DISSERTATION SYNAPTOTAGMIN IN ASYNCHRONOUS NEUROTRANSMITTER RELEASE AND SYNAPTIC DISEASE Submitted by Mallory Catherine Shields Department of Biomedical Sciences In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2018 Doctoral Committee: Advisor: Noreen Reist Deborah Garrity Michael Tamkun Susan Tsunoda   Copyright by Mallory Catherine Shields 2018 All Rights Reserved      ii   ABSTRACT SYNAPTOTAGMIN IN ASYNCHRONOUS NEUROTRANSMITTER RELEASE AND SYNAPTIC DISEASE The majority of cell-to-cell communication relies on the stimulated release of neurotransmitter. Two forms of Ca2+-dependent stimulated release, synchronous and asynchronous, have been identified. Synchronous release is the initial release that occurs within milliseconds of stimulation. Critical for efficient synaptic communication, synchronous release is the dominant form of release at most synapses. Alternatively, asynchronous release occurs over longer time periods, with implications in synaptic plasticity and development. However, its mechanisms are poorly understood. Both synchronous and asynchronous release rely on Ca2+ sensors to confer their distinct characteristics. Synaptotagmin 1 is widely accepted as the Ca2+ sensor for fast, synchronous release, but its role in asynchronous release is unclear. Previous studies have led to the hypothesis that synaptotagmin 1, particularly Ca2+ binding by its C2A domain, is needed to inhibit aberrant asynchronous fusion events. However, recent studies have raised questions regarding the interpretation of the results that led to this conclusion. In chapter 2, I have directly tested the effect of Ca2+ binding by synaptotagmin 1’s C2A domain on asynchronous release utilizing an alternant Ca2+-binding mutant. This novel mutation was designed to block Ca2+ binding without introducing the artifacts of the original Ca2+-binding mutation. By investigating asynchronous events in vivo at the Drosophila neuromuscular junction, I found no significant effect on asynchronous release when C2A Ca2+ binding was      iii   blocked. Thus, I conclude that Ca2+ binding by synaptotagmin’s C2A domain is not needed for regulation of asynchronous release, in contrast to the previous study that inadvertently introduced an artifact described below. To prevent Ca2+ binding, the original aspartate to asparagine mutations (sytD-N) removed some of the negatively-charged residues that coordinate Ca2+. This simultaneously introduced aberrant fusion events, because it also interrupted the electrostatic repulsion between synaptotagmin’s negatively-charged C2A Ca2+-binding pocket and the negatively-charged presynaptic membrane which is required to clamp constitutive SNARE-mediated fusion. Previous Reist lab results demonstrate that the sytD-N mutations in the C2A domain are likely behaving as ostensibly constitutively bound Ca2+. Indeed, I report that the sytD-N mutation displays slower release kinetics. To directly test if this mutation is the cause of the increase in asynchronous events, I generated additional mutations that prevent interactions with the presynaptic membrane coupled to the originally published sytD-N mutations. In chapter 3 of this dissertation, I investigated these novel mutations at the Drosophila neuromuscular junction. I reported no increase in asynchronous release relative to control, providing evidence that the increased asynchronous events in sytD-N mutants are a result of the original mutation acting as an asynchronous sensor. Together, my results contradict the current hypothesis in the field and provide the likely mechanism for the increased asynchronous release observed in the original study. This dissertation also investigated the relatively new role for synaptotagmin mutations in the etiology of neuromuscular disease. With increased availability of high-throughput sequencing, over 20 candidate genes have been implicated in different forms of congential myasthenic syndromes. These inherited disorders are caused by mutations in genes needed for      iv   effective neuromuscular signaling. Two families, presenting with similar myasthenic syndromes, carry point mutations in the C2B Ca2+ binding pocket of synaptotagmin, expressed as an autosomal dominant disorder. One of theses families contains a proline to leucine substitution (sytP-L) a residue that had not been previously investigated for synaptotagmin function. In chapter 4, I investigated the functional importance of this mutation and created a disease model for this familial condition by driving the expression of a homolous proline-leucine synaptotagmin substitution in the central nervous system of Drosophila. I demonstrated that the proline residue plays a functional role in efficient transmitter release by testing its function in an otherwise synaptotagmin null genetic background. Additionally, this mutation displayed characteristics similar to the human disorder when expressed in a heterozygous synaptotagmin background, similar to the familial expression. Namely, the sytP-L mutants exhibited a decreased release probability, which resulted in decreased evoked responses that facilitate upon high frequency stimulation, a rightward shift in Ca2+ sensitivity, and behavioral deficits, including decreased motor output and increased fatigability. Thus, these studies establish the causative nature of the sytP-L mutation in this rare form of congenital myasthenic syndrome and highlight the utility of the Drosophila system for disease modeling.      v   ACKNOWLEDGEMENTS As my time at Colorado State University comes to a close, I look back with overwhelming gratitude to the people in my life who have been there to support this dissertation. First and foremost, I acknowledge Dr. Noreen Reist, the quintessential fearless boss lady that all young women scientists dream of becoming. Noreen, your honesty, support, critical intellectual feedback, and overall “science mom” mentality has made my time here unforgettable and nothing short of the best job I can imagine. I feel prepared to face my next endeavor with confidence, and I am proud to say a bit of your fearlessness has worn off on me. I thank you for everything. Thank you to my committee comprised of Drs. Michael Tamkun, Susan Tsunoda, and Deborah Garitty, for providing feedback and guidance throughout my graduate carreer. I appreciate your edits and suggestions, and of course, your approval signatures. For the past and current members of the Reist lab: it has been a pleasure to work with each and every one of you. Ben Johnson, Alexa Mumm (Navarro), and Remi Boudreau: you taught me that electrophysiology and western analyses take patience. More importantly, you taught me game nights, bowling Thursdays, pie, birthday cupcakes, and terrible jokes make a new girl feel supported and included. Matt Bowers: I am so grateful you decided to join the Reist lab. Your positive attitude, willingness to grab a beer at the end of a long day, visual eye for figure formatting, and scientific feedback are invaluable. I am proud to call you my colleague and friend. Jasmin Hicks, you make a great new fit in the Reist lab and are a wonderful new friend. I know you will go on to do powerful things.      vi   My family is my main source of unwavering support and love. Mom, Dad, James, Grandmother, Uncle Jay, and Aunt Marilyn: Thank you for hearing the ups and downs of this process and keeping me inspired to finish. To my Pawpaw, Grandmamma, and Grandfather who have passed away: I wish you were still here today. You are missed. I love you all very much. The friends I have made have made this PhD a whole lot of fun. My roommates Rachel West, Ashley Turnidge, Adam Heck, and Mike Mangalea: You guys are just the best. There are countless other graduate students and post-docs and their families who have stamped a place on my heart, including An Dang, Kristen Brown, Krystle Frahm, Elizabeth Akin, Eric Marr, Nathan Byers, Sarah Wooldridge, Laura Sole, Max Vallejos, Coreen Frawley, and Alli and Jordan Werner. Thanks for all the dress-up parties, ski trips, commiserating, and bike brewery tours. A special thank you to Erin Bisenius for keeping my paperwork in line and for witty cards and chocolates on my birthday, and Karen Soloman and Shazette Pierce for handling my day-to-day administrative questions. You three made this process run smoothly, and your pleasant personalities make going to work feel less like work. None of this is possible without funding. Noreen’s National Science Foundation grants (IOS-1257362 and IOS-1025966) kept me afloat for years, but there are a few additional sources of funding that are appreciated: the Molecular, Cellular, and Integrative Neuroscience (MCIN) Program for funding my first year, the MCIN travel grants to help support my participation in various meetings, the Thornburg Graduate Excellence Award, and finally, the Vice President for Research Fellowship, which not only supplemented my funding and provided monetary support for meetings, but also offered leadership training throughout this last year. I had the pleasure to collaborate with both national and international scientists, and would like to address each of them. Rita Horvath, thank you for introducing us to the congenital      vii   myasthenia project and forging our collaboration, Roger Whittaker, for your clinical perspective and feedback, Alysia Vrailes-Mortimer, for opening the doors to your lab and teaching me the Drosophila Activity Monitoring Assay and your edits on our manuscript. Moto Yoshihara, thank you and your lab for the Drosophila line that spurred my thesis projects. The experts in the Colorado State University Statistics department are unparalleled. Ann Hess and Jim Zumbrunnen, thank you for helping me analyze mounds of data. Thanks to the Cold Spring Harbor Neurobiology of Drosophila course and to the instructors, especially Kate O’Conner-Giles, Greg Macleod, and Adrian Rothenfluh for their time and expertise, and to Stefan Pulver and Richard Daniels for their specialized instruction on two-electrode voltage clamp electrophysiology.      viii   TABLE OF CONTENTS ABSTRACT ................................................................................................................................... ii ACKNOWLEDGEMENTS .............................................................................................................v CHAPTER 1: OVERVIEW OF SYNAPTIC TRANSMISSION AND NEUROMUSCULAR DISEASE ........................................................................................................................................1 1.1 Overview ....................................................................................................................................1 1.2 Neuronal structure ......................................................................................................................2 1.3 Cellular membranes ..................................................................................................................3 1.4 Action potentials ........................................................................................................................3 1.5 Quantal release ..........................................................................................................................4 1.6 Modulation of the chemical synapse ..........................................................................................5 1.7 Active zones ...............................................................................................................................7 1.8 Synaptic vesicle cycle ..............................................................................................................10 1.9 Synaptic vesicle priming ..........................................................................................................11 1.10 Synaptic vesicle fusion ..........................................................................................................12 1.11 Ca2+ sensors for synchronous and asynchronous release ......................................................14 1.12 Synaptotagmin 1 in synchronous release ...............................................................................17 1.13 Synaptotagmin 1 in asynchronous release .............................................................................18 1.14 Clarification of synaptotagmin’s role in asynchronous release .............................................20 1.15 Neuromuscular disorders .......................................................................................................22 1.16 High throughput sequencing ..................................................................................................25 1.17 Synaptotagmin-related disorders ...........................................................................................26 1.18 Drosophila as a model system ...............................................................................................27 1.19 Investigation of a synaptotagmin disorder .............................................................................28 WORKS CITED ............................................................................................................................29 CHAPTER 2: CLARIFICATION OF CA2+ BINDING IN SYNAPTOTAGMIN 1’S C2A DOMAIN IN ASYNCHRONOUS NEUROTRANSMITTER RELEASE ...................................36 2.1 Summary ..................................................................................................................................36 2.2 Introduction ..............................................................................................................................37 2.3 Results ......................................................................................................................................39 2.4 Discussion ................................................................................................................................53 2.5 Conclusion ...............................................................................................................................56 2.6 Materials and Methods .............................................................................................................57 WORKS CITED ...........................................................................................................................62 CHAPTER 3: HYDROPHOBIC RESIDUES IN SYNAPTOTAGMIN 1’S C2A DOMAIN CLARIFY ITS ROLE IN ASYNCHRONOUS NEUROTRANSMISSION ................................65 3.1 Summary ..................................................................................................................................65 3.2 Introduction ..............................................................................................................................65 3.3 Results ......................................................................................................................................70 3.4 Discussion ................................................................................................................................81 3.5 Conclusion ...............................................................................................................................82 3.6 Materials and Methods .............................................................................................................83 WORKS CITED ...........................................................................................................................88      ix   CHAPTER 4: DROSOPHILA STUDIES SUPPORT A ROLE FOR A PRESYNAPTIC SYNAPTOTAGMIN MUTATION IN A HUMAN CONGENITAL MYASTHENIC SYNDROME .................................................................................................................................91 4.1 Summary ..................................................................................................................................91 4.2 Introduction ..............................................................................................................................92 4.3 Results ......................................................................................................................................95 4.4 Discussion ..............................................................................................................................109 4.5 Conclusion .............................................................................................................................114 4.6 Materials and Methods ...........................................................................................................115 WORKS CITED ..........................................................................................................................125 APPENDIX 1: SUPPLEMENTARY FIGURES ........................................................................128 APPENDIX 2: SUPPLEMENTARY TABLES .........................................................................130 APPENDIX 3: SYNAPTIC TRANSMISSION CHAPTER, CELL PHYSIOLOGY SOURCEBOOK, 5TH EDITION ..................................................................................................134      1   CHAPTER 1. OVERVIEW OF SYNAPTIC TRANSMISSION AND NEUROMUSCULAR DISEASE   1.1 Overview Nervous system function relies upon the transmission of signals between neurons. Neurons transmit information across long distances through action potentials, which generally do not propagate between spatially distinct cells. To cross the gap between nerve cells, the majority of cell-to-cell signaling relies on release of neurotransmitters. The majority of neurotransmitter release from active neurons occurs in a Ca2+ dependent manner at synapses. Within synapses, small, synaptic vesicles filled with a quantum of neurotransmitter fuse with the presynaptic membrane in a tightly regulated series of steps. There are two distinct forms of stimulated Ca2+-dependent fusion and subsequent neurotransmitter release, synchronous and asynchronous release. These release events are dependent on Ca2+ sensors. One essential protein, synaptotagmin 1, is widely accepted as the Ca2+ sensor for synchronous release. Previous studies implicate a regulatory role for synaptotagmin 1 in the other form of stimulated release, asynchronous release, but its precise mechanism is unclear. This dissertation investigates whether synaptotagmin 1 plays a regulatory role in the asynchronous form of Ca2+-dependent release. This dissertation also investigates the role of synaptotagmin in a newly characterized congenital myasthenic syndrome, a rare subset of neuromuscular disorders caused by genetically inherited synaptic protein mutations. As synaptic transmission is tightly regulated, dysfunction of any protein needed for release or response of neurotransmitter can result in human disease. Multiple diseases at the neuromuscular junction result from dysfunctional synaptic proteins. Clinicians diagnose neuromuscular disorders using nerve conduction studies, exercise      2   testing, and genetic sequencing. However, the need to uncover mechanisms mediating these disorders is currently unmet. Drosophila offer a quick, economical, and genetically flexible method for investigation of rare human diseases. By modeling this newly characterized synaptotagmin congenital myasthenic disease using Drosophila, I investigate a causative role in a synaptotagmin point mutation in the etiology of disease symptoms and uncover some of its mechanistic underpinnings. 1.2 Neuronal structure Neuronal structure is highly variable, from long projection neurons in the motor system to small bipolar cells of the retina. However, the canonical structure consists of three major structures: dendrites, the cell body, and the axon. Dendrites typically project out from a cell body to neighboring cells and receive neurotransmitter signals from the presynaptic cell terminal. These dendrites respond with small depolarizations or hyperpolarizations that spread passively to the axon hillock. The neuronal cell body houses many components typically found in other cells, such as the nucleus, smooth and rough endoplasmic reticulum, mitochondria, and golgi apparatus. The axon contains the axon hillock, which integrates all depolarizing and hyperpolarizing signals received from the dendrites. If the incoming summative signal reaches the threshold potential, the cell will fire a self-propagating electrical signal, or action potential, down the neuron’s axon, stimulating neurotransmitter release at the axon terminal. This release communicates a chemical signal onto the next cell.      3   1.3 Cellular membranes The interior and exterior of a neuron is separated by a cellular membrane, which consists of a lipid bilayer with outer hydrophilic head groups and inner hydrophobic fatty acid tails. This lipid bilayer is impermeable to ions. It is responsible for maintaining ionic and electrical gradients between the cytosol and extracellular space. In this way, cellular membranes act as capacitors separating charge. Controlled ion movement across the membrane is important for electrical activity. Hence, the cellular membrane contains many proteins, which allow ions or other proteins access across the membrane. These gatekeepers can either be passive, or they can use energy to pump ions across the membrane, called active transport. Passive gatekeepers include ion channels, which are gated pores that have some selectivity to ions. Some channels allow specific ions to cross the membrane and others allow more general ion flux. They can be voltage-gated, which only allow ionic flow when the membrane potential is depolarized or hyperpolarized, while others require binding of a ligand before allowing ion passage. Channel properties allow the cell to regulate its ionic concentrations and thus its electrical signaling. Diseases may arise when these channels lose their ability to function correctly, discussed later in this dissertation. 1.4 Action potentials An action potential propagates down the axon until it reaches the axon terminal. Here, the depolarization opens voltage-gated Ca2+ channels. The concentration of Ca2+ outside the cell is much greater than inside. Upon Ca2+ channel opening, Ca2+ enters the presynaptic terminal and results in the fusion of neurotransmitter-filled synaptic vesicles with the presynaptic terminal membrane. This results in the release of neurotransmitter into the synaptic cleft where it can then      4   generate signals in the next cell. Ca2+-dependent neurotransmitter release will be discussed in detail throughout this dissertation. 1.5 Quantal release Identification of individually fusing synaptic vesicles as the source of neurotransmitter release was groundbreaking in our understanding of synaptic transmission. Katz and colleagues proposed the quantal release hypothesis in the early 1950s [1-3]. Their studies at the frog neuromuscular junction identified spontaneous, small postsynaptic voltage changes that occurred in the absence of neural activity. These depolarizations resemble end plate potentials (EPPs) following an action potential, but on a smaller scale. These events are referred to as miniature end-plate potentials (mEPPs). The mEPPs appear to be random and between 0.3 and 0.5 mV. This suggests neurotransmitter is being released in discrete packets. Furthermore, they hypothesized that an action potential causes the synchronous release of a large number of these packets and generates a large EPP. The concept that synaptic transmission could be explained by the quantized release of neurotransmitter was proposed around the same time that small membranous vesicles within nerve terminals were first identified by electron microscopy [4, 5]. Together, these findings led to the vesicular hypothesis for neurotransmitter release, which states that each synaptic vesicle contains a similar amount of neurotransmitter (1 quantum), and that the transmitter release following an action potential results from a discrete number of vesicles fusing synchronously with the plasma membrane.      5   1.6 Modulation of the chemical synapse The response of a given postsynaptic cell to the same level of activity in a presynaptic cell can vary depending on the efficacy of the synapse. Synaptic efficacy is based on many factors, including the quantal content (number of neurotransmitter-filled synaptic vesicles that fuse in response to a given stimulus) and the type and number of postsynaptic receptors. Importantly, these pre- and postsynaptic parameters can be regulated by activity patterns. The capacity to modulate synaptic efficacy is an important mechanism underlying learning and memory. These activity-dependent changes in synaptic strength are known as synaptic plasticity. Experimentally, synaptic plasticity is measured both pre- and postsynaptically. Synapses may have high or low quantal content (m) relative to other synapses. m depends on the number of vesicles available to fuse (n), and the release probability of each vesicle (p). The release probability is dependent on the magnitude and duration of Ca2+ influx, the speed of Ca2+ clearance, and the proximity of the Ca2+ sensor to the channel. Presynaptically, the amount of neurotransmitter released can be decreased or increased, leading to depressed or facilitated responses. Experiments in which two closely spaced stimuli are administered, or paired pulse experiments, are commonly used to monitor changes in release probabilities. When the ratio of the second response to the first response, or paired pulse ratio (PPR) decreases, it is indicative of increased release probability, and decreases are consistent with decreased release probability [6]. High quantal content synapses result in large responses in the postsynaptic cell upon single stimulations, barring any postsynaptic adaptations. Alternatively, low quantal content synapses have the opposite characteristics and result in smaller postsynaptic responses. Depression can be observed in high quantal content synapses during paired pulse experiments, in which two closely spaced stimuli are administered to a cell [7, 8]. At high      6   quantal content synapses, the first stimulation is sufficient to release a large fraction of the readily releasable vesicles. Accordingly, there are fewer vesicles available to fuse during the second stimulus, which results in a reduced postsynaptic response. Therefore, the first response is robust, while the second response is reduced. Another example of synaptic depression occurs during presynaptic high-frequency stimulus trains [9, 10]. Since exocytosis is more rapid than endocytosis, such stimulation depletes the readily releasable pool of synaptic vesicles, even at low quantal content synapses, and quantal content is decreased. Facilitation is an incremental increase in the postsynaptic response to a given presynaptic stimulation observed in paired pulse experiments at low quantal content synapses. Since the first stimulus at such a synapse results in the fusion of only a small fraction of the readily releasable pool, the number of vesicles available for fusion following the second impulse is not significantly reduced. If the delay between the two stimuli is within tens of milliseconds (ms), not all the Ca2+ that entered during the first pulse has been removed by the time of the second pulse. Therefore, the [Ca2+] experienced by the release machinery during the second pulse is higher and triggers a greater number of synaptic vesicles to fuse. If the delay between stimuli is long enough to allow the Ca2+ from the first pulse to be removed, the second response is not facilitated. This is known as the residual Ca2+ hypothesis [11-13], and this form of presynaptic facilitation is very short-lived. Postsynaptically, changes in synaptic activity can lead to changes in the morphological makeup of the postsynaptic membrane through insertion or removal of receptors. Any change in receptor density results in a change in response strength to a given signal. These changes are referred to as either long-term potentiation, which is characterized by a long-lasting facilitated response in the postsynaptic cell following a brief tetanic stimulation of a strong input or      7   simultaneous stimulation of multiple weaker inputs, or long-term depression, which is a long- lasting, activity-dependent depressed postsynaptic response induced by prolonged low-frequency stimulation [14]. 1.7 Active zones Functionally, the role of the active zone is to transduce an electrical nerve terminal depolarization into neurotransmitter release. Couteaux and Pecot-Dechavassinein coined the term “active zone” in 1970 when they noticed docked vesicles at the frog neuromuscular junction adjacent to electron dense material at the presynaptic membrane in electron micrographs [15]. Subsequent ultrastructure studies revealed similar morphologies across organisms. The structure of the active zone material (AZM) varies among different types of synapses, but must include the requisite protein machinery for vesicle docking, priming, and fusion. Fusion machinery, Ca2+ channels, and Ca2+ sensors are all found at active zones. Fusion machinery The minimum machinery required to fuse a vesicle with its target membrane is the SNARE (SNAP [soluble NSF (n-ethlymaleimide-sensitive factor) attachment protein] receptor) fusion complex, which is comprised of a vesicle-associated SNARE protein (vSNARE) and target-membrane associated SNARE proteins (tSNAREs). At the synapse, the vSNARE is synaptobrevin-2, also known as vesicle-associated membrane protein 2 (VAMP2) and the tSNAREs are syntaxin-1 and synaptosomal-associated protein of 25 kDa (SNAP-25). The discovery of SNAREs as the fusion machinery in the late 1980’s and early 1990’s have been pivotal in our understanding of neurotransmission. Rothman’s group originally      8   discovered the cytosolic proteins NSF [16] and SNAP [17] in the late 1980’s and suggested their importance for membrane fusion [18, 19]. They determined interactions between both NSF, SNAP, and an unknown integral membrane protein needed for their interaction [20]. Simultaneously, synaptic vesicle and target membrane proteins were being identified [21-25] and implicated in neurotransmission through the use of neurotoxins [26-30]. Together, the seminal study using NSF and SNAP proteins to extract SNAP receptors, or SNAREs, from brain tissue was performed. Once purified, the proteins were identified as SNAP25, synaptobrevin, and syntaxin [31]. Furthermore, it was shown that without NSF and SNAP, the SNAREs form stable complexes [32] that bind much more efficiently if all three are present [33]. Each SNARE protein contains at least one SNARE motif of ~65 residues with a propensity to form coiled coils [34]. Synaptotobrevin and syntaxin each contain one SNARE motif, and SNAP-25 contains two [35]. Prior to vesicle docking, syntaxin is found in a stable, closed conformation, with its SNARE motif bound to a three-helix-bundle regulatory domain, making it inaccessible for interactions with other SNARE proteins [36]. Syntaxin undergoes a conformational change, which allows its SNARE motif to interact with the SNARE motifs of synaptobrevin and SNAP-25. This creates a trans-SNARE complex associated with both the vesicular membrane and the presynaptic plasma membrane. The formation of multiple SNARE complexes [37] contributes to the energy required to drive fusion of the vesicle and target membranes [32, 38]. SNARE complexes mediate both constitutive and regulated vesicle fusion events throughout cells. The basic concept of the minimal machinery necessary to fuse a vesicular with its target membrane is as follows: SNARE proteins on both the vesicular and target membranes associate      9   to form a tight coiled-coil configuration. When maximally coiled, trans-SNARE complexes force the two membranes together, thereby destabilizing the membranes’ natural curvature. Together with additional proteins such as Ca2+ sensors, fusion of the two membranes occurs. Once fused, the vSNAREs and tSNAREs form a cis-SNARE complex, and the vesicular contents are released. Ca2+ channels and sensors The presence of voltage-gated Ca2+ channels at active zones is necessary to couple neuronal depolarization to vesicle fusion events. Not only do these channels need to be present at active zones, their precise localization within an active zone can have immense effects on the efficacy of synaptic transmission. Ca2+ channels are located within 50 nm from docked vesicles [39-41]. When the cell is depolarized, the Ca2+ channels briefly open, creating a nanodomain of particularly high [Ca2+] immediately adjacent to each channel. This [Ca2+] reaches hundreds of uM at the mouth of the channel [42] and drops off rapidly with distance [43-46]. As the availability of Ca2+ is limited temporally and spatially, the closer the channel is located to a Ca2+ sensor, the greater the odds of saturating sensor binding and triggering vesicle fusion. Even a 5 nm change in the distance between the channel and the sensor has profound effects on vesicle release probability [47]. There are multiple Ca2+ sensors that transduce Ca2+ influx into neurotransmitter release, such as some synaptotagmins and double C2-containing protein (Doc2). These Ca2+ sensors exhibit varying Ca2+ affinities and release kinetics and result in differential forms of release, which is a major subject of this dissertation. In particular, I focus on synaptotagmin 1, a Ca2+-sensor postulated to perform distinct roles in multiple forms of Ca2+- dependent release.      10   1.8 Synaptic vesicle cycle Before transmitter release, synaptic vesicles must undergo a distinct, tightly regulated series of steps. First, the vesicles are loaded with neurotransmitter and are then transported to the active zone. Vesicles are docked to the presynaptic membrane at the active zone by way of specific molecular interactions, but only a subset of docked vesicles are fusion competent at any given time; namely, those that are maximally primed. In a resting terminal, individual maximally primed vesicles can spontaneously fuse with the presynaptic membrane, resulting in the release of a single quantum of neurotransmitter. In an activated neuron, the influx of Ca2+ through voltage-gated Ca2+ channels initiates a series of steps essential for the evoked release of neurotransmitter. Ca2+-sensing proteins activate the fusion machinery, which mediates the fusion of multiple maximally primed vesicles with the presynaptic membrane. These fusion events result in the release of neurotransmitter into the synaptic cleft. After neurotransmitter release, the fusion machinery is disassembled and recycled for subsequent use. Concurrently, the synaptic vesicle membrane and associated proteins are retrieved through endocytosis to form new synaptic vesicles. This series of distinct events is known as the synaptic vesicle cycle (Fig 1.1). This dissertation will focus on the priming and fusion steps.      11   Fig 1.1. Cartoon depiction of the synaptic vesicle cycle. Each tightly regulated step is represented with a letter, including vesicle loading with neurotransmitter (a), vesicle docking (b), variable vesicle priming (c), maximally-primed, docked vesicle (d), depolarization-triggered Ca2+ entry (e), vesicle fusion with presynaptic membrane (f), vesicle membrane collapse (g), endocytosis (h), and recycling endosome (i). 1.9 Synaptic vesicle priming After a vesicle is docked to the presynaptic membrane, additional interactions increase the probability of vesicle fusion. These interactions are termed “priming.” Munc13, an essential active zone protein, is thought to orchestrate the first step in fusion machinery assembly (note: Munc13 should not to be confused with the SM protein, Munc18). Munc13 binds to the closed conformation of syntaxin and facilitates the conformational change in syntaxin from its “closed” to its “open” state [36, 48]. Only then can syntaxin’s SNARE motif interact with the SNARE motifs of SNAP-25 and synaptobrevin to form the trans-SNARE complex. Recent work suggests that vesicle priming is in a state of dynamic equilibrium, such that only docked vesicles with the largest area of contact with the presynaptic membrane are maximally primed and ready to fuse upon Ca2+ influx [49]. One mechanism of accomplishing variable priming would be a balance between the repulsive forces of the vesicular and presynaptic membranes that result in SNARE complex uncoiling to favor minimal priming and SNARE complex coiling to favor maximal priming [49]. At any given moment, only a subpopulation of docked synaptic vesicles has a sufficient number of maximally coiled trans- SNARE complexes to produce maximal SV-PM contact area (Fig 1.2). These synaptic vesicles constitute the maximally primed population that is most likely to fuse upon Ca2+ influx.      12   Fig 1.2. Variable priming of synaptic vesicles. Left, schematic depicting a docked, minimally primed vesicle at the presynaptic membrane, defined by the small area of contact between the vesicle and plasma membranes. Right, SNARE-coiling pulls the vesicle and presynaptic membranes together, increasing the area of contact, resulting in a maximally primed vesicle that has the highest fusion probability. The degree of priming is in a state of dynamic equilibrium (arrows). 1.10 Synaptic vesicle fusion At the synapse, fusion events can occur in the absence of stimulation or can be evoked when the arrival of an action potential depolarizes the presynaptic terminal. Here, fusion is defined as any time a vesicle membrane combines with the presynaptic membrane, releasing its contents into the synaptic cleft. Evoked release results from depolarization-dependent Ca2+ influx that triggers the fusion of maximally primed vesicles with the presynaptic membrane. Miniature release Miniature release occurs when a single vesicle fuses with the presynaptic membrane in the absence of experimental stimulation. These events are thought to result from low probability conformational changes that complete coiling of the SNARE proteins. As these spontaneously fusing vesicles are in a maximally primed, fusion-competent state, less energy is required to overcome the fusion barrier. Discovered by Katz and colleagues and deemed “spontaneous miniature end plate potentials” at the neuromuscular junction [1, 2], these spontaneously occurring events are      13   observed both peripherally and centrally. Central synapses are innervated by many upstream signaling sources, so all stimulation-independent responses (spontaneous events) are difficult to differentiate from the events that result from one vesicle fusing with the presynaptic membrane (miniature events). To distinguish all spontaneous events from miniature events, researchers employ the use of tetrodotoxin, which blocks voltage-gated Na+ channels and prevents propagation of upstream action potentials. In this way, only miniature events are recorded. At the neuromuscular junction, the innervating nerve is isolated and severed to prevent upstream spontaneous signals. Therefore, all recorded events in the absence of stimulation are considered miniature events. To support this, the effect of tetrodotoxin to distinguish spontaneous events and miniature events at the neuromuscular junction in both invertebrate and mammalian systems has been investigated. At these synapses, all recorded spontaneous release is largely analogous to miniature events [50, 51]. As the remainder of this dissertation focuses on events at the neuromuscular junction, spontaneous release and miniature release will be used interchangeably. Synchronous fusion events Fast, synchronous release of neurotransmitter is the multiquantal stimulated release that occurs within ms of depolarization. Synchronous fusion events are Ca2+ dependent, relying on the large Ca2+ influx of open Ca2+ channels at active zones. This release is dominant in the majority of synapses, and is the canonical release type typically described in textbooks. It is the dominant form of release at Drosophila neuromuscular junctions, but becomes less apparent in certain synaptic mutants. Synaptotagmins 1 and 2 are Ca2+ sensors responsible for synchronous fusion events.      14   Asynchronous fusion events Asynchronous release is another form of Ca2+-dependent release that continues for 100’s of ms up to a second following stimulation. While most synapses exhibit little to no asynchronos release following a single action potential, it  is  still  observed  in  many  synapse  types  [52-­‐61]  and   has  implications  in  synaptic  plasticity  [62-­‐65]  and  development  [66,  67].  In some specialized synapses, such as specific interneurons of the hippocampus and brainstem, asynchronous release is predominant [59-61]. Interestingly, asynchronous release is commonly triggered by a single action potential at synapses in synaptotagmin 1/2 knockouts, when fast, synchronous release is nearly abolished [68]. The total amount of neurotransmitter released remains similar between wild type and synaptotagmin knockout neurons, although the method of release has shifted from predominantly synchronous to predominantly asynchronous [69, 70]. Asynchronous neurotransmitter release is observed in many neuronal types under particular circumstances, such as during and following extended high frequency activity. More research is needed for a full understanding of this release mechanism. 1.11 Ca2+ sensors for synchronous and asynchronous release Synchronous and asynchronous release phases require Ca2+ sensors with distinct characteristics. Synchronous release requires a Ca2+ sensor with fast Ca2+-binding kinetics to trigger the fusion of multiple quanta within a few ms, thus nearly simultaneously. The tradeoff for these fast binding kinetics is a lower affinity for Ca2+, so more Ca2+ is needed to saturate the Ca2+ sensors. Therefore, this Ca2+ sensor only triggers vesicle fusion when [Ca2+] is high. Alternatively, asynchronous Ca2+ sensors must exhibit high affinities for Ca2+ with slower Ca2+ interaction kinetics, triggering fusion events over a long period of time when [Ca2+] is decreased.      15   The Ca2+ sensors for fast, synchronous release are synaptotagmins 1 and 2. They are both located on synaptic vesicles and play functionally homologous roles in different parts of the nervous system. In mammals, synaptotagmin 1 is expressed predominantly in the cerebral hemispheres, while synaptotagmin 2 is expressed predominantly in the brainstem and spinal cord [71, 72]. Drosophila do not have a synaptotagmin 2 gene and synaptotagmin 1 (Dsyt1) is responsible for triggering fast, synchronous neurotransmitter release throughout the nervous system. Synaptotagmins 1 and 2 are low-affinity Ca2+ sensors and only bind Ca2+ when intracellular [Ca2+] is very high, as found in Ca2+ nanodomains. Without Ca2+, synaptotagmin interacts with SNARE proteins. In the Ca2+-bound state, synaptotagmin interacts with both SNARE proteins [73-78] and negatively charged phospholipid membranes in vitro [79-81]. Upon Ca2+ binding in vivo, synaptotagmins 1 and 2 trigger the fast, synchronous phase of neurotransmitter release [82]. These precise mechanisms are covered in more detail later in this chapter. The identity of the Ca2+ sensor for asynchronous release is still debated. Synaptotagmin 7 and Doc2 are currently the mammalian candidates for the Ca2+ sensor for triggering asynchronous neurotransmitter release. They are both enriched at synapses and exhibit high Ca2+ affinity and slow Ca2+ interaction kinetics [83-85]. The absence of either Doc2 or synaptotagmin 7 attenuates asynchronous release in synaptotagmin 1 knockout models, where asynchronous release is usually elevated [83, 84]. Even in the presence of synaptotagmin 1, synaptotagmin 7 knockouts attenuate asynchronous release under prolonged stimulation [84], although synaptotagmin 7 null mice are viable and fertile [86]. Recently, investigators report synaptotagmin 7’s role as a regulator of      16   asynchronous release at granule cell synapses, also [87]. However, the effect of Doc2 mutants is controversial [88, 89]. Doc2 knockout models are not yet available, but rabphilin, a Doc2-like protein that contains an additional N-terminal zinc finger domain, knockout mice display no obvious phenotype [90]. Additionally, some studies did not observe decreased asynchronous release upon Doc2 knockdown, although differences in experimental procedures can complicate results [88, 89]. The invertebrate candidate for asynchronous release is unknown. There is very little research on the identity of the invertebrate asynchronous sensor. Homologues of mammalian candidates Doc2 and synaptotagmin 7 are intuitive candidates. However, Doc2 is not present in invertebrate systems, and synaptotagmin 7, although present in Drosophila, displays expression patterns not consistent with a role as the Ca2+ sensor needed at active zones for asynchronous release. In third instar Drosophila larvae, synaptotagmin 7 is relegated to neuronal cell bodies in the central nervous system, and not detectable at the neuromuscular junction [91]. In double knockdowns of synaptotagmin 4 and 7, asynchronous release remains intact, eliminating it as the asynchronous sensor [92]. In C. elegans, synaptotagmin 7, together with synaptotagmin 4, is implicated in somatodendritic dopamine release, but not vesicle release at synaptic terminals [93]. Within the last year, a C. elegans neuronal Ca2+-sensing protein (NCS-2) has been implicated in asynchronous cholinergic release [94]. However, its Drosophila homologue frequenin, a high-affinity Ca2+ sensor present throughout the nervous system, has not been investigated for this function.      17   1.12 Synaptotagmin 1 in synchronous release Synaptotagmin 1’s role in synchronous release is well elucidated. Originally discovered and called p65 [95], Sudhof’s group suggested it plays a role in membrane interactions during exocytosis by calling attention to p65’s cytosolic domain that appeared homologous to the C2A domain of protein kinase C and could interact with membranes [96]. A year later, they showed that p65 (now deemed synaptotagmin) is highly conserved across species, including Drosophila [97]. Much like the studies used to isolate the SNARE proteins, they utilized neurotoxins. Using the spider venom alpha-latrotoxin, which increases neurotransmitter release, Petrenko et al showed the toxin receptor binds specifically to synaptotagmin, suggesting its role in neurotransmitter release [98]. Synaptotagmin’s C2 domains bind Ca2+ at physiological levels in a complex that contained negatively charged membranes, providing evidence that synaptotagmin is a Ca2+ sensor associated with release [99]. Importantly, synaptotagmin was directly linked to stimulating fast, synchronous release in 1994, when hippocampal cultures from synaptotagmin 1 null mice display abolished synchronous release, but asynchronous and spontaneous release persisted [100]. Synaptotagmin 1 acts as a synchronous Ca2+ sensor in stimulated release. It binds Ca2+ by its two C2 domains, C2A and C2B. These Ca2+ binding domains are lined with highly conserved negatively charged aspartate residues, resulting in a net negative charge before Ca2+ is bound. Ca2+ binding neutralizes the negative charge of the Ca2+ binding pockets and results in a net positive charge. This electrostatic change enhances interactions with the negatively charged presynaptic membrane and potentially with trans-SNARE complexes [73, 79, 101, 102]. Thus, Ca2+ binding allows synaptotagmin 1 to act as an “electrostatic switch” [103-106] from repulsion at rest to attraction upon Ca2+ influx.      18   Switching from electrostatic repulsion to electrostatic attraction permits hydrophobic residues in synaptotagmin 1’s C2 domains to escape the aqueous cytosol by penetrating into the hydrophobic core of the presynaptic membrane [101, 107]. One of these hydrophobic membrane- penetrating residues in C2A has been studied in vivo and results in a 50% knockdown of function when this phenylalanine (F) is mutated to a glutamate (E, P[sytF-E]) [108]. Moreover, when the hydrophobic residue found in C2B is mutated to a glutamate, the result is embryonic lethality. Therefore, the membrane interactions via these hydrophobic residues are critical for efficient synchronous fusion [108]. The insertion of these hydrophobic residues is thought to enhance fusion by two mechanisms. First, the inserted side chains occupy space, which induces positive curvature in the presynaptic membrane, bringing it closer to the curvature needed for fusion. Second, the insertion destabilizes the presynaptic membrane, again favoring fusion [81]. Therefore, synaptotagmin 1 can be thought of as providing the final push, much like popping a tightly inflated balloon, to trigger fusion. 1.13 Synaptotagmin 1 in asynchronous release The role of synaptotagmin 1 in asynchronous release remains unclear. It was originally postulated that the Ca2+ binding C2A domain of synaptotagmin 1 was actively regulating, or inhibiting, asynchronous release. This hypothesis is based on experimental results from two synaptotagmin mutants. Initially, studies reported increased asynchronous release in synaptotagmin 1 knockouts [82, 109-112]. Additionally, another study investigated the role of the C2A domain in both synchronous and asynchronous release [113]. In this study, Ca2+ binding in synaptotagmin’s C2A domain was blocked by mutating two negatively charged aspartates (D) found in the C2A Ca2+-binding pocket to neutral asparagines (N), here after called the P[sytD-N]      19   mutation. This mutation effectively prevents Ca2+ binding by altering the charge profile of the binding pocket. This P[sytD-N] mutant study reports no decrease in evoked release and an increase in asynchronous release. This study and the synaptotagmin 1 knockout studies that report increased asynchronous release led to the hypothesis that Ca2+ binding in synaptotagmin 1’s C2A domain is not necessary for fast, synchronous release, but is necessary to regulate, or inhibit, the asynchronous release sensor. I will call this hypothesis the inhibition hypothesis. Subsequently, Striegel et al [106] determined Ca2+ binding by synaptotagmin 1’s C2A domain is needed for efficient synchronous release, contrary to the results in the P[sytD-N] study. Striegel generated an alternative C2A Ca2+-binding mutation in which an essential aspartate (D) was mutated to a negatively charged glutamate (E), here after called the P[sytD-E] mutant. This mutant maintains the negative charge of the pocket while structural inhibiting C2A Ca2+ binding [106] and exhibits a significant ~80% decrease in synchronous release. A major function of synaptotagmin is to act as an electrostatic switch, which alters interactions with SNARE complexes and triggers an interaction with presynaptic membranes. The P[sytD-N] mutation effectively trips this electrostatic switch by neutralizing the Ca2+ binding pocket [106]. In this light, it is not surprising that in the presence of a wild type C2B domain, the P[sytD-N] mutant exhibits no impact on synchronous release since it is mimicking Ca2+ binding [106]. Additionally, the P[sytD-N] mutant responds more robustly at lower [Ca2+] for both synchronous and asynchronous release [113], indicating an increased affinity for Ca2+. Since the P[sytD-N] mutation mimics Ca2+ binding for synchronous release, its reported effect on asynchronous release also comes into question. An increase in Ca2+ affinity and mimicking bound Ca2+ may result in increased asynchronous release by triggering release events at lower [Ca2+]. These aberrant asynchronous events would be an artifact of the P[sytD-N]      20   mutation, and not the actual role of C2A in regulating asynchronous release. This sheds doubt onto the importance of synaptotagmin C2A Ca2+ binding in the regulation of asynchronous release. Increases in asynchronous events seen in the P[sytD-N] mutant may be a result of the P[sytD-N] mutation mimicking the asynchronous sensor itself. Asynchronous Ca2+ sensors exhibit slower binding and unbinding kinetics for Ca2+, permitting release on a longer timescale [53]. The P[sytD-N] mutants exhibit release events over longer time scales [113]. Asynchronous sensors exhibit higher affinities for Ca2+, which would allow the asynchronous sensor to bind Ca2+ at lower [Ca2+]. Indeed, the P[sytD-N] mutation exhibits increased affinity for Ca2+ [113]. In contrast, P[sytD-E] mutants exhibit decreased Ca2+ affinity [106], again providing evidence that P[sytD-N] mutant may be mimicking an asynchronous sensor. 1.14  Clarification  of  synaptotagmin’s  role  in  asynchronous  release   In chapter 2 of this dissertation, I directly test the role of Ca2+ binding in C2A in asynchronous release in vivo by utilizing the P[sytD-E] mutation, which prevents C2A Ca2+ binding while maintaining C2A’s inherently negative charge. If C2A Ca2+ binding is clamping the asynchronous Ca2+ sensor, an increase similar to that seen in the P[sytD-N] mutant should occur in the P[sytD-E] mutant. Such a result would corroborate the current hypothesis in the field. If, however, Ca2+ binding in C2A is not needed to regulate asynchronous events, the P[sytD-E] mutation will exhibit no increase in asynchronous events. I compare asynchronous release events in both the C2A P[sytD-N] and P[sytD-E] transgenic mutants to a transgenic control in a synaptotagmin null background of Drosophila.      21     In chapter 3, I test the possibility that the P[sytD-N] mutant is mimicking the asynchronous Ca2+ sensor in vivo by generating Drosophila mutants that contain the original P[sytD-N] mutation with additional mutations of the hydrophobic residues to prevent interactions with the presynaptic membrane. I generate a P[sytD-N] mutation coupled to a previously studied P[sytF-E] mutation (P[sytD-N,F-E]). I also generated the P[sytD-N,F-E] mutation with an additional hydrophobic mutation, methionine (M) 222, to a glutamate (E), hereafter called P[sytM-E,D-N,F-E]. If the P[sytD-N] mutation is simply mimicking the high-affinity asynchronous sensor, then preventing the downstream membrane interactions needed for triggering fusion events should prevent any aberrant increase in asynchronous release caused by P[sytD-N]’s neutralization of the pocket. Conversely, if the synaptotagmin P[sytD-N] mutation is not mimicking an asynchronous sensor, but is regulating the asynchronous sensor, consistent with the inhibition hypothesis, the increase in asynchronous release seen in the P[sytD-N] mutation should remain. From these studies, I conclude that C2A Ca2+ binding is not needed to regulate asynchronous release. The P[sytD-E] mutation does not exhibit increased asynchronous events. Moreover, I report no changes to asynchronous release in either the P[sytD-N,F-E] or P[sytM-E,D- N,F-E] mutations, supporting the conclusion that the P[sytD-N] mutant is mimicking an asynchronous sensor. These results contradict the inhibition hypothesis. Instead, together these results support an alternative hypothesis, the competition hypothesis, which suggests that synaptotagmin blocks access to SNARE interactions with the asynchronous sensor. The previous synaptotagmin knockout studies also support this hypothesis, as synapses without synaptotagmin 1 would provide more space for asynchronous sensor release interactions.      22   1.15 Neuromuscular disorders Many tightly regulated mechanisms are needed to result in efficient release of neurotransmitter and subsequent responses in the postsynaptic cell. When proteins needed for these processes are dysfunctional, synaptic disease may arise. For this dissertation, I will focus primarily on disorders associated with proteins found at the neuromuscular junction. These disorders can be categorized by location of the affected structures, be it presynaptic, synaptic cleft, or postsynaptic. Conditions such as myasthenia gravis affect synaptic proteins in the postsynaptic muscle cell, while neuromyotonia and Lambert-Eaton myasthenic syndrome affect proteins in the presynaptic neuron. They can also be categorized by origin of dysfunction, such as an immune disorder or a congenital disorder. Myasthenia gravis and Lambert-Eaton myasthenic syndrome are both autoimmune disorders, while congenital myasthenic syndromes are associated with rare familial mutations and can exhibit their own distinct symptoms. Neuromuscular diseases in human patients can be diagnosed using a combination of nerve conduction studies, exercise testing, immunological tests, and genetic sequencing. Nerve conduction studies Nerve conduction studies (NCS) are performed on patients with clinical presentations that are consistent with neuromuscular disorder. These tests help delineate neuromuscular junction diseases from one another. Clinicians perform NCS by placing surface electrodes over a muscle and the nerve that innervates it. The clinician will stimulate the nerve and progressively increase the stimulus intensity until the muscle response does not increase. This is the supramaximal stimulation used. Once the largest response of the muscle fiber is recorded, one can conclude that all motor nerve fibers are recruited, and therefore all the innervated muscle      23   fibers within the muscle are responding. This muscle response is called the compound muscle action potential (CMAP) [114]. CMAP amplitude is dependent on two major factors: 1.) the number of innervated muscle fibers available to respond to a given stimulus and 2.) amount of neurotransmission at the neuromuscular junction. The CMAP can detect a gross loss of motor unit innervation to the muscle, indicative of a progressive neurogenic disorder. If this is the case, the CMAP amplitude will be decreased. During the early phases of these disorders, however, the CMAP may appear normal due to compensatory reinnervation. As one motor unit becomes deinnervated, other motor axons send compensatory motor terminal branches to the muscle. If the new motor branches can maintain a similar innervation level to the rate of deinnervation, there may be no detection of deinnervation. The second factor that affects CMAP amplitude is the efficacy of neurotransmission at the neuromuscular junction. In this case, if there is a normal amount of innervated muscle fibers, but signal transmission from the pre- to postsynaptic cell is hindered, the result is a decreased CMAP amplitude. Unlike progressively deinnervating diseases, the deficits in neuronal transmission can be modulated and recorded through repetitive nerve stimulation tests. These tests are similar to single evoked CMAPs during baseline nerve conduction studies, but stimulations at varying frequencies are administered. These tests can help to localize the deficit. Facilitated amplitudes in response to high frequency stimuli (30 – 50 Hz) indicate a presynaptic deficit, while small amplitudes without facilitation suggests postsynaptic issues.      24   Lambert-Eaton myasthenic syndrome Lambert-Eaton Myasthenic Syndrome (LEMS) is an autoimmune disease in which patients experience muscle weakness due to a decrease in Ca2+-triggered neurotransmitter release at the neuromuscular junction [115]. This is the second most common neuromuscular disease, but is still 20 times less prevalent than the other immune-mediated neuromuscular junction disease, myasthenia gravis. As an immune-mediated disorder, IgG antibodies attack the presynaptic voltage-gated Ca2+ channels needed for efficient neurotransmission in most patients [116]. Patients experience the most muscle weakness after periods of rest. Strength improves with brief exercise, but does not sustain long periods of activity. Like myasthenia gravis, this disorder is also often co-morbid with cancer. Sixty percent of patients with LEMS experience small cell carcinoma [117]. LEMS usually affects patients over 40 years old. Nerve conduction studies of LEMS patients result in decreased CMAP amplitudes. Repetitive nerve stimulation (RNS) tests exhibit depression at low frequencies (2-3 Hz) and facilitation at high frequencies (30-50 Hz). A classic hallmark of LEMS is a facilitated CMAP amplitude response after 10 seconds of voluntary muscle contraction [118]. Interestingly, approximately 20% of LEMS patients express antibodies against the intravesicular domain of synaptotagmin, the only portion of the protein exposed to antibodies in the synaptic cleft upon vesicle fusion [119]. This is the first indication that synaptotagmin could be a candidate for synaptic disease.      25   Congenital myasthenic syndromes Congenital myasthenic syndromes (CMS) are genetically linked defects in one or multiple proteins found at the neuromuscular junction, including presynaptic, cleft, and postsynaptic proteins [118]. As of 2018, over 20 CMS genes were identified [120]. Some of the affected proteins and their importance have been discussed in this dissertation, including SNAP- 25, acetylcholine receptors, postsynaptic Na+ channels, and cleft enzymes that breakdown acetylcholine (acetylcholinesterase). Importantly for this dissertation, synaptotagmin 2, the fast, synchronous Ca2+ sensor at mammalian neuromuscular junctions, has also been implicated in CMS etiology [121, 122]. With the wide variety of proteins, nerve conduction tests, and clinical presentations, clinicians can point to candidate genes. Once candidate genes are identified, genetic testing aides in diagnosis and treatment plans. In patients, however, there is no way to definitively link a mutation to observed symptoms, necessitating the use of model systems to positively identify disease-causing mutations. 1.16 High throughput sequencing The decreasing cost and increasing availability of DNA sequencing has led to an increased incidence of genomic sequencing during patient diagnoses. This ever-increasing process will undoubtedly lead to the discovery of additional synaptic mutations and associated genetic disorders. The need for a relatively quick and cost effective method to begin elucidating the molecular mechanisms underlying these synaptic disorders is vital.      26   1.17 Synaptotagmin-related disorders It is only recently that mutations in the syt1 and syt2 genes have been directly implicated in human disease [121-123]. A patient with a de novo mutation in the human syt1 gene presented with an early onset dyskinesia, severe motor delay, and profound cognitive deficits attributed to a single amino acid substitution in the C2B Ca2+-binding pocket: an isoleucine to threonine (I-T) substitution [123]. This hydrophobic isoleucine residue had been shown previously to mediate Ca2+-dependent membrane penetration by synaptotagmin [79, 81, 124, 125]: an effector interaction critical for coupling Ca2+ influx with neurotransmitter release from neurons [83, 108]. Since this syt mutation results in the most severe deficits in an animal system [108] and is located in the syt1 gene, which is preferentially expressed in the cerebral hemispheres [71, 72], this patient experienced extreme cognitive deficits and the most severe motor deficits observed in a synaptotagmin disease to date [123]. Two additional mutations in the human syt2 gene were implicated in the etiology of congenital myasthenic syndrome patients shortly after: a proline to leucine (sytP-L) mutation and an adjacent aspartate to alanine (sytD-A) mutation. Both of these highly conserved residues are located in synaptotagmin’s C2B Ca2+-binding pocket [96, 126, 127]. The sytP-L mutation had never been studied. However, previous studies have demonstrated that the C2B domain Ca2+- binding aspartate (D) residues are essential for synaptotagmin function [82, 121, 125, 128], at times resulting in lethality in animal models [82, 121]. For the sytD-A familial mutation, expression of the mutant syt from a transgene in syt Drosophila heterozygotes resulted in lethality in 4 of 6 independent transgenic lines and a dramatic decrease in evoked response in the remaining two lines [121]. It is noteworthy, then, that deficits in the affected human family are comparatively mild [121, 122].      27   The proline residue affected by the second family is located directly adjacent to one of these previously studied aspartates residues. We speculate that this proline residue may provide conformational rigidity important for stabilizing the C2B Ca2+-binding pocket. By mutating the proline to a leucine, the rigid R group found in the proline residue would be lost, potentially affecting the precise conformation of the pocket and impacting the ability of the adjacent aspartate to bind Ca2+. Such a mechanism could result in a decreased, albeit not demolished, ability of the C2B domain to bind Ca2+ [82, 108, 113, 121-123]. A decreased ability of synaptotagmin to bind Ca2+ would hinder the efficacy of neurotransmission, and potentially to myasthenic disease symptoms. This mutation could be causing the symptoms seen in the affected family, but the correlations between those with the disorder and the mutation provide no direct evidence that the proline mutation leads to disease symptoms. 1.18 Drosophila as a model system One approach to directly assess the responsibility of the proline-leucine mutation in this congenital myasthenia is to leverage the power of Drosophila genetics. Rare disorders such as this synaptotagmin-implicated myasthenia are often underfunded and overlooked, as the need for medical research of these diseases does not affect the masses and receive little attention. However, the advances in human genetic sequencing are uncovering more of these rare genetic disorders. Drosophila are an ideal organism to investigate these mechanisms. Drosophila are economical, rapidly regenerate, and possess a genetic toolbox that allows us to drive the mutation of interest in subsets of cells. By creating a homologous mutation in Drosophila syt1, emergence of disease symptoms would create a direct link between the proline-leucine mutation and this congenital myasthenia. Electrophysiological tests where an intracellular electrode is      28   inserted directly into the fly body wall muscle fiber and its innervating nerve is stimulated can uncover mechanisms of disease etiology. The amplitude of the response can be measured much like a CMAP. Unlike a CMAP, the Drosophila tests examine a single muscle fiber, so deficits in the number of innervating nerve cells onto the muscle are not distinguished. This allows a direct investigation of synaptic transmission deficiency. If similar symptoms are detected, researchers can examine the mechanisms mediating this disorder more completely, as there would be a disease model readily available and a causal link between the mutation and disease symptoms. 1.19 Investigation of a synaptotagmin disorder In chapter 4 of this dissertation I directly test the importance of the previously uninvestigated proline residue in vivo by creating a homologous mutation in Drosophila. Our in silico modeling predicts a conformational change in the C2B Ca2+-binding pocket, potentially affecting synaptotagmin’s efficiency. I report homologous symptoms between the Drosophila mutation and human disorder, including: 1) decreased neurotransmission, 2) facilitated neurotransmission in response to high frequency stimulation, and 3) muscle fatigability. This study also provides mechanistic insight into this disease, as the mutation exhibits results consistent with decreased release probability and decreased Ca2+ affinity. 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Augustine, Dual roles of the C2B domain of synaptotagmin I in synchronizing Ca2+-dependent neurotransmitter release. J Neurosci, 2004. 24(39): p. 8542-50.      36   CHAPTER 2: CLARIFICATION OF CA2+ BINDING IN SYNAPTOTAGMIN 1’S C2A DOMAIN IN ASYNCHRONOUS NEUROTRANSMITTER RELEASE1 2.1 Summary Following nerve stimulation, there are two distinct phases of Ca2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca2+ binding by its C2A and C2B domains. It has also been implicated in the regulation of asynchronous neurotransmitter release. As blocking Ca2+ binding by the C2A domain of synaptotagmin 1 results in a dramatic increase in asynchronous release, C2A Ca2+ binding is postulated to directly inhibit asynchronous release. However, the mutation used to block Ca2+ binding in the previous experiments had the unintended side effect of mimicking Ca2+ binding, raising the possibility that the increase in asynchronous release was an artifact of ostensibly constitutive Ca2+ binding. To test whether Ca2+ binding by C2A is required for the direct regulation of asynchronous release, we utilize an alternate C2A mutation that we designed to block Ca2+ binding without mimicking it. Analysis of both the original mutation and our alternate mutation at the Drosophila neuromuscular junction shows opposite effects on: spontanteous release frequency, synchronous release kinetics, and asynchronous release events. Importantly, we found that asynchronous release is not increased in our novel mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca2+-evoked                                                                                                                           1  Authors: Mallory Shields, Matthew Bowers, McKenzie Fulcer, Lara Perinet, Marissa Metz, Noreen Reist      37   synaptic transmission and demonstrates that Ca2+ binding by the C2A domain of synaptotagmin 1 does not actively inhibit asynchronous neurotransmitter release in vivo. 2.2 Introduction Following nerve stimulation, there are two phases of Ca2+-dependent neurotransmitter release. Fast, synchronous release is the large burst of neurotransmitter release that occurs within milliseconds (ms) of the arrival of the action potential. At most healthy synapses, the majority of release occurs during the synchronous phase [1]. Synaptotagmin 1, which contains two Ca2+- binding C2 domains, C2A and C2B [2], is essential for coupling Ca2+ binding to efficient, synchronous release [3-6]. Asynchronous release can last from 10’s of ms to 10’s of seconds (s) [7], and has  been   functionally  implicated  in  synaptic  plasticity  [8-­‐11]  and  development  [12,  13]. While most synapses exhibit little to no asynchronous release, it  is  observed  in  many  synapse  types  [7].  In some specialized synapses, such as specific hippocampal and brainstem interneurons, asynchronous release is predominant [14-16]. In addition to being the Ca2+ sensor for fast, synchronous release, synaptotagmin 1 is proposed to directly regulate asynchronous release. Increases in asynchronous release are reported in synaptotagmin 1 null mutants [17, 18] and in a synaptotagmin 1 point mutant in which Ca2+ binding by the C2A domain is blocked [19]. Importantly, synchronous release remains intact in this C2A point mutant. Thus, the authors conclude that Ca2+ binding in C2A is not needed for efficient synchronous release, but does play a role in preventing asynchronous neurotransmission [19]. Together, these studies result in the inhibition hypothesis: that Ca2+      38   binding by the C2A domain of synaptotagmin is directly inhibiting asynchronous neurotransmitter release. More recently, our group demonstrated that Ca2+ binding by the C2A domain is required for efficient synchronous release [4], contrary to previous studies [20-22]. The original point mutations used to block Ca2+ binding by C2A removed negative charge from the Ca2+ binding pocket; key, negatively-charged aspartate residues (D) essential for coordinating Ca2+ were replaced with neutral asparagines (N), sytD-N. Since synaptotagmin 1 functions as an electrostatic switch [23, 24], removing negative charge may mimic Ca2+ binding and permit downstream effector interactions [20]. To directly test this hypothesis, we generated a novel Ca2+-binding mutation where an essential C2A aspartate was mutated to a negatively-charged glutamate (E) [4]. The sytD-E mutation maintains the negative charge of the pocket but prevents Ca2+ binding by steric hindrance resulting in an ~80% decrease in synchronous neurotransmitter release. This finding demonstrated that an intact C2B Ca2+-binding domain is sufficient to trigger the electrostatic switch in the absence of C2A Ca2+ binding only if the C2A Ca2+-binding pocket was neutralized. Thus, the failure of sytD-N mutations to impair synchronous release is an artifact of removing the electrostatic repulsion of the presynaptic membrane. The current interpretation, that a sytD-N mutation fails to inhibit asynchronous release because it cannot bind Ca2+, may be subject to the same artifact. By comparing a sytD-N mutation with the sytD-E mutation in Drosophila, we test whether Ca2+ binding by the C2A domain of synaptotagmin 1 is required to regulate asynchronous release events. If C2A Ca2+ binding clamps asynchronous release, the increased asynchronous release seen in the sytD-N mutation should also occur in the sytD-E mutation. However, if increased asynchronous release is an artifact of      39   ostensibly constitutive Ca2+ binding in sytD-N, then the sytD-E mutation should not result in increased asynchronous release. We now show the sytD-E mutation had no impact on asynchronous release, demonstrating that C2A Ca2+ binding does not regulate asynchronous neurotransmitter release. 2.3 Results Synaptotagmin mutations To test the function of Ca2+ binding by the C2A domain of synaptotagmin during vesicle fusion events, we completed a direct comparison of two disparate mutations that both block Ca2+ binding (Fig 2.1). Mutating the third and fourth of the Ca2+-binding Ds to Ns (sytD-N, Fig 2.1B) blocks Ca2+ binding and Ca2+-dependent C2A interactions by removing some of the negative charges required to coordinate Ca2+. Mutating the second D to an E (sytD-E, Fig 2.1C) in C2A blocks Ca2+ binding by steric hindrance while maintaining the negative charge of the pocket. In all experiments, synaptotagmin 1 was expressed as a transgene (P[syt]) in the absence of native synaptotagmin 1.      40   Fig 2.1. C2A domain of wild type and mutant synaptotagmin and their interactions with the negatively-charged presynaptic membrane. A) Cartoon depicting the C2A Ca2+-binding pocket of wild type synaptotagmin (sytWT) repeling the negatively-charged presynaptic membrane prior to Ca2+ entry due to 5 negatively-charged aspartate residues (magenta, left) and penetrating the presynaptic me