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EFFECTS OF ETHANOL EXPOSURE AND WITHDRAWAL ON EX VIVO CEREBELLAR SLICES

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004853/00001

Material Information

Title: EFFECTS OF ETHANOL EXPOSURE AND WITHDRAWAL ON EX VIVO CEREBELLAR SLICES
Physical Description: Book
Language: English
Creator: Roudabush, Stacy
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2013
Publication Date: 2013

Subjects

Subjects / Keywords: Ethanol
Biochemistry
Cerebellum
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Alcoholism is an addiction to ethanol (EtOH) that is marked by cyclic periods of chronic EtOH intake, withdrawal, attempts at abstinence, and relapse. Research into the excitotoxic properties of EtOH in the central nervous system (CNS) dominates the current scientific field, while studies are less focused on the mechanisms underlying brain atrophy and damage following chronic EtOH exposure and withdrawal periods. The current thesis attempted to explore the mechanisms following EtOH exposure and withdrawal in an in vitro model using adult (ten- and eleven-week old) 250 μm mouse cerebellar slices. Myelin and axon-associated proteins and actin fibers were significantly degraded following EtOH exposure, consistent with previous literature. However, EtOH exposure significantly decreased the primary metabolic enzyme GAPDH, as well as the water transporter protein AQ4. EtOH-W (4 h EtOH exposure, 20 h control serum) had differential and unexpected effects only on five proteins: degrading three (NFL, Bcl-2, Calpain-1), while increasing two (β-actin, CNPase). Control samples underwent significant protein degradation in the model compared to naive samples that were not exposed to the culture medium, and combined with propidium iodide staining measurements, suggests that a level of basal damage occurred in the plated samples. Model exposure did not completely destroy cerebellar slices, as indicated by PCR products of 4 genes (β-actin, Calpain-1, Calpain-2, Calpastatin). Future studies on in vitro models of adult myelin and axon in chronic alcoholism are needed to elucidate the role of proteases and apoptosis in degradation, but should also account for initial protease degradation.
Statement of Responsibility: by Stacy Roudabush
Thesis: Thesis (B.A.) -- New College of Florida, 2013
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Walstrom, Katherine

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2013 R8
System ID: NCFE004853:00001

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004853/00001

Material Information

Title: EFFECTS OF ETHANOL EXPOSURE AND WITHDRAWAL ON EX VIVO CEREBELLAR SLICES
Physical Description: Book
Language: English
Creator: Roudabush, Stacy
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2013
Publication Date: 2013

Subjects

Subjects / Keywords: Ethanol
Biochemistry
Cerebellum
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Alcoholism is an addiction to ethanol (EtOH) that is marked by cyclic periods of chronic EtOH intake, withdrawal, attempts at abstinence, and relapse. Research into the excitotoxic properties of EtOH in the central nervous system (CNS) dominates the current scientific field, while studies are less focused on the mechanisms underlying brain atrophy and damage following chronic EtOH exposure and withdrawal periods. The current thesis attempted to explore the mechanisms following EtOH exposure and withdrawal in an in vitro model using adult (ten- and eleven-week old) 250 μm mouse cerebellar slices. Myelin and axon-associated proteins and actin fibers were significantly degraded following EtOH exposure, consistent with previous literature. However, EtOH exposure significantly decreased the primary metabolic enzyme GAPDH, as well as the water transporter protein AQ4. EtOH-W (4 h EtOH exposure, 20 h control serum) had differential and unexpected effects only on five proteins: degrading three (NFL, Bcl-2, Calpain-1), while increasing two (β-actin, CNPase). Control samples underwent significant protein degradation in the model compared to naive samples that were not exposed to the culture medium, and combined with propidium iodide staining measurements, suggests that a level of basal damage occurred in the plated samples. Model exposure did not completely destroy cerebellar slices, as indicated by PCR products of 4 genes (β-actin, Calpain-1, Calpain-2, Calpastatin). Future studies on in vitro models of adult myelin and axon in chronic alcoholism are needed to elucidate the role of proteases and apoptosis in degradation, but should also account for initial protease degradation.
Statement of Responsibility: by Stacy Roudabush
Thesis: Thesis (B.A.) -- New College of Florida, 2013
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Walstrom, Katherine

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2013 R8
System ID: NCFE004853:00001


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EFFECTS OF ETHANOL EXPOSURE AND WITHDRAWAL ON EX VIVO CEREBELLAR SLICES BY STACY ROUDABUSH A Thesis Submitted to the Division of Natural Sciences New College of Florida in partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr. Katherine Walstrom Sarasota, Florida May 2013

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! ii Dedication I dedicate this thesis to persons suffering from symptoms of chronic alcohol abuse and to my aunt, Susan Stanfield, who suffered the most psychologica l, emotional, and severest of symptoms of alcohol withdrawal by taking her own life on Feburary 4, 2010.

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! iii Acknowledgments I would first like to thank the foundation that allowed me to begin my New College adventure, my strong and support ing family Mom, Dad, and Connor. Thank you for placing so much value in my education and for being the best role models I could ask for Coming to New College, I had only visited once during a winter break with my mom and really had no idea what I was getting into but I would not trade this experience for any other undergraduate education. New College greeted me with intellectual exploration lead by truly inspiring professors full of encouragement, knowl edge, and vigor for their work. I am grateful fo r Dr. Walstrom, Dr. Shipman, and Dr. Beulig for not only being amazing teachers in their courses, but also for the assistance and encouragement during the thesis process. I leave New College truly impacted by the people I met, including but never limited to, Ivery in Ham Center my best friends Hayden and Ruth, and the numerous gif ted students who will graduate with me. There are very special places in my heart for the people I have met in passing at New College and I can't wait to see what my peers will become in the future (Sarah O'Connor, I'm looking at you). I had the opportunit y to work at the New College Child Center for three out of my four years here and would like to thank Mr. T odd and all the wonderful children who made me smile and marvel at the simple joys of life on a constant basis. There were many days where I woul d run to lab from working with the children and blissfully realize I was already doing what I want to do for the rest of my life help children and work in science. I would also like to thank Dr. Naren L. Banik for the opportunity to join Dr. Supriti Saman taray and Dr. Vardhu i Knaryan at the Medical University of South Carolina, where this thesis work was performed. I will be fo rever grateful for t he scientific training and knowledge I received in this lab, as well as the fond memories and stories shared with our great lab group.

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! iv Table of Contents Dedication ii Acknowledgments i i i Table of Contents .iv List of Figures ..viii List of Ta bles ..x Abstract .xii Abbreviations ..1 Chapter 1: Introduction ..2 1.1 Alcoholism..........2 1.1.1 An O ver view of the Brain and Alcoholism .......................2 1.1.2 Ethanol Withdrawal in the CNS ...4 1.1.3 Development of the Current Model ...........6 1.2 Cerebellar Cell Type and Organization ..9 1.2.1 Cerebel lar Cell Types, EtOH, and Slice Culture.11 1.3 ETOH Toxicity on Myelin Proteins .. ...............12 1.4 Astrocytes and EtOH Toxicity .. ......13 1.4.1 GFAP.....14 1.4.2 Aquaporin 4...15 1.5 EtOH Toxicity on Cytoskeletal Components of the CNS... ...... .16 1.5.1 Neurofilaments .16 1.5.2 N F Structure ......16 1.5.3 Microtubules .....17 1.5.4 Actin Filaments.18

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! v 1.6 Apoptosis and EtOH. ... 19 1.6.1 Intrinsic Apoptosis ....20 1.6.2 Bcl 2 Proteins in Apoptosis.....21 1.6.3 Caspase independent Cell Death.....22 1.6.4 Extrinsic Apoptosis ...23 1.6.5 Lysosomal Involvement in Apoptosis... .24 1.6.6 Dramatic Increases of Intracellular Ca ++ Triggers Activation of Calpain s................25 1.6.7 Targets of Calpain in the Brain...26 1.6.8 The Calpain Inhibitor, Calpastatin ..26 Chapter 2: Materials & Methods ....27 2.1 Preparation of Cerebellar Slices ...27 2.2 Cell Viability with Propidium Iodide...27 2.3 Experimental Design ...30 2.3.1 Ethanol Exposure Paradigm ..2 9 2.3.2 Ethanol Withdrawal Paradigm ...30 2.4 Western Blot Analysis .30 2.4.1 Sample Preparation ....30 2.4.2 Western Blot .31 2.4.3 Reprobing Gels ... .31 2.5 Statistical Analysis...32 2.6 Semi Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT PCR) .............................32 2.6.1 Total RNA Isolation from Mice Cerebellum Slices.32 2.6.2 cDNA Preparation...33 2.6.3 RT PCR Amplification.33

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! vi 2.6.4 PCR Product Resolution....34 Chapter 3: Results ....35 3.1 Myelin Proteins.35 3 .2 Cytoskeletal Protein Degradation..39 3.3 GAPDH..48 3.4 Astrocytic Profile..49 3.5 Mechanisms of Degradatio n..55 3.6 RT PCR Results .. 70 Chapter 4: Discussion .....72 4.1 Myelin Protein Alterations in Response to EtOH....72 4.2 EtOH Selectively Perturbed Cytoskeletal Proteins.72 4.2.1 Neurofilaments.73 4.2.2 Tubul in..73 4.2.3 Actin Fibers...74 4.3 EtOH Induced Metabolic Disturbances.74 4.4 Selective Degradation of Astrocytic Proteins in Response to EtOH75 4.5 Mechanisms of Degradation. .76 4.5.1 Partial Activation of Caspases in Cerebellar Slices Exposed to EtOH......76 4.5.2 Bax and Bcl 2 Degradation Followed EtOH Exposure..77 4.5.3 Immature Cathepsin D Decreased with EtOH Exposure..78 4.5. 4 Calpain Degradation Followed EtOH Exposure.79 4.5.5 Calpastatin Activation in Cerebellar Slices..80 4.6 Naive Samples.....80 4.7 Future Improvements..81 Chapter 5: Conclusion ....84

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! vii Appendix A: Materials & Methods ....86 Appendix A.1: Cerebellar Slice Yield ..86 Appendix A.2: Western Blot Supplemental Information ...87 Appendix B: Results .. ..89 Appendix B.1: Full Western Blots ....89 References ......107

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! viii List of Figures Figure 1.1: Cerebellar cellular architecture .10 Figure 1.2: Confocal immunoflourescen t images of rat cerebellum...............17 Figure 1.3: Intrinsic apoptosis....................................21 Figure 1.4: Extrinsic apoptosis pathways.................................24 Figure 3.1: MBP protein expression .....36 Figure 3.2: CNPase protein expression .. ..... ..37 Figure 3.3 CNPase protein expression following withdrawal treatment .....38 Fig ure 3.4: NFL protein expression. ..... 40 Fi gure 3.5 NFH protein expression.... .... .41 Figure 3.6: NFL protein expression following withdrawal treatment ..... ..42 Figure 3.7: tubulin protein expression ..... ..43 Figure 3.8: actin protein expression .. .... .44 Figure 3.9: actin protein expression following withdrawal treatment ...45 Figure 3.10: GAPDH protein expression ....48 Figure 3.11: GFAP protein expression ...50 Figure 3.12: AQ4 protein expre ssion ..51 Figure 3.13: GS protein expression ....54 Figure 3.14: GS protein expression following withdrawal treatment ...55 Figure 3.15: Bax protein expression ...............59 Figure 3.16 : Bcl 2 protein expression ..58 Figure 3.17: Bcl 2 protein expression following withdrawal treatment .......59 Figure 3.18: Pro Caspase 8 protein expression ....60 Figure 3.19 : Pro Caspase 3 protein expression ...61 Figure 3.20: Calpain 1 protein expression ..64

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! ix Figure 3.21: Calpain 2 protein expression .. ..... ...65 Figure 3.22: Calpain 1 protein expression following withdrawal treatment ..............66 Figure 3.23: Calpastatin protein expression ................67 Figure 3.24: Cathepsin D protein expression .... 70 F igure 3.25: Calpain 1 and 2, C alpastatin, and actin RT PCR expression .. .... 71 Figure B.1: MBP full Western blot ................................89 Figure B.2: CNPase full Western blot ...............................90 Figure B.3: NFL full Western blot .............. ....................91 Figure B.4: NFH full Western blot .........................................92 Figure B.5: tubulin full Western blot ..................................93 Figure B.6: actin full Western blot ......... ............................94 Figure B.7: GAPDH full Western blot ...................................95 Figure B.8: GFAP full Western blot ......................................96 Figure B.9: AQ4 full Western blot ........ .................................97 Figure B.10: GS full Western blot .........................................98 Figure B.11: Bax full Western blot .......................................99 Figure B.1 2 : Bcl 2 full Western blot ...............................100 Figure B.13: Caspase 8 full Western blot .............................101 Figure B.14 : Caspase 3 full Western blot ..............................102 Figure B.15: Calpain 1 full Weste rn blot ...............................103 Figure B.16: Calpain 2 full Western blot ...................................104 Figure B.17: Calpastatin full Western blot .................................105 Figure B.18: Cathepsin D full Western blot ................................106

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! x List of Tables Table 2.1: Optical density measurement of propidium iodide cell viability test in ten week old mice cerebellar slices. .......28 Table 2.2: Optical densi ty measurement of propidium iodide cell viability test in eleven week old mice cerebellar slices...29 Table 2.3: Ethanol exposure and withdrawal experimental design.....30 Table 3.1: Myelin associated protein measurements follow ing withdrawal treatment .38 Table 3.2: Control versus naive measurements for axon and myeln proteins in cerebellar slices ........39 Table 3.3: Myelin associated protein measurements in control and EtOH 25 100 mM samples. ..39 Table 3.4: Control versus naive measurements for cytoskeletal proteins in cerebellar slices....46 Table 3.5: Cytoskeletal protein measurements in control and EtOH 25 100 mM samples .... ...47 Table 3.6: Cytoskeletal protein measurements following withdrawal treatment 47 Table 3.7: GAPDH measurements in control and EtOH 25 100 mM samples ..49 Table 3.8: Control versus naive measurements for GAPDH in cerebellar sl ices ..49 Table 3.9: GAPDH protein measurements following withdrawal treatment ...49 Table 3.10: Control versus naive measurements for GFAP and AQ4 proteins in ce rebellar slices.... ...52 Table 3.11: GFAP and AQ4 protein mea surements in control and EtOH 25 100 mM samples .......52 Table 3.12: GFAP and AQ4 protein levels following withdrawal treatment 53

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! xi Table 3.13: GS measurements were not significantly altered across all treatment groups ....... .55 Table 3.14: Control versus naive measurements for apoptoti c proteins in cerebellar slices....62 Table 3.15: Apoptotic protein measurements in con trol and EtOH 25 100 mM samples.. ..62 Table 3.16: Apoptotic protein measurements following withdrawal treatment ..63 Table 3.17: Calpain related protein measurements in control and EtOH 25 100 mM samples. ......68 Table 3.18: Calpain related protein measurements following withdrawal treatment 68 Table 3.19: Control versus naive measurements for calpain relate d proteins in cerebellar slices........69 Table A.1: Percent yield of cerebellar s lices ...86 Table A.2: Antibody supplemental information for Western blot analysis ..87 Table A.3: Developing time and reagents for Western blot analysis ..88

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! xii EFFECTS OF ETHANOL EXPOSURE AND WITHDRAWAL ON EX VIVO CEREBELLAR SLICES St acy Roudabush New College of Florida, 2013 ABSTRACT Alcoholism is an addiction to ethanol (EtOH) that is marked by cyclic periods of chronic EtOH intake, withdrawal, attempts at abstinence, and relapse. Research into the excitotoxic properties of EtOH in the central nervous system (CNS) dominates the current scientific field, while studies are less focused on the mechanisms underlying brain atrophy and damage following chronic EtOH exposure and withdrawal periods. The current thesis attempted to explore th e mechanisms following EtOH exposure and withdrawal in an in vitro model using adult ( ten and eleven week old) 250 m mouse cerebellar slices. Myelin and axon associated proteins and actin fibers were significantly degraded following EtOH exposure, consistent with previous literature. However, EtOH exposure significantly decreased the pri mary metabolic enzyme GAPDH, as well as the water transporter protein AQ4. EtOH W (4 h EtOH exposure, 20 h control serum) had differential and unexpected effects only on five proteins: degrading three (NFL, Bcl 2, Calpain 1), while increasing two ( actin CNPase). Control samples underwent significant protein degradation in the model compared to naive samples that were not exposed to the culture medium and combined with propidium iodide staining measurements suggests that a level of basal damage occurre d in the plated samples.

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! xiii Model exposure did not completely destroy cerebella r slices, as indicated by PCR products of 4 genes ( actin, Calpain 1, Calpain 2, Calpastatin). Future studies on in vitro models of adult myelin and axon in chronic alcoholism are needed to elucidate the role of proteases and apoptosis in degradation, but should also account for initial pr otease degradati on ________________________ Dr. Katherine Walstrom Division of Natural Science

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! 1 A bbreviations AA alko, alcohol AQ4 aquaporin 4 BBB blood brain barrier CaM calmodulin cAMP cyclic adenosine monophosphate CICD caspase independent cell death CIE chronic intermittent exposure CNS central nervous system EtOH ethanol, ethyl alcohol FAS fetal alcohol syndrome GABA gamma aminobutyric acid GAPDH gylceraldeyhde 3 phosphate dehydro genase GDP guanosine diphosphate GFAP glial fibrillary acidic protein GS glutamine synthetase GTP guanosine triphosphate IF intermediate filament IR immunoreactivity KO knock out LTP long term potentiation MAPK mitogen activated protein kinase MBP myelin b asic protein ML molecular layer MT microtubule NF neurofilament NFH neurofilament heavy NFL neurofilament light NFM neurofilament medium NMDAR N methyl D aspartate receptor s NT neurotransmitter OAP orthogonal array of particles PKC protein kinase C PNS pe ripheral nervous system PM plasma membrane ROS reactive oxygen species RT PCR reverse transcriptase polymerase chain reaction

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! 2 Chapter 1: Introduction 1.1 Alcoholism Alcoholism is an addiction to ethanol (EtOH), characterized by chronic consumption per iods followed by periods of addictive need for EtOH. Low concentrations of EtOH are associated with euphoria and behavioral excitation, while high concentrations can result in ataxia, drowsiness, slurred speech, stupor, and coma. Alcoholism is estimated to be the third leading cause of death in developed countries and is the number one risk factor for male death in the 15 59 age category (World Health Organization, 2004, 2008). EtOH is one of the most ancient addictive substances still in use today. At pres ent, there is no known cure for alcoholics, and current alcohol research is focused on dealing with EtOH as a psychoactive drug. Therapeutical treatments for alcoholics are limited; the primary treatment, benzodiazepines, has side effects including respira tory inhibition and/or development of dependency (Jung & Metzger, 2010). Chronic alcoholics have marked cerebral damage and function, but can also experience vitamin deficiencies, liver damage due to toxic EtOH metabolites, as well as major cardia c defects (Jung & Metzger, 2010; Alfonso Loeches & Guerri, 2011; WHO, PJ Mullholland, 2005). 1.1.1 An Overview of the Brain and Alcoholism The brain is a major target of chronic EtOH consumption and abuse, and even in individuals without confounding liver and heal th problems, EtOH can cause significant alterations to brain structure and brain physiology, as well as impaired cognitive function (Alfonso Loeches & Guerri, 2011). In order to investigate the effects of EtOH on the brain, we must first consider the compl ex constituents of the organ. The essential cellular element in the brain is the neuron, a highly specialized cell that communicates to other neuronal cell subtypes in the central nervous system (CNS) and peripheral

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! 3 nervous system (PNS). Three main regions morphologically define neurons: a cell body, numerous dendritic branches, and an axon. The cell body contains the nucleus, cytoplasm, and major cytoplasmic organelles, such as the energy producing mitochondria The cytoplasm refers to the liquid cytosol i nterspersed with three types of cytoskeletal proteins: microtubules (MTs, 25 nm diameter), microfilaments (8 nm diameter), and intermediate filaments (IFs, 10 12 nm diameter; Karp, 2010). The specific properites of cytoskeletal proteins and their roles in EtOH toxicity will be considered later. Emanating from the cell body are branched projections, or dendrites, that extend to other neuronal cell bodies and capillaries. Dendrites and cell bodies receive input from other neuronal cell type areas, while axons are responsible for the transportation of messages away from the cell body. Neurons communicate with other neurons and non neuronal cells at the synaptic cleft, which involves the axon of a neuron, the release of neurotransmitters (NT, such as glutamate) into the synaptic cleft, and the binding of NT to receptors on the postsynaptic side of the other cell. In the brain, neurons are organized into two visually distinct groups: white matter (myelinated axons and glial cells) and gray matter (cell bodies and capillaries). White matter appears white in brain tissue due to the high lipid content of myelinated axons. Myelin is a specialized compact membrane around axons that is formed from the membranous processes of specialized Schwann cells in the PNS, and by oligodendrocytes in the CNS ( Squire et al., 2008; Raval Fernandes & Rome, 1998). Oligodendrocytes and Schwann cells are two examples of glial cells, which are active supporter cells of neurons accounting for almost 60% of brain volume. The process of myeli nation by these cells results in axons enclosed with a lipid rich, multilayered myelin membrane that increases the conduction velocity of neuronal impulses from the neuronal cell body to a target cell (Raval Fernandes & Rome, 1998). Reduced brain

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! 4 weight an d volume have been observed in post mortem studies of human alcoholic tissues, which can primarily be attributed to the degeneration and atrophy of white matter and loss of neuronal gray matter (Alfonso Loeches & Guerri, 2011; Harper et al., 1985; Kril et al., 1997). Demyelination of axons is one of the principal morphological markers of alcoholism in nervous tissues, and is marked by primary degradation of myelin and/or supporting cells, followed by axonal degeneration (Chopra & Tiwari, 2012). In addition EtOH is associated with increased reactive oxygen species (ROS), which are reactive molecules that play physiological roles in cells but when overproduced can exert harmful effects on cellular components (Eysseric et al., 2000). Specifically, the metabol ism of EtOH by the class of cytochrome P450 (CYP) enzymes that exist in both the liver and the brain is thought to mediate ROS generation (Ferguson & Tyndale, 2011). Alcoholics have higher levels of certain CYP isoforms, one of which (CYP2E1) is expressed in the cerebellum and localized to glial cells of the brain (Hansson et al., 1990). Excessive ROS generation combined with neuroinflammatory events following EtOH exposure are thought to be the primary players in demyelination events, however the pathway by which neurons recognize these substances and then signal for myelin degradation remains elusive. 1.1.2 Ethanol Withdrawal in the CNS The abrupt withdrawal from long term and heavy EtOH consumption (EtOH Withdrawal, EtOH W) is associated with physiolog ical symptoms of depression and anxiety as well as physical signs such as a tremor, convulsion, coma and possibly death (Jung & Metzger, 2010). Alcoholics experience these withdrawal symptoms with increasing severity each time one tries to quit, and no cur rent therapeutic option is available without adverse side effects. The physical and behavioral indicators that arise

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! 5 from chronic EtOH consumption and abuse are associated with neuroadaptive changes that have a molecular, cellular, and structural basis. S ynaptic plasticity and long term potentiation (LTP) are the intimate learning processes that occur in the brain, in which the continual stimulation of certain nerves results in a molecular change in activity of these NT receptors. A similar process occurs following EtOH exposure and is thought to be responsible for the physical and mental manifestations of withdrawal and addiction. EtOH exposure can induce excitoxicity in neurons, which is marked by excitatory (glutamate) signaling cascades outweighing inhi bitory (gamma aminobutyric acid, GABA ) signaling (Iorio et al., 1993; Lovinger 1993). Excessive glutamate binds to NMDAR (N methyl D aspartate receptors), allowing a Ca ++ influx into the neuronal cytoplasm and further signal propagation that can result in neuronal degeneration and cell death. While acute doses of EtOH are inhibitory to NMDAR at first, chronic EtOH consumption resulted in an increased number of NMDAR in hippocampal and cerebellar t issues (Hoffman PL et al., 1995; Iorio et al., 1993; Iorio et al., 1992). The increase of NMDAR and NT receptors following EtOH exposure produces a greater expectation for receptor binding, while also altering the number of excitatory and inhibitory channels in the CNS. Seizures can occur in humans and animal models of alcoholism when inhibitory signals outweigh excitatory, and occur in more severity following EtOH W than EtOH exposure alone (Grant et al., 1990; Jaatinen & Rintala, 2008). Investigation of EtOH W effects on the CNS is becoming increasingly researched with the help of EtOH exposure paradigms that include exposure and withdrawal periods repeated a number of times in a cyclic manner. In a well developed chronic intermittent exposure (CIE) model of 16 h EtOH vapor exposure followed by 8 h withdrawal perio ds (repeated one to nine times in experiments), more severe EtOH W seizures were observed in mice following multiple

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! 6 withdrawal experiences, compared to mice tested after a single withdrawal experience (Becker & Hale, 1993; Becker et al., 1997). In male ra ts exposed to continuous or intermittent EtOH through drinking water for 5 months, reduced neuronal density and number in sympathetic neurons (Riikonen et al., 1999), and increased microglia cell content were observed only in the intermittent group that experienced two EtOH W per week (Riikonen et al., 2002). Microglia cells are responsible for immune responses in the brain and have been implicated in immune activation and in neurodegenerative diseases not primarily involving the immune system, including alcoholism ( Squire et al., 2008; Alfonso Loeches et al., 2012). Again, EtOH W, but not exposure alone, resulted in atrophy and shrinkage of neuritic processes and cell bodies in primary cortical neurons of 17 day old rat embryos (Nagy et al., 2001). From t hese and other studies, the idea that EtOH W may mediate a greater deal of toxicity than chronic exposures of EtOH alone is gaining more ground in the study of alcoholism. 1.1.3 Development of Current Model The study of the complex nature of myelinated neurons and neuronal interactions in the CNS requires a comprehensive model that exemplifies the intricate connections and constituents of the brain. A number of animal models have developed to study EtOH toxicity, such as the breeding of alcohol preferri ng P rats (from Indiana University; Knapp & Breese, 2012). Animal subjects can be provided EtOH as drinking water or be exposed to EtOH vapor, in inhalation chambers, at varying concentrations and lengths of time. As rodents do not generally prefer the ta ste of EtOH, the sensitization to EtOH is important in obtaining high blood EtOH concentrations comparable to human values and therefore, for modeling chronic EtOH consumption. The aforementioned CIE pattern of EtOH exposure and withdrawal times were eluci dated in studies where sensitization to voluntary EtOH intake following initial EtOH exposure was achieved in a specific strain of

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! 7 male rats (C57BL/6J; Lopez & Becker, 2005; Becker & Lopez, 2004). Exposure and withdrawal times from the CIE model were utili zed in various cell culture experiments in the lab where this thesis work was done, and serves as an ideal paradigm for future studies. However, these cell culture studies are limited in exploring the effects of alcoholism, as they do not encompass the mul tiplex of neuronal cell types and connections present in the human brain. A current method to study EtOH effects on the brain is organotypic slice culture, which involves the extraction of brain matter from embryonic or postnatal mice or rat pups, with the intention of maintaining viability and allowing the development of neuronal circuitry and myelination in vitro GŠhwiler (1981 1984 ) initially developed a monolayer n ervous tissue culture method, which Notterpek et al. (1993) built upon and observed e ndogenous myelination in embryonic cerebellar slice cultures two to four weeks after preparation. The use of cell culture inserts in 6 well plates allows for appropriate oxygen flow and nutrient supply to slices (Stoppini et al., 1991). Additional scientif ic advancements have allowed for isolation of postnatal day 6 9 mice or rat pup brain slices that are viable for up to six months in vitro (Gogolla et al., 2006). The use of embryonic or postnatal tissue, particularly those from mothers who chronically ing ested EtOH, is common in current literature and intended for the study of a spectrum of disorders resulting from fetal alcohol syndrome (FAS). By using younger mice, slice culture experiments are able to promote the continued growth of synapses, and a grea ter number of synapses in vitro than in vivo have supported this. Complete neuronal circuitry in organotypic slice culture studies have allowed for experiments in electrophysiology, time lapse imaging of dendrite and axon outgrowth, electron microscopy and single cell genomi c studies (Gogolla et al., 2006; Kapfhammer 2010). In the current study, the idea of organotypic slice culture was applied to a shorter time period and utilized adult mice, as the preparation and maintenance of long term

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! 8 slices exceede d the time available to complete an undergraduate thesis. The main goal of this thesis was to develop a model to study EtOH exposure and withdrawal on adult axon myelin integrity in the cerebellum. The cerebellum, Latin for "little brain", is located poste rior and below the cerebrum and is responsible largely for coordination and motor movements in humans. The cerebellum was chosen for development of the current model due to ease of isolation, expansive number of neuronal cell types, and previous implicatio ns in EtOH toxicity. Differing from current organotypic slice culture experiments, ten and eleven week old mice were used in the current study to model adult axon and myelin Cell migration in the cerebellum of developing rodent brain is complete after three weeks, and by the end of the fourth week cellular morphology has fully developed and synaptic connectivity has matured ( Squire et al., 2008 ). Preparation of slice culture from older mice has been little utilized due to the high lipid content of matur e myelin structures, and t his study marks one of the few attempts at a short term, older mouse model. The first hypothesis of this thesis is that this model will allow the study of the effects of EtOH exposure and withdrawal on adult axon and myelin consti tuents. In order to explore the effects of EtOH on an ex vivo system containing adult myelin and neuronal structures, a number of proteins associated with homeostasis, structure, motility, metabolic function and cellular death pathways were measured. A r eview of cerebellar morphology and current literature related to cellular components following EtOH exposure and/or withdrawal follows. Based on these studies and previous cell culture work in the Samantaray lab, it is hypothesized that the neural structur al and functional integrity will be compromised due to upregulated cell death and protein proteolysis following 24 h EtOH exposure.

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! 9 1.2 Cerebellar Cell Type and Organization The cerebellum is the "little brain" in the back of the neck that is responsible for a large majority of motor activities and some non motor activities. The cerebellum is responsible for the integration of signals and the coordination of the movement of different joints during movement, as well as motor learning ( Squire et al., 2008 ). Non motor activities suggested to occur through the cerebellum include acquisition of sensorial data, memory and emotion (Squire et a l., 2008; Schmahmann, 1991). Cerebellar atrophy is the most widely recognized structural change in all alcoholics and can also be accompanied by volume loss in certain regions of the cerebellum (Kril et al., 1997; Jaatinen & Rintala, 2007). The degradation of cerebello cortical circuits, such as impaired visuospatial abilities and problem solving, has long been observed in al coholics, presumably following EtOH degradation of axon myelin connec tions (Jaatinen & Rintala, 2007; Fitzpatrick et al., 2008). The cerebellum has two distinct lateral hemispheres that are connected to an enlarged cerebral cortex in primates. A three laye red cortex folded in thin, parallel strips, called folia, encompasses the outwar d surface of the cerebellum making it visually distinctive from the brain. There are three levels of the cerebellar cortex, thus named medial, intermediate, and lateral, that contain a total of seven known types of cells with intricate connections granule cells, Purkinje cells, Golgi cells, mossy and climbing fibers, unipolar brush cell, and stellate/basket cells ( Squire et al., 2008 ). The cells comprising the cerebellum are classified into four layers: the molecular cell layer (ML), Purkinje layer, granule cell layer, and white matter most superficially (see Figure 1.1, next page ).

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! 10 Figure 1.1: Cerebellar cellular architecture. A cross section view of the interactions of th e four cellular layers of the cerebellum molecular, Purki nje, granular, and white matter (Figure 32.3 Squire et al., 2008 ). The cerebellar cortex receives three types of extra cerebellar afferents, or nerves entering the CNS: mossy fibers, climbing fibers, and a diffusely organized array of mono aminergic and cholinergic afferents. Neuronal communication in the brain is mediated via feed forward or feed backward pathways, which are pathways in which the signal generated acts in an inhibitory or excitatory f ashion towards the signal(s) that propagated it. The mossy fibers contact several granule cells in glomeruli, and Golgi cells act as feed backward inhibition to these granule cells, meaning upon activation Golgi cells produce a signal that inhibits granul e cells. Each granule cell receives input from approximately four mossy fibers, and the near stimulation input of all are required to produce an action potential in granule cells to overcome tonic excitatory currents ( Squire et al., 2008 ). Ascending granul e cell axons make few synaptic contacts onto Purkinje cell layers before bifurcating in the outer most ML to form parallel fibers. Parallel

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! 11 fibers, aptly named because they run parallel to the folia, extend 5 to 10 mm through the cortex and release glutama te onto the dendritic spines of 2000 to 3000 Purkinje cells ( Squire et al., 2008 ). Parallel fibers terminate on the distal branches of Purkinje cells, while their smooth branches are connected to a single climbing fiber (Voogd & Glickstein, 1998). Purkinj e cells receive a wide array of input from the three cerebellar zones that concern sensory conditions, internal and external states, and plans of the organism ( Squire et al., 2008 ). Purkinje cells are thought to constitute the majority of coordination outp ut, as Purkinje neuron knockout ( KO ) mice displayed more extreme motor deficit in a Rotarod test than granule KO mice (Levin et al., 2006). Other constituents of the ML include two inhibitory interneurons, basket and stellate cells, that are excited by pa rallel fibers and form a feed forward inhibitory circuit with synapses of Purkinje cells. The seventh, most recently discovered cell type of the cerebellum is the unipolar brush cell found in vestibular regions of the cerebellum ( Squire et al., 2008 ). 1.2 .1 Cerebellar Cell Types, EtOH, and Slice Culture Research into the toxicity of EtOH on Purkinje cells is extensive due to the multiplex nature of the cells. Cerebellar atrophy following chronic EtOH exposure and withdrawal is modulated by a significant d ecrease in, atrophy, or loss of Purkinje cells, along with degradation of dendritic and synaptic connections in the ML (Jaatinen & Rintala, 2008). Et OH concentrations as low as 10 M, applied for no more than five minutes, in 20 and 30 day old rat cerebellar slices decreased synaptic plasticity between Purkinje neurons and climbing fibers, as measured by whole cell patch clamp electrophysiology (Carta et al., 2006). Additionally, d ecreased Purkinje cell numbers were observed in rats fed a liquid diet of EtOH for five weeks, followed by a two week

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! 12 withdrawal period (Jung ME et al., 2002), in rats fed EtOH for four weeks followed by four weeks of withdrawal, and in r ats exposed to EtO H vapors for six weeks ex posure interspersed with a total of four weeks of withdrawal (Phillips & Cragg, 1984). Most typically in cerebellar organotypic slice culture preparations, sagittal slices are performed and allow the preservation of granule cell Pu rkinje cell deep cerebellar nuclei cel lular connections (Mouginot & GŠhwiler 1995; Tanaka et al., 1994). Since the brainstem is removed from isolated cerebella in slice culture prep, all extrinsic afferents projecting to the cerebellum and efferent axons projecting out of the to the cerebellum are completely transected. Some studies have been able to combine cerebellar samples with other brain regions or the brain stem to maintain these external connections; however, myelin and axon integrity were the mai n targets of study in the current thesis. 1.3 EtOH Toxicity on Myelin Proteins Secondary demyelin ation of axons in white matter is a common marker of atrophy in brains of chronic alcoholics and is characterized by loss of function of myelin associated pr oteins. In order to confirm this in the present study of adult myelin structures, two myelin associated proteins were measured. CNPase, or 2', 3' cyclic nucleotide 3' phosphodiesterase, is an enzyme associated with the myelination of neurons in the CNS who se exact function has yet to be fully elucidated. Although not a major component of compact myelin, CNPase has been observed in concentrated regions in the myelin sheath associated with the cytoplasm of non compact myelin and in cells committed to forming myelin (Trapp et al., 1988; Siegel et al., 1999). Transgenic mice overexpressing CNPase up to six fold greater than normal levels had aberrant oligodendrocytes and myelin sheaths as measured by immunocytochemistry, suggesting CNPase is necessary, in some way, for correct myelination events in oligodendrocytes (Gravel et al., 1996). Other theories suggest a variety of CNPase interactions from RNA

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! 13 metabolism within myelin to interactions with calmodulin (CaM), a ubiquitous calcium sensor (Myllykoski et al., 2012), and cytoskeletal proteins (De Angelis & Braun, 1996). CaM has also been seen to interact with another myelination protein, myelin basic protein (MBP). MBP makes up approximately 30% of total protein in the CNS and is located on the cytoplasmic sid e of myelin membranes (Siegel et al., 1999). In mice, four known isoforms of MBP arise from a gene encoding at least seven exons. The 21.5 kDa MBP contains all seven exons, while 18.5, 17.0, and 14 kDa isoforms are the result of alternative splicing of a c ommon mRNA precursor (Siegel et al., 1999). In mice fed 10% v/v EtOH for 5 months, disruptions to myelin morphology and alteration of MBP and CNPase were confirmed with histochemistry, immunohistochemistry and electron micrography techniques (Alfonso Loec hes et al., 2012). 1.4 Astrocytes and EtOH Toxicity Astrocytes are important glial cells that maintain normal brain physiology in a number of ways formation and regulation of the blood brain barrier (BBB), metabolism of NT s, and maintenance of the ionic balance of the extracellular medium to name a few (Gomes et al., 1999; Middeldorp & Hol, 2011). Astrocytes appear star shaped cells when stained with reagents that highlight their complex bundles of IFs Astrocytes also have large glial end feet on capill aries and ensheath dendrites and synapses ( Squire et al., 2008 ). Astrocytes are present throughout the brain and spinal cord, and are intimately involved in synaptic cleft transfer and storage of information (Middeldorp & Hol, 2011). Astrocyte and neuron interaction is mediated by the transport of the NT, glutamate. Astrocytes are considered the primary cell type responsible for glutamate uptake, where it is metabolized into glutamine and used in the production of additional glutamate (Middeldorp & Hol, 20 11, Squire et al., 2008 ). The enzyme glutamine

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! 14 synthetase (GS) is responsible for the recycling of glutamate and ammonia into glutamine, an imperative protein building block and nitrogen source. The importance of astrocytes, their localizations in the CNS, and the complex nature of their constituents have only recently come to light with the assistance of the genome era, and generation of better visualization and immunostaining techniques. In order to assess the integrity of astrocytes in adult cerebellar s lices, GS and common cellular proteins of astrocytes were investigated. 1.4.1 GFAP The IF vimentin is present in immature astrocytes throughout development, until the IF glial fibrillary acidic protein (GFAP) progressively replaces it in mature astrocytes (Gomes et al., 1999). Although GFAP is present throughout other cells in the body, it is a primary marker of mature astrocytes in many studies GFAP is thought to be the primary IF responsible for astrocyte migration, motility, cell shape, and structure (Middledorp & Hol, 2011). Studies utilizin g the development of GFAP KO mice showed morphological and functional alterations in BBB, characterized by loss and disorganization of myelin in cerebellar white matter and granular cell layer (Middledorp & Hol, 20 11). The age dependent expression of GFAP has made for varying results concerning EtOH toxicity on mature astrocytes. For example, a dose dependent decrease of GFAP immunoreactivity (GFAP IR) was observed only in specific regions of the cerebellum in aged female mice in response to a 21 month long period exposure of EtOH as drinking water (Rintala et al ., 2001). Both young and old (3 and 24 month old) Alko, Alcohol rats (AA, bred to prefer EtOH) were exposed to 10 12% vol/vol EtOH for 21 months as their d rinking water, were wi thdrawn from EtOH exposure for one week before sacrificing and analyzed using immunoreactivity experiments. In this study, an age related decrease in GFAP IR after EtOH exposure differs from the numerous results implicating an increas e in GFAP IR and mRNA in regions of rodents and human brain (Rintala et al., 2001).

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! 15 Furthermore, the withdrawal period in the study by Rintala and colleagues (2001) was uncharacteristically not followed by withdrawal seizures in the mice, but the withdraw al period may account for these subtle differences in GFAP IR. Different EtOH exposure times, models of EtOH exposure, and measurements of GFAP (mRNA, IR, Western blot) have not assisted the research into the mechanism by which EtOH propagates toxicity in astrocytes either. EtOH toxicity may propagate by directly modifying GFAP transcription (Vall Ž s et al., 1997; Rintala et al., 2001), or indirectly, as EtOH metabolism can also generate ROS and toxic amounts of acetaldehyde (Eysseric et al., 2000). 1.4.2 A quaporin 4 Aquaporins are a class of 13 transmembrane proteins responsible for water transport across the cell membrane. Aquaporin proteins exist in ~30 kDa monomers, containing six membrane spanning helical domains that surround a narrow, a queous pore (Sm ith & Agre, 1991; Preston & Agre, 1991). The monomers can oligomerize to tetramers in the cell membrane, while still remaining independently functional (Iacovetta et al., 2012; Verkman et al., 2012). Aquaporin 4 (AQ4) is the predominant brain aquaporin, ca n exist in two forms named M1 and M23 (Jung et al., 1994), and is seen to polarize at the membrane edge in astrocytes (Nielsen et al., 1999). In immunocytochemistry studies of glial cells, AQ4 was present in the greatest density near capillaries and least dense near neurons (Nielsen et al. 1999). The localization of AQ4 in a type of glial cell lining the epithelial membrane, or ependymal cells, of the subfornical organ (a highly vascularized, sensory organ) and localization in astrocytes is important for me diating water transport between glial cells and the fluid between capillaries (Nielsen et al., 1999).

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! 16 1.5 Ethanol Toxicity o n Cytoskeletal Components of the CNS 1.5.1 Neurofilaments IFs are tissue and developmentally specific and classified into ten gr oups (I X, Gomes et al., 1999). Of relevance in the current study are the IFs present in neuronal tissues (neurofilaments) and in astroglial cells (i.e, GFAP). Class IV IFs are the neurofilaments and internexin, which are specifically expressed in matured neuronal tissue. Neurofilaments (NFs) are viscoelastic structures that can stretch 3x greater than a native state, while also interacting with neighboring microtubules (MT) and other NFs (Perrot et al., 2008). NFs are aligned parallel to the axon, making them important determinants in axonal diameter and therefore, the speed of conductance of the action potential along myelinated neurons (Kumar et al., 2002; Hoffman PN et al., 1987). N Fs are classified int o three isoforms: neurofilament light (NFL), neurof ilament medium (NFM), and neurofilament heavy (NFH). The predicted molecular weight from human DNA sequence of amino acids (NFL 61. 5 kDa, NFM 102.5 kDa, NFH 112.5 kDa ) differs from the molecular PAGE SDS weight (NFL 60 kDa, NFM 160 kDa, NFH 205 kDa; Ev rard & Brusco, 2011). This difference is due to post translational modifications combined with a high content of negatively charged amino acids. In the present study of EtOH exposure and withdrawal on cerebellar slices, NFL and NFH were measured to chara cterize axonal integrity. 1.5.2 NF Structure The three main neurofilaments share a common tripartite structure with varying head and tail regions, the latter of which interacts with other NFs. Neurofilament assembly occurs in the cell body in a manner tha t is dependent upon ionic strength, pH and temperature (Perrot et al., 2008; Angelides et al ., 1989 ). The three filaments form a mature protein in adult rat wit h a ratio of 2:2:1 (NFH:NFM:NFL; Scott et al., 1995). Furthermore, NFL can self assemble into a filament structure, while the remaining

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! 17 filaments require NFL to co assemble (Perrot et al., 2008). NFH and NFM have cytoplasmic tails that protrude from the core cylindrical structure and may facilitate filament filament interaction (Kumar et al., 2002). Phosphorylation modifications of NFM and NFH are necessary for interaction with kinesin and dy n e in and are involved in cross bridges with other NF or MT (Perrot et al 2008). The disruption of the cohesive structure of NFs and proteins in contact with NFs (Figure 1.2) would greatly alter axonal caliber, and trafficking and signaling events in the CNS. Figure 1.2: Confocal immunoflourescent images of rat cerebellum. Using confocal microscopy, immunoflourescent images of rat cerebellum display the distribu tion of NFL (red, Alexa Flour 555 Conjugate), GFAP (green, Mouse mAb #3670) and nuclear bodies (blue pseudo col or, fluorescent DNA dye DRAQ5; i mage and product details from www.cellsignaling.com) 1.5.3 Microtubules Microtubules (MTs) are the large diame ter, hollow, and rigid proteins involved in a diverse array of cytoskeletal structures, including those needed for cell division and movement. MTs have a wall thickness of approximately 4 nm that is composed of alternating alpha and beta protofilaments. Th irteen protofilaments rows are arranged in a cy lindr ic al fashion by noncovalent bonds, which allows for the quick assembly and

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! 18 disassembly of MTs. Normally, guanosine triphosphate (GTP) is bound to protofilaments in a straight microtubule, which increases MT stability and is thought to promote polymerization. During growth, rapid polymerization to the positive, or beta end, of MT, occurs faster than GTP can be hydrolyzed into guanosine diphosphate (GDP), resulting in a cap formation. When growth slows, GTP is hydrolyzed and GDP bo und protofilaments destabilize bonding in the MT, resulting in rapid depolymerization of the MT (Karp, 2010). MT s are often associated with microtubule associated proteins (MAPs) that embed into MTs and facilitate interaction with o rganelles and other filaments (Karp, 2010). Alpha and beta MT heterodimers have multiple isoforms from different genes, and ongoing research is focused on the functional significance and localization of tubulin. Excluding one isotype, all known beta isot ypes exist throughout the body and brain and differ in a 15 amino acid carboxy terminal region (Burgoyne et al., 1988). The carboxy terminal tail is thought to be important for interaction with MAPs, the later of which have restricted and characteristic lo calization in parts of the cerebellum (Burgoyne et al., 1988). During early cerebellar development, class III tubulin is expressed in the granule layer while taking up to a year to appear in Purkinje cells (Katestos et al., 1993). tubulin is currently used as a neuronal marker in developing rodents, as it is not expressed in glial cells (Katestos et al., 1993; 2003). 1.5.4 Actin filaments The smallest diameter filaments comprising the cytoskeleton are the microfilaments, which are composed of the actin protein. In the presence of ATP, actin monomers can polymerize into an 8 nm diameter, two stranded helical st ructure that is responsible for the motility of cells and the intracellular motility of various organelles (Karp, 2010). In humans, actin exists in three isoforms (alpha, beta, and gamma) that

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! 19 share common amino acid sequences (Karp, 2010). Altered actin f ilaments appear profusely disorganized and result in contracted and disturbed cell capacity in response to EtOH in humans and in cellular models of FAS (Evrard & Brusco, 2011). In C6 glioma line cells, acute EtOH exposure (100 or 200 mM for 23 h) caused significant loss of stress fibers and a more diffuse organization in immunostaining for act in filaments, via a proposed ROS induced pathway (Loureiro et al., 2010). 1.6 Apoptosis and EtOH A variety of cellular signaling cascades are implicated in contribu ting to the atrophy of axon and myelin in the CNS following EtOH exposure, varying from ROS generated toxic proteins to neuroinflammatory events that ultimately result in neuronal cell death. Apoptosis is the cell death mechanism characterized by decreased cell volume, chromatin condensation, nuclear fragmentation, and may result in eventual engulfment by resident phagocytes (Kroemer et al., 2009; Galluzzi et al., 2012). Damaged or long lived cytoplasmic organelles are sequestered into autophagosomes, whic h are then fused with lysosomes for degradation or release into the ECM in the process of autophagy. Autophagy is a physiologically relevant process, but is also associated with the recycling of degraded organelles, or apoptotic bodies' following apoptosi s. Apoptosis is naturally occurring at regulated and constant rates in the human body at any momen t, acting to maintain the extra cellular matrix (ECM), maintain and monitor embryonic development, as well as tissue and immune homeostasis (Repnik & Turk, 201 0). However, apoptosis has been implicated in the neuronal cell death that occurs in numerous neurological disorders following myelin degeneration, including Parkinson's disease, stroke, and Alzheimer's disease (Tait & Green, 2010; Mattson, 2000). Therefo re, it is not unreasonable to hypothesize the same neuronal cell death

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! 20 mechanism is responsible for EtOH induced myelin degradation. Apoptosis can be initiated and follow two main pathways intrinsic and extrinsic apoptosis which differ by localization of cell death signal and can activate the family of caspases (cysteine aspartic proteases; Repnik & Turk, 2010; Galluzzi et al., 2012). 1.6.1 Intrinsic Apoptosis Intrinsic apoptosis is the mitochondria centered pathway that occurs in response to intracellul ar signals such as DNA damage, cytosolic Ca ++ overload, oxidative stress, and excitotoxicity (Galluzzi et al., 2012). These intracellular signals have been implicated in EtOH toxicity, along with mitochondrial impairments in the nucleus, lysosomes, endopl asmic reticulum, and cytoplasm of cells in the adult rat brain (Jaatinen & Rintala, 2007; Chopra & Tiwari, 2012). Following intracellular stress, pro and anti apoptotic proteins are targeted to the mitochond rial outer membrane. If pro apoptotic cascades dominate the membrane becomes permeabilized in a process known as mitochondrial outer membrane permeabilization (MOMP). This results in the release of intermembrane space (IMS) proteins, dissipation of the mitochondrial transmembrane potential, decreased A TP synthesis, and inhibition of the respiratory chain (Galluzzi et al., 2012). Apoptosis inducing factor (AIG) and endonuclease G (ENDOG), two IMS proteins, translocate to the nucleus and mediate large scale DNA fragmentation in a Caspase independent path way, which is discussed in more detail later. Cytochrome c is also released from the IMS and is able to activate and bind to the cytosolic protein APAF1. Along with dATP, cytochrome c induces the oligomerization of APAF1 to form a large seven ring structur e known as the apoptosome. The apoptosome recruits pro Caspase 9 and releases active Caspase 9 that can go on to activate the Caspase 3 proteolytic cascade (Figure 1.3, next page; Galluzzi et al., 2012).

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! 21 Figure 1.3: Intrinsic a poptosis. Intracellular s tress activates proteins to permeabilize the mitochondrial outer membrane in MOMP, resulting in the release of intermembrane space (IMS) proteins. The release of cytochrome c from the IMS activates the formation of the apoptosome complex containing APAF1 ( a cytoplasmic adaptor protein), dATP, and pro Caspase 9. Upon activation at this complex, Caspase 9 act ivates the effector Caspase 3. Other IMS proteins direct IAP binding protein with low pI (DIABLO), high temperature requirement protein A2 (HTRA2), apopt osis inducing factor (AIF), and endonuclease G (ENDOG) contribute to Caspase independent apoptosis ( Image from Galluzzi et al., 2012). 1.6.2 Bcl 2 Proteins in Apoptosis The pro and anti apoptotic proteins that modulate MOMP are the Bcl 2 protein family. The B cell lymphoma 2 gene encodes Bcl 2 proteins and was originally discovered as a proto oncogene able to induce cell survival under deleterious growth conditions, instead of promoting cell proliferation (Jourdain & Martinou, 2009). Anti apoptotic prot eins (Bcl 2, Bcl Xl, Bcl W, and Mcl 1) have four Bcl 2 homology (BH) domains, where pro apoptotic proteins (Bax, Bak, Bid, Bad, Bim, Noxa, and Puma) have

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! 22 three, or only one, BH domain (Jourdain & Martinou, 2009). Bax is located in the cytosol of healthy ce lls, but upon activation of Caspase 8 and subsequent truncation of Bid (tBid), Bax undergoes structural changes and becomes localized to the mitochondrial outer membrane (Jourdain & Martinou, 2009). The specific interactions between pro and anti apoptoti c Bcl 2 proteins at this point in signaling are still debated and researched extensively. In most cases, the ratio between anti and pro apoptotic proteins can determine the extent to which apoptosis is carried out. In the present study, pro apoptotic Bax and anti apoptotic Bcl 2 proteins were measured. 1.6.3 Caspase independent Cell D eath Following MOMP, the decline in ATP production and mitochondrial membrane potential can activate Caspases that result in large scale cleavage of proteins and eventually c ell death. However, in certain cells, Caspase inhibition after MOMP still results in cell death, albeit at a slower rate, known as Caspase independent cell death (CICD). CICD results when pro apoptotic signals outweigh anti apoptotic signals and is mediate d by the extent of release of IMS proteins. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) catalyzes the sixth step in glycolysis and is commonly measured to probe for metabolic irregularities in a variety of experiments. However, recent reports show tha t GAPDH also can play dynamic roles in cell death pathways. GAPDH has also been found to accumulate in the mitochondria and nucleus following a number of apoptotic stimuli (Tarze et al., 2006; Chuang, 2004), where it may be involved in DNA repair and regul ating transcription (Ou et al., 2011). Autophagy occurs at basal levels in cells to sequester damaged or aged cellular constituents within autophagosomes, which are then fused with lysosomes for degradation or release into the ECM. The overexpression of G APDH in HeLa cells treated with an apoptosis inducer and Caspase inhibitor (to model CICD) had higher

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! 23 basal ATP levels and was associated with increased autophagy (Colell et al., 2007). This suggests that GAPDH may assist in cell recovery following MOMP by increasing energy levels, although full recovery required 7 10 days in HeLa cells mentioned above (Colell et al., 2007). This pathway may also present following EtOH toxicity, as AA rats exposed to 75 mM EtOH for four weeks had significant GAPDH upregula tion in preparations of the prefrontal cortex, as measured by both Western blot and mRNA expression (Ou et al., 2011). 1.6.4 Extrinsic Apoptosis Extrinsic apoptosis is initiated by the binding of death receptor ligands to appropriate receptors in the plas ma membrane (PM), which signals for the cytoplasmic tails of the receptors to recruit other proteins. Specifically, FAS ligand binding to FAS results in the formation of a supramolecular platform, the death inducing signaling complex (DISC) that includes p ro Caspase 8 or 10. If death signaling cascades prevail, Caspase 8 becomes activated and goes on to activate effector Caspases. Cross talk between the two pathways occurs when Caspase 8 cleaves a Bcl 2 protein Bid into tBid (truncated Bid), which can acti vate MOMP. Other receptors in the PM, such as dependence receptors can relay lethal signals in the absence of their ligand and facilitate MOMP by activation of Caspase 9 (Figure 1.4 next page ).

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! 24 Figure 1.4. Extrinsic apoptosis pathways. Extrinsic apopto sis signaling as mediated by the binding of FAS ligands (FASL) to FAS receptors or by the absence of ligand binding to dependence receptors DCC or UNC58. In the first instance, cytoplasmic heterotrimer tails recruit Fas associated protein with a death doma in (FADD), cFLIPs, cellular inhibitor of apoptosis proteins (cIAPs), and pro Caspase 8 or 10 to form the death indu cing signaling complex (DISC). In the second instance, lack of signaling promotes formation of a pro Caspase 9 activating complex with DRL, TUCAN, DAPK death associated protein kinase 1 (DAPK1) and/or UNC58 bound protein phosphatase 2A (PP2A) proteins. Caspases can go on to initiate MOMP (via tBid) or act ivate other effector Caspases ( Image: Galluzzi et al 2012). 1.6.5 Lysosomal Involvement in Apoptosis Lysosomes are the terminal compartments in the endocytic pathway containing acid hydrol ase enzymes that degrade org anelles and proteins. Cathepsin D (Cath D) is one of the many aspartic endoprotease s expressed in lysosomes that functions opti mally at acidic pH's (Liaudet Coopman et al., 2006). Cath D is involved in general protein turnover, cell growth and homeostasis events, and antigen presentation. Cath D synthesis occurs on the rough endoplasmic reticulum and yields a pre pro enzyme that i s co translationally modified to remove signaling peptides. This pro Cath D is

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! 25 glycosylated at two N linked sites after which it is transported to the Golgi Stacks as a 52 kDa intermediate. Pro Cath D can then bind to mannose 6 phosphate (MP6) receptors, w hich targets it to lysosomes as a 48 kDa single chain molecule (Liaudet Coopman et al., 2006). This activation most likely depends upon other cysteine lysosomal and/or aspartic proteases (Samarel et al.,1989). In lysosomes, Cath D is then cleaved again by cysteine proteases into a mature enzyme containing a light (14 kDa) amino terminal domain chain and a heavy (34 kDa) carboxyl terminal domain chain (Gieselmann et al., 1985). When Cath D is activated and released from the lysosomal compartment in research settings, apoptosis and degradation of the ECM have been observed (Liaudet Coopman et al., 2006). 1.6.6 Dramatic Increases of Intracellular Calcium Triggers Activation of Calpains Increases of intracellular Ca ++ mediated by the response of activation of pl asma membrane receptor channels, or released from intracellular Ca ++ storages can activate C alpains. Calpains, or calcium activated neutral proteases, exist in two isoforms in mammals Calpain 1 and Calpain 2, encoded by CAPN1 and CAPN2 genes, respectively Calpains are heterodimers composed of a large 80 kDa catalytic subunit and a smaller 30 kDa regulatory unit that dissociate from one another after autolysis induced by Ca ++ binding (Ono & Sorimachi, 2012). Calpain 1 and 2 have been observed in both g lia and neuronal cells, where they are found within the soma, axons, synaptic terminals, and dendritic spines (Zadran et al., 2010 and Wu & Lynch, 2006). Calpain interacts with substrates ranging from membrane receptors to cytosolic proteins. Calpain 1 a nd 2 differ in the amount of Ca ++ required for activation: 1 20 # M and 0.250 0.750 mM, respectively ( DeMartino et al ., 1986)

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! 26 1.6.7 Targets of Calpain in the Brain An assortm ent of proteins is degraded by C alpains, in both a homeostatic manner and in a deleterious manner during time s of neuronal stress. Calpains are thought to contribute to both the developmental regulation and adult homeostasis of cell adhesion by modifying integrins, cadherins and cytoskeletal proteins (Saido et al., 1994). Calpain activation has been implicated in numerous pathological states involving myelin, such as stroke, epilepsy, and neurodegenerative diseases (calpain.org, Tait & Green, Samantaray et al., 2011). I n the cerebellum and cortex of one month old rats fed 13% EtOH in drinking fluid for 12 weeks, increased Calpain activity, as measured by spectrophotometry, and Caspase 3 activation was observed (Rajgopal & Vermuir, 2002). 1.6.8 The Calpain Inhibitor, Calpastatin Calpastatin is an endogenous protein ubiquitously exp ressed in glial cells and neurons with cel lular localizations similar to C alpain (Saido et al., 1994). Calpastatin is one of the few known endogenous inhibitors of C alpain, and blocks Calpain substrate binding in a reversible manner after colocalizing in t he cytosol. Calcium influx in PC12 and neuroblastoma cell lines was seen to induce calpastatin movement from nuclear invaginations to the cytosol (Wu & Lynch, 2006). A tight ratio of Calpain to calpastatin proteins is required for homeostatic function, bu t may be altered during deleterious processes.

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! 27 Chapter 2: Methods & Materials 2.1 Preparation of Cerebellar Slices Materials and methods pertaining to model exposure were adapted from previous organotypic model set ups by PJ Mulholland (2005, 2010) a nd Gogolla et al. (2006) Male C57BL/6 ten week old mice from Charles Rivers Lab were decapitated, brains removed and placed in dissection medium. Dissection medium contained Minimum Essential Medium Eagle (Mediatech) with 25 mM HEPES (Sigma), 2 mM Glutama x (Invitrogen), a nd 100 g/mL penicillin streptomycin (Cellgro). Cerebella were further dissected from the brain, separated from the brain stem, and placed on the McIlwain Tissue Chopper (Mickle Lab Engineering Co. Ltd.) and 250 m sagittal sections taken. Sections were s eparated under a dissection microscope (Nikon SMZ800) and 2 3 slices place d on one insert (Millipore 0.4 m, 30 mm diameter) using transfer pipettes. Excess medium, if any, was removed from the inserts. Six well plates (Corning Incorporation) with inserts contained 1 mL culture medium per well. The percent yield [(cerebella length slices obtained)/slice width) 100] of slices obtained from the third set of mice sacrificed was recorded for experimental improvement (Appendix A Table 2). Culture medium con sisted of dissection medium supplemented with 36 mM glucose and 25% v/v Horse Serum: Platelet poor plasma (Sigma). Plates were incubated at 37 o C in a humidified atmosphere of 95% air and 5% CO 2 2.2 Cell Viability with Propidium Iodide Viability of the fi rst sets of cerebellar slices prepared was tested with 1 mL propidium iodide (PI) after days 1, 2, and 3. Application time and washing methods were experiments to obtain minimal cell death during model exposure. Ten week old mice cerebellar slices were sta ined for 16, 40, and 60 h with 1 mL PI (2.5 g/mL) in culture medium, washed with 1 mL of culture medium three x for 5 min, and images of individual

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! 28 cerebella slices captured at 4 95 nm wavelength usi ng an Evos LCD microscope (Fish er Sci; 2X magnification, 80% light intensity) Eleven week old mice cerebellar slices were stained at 16 and 24 h time points, but for 8 h, with 1 mL of PI (1 g/mL) in culture medium, washed with 1 mL of culture medium three x for 5 min, and imaged as described above. Resulting TIFF images (600 pi xel resolution) were measured for optical density using ImageJ (NIH, Bethesda, MD). Averages were calculated for each hour, but did not indicate a significant time of extreme cell death, so 24 h exposure was decided upon (Tables 2.1 & 2.2, next page). Mou se Number PI Staining Time 16 h Optical Density PI Staining Time 40 h Optical Density PI Staining Time 60 h Optical Density Mouse 1 Slice 1 6.675 Slice 1 6.863 Slice 1 3.301 Slice 2 9.475 Slice 2 7.094 Slice 2 6.225 Slice 3 10.334 Slice 3 4.235 Slic e 3 4.567 Slice 4 7.741 Slice 4 3.712 Average 8.828 Average 6.483 Average 4.451 Mouse 2 Slice 1 4.32 Slice 1 7.707 Slice 1 4.403 Slice 2 6.553 Slice 2 6.592 Slice 2 2.631 Slice 3 4.767 Average 5.213 Average 7.149 Average 3.517 Mous e 3 Slice 1 4.101 Slice 1 6.272 Slice 1 5.553 Slice 2 4.547 Slice 2 6.993 Slice 2 5.623 Average 4.324 Slice 3 5.683 Average 5.588 Average 6.316 Total Average s 16 h average 6.346 40 h Average 6.575 60 h average 4.614 Table 2.1: Optical density measurement of propidium iodide cell viability test in ten week old mice cerebellar slices. Optical density measurements of PI staining for 16 h, 40 h, and 60 h of Mouse 1, 2, and 3 respectively. Average optical density measurements for each day of staining, as well as total averages, are presented.

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! 29 Mouse Number PI Staining Time 16 h Optical Density PI Staining Time 24 h Optical Density Mouse 1 Slice 1 2.614 Slice 1 7.413 Slice 2 4.826 Slice 2 5.409 Slice 3 2.63 Slice 3 2.677 Avera ge 3.35 7 Average 5.166 Mouse 2 Slice 1 2.694 Slice 1 4.686 Slice 2 5.725 Slice 2 4.041 Slice 3 4.917 Slice 3 4.975 Average 4.445 Average 4.567 Mouse 3 Slice 1 9.128 Slice 1 2.702 Slice 2 6.02 Slice 2 6.677 Slice 3 4.777 Slice 3 3.269 Av erage 6.64 2 Average 4.216 Total Averages 16 h average 4.815 24 h average 4.650 Table 2.2: Optical density measurement of propidium iodide cell viability test in eleven week old mice cerebellar slices. Optical density measurements of PI staining of el even week old mice cerebellar slices after 16 and 24 h. Average optical density measurements for each day of staining, as well as total averages, are presented. 2.3 Experimental Design Two to three 250 m cerebellar slices were randomly placed on one ins ert in a 6 well plate. Cerebellar slices were allowed to acclimatize for one hour after dissection. Tissues that were not intact after dissection were saved and stored at 80 o C; these are called the naive samples. A 24 h time period for the model was chose n to include EtOH withdrawal periods, based on CIE model exposure times (Becker, 1993, 1997, & 2004) and previous EtOH exposure times used in the Banik lab, after which slices were stored at 80 o C until sample preparation. 2.3.1 Ethanol Exposure Paradigm A fter acclimatization, cerebellar slices were exposed to 25 mM, 50 mM and 100 mM concentrations of EtOH (200 proof, Pharmco AAPER) for 24 h. The wells surrounding the sample wells were filled with a 10% greater EtOH concentration to create a microenvironmen t. Control samples were refreshed after acclimatization and exposed to culture medium for 24 h

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! 30 2.3.2 Ethanol Withdrawal Paradigm After acclimatization, cerebellar slices were exposed to EtOH 100 mM for 4 h (EtOH 100 mM Withdrawal). At the same point, cont rol withdrawal (control w) samples were refreshed with culture medium for 4 h. After 4 h, both sets were treated with culture medium for 20 h (Table 2.3) Group 0 h 4 h 24 h Control +Medium EtOH Exposu re +EtOH (25, 50, 100 mM) Medium Control W +Medium All +Fresh Medium EtOH 100 mM W +EtOH (100 mM) Medium All +Fresh Medium Table 2.3: Ethanol exposure and withdrawal experimental d esign. Twenty four hour overview of EtOH exposure (25 100 mM) and cultu re medium treatment of cerebellar slices. Naive samples were stored at 80 o C and were not exposed to the above model. 2.4 Western Blot Analysis 2.4.1 Sample Preparation Tissues were diluted in 1 mL of buffer containing 50 mM Tris HCL (pH 7.4), 1 mM pheny lmethylsulfonyl fluoride (PMSF; Beteshda Research Laboratories, Gaithersburg, MD), and 5 mM EGTA (Sigma Aldrich), homogenized using Polytron batch homogenizer (Kinematica) for 3 x 10 seconds, vortexed for 10 s and allowed to further dissociate on ice for 1 5 minutes. Protein estimation was performed using a Coomassie Plus Protein Assay Reagent (Pierce) and measured at 595 nm by spectrophotometric analysis ( Spectronic). Samples were then diluted 1:1 v/v with sample buffer (62.5 mM Tris HCL (pH 6.8), 2% SDS, 5 mM mercaptoethanol, 10% glycerol) and boiled for 10 min. Samples were equilibrated to a final protein concentration of 1.5 mg/mL with a 1:1 v/v mix of sample and homogenizing buffer, containing bromophenol blue dye (0.01%; Sigma Aldrich), and stored at 4 o C.

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! 31 2.4.2 Western Blot Equal volumes of prepared samples (10 # L) were loaded on 4 20% gradient gels (Biorad). Gels were electrophoresed for 1 hr at 100 V and resolved proteins were transferred to Immobiolon TM P polyvinylidene fluoride micro porous membranes (Millipore) in a Genie transfer apparatus (Idea Scientific). Next, membranes were blocked in 5% nonfat milk in Tris/Tween buffer (20 mM Tris HCl (pH 7.6), 137 mM NaCl, and 0.1% Tween 20) for 1 hr at 4 o C. Eighteen primary IgG antibodies were used, both mon oclonal and polyclonal. The specifications of these antibodies including antibody targeted molecular weight, company, catalog number, and dilution used are presen ted in the Appendix in Table A.2 After overnight incubation of primary antibody at 4 o C, membr anes were incubated with horseradish peroxidase (HRP) conjugated goat anti mouse and anti rabbit secondary antibody (ICN Biomedicals) for 1 hr at room temperature. Between all steps, membranes were washed three times x 5 min with Tris buffer. After seconda ry incubation, membranes were incubated with enhanced chemiluminescent (ECL or ECL Plus) reagent (Amersham) and imaged on a FluorChem FC2 Imaging System (Alpha Innotech/Cell Biosciences). Based on previous research and optimization in Banik lab, blots we re developed and imaged at times varyi ng from 30 s to 4 min (Table A.3 ). 2.4.3 Reprobing Gels GAPDH and GFAP immunoblots were reprobed for Bax and Bcl 2, respectively, after being washed three times x 5 min in Tris buffer and washed again over night at 4 o C. The blots were brought to room temperature with a final wash three times x 5 min before developing.

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! 32 2.5 Statistical Analysis Western blots were imaged using Photoshop and optical density (OD) of each band was measured with ImageJ (NIH, Bethesda, MD) Grayscale TIFF images (300 pixels/inch, 8 bits/channel) were analyzed after 50 pixel background subtraction in ImageJ. Each band represents a minimum of three slices from three mice, which was performed in triplicate, as three bands were run for each cat egory. Results were assessed by InStat GraphPad software (GraphPad Company). Since data were not normally distributed, data were compared by ranks using Kruskal Wallis (control v. EtOH 25 100 mM) or Mann Whitney (control W v. EtOH 100mM W and naive v. cont rol). The difference between two conditions was considered significant at p < 0.05. Post tests were performed for significance from Kruskal Wallis by Dunn's Multiple Comparison's test. Box plots were constructed in Origin (OriginLab Corporation, Northampton, MA). 2.6 Semi quantitative Reverse Transcriptase Polymerase Chain Reaction ( RT PCR ) 2.6.1 Total RNA Isolation from Mice Cerebellum Slices After appropriate exposure, mice cerebella tissue slices were harvested from each category into 1 mL Trizol Reagen t (Applied Biosystems) following manufacturer's protocol with slight adjustment. Samples were homogenized (Kinematic Polytron Homogenizer, PT 1200E) for 3 x 10 seconds, followed by 5 min incubation at room temperature. Samp les were centrifuged for 10 min, after which supernatant was transferred to fresh tubes and allowed to equilibrate for 5 min at room temperature. Next, 200 L of chloroform (Sigma) was added to each sample, vortexed for 15 sec, incubated at room temperature for 10 min, then centrifuged fo r 10 min, allowing distinct separation of DNA, RNA, and protein. The upper aqueous layer of RNA (400 L) was transferred to fresh tubes, to which 500 L of isopropanol (Sigma, 99%) was added. Samples were vortexed for 10 seconds, incubated at room temperat ure for 10 min, and centrifuged for

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! 33 8 min. The supernatant was removed, 1 mL of 75% EtOH was added to the RNA pellet, and centrifuged for 10 min. The EtOH was removed and the RNA pellet allowed to air dry. The RNA pellet was rehydrated with 10 L of Nuclea se free water (Ambion Invitrogen), and samples stored at 80 o C. All centrifugations occurred at 12,000 x g at 4 o C in an Eppendorf centrifuge (Germany). RNA concentration was determined using a 1:500 dilution of samples with TE Buffer (10 mM Tris (Fischer S ci.), 1 mM EDTA (Fischer Sci.), pH to 8.0). RNA concentration was determined based on the absorption at 280:260 ratio and dilution factor, as measured in a Biomate 3S UV Visible Spectrophotometer (Thermo Scientific). RNA concentrations were adjusted corres pondingly to 0.4 g/10 L with nuclease free water (Ambion Invitrogen). 2.6.2 cDNA preparation Components from cDNA kit (Applied Biosystems) were added to 0.4 g of RNA, adjusted based on concentration. Reactions were added to an Eppendorf Mastercycler for four stages 10 min at 25 o C, 120 min at 37 o C, 5 min at 85 o C and held at 4 o C with a lid temperature set at 105 o C. 2.6.3 RT PCR Amplification cDNA samples and PCR kit components were gently thawed on ice. A master mix containing PCR Buffer II, 25 mM MgCl 2 10 mM dNTP Mix, 10 M Forward Primer, 10 M Reverse Primer, AmpliTaq Gold Polymerase, and Nuclease Free Water was prepared and 48 L of the mix was added to new 0.5 mL PCR tubes. To the master mix, 2 L of cDNA was added and four genes amplified Calpain 1, Calpain 2, Calpa statin, and actin (IBT Technologies). Reactions occurred in a thermal cycler (Eppendorf Master Cycler) for four stages 1 cycle at 95 o C for 10 m, 40 cycles of 95 o C x 30 s, 62 o C x 1 min, and 72 o C for 1 min, 1 cycle of 72 o C for 10 m, and held at 4 o C.

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! 34 2.6. 4 PCR Product Resolution PCR products (10 L) and 100 bp ma r ker (Promega, 10 L) were diluted with 2 L 6x loading buffer and loaded into 10% Mini Protean TBE Gels (Bio Rad). Products were electrophoresed at 100 V for 2 h, stained with SYBR Safe (Invitroge n) diluted in TBE Buffer (0.089 M Tris, 0.089 M Boric Acid, 0.0025 M EDTA, pH 8.8), and imaged for 800 ms at 340 nm using an Alpha Innotech Flourchem FC2 (San Leebro, CA).

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! 35 Chapter 3: Results In order to study EtOH exposure and withdrawal effects in the developed cerebellum, 250 # m thick sagittal cerebellar slices from ten and eleven week old mice were incubated with or without EtOH for 24 h. A time course of 24 h was used in the current study to allow EtOH W time and sufficient chronic e xposure of doses of physiologically relevant concentrations of EtOH (25, 50, 100 mM). After appropriate exposure (Table 2.1) of cerebellar slices, protein extraction was performed and protein from two or three slices was loaded in each lane for Western bl ot analysis. Naive samples were stored directly at 80 o C after dissection and separation, and compared to control groups in order to characterize the model. Primary and secondary antibodies allowed for the identification of respective protein bands. Optica l density was measured and compared between appropriate group using statistical analysis methods (see Materials & Methods). Full gels with appropriate bands identifie d are available in Appendix B Following Western blot analysis, the molecular levels of actin, Calpain 1 and C alpain 2 were probed using reverse transcriptase PCR (RT PCR), however, results were inconsistent among replications and are briefly discussed in the last section of the chapter. To begin, a number of axon, myelin, cytoskeletal, and h ousekeeping proteins that have previously been targets of EtOH toxicity were measured. At the end of each section, tables containing median OD measurements, percent change, and significance values of the proteins measured are presented. 3.1 Myelin Protei ns The integrity of characteristic proteins comprising myelin was probed, as EtOH induced cerebellar damage is associated with the demyelination and atrophy of axons

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! 36 (Chopra & Tiwari, 2012). Myelin basic protein (MBP) is expressed in mature oligodendrocyte s, involved in myelination of the nervous system, and makes up roughly 30 % of total protein in the CNS (Siegel et al., 1999) MBP levels were significantly decreased in control samples compared to naive samples, and in cerebellar slices exposed to 100 mM E tOH compared to control samples (Figure 3.1). Figure 3.1: MBP protein expression. Representative Western blot (A) and median box plots with statistical significance of 21.5 kDa MBP in cerebellar slices. Naive samples were stored at 80 o C. Optical densit y measurements were compared between (Ai) control vs. naive, and (Aii) control to EtOH 25 100 mM. Total N values are as follows: naive (n=9), control (n=22), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=22), where ** P<0.0001 and P<0.001.

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! 37 CNPase another important enzyme implicated in myelination in the PNS and CNS, was also significantly decreased in control samples compared to naive samples but unperturbed by EtOH exposure (Figure 3.2). Figure 3.2: CNPase protein expression. Representative W estern blot (A) and median box plots of 48 kDa CNPase in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive, and (Aii) control to EtOH 25 100 mM. Total N values are as follows: n aive (n=9), control (n=11), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=11), where P<0.001. While no significant CNPase change was observed following EtOH exposure, withdrawal samples treated with 100 mM EtOH for 4 h had significantly greater C NPase than control withdrawal samples (Figure 3.3, Table 3.1 next page ).

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! 38 Figure 3.3: CNPase protein expression following withdrawal treatment. Representative Western blot (B) and median box plot with statistical significance (Bi) of 48 kDa CNPase in ce rebellar slices. Optical density measurements were compared between control W (n=11) vs. EtOH 100 mM W (n=11), where P<0.001. Myelin associated Protein Control W EtOH 100 mM W MBP (OD) 17.586 17.235 Percent Change (%) 1.9 CNPase (OD) 78.358 92.258 Significance p<0.001 Percent Change (%) +17.7 Table 3.1: Myelin associated protein measurements following withdrawal treatment. Median OD measurements, percent change (%), and significance of axon and myelin protein measurements between control W and EtOH 100 mM W cerebellar samples.

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! 39 While CNPase increased following EtOH W treatment, MBP was not affected. Tables 3.2 and 3.3 provide an overview of median OD measurements for CNPase and MBP. Axon and Myelin Protein Nai ve Control MBP (OD) 25.012 18. 861 Significance P<0.0001 Percent Change (%) 98.7 CNPase (OD) 136.709 114.175 Significance P<0.001 Percent Change (%) 16.5 Table 3.2: Control versus naive measurements for axon and myelin proteins in cerebellar slices. Median OD measurements, p ercent change (%), and significance of axon and myelin protein measurements between control and naive cerebellar samples. Myelin associated Protein Control EtOH 25 mM EtOH 50 mM EtOH 100 mM MBP (OD) 18.861 19.537 17.878 15.044 Significance P<0.001 P ercent Change (%) +3.6 5.2 20.2 CNPase (OD) 114.175 110.795 104.316 100.958 Percent Change (%) 2.9 8.6 11.6 Table 3.3: Myelin associated protein measurements in control and EtOH 25 100 mM samples. Median OD measurements, percent change (%), and s ignificance of axon and myelin protein measurements between control and EtOH 25 100 mM cerebellar samples. 3.2 Cytoskeletal Protein Degradation Cytoskeletal proteins are involved in the integrity, motility, and structure of neurons and were probed next fo r structural integrity of the cerebellar slices. Three main types of proteins comprise the neuronal cytoskeleton microtubules (MT s ), intermediate filaments (IF s ), and actin filaments (Perrot et al., 2008).

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! 40 NFs are the most abundant neuronal IF and exist in three isoforms NFL, NFM, and NFH. NFs determine axon diameter, and can also cross link with one another and with MTs (Hoffman PN et al., 1987; Perrot et al., 2008). Levels of NFL and NFH (Figure 3.4 and 3.5, respectively) were both significantly decre ased ~50% in control cerebellar slices compared to naive. A dose dependent decrease in NFL and NFH was observed in response to EtOH, but only NFL degradation was to the point of significance (Figure 3.4). Figure 3.4: NFL protein expression. Representati ve Western blot (A) and median box plots with statistical significance of 68 kDa NFL in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive and (Aii) control to EtOH 25 100 mM. To tal N values are as follows: naive (n=9), control (n=11), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=11), where P<0.001 and P<0.05.

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! 41 Figure 3.5: NFH protein expression. Representative Western blot (A) and median box plots with statistical significance of 200 kDa NFH in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive and (Aii) control to EtOH 25 100 mM. Total N values are as follows: naive (n=9), control (n=11), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=11), and P<0.001. NFL was significantly decreased in EtOH 100 mM W samples compared to control W samples (Figure 3.6 next page ).

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! 42 Figure 3.6: NFL protein expression following withdrawal treatmen t. Representative Western blot (B) and median box plot with statistical significance plot (Bi) of 68 kDa NFL in cerebellar slices exposed to EtOH 100 mM (EtOH 100 mM Withdrawal) or culture medium (control Withdrawal) for 4 h, at which point refreshed with fresh culture medium (see Materials & Methods). Optical density measurements were compared between control W (n=11) vs. EtOH 100 mM W (n=11), where P<0.05. tubulin is a tubulin isoform found in MTs in neuronal cells. While tubulin protein levels were not found to be significantly altered in EtOH treated cerebellar samples, tubulin was significantly degraded in control samples compared to naive samples (Fi gure 3.7 next page ).

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! 43 Figure 3.7: tubulin protein expression Representative Western blot (A) and median box plots with statistical significance of 46 kDa tubulin in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive, and (Aii) control to EtOH 25 100 mM. Total N values are as follows: naive (n=9), control (n=22), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=22), where P<0.001. Actin filaments are 8 nm in diamete r, and assemble into double helical polymers ( Karp, 2010) Actin filaments are involved in variety of cellular functions including cell motility and axonal growth. actin was significantly decreased in cerebellar samples treated with 25 and 100 mM EtOH compared to control samples (Figure 3.8 next page ).

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! 44 Figure 3.8: actin protein expression Representative Western blot (A) and median box plots with statistical significance of 42 kDa actin in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive and (Aii) control to EtOH 25 100 mM. Total N values are as follows: naive (n=9), control (n= 22), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=22), where ** P<0.0001, P<0.001, and P<0.01. Interestingly, actin was significantly increased in withdrawal samples treated with EtOH (100 mM) compared to control withdrawal samples (Figure 3 .9 next page ).

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! 45 Figure 3.9: actin protein expression following withdrawal treatment Representative Western blot (B) and median box plot with statistical significance (Bi) of 42 kDa actin cerebellar slices exposed to (EtOH 100 mM Withdrawal) or cult ure medium (control Withdrawal for 4 h, at which point refreshed with fresh culture medium (see Materials & Methods). Optical density measurements were compared between control W (n=11) vs. EtOH 100 mM W (n=11), where P<0.01. Overall, protein measureme nts for the cytoskeletal proteins NFL, NFH, tubulin and actin were significantly decreased in control samples compared to naive samples (Table 3.4, next page).

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! 46 Cytoskeletal Protein Naive Control NFL (OD) 113.64 62.292 Significance P<0.001 Percent Change (%) 45.2 NFH (OD) 138.808 74.124 Significance P<0.001 Percent Change (%) 46.6 tubulin (OD) 129.175 19.482 Significance P<0.001 Percent Change (%) 84.9 actin (OD) 96.299 48.703 Significance P<0.0001 Percent Change (%) 49.4 Table 3.4: Control versus naive meas urements for cytoskeletal proteins in cerebellar slices Median OD measurements, percent change (%), and significance of cytoskeletal protein measurements between control and naive cerebellar samples. While there was not a significant decrease for all cy toskeletal proteins following EtOH treatment, there is an observable downward trend in OD measurements (Table 3.5, next page). Actin and NFL were the only cytoskeletal proteins significantly altered by EtOH W treatment, and responded in an opposing manner (Table 3.6, next page).

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! 47 Cytoskele tal Protein Control EtOH 25 mM EtOH 50 mM EtOH 100 mM NFL (OD) 62.292 47.002 33.0 08 8.144 Significance P<0.05 P<0.001 Percent Change (%) 24.5 47.0 86.9 NFH (OD) 74.124 71.722 57.867 54.713 Percent Change (%) 3.2 21.9 26.2 tubulin (OD) 19.482 18.656 19.291 18.089 Percent Change (%) 4.2 0.9 7.2 actin (OD) 48.703 38.33 43.761 34.0695 Significance P<0.0 0 1 P<0.01 Percent Change (%) 21.3 10.1 30.0 Table 3.5: Cytoskeletal protein measurements in control and EtOH 25 100 mM samples. Median OD measurements, percent change (%), and significance of cytoskeletal pr otein measurements between control and EtOH 25 100 mM cerebellar samples. Cytoskeletal Protein Control W EtOH 100 mM W NFL (OD) 39.008 8.883 Significance p<0.0 5 Percent Change (%) 77.2 NFH (OD) 44.574 46.727 Percent Change (% ) +4.8 B tubulin (OD) 8.261 8.986 Percent Change (%) 8.7 B actin (OD) 34.088 41.176 Significance P<0.01 Percent Change (%) +20.8 Table 3.6: Cytoskeletal protein measurements following withdrawal treatment. Median OD measurements, percent change (%), and significance of cytoskeletal protein measurements between control W and EtOH 100 mM W cerebellar samples.

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! 48 3.3 GAPDH Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) catalyzes the reversible oxidative phosphorylation of glyceraldehyde 3 phos phate, an important step in carbohydrate metabolism. The expression of GAPDH was significantly decreased in treated cerebellar slices exposed to 100 mM EtOH, suggesting impaired metabolic activity (Figure 3.10, Table 3.7, next page). Figure 3.10: GAPDH protein expression Representative Western blot (A) and median box plots with statistical significance of 37 kDa GAPDH in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h or naive samples c ompared to control samples. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive, and (Aii) control to EtOH 25 100 mM. Total N values are as follows: naive (n=9), c ontrol (n=11), EtOH 25 mM (n=11), E tOH 50 mM (n=11), EtOH 100 mM (n=11), where *P<0.001 and P<0.01.

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! 49 Housekeeping Protein Control EtOH 25 mM EtOH 50 mM EtOH 100 mM GAPDH (OD) 46.433 28.802 30.35 21.399 Significance P<0.01 Percent Change (%) 37.9 34.6 53.9 Table 3.7: GAPDH m easurements in control and EtOH 25 100 mM samples Median OD measurements, percent change (%), and significance of cytoskeletal protein measurements between control and EtOH 25 100 mM cerebellar samples. Significant degradation of the enzyme was also obse rved in control samples compared to naive, but EtOH W did not significantly alter GAPDH OD levels (Tables 3.8, 3.9). Table 3.8: Control versu s naive measurements for GAPDH in cerebellar slices. Median OD measurements, percent change (%), and significance of housekeeping protein measurements between control and naive cerebellar samples. Housekeeping Protein Control W EtOH 100 mM W GAPDH (OD) 16.268 14.442 Percent Change (%) 11.2 Table 3.9: GAPDH protein measurements following withdrawal treatment. Median OD measurements, percent change (%), and significance of housekeeping proteins between control W and EtOH 100 mM W. 3.4 Astrocytic profi le Glial cells are supporter cells that account for more than half of the brai n cell content and comprise many cell types, including astrocytes and Schwann cells. Astrocytes act to protect neurons by removing neurotransmitters, such as glutamate, from syn aptic clefts following neurotransmission ( Squire et al., 2008 ). Housekeeping Protein Nai ve Control GAPDH 128.243 46.433 Significance P<0.001 Percent Change (%) 63.8

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! 50 Glial fibrillary acidic protein, GFAP, is a characteristic intermediate filament of differentiated astrocytes. While GFAP was not significantly altered by EtOH treatment in cerebellar slices, a prominent dose dependent decrease in optical density measurements was observed (Figure 3.11, Table 10). About half (53%) of GFAP was lost in control slices compared to naive GFAP values (Figure 3.11 next page ). Figure 3.11: GFAP protein expression. Representative Western blot (A) and median box plots with statistical significance of 51 kDa GFAP in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive, and (Aii) control v. EtOH 25 100 mM. Total N values are as follows: naive (n=9), control (n=11), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=11), where P<0.05. Aquaporin 4 (AQ4) is the predominant brain water channel protein that is expressed in glial cells and to a l esser extent in ependymal cells lining ventricles (Nielsen et al, 1990). AQ4 is significantly degraded in cerebellar slices exposed to EtOH (50 and 100 mM) compared to control samples (Figure 3.12 next page ).

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! 51 Figure 3.12: AQ4 protein expression Repre sentative Western blot (A) and median box plots with statistical significance of 35 kDa AQ4 in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive, and (Aii) control to EtOH 25 10 0 mM and. Total N values are as follows: naive (n=6), control (n=11), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=11), where P<0.001 and p<0.05.

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! 52 Both AQ4 and GFAP control samples were significantly decreased compared to naive samples ( Table 3.10). Astrocytic Protein s Nai ve Control GFAP (OD) 132.892 61.68 Significance P<0.05 Percent Change (%) 53.9 AQ4 (OD) 45.213 36.859 Significance P<0.05 Percent Change (%) 18.5 Table 3.10: Control versus naive measurements for GFAP and A Q4 proteins in cerebellar slices Median OD measurements, percent change (%), and significance of astrocytic proteins between naive and control samples. Both AQ4 and GFAP underwent dose dependent decreases in OD following EtOH treatment (Table 3.11), but were not significantly altered after withdrawal treatment (Table 3.12, next page). Astrocyte Protein s Control EtOH 25 mM EtOH 50 mM EtOH 100 mM GFAP (OD) 61.68 35.147 38.7 37.97 Percent Change (%) 43.0 37.3 53.9 AQ4 (OD) 36.859 37.55 28.102 27.479 Significance P<0.05 P<0.001 Percent Change (%) +1.9 23.8 25.4 Table 3.11: GFAP and AQ4 protein measurements in control and EtOH 25 100 mM samples. Median OD measurements, percent change (%), and significance of astrocyte proteins between control an d EtOH 25 100 mM.

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! 53 Astrocyte Protein Control W EtOH 100 mM W GFAP (OD) 11.838 12.897 Percent Change (%) +8.9 AQ4 (OD) 23.44 24.446 Percent Change (%) +4.3 Table 3.12: GFAP and AQ4 protein levels following withdrawal treatment Median OD me asurements and percent change (%) of astrocytic proteins between naive and control samples. Glutamine synthetase (GS) is an enzyme that catalyzes the reaction of excess ammonia and glutamate to produce glutamine, making it an important regulatory protein in astrocytes. There was no significant change in GS protein across all groups of cerebellar samples (Figure 3.13 & 3.14, Table 3.13, next page).

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! 54 Figure 3.13: GS protein expression. Representative Western blot (A) and median box plots of 4 2 kDa GS OD in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive, and (Aii) control to EtOH 25 100 mM, both found to be not significant. Total N values are as follows: naive (n= 9), control (n=11), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=11).

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! 55 Figure 3.14: GS protein expression following withdrawal treatment. Representative Western blot (B) and median box plot (Bi) of 42 kDa GS in cerebellar slices exposed to EtOH 1 00 mM (EtOH 100 mM W) or culture medium (control withdrawal) for 4 h, at which point refreshed with fresh culture medium (see Materials & Methods). Optical density measurements were compared between control W (n=11) vs. EtOH 100 mM W (n=11), found to be no t significant. Astrocytic Protein Naive Control EtOH 25 mM EtOH 50 mM EtOH 100 mM Control W EtOH 100 mM W GS (OD) 7.7705 10.062 7.639 10.204 10.682 18.463 19.102 Percent Change (%) +29.5 24. 1 +1.4 +6. 2 +3.5 Table 3.13: GS measurements were not signi ficantly altered across all treatment groups. Median OD measurements from Western blot analysis were compared (i) naive v. control, (ii) control v. EtOH 25 100 mM, and (iii) control W v. EtOH 100 mM W. Percent change (%) was calculated in the same manner b etween groups and no significant change was found across all treatment groups. 3.5 Mechanisms of Degradation After observing widespread decreases of structural, myelination, and astrocytic proteins following EtOH exposure, the path to degradation was prob ed by measurement of apoptotic and Calpain associated proteins.

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! 56 The intrinsic pathway o f apoptosis is characterized by activation of Bcl 2 family proteins, including Bax and Bcl 2. This family contains both pro and anti apoptotic proteins whose ratio can determine the magnitude to which apoptosis occurs. Upon activation, pro apoptotic Bax translocates from it's endogenous location in the cytosol to the mitochondrial outer membrane. Research still remains as to how direct interaction of Bcl 2 proteins occ u rs in this pathway, but if pro apoptotic signals outweigh anti apoptotic cascades, the mitochondrial outer membrane is permeabilized in a process known as mitochondrial outer membrane permeabilization (MOMP). Subsequent release of intermembrane space (IMS) proteins, such as cytochrome c and Ca ++ storages, can be released and attenuate apoptotic cascades (Tait & Green, 2010). In the current analysis of cerebellar slices, pro apoptotic Bax significantly decreased with each occurring dose of EtOH (25, 50, 100 mM) compared to control samples. Pro apoptotic Bax was the only apoptosis related protein to be significantly decreased in control samples compared to naive samples (Figure 3.15, next page).

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! 57 Figure 3.15: Bax protein expression Representative Wester n blot (A) and median box plots with statistical significance of 23 kDa Bax in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive and (Aii) control v. EtOH 25 100 mM. Total N val ues are as follows: naive (n=9), control (n=11), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=11), where P<0.001 and p<0.05.

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! 58 Anti apoptotic Bcl 2 was significantly decreased following 100 mM EtOH exposure for 24 h, suggesting the anti apo ptotic cascade was not properly a ctivated (Figure 3.16 ). Figure 3.16: Bcl 2 protein expression. Representative Western blot (A) and median box plots with statistical significance of 28 kDa Bcl 2 in cerebellar slices. Naive samples were stored at 80 o C. O ptical density measurements were compared between (Ai) control vs. naive and (Aii) control to EtOH 25 100 mM. Total N values are as follows: naive (n=9), control (n=22), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=22), where P<0.001. Bcl 2, bu t not Bax, had signific ant degradation in EtOH 100 mM w ithdrawal samples compared to control w (Figure 3.17, next page).

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! 59 Figure 3.17: Bcl 2 protein expression following withdrawal treatment. Representative Western blot (B) and median box plot with statis tical significance (Bi) box plot of 28 kDa Bcl 2 in cerebellar slices exposed to EtOH 100 mM withdrawal) or culture medium (control withdrawal) for 4 h, at which point refreshed with fresh culture medium (see Materials & Methods). Optical density measureme nts were compared between control W (n=11) vs. EtOH 100 mM W (n=11), where P<0.05. Next, the extrinsic apoptotic pathway was probed for activation in cerebellar slices. The binding of ligands to death receptors activates the formation of pro Caspase 8 a nd adaptor proteins to form an aggregate known as the death inducing signal complex (DISC; Galluzzi et al., 2012). Proteolysis of pro Caspase 8, originally a 55 kDa protein, results in the activated p18 and p10 Caspase 8 heterodimers. These activated Caspa se 8 protein bands were no t observed (Appendix Figure B.13 ). Pro Caspase 8 bands at 55 kDa were instead measured, with a significant decrease observed in cerebellar samples treated with 50 mM EtOH compared to control samples (Figure 3.18, next page).

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! 60 Fi gure 3.18: Pro Caspase 8 protein expression Representative Western blot (A) and median box plots with statistical significance of 50 kDa pro Caspase 8 in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared bet ween (Ai) control vs. naive and (Aii) control to EtOH 25 100 mM. Total N values are as follows: naive (n=6), control (n=22), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=22), where P<0.001. Apoptosis can occur via intrinsic and extrinsic pathwa ys, which converge on Caspase 3 as an effector Caspase. Caspase 3 activation involves the proteolysis of pro Caspase 3 (32 kDa) into 11, 17, and 20 kDa active heterodimers that perpetuate the apoptotic cascade. Activated Caspase 3 bands were faintly observ ed and unable to be quantified (Appendix Figure B.14 ); therefore, pro Caspase 3 bands at 32 kDa were

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! 61 analyzed. There were no significant changes in pro Caspase 3 levels across all groups (Figure 3.19). Figure 3.19: Pro Caspase 3 protein expression. Repr esentative Western blot (A) and median box plots of 32 kDa pro Caspase 3 in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h or naive samples compared to control samples. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive, and (Aii) control vs. EtOH 25 100 mM. Total N values are as follows: naive (n=9), control (n=22), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=22), found no t to be significant The incomplete activation of the effector Caspase 3 is consistent with the upstream missing activation of Caspase 8 and decrease of Bcl 2 proteins following EtOH exposure. The median OD measurements, along with significance and percen t change for pro Caspase 8 and the above mentioned apoptotic proteins ar e reviewed in Tables 3.14 3.16 ( next page )

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! 62 Apoptotic Protein Control Naive Pro Caspase 3 (OD) 67.7895 110.808 Percent Change (%) +63.5 Pro Caspase 8 (OD) 74.973 70.889 Percent Ch ange (%) 5.4 B ax (OD) 50.021 43.037 Significance P<0.05 Percent Change (%) 13.9 Bcl 2 (OD) 51.661 33.848 Percent Change (%) 34.5 Table 3.14: Control versus naive measurements for apoptotic proteins in cerebellar slices. Median OD measuremen ts, percent change (%), and significance of apoptotic proteins between control and naive samples. Apoptotic Protein Control EtOH 25 mM EtOH 50 mM EtOH 100 mM Pro Caspase 3 (OD) 110.808 128.475 130.847 112.513 Percent Change (%) +15.9 +18. 1 +1.5 Pro Ca spase 8(OD) 70.8895 55.784 49.282 69.074 Significance P<0.001 Percent Change (%) 21.3 30.5 2.6 Bax (OD) 43.037 38.92 26.728 25.105 Significance P<0.05 P<0.001 P<0.001 Percent Change (%) 9.6 37.9 41.7 Bcl 2 (OD) 33.848 27.409 26.723 23.707 Significance P<0.001 Percent Change (%) 19.0 21.0 29.9 Table 3.15: Apoptotic protein measurements in control and EtOH 25 100 mM samples. Median OD measurements, percent change (%), and significance of apoptotic protein measurements between contr ol and EtOH 25 100 mM cerebellar samples

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! 63 Apoptotic Protein Control W EtOH 100 mM W Pro Caspase 3 (OD) 91.799 105.559 Percent Change (%) +14.9 Pro Caspase 8 (OD) 53.92 62.521 Percent Change (%) +15.9 B ax (OD) 24.157 22.107 Percent Change (%) 8.5 Bcl 2(OD) 46.703 41.675 Significance P<0.05 Percent Change (%) 10.7 Table 3.16: Apoptotic protein measurement following withdrawal treatment Median OD measurement and percent change (%) of apoptotic protein measurements between control W and EtOH 25 100 mM W. Other sources of cytoskeletal degradation and proteolytic activation following cell damage are Calpain s, or Ca ++ activated neut ral proteases. Two isoforms of C alpain a re expressed in neural tissue, Calpain 1 and C alpain 2, which res pond to micro and milli molar concentrations, respectively, of Ca ++ flux in the cell. Calpains have been seen to be responsible for degradation of MT and cytoskeletal proteins (Wu & Lynch, 2006). However, both C alpain 1 and Calpain 2 were significantly d egraded in cerebellar slices treated with EtOH (25, 50, 100 mM), suggesting that they are not responsible for EtOH induced axon and myelin degradation (Figures 3.20 and 3.21, next page).

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! 64 Figure 3.20: Calpain 1 protein expression. Representative Western blot (A) and median box plots with statistical significance of 76 kDa Calpain 1 in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive, and (Aii) control to EtOH 25 100 mM and. To tal N values are as follows: naive (n=9), control (n=11), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=11), where P<0.001 and P<0.05.

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! 65 Figure 3.21: Calpain 2 protein expression. Representative Western blot (A) and median box plots with statist ical significance of 76 kDa Calpain 2 in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) control vs. naive, and (Aii) control to EtOH 25 100 mM. Total N values are as follows: naive (n=9), cont rol (n=11), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=11), where P<0.001 and P<0.05. Only Calpain 1 was significantly decreased in EtOH 100 mM withdrawal cerebellar slices compared to control w samples (Figure 3.22, next page).

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! 66 Figure 3.22: Calpain 1 protein expression following withdrawal treatment. Representative Western blot (B) and median box plot with statistical significance (Bi) of 76 kDa Calpain 1 cerebellar slices exposed to EtOH 100 mM (EtOH 100 mM Withdrawal) or culture mediu m (control Withdrawal) for 4 h, at which point refreshed with fresh culture medium (see Materials & Methods). Optical density measurements were compared between control W (n=11) vs. EtOH 100 mM W (n=11), where P<0.05. Interestingly, the endogenous Calp ain inhibitor, Calpastatin, was found to be significantly increased approximately 53% more in cerebellar control samples compared to naive samples (Figure 3.23, next page).

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! 67 Figure 3.23: Calpastatin protein expression Representative Western blot (A) an d median box plots with statistical significance of 126 kDa C alpastatin in cerebellar slices. Naive samples were stored at 80 o C. Optical density measurements were compared between (Ai) naive v. control, and (Aii) control to EtOH 25 100 mM. Total N values are as follows: naive (n=6), control (n=11), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=11), where P<0.001. With an over 50% increase in Calpastatin OD measurements from naive to control samples, control and EtOH treated samples remained at si mi lar levels (Tables 3.17 & 3.18, next page). The levels of Calpain 1 and Calpain 2 did not significantly alter between control and naive samples (Table 3.19, next page).

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! 68 Calpain related Protein Control EtOH 25 mM EtOH 50 mM EtOH 100 mM Calpain 2 (OD) 72.117 56.293 46.518 52.937 Significance P<0.05 P<0.001 P<0.001 Percent Change (%) 21.9 35.5 26.6 Calpain 1 (OD) 59.328 42.313 34.263 38.541 Significance P<0.05 P<0.001 P<0.001 Percent Change (%) 28.7 42.2 35.0 Calpastatin (OD) 20.732 21.9 29 15.935 17.857 Percent Change (%) +5.7 23.1 13.9 Table 3.17: Calpain related protein measurements in control and ETOH 25 100 mM samples. Median OD measurements, percent c hange (%), and significance of C alpain related protein measurements between con trol and EtOH 25 100 mM cerebellar samples. Calpain Related Protein Control W EtOH 100 mM W Calpain 2 (OD) 30.024 32.718 Percent Change (%) +8.9 Calpain 1 (OD) 32.36 27.086 Significance P<0.05 Percent Change (%) 16.3 Calpastatin (OD) 25.303 19.8 16 Percent Change (%) 21.7 Table 3.18: Calpain related protein measurements following withdrawal treatment. Median OD measurements, percent c hange (%), and significance of C alpain related protein measurements between control W and EtOH 100 mM W cerebe llar samples.

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! 69 Calpain Related Protein Control Naive Calpain 2 (OD) 77.809 72.117 Percent Change (%) 7.3 Calpain 1 (OD) 60.77 59.328 Percent Change (%) 2.4 Calpastatin (OD) 13.463 20.732 Significance P<0.001 Percent Change (%) +53.9 Tabl e 3.19: Control versus naive measurements for Calpain related proteins in cerebellar slices Median OD measurements, percent change (%), and significance of Calpain related protein measurements between control and naive cerebellar samples Lysosomal invol vement of protein degradation was probed by measuring a lysosomal aspartic protease that functions best in acidic environments, Cathepsin D. Cath D is activated by Caspases into a mature 34 kDa isoforms, which is hypothesized to be an initiator of apoptosi s since it's release into the cytosol triggers cleavage of Bcl 2 proteins (Liaudet Coopman et al., 2006). In the current study, Cath D was significantly degraded in cerebellar samples treated with 50 mM compared to control samples, although blotting error s do not allow for a correct 100 mM protein estimation (Figure 3.24, next page).

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! 70 Figure 3.24: Cathepsin D protein expression. Representative Western blot (A) and median box plots with statistical significance of 48 kDa intermediate Cathepsin D in cerebe llar slices. Optical density measurements were compared between (Ai) control vs. naive, and (Aii) control to EtOH 25 100 mM. Total N values are as follows: Naive (n=6), Control (n=22), EtOH 25 mM (n=11), EtOH 50 mM (n=11), EtOH 100 mM (n=22), where P<0.0 01. 3.6 RT PCR Results Following Western blot analysis, three sets of RT PCR reactions were performed for Calpain 1 and 2, Calpastatin, and actin, however, products were inconsistent among sets and could not be analyzed for EtOH effects. The RT PCR products of actin contain a residual ~400 kDa double band product, which could be due to non specific annealing of primers. Non specific annealing of primers, incorrect annealing

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! 71 temperature, and a number of other procedural errors could account for inc onsistencies. The two gels with the greatest number of RT PCR products are presented (Figure 3.25). Figure 3.25: Calpain 1 and 2, Calpastatin, and actin RT PCR expression. RT PCR products for Calpain 1 and 2 (CAPN1 and CAPN2, respectively; A) and Cs alpastatin (calpst) and actin (B) were separated by a 100 bp maker (M) with respective expected product lengths. Samples were run in the same order: naive (N), control (C), control w (CW), EtOH 25 mM (25), EtOH 50 mM (50), EtOH 100 mM (100), EtOH 100 mM W (100 w).

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! 72 Chapter 4: Discussion Alcoholism is an addiction to ethanol (EtOH) that is characterized by alterations to brain morphology and degradation of im portant cerebral circuits Current research on alcoholism is focused on excitotoxic pr op erti es of EtOH and less on the impact EtOH can have on myelin and axon constituents. In the current thesis, ten and eleven week old cerebellar mice samples were exposed to EtOH for 4 or 24 h and compared to respective control samples. Eighteen proteins were measured by Western blot analysis and a discussion of EtOH exposure or withdrawal on these proteins follows. 4.1. Myelin Protein Alterations in Response to EtOH Myelin atrophy is a common pathological marker in alcoholics and loss of myelin predispo ses axons to consequent pro inflammatory events and proteolytic cascades (Alfonso Loeches et al., 2012). Two myelin associated proteins, 2', 3' cyclic nucleotide 3' phosphodiesterase (CNPase) and myelin basic protein (MBP), were analyzed in the current mou se cerebellar slices. The 21.5 kDa isoform of MBP was significantly degraded following 24 h EtOH exposure (Figure 3.1), supporting the hypothesis that EtOH targets myelin proteins. CNPase levels were not altered by EtOH exposure, but were significantly inc reased 17% in EtOH 100 mM W samples (Figure s 3.2 & 3.3). This is an unexpected result and could be supported by the idea that the slices were attempting to recover following assault. 4.2 EtOH Selectively Perturbed Cytoskeletal Proteins Cytoskeletal protei ns are involved in integrity, motility, and structure of the neuron. The three main types of proteins that comprise the cytoskeleton microtubules (MTs), intermediate filaments (IFs), and actin filaments had different responses to EtOH exposure and withdra wal in the current study.

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! 73 4.2.1 Neurofilaments NFs are vital in neuronal structure and integrity, increasing axonal caliber, and interactions with other MTs and NFs. There are three polypeptide subunits that comprise the NF classification: NF proteins lig ht (60 kDa), medium (160 kDa), and heavy (200 kDa). In the present cerebellar slices, both EtOH exposure and withdrawal of EtOH resulted i n a decrease of NFL (Figures 3.4 & 3.6), but did not significantly affect NFH (Figure 3.5), supporting the hypothesis that EtOH targets axonal components. This selective propensity for an over 80% NFL degradation compared to only a 27% decrease in NFH OD after 100 mM EtOH exposure may be due to the fact that NFL is able to self assemble, while NFH and NFM require vimentin or NFL for NF assembly (Perrot et al., 2008). NFH and NFM also undergo post translation phosphorylation modifications that may have protected NFH from degradation (Perrot et al., 2008). However, in albino mouse allowed to intake EtOH chow for 4 weeks, th e selective degradation of NFH was observed (Tikhomirov et al., 2008), suggesting NFH may require more chronic doses of EtOH to be degraded. 4.2.2 Tubulin Tubulin proteins are the larger diameter, rigid and hollow cytoskeletal filaments responsible for t he structural integrity and motility of cells. In the present study, tubulin proteins were not significantly altered with EtOH exposure or withdrawal (Figure 3.7). Altered microtubule organization and polymerization were observed in cultured astrocytes e xposed to 100 mM for 7 days (Tom ‡ s et al., 2005), while, Loureiro et al. (2011) found no significant degradation of the $ tubulin protein exposed to 100 mM EtOH for 23 h in C6 cells. Additionally, PC12 cells exposed to 100 mM EtOH for 96 h had an increased polymerized tubulin content, but did not have a significant change in tubulin expression following four days of exposure (Reiter Funk & Dohrman, 2005).

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! 74 These previous findings combined with the current study suggest that chronic exposure longer than 24 h may be required to affect microtubule stress fibers. 4.2.3 Actin Fibers actin fibers were significantly degraded when exposed to 25 mM EtOH and 100 mM EtOH (Figure 3.8). This degradation is consistent with the rearrangement and loss of cytoskeletal p roteins actin, RhoA and vinculin, which were observed in C6 cells exposed to 100 mM and 200 mM EtOH for 23 h (Loureiro et al., 2011). actin is commonly used as a loading standard, and the decline of actin fibers due to EtOH exposure may have been overl ooked when it was measured in previous studies. Indeed, Dittmer & Dittmer (2006) observed that actin antibodies did not accurately predict the amount of protein loaded, and combined with the current thesis, warrants a reconsideration of actin as a loadi ng control. Opposing this, actin filaments were significantly increased when exposed to EtOH 100 mM for 4 hours (Figure 3.9), which may be explained by cytoskeletal rearrangement that occurs following EtOH exposure, where actin stress fiber are less dens e and/or become packed in peripheral actin bundles. It may be possible that the slices attempted to reorganize these cytoskeletal changes following 4 h EtOH exposure and were able to increase actin content 20% in EtOH 100 mM W cerebellar slices. 4.3 EtO H induced Metabolic Disturbances Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) catalyzes the sixth step in glycolysis (Berg, 2011) and is commonly measured to probe for metabolic irregularities in a variety of experiments. GAPDH has been found to be ov erexpressed in the nucleus and mitochondria when apoptosis is induced by a range of assaults in different cell lines (Chuang et al 2004). During Caspase independent cell death (CICD),

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! 75 GAPDH overexpression can mediate cell survival by increasing glycolysi s and autophage events (Song & Finkel, 2007; Colell et al., 2007). GAPDH was observed to increase in the prefrontal cortex of EtOH preferring rats fed an EtOH diet for 4 weeks and in neuroblastoma and glioblastoma cell lines following 75 mM EtOH exposure ( Ou et al., 2010). In plated cerebellar slices, GAPDH significantly decreased following 100 mM EtOH exposure (Figure 3.10), which disagrees with the previous experiment where rats ingested EtOH. Furthermore, GAPDH protein levels in plated control samples we re 60% lower than in naive samples (Figure 3.10). The significant degradation in control samples compared to naive samples suggests that the model impaired metabolic function that was further attenuated by EtOH exposure. 4.4 Selective Degradation of Ast rocytic Proteins in Response to EtOH Astrocytes are the neuron supporter cells that are responsible for the removal of NT s in the synaptic cleft and for maintenance of neuronal ion balance and the blood brain barrier Astrocytes are essential for neuronal glucose metabolism as alterations to glucose transport in these cells compromise neuronal function and brain development (Wiesinger et al. 1997; Magistretti & Pellerin, 1999; Tom‡s et al. 2002). GFAP is a class III intermediate filament characteristic of astrocytes and helps determines cel l shape (Middeldorp & Hol, 2011; Evrard & Brusco, 2011). GFAP was not significantly altered in the present study of cerebellar slices following EtOH exposure and withdrawal, but trended downward with an almost 60% loss in EtOH 100 mM samples (Figure 3.11). GFAP is known to respond in a time and dose dependent manner to EtOH, as studies by Franke et al. (1997) reported different GFAP changes in the hippocampus of mice exposed to 10% EtOH for 4 weeks and 36 w eeks GFAP i mmunoreactivity (IR) measurements of 4w EtOH treated mice after were significantly greater than control mice, but this immunoreactivity decreased in mice exposed for 36w (Franke et al., 1997).

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! 76 Furthermore, the hippocampus and cerebral cortex of rats expose d to up to 15% EtOH for 12 w eeks had decreased GFAP protein levels, but cerebellar preparations did not (Tikhomirov et al., 2008). Together, these studies and the current results suggest chronic (> 24 h ) EtOH is needed to induce GFAP protein level changes. Aquaporin 4 (AQ4) is a membrane bound tetramer that is integral in the response of astrocytes to extracellular and intracellular concentration perturbations with the help of various membrane bound proteins (Iacovetta et al 2012). Concentrations of 50 mM and 100 mM EtOH significantly decreased AQ4 proteins in the current cerebellar slices (Figure 3.12). There is also a loss of AQ4 in control samples compared to naive samples. Together, these results suggest specific degradation of this integral membrane p rotein, and therefore reduced water transport, in astrocytes due to the culturing conditions as well as to EtOH exposure. 4.5 Mechanisms of Degradation 4.5.1 Partial Activation of Caspases in Cerebellar Slices Exposed to EtOH Apoptosis is a cellular proc ess that results in cell death and can be propagated by extracellular localization of death ligands (extrinsic pathway) or by intracellular signals (intrinsic pathway). The intrinsic pathway is activated by intracellular signals and is marked by substantia l decline in mitochondrial function. Death signaling ligands can modulate the extrinsic pathway of apoptosis following ligation to their receptors on the PM. Caspase 8 was probed for activation of extrinsic apoptotic cascades. Caspase 3 is the effector Cas pase that is activated downstream of both pathways and was probed for this reason. The polyclonal antibodies used identified both pro and activated forms of both Caspases (see Appendix Table A.2 ).

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! 77 Death receptor ligand binding facilitates the formation o f a 'death inducing signal complex' (DISC) that recruits proteins and pro Caspase 8 (or 10). In the current study, Caspase 8 was not cleaved into an active 18 kDa isoform, and only the pro Caspase 8 50 kDa band wa s observed (Appendix Figure B.13 ). A signi ficant degradation of pro Caspase 8 due to EtOH exposure at 50 mM, but not 100 mM was observed (Figure 3.18). This suggests that EtOH exposure did not activate the extrinsic apoptosis pathway in the current study. 4.5.2 Bax and Bcl 2 Degradation Followed EtOH Exposure Mitochondrial outer membrane permeabilization (MOMP) is characterized by release of cytochrome c, increased intracellular Ca ++ flux, and decreased oxidative phosphorylation pathway activity, all of which can perpetuate apoptotic signals. MOMP is facilitated by Bcl 2 family proteins, which act in both anti and pro apoptotic events by localization to the mitochondrial outer membrane. In the present study, the pro apoptotic 23 kDa Bax was significantly degraded with increasing concentration of 2 4 h EtOH exposure (Figure 3.15), as was anti apoptotic Bcl 2 protein following 100 mM EtOH exposure (Figure 3.16). The pro apoptotic protein Bax was decreased approximately 40% following 100 mM EtOH exposure whereas anti apoptotic Bcl 2 had only a 20% de crease when exposed to 100 mM EtOH (24 h). Cerebellar EtOH w samples had 10% l ess Bcl 2 protein than control w samples (Figure 3.17), but Bax expression was not altered Additionally, Caspase 8 can go on to activate Bid to tBid, which recruits Bax to the m itochondrial outer membrane. The decrease in Bax and Bcl 2 following EtOH exposure is consistent with the hypothesis that extrinsic apoptosis was not fully activated, and additionally indicates intrinsic apoptosis via MOMP was not activated either.

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! 78 Active Caspase 3 bands at 11/17/20 kDa were too faint to be quantified; instead prominent pro Caspase 3 bands at 32 kDa were analyzed and not significantly affected by EtOH exposure of withdrawal (Figure 3.19, Figure B.11). Full activation of Caspase 3 has been o bserved in one month old rats consuming EtOH for up to 8 weeks (Rajgopal et al., 2003). The absence of activated Caspase 3 is consistent with the inactivation and degradation of the extrinsic Caspase 8 and degradation of MOMP inducing Bax and Bcl 2. 4.5.5 Immatu re Cathepsin D Decreased with EtOH Exposure Other sources of NF degradation include lysosomal proteases, such as Cathepsin D. After release from the lysosomal compartment, Cath D can initiate certain apoptotic signaling cascades and the degradation of the extracellular matrix (ECM). Cath D has been observed in both pro and anti apoptotic signaling in gene knockout (KO) experiments and breast cancer cell lines, respectively (Liaudet Coopman et al ., 2006). Mature 34 kDa Cath D is hypothesized to be an initiator of apoptosis since it's release into the cytosol triggers cleavage of Bcl 2 proteins, resulting in cytochrome c release from mitochondrial sources and activating apoptosis signals (Liaudet Coopman et al., 2006). Cath D was significantly decre ased in mouse cerebellar slices treated with 50 mM EtOH compared to control samples (Figure 3.24). Cath D measurements were from the intermediate 48 kDa Cath D forms, with very faint bands for the active 34 kDa form (Appendix Figure B.18), supporting the t heory that activation of proteases has yet to occur in these slices since the caspase family cleave s the 48 kDa Cath D into the 14 and 34 kDa forms following lysosomal release.

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! 79 The microenvironment of the cytosol is tightly regulated, yet can be altered b y changes in metabolism and mitochondrial electron transport. Gottlieb et al. (1996) observed that apoptosis was preceded by intracellular acidification in Jurkat cells and that Cath D was active around a pH of 6.2 6.8. In the present study, GAPDH was sign ificantly degraded with 100 mM EtOH treatment, indicating loss of enzymatic activity in the metabolic system, possibly resulting in a build up of glucose reactants and products that could alter the pH of the cytosol. Additionally, in GFAP and vimentin KO m ice, endosome and lysosome mobility was compromised without proper IF mediated activity (Middeldorp & Hol, 2011), which suggests that the already damaged cytoskeleton may be responsible for Cath D inactivation in the current cerebellar slices. 4.5.3. Calpa in Degradation Followed EtOH Exposure Two isoforms of calcium dependent cysteine proteases, Calpain 1 and Calpain 2, exist in neuronal tissues and act as modulator proteases in a range of physiologically relevant signaling cascades. Calpain 1 and Calpain 2 are activated by micro and milli molar Ca ++ concentrations, respectively, which induces autolytic cleavage and dissociation into active 76 80 kDa catalytic and 29 kDa regulatory subunits (Ono & Sorimachi 2012). Measurements of the 76 kDa subunits of Cal pain 1 and 2 were significantly decreased with increasing concentrations of EtOH exposure (Figures 3.20, 3.21). Calpain upregulation has been implicated in neuronal degeneration, suggesting that EtOH induced degradation of cytoskeletal proteins proceeded via calpain independent pathways. There was also a 16 % significant degradation of C alpain 1 in withdrawal samples, but no significant change in C alpain 2 levels. Activat ed large and small subunits of C alpain remain sensitized following Ca ++ removal (Kitaga ki et al., 1996), suggesting that C alpain 1 may have been sequestered by it's endogenous inhibitor, C alpastatin, following withdrawal from EtOH.

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! 80 4.5.4 Calpastatin Activation in Cerebellar Slices Calpastatin is expressed in glial cells and neurons with cel lular localizations similar to calpain s (Saido et al 1994). Calpastatin is one of the few endogenous proteins that is able to inhibit both forms of cal pain. An over 200% increase in C alpastatin levels was observed in control samples compared to naive sam ples (Figure 3.23). There was no significant effect of EtOH exposure or withdrawal on Calpastatin levels, suggesting that Calpastatin may have been activated initially in treated slices and remained at those levels throughout EtOH exposure. The upregulati on of Calpastatin in control samples compared to naive samples indicates Calpastatin activatio n and possible sequestering of Calpain proteins. In one month old rat cerebellum following a 12 w EtOH diet, decreased Calpain 2 and unchanged levels of Calpastat in were reported (Rajgopal & Vemuri, 2002). Rajgopal and Vemuri (2002) proposed that the observed protei n measurements are a result of C alpain 1 autoproteolysis, the products of which Calpastatin preferentially binds to. Indeed, the unaltered levels of Cal pastatin in the current study may be responsible for sequestering Calpain s during EtOH exposure and/or withdrawal. Overall, the current cerebellar slices underwent significant myelin and cytoskeletal degradation that could not be attributed to apoptotic pa thways measured. EtOH toxicity has also been known to propagate via numerous pathways that were not measured, including ROS generation and pro inflammatory pathways (Jung & Metzger, 2010; Evrard & Brusco, 2011 ). 4.6 Naive samples Although other routes o f cytoskeletal and myelin degradation may have occurred in the samples, it is also important to consider the naive category. Naive samples were

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! 81 stored at 80 o C directly following dissection and were compared to control samples that were exposed to control medium for 24 h and then stored. The naive samples portray the original amount of protein present in the animals, while the control samples portray the amount of protein following model exposure. Ten of the eighteen proteins measured by Western blot analy sis, including all of the cytoskeletal proteins, GAPDH, Bax, and AQ4, decreased significantly between naive and control sample. While pro apoptotic Bax underwent significant degradation, Calpastatin was significantly increased in control samples compared t o naive samples. The proteins that did not significantly alter between naive and control samples were the majority of the apoptotic proteins (pro Caspase 3, pro Caspase 3, Calpain 1, Calpain 2, and Cath D) and GS. This result is important and suggests that some basal level of damage is occurring in the slices, and this must be taken into account when considering the results. Indeed, in 20 30 day old male hippocampal organotypic slices, propidium iodide (PI, permeable to dying cells and stains DNA) values we re significantly greatest in t he first ten days in vitro (DIV; Xiang et al., 2000). However, from 10 80 DIV, PI staining was never observed (Xiang et al., 2000), supporting the idea of initial slice degeneration followed by a stabilization. Although the PC R data was inconsistent among trial replications, some replications did show naive and control products (Figure 3.25), suggesting that the model did not completely destroy molecular machinery. Overall, this implies that the current results concerning EtOH exposure and withdrawal on proteins may still be valid, yet the model should greatly be improved to account for the degradation occurring upon culturing of cerebellar slices. 4.7 Future Improvements Numerous aspects of the current study could be altered according to varying organotypic materials and methods. While the current study utilized common media

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! 82 constituents prevalent in the literature, there are always other options available. For example, serum free medium increased neuronal hippocampal cell su rvival from 3 5 month old mice for 30 days (Kim et al., 2013), but did not allow for physiologically relevant process of neurogenesis, or the creation of new synapses. Utilizing protease inhibitors in the medium of the model was not an option for this thes is due to the downstream protease cascades that were studied, but inhibitors could be used in future studies of myelin associated proteins. Propidium iodide (PI) is the fluorescent, membrane impermeable dye that intercalates between nucleic acids of dead a nd/or dying cells, and was utilized to indicate a pertinent time of cell death in the development of the current model. This experiment was repeated twice with slightly different protocols; however, a significant time of cell death was not obvious (Tables 2.1 & 2.2). It is also possible PI was not able to permeabilize 250 m of rat cerebellar matter, as a recent study implicated post fixative staining of PI in bright field microscopy as a better visualization of cytoarchitecture following brain trauma than pre fixative staining (Hezel et al., 2012). A better indicator of ce ll death occurring in the slices may have alleviated the significant degradation that occurred in control samples. Other staining procedures, such as NeuN (specifically stains neuronal cell bodies) or TUNEL (DNA fragmentation) staining, were not performed due to time constrictions in the current study but would have greatly assisted in assessing the functional and structural integrity of the slices both in naive preparations and following model exposure. This could also be accomplished by taking electrophy siology or lactate dehydrogenase measurements before model exposure, which would show ion channel and anaerobic metabolic activity, respectively. The current study aimed to model mature myelin and axonal constituents, which may be better visualized with no n invasive imaging, such as the relatively new method of diffusion tensor imaging (DTI). By measuring the diffusion of water in the brain, DTI is a

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! 83 sensitive probe for cellular structure and has been used in neurosurgical planning and investigation, as wel l investigation of neurodegenerative diseases and conditions (O'Donnell and Westin, 2011).

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! 84 Chapter 5: Conclusion In conclusion, the current thesis attempt ed to develop and characterize an ex vivo model of adult axon and myelin in order to order to investigate the effects of EtOH exposure and withdrawal. The expression levels of eighteen proteins previously implicated in EtOH degradation were probed by Western blot analysis. Myelin and axon associated proteins and cytoskeletal proteins were degraded by EtOH exposure, consistent with previous literature and supporting our first hypothesis. However, Calpain 1, Calpain 2, pro Caspase 8, Bax and Bcl 2 proteins were degraded following EtOH exposure, disproving our hypothesis that EtOH induced neur onal damage was due to apoptosis and proteolysis. EtOH W resulted in the degradation of three proteins (NFL, Calpain 1, and Bcl 2) but increased two ( actin and CNPase), which are unexpected and novel results. Furthermore, ten out of eighteen proteins, including metabolic and cytoskeletal proteins important in neuronal maintenance, underwent significant degradation in control samples compared to naive samples that were not exposed to the model. This indicates a level of basal damage occurring in cultured slices, coupled with metabolic and structural degradation, and disproves the hypothesis that the current model can be utilized to study EtOH exposure a nd withdrawal. Other models to study the effects of EtOH exposure and withdrawal on adult myelin could include a more time and labor intensive set up exactly like organotypic materials utilizing protease inhibitors, or a long term CIE EtOH model in roden ts, perhaps followed by non invasive imaging of myelin structures. Future studies that do not utilize protease inhibitors, but can account for initial protease activation in intact brain matter, would allow for the study of protease pathways in alcoholism. Apoptosis and activation of Calpain s have been implicated in a number of neurodegenerative diseases (Tait & Green, 2010; Mattson, 2000; Banik et al., 1997; Samantaray et al., 2011), and

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! 85 therefore should not be ruled out as in vivo pathways of EtOH induced myelin and axonal degeneration.

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! 86 Appendix A: Materials & Methods Appendix A.1 : Percent Yield of Isolated Cer e bellar Slices Mouse Cerebellum Width Slice Width Slices obtained Percent Yield 1 0.8 cm 250 m 10 31.25% 2 0.8 cm 250 m 15 46.875% 3 0.8 cm 250 m 10 31.25% 4 0.8 cm 250 m 4 12.5% 5 0.9 cm 250 m 8 22.2% 6 0.8 cm 250 m 13 40.7% 7 0.8 cm 250 m 14 43.75% 8 0.9 cm 250 m 10 27.8% 9 0.9 cm 250 m 13 36.1% 10 0.8 cm 250 m 15 46.875% 11 0.9 cm 250 m 10 27.8% Table A.1: Percent yield of Cerebella Slices Percent yield [(cerebella length slices obtained)/slice width) 100] of slices obtained from eleven week old mice for Western blot analysis.

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! 87 Appendix A.2 : Western Blot Supplemental Antibody Targ et Mouse /Rabbit Poly/ Mon oclo nal Target kDa Source Catalog # Dilution Reprobed Bax Mouse Mono 23 Santa Cruz SC 7840 1:250 Calpastatin Rabbit Poly 120 Santa Cruz SC 20779 1:250 Aquaporin 4 Rabbit 35 kDa AbCam 46182 1:250 Glutamine Synthetase Mouse M ono 42 AbCam 64613 1:1000 Glial Fibrillary Acidic Protein Mouse Mono 51 Millipore Mab 360 1:400 Reprobed for Bax GAPDH Mouse Mono 37 Santa Cruz SC 137179 1:250 Reprobed for Bcl 2 Tubulin Mouse Mono 46 Sigma T8660 1:1000 Calpain 1 Rabbit Poly 80 and 76 Santa Cruz/Banik Lab 1:250 Reprobed for Calpain 2 CNPase Mouse 46 and 48 kDa Sigma C5922 1:500 NFH Rabbit 200 Sigma N4142 1:1000 NFL Mouse Mono 68 Sigma N5139 1:1000 MBP Mouse 21.5 Santa Cruz 1:500 11/17/20 Mat ure Caspase 3 Rabbit Poly 32 Pro Santa Cruz Sc 7148 1:250 18 Mature Caspase 8 Rabbit Poly 50 Pro Santa Cruz SC 7890 1:250 Bcl 2 Mouse 28 Santa Cruz SC 7382 1:250 52 60 Immature 46 48 Intermedi ate Cathepsin D Rabbit Poly 33 Mature Santa Cruz 10725 1:250 Actin Mouse Mono 42 Sigma A5441 1:10,000 Calpain 2 Mouse Mono 80 and 76 Santa Cruz SC 373966 1:250 Table A.2: Antibody supplemental information for Western blot analysis. Poly and mono clonal primary IgG antibodies utilized with respective expected molecular weight, animal of origin, company and catalog number, antibody dilutions used, and reprobe status, if applicable.

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! 88 Reagent Application Time Dry Time Develop Time Antibody 4 min AQ4, Calpastatin, Bax, Bcl 2, GFAP, GAPDH, Calpain 1, Calpain 2, tubulin, Caspase 8 2 min NFL 1 min GS ECL Plus 1 min 1 min 30 s CNPase 4 min NFH, Caspase 3 2 min actin, MBP ECL Table A.3: Developing time and reagents for Western blot: ECL or ECL Plus Reagents and development time of Western blots presented in the current thesis. Other times of development have been experimented with and the best resolution was chosen for figures, while other development times were based on previous development methods in Banik lab.

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! 89 Appendix B : R esult s Appendix B.1 : Full Western Blots Figure B.1: MBP full Western blot Protein expression of probed for MBP in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h compared to control samples i n culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 90 Figure B.2: CNPase full Western blot Protein expression of 48 kDa CNPase in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel co ntains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 91 Figure B.3: NFL full Western blot Protein expression of 68 kDa NFL i n cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture m edium (control withdrawal) for 4 h, at which point refreshed with fresh culture medium for remaining 20 h.

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! 92 Figure B.4: NFH full Western blot Protein expression of 200 kDa NFH in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to con trol samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 93 Figure B.5: Tubulin full Western blot Protein expression of 46 kDa tubulin in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h. Naive samples were stored at 80 o C The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium (control withdrawal) for 4 h, at which point refreshed with fresh culture medium for remaining 20 h

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! 94 Figure B.6: actin full Western blot. Prot ein expression of 42 kDa actin in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (Et OH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 95 Figure B.7: GAPDH full Western blot Protein expression of 37 kDa GAPDH in cerebe llar slices exposed to EtOH (25 50, 100 mM) for 24 h compared to control s amples in culture medium for 24 h compared to control samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or cu lture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 96 Figure B.8: GFAP full Western blot Protein expression of 51 kDa GFAP in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to cont rol samples in culture medium for 24 h compared to control samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 97 Figure B.9: AQ4 full Western blot Protein expression of 35 kDa AQ4 in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium fo r 24 h. Naive samples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 98 Figure B.1 0: GS full Western blot Protein expression of 42 kDa glutamine synthetase in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contains sa mples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 99 Figure B.11 : Bax full Western nlot. Protein expression of 23 kDa Bax in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 100 Figure B.12 : Bcl 2 full Western blot. Protein expression of 28 kDa Bcl 2 in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compare d to control samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh cultur e medium for remaining 20 h.

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! 101 Figure B.1 3 : Caspase 8 full Western blot. Protein expression of 50 kDa active Caspase 3 in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h. Naive samples wer e stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 102 Figure B.14 : Caspase 3 full Weste rn blot. Protein expression of 32 kDa pro Caspase 3 and 20 kDa active Caspase 3 in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contai ns samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 103 Figure B.15: Calpain 1 full Western blot. Protein expression of 80 k Da pro and 76 kDa active Calpain 1 in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 104 Figure B.16: Calpain 2 full Western b lot. Protein expression of 80 kDa pro and 76 kDa active Calpain 2 in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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! 105 Figure B.17: Calpastatin full Western blot. Protein expression of 126 kDa Calpastatin in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compare d to control samples in culture medium for 24 h. Naive samples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh cultur e medium for remaining 20 h.

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! 106 Figure B.18: Cathepsin D full Western blot Protein expression of 48 kDa immature cathepsin D in cerebellar slices exposed to EtOH (25, 50, 100 mM) for 24 h compared to control samples in culture medium for 24 h. Naive sam ples were stored at 80 o C. The second gel contains samples that were exposed to EtOH 100 mM (EtOH 100 mM withdrawal) or culture medium for 4 h (control w), at which point refreshed with fresh culture medium for remaining 20 h.

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