Transport and regulation of presynaptic assemblies

Neurological Disorders And Stroke

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Abstract

Synaptic function comprises the seamless integration of transport, assembly, and regulation of protein components from which synaptic transmission and higher brain physiology can arise. We are focusing our investigation on two molecular and cellular frontiers of synaptic function: the transport mechanisms by which mobile cargo vesicles containing synaptic components link to motor proteins for delivery to synapses, and how regulation of specific assembly of release machinery may govern synaptic vesicle priming and exocytosis. To this end, using a multi-disciplinary approach, we have been systematically identifying the molecules and regulatory pathways involved in both processes: the transport of synaptic components to nerve terminals and the priming of docked vesicles for regulated exocytosis. Snapin was first identified in our laboratory as a SNAP-25 binding protein that associates with the SNARE complex and enhances the association of synaptotagmin with the SNARE complex. Our studies in chromaffin cells cultured from snapin knockout mice showed that deletion of snapin resulted in a depletion of a readily-releasable pool of vesicles, and that this inhibitory effect could be fully rescued by overexpression in the mutant cells of a wile-type transgene, indicating that Snapin is an important modulator for priming of exocytosis. Identification of proteins that regulate synaptic vesicle priming and exocytosis are critical for understanding the mechanisms underlying calcium-dependent neurotransmitter release. Our biochemical and electrophysiological analysis of snapin knockout mice suggests that Snapin plays a critical role in neurosecretion in chromaffin cells by stabilizing release-ready vesicles and contributing to the structural coupling of synaptotagmin, a calcium sensor, with the SNARE protein complex. However, a number of key questions remain to be addressed. How does Snapin regulate the interaction of synaptotagmin with the SNARE complex? Does Snapin play a similar role in synaptic vesicle priming at CNS synapses? Does Snapin participate in general intracellular membrane trafficking by promoting interactions of other synaptotagmins with non-neuronal SNARE complexes? Addressing these questions by further analyzing snapin KO mice will be the focus of our present research. Syntabulin is another protein identified in our lab, which binds to syntaxin-1, a SNARE protein critical for synaptic vesicle priming and exocytosis. Combining live cell imaging with loss-of-function approaches employing siRNA and dominant negative transgenes, we demonstrate that syntabulin is a cargo adaptor protein linking syntaxin-cargo vesicles to the Kinesin family motor protein KIF5, and further show that this interaction is essential for the anterograde transport of syntaxin-1 into and along neuronal processes. The neuronal transport represents the intracellular trafficking route for proteins and organelles involved in synaptic transmission and plasticity. We propose to further characterize the role of syntabulin in synaptic transport via the secretory trafficking pathway and trans-Golgi-network (TGN)-derived carriers that together contribute to two key processes of developmental plasticity (1) synaptogenesis and (2) neuritogenesis. We believe the continued application of protein interaction studies, somatic cell genetics, and live cell imaging in combination with a multi-disciplinary analysis of genetically engineered mice will allow us to unravel the molecular mechanisms of microtubule-based transport of synaptic components to synapses.