Sandra M. Bajjalieh - US grants

THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OR AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEND ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE ABSTRACT BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. DESCRIPTION: The identification of the molecular events producing and regulating neurotransmitter release is a central goal of neurobiology. An understanding of this phenomenon is crucial to several applied fields of neuroscience, including the study of learning and memory and the etiology of nervous system pathologies. The release of neurotransmitters is the summation of many events, each of which is a potential site of regulation. The larger goal of Dr. Bajjalieh's laboratory is to identify the proteins that mediate neurosecretion, their activity, and how their action is regulated. The work proposed here addresses the function of Synaptic Vesicle Protein 2 (SV2), a component of all synaptic vesicles. Sequence analyses indicate that SV2 belongs to a family of transporter proteins. Based on this similarity and analyses of SV2 isoform expression, it has been hypothesized that SV2 serves as a transporter of a common vesicle constituent, as a plasma membrane sugar transporter, or as a fusion pore. Early attempts to identify the action of SV2 by antibody neutralization and expression in non-neuronal cells have been inconclusive. The activity of SV2 remains unknown. The following studies are designed to identify proteins and post-translational modifications required for SV2 activity and to test hypotheses of its function. These studies will ask: 1) What proteins interact with SV2? The identification of associated proteins is often a first step in determining a protein's function. These studies will provide information on SV2's action by identifying co-factors and regulators. 2) What kinases and phosphatases act on SV2? Preliminary experiments indicate that SV2 is phosphorylated in nerve terminals and that its phosphorylation may be regulated by synaptic activity. These studies will provide information on the regulation of SV2 that may be crucial to a direct demonstration of its activity. 3) How does depletion of SV2 expression affect secretion in cultured neuroendocrine cells? Stable cell lines which express low levels of SV2 will be generated. These cell lines will be used to test several hypotheses of SV2 function.

DESCRIPTION Calcium-regulated secretion of neurotransmitters is the primary mode of communication in the nervous system. Neurotransmitter secretion is mediated by a unique organelle, the synaptic vesicle, which undergoes repetitive rounds of filling, targeting, priming, exocytosis and endocytosis in the presynaptic terminal. Several of these stages are accomplished via the sequential formation and dissociation of protein complexes. Identifying the protein complexes involved, determining where in the vesicle cycle each complex acts and describing how calcium regulates their action is therefore central to understanding the molecular mechanisms of neurotransmitter release. The applicant and her students have identified a protein complex that contains the synaptic vesicle proteins SV2 and synaptotagmin. SV2 is a transporter-like membrane glycoprotein hypothesized to function either as a small molecule transporter or as a component of the fusion pore that initiates transmitter release. Synaptotagmin is a calcium-binding protein required for normal calcium-regulated exocytosis. The presence of these proteins in all synapses, together with the observation that calcium regulates their interaction, suggests that the SV2 synaptotagmin complex plays a crucial role in neurotransmitter secretion. To identify that role the applicant proposes four aims designed to determine the physiological function of the complex and how that function is regulated. 1. The applicant will determine the components of the SV2 synaptotagmin complex. 2. The applicant will test the effect of disruption of SV2-synaptotagmin interaction on regulated secretion in PC12 cells. 3. The applicant will test whether all eight isoforms of synaptotagmin demonstrate calcium-dependent interactions with SV2. 4. The applicant will determine whether phosphorylation regulates the interaction of SV2 and synaptotagmin. These studies will provide basic information required to understand the changes in synaptic efficacy that underlie both learning and memory as well as multiple neuropathologies.

laboratory mouse; secretion

DESCRIPTION: (provided by the applicant) Chronic drug use leads to changes in neuronal functioning that ultimately result in addiction and tolerance. Like other forms of neuronal plasticity, drug-induced adaptations include modifications of synaptic efficacy. An understanding of synaptic functioning at a molecular level is therefore a crucial first step towards identifying the molecular changes that produce addiction and tolerance. Research into the molecular basis of synaptic transmission has focused largely on proteins of the synapse. However, as in other cellular processes, lipids also play a crucial role. The larger goal of this project is to elucidate the role of sphingolipids in neurotransmitter secretion, a role suggested by the presence of a ceramide (N-acyl sphingosine) kinase on synaptic vesicles. cDNAs encoding a ceramide kinase of any kind have yet to be identified. This Stage I project aims to 1) isolate cDNAs encoding brain ceramide kinase and 2) to identify the tissue expression patterns and intracellular location of brain ceramide kinase. This work will lay the foundation for biochemical and genetic studies of the role of ceramide kianse, its substrate and its product in neurotransmission and how changes in its activity contribute to synaptic plasticity.

DESCRIPTION (provided by applicant): The precise control of neurotransmitter release is mediated by proteins specific to synapses. One family of essential regulators is SV2, a family of transporter-like proteins that have emerged as a target of new therapies to treat nervous system disorders. Synaptic vesicles also contain a related protein, SVOP (SV2-like protein), about which very little is known. To study the role of SVOP in neurotransmission, we propose to establish mouse lines in which SVOP expression can be universally or selectively disrupted. These mice will be a crucial reagent in determining both how SVOP contributes to neuronal functioning and whether it holds promise in the development of new therapeutics. PUBLIC HEALTH RELEVANCE: This pilot project will generate a genetically modified mouse line that will be used to identify the function of SVOP, a transporter-like protein present on synaptic vesicles about which very little is known. SVOP has significant similarity to SV2, a small family of synaptic vesicle proteins that are essential regulators of neurotransmitter release. SV2A is the binding site of a new class of antiepileptic drugs that are also finding use in the treatment of other nervous system disorders including neuropathic pain and dyskinesias. The proposed mouse line will play a crucial role in vetting SVOP's potential as an alternate drug target.

DESCRIPTION (provided by applicant): In response to RFA-MH-12-140, Development of Tools to Explore the Synaptome, we propose to develop a procedure to trap and isolate recycling synaptic vesicles as an essential first step in analyzing how vesicle protein composition varies with changes in synaptic activity, drug treatment, and under pathological conditions. The ability of synaptic vesicles to undergo multiple rounds of fusion is crucial to maintaining fast, efficient neurotransmission. Synaptic vesicles are formed and recycle via endocytosis. At many synapses, a small proportion of the total vesicle population mediates the majority of neurotransmission while the larger portion remains untapped in a reserve pool. Therefore, proteomic analyses using traditional vesicle purification schemes cannot track changes in the protein composition of the recycling pool. To address this we will develop a scalable, readily applied purification protocol for recycling synaptic vesicles that relies on transiently trapping recycling vesicles as a means of separating them from the total vesicle pool. This approach will open a new field of investigation into the complex endocytotic machinery that regulates vesicle recycling. It will provide a means to test the effects of drugs, genetic manipulations, and pathological conditions on this very important process.

? DESCRIPTION (provided by applicant): Lipids are essential signaling molecules that regulate all aspects of cellular function. Unlike proteins, whose location can be tracked in situ with antibodies or expression of tagged fusion proteins, our ability to track signaling lipids in situ i still primitive and constitutes a major impediment to understanding their role in cell function. Ths is especially true for the signaling lipid ceramide 1- phosphate (C1P), which has been implicated in multiple cellular functions including transmitter exocytosis, inflammation, cell proliferation, protein function regulation, and cytoskeletal dynamics. To overcome this technological challenge we will use deep scanning mutagenesis of the C2 lipid-binding motif, combined with deep sequencing and sequence analysis algorithms, to generate proteins with specific, high-affinity binding to C1P. The resulting proteins will serve as the basis for fluorescent biosensors to track C1P in situ. In addition, the novel protein engineering platform we assemble will be a powerful new strategy for developing new sensors for other lipids.