SNAP-25 EXPRESSION AND HYPERACTIVITY IN COLOBOMA MICE

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The regulated expression of presynaptic nerve terminal proteins is critical both in developing the circuitry and in controlling neurotransmitter signaling that underlies nervous system function and ultimately behavior. The long-term goal is to define the molecular mechanisms governing the regulation and interactions between these synaptic proteins, and how this contributes to the diversity and plasticity of normal synaptic transmission, and how dysregulation of these processes leads to neurophysiological deficits. Specifically, this investigation addresses the function of SNAP-25, a protein that plays a key role in vesicle docking and regulated exocytosis of neurotransmitters. The mouse mutant, coloboma, bearing a contiguous gene defect and deficient in SNAP-25 expression, implicates dysregulation of SNAP-25 in spontaneous hyperactivity, and with delayed neurobehavorial development, deficits in learning and memory, and at the cellular level in abnormal hippocampal physiology and neurotransmitter release. Mutations affecting SNAP-25 expression may serve as effective models of hyperkinesis, a prominent component of Attention Deficit Hyperactivity Disorder, Tourette syndrome and other neurophysiological disorders. The proposed studies will test the hypothesis that SNAP-25 is involved in these abnormalities, and that two developmentally regulated isoforms of the protein have specialized roles that contribute differently to neural development and mature physiology of synaptic transmission. Towards this goal an integrated approach incorporating the following Specific Aims is proposed: 1) to determine if the phenotypic effects ascribed to the coloboma mutation are specific to SNAP-25. These studies will use Snap gene "rescued" and homologous recombinant null mutants to characterize deficits in neurobehavioral development and learning, in hippocampal electrophysiology, including long-term potentiation and theta EEG activity, and in transmitter release using in vitro synaptosomal preparations. 2) to determine the molecular specificity of SNAP-25 isoforms. Experiments using yeast expression systems and in vitro protein binding assays will characterize the differential interactions of SNAP- 25a and b isoforms with syntaxin and other presynaptic proteins involved in regulated vesicular exocytosis. 3) to define the specific role of the SNAP-25b isoform in neurotransmission. SNAP-25b will be over-expressed in neural cell lines deficient in this isoform, and the cells will be assayed for synaptic vesicle cycling and acetylcholine release as indexes of SNAP-25b function in synaptic transmission. 4) to establish the function of SNAP-25b in the intact nervous system, a homologous recombination strategy will be used to generate mutant mice that are limited to SNAP-25a isoform expression. Analysis of these partial loss- of-function mutants at behaviorial, electrophysiological and neurochemical levels will determine if deficits in mature neurophysiology and behavior result from deficiencies in specialized functions of SNAP- 25b. Through these studies, a better understanding of the molecular processes of neurotransmission will be achieved and, importantly, novel well-defined animal models will be established for the design and characterization of therapeutics targeted to hyperactivity in human neuropsychiatric disorders.