Janet Richmond - US grants

DESCRIPTION: (Applicant's Abstract) The process of synaptic transnuission. appears to be highly conserved across species, allowing functional questions about the vertebrate central nervous system to be addressed in more tractable systems. For example, over 30 genes implicated in synaptic transmission have been identified in the nematode, Caenorhabditis elegans, and homologs of these genes have subsequently been identified in vertebrates. Defects in neurotransmission due to neurodegeneration can lead to severe deficits in nervous system function evident in disorders such as Parkinson's and Alzeihmer's diseases. One of the most powerful approaches to study the roles of synaptic proteins is to examine synaptic physiology in mutant animals lacking these proteins. A preparation suitable for detailed electrophysiological analysis of synaptic function in C.elegans has not previously been available, primarily due to the technical difficulties of dissecting such a small organism. Preliminary data provided in this proposal demonstrate the feasibility of whole-cell, voltage-clamp recording synaptic activity in a newly developed C. elegans body wall neuromuscular preparation. In order to use this preparation to probe synaptic transmission in mutant animals, a baseline characterization of the wildtype synapse is required. The C. elegans neuromuscular junction is innervated by both GABAergic and cholinergic motor neurons. In order to characterize release at this synapse it will be necessary to study these two inputs separately. Aim I will identify pharmacological tools to separate the two synaptic inputs. Aim 2 will use these pharmacological tools to characterize spontaneous GABAergic and cholinergic synaptic activity. Aim 3 will develop methods to evoke GABA and ACh release at the neuromuscular junction. In aim 4, the Ca 2+-dependence of evoked release will be quantified. Aim 5 will test the synaptic plasticity of the neuromuscular synapse using protocols to elicit paired-pulse and short-term facilitation, augmentation and post-tetanic potentiation. This preparation is expected to provide a valuable new tool in which to combine the strengths of C. elegans genetics and molecular biology with electrophysiological analyses. Completion of this project is expected to lay the foundation for future evaluation of over 30 proteins implicated in synaptic transmission.

DESCRIPTION (provided by applicant): Neurons release neurotransmitter into the synaptic cleft by exocytosis of synaptic vesicles. The proteins that mediate exocytosis are members of conserved protein families involved in general intracellular fusion events. Critical proteins in this process include the SNARE proteins, syntaxin, SNAP-25 and synaptobrevin, as well as UNC-18 and UNC-13. Although we know that UNC-18 and UNC-13 interact with the essential SNARE protein syntaxin, the precise role and sequential order in which these proteins regulate fusion have yet to be determined. In my laboratory we combine genetic and molecular approaches with a newly developed electrophysiological technique to study exocytosis in the nematode Caenorhabditis elegans. Using these techniques we have demonstrated that exocytosis is arrested at a late stage in unc-13 mutants. We now propose to examine the role of UNC-18 in C. elegans synaptic transmission. UNC-18 binds to a closed conformation of syntaxin, which excludes the formation of the SNARE complex. Several models have been proposed for UNC-18 function: (1) UNC-18 dissociation may promote or maintain syntaxin in an open state, enabling SNARE complex assembly and fusion. (2) UNC-18 may maintain syntaxin in the closed conformation to prevent SNARE complex formation. (3) UNC- 18 and syntaxin may mediate the fusion step directly. To test these models, we propose to: 1) Determine whether UNC-18 promotes exocytosis after vesicles have docked to the plasma membrane. 2) Test whether the UNC-18-syntaxin interaction plays an inhibitory role in exocytosis. 3) Test whether constitutively open syntaxin bypasses the requirement for UNC-18. 4) Identify other proteins that act downstream or in parallel with UNC-18 by a) mapping and identifying a recently isolated unc-18 mutant suppressor and b) conducting a genetic screen to identify other unc-18 suppressors. These experiments may contribute to our understanding of Alzheimer's disease, stroke and vesicle trafficking disorders.

DESCRIPTION (provided by applicant): Synaptic vesicle exocytosis is a highly specialized vesicle trafficking process in which calcium triggers fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release. SNARE complex assembly between synaptobrevin, SNAP-25 and syntaxin is a critical requirement preceding this vesicle fusion event. Several SNARE-interacting proteins have been shown to profoundly influence the strength of synaptic transmission, through their regulatory effects on the SNARE complex. Recently, a new SNARE binding partner, tomosyn was isolated from rat brain cytosol. Tomosyn has a SNARE binding domain that can compete with synaptobrevin for assembly into a tomosyn SNARE complex with syntaxin and SNAP-25. Based on these biochemical observations as well as tomosyn overexpression data, tomosyn is proposed to regulate vesicle release through an undefined mechanism. There are presently no loss-of-functions mutants available in any organism other than C. elegans. Therefore, we intend to examine the mechanism of tomosyn action at synapses in this powerful genetic model organism. Aim 1) Characterize the synaptic phenotype of tom-1 deletion mutants. We have obtained two tom-1 deletion mutants that have phenotypes consistent with increased synaptic transmission. We will conduct a detailed characterization of these tom-1 mutants including behavioral, cytoarchitectural, pharmacological, electrophysiological and ultrastructural analyses. Aim 2) Determine which TOM-1 isoforms regulate synaptic transmission. C. elegans tom-1 encodes three isoforms. The isoform expression patterns will be ascertained and mosaic analysis and tissue specific rescue experiments will be performed. Aim 3) Genetic analysis of TOM-1 function. We hypothesize that tomosyn regulates the priming step of exocytosis. To test this model we will generate and characterize double mutants between tom-1 and several mutants known to affect the vesicle primed pool (unc-13, unc-10, open-syntaxin and unc-18). Aim 4) Identify TOM-1 domains required for the regulation of synaptic transmission. TOM-1 protein domains essential for the regulation of exocytosis will be identified using a genetic screen for mutants that fail to complement the tom-1 mutation. These experiments are likely to further our understanding of neurotransmission, a foundation that may contribute to our understanding of neurological diseases and vesicle trafficking disorders. PUBLIC HEALTH RELEVANCE: Information flow within nervous systems occurs via specialized cell-cell contacts called 'synapses'. The mechanisms controlling information flow through synapses are incompletely understood. Here we propose supplemental work to study a protein called 'tomosyn', which our previous work, as well as work from other labs, has shown is an important regulator of synapse function. Specifically, our supplemental work will extend our previously funded work from worms into another genetic model organism, the fruitfly (Drosophila melanogaster), where we can probe the mechanism by which tomosyn works in more detail, and explore whether tomosyn plays a role in learning. A molecular understanding of tomosyn could contribute to treatments for a variety of neurological and learning disorders.

DESCRIPTION (provided by applicant): Synaptic vesicle exocytosis is a highly specialized vesicle trafficking process in which calcium triggers fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release. SNARE complex assembly between synaptobrevin, SNAP-25 and syntaxin is a critical requirement preceeding this vesicle fusion event. Several SNARE-interacting proteins, have been shown to profoundly influence the strength of synaptic transmssion, through their regulatory effects on the SNARE complex. Recently, a new SNARE binding partner, tomosyn was isolated from rat brain cytosol. Tomosyn has a SNARE binding domain that can compete with synaptobrevin for assembly into a tomosyn SNARE complex with syntaxin and SNAP-25. Based on these biochemical observations as well as tomosyn overexpression data, tomosyn is proposed to regulate vesicle release through an undefined mechanism. There are presently no loss-of-functions mutants available in any organism other than C. elegans. Therefore, we intend to examine the mechanism of tomosyn action at synapses in this powerful genetic model organism. Aim 1) Characterize the synaptic phenotype of tom-1 deletion mutants. We have obtained two tom-1 deletion mutants that have phenotypes consistent with increased synaptic transmission. We will conduct a detailed characterization of these tom-1 mutants including behavioral, cytoarchitectural, pharmacological, electophysiological and ultrastructural analyses. Aim 2) Determine which TOM-1 isoforms regulate synaptic transmission. C. elegans tom-1 encodes three isoforms. The isoform expression patterns will be ascertained and mosaic analysis and tissue specific rescue experiments will be performed. Aim 3) Genetic analysis of TOM-1 function. We hyptheisize that tomosyn regulates the priming step of exocytosis. To test this model we will generate and characterize double mutants between tom-1 and several mutants known to effect the vesicle primed pool (unc-13, unc- 10, open-syntaxin and unc-18). Aim 4) Identify TOM-1 domains required for the regulation of synaptic transmission. TOM-1 protein domains essential for the regulation of exocytosis will be identified using a genetic screen for mutants that fail to complement the tom-1 mutation. These experiments are likely to further our understanding of neurotransmission, a foundation that may contribute to our understanding of neurological diseases and vesicle trafficking disorders.

DESCRIPTION (provided by applicant): Developing neural circuits are actively remodeled as synapses are created in new locations and dismantled in others. These dynamic events are regulated by neuronal activity to produce mature circuits with specific physiological functions. This phenomenon has been observed throughout animal phylogeny which suggests that the underlying pathways are conserved. However, the molecular mechanisms that drive synaptic remodeling are largely unknown. Here we propose a strategy that exploits the simple model organism, C. elegans, to define a development program that remodels the synaptic architecture of a GABAergic circuit. Ventral synapses for DD class GABA neurons are relocated to new sites on the dorsal side during larval development. This synaptic remodeling program is blocked by the UNC-55/COUP-TF transcription factor in VD motor neurons which normally synapse with ventral muscles. We exploited this UNC- 55 function in a powerful cell-specific profiling strategy that identified 19 conserved genes with roles in synaptic remodeling. We have now shown that one of these UNC-55 targets, the DEG/ENaC cation channel, UNC-8, promotes synaptic remodeling in a mechanism that is activated by GABAergic signaling. This finding is important because DEG/ENaC proteins have been implicated in learning and memory but the mechanism that links DEG/ENaC function to synaptic plasticity is poorly understood. Specific Aim 1 tests the key prediction that UNC-8 is closely associated with GABAergic synapses that are remodeled by UNC-8 activity. Specific Aim 2 is designed to test the novel hypothesis that a Ca2+-dependent mechanism links neural activity to UNC-8 cation transport in a feedback loop that dismantles the presynaptic machinery. Specific Aim 3 defines the cellular origin and molecular components of the proposed activity-dependent pathway that regulates UNC-8 and promotes GABAergic synaptic remodeling. Together, these approaches offer a powerful opportunity to delineate an intricate molecular pathway that controls synaptic plasticity. Moreover, the conservation of these remodeling components in mammals argues that the results of this work are likely to reveal fundamental mechanisms that regulate synaptic plasticity in the human brain.

Developing neural circuits are actively remodeled as synapses are created in new locations and dismantled in others. These dynamic changes are driven by the combined effects of genetic programs and neural activity that together shape the architecture and function of mature circuits. Synaptic plasticity has been observed throughout animal phylogeny which suggests that the underlying pathways are conserved and thus can be investigated in simple model organisms that are amenable to experimental analysis. Here we propose to use the nematode, C. elegans, to define a development program that remodels the synaptic architecture of a GABAergic circuit. During early larval development, DD-class GABAergic neurons undergo a dramatic remodeling program in which the presynaptic apparatus exchanges locations with postsynaptic components within the DD neuronal process. To reveal the mechanism of this effect, we are investigating the functional roles of ~20 conserved genes that we have determined are transcriptionally regulated to drive GABA neuron remodeling. Our work has shown that two of these targets, the DEG/ENaC cation channel protein, UNC-8, and ARX-5/p21, a conserved component of the Arp2/3 complex, function together in an activity-dependent mechanism that dismantles the presynaptic domain. Aim 1 tests the hypothesis that UNC-8 cation transport elevates intracellular calcium to drive presynaptic disassembly and that this effect is regulated by calcium- dependent phosphorylation. This goal is important because members of the DEG/ENaC protein family have been implicated in learning and memory but the mechanism that links DEG/ENaC function to synaptic plasticity is poorly understood. Aim 2 tests the hypothesis that the UNC-8 function triggers an actin-dependent endocytic mechanism that recycles presynaptic components for reassembly at new locations. These experiments derive from our surprising discovery that a key functional protein of the Arp2/3 actin-branching complex is transcriptionally regulated to effect synapse removal and that newly identified components of an endocytic recycling pathway are involved. Together, these approaches offer a powerful opportunity to delineate intricate molecular pathways that link neural activity to genetic programming in the execution of a synaptic remodeling mechanism. Moreover, the conservation of C. elegans remodeling components in mammals argues that this work is likely to reveal fundamental mechanisms that regulate synaptic plasticity in the human brain.