,000g for 15 minutes at 4 o C. The supernatant was removed, dried in speed vacuum and pellets resuspended in 100 L DEPC H 2 O/tube by alternately heating to 50 o C and vortexing until pellet dissolves. The

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89 combined samples were quantitated using a dilution of 2 L in 98 L DEPC H 2 O with the Bio-Rad SmartSpec 3000. Reverse Transcription-PCR and Cloning of PCR Product Using the total RNA isolated above, 3 g was reversed transcribed with the GIBCO-BRL SuperScript Preamplification System (Life Technologies) in 20 L of reaction. The resulting cDNA was amplified by PCR with the time and reaction temperature conditions estimated to be optimal for 35 cycles. Primers to amplify the rat and human caspase-12 were selected from the mouse caspase-12 cDNA sequence (GenBank sequence Y13090). The amplified cDNA was analyzed in 1% agarose electrophoretic gels containing 3.75 L of ethidium bromide. UV light gel images were captured and analyzed by the Alpha Imager 2200 Documentation and Analysis System. The housekeeping gene GAPDH was used as the PCR positive control because its levels tend to remain constant for different tissues and in different model organisms. The PCR products were colony purified or gel purified and subcloned using the pCR2.1 -TOPO TA cloning vector system (Invitrogen) following the supplier’s recommendations. TOPO TA Cloning The PCR products were TOPO TA cloned as per the recommendations of the manufacturer (Invitrogen). The clones were plated on LB plus ampicillin and the bacteria were grown overnight. Individual colonies were selected from the plate, grown in liquid LB plus ampicillin, which is then used as a template for PCR using vector specific primers. The PCR products were confirmed by agarose gel electrophoresis before preparation for sequencing.

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90 Sequenced Results on CEQ 2000 DNA Analysis System Following colony or gel purification, the PCR products were purified by solid phase reversible immobilization (SPRI). Sequencing reaction was prepared by pipetting the CEQ Dye Terminator Cycle Sequencing Kit (Beckman Coulter) reagents into a thin-wall thermal cycling tube along with an aliquot of the cDNA template and the selected primers. The thermal cycling program of 30 cycles was used as described by the supplier’s protocol. In order to remove excess dye terminators the PCR product were centrifuged through G-75 sephadex in a liquid slurry, vacuum dried and then resuspended in deionized formamide. The samples were transferred to the appropriate wells of the sample plate and sequenced in the CEQ 2000 DNA Analysis System. Automated four-color detection, data analysis and base calls of the sequencing reaction product in each capillary was performed and viewed using the supplier-supplied software CEQ Data Analysis Module (Beckman Coulter). Extending Sequence Using the 3’-Rapid Amplification of cDNA Ends (RACE) To facilitate the recovery of additional coding sequences of the cDNA an in vitro method of rapid amplification of cDNA ends was undertaken (Figure 5-1). Using the supplier’s recommendations for the SMART RACE cDNA Amplification Kit (Clontech), the 3’-rapid amplification of cDNA ends was initiated. It began with the creation of a 3’-RACE-Ready first strand cDNA from the total RNA isolated from the selected rat. First strand cDNA synthesis is initiated at the poly(A) tail of mRNA using the adapter primer (AP). After first strand cDNA synthesis, the original mRNA template is destroyed with RNase H, which is specific for RNA:DNA heteroduplex molecules. Amplification was performed, without intermediate organic extractions or ethanol precipitations, using two primers: one is based upon the partial sequence obtained from

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91 previous work annealed to a site located within the cDNA molecule; the other was a universal amplification primer that targets the mRNA of the cDNA complementary to the 3 end of the mRNA. The universal amplification primer (UAP) provided with the system is designed for the rapid and efficient cloning of RACE products using the uracil DNA glycosylase cloning method. The cDNA was PCR amplified according to the supplier’s recommended protocol using a PCR touchdown thermal cycling program. The annealing temperature and the number of cycles depended on the T m for the primers and the abundance of the template to be amplified. Figure 5-1: Summary of the 3’ RACE system procedure.

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CHAPTER 6 CONCLUSION AND FUTURE DIRECTIONS Traumatic brain injury is a serious health issue in the United States as well as other nations. According to the Centers for Disease Control and Prevention, USA, traumatic brain injury is frequently referred to as the silent epidemic because the problems that result from it (e.g., impaired memory) often are not visible. The tragedy is that despite about 2% of the United States population suffering from some form of injury impairment there is, as of June, 2004, no known pharmacological treatment available. TBI causes progressive neuronal degeneration resulting from acute and delayed cell death in part by apoptotic inducing caspases. Programmed cell death, apoptosis, a conserved active molecular process often requires active transcription and translation of proteins for initiation of the molecular program that eradicates excess or potentially dangerous cells. This regulated program allows multicellular organisms to tightly control cell numbers, tissue size and self protection from rogue cells that threaten homeostasis. The study of apoptosis has recently taken a remarkably new direction. Two widely studied apoptotic pathways have previously been implicated in programmed cell death following TBI – the intrinsic or mitochondrial pathway requiring caspase-9 and the extrinsic or receptor-mediated signal transduction pathway requiring Fas or TNFand caspase-8 or -10. The discovery that caspase-12 is linked to the endoplasmic reticulum (ER) and activated only upon ER perturbation suggests a third apoptotic pathway. One important consequence of the loss of ER calcium homeostasis following central nervous system injury is the activation of the unfolded protein response (UPR), 92

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93 which determines whether cells survive or undergo apoptosis. This activation induces and activates caspase-12 and, it is believed, apoptotic cell death. As I have discussed in chapters 2 and 3, data from our laboratory suggest that caspase-12 and caspase-7 are involved in this process where caspase-7 may play a role in the activation of caspase-12 and then in turn may be activated by caspase-12. Our published data (as presented in Chapter 2) show that caspase-12 is up-regulated and processed following TBI. Currently we are in the process of publishing the data, discussed in Chapter 3, showing a similar response by caspase-7. Caspase-7 involvement is an exceptionally novel finding since this caspase has been generally characterized as not being present in the brain or at least not active. Chapters 2 and 3 of this dissertation critically examined the participation of two members of the caspase family in rats following traumatic brain injury. While others have shown the interaction of caspase-12 and caspase-7 with the finding that caspase-7 activates caspase-12 (Rao et al., 2001) the data of this dissertation are the first to show that they are activated following brain trauma in rats. For caspase-12 the results showed caspase-12 is up-regulated and processed in rat brains after TBI. This study examined rat caspase-12 expression using the controlled cortical impact TBI model. Immunoblots of fractionalized cell lysates found elevated caspase-12 proform and processed form with peak induction observed within 24 hours post-injury in the cortex (418% and 503%, respectively). Hippocampus caspase-12 proform induction peaked at 24 hours post-injury (641%), while processed form induction peaked at 6 hours (620%). Semi-quantitative RT-PCR analysis confirmed elevated caspase-12 mRNA levels after TBI. Injury severity of 1.6 mm compression was associated with increased caspase-12 mRNA expression,

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94 peaking at 5 days in the cortex at 1,259% and at 6 hours in the hippocampus at 460%. Immunohistochemical analysis revealed caspase-12 induction in neurons in both the cortex and hippocampus, as well as in astrocytes at the cortical contusion site. The time series for our study of cortical mRNA levels ended after 5 days at their highest point, additional research is required to understand the reason for the continued elevated levels when the protein levels have ostensibly returned to normal, how far into the future it continues to remain elevated, and the consequences of these elevated levels. For caspase-7 our data also showed the up-regulation and activation of caspase-7 in rat brains after TBI. This study also used the controlled cortical impact TBI model. Immunoblots of fractionalized cell lysates found elevated caspase-7 proform, pre-active, and active forms with peak induction observed at 3 days, 7 days, and 5 days post-injury, respectively in the cortex (131%, 371% and 407%, respectively). Hippocampus caspase-7 proform induction remained relative stable but peaked at 5 days post-injury for the pre-active and active forms at 1,519% and 766%, respectively. Semi-quantitative RT-PCR analysis confirmed elevated caspase-7 mRNA levels after TBI. Injury severity of 1.6 mm compression was associated with increased caspase-7 mRNA expression, peaking at 5 days in the cortex at 515% and at day 1 in the hippocampus at 650%. Immunohistochemical analysis revealed caspase-7 induction in neurons and astrocytes in both the cortex and hippocampus. The time series for our study of cortical mRNA levels ended after 5 days at their highest point suggesting that additional research is required, like for caspase-12, to understand the reason for the continued elevated levels when the protein levels have ostensibly returned to normal, how far into the future it continues to remain elevated, and the consequences of these elevated levels. This is relevant when we

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95 consider the fact that caspase-7, normally considered a downstream executioner, targets caspase-12, an upstream initiator, for activation yet we found the activated protein levels of caspase-7 peaking after caspase-12. This seemingly contradictory pattern of activation requires serious future study. One of the known properties of TBI is that the lesion size that forms after injury has been reported to continue to increase in size over the life of the patient. If there is really a feedback loop between caspase-12 and caspase-7 this is important to understanding the neuropathological progression of the disease and to offer the researchers a therapeutic target for treatment. An added complexity that increases the difficulty of studying the caspase-12/caspase-7 story is the number of alternative spliced mRNAs that may exist for each caspase. Each isoform may perform differently and may even have opposing reactions to trauma as was discussed in chapter 4 for several other caspases including caspase-2. As was pointed out in chapter 3, caspase-7 has been reported to have 3 isoforms. To date no one has examined the individual role of any of these three. Caspase-12 in humans has been cited to have 9 isoforms. For the rat, our model TBI organism, it appears that a second isoform has been uncovered as was discussed chapter 4, but that is suggestive that there may be more. When you begin to consider the possible combination of reactions that may occur among the different isoforms the difficulty of the study can only then be appreciated. The novel and important finding of this dissertation is that the systematic examination of the response of caspase-7 and caspase-12 in relation to the unfolded protein response following ER stress due to traumatic brain injury in rat is critical to understanding programmed cell death. TBI has multifactor characteristics including

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96 primarily mechanical deformation and secondary hypoxia and ischemia, all of which could result in the release of excitotoxic levels of glutamate and like agents. Since apoptosis occurs following TBI in animals and humans, understanding the biochemical and molecular mechanisms of apoptotic cell death is important to finding the means to assess and treat patients with pathological traumatic brain injury.

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APPENDIX A RAT CASPASE-12 ISOFORM GENETIC CDNA SEQUENCE NM_130422_Rattus_norvegicus CCGGGCAGGTCTGCC ATG GCTGCCAAGAGAACACATGAAAGGGATCCAAT “In House” Rat Casp12 Iso -----------------------------GACACATGAAAGAGATCCAAT *********** ******** Start Codon (ATG) NM_130422_Rattus_norvegicus CTACAAGATCAAAGGTTTGGCCAAGGACATGCTGGATGGAGTTTTTGATG “In House” Rat Casp12 Iso CTACAAGATCAAAGGTTTGGCCAAGGACATGCTGGATGGAGTTTTTGATG ************************************************** NM_130422_Rattus_norvegicus ACCTGATGGACAAAAATGTTTTAAATGGCGATGAGTTACTCAAAATCGGA “In House” Rat Casp12 Iso ACCTGATGGACAAAAATGTTTTAAATGGCGATGAGTTACTCAAAATCGGA ************************************************** NM_130422_Rattus_norvegicus GAAGGAGCGAGCTTAATCCTGAGCAAAGCTGAGAACCTGGTTGAAAGTTT “In House” Rat Casp12 Iso GAAGGAGCGAGCTTAATCCTGAGCAAAGCTGAGAACCTGGTTGAAGGTTT ********************************************* **** NM_130422_Rattus_norvegicus CTTTGAGAAAACAGAAATGGCAGGAAAAATATTTGCAGGTCACATTGCCA “In House” Rat Casp12 Iso CTTTGAGAAAACAGAAATGGCAGGAAAAATATTTGCAGGTCACATTGCCA ************************************************** NM_130422_Rattus_norvegicus ATTCCGACAAACAGCTGAGTTTACAATTTCCTTCTGATGATGAAGAGGAT “In House” Rat Casp12 Iso ATTCCGACAAACAGCTGAGTTTACAATTTCCTTCTGATGATGAAGAGGAT ************************************************** NM_130422_Rattus_norvegicus GAACTTCAGAAAATGTTTACACCATCTTCTGCATCAG-----------“In House” Rat Casp12 Iso GAACTTCAGAAAATGTTTACACCATCTTCTGCATCAGATTCTGAAAAAG ************************************* NM_130422_Rattus_norvegicus -------------------------------------------------- “In House” Rat Casp12 Iso AAATTAAAGTCAACAAAAATGAGGGACAAGATTTATTGAAGCAGATAATG NM_130422_Rattus_norvegicus ----------------AATCCAGGGGAAAAGTAGAAGATGAAGAAATGGA “In House” Rat Casp12 Iso CCTTTTTCTCTGATGGAACCCAGGGGAAAAGTAGAAGATGAAGAAATGGA ** ******************************* NM_130422_Rattus_norvegicus GGTAAATGTTGGAGTGGCCCATGCATCACATCTAATGCTTACAGTTCCTC “In House” Rat Casp12 Iso GGTAAGTGTTGGAGTGGCCCATGCATCACATCTAATGCTTACAGTTCCTC ***** ******************************************** NM_130422_Rattus_norvegicus AGGGAATCCAGAGCACAGAAGTACAGGATTCACTGAAGCTATGTTCTCGT “In House” Rat Casp12 Iso AGGGAATCCAGAGCACAGAAGTACAGGATTCACTGAAGCTATGTTCTCGT ************************************************** NM_130422_Rattus_norvegicus GATTGGTTTTGTACGATGAAGACAGAAAGGGCAGAAGAGATATATCCAGT “In House” Rat Casp12 Iso GATTGGTTTTGTACGATGAAGACAGAAAGGGCAGAAGAGATATATCCAGT ************************************************** NM_130422_Rattus_norvegicus GATGGAGAAGGAAGGCCGAACCCGCCTGGCCCTCATCATCTGCAACAAAA “In House” Rat Casp12 Iso GATGGAGAAGGAAGGCCGAACCCGCCTGGCTCTCATCATCTGCAACAAAA ****************************** ******************* NM_130422_Rattus_norvegicus AGTTTGACTATCTTTTTGATAGAGATGATGCTGAGACTGACATTTTGAAC “In House” Rat Casp12 Iso AGTTTGACTATCTTTTTGATAGAGATGATGCTGAGACTGACATTTTGAAC ************************************************** 97

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98 NM_130422_Rattus_norvegicus AGTTTGACTATCTTTTTGATAGAGATGATGCTGAGACTGACATTTTGAAC “In House” Rat Casp12 Iso AGTTTGACTATCTTTTTGATAGAGATGATGCTGAGACTGACATTTTGAAC ************************************************** NM_130422_Rattus_norvegicus ATGAAAGAACTACTTCAAAATCTTGGATACTCAGTGGTGATAAAGGAAAA “In House” Rat Casp12 Iso ATGAAAGAACTACTTCAAAATCTTGGATACTCAGTGGTGATAAAGGAAAA ************************************************** NM_130422_Rattus_norvegicus CCTTACAGCTCAGGAAATGGAGACAGAATTAATGAAGTTTGCTGGCCGTC “In House” Rat Casp12 Iso CCTTACAGCTCAGGAAATGGAGACAGAATTAATGAAGTTTGCTGGCCGTC ************************************************** NM_130422_Rattus_norvegicus CAGAGCACCAGTCCTCCGACAGCACATTCCTGGTCTTTATGTCCCACGGC “In House” Rat Casp12 Iso CAGAGCACCAGTCCTCCGACAGCACATTCCTGGTCTTTATGTCCCACGGC ************************************************** NM_130422_Rattus_norvegicus ATCCTGGAAGGAATCTGTGGGGTGAAGCACAGAAACAAAAAGCCAGATGT “In House” Rat Casp12 Iso ATCCTGGAAGGAATCTGTGGGGTGAAGCACAGAAACAAAAAGCCAGATGT ************************************************** NM_130422_Rattus_norvegicus TCTTCATGATGACACTATCTTCACCATTTTCAACAATTCTAACTGTCCGA “In House” Rat Casp12 Iso TCTTCATGATGACACTATCTTCACCATTTTCAACAATTCTAACTGTCCGA ************************************************** NM_130422_Rattus_norvegicus GTCTGAGAAACAAACCCAAGATTCTCATCATGCAAGCCTGCAGAGGCAGA “In House” Rat Casp12 Iso GTCTGAGAAACAAACCCAAGATTCTCATCATGCAAGCCTGCAGAGGCAGA ************************************************** NM_130422_Rattus_norvegicus CATACTGGTACTATTTGGGTATCCACAAGCAAAGGGATAGCCACTGCTGA “In House” Rat Casp12 Iso CATACTGGTACTATTTGGGTATCCACAAGCAAAGGGATAGCCACTGCTGA ************************************************** NM_130422_Rattus_norvegicus TACAGATGAGGAATGTGTGTTGAGCCATAGATGGAATAATAGTATAACAA “In House” Rat Casp12 Iso TACAGATGAGGAATGTGTGTTGAGCCATAGATGGAATAATAGTATAACAA ************************************************** NM_130422_Rattus_norvegicus AGGCCCATGTGGAGACAGATTTCATTGCTTTCAAATCTTCTACCCCACAT “In House” Rat Casp12 Iso AGGCCCATGTGGAGACAGATATCATTGCTTTCAAATCTTGTACCCCACAT ******************** ****************** ********** NM_130422_Rattus_norvegicus AACATTTCTTGGAAGGTAGGCAAGAGTGGCTCTCTCTTCATTTCCAAACT “In House” Rat Casp12 Iso AACATTTCTTGGAAGGTAGGCAAGAGTGGCTCTCTCTTCATTTCCAAACT ************************************************** NM_130422_Rattus_norvegicus CATTGACTGCTTCAAAAAGTATTGTTGGTGTTATCATTTGGAAGAAATTT “In House” Rat Casp12 Iso CATTGACTGCTTCAAAAAGTATTGTTGGTGTTATCATTTGGAAGAAATTT ************************************************** NM_130422_Rattus_norvegicus TCCGAAAGGTTCAATACTCATTTGAGGTCCCAGGTGAACTGACCCAGATG “In House” Rat Casp12 Iso TCCGAAAGGTTCAATACTCATTTGAGGTCCCAGGTGAACTGACCCAGATG ************************************************** NM_130422_Rattus_norvegicus CCCACCATTGAGAGAGTATCTATGACACGCTACTTCTACCTTTTCCCTGG “In House” Rat Casp12 Iso CCCACCATTGAGAGAGTATCTATGACACGCTACTTCTACCTTTTCCCTGG ************************************************** NM_130422_Rattus_norvegicus AAAT TAA CACAGCCTACTCTTGCAAATAAAT------------------“In House” Rat Casp12 Iso AAAT TAA CACAGCCTACTCTTGCAAATAAATGTATGTTAACATGTATTAT ******************************* Stop Codon (TAA) “In House” Rat Casp12 Iso TTCTTGTTAATAAATATATGTGAGGAGTGGATCTGGGAACCCATTTATAG “In House” Rat Casp12 Iso TCTGTATAAAATCTGAATAGACTAGAAAATGATGGTGATCCACCAGCTTC “In House” Rat Casp12 Iso CTGTTACTGTGGATATTACTTAATGAACCTGGGGAAAACCTTGGTTTTAT “In House” Rat Casp12 Iso TTGCTTTCATTGTAGTATAATGAGTGCATTGTTAGAAATCATTAGTAGGG “In House” Rat Casp12 Iso GTTGGGAATTTAGCTCAGTGGTAGAGCGCTTGCCTAGCAAACTCAAGGCC “In House” Rat Casp12 Iso CTGGGTTCGGTCCCCAGCTCCGAAAAAAAAGAAAAA Poly(A) Tail

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APPENDIX B RAT CASPASE-12 ISOFORM PROTEIN SEQUENCE The amino acids in blue at the beginning of the sequence represent the amino acids of the uncompleted “In house” sequence. The amino acids in red in the middle represent the additional exon for this isoform. The underlined amino acids SHG and QACRG are the enzymatic site. The bold amino acids KT represent the suggested beginning of the large active subunit. Caspase-12 Published and “In House” Isoform Protein Sequence Alignment Publ. Rat Casp12 cDNA MAAKRTHERDPIYKIKGLAKDMLDGVFDDLMDKNVLNGDELLKIGEGASLILSKAENLVE “In house” Casp12 Isof -----THERDPIYKIKGLAKDMLDGVFDDLMDKNVLNGDELLKIGEGASLILSKAENLVE ******************************************************* Publ. Rat Casp12 cDNA SFFEKTEMAGKIFAGHIANSDKQLSLQFPSDDEEDELQKMFTPSSAS------------“In house” Casp12 Isof GFFEKTEMAGKIFAGHIANSDKQLSLQFPSDDEEDELQKMFTPSSASDSEKEIKVNKNEG .********************************************** Publ. Rat Casp12 cDNA -------------ESRGKVEDEEMEVNVGVAHASHLMLTVPQGIQSTEVQDSLKLCSRDW “In house” Casp12 Isof QDLLKQIMPFSLMEPRGKVEDEEMEVSVGVAHASHLMLTVPQGIQSTEVQDSLKLCSRDW ***********.********************************* Publ. Rat Casp12 cDNA FCTM KT ERAEEIYPVMEKEGRTRLALIICNKKFDYLFDRDDAETDILNMKELLQNLGYSV “In house” Casp12 Isof FCTM KT ERAEEIYPVMEKEGRTRLALIICNKKFDYLFDRDDAETDILNMKELLQNLGYSV ************************************************************ Publ. Rat Casp12 cDNA VIKENLTAQEMETELMKFAGRPEHQSSDSTFLVFM SHG ILEGICGVKHRNKKPDVLHDDT “In house” Casp12 Isof VIKENLTAQEMETELMKFAGRPEHQSSDSTFLVFM SHG ILEGICGVKHRNKKPDVLHDDT ************************************************************ Publ. Rat Casp12 cDNA IFTIFNNSNCPSLRNKPKILIM QACRG RHTGTIWVSTSKGIATADTDEECVLSHRWNNSI “In house” Casp12 Isof IFTIFNNSNCPSLRNKPKILIM QACRG RHTGTIWVSTSKGIATADTDEECVLSHRWNNSI ************************************************************ Publ. Rat Casp12 cDNA TKAHVETDFIAFKSSTPHNISWKVGKSGSLFISKLIDCFKKYCWCYHLEEIFRKVQYSFE “In house” Casp12 Isof TKAHVETDIIAFKSCTPHNISWKVGKSGSLFISKLIDCFKKYCWCYHLEEIFRKVQYSFE ********:*****.********************************************* Publ. Rat Casp12 cDNA VPGELTQMPTIERVSMTRYFYLFPGN “In house” Casp12 Isof VPGELTQMPTIERVSMTRYFYLFPGN ************************** * single, fully conserved residue : conservation of strong groups . conservation of weak groups no consensus 99

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100 Caspase-12 Isoform Gene to Protein Sequence Translation T H E R D P I Y K I K G L A K D M L D G 2 acacatgaaagagatccaatctacaagatcaaaggtttggccaaggacatgctggatgga 61 V F D D L M D K N V L N G D E L L K I G 62 gtttttgatgacctgatggacaaaaatgttttaaatggcgatgagttactcaaaatcgga 121 E G A S L I L S K A E N L V E G F F E K 122 gaaggagcgagcttaatcctgagcaaagctgagaacctggttgaaggtttctttgagaaa 181 T E M A G K I F A G H I A N S D K Q L S 182 acagaaatggcaggaaaaatatttgcaggtcacattgccaattccgacaaacagctgagt 241 L Q F P S D D E E D E L Q K M F T P S S 242 ttacaatttccttctgatgatgaagaggatgaacttcagaaaatgtttacaccatcttct 301 A S D S E K E I K V N K N E G Q D L L K 302 gcatcagattctgaaaaagaaattaaagtcaacaaaaatgagggacaagatttattgaag 361 Q I M P F S L M E P R G K V E D E E M E 362 cagataatgcctttttctctgatggaacccaggggaaaagtagaagatgaagaaatggag 421 V S V G V A H A S H L M L T V P Q G I Q 422 gtaagtgttggagtggcccatgcatcacatctaatgcttacagttcctcagggaatccag 481 S T E V Q D S L K L C S R D W F C T M K 482 agcacagaagtacaggattcactgaagctatgttctcgtgattggttttgtacgatg aag 541 T E R A E E I Y P V M E K E G R T R L A 542 aca gaaagggcagaagagatatatccagtgatggagaaggaaggccgaacccgcctggct 601 L I I C N K K F D Y L F D R D D A E T D 602 ctcatcatctgcaacaaaaagtttgactatctttttgatagagatgatgctgagactgac 661 I L N M K E L L Q N L G Y S V V I K E N 662 attttgaacatgaaagaactacttcaaaatcttggatactcagtggtgataaaggaaaac 721 L T A Q E M E T E L M K F A G R P E H Q 722 cttacagctcaggaaatggagacagaattaatgaagtttgctggccgtccagagcaccag 781 S S D S T F L V F M S H G I L E G I C G 782 tcctccgacagcacattcctggtctttatg tcccacggc atcctggaaggaatctgtggg 841 V K H R N K K P D V L H D D T I F T I F 842 gtgaagcacagaaacaaaaagccagatgttcttcatgatgacactatcttcaccattttc 901 N N S N C P S L R N K P K I L I M Q A C 902 aacaattctaactgtccgagtctgagaaacaaacccaagattctcatcatg caagcctgc 961 R G R H T G T I W V S T S K G I A T A D 962 agaggc agacatactggtactatttgggtatccacaagcaaagggatagccactgctgat 1021 T D E E C V L S H R W N N S I T K A H V 1022 acagatgaggaatgtgtgttgagccatagatggaataatagtataacaaaggcccatgtg 1081 E T D I I A F K S C T P H N I S W K V G 1082 gagacagatatcattgctttcaaatcttgtaccccacataacatttcttggaaggtaggc 1141 K S G S L F I S K L I D C F K K Y C W C 1142 aagagtggctctctcttcatttccaaactcattgactgcttcaaaaagtattgttggtgt 1201

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101 Y H L E E I F R K V Q Y S F E V P G E L 1202 tatcatttggaagaaattttccgaaaggttcaatactcatttgaggtcccaggtgaactg 1261 T Q M P T I E R V S M T R Y F Y L F P G 1262 acccagatgcccaccattgagagagtatctatgacacgctacttctaccttttccctgga 1321 N * 1322 aattaacacagcctactcttgcaaataaatgtatgttaacatgtattatttcttgttaat 1381 1382 aaatatatgtgaggagtggatctgggaacccatttatagtctgtataaaatctgaataga 1441 1442 ctagaaaatgatggtgatccaccagcttcctgttactgtggatattacttaatgaacctg 1501 1502 gggaaaaccttggttttatttgctttcattgtagtataatgagtgcattgttagaaatca 1561 1562 ttagtaggggttgggaatttagctcagtggtagagcgcttgcctagcaaactcaaggccc 1621 1622 tgggttcggtccccagctccgaaaaaaaagaaaaa 1656

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APPENDIX C THE COMPLETE RAT CASPASE-12 GENE The exons are underlined with the additional exon representing the new isoform also in red. There is some discrepancy between the starting codon and the next 3 bases of the cDNA (see Appendix A) and the starting 6 bases of this gene sequence taken from the supercontig representing the entire rat CASPASE-12 gene. This is a problem the editors of the sequence at NIH GenBank need to resolve. That is beyond the scope of this report. The four DNA bases are ATCG. The N represents bases the sequencers were unable to resolve. >ref|NW_047797.1|Rn8_2321:2083888-2111000 Rattus norvegicus chromosome 8 WGS supercontig ATGTCTAAATGCTATTATTTTACA GCCAAGAGAACACATGAAAGGGATCCAATCTACAAGATCAAAGGTTT GGCCAAGGACATGCTGGATGGAGTTTTTGATGACCTGATGGACAAAAATGTTTTAAATGGCGATGAGTTAC TCAAAATCGGAGAAGGAGCGAGCTTAATCCTGAGCAAAGCTGAGAACCTGGTTGAAAGTTTCTTTGAGAAA ACAGAAATGGCAGGAAAAATATTTGCAGGTCACATTGCCAATTCCGACAAACAGCTGAGTTTAC GTGAGTAGTTCTGGCCAAAACACCATCAATTCCTTCTTTTCCACTGCCTACAAGGCTTCTATGTTTACTTCTCTTGCTCTTAAGGAAATCTAACCTCTCCATATCCTGTAGGCTGTTGCTTATCTATTTGTTTAACAGATGAAAAAGGCCCAGATTCACCCAAAGTACATATTATTCCTGAATATTTTATTTTTCCCTTAACAATTGCACCATTGATTACTCCAGGAAATTTACACATTCATTAGATTATTGTATTTCCCTGTCCTAAAACACGGTCATGAGAATACATATTATGAATACATATTAATAACGTAATAAATGACTGTGACTGATGGCAGGTACTCAGCAAATATTCAGTCATATTGATGAAGAAAGAGCAGGGATTTTTAAAAAAAATTTTATTTTCTTTTTTATTGGCTATTTTATTTGTTTACATTTCAAATGTTGTCCTCCTTCCAGATTTCCCTCTCTGCAAGCCCCCTATCCTACCACCCCTTGCCTGCCTCTATGAGGGTGCTCTCTCACCTCACCATCCCAGGATCCCCCTATACTGGGTCATCAAGCATCTACAGGACCAAGGGCCTCCCCTCCGATTGATAACAGATAATGCACTTCTCTGCTACATCTGCGGTGGGAACCATGGGGTCTCTCCATATGTAATCTTGGTGGTTTAATCCCTGGAAACTCTGGGAGGCCCAGTTAGTTGATGTTCTTCCTGTGGAGTTTCAATCCCCTTCAACACCTTCAGCTCCTGCTCCTATTCTCTTGGGGTGTCCATGCTCAGCCTTACAACAGGCTGTGAGCCCCTGTATCTCTATTGATCAGGCTCTGGCAGGGCCTCTTGGGGAACAGCTATGCCAGACTTCAGTCACTCAGAGCTTCTTGGCATCAGCAATAGTGTCTATATTTGGCATCTGCAGATGGGATGGATCCCTGGATGGGGTAGTCTCTGCATGACATTTCCTTAATTTCTGCTTCATTCTTAGTCCCTGTGTTTTTTTGAGACAGGGGCTAGACAATTCTGGGTTAAAATTTTTGAGATTTGTTGGTGGCCCCATCCCTCAACCAGGGCCTGTGCCTATCCACTAGACATTGTCTCTACAGGTTCTACCACTCCTATGTTGGGTATTTTTGTTTCCCATTGGGTCCTTGGAACCTCTTGCTTCCCTGGCATCTGGGACTTTCTAGTGGCTATCCTGAGTTCTTCATCCATCACTGCTACATACCTCCATTCAATTTCCTGATCCTCTGTACTTCTCCCCTGTCTCTTCCCACACCTGATCCTGATCCTGTCTCCCCTTTAATTGTGATCTAAGTTTCTGGTCACATCCTGGAGATTTTAGGTTCAAACAAACAAAAAATCTCCAAATTTCAAGTGTAGTGGATTATTAATTTATCTCTAAAAACAAGAAGTATTGTTAGATTTTGAGTATGGAGCTGTCGCATTCAGGCCCCAGGTTTTGTATTTTGGTAGGGCAGCATGAATCACTGAAGGTCCACGATCATACCCTAGCAGTTGATACAGAAAATGTATACGTTAATTGTTAACATTTTCATCGTGTATTTGATGCTGTATAAAAACAACTTTAATACAGTCAAGTATATCTATGTTCTTAGAAAAATTATTATTACAAAGAAAC 102

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103 TCTGGTAAAGGCTTTGTCTACATCTTTATATAAAAATCAAAGAAAAGTTTTTGTTTTATAGAATCTGTGCCTCCATTTTTTATTTGGAAAAGTCTAGGATGGTTATAAACAAAACTGAAGCTTGGAAAACATTGGAATACCCATGTTTGAAAGAGCAACTTGGGCATTTATTTATCAGTGCTTTGAGAGGGTTTTCATGCATATATGGCCTAGAAATGTTGTTGTATCTGCCCCCAAAATCTGCTGTTCAGCCCGTTCAGAGCCTGCCCCACATGTGGCCCAAACATATACAGCCACCAAAATAGATAAGATGGATGAAGTGCAAGCTGACAGGAACCAGATGTAGATCTCTCTTGAGAGACGCAGCCAGAATATGGCAAATACATAGGTGATGCCAGCAGGAAACCACTGAACTGAGAACGGGACCCTCGTTGAAGGAATCAGAGAAAGGACTGGAAGAGCTTGAAAGGTCTCAAGACCCCATATGAATAACAATGCCAACCAATCAGAGCTTCCAGAGACTAAGCCACTAACTACCCAAAGACTATACATGGACTGACCCTGGACTCTGACCTCATAGGTAGCAGTGAATAGCCTAGTAAGGGCACCAGTGGAAGGGGAAGCCCTTGGTCCTGCCAAGACTGATCCCCCAATGAACGTGATTGTTGCGGGGATGGTGGTAATGGGAGGAAGATGGGGAGGGGAACACTCATATAGAAGAGGAGGAGAANGGGGTTAGGGGGATGCTTGCCTNGNAAACCGGGAAAGGNTAATAACAACTGNAAGGNAAATAAGAAATACNNCCTNCNNTATGTATGGNGAAAAGAANNNGAAAAAAAAGAANNNNCTGCTGNNCATATTGTTTCAG AATTTCNTTCTGATGATGAAGNNNATGAACTTCTGNNAATGTTTACGCNANCNTC TGCATCAG GTNATTANNTCTATGTGACAGACTGCAACAGAAACCATTTGATCTCTAGGTTTTTGTTGGATATTTTTATATTTGCATTTCAAATGTTATCCCCTTTCTCAGTTTCCATTCATAAACACCNNATTCCATCCCCTGCCAGCCTGCTTCTATGAAGGTATTCACTCACCCACCCACCNACCCGTTCACGCCACCCTGCCCTGACATTCCCCTACACTAGGGCATAGAGCCTTGGCAGGTCTAATAGCTTCTCCTCCCATTGATGCCGAGCAGAGGCATCTTCTGGTACATATGCACCTGGAGCCATGGGTCTGTGCATGTGTACAATTTGGATGGTGGTTTAGTCCCTGGGAGCTCTAGTTTTTTTGGTATTGTTGTACTTATGGGTTAGAAACCCATTAATCTCCTTCAATCCTTTCTCTAAGTTCTCCATTGGAGACCCCATTCTCAGTTCAATGGTTGGTTGCAAGCAGGTTGGCTGCAAGCATCTTCCTCTGTATTTGTCAGGCTCTGGCAGAGTCTCTCAGGAGACAGCTATATCAGGCATCTGTCAGCATGCACTTCTTGGCATCAACAATATTGTCTGAGTTTGGGAGTTATATATGAGCTGGATCCTCAGGTGTGATAGTCTAAAAATGGCTGCTCCTTCATGCTCTGCTCCAAACTTTGTCTACATATCTCCTCCTAGGAATATTTTTGTTACCCCTTCTAAGAAGGACTAAAGCATTTGCAATTTGGTCATTCTTCTTCCTGAGCTTCATGTGGTCTATGGATTGTTTCTTGGTAATTGAGATTTTGGGCCAATAACCACTTATCAATGAGTGCATACAATATGTGTCTTTTTGCAATTGGGTTGCCTACTCAAGATTTTCTAGTTCCATCCAATTCGCTATGAATTTCTGAAGTAACTGTTGTTAATAGCTGAAAAGTATTCCTTTGTGTAAATGTATCACATTTTCTGTATCCATTTGTCTGTGGAAGGGCATCTGGATTCATTCCAACTTCTGACTATTATAAACAAGGCTGCTATGAACATAGTGGAGCATGTATCCTTGTTATATGTTGGAGCATCATTTGGTTATATGCCCAGGAGTGGCATAGCTGGGCCTTCAAGTAGTACTATGTCCAATTTTCTTTTTTTCCCATCTTTATTAAATTGGGTATTTCTTATTTATATTTCTAAGGAACCTCCAGACTGATGTCCAGAGTGGTTGTACCAGCTTGCAATCCCACCAAAATGGAGGAGTGCTCCTCCACCTCCACATACCCACCAGCATCTGTTCTCAGCTGAGTTTTGAACTTAGCCATTCTGACTGTTGTGAGGTGGAATCTCAGGGTTGTTTTGATTTGCATTTCCCTGATGACTAAGGATGTGAAACATTTCTGTAGGTGCTTCTCAGCCATACAATATTCGTCAGTGGAGATTTTTTTTTTGTTTACCTCTGTTCCCCATTTTAATGGGTTATTTGATTCTCTGAATTCTATCTTCATGAGTTCTTTGTATATATTGAATATTAGCCCTCTATCAATTGTAGGATTGGTAAAAATAGGGGTTGGGGATTTACCTCAGTGGTAGAGCACTTGCCTAGCAACCACAAGGCCCTGGGTTCAGTCCCCAGCTCCGAATTAAAAAAAAAAAAAGAGGATTGGTAACAATATGTTTACAAGCTGTTGGATGCCATTTTGTCCTAATGACAGTATCCTTTGCCTTACAGGAGTTTAGCAAATTTATGAAATCACATTTCTTGAATCTTGATCAATGAGCGTAAGCCATTGGTGTTCTGTTCAGGAAATTTTCCCCAGTGCCCATGGGTTTGAGGATGTTGCCCAGTTTTTCTTCTATTAGTTTTTGTGTATTTGGTTTTATGTGGAGGTTCTAGATTCACTTAGACTTGAGCTTTTACAGGGCAATAAGAATGGATTGTTCTGCATTCCCTACATGATGATCTGCAATTGAACCAGCACCATATGTTGAAAATACTCTTTACTCCACTGGGTGGTTTTAGCTTCTTTGTCAGAGATCAAGTGAGCATAGGTGTGTGGGGTTATTCTGGGTCTTCAATTATATTCCATTGACATACATGCCTCTTTCTGTATGAATACCATATAGCTTTTATTACAGTTGCTCTGTAATGCAGCTTGAGGTCAGGGATGGTGATTGACTGATAATTTCTTTTCTTGTTGAGAATAGGTTTTGCTATCCTGGGTTTTTTGATATTCCAAATGAAGTTGCTAATTGCTTTTTCGAACTTTATGGAGAATTTAGTGGGAATTTTAATGGGGATTGCATTGAATCTGTAGATTGCTTTTGCCAGGAAGGTCATTTTACAATTTTAATCCTGCCAATCAATGAGCATGGGAGATCTTCTTCAATGTCTTTCTCCAGAGGCTTGAAGTTCTTGTCATACAGATCTTTCACTGACTTCGTTATAGTCACACCAAGGTATTTTATATTATTTGTGATTATTGTGAAGGGTGACTTTTCCCTAATTTCTTTCTCAGCCTGTTTATCATTTGAGTAGAGGAAGGCTACTCATTGGTTTGAGTACAATCTATATCCAACCATGTTGCAGAAGTTGTTTATCATGTTTAGGAGTTCTCTGGTGGAATTTTTTGGGGTCATTTAATTGAACTATTATATCATCTGCAAAATAGTGATATTTTGAATACTTCCTTTCCAATTTGTATTCCTTTGACCTACTTTTGTTGTCTGATTGCTTTGGCTAGGGCTGCAAGTACTATATTGAATAGGTAGGGAGAGAGTGGGCAGCCTTGTCTAGTCCCTGATTTTAGTGGGATTGCTTCAAGCTTCTCTCCATTTAGTTTTATGTTGGCTACTGGTTTGCTATATATTGCTTTTACTATGTTTAGGTATAGGCCTTGAATTCCTGATCTTTCCAAAGCTTTTAACATGAAAGGGTGTTGAATTTTGTGAAATGCTTTCTCAGCATCTAATGAGATGATGATGTGTTTTTTTTCCCTTTCAGTTTGTTTATAAAGTGGATTACATTGGTGTGTTTCCATATATTAAACCATCCCTGCGTGCCTACAATGAAGTCTACTTAATCATGATGGATGATCCTTTTGAGGCGTTCTTTGATTCAGATTGTGAGAATTTT

PAGE 115

104 ATTGACTATTTTTGCTTCAATATTCATAAGGGAAATTGGTCTGAAATGCTCTTTGTTGGGTTTTTCTGTGGTCTAGGTATAAGCATAATTGTGACTTCACAGAAGGAATTGGGTAATGTTCCTTCTGTTTCTATTTTGTGGAGTAGTTTGGAGAGTATTGGTATTAAGTGTTCTACGAAGGTCTGATAGAATTCTGTACTAAACTCATCTCCTCCTGGGCTTTTAGTTGTTCTTGTTTCGGTCAACTTTAAATGACTGTTTCTATTTCTTTAGGAGTTATGGTACTGGTTTAATGGTTTATCTCTTCCTAATTTAACTTTGGTAACTGGTGCAGGTCAGGAAAATTGTCCATTTAATCCGGATTTTCCAGTTTTATTGAATATAGTCTTTTGTAGTAGGAATTCCTGATTTTTTTTTTAATTTCCTCAGATTCTGTTGTCATGTCTCTCTTTTCATGTCTGATTTTGTTAATTTAGATATTCTCTTTGTGCCCTCTAGTTAGTTTGGCTAAGGGTTTATCTATCTTGTTGATTTTTCTCACAGGATCAGATCCTGGTTTTGTCAATTCTCTCTATAGTTCTTTTTGTTTCTACTTAGTTGACTTCAGCCCTGAGATTGATTAATTCTTGCTGGCTACTCCTCTGGGTGTATTTGCCTTTTTTGTTCTAGAGCTTTTAGGCTCTCTCCAGTTTCATGTTGGAGGCATTCAGCTGCTGACACATGCTCTTTCCTGTTTCTTTCTGCAGGCACTCAGCGCTATGAGATTTCCTCTTAACACTGCTTTCATTGTGTCTCATCAGTTTGGGTATGGTGTGCCTTCATTTTCATTAAATTCTAAAAAGTCTTTAATTTCTTTCTTTATTTCTTCTCTGACCAAGATGTCATTGTGATGAGCATTGTGCAGCTTCCTGGTCCATGGCAATCTGATAGGATGCATGGGATTATTTCAATCTTCTTGTATCTGCTGAGGCCTGTTTTGTGACTAGTTTTTTTTTCTTTTCTTTTCTTTTTTTTTCTTTCTTTTTTTTTTTTTTTCGGAGCTGGGGATTGAACCTAAGGCCTTGCGCTTGCTAGGCAAGTGCTCTACCACTGAGCTAAATCCCCAACCCTTTGTGACTAGTTTTACGGTCAGTTTTTGAGAAGGTAGCATGAGGTGCTGAGAAGTAGATATATTGTTTTGTTTTAAGATGAAATGTTCTATAGATATCTGTTAAATCCATTTGTTCATAACTTTTGTTAATTTCTCTGTGTCTCTGTTTAGTTTTTATTTCCATAATCTGTCCATTGACGAGAATGGGGTGTTGATGCCACCCACTATTATTGTATGGAGTGGAATGTATACCTTGAGCTTAAGTAAGGTTTCTTTTATGGATGTAGGGGCCCTTGCATTTGGAGCATAGATGTTCAGGATTGAGAGTTCATCTTGGTGGATTTTTTCATTGATCAATATGAAGTGTCCTTCTTTAGCCTTTTTGATAACTTTTGATTGAAAGTTGATTTTATTCGATATTAGAATGGCTACACCAGCTGGTTTCTTGGGACCACATGCTTGGAAAATTGTTTCCCAGCCTTTTATTCTGATATAGGGTCTGTCTTTGTCACTAAGCTGTGTTTCTTATTTGCAGCAAAATGCTCTGTCCTATTTACATATACATTCTATTATTCTATGTCTTTTTACTGGGAAGTTGAGTCCATTGATACTGAAAGACATTATGGACCAACGATTGTTGTTTTCTGTTATTTTTGTTGTTAGGGGAGGGATTATGTTTGTGTGGCTTTCTTCTCTTGGGTTATTTGAAAGAAGATTATGTTCTTGCTTTTTGATCATGTAGTTTCCCTCATTGTGTTGGAGTTTTCCATCATTATTCTTTTTTAAGGCTGAATTTGTGGAAAGATATTTTGTAAATTTGGTTTTGTCATGGAGTATCTTGGCTTCTCCATCTATGTCCATGAGAGTTTTGCTGGGCTGAGATTTATGTTTTCTTAGAGACTGTATGACATCTGCTAAGGATCTGTTTTTCATAGTCCTTGTTGAGATGTCTGGTGTGATTCTGATACATCTGCCTTCACATAATACATGGGCTTTTTCAATTACTGCTTTTAATATTCTTTCTTTGTTTTGTCCATTTGCTGTTTTGACTACCATGTGATGATGGGAGAAATTACTTTTCTGGTCCAATCTATTTAGAGTTCTGTAGGCTTCTTGTACATTTATGGGCATACTCTGTAGGTTAGGGAAATTTTCTTCTATAATTTTGTTGAAGATATTTACTGGCGCCTTAAGTTGGGAATCATCGTTTTCTTCTATACCTATTATCTGTAGGTTTTATCTTTTCATTGTGTCTTTGATTTCCTGTATTAGGACAGGAATTTTTTCATTTTCTTTTATTTTTTTAATTTTGATATTTTTATTTACATTTCAAATGTTATTTCCTTTCCCCCTTTTCCATTTATAAGCCTCCTATCCCATCCCCATCCCCCTTTTTATAAGGGTGTTCCCCCTCCCCAAACAACCCCCTTCCTGCCTCCCCACCCTCACATTCCCCTACACTGGGGGAGTCCATCCTTGGTAGGACCAAGAGCTTCTCCTCCCATTGATGGCCAACAAGGCCATCCTCTGCTAAATGAAGCAGGAGCCATGGGTCTGTCCATTTGTAGTCTTTGTGTAGTGGTTTAGTCTCTAGGAGCTCTGATTTGTTGGTATTGTTGTTCTTACGGGGTTGCAATGCACTTCTTGGTTTCATCAATAGTGTCTAGTTTTGTTGGCTGTATATATAAGGCCTGGATCCCCTGGTGGGGGAGGTTCTGAATGGCCATTCCTTCAGTCTCTGCTCCAAATTTTGCCTCCATGTCAACTTCTATGAATACTTTTGTTCCCCCTTTTAAGGAGGACTGAAGCATCCACACTCTGGTTGTCTTTCTTCTTGTGTTTCATTTGGTCTGTAGATTGCATCTTGGGTAATTAGAGCTTTTGAGTGTTTTGCATTTTCTTTGCTTTTGACAATGATTTCTATGGTGTCTTCTTTCATGAGATTCTATCTTCTGTCTCTTGTATTCTGTTGGTAATGCTTGTGTCTATGACTCCTGTTCTCATTTCTATGTTTTCTATGTCCAGGGTTGTCTTTCATTGTGATTTCTTTATTGTTCCTATTTCAATTTTTAGATCCTGGATCATTTTTGTTCATTCCTTAATCTGTTTGTTTGTGTTTCCTATAATTCTTTAAGGGATTTTTATGATACCTCTTTAATGTGTTCTCCCTGTTTACCTATGTTGTCCTATATATCTTTCTTTCTTTTTTTTTTTTATTAACTTGAGTATTTCTTATTTACATTTCGAGTGTTATTCCCTTTCCCGTTTTCCGGGCAAACATCCGCCTAATCCCTCCCCCTCCCCTTCTTTATGGGTGTTCCCCTCTCCATCCTCCCCCCATTGCCGCCCTCCCCCCAACAATCACGTTCACTGGGGGTTCAGTCTTAGCAGGACCAAGGGCTTCCCCTTCTACTGGTGATCTTACTAGAATATTCATTGCTACCTATAAGGTCAGAGTCCAGGGTCAGTCCATGTANTAGTCTTTAGGTAGTGGCTTAGTCCCTGGAAGCTCTGATTGCTTGCCATTCTTGTTCATAAGGAGTCTCGAGCTCCTTCAAGCTCTTCCACTTCTTTCTCTGGTTCCTTCAACGGGATTCCCGTTCTCAGTTCAGTGGTTTGCTGCTGGCATTCGCCTCTGTATTTGCTGTATTCTGGCTGTGTCNNNCAGGAGCGATCTACATCCAACTACNNTCGGCCTGCACTTCNNNGCNNCATNNANCNTGNNNAATGGAAGGCTGTATAAGTATGGNCCACATGTGGGGCAGGATCTTAAGGGGTTTCCTTCAGTCTCTGTTTTAATCTTTGCCTCTCTATTCCCTGCCAAGAGTATTCTAGTTCNCCTTTTAAAGAAGGAGTGAAGCATTGACATTTTGATCATCTGTCGTGAGTTTCATTTGTTCTAGGCATCTAGGGTAATTCAAGCATTTGGGCTAATAGCCACTTATCAATGAATGCAT

PAGE 116

105 ACCATGTGTGTTTTTCTGTGATTGGGTTACCTCACTCAGGATGATATTTTCCAGTTCCAACCATTTCCCTACGAATTTCATAAAGTAATTGTTTTTGATAGCTGAGTAATATTACAATTGTGTAGATGTACCACATTTTCTGTATCCATTCCTCTGTTGAAGGGCATCTGGGTTCTTTCCAGCTTCTGGCTATTATAAATAAGGCTGCGATGAACATAGTGGAGCACGTGTCTTTTTTATATGTTGGGGCATCTTTTGGGTATATGCCCAAGAGAGGTATAGCTGGATCCTCAGGCAGTTCAATGTCCAATTTTCTGAGGAACCTCCAGACTGATTTCTAGAATGGTTGTACCAGTCTGCAATCCCACCAACAATGGAGGAGTGTTCCTCTATCTCCGCATCCTCGCCACCATTTGCTGTCACCTGAGTTTTTGAGCTTAGCCATTCTCAGTGGTGTGAGGTGAAATCTCAGGGTTGTTTTGATTTGCATTTCCCTTATCACTAAAGATGTTGAACATTTATTTAGGTGTTTCTCGGCCATTCGGCATTCCTCAGCTGTGAATTCCTTGTTTACCTCTGAACTCCATTTTTTAATAGGGTTATTTGTTTCCCTGCGGTCTAACTTCTTGAGTTCTTTGTATATTTTGGATATAAGGCCTCTATCTGTTGTAGGATTGGTAAAGATGTTTTCCCACACTGTTGGTTGCCATTTTGTCCTAACCACAGTGTCCTTTGCCTTACAGAAGCTTTGCGTTTTATGAGATCCCATTTGTCAATTCTTGATCTTAGAGCATAAGCCATTGGTGTTTTGTTCAGGAAATTTTTTCCAGTGCCCATATGTTCCAGATGCTTCCCTAGTTTTTCTTCTATTAGTTTCACTGTATCTGGTTTGATGTGGAGGTCCTTGATCCACTTGGACTTAAGTTTTGTACAGGGTGATAAGCATGGATTGATCTGCATTCTTCTACATGTTGACCTCCAGTTGAACCAGCACCATTTGCTGAAAATGCTATCTTTTTTCCATTGGATGGTTTTGGCTCCTTTGTCAAAAATCAAGAGTCCATAGGTGTGTGGGTTCATTTCTGGGTCTTCAATTCTGTTTCATTGGTCTATCTGTCTGTCTCTGTACCAATACCATGCAGTTTTTATCACTAATGCTCTGTAATATTGCTTGAGTTCAGGGAGTGATTCCCCCTGAAGTCCTTTTATTGTTGAGGATAGTTTTAGCTATCCTGGGTTTTTTGTTATTCCAGATGAATTTGCAAATTGTTCTGTCTAACTCTTTGAAGAATTGGATTGGTATTTTGATGGGGATTGCATTGAATCTGTAGATCGCTTTTGGTAAAATGGCCATTTTTACTATATTAATCCTGCCAATCCATGAGCATGGGAGATCTTTCCATCTTCTGAGGTCTTCAATTTCTTTCTTCAGTGTCTTGAAGTTCTTATTGTACAGATGTTTTACTTGCTTGGTTAAAGTCTCACCGAGGTACTTTATATTATTTGTGTCTACTATGAACGGTGTCGTTTCCCGAATTTCTTTCTCAGGTTTTTTTTTTTTTTTTTTTTTTTTTTGGGGAGAGGAAGGCTACTGATTTATTTGAGTTAATTTTATACCCAGCCACTTTGCTGACGTTGTTTATCAGCTTTAGTAGTTCTCTGGTGGAACTTTTGGGATCACTTAATTATACTATCATATCATCTGCAAATAGTGATATTTTGACTTCCTCTTTTCCGATCTGTATCCCCTTGACCTCCTTTTGTTGTCTGATTGCTTTTGCTAGAACTTCAAGAACTATATTGAATAAGTAGGGAGAGAATGGGCAGCCTTGTCTAGTCCCTGATTTTAGTGGGATTGCTTCAATTTCTCTCCATTTAGTTTAATGATAGCAACTAGTTTGCTGTATATGGCTTTTACTATGTTTAGGTATGGGCCCTGTATTCCTATTCTTTCCAGGACTTTTATCATGAAGGGGTGTTGAATTTTGTTAAATGCTTTCTCAGCATCTAATGAAATAATCATGTGTTTTTTTTCCTTTCAGTTTGTTTATATAATGGATCACGTTGATGGTTTTCCGTATATTAAACCATCCCTGCATGCCTGGGATGAAGCCTACTTGATCATGGTGGATGATTGTTTTGATGTGCTCTTGGATTTGGTGTGCCAGAATTTTATTGAGTATTTTTGCATCGATATTCATAAGGGAAATTGGTCTGAAGTTCTCTTTCTTTGTTGGGTCTTTGTGTGGTTTAGGTTTAAGAGTAATTGTGGCTTCATAGAAGGAATTCGGTAGTGCTCCTTCTGTTTCAATTTTGTGAAATAGTTTGGATAATATTGGTGTGAGGTCTTCTATGAATGTCTGATAGAATTCTGCACTAAACCCGTCAGGACCTGGGCTCTTCATTGTTGGGAGACCTTTAATGACTGCTTCTATTTCCGTAGGAGTTATGGGGTTATTTAACTCGTTTATCTCTTCCTGATTTAACTTCGGTATCTGGTATCTGTCTGGGGAATTGTCCATTTCCTCCAGATTTTCAGGTTTTGTTGAATATAGGAATTATAGTAAGATCTGATGATTTTTTGAATTTCCTCTGAATCTGTAGTTATGTCTCCCTTTTCATATCTGGCTAAGGGTTTATCTATCTTGTTGATTTTCTCAAAGAACCAACTTTTGGTTCTGTTGATTCTTTCTATGGTTCTTTTTGTTTCTACTTGGTTGATTCAGCTCTGAGTTTGATTATTTCCTGCCTTCTACTCCTCCTGGGTGTATTTGCTTCTTTTTGTTCTAGAGCTTTTAGGTGTGTTGTCAAGCTGCTGACATATGCTCTTTCCTGTTTCTTTCTGTAGGCACTCAGAGCTATGAGTTTTCCTCTTAGCACAGCTTTCATTGTGCCCCATAATTTTGGGTATGTTGCACCTTCATTTTCATTAAATTCTAAAAAGTCTTTAATTTCTTTCTTTATTTCTTCCTTGACCAGGTTATCATTGAGTAGAGCATTGTTCAATTTCCACGTATATGTGGGCATTCTTCCCTTATCGTTATTGAAGACCAGTTTTAGGCCGTGGTGGTCTGATAGCACATATGGGATTATTTCTATCTTTCTGTACCTGTTGAGGCCCGTTTTTTGACCAATTATATGGTCAATTTTGGAGAAAGTACCATGACGAGCTGAGAAGAAGGTATATCCTTTTGTTTTAGGATAGAATGTTCTATAAATATCTGTTACGTCCATTTGGCTCATGACTTCTCTTAGTCTGCCTACGTCTCTGTTTAATTTCTGTTTCCATGATCTGTCCATTGATGAGAGTGGGGTGCTGAAATCTCCTACTATTACTGTGTGAGGTGCAATGTGTGTTTTGAGCTTTAGAAAGGTTTCTTTTACGTATGTAGGTGCCCTTGTATTTGGGCATTTTAGGATTGAGAGTTCATCTTGTTGGATTTTTCCTTTGATGAATATGGTGTCCTTCCTTAACTTTTTTGATGACTTTTAGTTGAAAATTGATTTTATTTGATATTAGAATGGCTACTCCAGCTTGCTTCTTCCGACCATTTGGTTGGAAAGTTGTTTTCCAGCCTTTCACTCTGAGGTACTGTCTGTCATTGTCTTTTAGGTGTGTTTCCTGTAGGCAGCAGAATGCAGGGTCCTCGTTCATATCCAGTTTGTTAATCTATGTTTTTTTATTGGGGAGTTGAGACCATTGATGTTGAGAGATATTAAGGAATAGTGATTATTGCTTCCTGTTATATTCATATTTGGATATGAGGTTATGTTTGTGTGCTTTTCTTCTCTTTGTTTTGTTGGCAAGACGATTAGNNTCTTGCNTCTTCTATGGTATAGCTTCCCTCCTTGTGTTGGGCTTTACCATTTATTATCCTTTGTAGTGCTGGATTTGTAGAAAGATATTGTGTAAATTTGGTTTTGTCATGGAATATCTTGGTTTCTCCATCTATGNTTAATTGAGAGTTTTGCAGGATACAGTAACCTGGGCTGGCATTTTTGTTCTTTTAAGGTCTGTATGGCATCTGTCCAGGATCTTCTGGCTTT

PAGE 117

106 CATAGTTTATGGCGAAAAGTCTGGTGTGATTCTGATACGTCTGCCTTTATATGTTACTTGACCTTTCTCCCTTACTGCTTTTAATATTCTTTCTTTATTTTGTGCCTTTGGTGTTTTGACTATTATGTGACGGGAGGAGTTTCTTTNCTGGTCCAATCTATTTGGAGTTCTGTAGGCTTCTTGTATGCTTATGGGCATCTCTTTCTTTAGGTTAGGGAAGTTNNCTTCTATGACTTTGCTGAAGATTTTTACTGGTCCTTTGAGCTGGGAGTCTTCACTCTCTTCTATACCTANNNTNNTTAGGTTTGATCNTCTCATTGAGTCCTGGATTTCCTGTATGTTTTGGACCAGTAGCTTTTTCCACTTTACATTATCTTTGACAGTTGAGTCGATGATTTCTATGGAATCTTCTGCTCCTGAGATTCTCTCTTCCATCTCTTGTATTCTGTTGGTGAAGCTCGTATCTACAGCTCCTTGTCTATTCTTTTGGTTTTCTATATCCAGGGTTGTTTCCATGTGTTCTTTCTTGATTGCTTCTATTTCCATTTTTAATTCCTTCAACTGTTTGATTGTGTTTTCCTGGAATTCTTTCAGGGATTTTTGTGACTCCTCTCTATGGGCTTCTACTTGTTTATTTATGTTTTCCTGGAATTCTTTTAGGGATTTTTGCGATTCCTCTCTGTAGGCTTCTACTTGTTCTCTAAGGGATTTCTTCACGTCTTTTTTGAAGTCCTCCAGTATCATGATAAAATATGATTTTGAAACTAGATCTTGCTTTTCTGGTGTGTTTGGGTATTCCGTGTTTGCTTTGGTTGGAGAATTGGGTTCTGATGATGCCATGTAGTCTTGGTTTCTGTTGCTTGGGTTCCTGCGCTTGCCTCTCACCCTCATTCTAGTGTTACTTTGTTCTGCTATTTCTGACAGTGGCTAGACTGTCCTATAAGCCTGTGTTTCAGGAGTGCTGTAGACCTGTTTTCCTGTTTCCTTTCAGCCAGTTATGGGGACAGAGTGTTCTGCTTTCGGGCGTGTAGTTTTTCCTCTCTACAGGTCTTCAGCTGTTCCTGTGGACCTGTGTCTTGAGTTCACCGGGCAGGTCGCTTGCAGCAGAAAAGTTAGTCTTACCTGTGGTCCCGAGGCTCAAGTTTGCTCCTGGGGTGTTGCTTATGAGTTCTCTGCGGCCACAGCAACCAGGAAGATCTGGGCCGCCCTTTCCAGGAGCTTCTGTGCACCAAGGTTCCAGATGGTGTTTGGTGTTTTCCTCTGGAATCAGTAATGTGTGCAGAGTGCAGTCTCTTCTGGTTTCCCACGTGTGTCTGCCTCTCTGAAGGTTTAGCTCTCCCTCCCATGGGATTTGGGTGCAGAGAACTGTTTATCCGGTCAGTCCCTTCAGGTTCCAGTGGTGTCTCAGACGCAGTGGACCTGCTGTTCCTGGGTCCTCCTCTACGGGAACCTAGAGGCTGTATCTGTCCTATATTTCTTTAAGGGAGTTATTTATGTCTTTTTTTTCCAGGTAACTTAGTATTTATTTATTTATTTATTTATTTATTTATTTATTTATTTATTAGTTTTTTCCATCTTCATTAAATTGGGTATTTTTTATTTACATTTCAAATGTAATTTCCTTTTCCGGTTTCCAATCCAAAATTCCCCTAATCTCTCCCCCTCCCCCCTTCTTTTTAGTGTATTTATTTTTATTTTTTTCTCCATCTTTATTAAATTGGGTATTTCTCATTTACATTTCAATCGTTATTATTTTTCCCAGTTTCCAGGCCAACATCCCCCTAATCCATTCCCCTCCCCTTCTATATGGGTGTTTCCCTCCCATATACACATTGGAATACTACTCAGCTATCAAAAACAATGACTTTATGAAATTTATAGTCAAACAGATGGACTGGGAAATATCATCCTGAGTGAGGTAACCCAATCACAGAAAAATACCCATGGTATGCACTCACTGATAAGTGGATATTAGCCCAAATGCTCGAATTACCCAAGATGCATAGAACACATGAAATTCAAGAAGGATGACAAAAATGCAGATGCTTCACTCCTTCTTTAAAAGGGGAACAAAAATACCCTTGGGAGGGAATAGGGAGGCAAAGTTTAGAACAGAGGCTGAAGGAACGCCCATTAAGAGCCTGCCCCACATGTGGCCCATTAATATACAGCCACCAAACTAGATAAGACAGATGAAGCAAAGAAGTGCAGGCTGACAGGGACCGGATGTAGATCTCTCCTGAGAGACACAGCCAGAATATGGCAAATACGTAGGCAAATGCCAGCAGCAAACTACTGAACTGAGAATAGGACCCCCGTTGAAGGAATCAGAGAAAGGACTGAAAGAGCTTGAAGGGGCTTGAGACCCCATATGGACAATGCCAACCAATCAGAGCTTCCAGGGACTAAGCCACTACCCAAAGATTATATATACATGGACTAATCCTGGGCTCCAACTGCATGAATAGCCTATAAGGGCACCAGTGGAAAGGGAAGACCTTGGTCCTGCCAAGACTGAACCTCTAGTGAATGGGATTCTGGAGGGGAAAGTGGTAATGGGGGGAAGATGTGGACGGGAACACCCATATAGAAGGAAAGTCCTCTAACGTCAACATGAGTTGTGCTTTTAAATCAAAATCTTGCTTTTCCCAACGCATCGGGCTATCCAGGGGTTGCTGTGGTTGTGATAACTGGATTCTGATGATGCCAAGTGGACTTGGTTTCTGTTGCTTAGGTTCTTGCCCTTGCCTCTTTCCATGTATTTACCTCTGATATTAGCTGGTCTTGCTGTGTCTGACAGTGGTTGACCCTCCTATAAACTTGATTATCAGCACTACTGGTGAACCAGCTCTCTCCTAGCAGGATCTGGGTACAGAGAACTGTGGCACAGGGCCAGCTCAGAGCACAGACAGAAACCAGAAGGATCCTGTCGCAGACTGCTCCTCAATCCCTGTGTCCTGTGTGTTCTAGGCAGGTTCTTCTTGGGCAAGGGATGTGAGTAGAAATGTTTGTCTTATCTGTGCTCTCTCATGTGTTAGTCCTGGGAGACCAGCTTTTTTCCAGTGGGATCTGAGCACAGAGAGCTGTGGCACAGTGTCTGCTTTGCGCACAGGTGAAAACCAGAAGCTTATCTCTATTTTTATGTCCCAGGAAAGACTCAAGGTAATGTAGTCTTCTACATTTTCCTTCTCAGAAGTGTCAACAGTATATAACTGCTGAGTTAGACAGAGTCACTTGACACTGGAGACATAGCCATTGGTTCCTCATATCCAGTGGACGACCTCTCAGCCTTCAGCAAATAGACAACAGAATTTGGATTCAGGCATTACTAATAATAATACACTATAAAATAAGAAAAGGAGGAGATGACACTAGAGGTTAAGGAATGGGGACACAAGCAGGAGTTTAAAAGAAATAGTGTTAAACCTGAGAATTGTAAGGGGAAAATTTCACATGCACAATTGTCTAGTTGAGCTTTCCTTTGGGGTGCACCAGGGATTGGCAAACCACCTACCTCACCCTAAATAGAGATTTGACAGATGTTCCACTGCCAGTCTCTTGAGATCCTGTGTATAGTAGAGATGAGATATTCCCAGGGTTGAGTGAGTTAGCTGTTATATACTGATAGAAAAATAAAATGCAAGGGAAGGAACAAAGGCAAGGCCATATACAAATCATGTGAATGGGGCTGCAAGTTTAGTGTAGATTAAAACCTATGTTTTTGCAATTTATATTATGCATAAGAAACAGGAAAATCATGAATTTATTCATTGCCCATCTTAGCTGTAAACAGAGCCCTTAGCGTATAAAGCATATTATTCCTGTTTTGGACTGAATAGTGGTGCACTGGCATGATCACAATGCAATGTATCAATATACTGAAGTTTCAAAAAAACAAATAAAAATTATATTTGAAACAGAATACTGATTCTCCTTTTCTTTGTNTACTCCAG ATTCTGAAAAAGAATTAAAGTNA AAAAAATGANGGANNAGATTTATTGAAGNANATAATNNNTTTTTCTCTGATNN NNNNNNGNNNNANANNNN

PAGE 118

107 NNNNNNNATNNNNCTTGNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAANNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGTTTTCAAATCCTGGTTTTCAACTCTCTTTCAG AATCCAGGGGAAAAGTAGAAGATGAAGAAATG GAGGTAAATGTTGGAGTGGCCCATGCATCACATCTAATGCTTACAG GTAATGTCAATTCCAGGGTGTATGCTGATGCCATTTGCGAGAAAGTAAATTTTCGAAGCTTTTGTGGAATCTGGAACTAGGGCCACAGTCAAGAAGGCCTTACTGCAAGGCCATCATTATCACTGACATGTGCTGTCATTGCCCATGTTTTAAAATGATGTTAATAACATCTTTAAATTTATAAAACAGTAGAGGCTTAAGAAATTACTAGCACAAACAAGATGTAGTTAAGTATGTTTGTATACTGCTCTTTAAATTTTATATATAATTAAATAAAAATACCTAACATTACATAAGTCACATTCTTTGTGTTCACCAAAGAAATTGATGAACTAGTGTAGTTTATTAAGGAATTACACTTATCTAGAAAGGGGAGTTATAGGAGAATTTCAGGGCAGGGAGGTGGGAGGAAGTGAGTAGGTGAGGGAACACCCTCACAGAAGCATGGTGGAGAAAGGGATAGTAGGTTTCTGGAGGGGAAACCAGAAAAGGGAATAACATTTAAAATGTAAATAAAAAATCTAAAACAAGAAAAAGAATGCTATATTTTTAGAAGTTCTGACAAACTAAGGAATGAAACGTTTGTGAATTTTTTTTTATGTTTGCATGCCTACACCATACATTAAATGATATACTGAATTTCTGTTCTCAATACTTGGTTTATATGGCAGCAAGAATGATAGATCAAGACCCCTTAATACCATTGAGTTTATAAATTAGTGAGTGGTGGTATACCATAAACATGGAAAAAGTAGTATAAATTAATTTAACAGATAAAAGAAAGAGAATTAGACTAGAGAAAGCAATTGCATAGGTAAAATGGGTTGAGGCTTGAGGATGTTTAGTCAAGCTCACATCTATTGAGGAGCTATATTAGAATATACCTCCAAGGAGCAAAGGAAATTTGTCCAAAACTGTTGTACAAAACTAAAGGCTAGGGTAGACCTGATACAAGAATGACCTTGTCATGTCCAAAGTCCTTACGGAAGGTTATAATCTCACAGGGAAGTAGGAAATTGAATGCGAAATGAGTAAAGCCATGAGTGTTAGGTACAGAGTCTGACATATGATGCCTATGCCTTCTTTTCAGTGGAAAAGGAGTCTACTATGGTAGACGTGTACATGCATATGTGCTAATTTGTGTGCATGCATGTGTGTGCATGAGTATGAATACAAGTATTCACATGTGTGTGCATGCTTGTGTATGTGTGTGCATGTGTGCATACATGTGTGCATGTGTATGTATGGGTGTGTGTAAATGAATGTGTGCATAGGTGTGAATATGCATGTAGGCATGAATGTATGTATGCATGTATGTGTTTCCTGCGAAATTGCTTTCATTGAGAGTTGTCATTCCCTAAAACTTTGATCAAATATAGACCCACTGATTAGTCCGGTCATCTCTGAGAGTTAAAAAACACCCAACTGTGTCCTAAGCAGATGCAAATAGCAAGAAATCCTTTATGTGTAAGATGTTTCTGTAATCTCAGCTTACACATCATAAATCAGCAAAAAGAGACAAGAGACTGTTGATAGAGCCAAACATTATTATGAAAATACAAGAAGAGCAGAGGGAGAGAGGAGGAGAGCAAGATGCAAAATTCTAAGTAAATGATCTACCAAAATTCTTGTGAGGGATTGAGAAGGTTTGGGGTTCAAAGCAGAAAGGGAGGCAGTGTTCTAAGCAAGGATATTCACTTCCTTGTAGTATGAGAGAAAACGAATAGCGGCCAAGGTAAATAAATGATTAGTGCTTAAAAAGATCAGGAGGGTCACTGTCCTTTGGAAAATCAAGGACATAAGAGCAAATGTTTTAAAGAAGTTGAAAATATTCAATTATTAGTAATGAAGTGGTTTTCTTCTCCGGTGCATCAAACTAGATTACGATTAAAGACTTCTTTTGTTTTTATATTTACAAGCAGTAAATATGGAATAATTCATGAGGTCATTATTAAGGTGTTAAATGTTATCTACATGGTCCAGATATAAAGGTGTATATTTTGCAATATATAGAACTGTAAAGAACCACTAAACCGATGTGGGCTTCCTGTCACTGAAATGGCCTGAAAGGGTAGAAAAATCAGAATATTAACTACCTATTTAAGTCAACTAAATAAAGCTTAACTTTCTTACAAAGTTTAGATCCCTAAGTTTATGTGATTTTAATTTATTGAACTTGAAAGACTAGACAATGTTGGCCACACTAGAAAAGCACAGTGTCAGTGATAAACAGAAAACAAAAGAAAGAAAAAATCAAGAGCTTTGCTTCAGTCATTTATATATGAATTTGTTTTTTTTTCTCATTACTTTTTGAGGAGCATCAATGAGCACTCCAGAATTATAAACTGGGGGCTGTGACATGTGCATGACTTACAGAGTTGTGTCAGTTCTTCGTTTCTGTTTTCTCTTTCAG TTCCTCAGGGAATCCAGAGCACAGAA GTACAGGATTCACTGAAGCTATGTTCTCGTGATTGGTTTTGTACGATGAAGACAGAAAGGGCAGAAGAG GTGATTAGCTTTGGAACCAGGGGACTAGAGTTGGCCCATTCAGGCTATGCACACAGATTCCTTCAGATTAAATGAAAGAAAATGAACTTTATCTAATCAAGTGGTTCTCTTCCTAAATTTTTTCCCATATTTAAATCTTATCTCAAAGTATGTCCTCAAATCCACCCCACCAGATAATCCAAGCCATGTCACTAGTTGAAATTTATACTCTATTCAGGCAACGATATATGTTTTTAGTCTCACATTCTATCATTTCGTCAACACAGCATAACTTTATCAGCAACATTCTGTTGTTCACAACTACTTCCGTCCTCATTGTACTGATTGTAGAGATATGAAGAGGACCCTTTCTTAAATTCCATGTGTCTGATAACTGCAG ATATATCCAGTGATGGAGAAGGAAGGCCGAACCCGCCTGGCCCTCATCA TCTGCAACAAAAAGTTTGACTATCTTTTTGATAGAGATGATGCTGAGACTGACATTTTGAACATGAAAGAA CTACTTCAAAATCTTGGATACTCAGTGGTGATAAAGGAAAACCTTACAGCTCAG GTAATGGCCCCAAAGATGGGGGCAACCTTCCCTGTGCTATTGTCAACCTATCTTTGGGTAGCAACCAGAACAAATTGTATTTGCTCCAGAGCAGGATTTCCTGGGTTTTGACCTTGTCCAAAGTGTAGTCTCATAAAAAGTATTCACAGAATGCTTTGCAACATTATTTCAACAAAACAGGAGGCATTTCTTAATTGTTAGTCTGGATGTTGCTATTAATTGATGTTCTAAGAGAAGCATAAGCTCTGAGTTTTGAAACTTGCAGTACCTAGAGAATAAACATATAAAAATGTAACAATATCGGAAAAATATGCCTAATTATTGTGAGTTATACAAAGGTTATAATCTCAATTCTGTCCAAACAACCTAAGAAAATTTGATTAAAACATGCAAAACTTGATATTGTTTGATAGCAAAAAAAATTTATATTTTCAATTTTTTCA

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108 ATATAAACTCTGTGTATCAGTTCTTTTAATGAGAATTTTCTAAAACCTAATTGTAATAAAGTTGGTTTTGATTATATTCTATCTCTCAGGTTCCTTAAAAATCCAAGATTCTCCTTAAAGACATAGGAATGAAAGCTATGTTTAATAAAAAACAAAAGAATAATGTTCATATCATTTTCTGACTTGATGTAAAAAATGTTACTTATCTTACCACAGCACTCTGCCTAAATTACTTGATTCCTAGAAGTTTAGTTTTTGTGAACTTCCTCTGTCACTCAGATACAAACTTTATTTGGAGAATACATTGGGAATGACTCTGCAGTCTTTTTAG GAAATGGAGACAGAATTAATGAAG TTTGCTGGCCGTCCAGAGCACCAGTCCTCCGACAGCACATTCCTGGTCTTTATGTCCCACGGCATCCTGGA AGGAATCTGTGGGGTGAAGCACAGAAACAAAAAGCCAGATGTTCTTCATGATGACACTATCTTCACCATTT TCAACAATTCTAACTGTCCGAGTCTGAGAAACAAACCCAAGATTCTCATCATGCAAGCCTGCAGAGGCAG TGAGTCAGAGTAAAGGATCTAGCATGTGGTTCTGAACTTTCTTTTATGAGTCTGGCATATTCCAGTAGACAAATGGGGGAAGGACATTTTAGTACCTTTTGTGTGTACAGTCCAAAAGATACTATCTTACATTTTATGTTTTAAAGAAACAATAAGTATCCTGGGATAATAAGAAATGCCCCAGTCATTTGTCAACATTGTGGGTGTTGTATTGTTCTTGTTCACCGGGGCTTTTTATATTTTTGAGGCCAGGAAGATGGCTTAGTGAGTAGAAGTGCTCACTGTGCAAGAACAAATCCCAAGGTCCTATGTGCAAAGTTAGGCACAGCATGTCTTCACACAACCCCAGGATCATAGATGGTAGACACAGGCAAGATTACTGATGCTTTGCTAGCAAGTAAGCTCCAGGTTCACAGACAAATCCTGTCAAAAGGGTAAGGTGAAGAGTGATAGAGCAAAATCACTGTCATATTTTTTCTTCAGCCTCTGAGTGTGCAAAAGGACATTGATAACTTGCCCTCATATATGTATATGATTCATACACATGAAACATGTGCACATCAATATTTGCATTTTCAATTAAATCATAATGCAATGTAGCAAATAAAGTATATTATAGCATTTGTATATTTTATGGACTATGCAACAGTGAATGTGTGTTACATTTGTTTAAGAAGTGTCATCTCTCCTAGG ACATACTGGTACTATTTG GGTATCCACAAGCAAAGGGATAGCCACTGCTGATACAGATGAGGAATGTGTGTTGAGCCATAGATGGAATA ATAGTATAACAAAGGCCCATGTGGAGACAGATTTCATTGCTTTCAAATCTTCTACCCCAC GTAAGTGACTTCAGATGTCCTGACTCTATTAGTAGTAGTTTTTAATATAACATGCTAGGCTGAGATAATATATTTTCAATAAACTGTCTATGTCTAATTATTGGGATGCTTTTGGTGGTAGAGATATTTAAAGTATTTCAGTTTTCATGATTAGATTTGCTTTAGAAATGATTAATGTGGACAGGTAATTTTGGGGAATCCAAAATTCAGATATTTTCACTATTAAAATAAATGCTATTTCACTTTGGTTTTCTTCTTAAACATGTTAGTGACTTATCAATGCATAACCTATAAGATCCCAAAATTCTACCAGTAAAAACAATCCTAAGCCTACAGCACGTACTGTGGAAACTATTAGCCTATTATTTTAATCTAATTTTTCGAAACTACTTAACACTGAAATGTTTGCCTTCATATTAATATAGGCAACAGTGGTTAATGATCTGTCTGTGACAGAGAAACTAGAGCAAATATGACATACAGCACAGAAACAAAACAATCCCAGTAGCTGAATGTCAATTTGGGCAAACATCCTTTTAAACAGTCATCTACAACAGGGCTCTACGAAGGATTTATAGAACAAGGCAGCTTGTGACAAGTTTGTACAAATCAGGATTCCTTGCATTCACATACTATGAGTATAAGGTCTCATGTGTGTATGAATCATTAGGAGATTGTGGTATGCAGAGTTCTGCTTCTGTGTGTATGCATTGAGCCCTGACATGCAACCTTTCTAATAAATCATAGTGATGTATCTGCAGCTGATGCATGGCCATATGTGATTTAAGAGAGATTAGCGGACTCTTTAAGTATAGACCAGAGAAGGAAAACTAAGAGTGGCCACCTACACCTAGATAAGGTGCATTTTTATTTATTTTATGTATTACAATTTTAATGAGATTTTATTTGAAAGAAAAATTCTATATATTTTACTGAAAAACAAGAAACTAAAAGTTATATGCAATCTGCACAGCATCTTTTATGGTAGTAACTTTGGAGAAATGTAAACACTGAAATGATGCAGATTTAACTTTTAAATTAAAGGAATGAAATGCAGTGTACTGAGGGTAGATGGAGTAAAGCAGGTTAGACTGCTATTGCAGAACTGAGACACAGAGATGATAATGATAACATGGAATGAGTCAAGTCATTGGCTCCAGTAAATTGTATGAGAAATGAACATGACTCATTGCTTTAGCTGATTTGACTGAAAAGAGATAGGGAATATGTAGGTGATACATTATCCAAAACCAGTACTACATTGAAATTACTTAATAGGATACTTTATTTTCCTAAAGATTGTACCATATTATTTCATGTCATAGTAATATTTTATTTAACAGAATCATGTATGGAAGGATGTACTATAAATATTCTCTGATATGTTATAAATTAACATGTGAATGAATGTTTTTAAGTTTTAAAGCGACAGTTTGAGATGGCATTGCCATAGCTGAAAGTTAAATGATGGATAAAAAGTTGAGAAATTGTTCATTTCCAGAAGATATCTGCCTTAACGGTATAGTTTGGGGAATTGTCAACTGCTAAAACAATATTTTCCAATGCTAAAGAGAGATAAAAACAATGGAAAATTGAGAATATTGTTAGAAATATTGTGGGGATGAGGTTGAAAATGCGCTTAAAAATGAAGACTAAGGATGAAAAAGGAATCCAGTGTGCTTGGCTTACAAACCAAAGGAAGAAAAACCAATGAATGAATTTCAGAGTTATTGGTAAGGCTCTCAGTGCTACCAGTTCTAATGTGAATGATAGGTCAAGTGCAGGTGAAGCTTTATAAATGAGTTTGACTTAGGGATTTAGAGATATTGGGGCAGGTGGCAGGCCCATTTTGTGACAGTGGTATAAGATCTATGAGAAGAAGCCAAATACAAGTAGAGTCTTGTGAAAGGAGCTGCCAAGGAAGGATAGCTTTAAATTCACTGAAGGAAAAAACTTAAGTGGCAGTTAGAGAACTGTTTCTCTCTGCTTTAACAACAGAAGCTCAGTTCAAAAGGCAGAGAAATTGTCTTCTTTAAGAGTATTACTCAATAGGAGAAATGTGCTAAAACAAATATAGCAGAGATGCTAGGACTGTTTACAGATGAGTTTTTCTTTACCATGAAGTGGATCCTGGAAGGGCCCTGTGACTTGCTTCACAGGATTTGCTTGCTGTGGACTGTAGCATTCAGCTCACCTATTAAAAAAAAAGAAAAACCTGTAGCTTTTTGATGGAGGATCTGCACGAGTACTCCATGTGTCTTATATGGAGCATTCTGCTTTACTCACCTTTGGCCCTCTGTCTTCTATGTAGTATCAGTCATACTTGGTTCCTAAAGTGTCATGCCATGAGTCTTCCACACTTCATTTTCATCTTTCCCATGCCTTCAATTACAG ATAACATTTCTTGGAAGGTAGGCAAGAGTGG CTCTCTCTTCATTTCCAAACTCATTGACTGCTTCAAAAAGTATTGTTGGTGTTATCATTTGGAAGAAATTT TCCGAA AGGTAAGGGTTTGGTGTTAATCTTTAATGTTCTCTAAAACTGCCATGTGCTAAGAGCCATAAAAAGTTCCATTCTGATTATACTTAACATCCTATTTTTTTAAAGAATGTTTTAGATTACCACATATAAGCATTGT

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109 GGTTTTTTTTTTTCTAAAAACCCTCTGATTTTTAGACGCTTGTGCAGACACTTTTCATAAGTTCCTAAGTAACTTTGCCTGCATTACATGCAGGGAAAACCTAAAAAAAATGTTATAGTACTCAGCCACAGTCCTAACATACATCACAACTTTTGTTGTCATCGTTGTTGTTGTTGTTGTTGTTGTTGTTGTTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGTTGGGTAGAGTAGCAAAAGCTGTTCCCTTCTTAGGAGAACTCTAAGGACTTTCTTTGCCACTAC AGGTTCAATACTCATTTGAGGTCCCAGGTGAACTGACCCAGATGCCCACCATTGA GAGAGTATCTATGACACGCTACTTCTACCTTTTCCCTGGAAATTAACACAGCCTACTCTTGCAAATAAAT GTATGTTAACATGTATTATTTCTTGTTAATAAATATATGTGAGGAGTGGATCTGGGAACCCATTTATAGTCTGTATAAAATCTGAATAGACTAGAAAATGATGGTGATCCACCAGCTTCCTGTTACTGTGGATATTACTTAATGAACCTGGGGAAAACCTTGGTTTTATTTGCTTTCATTGTAGTATAATGATTGCATTGTTAGAAATCATTAGTAGGGGTTGGGAATTTAGCTCAGTGGTAGAGCGCTTGCCTAGCAAACTCAAGGCCCTGGGTTCGGTCCCCAGCTCCGAAAAAAAAGAAAAAAGAAAAAAAAAAGAAAAAAGAAAGAAAAAGAAATCATTAGTAATCTGGTAGCCAAGCGGAAACCCACTGAGAACTAGAGTCTTCTTTCTGGAACTCAATCAGTGATATTTGATTTTCATGCAAGTACAACATTCTGTAATTTTAAGTACCACGCAGAGGAGTTATCTCTTAAGAAGAAGTAATT

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BIOGRAPHICAL SKETCH Stephen Frank Larner was born and raised in Aberdeen, Washington, USA. After high school he entered the University of Oregon (Eugene, OR) and earned his B.B.A. degree in international business. He followed that with a Master of Management from the Northwestern University (Evanston, IL). After passing the C.P.A. exam Stephen proceeded to spend the next 10 years in business, first working as an auditor for an international public accounting firm and then as a financial analyst for an international oil and gas exploration company, in both cases rising to management positions. During an economic downturn Stephen returned to college to pursue education that would take him completely into another field – science. He received his B.S. degree from Seattle Pacific University (Seattle, WA) where he majored in biology, minored in chemistry and computer science. At the same time, while at Seattle Pacific University, he completed a Master of Arts in Teaching and worked as a laboratory assistant. Upon completing his teaching internship he became certified to teach biology, chemistry, mathematics, and computer science. After making the decision to pursue a career in research he returned to school at the University of Washington (Seattle, WA) completing the requirements for a M.S. degree. While Stephen was completing work on his master’s thesis he began his study for a Ph.D. in the Interdisciplinary Program in Biomedical Sciences at the University of Florida, College of Medicine (Gainesville, FL). Stephen was awarded the Alumni Graduate Fellowship and the Bryan W. Robinson Neurological Foundation Grant-in-Aid Achievement Award three times (2000, 2003, and 2004). He joined the 127

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128 Department of Neuroscience in May of 2000 and the laboratory where he completed his doctoral degree under the mentorship of Dr. Ronald L. Hayes as part of the Center for Traumatic Brain Injury Studies and the Evelyn F. and William L. McKnight Brain Institute.