Konrad E. Zinsmaier - US grants

9604889 Zinsmaier The signaling pathway of depolarization-dependent neurotransmitter release is a product of trafficking synaptic vesicles through repeated cycles of secretion, membrane recycling, and vesicle genesis. Each stage of this cycle requires the cooperative action of proteins. This proposal studies the cysteine-string protein (csp) intrinsic to synaptic vesicles. The loss of csp protein in Drosophila mutants progressively reduces evoked transmitter release at higher temperatures, but not spontaneous release. Such a failure of neurotransmitter release in csp mutants could be due to a defect at any stage of the vesicle cycle. To gain a better understanding of csp function this proposal will determine which stage of the vesicle cycle requires csp function by imaging the dynamics of the synaptic vesicle cycle in the csp mutant flies. From earlier studies it has been proposed, that csp might serve as a cofactor to catalytically recruit an unknown protein to cooperate protein interactions critical for evoked neurotransmitter release. To explore this working hypothesis the proposal plans to identify protein domains of csp which are critical for its function by genetic engineering. In addition, a strategy has been designed to determine the molecular components that interact with csp combining the purification of interacting proteins and the isolation of mutations interacting with the csp mutation. The identification of critical parts of the csp protein and its interacting partners will provide the basis to develop more specific ideas to directly elucidate csp function. Given the numerous signaling pathways conserved from flies to mammals, Drosophila will serve as a model system to illuminate basic mechanisms of normal and abnormal synaptic transmission.

Two conditions appear essential for fast synchronous neurotransmitter release: First, an optimal adjustment of CA/2+ entry kinetics and second, and optimal assembly of the CA2+ entry site in close proximity to the Ca/2+ entry site in close proximity to the Ca/2+ sensor site to ensure a short diffusion passage for Ca/2+ ions. Our previous genetic analysis of cysteine string protein (Csp) mutations in Drosophila indicates that CSP mediates synchronous neurotransmitter release but not vesicle recycling. In vitro studies of vertebrate CSP suggest a cooperative interaction of CSP with bovine 70kD heat shock cognate protein (HSC70), which is suggested to mediate the uncoating of clathrin-coated vesicles during vesicle recycling Combining the genetic and biochemical evidence, we propose that CSP may cooperatively interact with HSC70 to mediate synchronous release by directing HSC70 to the synchronous release machinery and stimulating its activity. This model postulates a novel function for HSC70 in neurotransmitter release. The focus of this proposal will be to stringently test this hypothesis in vivo by analyzing synchronous neurotransmitter release in mutant Drosophila strains which lack HSC70 and/or CSP function in Drosophila, five distinct constitutively expressed heat shock cognate genes (HSC1-5) have been identified which all share a significant sequence homology with bovine HSC70. However, no fly mutations have been reported for either gene. Since it will be critical for our success to study the correct homologous protein, we propose in Aim 1 to identify the true HSC70 homologue by its biochemical interaction with CSP. To test some of the suggested functions of HSC70., we propose in Aim 2 to determine the in vivo role of HSC70 for synchronous release and for vesicle recycling. Therefore, we will study the effects caused by the loss of HSC70 function in mutant Drosophila. Specifically, we will use electrophsiological recordings and FM1-43 imaging at the larval neuromuscular junction to demonstrate any impairment of neurotransmission. In Aim 3 we propose to test whether CSP and fly HSC70 cooperatively interact in vivo. This will achieved by an analysis of synchronous release and vesicle recycling of double mutant flies which lack simultaneously CSP and HSC70 cooperatively interact in vivo. This will achieved by an analysis of synchronous release and vesicle recycling of double mutant flies which lack simultaneously CSP and HSC70 function. Alternatively, we will determine whether the over expression of HSC70 is able to rescue to neurotransmitter release defect caused by the loss of CSP function. This will also facilitate to determine the hierarchical order of CSP and HSC70 functions in transmitter release. The proposed work may significantly help to understand the molecular mechanism of synchronous neurotransmitter release which is one of the basic conditions mediating the functional plasticity of our nervous system. This understanding will help to fight the dramatic effects of synaptic dysfunction for human life.

Regulated neurotransmitter secretion mediates communication between nerve cells of the nervous system. A "sender" cell encodes its electrical signal into a chemical signal by using a neurotransmitter stored in synaptic vesicles. The neurotransmitter is secreted onto a "receiver" cell, which decodes the signal back into an electrical signal. Neurotransmitter secretion is accomplished by fusing the vesicle membrane with the cell membrane expelling its content. Little is known about the molecular machinery driving vesicle fusion. The goal of this proposal is to investigate these molecular mechanisms and, in particular, the role of cysteine-string protein (CSP). This project will test the hypothesis that CSP may mediate secretion by modulating Ca2+ channels, Ca2+ channel-vesicle linkage, and/or vesicle fusion. This will be achieved by employing genetically manipulated motor nerve terminals of the fruit fly Drosophila as a model system.
The extraordinary fast and precise communication of nerve cells is central to understand how the nervous system works - how we perceive, move, feel, learn, and remember. Failure of secretion has a fatal influence on human life. In most cases, the abnormalities are so severe that embryonic development will not proceed. Even subtle defects of neurotransmitter secretion severely impair higher brain functions like learning and memory. CSP has been recently implicated as an important factor during the treatment of manic depression in humans. Thus, the research being conducted has crucial implications on our basic knowledge of neuronal secretion and subsequently on human medicine to detect, to treat, and to possibly prevent neurological disorders.

Regulated neurotransmitter exocytosis is a fundamental process for the intercellular communication among neurons. It is widely accepted that neurotransmitter secretion is a tightly regulated form of constitutive vesicular exocytosis shared by all eukaryotic cells. At the nerve terminal, a depolarization-induced Ca2+ influx through ion specific channels triggers the fusion of synaptic vesicles, which expel their neurotransmitter cargo onto the postsynaptic cell.
A combination of biochemical and genetic approaches by many laboratories has lead to molecular models describing exocytotic and endocytotic mechanisms. In particular, recent advances uncovered the SNARE- or core complex driving constitutive membrane fusion. However, little is known about mechanism that mediate vesicle docking, arrest fusion-competent vesicles in the readily releasable pool, accomplish Ca2+ signaling from the sensor to the fusion machinery, adjust the probability of vesicle fusion, or regulate the size of readily releasable pool.
We have initiated a genetic screen to identify further components mediating Ca2+-triggered exocytosis at nerve terminals and identified several novel mutations, which potentially affect neurotransmission. Of these, the mutation B682 is especially interesting because it inhibits a unique activity-dependent loss of evoked release, which coincides with a simultaneous increase in spontaneous neurotransmitter release, suggestive of an impaired releasable vesicle pool. A unifying hypothesis suggests that B682 protein mediates a late step in synaptic vesicle maturation that accumulates fusion-competent vesicles in the "readily releasable vesicle pool". Specifically, B682 may "clamp" these fusogenic vesicles such that they await the fusion-triggering Ca2+ signal.
To test this hypothesis (and explore others if warranted), loss- and gain-of-function B682 mutations will be examined for their effects on synaptic function at NMJs. These effects will be examined by a multi-disciplinary approach including electrophysiology, Ca2+ imaging, FM1-43 imaging, confocal and electron microscopy, and biochemistry.
The first two Objectives will lay basic and essential groundwork for evaluating B682's synaptic role. Specifically, we will verify that B682 is indeed a presynaptic protein that affects a physiological step of synaptic transmission but not neuromuscular synaptogenesis. Once these points are fully established the third Objective will test the essence of the B682 protein hypothesis. Specifically, it will test the prediction that B682 mutations do not impair Ca2+ entry and/or extrusion. In addition, this objective will test whether the abnormal vesicle distribution in B682 mutants is caused by a defect in vesicle recycling, steps of vesicle trafficking to active zones and/or refilling the readily releasable vesicle pool. Together, these studies will help to better understand the molecular mechanisms that mediate the regulation of fast, synchronous neurotransmitter exocytosis at nerve terminals.

DESCRIPTION (provided by applicant): Neurotransmitter release is regulated at many steps, which in part confers upon synapses their plasticity, adaptability, and individuality. Two key steps, Ca2+ entry and vesicle fusion, appear to be regulated by the synaptic vesicle-associated cysteine-string protein (CSP) - but in "opposing" ways. Previous work supports the hypothesis that CSP reduces release by inhibiting presynaptic Ca2+ entry and increases release by promoting a downstream step of Ca2+-triggered fusion. Together with Hsc70, CSP might direct protein interactions among Ca 2v channels, G proteins, syntaxin, and synaptotagmin. To better understand CSP's action, we need to know: (1) whether CSP indeed promotes Gbeta/gamma inhibition of Ca2+ channels at nerve terminals, (2) which of the other known CSP interactions mediates which function of CSP, and (3) which functions are regulated by PKA-phosphorylation at nerve terminals. To resolve these issues, I propose to test the above hypotheses by exploiting the genetic model system Drosophila to examine the effects of systematically targeted CSP mutations on neurotransmission at neuromuscular junctions, accomplishing a complete in vivo structure/function analysis. Specifically, Aim 1 will (a) correlate protein interactions of the J-, L-, C-, and Ct-domain with CSP's synaptic roles, including Ca2+ entry, Ca2+-triggered fusion, short-term plasticity of release, and Ca2+ homeostasis. Aim 1 will also (b) resolve the significance of PKA-mediated phosphorylation of CSP at nerve terminals. Aim 2 will determine (a) whether CSP is critical for Gbeta/gamma inhibition of Ca2+ entry and/or (b) vesicular fusion. From this systematic analysis a substantial framework will emerge for understanding the apparently opposing actions of CSP. The proposed work will also expand our understanding of important regulatory mechanisms of synaptic transmission and their relation to the functional plasticity of the nervous system and human health.

[unreadable] DESCRIPTION (provided by applicant): Intracellular Ca2+ signals are an integral part of information processing by neuronal circuits facilitating pre- and postsynaptic functions including long-term potentiation (LTP) and depression (LTD) of synaptic transmission, the electrophysiological correlates of long-term synaptic plasticity. Since finely tuned changes in Ca2+ modulate a variety of intracellular functions, signal specificity requires a tight spatial and temporal control of the cytosolic Ca2+ signal. This need is most apparent for fast neurotransmitter release where voltage-dependent Ca2+ signaling triggers the fusion of synaptic vesicles at presynaptic terminals on a sub-millisecond scale. The endoplasmic reticulum (ER) can act as a Ca2+ store/sink or as a Ca2+ source and is likely to balance these dual roles at presynaptic terminals to meet the specific needs of the synapse for transmitter release. However, the contributions of ER to presynaptic Ca2+ signaling and neurotransmitter release are not well understood. We hypothesize that the ER acts as the primary presynaptic Ca2+ sink at fly NMJs. If so, the presynaptic ER may not only temporarily buffer cytosolic Ca2+ by SERCA pumping but also permanently remove Ca from synaptic terminals by "tunneling" luminal Ca2+ into axons. Limited ER Ca2+ release within terminals may modulate neurotransmitter release but mainly serve mitochondrial Ca2+ uptake activating mitochondrial energy production. We will test this hypothesis by exploiting genetically manipulated Drosophila and image depolarization-induced changes in presynaptic Ca levels of the ER lumen, mitochondria and the cytosol of larval NMJs. At the center of this proposal is the development of transgenic Ca2+ indicators that can faithfully report Ca2+ changes in the lumen of the ER and mitochondria, which we expect will also be of wide use for the genetic analysis of mitochondrial and/or ER Ca2+ dynamics in any cell of Drosophila. Specifically, we will determine the dynamics of ER-mediated Ca2+ uptake, Ca2+ release and/or Ca2+ diffusion (tunneling) upon repetitive nerve stimulation at presynaptic terminals of larval Drosophila NMJs (Aim 1) and determine the role of ER-mitochondria interactions for presynaptic Ca2+ homeostasis at larval Drosophila NMJs (Aim 2). From this systematic analysis a basic framework will emerge for better understanding the role of the ER in presynaptic Ca2+ signaling/homeostasis at presynaptic terminals expanding our understanding of important regulatory mechanisms of synaptic transmission and their relation to the functional plasticity of the nervous system and human health. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The efficient and stable transfer of information among neurons occurs at specialized cell-cell contact sites, called synapses but neither their structure nor their strength is static. Synapses are rearranged as neuronal circuitry is refined upon changes in neuronal activity throughout life. This process is generally believed to underlie learning and memory. Failure or even subtle changes in synaptic strength and/or wiring can disturb neuronal circuits and cause neurological, psychiatric, and/or neurodegenerative disorders. However, despite considerable progress, many molecular mechanisms that govern synaptic function are still poorly understood or not known. Using the model system Drosophila, we employed a forward genetic approach and identified a large number of gene candidates that may express critical and novel synaptic components. The major questions are now: (1) Which genes have been mutated and (2) where are the underlying proteins localized within a neuron? The proposed study is designed to answer these questions for 4 of our newly identified mutations, which all affect essential presynaptic mechanisms of synaptic transmission. The gained knowledge (molecular identity and localization of the mutated proteins and their significance for synaptic function) together with the newly produced tools (transgenes and antibodies) will then provide an essential foundation to successfully obtain large-scale federal funding to dissect the mutated molecular mechanisms underlying synaptic function. Specifically, Aim 1 will physically identify the gene locus that is mutated by the synaptic mutations B332, B689, B773, and B936. This will be achieved by genetically mapping the mutation to a small number of genes, which will then allow a molecular identification of the mutation and an association of the mutation with a particular gene. Aim 2 will resolve the tissue-specific and subcellular localization of the newly identified proteins. The proposed identification and subsequent functional analysis of new components governing synaptic structure and/or function will not only advance our basic biochemical knowledge but may also yield critical insights into the pathologies of homologous proteins in human and accelerate the development of new concepts for detecting, treating, and/or preventing neurological and psychiatric disorders. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Mitochondria are vital for aerobic respiration, the regulation of Ca2+ homeostasis, apoptosis, aging, and cancer. The intracellular distribution of mitochondria is adaptable to physiological stresses and changes in cellular activity. This plastic control is believed to be especially important in neurons where mitochondria are enriched at regions of intense energy consumption like synapses. Despite the significance of mitochondria for synaptic function, we still do not understand the molecular mechanisms controlling their delivery and targeting to synapses. A comprehensive understanding is urgently needed since abnormal mitochondrial transport, like abnormal mitochondrial function, is associated with various forms of muscular dystrophy, cardiomyopathy, neuropathy, paraplegia, and neurodegeneration. Our previous work suggests that the evolutionary conserved mitochondrial Rho-like GTPase Miro may act as a mitochondrial sensor that integrates intracellular signals to control long-distance transport of mitochondria. Specifically, loss of Drosophila Miro (dMiro) function prevents mitochondrial transport into axons and dendrites while gain of dMiro function leads to an abnormal accumulation of mitochondria at motor nerve terminals. Together, these results suggest dMiro may control anterograde axonal transport and the distribution of mitochondria to synaptic sites. To further test this hypothesis, we will take advantage of the model system Drosophila and genetically manipulate dMiro and other proteins of the mitochondrial transport machinery. Mutant effects on mitochondrial transport will be primarily examined in larval motor neurons, their axons and axon terminals by live imaging of GFP-tagged mitochondria to resolve the following key issues: Aim 1 will resolve whether dMiro promotes net-anterograde axonal transport by increasing the efficiency of microtubules (MT) plus end- or decreasing minus end-directed transport. Aim 2 will determine the role of dMiro's EF-hand Ca2+ binding domains for mitochondrial transport and/or the intracellular distribution of mitochondria. Aim 3 will test the molecular mechanisms by which dMiro may control mitochondrial transport. The proposed project is expected to reveal important molecular signaling mechanisms that regulate the long- distance transport of mitochondria and their use-dependent distribution into synaptic terminals. Uncovering these signaling pathways will significantly expand our understanding of basic mechanisms and accelerate the development of new concepts for detecting, treating, and/or preventing disorders that are caused by defective mitochondrial transport pathways and/or impaired mitochondrial function.

DESCRIPTION (provided by applicant): The efficient and stable transfer of information among neurons occurs at specialized cell-cell contact sites, called synapses. Even subtle changes in synaptic strength can disturb neuronal circuits and cause psychiatric, neurological, or neurodegenerative disorders. Effective synaptic transmission requires fast neurotransmitter secretion by Ca2+ triggered synaptic vesicle (SV) fusion and subsequently effective SV endocytosis. It has been theorized that "cone-shaped" lipids may aid the extreme membrane curvatures during SV fusion and fission. We hypothesize that at least some P4-ATPases may aid membrane curvatures during SV fusion and/or fission by locally translocating (flipping) specific lipids from the outer to the inner leaflet of the membrane. Our preliminary results suggest that this may be indeed the case. Taking advantage of the genetic model system Drosophila, we have identified mutations in the Drosophila ortholog (dATP8B) of the 4 paralogous human ATP8B1-4 flippases. Deletion of dATP8B impairs viability, locomotion and SV exo- and endocytosis, suggesting a critical for synaptic function. In addition, we obtained genetic evidence that ties dATP8B to the E3 ubiquitin ligase UBE3A, whose dysfunction causes Angelman Syndrome, an inherited neurological disorder leading to mental retardation. To gain critical data and tools for obtaining large-scale federal funding to test the synaptic role of dATP8B, we suggest in Aim 1 to generate antibodies and tagged transgenes to resolve the tissue-specific expression pattern and synaptic localization of dATP8B and its putative co-factor dCdc50. Aim 2 will establish in vivo and in situ "lipid flippase assays" to determine whether dATP8B mediates lipid flipping in cultured neurons and at synaptic terminals. Confirming flippase activity and a synaptic localization of dATP8B will provide a critical foundation to later test how lipid flipping promotes SV fusion or fission. Aim 3 will determine whether dCdc50 and the P4-ATPases dATP8A, dATP9, dATP10, and dATP11 are required for neuronal and/or synaptic function. This "survey" is justified since it is not known whether these P4-ATPases are required for neuronal function despite the association of some with Alzheimer's disease, Autism or Angelman syndrome. Together, these aims will provide critical preliminary data and tools like antibodies and transgenic animals to successfully obtain large-scale federal funding to rigorously test the significance and role of lipid flippases for neuronal and synaptic function. The proposed analysis of new components governing synaptic function will not only advance our basic knowledge but may also yield critical insights into the pathologies of homologous proteins in human brain disorders, like Autism and Angelman Syndrome. PUBLIC HEALTH RELEVANCE: Transmitting information from one nerve cell to another is critical for brain function. Successful completion of the project is expected to significantly advance our understanding of molecular mechanisms underlying nerve cell communication and provide insights into how failure of these mechanisms causes mental retardation. Results from this work are likely functionally relevant for understanding Angelman Syndrome (AS), an inherited neurological disorder that is characterized by mental retardation, minimal speech, difficulties in motor coordination, and other deficiencies.

How we perceive, move, feel, learn, and remember is a product of the ability of nerve cells to form circuits that allow an extraordinarily fast and precise communication processing information. Information is transferred from cell to cell through specialized cell-cell contact sites, termed synapses. Despite tremendous advances, understanding the molecular mechanisms facilitating, maintaining and/or re-arranging synaptic function and/or structure remains a key problem in contemporary neuroscience. Using a genetic approach, we identified a new protein (WD40A) that is very similar from fruit flies to humans. We hypothesize that WD40A may control the protein levels of another uncharacterized protein (ATP8B) that may control the lipid (i.e., fatty acid) composition of the synaptic membranes that facilitate information transfer from one nerve cell to another. To better support this hypothesis, the role and significance of WD40A for synaptic function and structure will be explored using electrical and optical recordings to examine the effects of genetic mutations in WD40A on synaptic function. In addition, genetic and biochemical approaches will be used to characterize exactly how WD40A controls ATP8B protein levels at synapses. The project will develop valuable research tools that will be made freely available to the research community and will provide research opportunities for both graduate and undergraduate students. This work is expected to characterize a fundamentally new molecular mechanism that induces local changes in the lipid environment of synapses aiding rapid communication among nerve cells. This knowledge will significantly advance our understanding of how the brain is able to compute information on a millisecond scale, and improve our ability to design better medical approaches to treat or prevent some neurological disorders in humans.

DESCRIPTION (provided by applicant): Supplying axons, dendrites, and synapses with mitochondria is vital for sustaining neuronal excitability and synaptic transmission. In contrast to most other cells, mitochondrial transport is critical for neuronal survival, as impaired transport causes the same pathology as impaired mitochondrial function. A combination of the large distance between soma and synapses, the complexity of neuronal branches, the need to relocate mitochondria in response to changes in neuronal activity, coupled with the need of mitochondria to eventually return to the cell body requires a transport system that is sensitive to pathways communicating the energetic state of the neuron and the state of the mitochondrion. However, we know little about the link between mitochondrial transport and mechanisms that maintain mitochondrial function in axons. A better understanding is urgently needed because even slight impairments of mitochondrial function and/or distribution can cause or intensify neuropathy, neurodegeneration, and/or paraplegia. The evolutionarily conserved mitochondrial GTPase Miro contains two Ca2+ binding domains sandwiched between Rho- and Rab-like GTPase (G) domains. Our genetic analysis shows that mutations in Miro have pleiotropic effects on the biology of mitochondria in axons. We hypothesize that Miro is a central integration node for multimodal signals that controls distinct mechanisms including mitochondrial transport, mitochondrial fusion & fission and autophagy. To test this further, we are taking advantage of the model system Drosophila to elucidate the multiple roles of Miro in neurons by genetically manipulating Miro and its interacting proteins. Aim 1 will characterize how Miro's G1 domain promotes the use of kinesin for anterograde transport and the use of dynein for retrograde transport in axons. Aim 2 will characterize the Ca2+-sensitive role of Miro for mitochondrial function in axons. Aim 3 will characterize the role of Miro for mitochondrial fusion & fission and/or autophagy. The project is expected to reveal critical insights into molecular signaling mechanisms that ensure mitochondrial function in axons. Uncovering these pathways will expand our understanding of logistical mechanisms that are critical for the long-term survival of neurons.

? DESCRIPTION (provided by applicant): The necessity of understanding causes of neurodegenerative diseases and developing potential treatments is increasing as life expectancy is extending. Parry disease (CLN4B) is an autosomal dominant form of Neuronal Ceroid Lipofuscinosis (NCL) with adult onset (ANCL). NCL comprises a group of inherited neurodegenerative diseases of children and occasionally adults that lead to physical deterioration, seizures, blindness, dementia, and premature death. NCL is morphologically characterized by degeneration of the cortex and cerebellum, and by lysosomal accumulations of lipofuscin. Parry disease (CLN4B) is caused by dominant lethal mutations in the DNAJC5 gene encoding CSP?, a well-studied synaptic vesicle protein that is neuroprotective and required to maintain synaptic function. Little is known about disease etiology besides biochemical evidence that the disease-causing dominant mutations in CSP? trigger the formation of large protein aggregates that contain mutant but also normal CSP?. However, whether these aggregates cause neurotoxic gain- and/or loss-of-function effects or, alternatively, are neuroprotective is no known. To gain a comprehensive understanding of mechanisms underlying Parry Disease, we face three challenges: The first is to systematically identify the nature(s) of the toxicity triggeing neuronal failure and neurodegeneration. The second is to identify the impaired molecular and cellular signaling pathways that are impaired by the toxic substrate or can counteract its effects. The third is to understand how the various signaling pathways interact with and feedback on each other to maintain homeostasis and prevent neuronal failure and neurodegeneration. We propose to establish the first animal model for Parry Disease by expressing disease-causing human CSP? in Drosophila. The fly is well suited to dissect the likely complex genetic nature of the disease since the neuroprotective and synaptic functions of fly and mouse CSP are well conserved, loss- and gain of function mutants of fly CSP are well studied, and numerous other sophisticated genetic tools aiding the analysis are available. To gain critical mechanistic insight into the etiology of Parry Disease, we suggest a rigorous and comprehensive analysis dissecting the genetic nature of the dominant mutations, the affected neuronal signaling pathways, and a genome-wide unbiased genetic identification of genes that positively or negatively contribute to the disease. Uncovering molecules and signaling pathways that are either impaired or able to counteract the effects of the disease-causing mutations in CSP will significantly expand our understanding of disease etiology and may accelerate the development of new therapeutic concepts treating Parry Disease.

The necessity of understanding causes of neurodegenerative diseases and developing potential treatments is increasing as life expectancy is extending. Neuronal ceroid lipofuscinoses (NCLs; also known as Batten disease) comprise a group of 14 monogenic neurodegenerative diseases with lysosomal pathology (CLN1-14). NCLs are typically due to recessive mutations in genes that mediate lysosomal function or ER-lysosomal trafficking with one atypical exception: the dominantly inherited NCL CLN4, which is caused by mutations in the synaptic vesicle (SV) protein CSP?. Normally, CSP? is critical to maintain synaptic function and prevent activity-dependent neurodegeneration. It also mediates the clearance of aggregating proteins like TDP-43 or ?-synuclein by unconventional secretion pathways. Little is known about CLN4 disease etiology besides biochemical evidence that CLN4-causing mutations induce the formation of ubiquitinated CSP? oligomers/aggregates. Whether and how the oligomeric or monomeric protein causes lysosomal failure, neurodegeneration, and premature death remains enigmatic. We have established the first animal models of CLN4 by expressing disease-causing human CSP? (hCSP?) or fly CSP (dCSP) in Drosophila neurons. Both models recapitulate the biochemical pathology of CLN4 post-mortem brains. Further analysis revealed a novel link between CLN4 mutant CSP and prelysosomal failure. Unexpectedly, we also found that the dominant CLN4 alleles act as hypermorphic gain of function mutations inducing the oligomerization of CSP, prelysosomal failure, neurodegeneration, and lethality. We suggest that hypermorphic CLN4 mutations increase the affinity for some or one of CSP?s protein interaction causing disease. Next to an exaggerated dimerization of CSP leading to oligomerization, CLN4 mutations increase interactions of CSP with the synaptically localized palmitoyl-transferase Hip14 that could lead to prelysosomal failure. Finally, increased interactions of CSP with Hsc70 on endosomes destined to form multivesicular bodies may interfere with their processing, sorting and/or trafficking. We propose to test these possibilities by genetic approaches to better understand both the mechanisms underlying CSP?s normal neuroprotective role, and the mechanisms underlying the hypermorphic CLN4 mutations causing protein aggregation, lysosomal failure, neurodegeneration, and premature death. Uncovering mechanisms underlying CLN4 may inform the future development of therapeutic interventions. In addition, a better understanding of CSP?s neuroprotective role is important for various other neurodegenerative diseases that may be attenuated by CSP?s clearance of misfolded proteins.