Transport and regulation of presynaptic assemblies

Neurological Disorders And Stroke

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Abstract

Our research program has been focused on the molecular and cellular mechanisms underlying: (1) the transport of synaptic components and organelles to mature and developing synapses and (2) the regulation of synaptic vesicle exocytosis. Using a combination of state-of-the-art live cell imaging, biochemistry, cell biology, and electrophysiology, we have identified three novel proteins named Snapin, syntaphilin, and syntabulin. With the generation of knockout mice, the physiological roles of Snapin in the regulation of synaptic vesicles exocytosis and of syntaphilin in controlling the motility of axonal mitochondria are being revealed. Using live cell imaging combined with loss-of-function approaches we are conducting a critical assessment of syntabulin's role in the dynamic trafficking of presynaptic components. Our studies provide a foundation for further studying the biological mechanisms underlying neuronal transport and neuroexocytosis. [unreadable] [unreadable] The role of syntabulin in the KIF5-mediated trafficking of synaptic components: [unreadable] The formation of new synapses or the remodeling of existing synapses requires the targeted delivery of synaptic components to the sites of axo-dendritic contact, a process that begins with the transport of synaptic carrier vesicles along the secretory pathway. Cargo vesicles and organelles must be attached to their transport motors with a high degree of specificity to preserve cargo/organelle identity and targeted trafficking. However, the mechanism underlying presynaptic cargo-motor interactions remains unresolved. [unreadable] We identified a novel syntaxin-binding and kinesin-1 motor (KIF5)-associated protein named syntabulin. Our studies with syntabulin loss-of-function analysis combined with live cell time-lapse imaging suggest that syntaxin-1 acts as a receptor of the cargo vesicles and syntabulin is a candidate adaptor capable of conjoining the KIF5 motor with the syntaxin-1 transport cargos, thus contributing to the trafficking and proper distribution of syntaxin along neuronal processes. We further demonstrated that syntaxin-1, syntabulin, and KIF5 are the molecular components of transport machinery critical for the axonal trafficking of presynaptic components. Syntabulin loss-of-function impairs the motility of the presynaptic cargo vesicles, reduces the axonal densities of both synaptic vesicle clusters and the activity-dependent loading of FM4-64 dye, and results in a marked reduction in the evoked synaptic transmission and the frequency of asynchronous quantal events in the hippocampal synapses. Our study suggests that syntabulin mediates the trafficking of synaptic components, thus contributing to the assembly of presynaptic terminals. [unreadable] In addition to its role in the trafficking of the syntaxin-containing cargos, the syntabulin-KIF5 complex is also an important component of the mitochondrial transport machinery. A significant portion of syntabulin associates with mitochondria via its carboxyl terminal tail. Syntabulin loss-of-function impairs anterograde but not retrograde transport of mitochondria along the neuronal processes of hippocampal neurons, resulting in a clustering of mitochondria in the soma. This provides evidence that syntabulin functions as an adaptor involved in KIF5-mediated trafficking of both syntaxin-containing cargos and mitochondria. As the efficient control of t-SNAREs and mitochondrial distribution at synapses is critical for proper synaptic function, our findings provide foundation to elucidate the mechanisms underlying axonal trafficking of synaptic components for the assembly of synapses and the modulation of synaptic transmission. [unreadable] [unreadable] The role of Snapin in the modulation of synaptic vesicle exocytosis: [unreadable] In neurons and neurosecretory cells, the fusion of synaptic vesicles and large dense-core vesicles (LDCVs) with the plasma membrane via exocytosis results in the release of neurotransmitters. Ca2+-triggered exocytosis depends on the presence of a pool of primed release-ready vesicles. The priming step corresponds to the assembly of the SNARE complex, in which the vesicle-associated VAMP interacts with plasma membrane-associated SNAP-25 and syntaxin-1. Maturation into a release-ready vesicle requires synaptotagmin-I, which provides Ca2+-dependent regulation of the fusion machinery. New evidence has emerged indicating that the interaction of synaptotagmin with SNAP-25 or the SNARE complex is critical for vesicle release and provides a clue as to how a calcium sensor is structurally and functionally coupled to the SNARE-based fusion machinery. However, the mechanisms underlying the regulation of this coupling during the vesicle priming remain unclear. [unreadable] Snapin was first identified in our lab as a SNAP-25 and synaptotagmin binding protein that enhances the association of synaptotagmin with the SNARE complex. The physiological role of Snapin in regulatory exocytosis was examined by microinjection of Snapin into presynaptic SCGNs in culture, and by overexpression of Snapin in both adrenal chromaffin cells and hippocampal neurons. To critically address the specific role of Snapin in exocytosis, we recently generated snapin knockout mice. The deletion of snapin leads to a marked reduction in the amount of the synaptotagmin-SNARE complex, which is consistent with our previous findings that recombinant Snapin enhances the interaction of synaptotagmin and SNAP-25 in vitro. Exocytosis of LDCVs in snapin (-/-) chromaffin cells displayed a selective reduction in the exocytotic burst without a change in the sustained component of release, suggesting a reduction in the pool size of release-ready vesicles. This inhibitory effect could be fully rescued by expressing a wild-type snapin transgene in the mutant cells. These studies using snapin KO mice in combination with genetic rescue experiments suggest that Snapin is an important modulator for vesicle exocytosis, possibly by stabilizing the structural coupling of synaptotagmin with the SNARE complex. [unreadable] [unreadable] The role of syntaphilin in the control of axonal mitochondrial motility: [unreadable] The proper transport and distribution of mitochondria in axons and at synapses is critical to neurotransmission and synaptic plasticity. Mitochondria in axons display distinct motility patterns and undergo saltatory bidirectional movements. While approximately one-third of axonal mitochondria are mobile with variable speed and direction, a large proportion remain in a prolonged stationary phase in coordination with axonal physiology. However, the mechanisms that mediate the stationary docking of mitochondria in axons and at synapses remain elusive. [unreadable] Syntaphilin (SNPH) is a neuron-specific and axon-targeted protein first identified in our lab as a syntaxin-binding protein. Our effort in generating SNPH KO mice has led to the discovery of a novel role for SNPH in the control of axonal mitochondrial motility. SNPH is required for maintaining a large portion of axonal mitochondria in a stationary state through an interaction with the microtubule-based cytoskeleton. Axonal mitochondria that contain either exogenously and endogenously expressed SNPH lose their motility, revealing a binomial distribution with a correlation between the SNPH-tagged mitochondria and stationary mitochondria. The microtubule-binding domain of SNPH is required for mitochondrial immobilization through an interaction with microtubules. Targeted deletion of the SNPH gene in mice markedly increases mitochondrial motility along axonal processes and consequently results in a reduction in the density of mitochondria within axons and at presynaptic terminals. Our studies reveal that SNPH is capable of immobilizing axonal mitochondria, thus providing a molecular explanation for complex behavior of mitochondria in axon.