The Prion Protein Binds to Syntaxin and Synapsin in the Presynaptic Neuromuscular Junction

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Title:
The Prion Protein Binds to Syntaxin and Synapsin in the Presynaptic Neuromuscular Junction
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english
Creator:
Herrera, Jose
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University of Florida
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Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Master's ( M.S.)
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University of Florida
Degree Disciplines:
Medical Sciences, Molecular Genetics and Microbiology
Committee Chair:
Fernandez-Funez, Pedro
Committee Members:
Foster, Tom
Kumar, Ashok
Carter, Christy S.
Kraft, John

Subjects

Subjects / Keywords:
prion -- protein -- synapse -- synapsin -- syntaxin
Molecular Genetics and Microbiology -- Dissertations, Academic -- UF
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Medical Sciences thesis, M.S.
Electronic Thesis or Dissertation
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )

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Abstract:
The prion protein(PrP) is a GPI-anchored glycoprotein located in the extracellular membrane of neuronal and glial cells of the developing and mature nervous system. Although PrP misfolding and aggregation are the causative factors in severa lneurodegenerative disorders, the physiological role of PrP is unknown. Previous studies have described the localization of PrP in the synapse, where itinteracts with synaptic proteins; however, the physiological relevance thereof remains unknown. My goal was to ascertain the function of PrP in the synapse,based on its interaction with known synaptic proteins. Using transgenic fliesthat express wild-type PrP, I first tested the co-localization of PrP with candidate synaptic proteins by immunofluorescence. Then, I utilizedco-immunoprecipitation to confirm the direct interaction of positive candidates. I found that the synaptic proteins, Synapsin and Syntaxin, co-localize and interact directly with the wild-type PrP. Moreover, I studied several mutant PrP fly strains,P102L, Y145X, V180I, and M206, 213S, to characterize phenotypic changes in synaptic morphology and protein distribution. Over time, wild-type and mutant strains of PrP exhibited significant losses in synaptic boutons and active zones. Taken together, these results suggest a possible role of neurotransmission for PrP in the presynaptic neuromuscular junction.
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University 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.
Thesis:
Thesis (M.S.)--University of Florida, 2012.
Local:
Adviser: Fernandez-Funez, Pedro.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31
Statement of Responsibility:
by Jose Herrera.

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lcc - LD1780 2012
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UFE0044772:00001


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1 THE PRION PROTEIN BINDS TO SYNTAXIN AND SYNAPSIN IN THE PRESYNAPTIC NEUROMUSCULAR JUNCTION By JOS HERRERA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Jos Herrera

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3 To my parents

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4 ACKNOWLEDGMENTS As I reflect upon the past two years of graduate w ork, what astounds me most is the amount of independence I have gained from working on my own project. I arrived at the Fernandez Funez and RinconLimas lab a shy and nervous 22 year old, without much knowledge of what was to be expected of me. Perhaps mos t daunting was Drosophila melanogaster itself, a new organism unfamiliar to me: I had spent most of my undergraduate years conducting research with mice and rats, but the fruit fly was a completely new animal model for me. Because of the significant differ ences between these organisms, I had to immerse myself in learning fly genetics, breeding, and brain anatomy for weeks after joining the lab. My first project entailed studying the relationship between the prion protein (PrP) and amyloidd at first to learn how to dissect adult brains, a meticulous task that requires the most dexterous of hands. Adroit as I am, it was challenging at first to habituate to the nuances of such dissections. In time, however, I was able to master the skill, and I grew confident in my ability to study Drosophila. Fly husbandry was also a bit of an awkward task for me, but once I learned the genetics behind the concept, I was able to master it, too. Fruitful as it was to excel at new challenges, my project had not been going well. flies co expressing both proteins should suffer the greatest neurodegeneration; however, what I was getting was quite the opposite. As I struggled each day to pr ove my thesis, it became clearer that I would have to drop the project, in search for a new. Discouraged and defeated, my mentor assigned me a new project: I was to assess the effects of PrP in the neuromuscular junction (NMJ) of the fruit fly, in order to find binding

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5 partners of PrP at the synapse. Unlike my previous project, I was much more successful than with my first project. This could not have been possible without my mentors, Drs. Pedro Fernandez Funez and Diego RinconLimas, as well as the brillia nt PrP expert in our lab, Dr. Jonatan Sanchez Garcia.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 2 MATERIALS AND METHODS ................................................................................ 18 Tissue Preparation .................................................................................................. 18 Larval NMJ Preparation .................................................................................... 18 Adult Abdominal NMJ Preparation ................................................................... 18 Immunofluorescence ............................................................................................... 18 Western blot analysis .............................................................................................. 19 Co Immunoprecipitation .......................................................................................... 20 Conjugation of Dynabeads ............................................................................... 20 Co Immunoprecipitation ................................................................................... 20 NaPTa/Sarkosyl Precipitation ................................................................................. 21 Fly Strains ............................................................................................................... 21 Climbing Assay ....................................................................................................... 22 Microscopy .............................................................................................................. 22 3 RESULTS ............................................................................................................... 23 Co localization at the Larval Neuromuscular Junction ............................................ 23 The Larval Neuromuscular Junction ................................................................. 23 Cell Adhesion Proteins ..................................................................................... 23 Structural Proteins ............................................................................................ 24 Synaptic Vesicle Cycle Proteins ....................................................................... 24 Co Localization at the Adult Neuromuscular Junction ............................................ 25 The Adult Neuromuscular Junction .................................................................. 25 Co loc alization of Synapsin and Syntaxin ......................................................... 25 Protein Interaction in the Adult Fly .......................................................................... 26 Interaction between PrP and Syntaxin ............................................................. 26 Interaction between PrP and Synapsin ............................................................ 27 Locomotor Dysfunction in PrP Adult Flies ............................................................... 27 The Climbing Assay ......................................................................................... 27

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7 Locomotor Dysfunction ..................................................................................... 28 Interaction between PrPSC and Synaptic Proteins .................................................. 28 PrPSC in the Presynapse .................................................................................. 28 4 DISCUSSION ......................................................................................................... 40 LIST OF REFERENCES ............................................................................................... 42 BIOGRAPHICAL SKETCH ............................................................................................ 48

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8 LIST OF TABLES Table page 3 1 Candidate synaptic proteins ............................................................................... 30

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9 LIST OF FIGURES Figure page 3 1 Localization of PrP in rel ation to cell adhesion proteins ...................................... 31 3 2 Local ization of PrP in rel ation to cell adhesion proteins ...................................... 31 3 3 Localization of PrP in relation to synaptic vesicle cycle proteins ....................... 32 3 4 Co local ization of PrP in the adult NMJ .............................................................. 32 3 5 PrP interacts directly with Syntaxin. .................................................................... 33 3 6 PrP and Syn apsin inter act in the adult NMJ ....................................................... 33 3 7 Significant loss of synaptic boutons through adulthood. Statistical significance p compared to the control group LacZ. 0.005
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10 LIST OF ABBREVIATION S BSA Bovine Serum Albumin CSP Cysteine String Protein DLG Discs large M2 M206, 213S Neur Neurofilament PBS Phosphate Buffered Saline PrPC Cellular Prion Protein PrP SC Scrapie Prion Protein ROP Repressor of Primer Syn Synapsin Syx Syntaxin TBS Tris Buffered Saline Int Integrin Tub Tubulin

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requir ements for the Degree of Master of Science THE PRION PROTEIN BINDS TO SYNTAXIN AND SYNAPSIN IN THE PRESYNAPTIC NEUROMUSCULAR JUNCTION By Jos Herrera D ecember 2012 Chair: Pedro Fern ndez F nez Major: Medical Sciences Translational Biotechnology The prion protein (PrP) i s a GPI anchored glycoprotein located in the extracellular membrane of neuronal and glial cells of the developing and mature nervous system. Although PrP misfolding and aggregation are the causative factors in several neurodegenerative disorders, the physi ological role of PrP is unknown. Previous studies have described the localization of PrP in the synapse, where it interacts with synaptic proteins; however, the physiological relevance thereof remains unknown. My goal was to ascertain the function of PrP i n the synapse, based on its interaction with known synaptic proteins. Using transgenic flies that express wildtype PrP, I first tested the colocalization of PrP with candidate synaptic proteins by immunofluorescence. Then, I utilized coimmunoprecipitati on to confirm the direct interaction of positive candidates. I found that the synaptic proteins, Synapsin and Syntaxin, colocalize and interact directly with the wild type PrP. Moreover, I studied several mutant PrP fly strains, P102L, Y145X, V180I, and M206, 213S, to characterize phenotypic changes in synaptic morphology and protein distribution. Over time, wildtype and mutant strains of PrP

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12 exhibited significant losses in synaptic boutons and active zones. Taken together, these results suggest a possible role of neurotransmission for PrP in the presynaptic neuromuscular junction.

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13 CHAPTER 1 INTRODUCTION The cellular prion protein (PrPC) is a GPI anchored glycoprotein located in the extracellular membrane of neuronal and glial cells of the developing and mature nervous system. Although PrPC is the causative factor in several neurodegenerative disorders, its physiological role remains unknown. Full appreciation of the functional roles of PrPC has likely been abashed by the striking lack of phenotype repor ted for the first Prnp knockout mouse (18) The contrast between the undisturbed phenotype and the spectacular change in the sensitivity to disease in these mice greatly advanced the field of prion pathology, whereas physiological roles of PrPC were largel y neglected (18) Confounding this are the extensive amounts of purported binding partners of PrPC, all of which are involved in unrelated cellular functions Prion diseases are fatal neurodegenerative disorders in humans and animals, which include Creutzf eldt Jakob disease in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep (4, 16) All of these disorders are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein PrPC, termed PrPSC, which constitutes the major component of infectious scrapie prions (3). Following the cleavage of its 22amino acid (aa) signal peptide, most PrPC is translocated to the cell surface as an N glycosylated, glycosylphosphatidylinositol (GPI) anchored protein of 208 2 09 aa (3). It is thought that the conversion to its infectious form occurs after this translocation, whereby several structural and biochemical properties change (5) helical regions of PrPC sheets, which renders PrPSC partially resistant to proteolytic digestion; however, the molecular mechanisms for this conversion remain enigmatic (3).

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14 Prion diseases contain four keystone features that characterize the degenerative tissue damage manifest in these conditions: amyloid plaque formation, astrocytosis, neuronal loss, and spongiform changes (7). Clinical manifestations in human vary, but most common are lack of coordination, rigid gait, and personality changes (7). Researchers have attributed these symptoms to the g radual degenerative loss of tissue, which has led to the tremendous effort in creating mouse models that lead to prion diseases (2, 7) Of note, one study in Drosophila found an increased number of synaptic boutons in flies expressing a common mutation of prion diseases, although the relative amount of active zones decreased when compared to wild type (9). Similar efforts have been placed to elucidate the physiological role of PrPC through the creation of the Prnp knockout mouse model (23). Several neuronal processes have been influenced by the Prnp knockout mouse model, including neuronal survival; neurite outgrowth; maintenance of myelinated fibers; and synapse formation, maintenance and function (10, 12, 14). Studies utilizing immuneelectron microscopy s uggest that PrP is localized in synaptic boutons, mainly of presynaptic origin (2, 14) Furthermore, some PrPC glycoforms can be selectively transported along axons, suggesting that these glycoforms may be specifically presynaptic (16) Others, however, have described a broader neuronal distribution of PrPC(10, 12). These studies hold that PrP is distributed more homogeneously among the neuron, and that it deposits both preand postsynaptically. Electrophysiological studies of CA1 hippocampal neurons from PrP knockout mice support the view that PrPC is an important protein in synapses (2, 19) Excitatory glutamatergic synaptic transmission, GABAA receptor mediated fast inhibition, long -

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15 term potentiation, and late afterhyperpolarization were reduced or absent in mice lacking PrP. Altered mechanisms are also characteristic of PrP deficient mice, as synaptic dysfunction may lead to changes in circadian rhythm, sleep, as well as impairment in spatial learning (20, 22, 23, 26) Other studies have observed PrP lo calization at the neuromuscular junction (17, 25) The administration of recombinant PrPC at nanomolar concentrations leads to the potentiation of acetycholine release from presynaptic terminals in mouse phrenic diaphragm. What remains unclear, however, is the role of PrP at the synapse. Of note is the interaction of PrPC with Synapsin. In their study, Spielhaupter and Schtzl performed a yeast twohybrid screen for possible PrPC interactors, using murine PrPC (amino acids 23 231) as bait to search a mouse brain cDNA expression library (62 ) After identifying several interaction partners, one of which was Synapsin I, they verified the in vivo interaction of these proteins by coimmunoprecipitation assays with recombinant and authentic proteins in mammalian c ells. The binding regions were then mapped using truncated PrPC constructs. They found that the D domain of Synapsin I can physically interact with both the N and C terminals of PrPC. These findings indicate that PrP has at least two distinct sites that e nable it to interact with Synapsin, suggesting a role in neurotransmission. Despite these findings, it remains unclear how a protein such as PrP, which is localized to the outer leaflet, could bind to an intracellular protein. For a GPI anchored protein such as PrPC, its complex would necessitate a transmembrane adaptor to induce intracellular signal pathways, which would activate the transduction of extracellular signals (18) Thus, PrPC may interact with a transmembrane protein or with

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16 a protein complex t hat mediates functional association with intracellular pathways (18) This theory has brought about a vast array of studies, which have attempted to discover putative binding partners by different methods, including coimmunoprecipitation, chemical cross l inking, and yeast two hybrid. These studies have yielded numerous interaction partners, which include membrane proteins, cytoplasmic proteins, and even nuclear proteins; however, the functional significance of most of these interactions remains unknown (1 8) The methods utilized to discover these interactions contain setbacks. Because PrPC is constrained to the outer leaflet, a yeast two hybrid may artificially expose PrPC to cytosolic compartments with disparate biochemical properties, which will result i n a high number of falsepositives. Studies employing this method would then need to use additional techniques to confirm any detected interaction. The selection of detergent conditions is crucial in coimmunoprecipiation, as stringent detergents can destr oy weak or transient proteinprotein interactions. The influence of the detergent used was exemplified in one study, in which the interactions between PrPC and the dystroglycan complex were studied as a function of the detergent used and on the integrity of lipid rafts (15) Thus, I set out to determine the function of PrPC in the synapse, based on its function with known synaptic proteins in Drosophila melanogaster Drosophila makes an excellent model in which to study neurodegenerative dis orders because of its shared repertoire of diseaseassociated genes with mammals. Furthermore, its tractability allows for an in vivo validation of interaction at the synapse, as well for the cellular pathways and compartments involved. Using this model I first chose c andidate

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17 synaptic proteins and used immunofluorescence to observe colocalization, after which I utilize d co immunoprecipitation to ver ify that any positive candidate(s) directly interact(s) with PrPC. I also tested whether any of these positive candidates interact with PrPSC over time, which could explain the locomotor dysfunction manifest in prion diseases. I then compared changes in synaptic morphology and protein distribution of my wildtype flies with those of four prion mutants, P102L, Y145X, V180I, and M206, 213S in order to characterize any phenotypic changes.

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18 CHAPTER 2 MATERIALS AND METHOD S Tissue Preparation Larval NMJ Preparation For analysis of the larval NMJ synapses, wandering third instar larvae were collected and placed on plates with 5% agar (agar + H20), temporarily stunned with HL3 medium (70 mM NaCl, 5 mM KCl, 0.2 mM CaCl2, 20 mM MgCl2, 10 mM NaHCO3, 5 mM trehalose, 115 mM sucrose, and 5 mM HEPES, pH 7.3), and pinned down on the anterior and posterior corporeal ends. The anterior porti on of the larvae was then cut towards the posterior, after which the body wall was stretched out and exposed. Adult Abdominal NMJ Preparation For analysis of the adult NMJ synapses, flies expressing PrP in motor neurons under the control of BG380Gal4 wer e collected at days 1, 15 and 30. Adults were first anesthetized in CO2, after which their abdomen was removed and dissected on plates with 5% agar (agar + H20). The abdomen was pinned down on its anterior and posterior ends, cut open to remove trachea, st retched out, and then exposed. Immunofluorescence Samples were fixed in 4% formaldehyde (10% formaldehyde, 1x phosphate buffered solution, H20) for 25 minutes, followed by three washes in 0.3% PBS T (PBS + Triton X 100) for 10 minutes. They were blocked w ith 1% BSA (bovine serum albumin in 0.3% PBST) for 30 minutes, and then incubated with the primary antibody overnight at 4C. After three washes in 0.3% PBS T and blocking with 1% BSA, the samples were incubated in secondary antibody for two hours, washed three times in 0.3% PBS T, and then mounted onto slides in Vectashield (Vector Laboratories, Burlingname, CA).

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19 Images were captured using a Zeiss ApoTome structured illumination microscopy system. The primary antibodies, anti Actin (1E12), anti BRP (NC8 2), anti CSP (6D6), anti DLG (4F3), anti FasII (1D4), anti Futsch (22C10), anti Integrin (CF.2C7), anti Integrin (CF.6G11), anti Neurofilament (4H6), anti ROP (4F8), anti SV2 (SV2), anti Synapsin (3C11), anti Synaptobrevin (SB1), anti Synaptotagmin (3H 2 2D7), anti Syntaxin (8C3), anti Tubulin (12G10), and anti Tubulin (E7) were all obtained from the Developmental Studies Hybridoma Bank, DSHB (University of Iowa, Iowa City, IA). Each of these antibodies was diluted at 1:10 in 1% BSA. The prion protein antibody 3F4 was obtained from Signet and was used at a 1:1500 dilution The monoclonal rabbit anti PrP antibody was obtained from Epitomics and was used at a dilution of 1:250. Immunoreactivity was visualized with FITC or Cy3 conjugated goat anti mous e/rabbit secondary antibodies (Biorad) at a 1:600 dilution. Western blot analysis Flies ubiquitously expressing PrP under the control of daGal4 were collected at days 1 and 30. Adult flies were homogenized in icecold homogenization buffer (RIPA + 1x protease inhibitor) for two minutes, and centrifuged at 13,000 rpm for one minute. The supernatant was then collected and transferred to a new tube with loading buffer (NuPAGE 4x). After incubation at 95C for five minutes, the samples were centrifuged at 5,0 00 rpm and run on 4 12% SDS PAGE gels. Proteins were then transferred from the gel onto a 0.45m nitrocellulose membrane for 90 minutes, blocked for 30 minutes in 5% milk (non fat dairy milk + 0.1 % TBS T), and incubated with primary antibody overnight at 4C. Membranes were then washed three times in 0.1% TBS T (tris buffered saline, Tween 20, H20) for 10 minutes, and incubated with secondary antibody for two hours. Membranes were again washed three times in 0.1% TBS T, treated with

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20 Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific), and exposed with film. AntiBRP (NC82), anti Synapsin (3C11), and anti Syntaxin (8C3) were diluted at 1:400 in 5% milk. The prion protein antibody 3F4 was diluted at 1:10,000. HRP conjugated goat anti mouse s econdary antibody (Biorad) was used at a dilution of 1:2000. Co Immunoprecipitation Conjugation of Dynabeads Dynabeads M 270 epoxy beads (Invitrogen) were conjugated with anti PrP (3F4). 6 mg of Dynabeads were washed in C1 solution, and the supernatant w as then removed. C1 solution was again added with 15 l of 3F4, mixed by pipetting, and then added to C2 solution. After an overnight incubation at 4C, the beads underwent the following washes: one wash with HB, one wash with LB, and two washes with SB. B eads were left incubating with a third wash of SB at RT for 15 minutes. Thereafter, the supernatant was removed, and 500 l of SB were added to the beads. For coupling with anti Synapsin (3C11) and anti Syntaxin (8C3), all steps were followed as previously described, save the concentration of antibody added to the beads: 67 l of 3C11 or 70.08 l of 8C3 were mixed with C1. Co Immunoprecipitation Flies ubiquitously expressing PrP under the control of daGal4 were collected at days 1 and 30. Adult flies wer e homogenized in extraction buffer (5x IP, 5M NaCl, 0.1M MgCl2, 1x protease inhibitor, DTT, H20) and centrifuged for one minute at 13,000 rpm. 30 l of supernatant were then transferred to a tube with 150 l of the conjugated beads, and they were left incubating overnight at 4C. After three washes with extraction buffer, the samples were incubated with last wash buffer (5x LWB + Tween

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21 20) for five minutes. The supernatant was discarded, and the samples were incubated with elution buffer (EB) for five minut es. Loading buffer (NuPAGE 4x) was then added. After heating the samples at 95C for five minutes, they were analyzed by Western blots. NaPTa/Sarkosyl Precipitation Flies ubiquitously expressing PrP under the control of daGal4 were collected at days 1 and 30. Adults were homogenized in homogenization buffer (1x phosphate buffered saline, 0.15M NaCl, 1% Triton X 100) for five minutes. 20% sarkosyl was then added, and samples were incubated at 37C for 30 minutes at 800 rpm. After a solution containing 0.1M MgCl2 and 1U/l benzonase was added to the samples, they were again incubated as previously described. 4% NaPTa was then added, and samples were left incubating at 37C for 30 minutes at 800 rpm. 30 l of each sample were then removed and placed in a new t ube with loading buffer (NuPAGE 4x). The remaining volume was centrifuged at 4C for 30 minutes at 13,000 rpm, and the supernatant was then removed and transferred to a new tube with loading buffer. The pellet was resuspended in homogenization buffer, aft er which loading buffer was added. Supernatant, pellet, and an equivalent aliquot of the total fraction were then analyzed by Western blots. Fly Strains The following constructs were obtained from the Bloomington Drosophila Stock Center: the P elements to induce overexpression of Syntaxin or Synapsin; the reporter strain UAS:LacZ, the neuronal Elav Gal4, motor neuronal BG380Gal4, and ubiquitous daGal4 drivers. All of the mutant strains for PrP, UAS:HaPrP M6, UAS:HaPrPM206,213S, UAS:HaPrP P102L, UAS:HaPr P V180I, and UAS:HaPrP Y145X, were

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22 constructed in the Fernandez Funez/RinconLimas lab. Crosses were kept at 25C, and their respective progenies were transferred to 28C, except for those expressing Elav Gal4, which were kept at 27C. Flies were raised in standard corneal medium at the indicated temperatures. Climbing Assay Flies expressing PrP under the control of Elav Gal4 were subjected to a climbing assay. There were two separate pools that underwent locomotor function analysis. The first consisted of all of the PrP strains, which were crossed with Elav Gal4. The reporter strain UAS:LacZ was used as a control. The second pool comprised flies expressing Synapsin or Syntaxin. These flies expressed either normal or upregulated levels of either protein. Adult flies were first transferred to an empty vial containing a fivecentimeter tape in place. The flies were then tapped firmly to the bottom of the vial, and the timer was started. Each trial lasted 10 seconds, for a total of eight trials per day. Scoring for each trial was based on the number of flies below the fivecentimeter tape after 10 seconds. Climbing ability was plotted as a function of age, wherein t he average of the eight daily trials was calculated. Microscopy Samples were imaged using a Zeiss ApoTome structured illumination microscopy system. Images were acquired as a Z stack, and then rendered as a maximum projection with orthoview. For larval samples, the entire NMJ synapses of muscles 6/7 were optically sectioned. All optical sections were i series of 1020 sections per synapse. Students T test was utilized for quantitative analysis.

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23 CHAPTER 3 RESULTS Co localization at the Larval Neuromuscular Junction The Larval Neuromuscular Junction To study co localization, I first looked at the neuromuscular junction (NMJ) of thirdinstar larvae. The larva proffers a good model for studying the synaptic system because the nervemuscle is thin and visible for microscopy, and the nerve terminals are large and easily ac cessible for manipulation. This system comprises eight hemisegments, each of which contains 30 muscles. A total of 31 motoneurons terminate at these muscles, with a pattern that is highly stereotypical per hemisegment. To gain a comprehensive appreciation of the possible role(s) PrP may play at the synapse, I chose candidate synaptic proteins involved in three disparate functions: cell adhesion, structural maintenance, and the synaptic vesicle cycle (Table 31). After dissecting thirdinstar larvae, I cost ained the samples with antibodies against PrP and a candidate synaptic protein. Cell Adhesion Proteins The cell adhesion proteins are located on the cell surface, where they bind to other cells or the extracellular matrix. I began my study by observing the co localization of Discs large (Dlg) in relation to that of PrP. Dlg is localized in the subsynaptic reticulum (SSR), where it helps remodel and sprout new neuromuscular synapses Figure 3 1, A demonstrates that Dlg surrounds PrP, which confirms that PrP is localized in the plasma membrane of the presynaptic terminal, and is not secr eted to the intersynaptic space. I Integrin and PrP share a complimentary expression pattern, but their failure to colocalize indicates that they do not play similar roles in the NMJ (Figure

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24 3 1, B ). Of note, a previous study that used a co immunoprecipitation assay discovered that mammalian NCAM, a neuronal cell adhesion molecule, interacts with PrP ( 3 ) However, the Drosophila orthologue of NCAM, Fasciclin II, did not co localize with PrP (Fi gure 3 1, C ). Overall, none of the tested cell adhesion proteins colocalized with PrP. This led me to conclude that PrP does not participate in the cell adhesion functions specific to the proteins tested. Structural Proteins The cytoskeleton helps to for m and maintain the shape of the NMJ. I first studied the Neurofilament protein, which acts through adaptor proteins to maintain cell cell adhesion. Rather than colocalize with PrP, Neurofilament wraps around it, suggesting that both proteins have disparat e subcomparementalization (Figure 3 2, A ). Interestingly, a study using chemical cross Tubulin and PrP in mice, although my study failed to corroborate this finding (Figure 3 2, B) PrP thus has no interaction wi th the tested structural proteins to form or maintain the cytoskeleton of the NMJ presynaptic terminal. Synaptic Vesicle Cycle Proteins The release and recycling of synaptic vesicles involves a complex cycle, for which a vast array of proteins exists. I ex amined proteins involved in both endoand exocytosis. In doing so, I discovered two positive candidates that colocalize with PrP: Synapsin and Syntaxin. Synapsin is located within the membrane of the synaptic vesicle, where it cross links vesicles to one another and cytoskeletal proteins Spielhaupter and Schtzl previously confirmed the interaction of Synapsin and PrP with a co immunoprecipitation assay. It remains unclear, though, how a cytosolic protein such as Synapsin could interact with PrP, which i s located in the outer leaflet of the

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25 plasma membrane. In my study, S ynapsin and PrP only partially colocalize, indicating that PrP does not mainly interact with Synapsin (Figure 3 3, A ). Conversely, no previous studies have reported the interaction of Pr P with Syntaxin. The latter protein is localized in the plasma membrane, where it binds to Synaptotagmin and assists in the fusion of the vesicle to the membrane. The finding that Synta xin co localized with PrP helped elucidate the possible subcompartmental location of PrP in the plasma membrane of the NMJ, in that Syntaxin is contained within the lipid rafts of the plasma membrane (Figure 3 3, B ). This suggest ed that PrP also lies within the lipid rafts of the presynaptic terminal, but it remained unclear whether this colocalization is preserved in adulthood. Co Localization at the Adult Neuromuscular Junction The Adult Neuromuscular Junction A limitation to the larval NMJ is its inability to conclude whether any protein interactions remain important in adulthood, as certain processes during metamorphosis can render proteinprotein interactions moot. I wanted to study whether PrP maintained its co localization with Syntaxin and Synapsin in the adult fly. To assess adult colocalization, I looked at the ab dominal NMJ of the adult. Similar to the larval model, the abdominal NMJ allows for simple visualization of the synaptic system because of its thin nervemuscles and large nerve terminals. Unlike the larval system, however, the synaptic terminals of the ab domen are much more highly stereotypical because they are similar in structure among seg ments, of which there are six. Co localization of Synapsin and Syntaxin Both Syntaxin and Synapsin remain functionally relevant proteins in adulthood: Syntaxin continues to play an important role in neurotransmitter release, aiding in the fusion of vesicle to membrane. Likewise, Synapsin continues to tether synaptic vesicles

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26 to the cytoskeleton, specifically at the reserve pool of the axon terminal. I evaluated whether P rP still co localizes with these functionally relevant proteins in adulthood. I dissected flies at day 1, and subjected them to the same fixation and staining protocol as I had utilized for the thirdinstar larvae. As seen in Figure 34, both Syntaxin and Synapsin maintain their colocalization with PrP. However, the colocalization does not appear as prevalent as in the larvae (Figure 34). Protein Interaction in the Adult Fly Interaction between PrP and Syntaxin After I discovered the colocalization of b oth Syntaxin and Synapsin with PrP, I utilized a coimmunoprecipitation assay to confirm their interaction in the Drosophila neuromuscular system I first precipitated Syntaxin from wholetissue lysates of adult flies, utilizing PrP conjugated beads. Flies ubiquitously expressing PrP under the control of da Gal4 were compared with nontransgenic yw flies, which served as a control. At day 1, I observed Syntaxin (35 kDa) only in flies expressing PrP, whereas this band was absent in yw flies; likewise, detect ion of Syntaxin failed to appear in beads precipitated without any tissue lysate (Figure 35, A Lane 1). I then performed the converse, immunoprecipitating PrP with Syntaxinconjugated Dynabeads, from wholetissue lysates of adult flies. This was to ensure that the detection of Syntaxin did not occur by mere chance. Similar to the results from Figure 35, A, I observed PrP (27 kDa) solely in those flies expressing PrP. Absent of a similarly sized band were both controls. This confirmed that PrP interacts di rectly with Syntaxin, in flies at day 1. I was also interested to study whether this interaction was preserved over time in adulthood. I compared flies at day 30 that ubiquitously expressed PrP, with those that lacked such expression. I followed the sa me protocol as previously used: I first

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27 precipitated Syntaxin from wholetissue lysates, with PrP conjugated beads. At day 30, Syntaxin (35 kDa) appears only in those flies expressing PrP, whereas all controls lack any band of similar size. I then corrobor ated my findings by again conducting the inverse. Only in the da Gal4 fly (Lane SOMETHING) did I detect the presence of PrP (27 kDa). Thus, not only does Syntaxin bind to PrP, but this interaction also remains conserved throughout the lifespan of the adult fly. Interaction between PrP and Synapsin I next precipitated Synapsin from wholetissue lysates of one day old adult flies, utilizing PrP conjugated Dynabeads. Synapsin (70 kDa) appears only in those flies expressing PrP, whereas all controls lack s uch a band (Figure 36, A). When I performed the converse experiment, only in those flies expressing PrP is there any detection of a band at 27 kDa (PrP) (Figure 3 6, B). This concludes that Synapsin binds to PrP in one day old flies. Flies at Day 30 also exhibited a similar interaction between the two proteins. Upon the precipitation of Synapsin with PrP conjugated beads, only a band (70 kDa) appears in daGal4 flies (Lane 4), which indicates that Synapsin interacts with PrP throughout adulthood. Locomotor Dysfunction in PrP Adult Flies The Climbing Assay To assess locomotor dysfunction in the adult fly, often investigators will utilize the climbing assay. This assay takes advatange of the fruit flys proclivity to stand above the base of a vial for a prolonged period of time; however, older flies do not exhibit this behavior as often because their ability to climb decreases as they age. The reasons behind this loss of function are manifold, one of which is the loss of synapses. With the gradual dec rease in synaptic number, the communication between neuronal and

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28 muscles cells is compromised, which leads to aberrant locomotor function. To investigate whether the observed decrease in synaptic number and density lead to a rapid decline in locomotor func tion, I performed a climbing assay, using flies expressing PrP in motoneurons. Locomotor Dysfunction As shown in Figure 310A, control females expressing cytoplasmic LacZ performed well i n climbing assays over 15 days and stopped climbing at around day 55 PrP flies, on the other hand, retain a 50% climbing ability only until Day 7 and stop climbing by Day 23 (Figure 310B). The decline in locomotor function, though, was more gradual than abrupt. The latter phenomenon has been observed in other flies also expressing PrP. Nevertheless, the decline in locomotor function exhibited by the PrP flies was significant when compared to the control females expressing cytoplasmic LacZ Since these flies exhibited an early locomotor dysfunction, I wondered if PrP was a ffecting motor neuron development overtime, possibly through the sequestration of either of these proteins by PrPSC. Interaction between PrPSC and Synaptic Proteins PrPSC in the Presynapse Because of the siginificant loss of active zones and overall boutons by day 15, I evaluated the possibility that PrPSC, the insoluble conformer of PrP, could be interacting with either or both Syntaxin and Synapsin. This would lead to a titration of the amount of protein necessary for neurons to communicate properly with the muscle cells they innervate. Aberrant signaling between the preand postsynapse would lead then to the observed, phenotypic changes. For this assay, I performed a co immunoprecipitation assay using 15B3, an antibody that recognizes a PrPSC like struc ture As seen in Figure

PAGE 29

29 3 11B, the PrP precipitated by the conjugate beads is faint at day 1 (Lane 3), but by day 30, the amount of PrP detected by this antibody increases. If PrPSC were binding to either of these proteins, then I would observe a similar p henomenon. Interaction between Synaptic Proteins and Insoluble PrP I first precipitated Synapsin, utilizing the 15B3 antibody. As seen in Figure 311A, however, a band at 70 kDa, which is of a similar molecular weight to that of Synapsin, appears in all lanes; moreover, the expression of this band is uniform in each lane, which led me to conclude that Synapsin does not seem to bind with the insoluble form of PrP. Likewise, the when I precipitated Syntaxin with the 15B3 antibody, I achieved similar results to those of Synapsin: a band of similar length to Syntaxin (36 kDa) appears in all lanes, irrespective of genotype or age (Figure 311C). Because it at first appeared that there were nonspecific bands of similar molecular weight that could be competing, and thus, binding to 15B3, I first blocked the antibody before precipitating it with either Synapsin or Syntaxin. This was to deter any possible non specific binding at both ~36 kDa and ~70 kDa; however, I still detected these bands in all lanes. This implies that neither Syntaxin nor Synapsin binds to PrPSC.

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30 Table 3 1 Candidate synaptic proteins Proteins Function(s) Co Localization Structural Neurofilament Interacts at the plasma membrane via adaptor proteins either for cell cell adhesion or cell matrix adhesion actin Forms microfilaments to support cells; involved in trafficking of vesicles tubulin Involved in microtubule formation tubulin Involved in microtubule formation Futsch Microtubule binding protein involved in synaptic growth Cell Adhesion Fasciclin II Prevents active zone and postsynaptic density from growing out of bounds Discs Large Synaptic localization of its synaptic binding partners, fasciclin II and shaker integrin Mediates attachment between a cell and its surrounding tissue integrin Interacts with integrin in signal transduction Synaptic Vesicle Cycle CSP Downregulates presynaptic calcium channels; renatures proteins during synaptic vesicle cycle Synapto tagmin Calcium sensor Synapsin Cross links vesicle to cytoplasmn (actin); involved in synaptic vesicle release + ROP Binds syntaxin; promotes syntaxin stability; controls spatially correct assembly of core complexes Syntaxin Binds synaptotagmin; pla ys a role in exocytosis + SV2 Synaptic vesicle phosphoproteins Synaptobrevin Fusion of the synaptic vesicle Bruchpilot In active zones, binds vesicle to plasma membrane or central core of cytoplasm

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31 A) B) Figure 31. Localization of PrP in relation to cell adhesion proteins A) PrP and DLG do not appear to coIntegrin and PrP do not appear to col ocalize. A) B) Figure 32. Localization of PrP in relation to cell adhesion proteins A) PrP and Neurofilament do not appear to colocalize. B) Tubulin and PrP do not appear to co localize.

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32 A) B) Figure 33. Localization of PrP in relation to synaptic vesicle cycle proteins A) PrP and Synapsin appear to co localize. B) Syntaxin and PrP appear to colocalize. A) B) Figure 34. Co localization of PrP in the adult NMJ A) PrP and Synapsin appear to colocalize. B) Syntaxin and PrP appear to colocalize.

PAGE 33

33 A) B) Figure 35. PrP interacts directly with Syntaxin. A) Syntaxin (36 kDa) precipitated from PrP conjugated beads detected only in Lane 3. B) PrP (27 kDa) precip itated from Syntaxin conjugated beads detected only in Lane 3 A) B) Figure 36. PrP and Synapsin interact in the adult NMJ A) Synapsin (70 kDa) precipitated from PrP conjugated beads detected only in Lane 3. B) PrP (27 kDa) precipitated from Synapsinconjugated beads detected only in Lane 3.

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34 Figure 37 Significant loss of synaptic boutons through adulthood. Statistical significance p compared to the control group LacZ. 0.005
PAGE 35

35 Figure 39. Locomotor dysfunction over a span of two months

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36 A) B) C) Figure 310. Insoluble PrP does not interact with either Synapsin or Syntaxin. A) A band with a similar length to Synapsin (70 kDa) is found in all lanes. B) A faint band of PrP (27 kDa) is detected at Day 1 in Lane 3, and a darker band (27 kDa) is seen in Lane 4. C) A band with similar length to Syntaxin (36 kDa) is detected in all lanes.

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37 A) B) C) Figure 311. Loss of synaptic boutons through adulthood. A) The average number of synaptic boutons at day 1. B) The average number of synaptic bouto ns at day 15. C) The average number of synaptic boutons at day 30. Statistical significance p compared to either LacZ or wildty pe (WT) PrP.* 0.005
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38 A) B) C) Figure 312. Loss of active zones per nerve through adulthood. A) The average number of active zones per nerve between all genotypes at day 1. B) The average number of active zones per nerve between all genotypes at day 1. C) The average number of active zones per nerve between all genotypes at day 1. All genotypes compared to either LacZ or wildtype (WT) PrP. 0.005
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39 A) B) C) D) Figure 313. Locomotor activity for mutants across two months A) Locomotor activity of the M2 mutants compared to the control group LacZ. B) Locomotor activity of the P102L mutants compared to the control group LacZ. Locomotor activity of theY145X mutants compared to the control group. LacZ. D) Locomotor activity of the V180I mutants compared to the control group LacZ.

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40 CHAPTER 4 DISCUSSION Considerable attention has been devoted in the last several decades to the discovery of putative binding partners of PrP (37, 45, 49). Researchers have long believed that the discovery of its interaction partners could lead to the elucidation of its physiological role, which still remains uncertain (37, 45, 46, 51). This search has mostly gone in vane, however, because of the vast amount of purported binding partners of PrP, w hich confer unto it a lita ny of possible functions. Future studies will need to consolidate the discrepancy inherent in this study: Why should soluble PrP bind to Syntaxin and Synapsin, without doing so once it becomes insoluble? Although I have atte mpted in this thesis to answer this same question, one area of possible further study could be attempting to study whether PrP, in the lipid raft, binds either to Syntaxin or Synapsin. For this, a lipid raft purification could be utilized, followed by a coimmunoprecipitation of this small fraction. Because insoluble PrP still remains at the lipid raft, it might follow that insoluble PrP binds to Syntaxin or Synapsin in this area. One might also attempt a chemical cross linking, because this is a much more sensitive assay for the detection of proteinprotein interactions. If the interaction between PrP and the two synaptic proteins, Syntaxin or Synapsin is weak, then it could be that the detergents utilized in the 15b3 coimmunoprecipitation assay may be destroying the interaction if these two proteins with PrP; thus, a chemical crosslinking may be able to keep the bond that these proteins have with PrP, since the assay is able to detect interaction that are physiologically weak. If the chemical c ross linking and lipid raft purification studies were to yield

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41 positive results, then we could conclude two things about the nature of the relationship between PrP and these two synaptic proteins. The first would suggest that this interaction occurs mainly in the lipid raft, at both the soluble and insoluble states of PrP. Moreover, it would imply that the interaction that PrP has with both of these proteins is a weak one, although still important. The likelihood of its being the only two proteins to which it binds, however, is unlikely. This would mainly be due to the fact that there mus be a stringer protein to which PrP binds.

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48 BIOGRAPHICAL SKET CH Jos Herrera was born in Hialeah, Florida, and schooled for most of his life in Miami. At the age of 18, he moved to Gainesville to attend the University of Florida, and pursued for the next four years a B.S. in p sychology. His interest in neuroscience led him to volunteer at the Foster lab, which studied the effect of age and memory. There, his tasks included running several behavioral assays in r ats and mice. He then pursued a n M.S. in b iotechnology, as his interest in researching the brain had grown while as an undergraduate.