Specific Aim 1. Retrograde transport regulates autophagy-lysosomal function. Maintaining cellular homeostasis in neurons depends on efficient intracellular transport. Late endocytic trafficking, which delivers target materials into lysosomes, is critical for maintaining efficient degradation capacities via autophagy-lysosomal pathways. However, the mechanisms regulating the autophagy-lysosomal system in neurons remain incompletely understood. Dynein-mediated retrograde transport can enhance late endocytic trafficking from distal processes to the soma, where lysosomes are predominantly localized, and drive late endosomes and lysosomes close enough to fuse with higher efficiency, thus ensuring proper autophagy-lysosomal function. Our recent study uncovered a critical role for Snapin in regulating late endocytic transport and membrane trafficking (Cai et al., Neuron 2010). Snapin acts as a motor adaptor by attaching dynein to late endosomes. Snapin (-/-) neurons exhibit aberrant accumulation of immature lysosomes, impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis, and impaired clearance of autolysosomes. Snapin deficiency leads to reduced neuron viability, axonal degeneration and developmental defects in the central nervous system. Reintroducing the snapin transgene rescues these phenotypes by enhancing the delivery of endosomal cargos to lysosomes and by facilitating autophagy-lysosomal function. Our study suggests that Snapin is a candidate molecular target for autophagy-lysosomal regulation. Our studies elucidate a new mechanism that regulates neuronal autophagy-lysosomal function by coordinating two dynamic cellular processes: dynein-mediated late endocytic transport and endosomal-lysosomal membrane trafficking. Such a mechanism is critical for cellular homeostasis and essential for neuronal survival and development. Snapin-deficient mice provide an important genetic tool for characterizing the role of impaired autophagy-lysosomal function in the clearance of aggregated proteins and dysfunctional organelles during neurodegeneratio (Cai and Sheng, Autophagy 2011;Zhou et al., 2011).
Specific Aim 2. Mechanisms regulating neurotrophin retrograde signaling. The neurotrophic signaling pathway from axonal terminals to cell bodies is crucial for dendrite growth and neuron survival. BDNF is one of the well-studied neurotrophic factors regulating dendrite outgrowth and branching. By binding to its receptor TrkB, BDNF triggers the internalization of ligand-receptor complexes into signaling endosomes, and activates signal transduction cascades, ultimately leading to retrograde signaling in the nucleus. While signaling endosome hypothesis is one of accepted models, the molecular machinery that drives retrograde axonal transport of BDNF-TrkB signaling endosomes is largely unknown. It also remains unclear whether retrograde axonal transport of BDNF-TrkB signaling endosomes has a direct impact on dendritic growth in CNS. Dynein motors are responsible for retrograde transport. However, mechanisms recruiting dynein to TrkB signaling endosomes have not been elucidated. In particular, a long-standing question is how BDNF-TrkB signaling complexes are delivered from axonal terminals to cell bodies. Our recent study provides mechanistic insights into the motor-adaptor machinery that drives the retrograde transport of TrkB signaling endosomes. Snapin acts as an adaptor recruiting the dynein motor to TrkB signaling endosomes via binding to DIC. Snapin-mediated and dynein-driven retrograde transport is essential to delivery activated BDNF-TrkB signaling complexes from axonal terminals to somas, and thus, is critical for dendritic growth of cortical neurons in vitro. Such a mechanism enables neurons to maintain an efficient response to neurotrophic factors at distal terminals. In snapin-deficient neurons, the recruitment of dynein motors to TrkB signaling endosomes was impaired resulting in the following three major defects: (1) fewer TrkB endosomes were transported from distal processes toward the soma;(2) the efficacy of BDNF-induced retrograde signaling in the nucleus was reduced;and (3) dendritic growth of cortical neurons was decreased. These phenotypes could be effectively rescued by reintroducing transgene expressing Snapin but not its mutant defective in DIC binding. The proposed Snapin- DIC mechanism was further tested in wild-type neurons expressing dominant-negative Snapin mutant. Disrupting Snapin-DIC interaction immobilizes TrkB signaling endosomes and reduces dendrite growth of cortical neurons in culture. Therefore, Snapin-DIC coupling is one of the primary mechanisms mediating BDNF-TrkB retrograde signaling in cortical neurons, thus providing new mechanistic insights into the regulation of neuronal growth and survival (Zhou et al., Cell Reports, 2012).
Specific Aim 3. Anterograde axonal transport regulates synaptic formation and plasticity. The formation of new synapses and remodeling of existing synapses play an important role in the various forms of synaptic plasticity and require the targeted delivery of newly synthesized synaptic components from the soma to the synaptic terminals. Thus, efficient axonal transport of newly synthesized synaptic components to nascent presynaptic boutons is critical in response to neuronal activity. However, the molecular identities of the motor-adaptor complex essential for assembling presynaptic terminals in developing neurons and in remodeling synapses of mature neurons in response to neuronal activity remain unknown. Our previous studies established that syntabulin is an adaptor capable of linking KIF5 motor and synaptic protein cargoes (Su et al., Nature Cell Biology, 2004). Syntabulin-KIF5 mediates axonal transport of synaptic components essential for presynaptic assembly. Syntabulin loss-of-function blocks formation of new presynaptic boutons in developing neurons. Our studies establish that kinesin-mediated anterograde axonal transport is another critical factor in the cellular mechanism underlying activity-dependent presynaptic plasticity (Cai et al., J Neuroscience 2007). Our recent study further demonstrated the critical role of syntabulin in the maintenance of presynaptic function and regulation of synaptic plasticity in well-matured sympathetic SCG neurons (Ma et al., J Neuroscience 2009). Conditional syntabulin knockout mice have been recently generated in the lab. We will use this mouse line to (1) determine whether deficiency in syntabulin/KIF5-mediated transport has any impact on synapse maintenance and plasticity in mature neurons and adult mice;(2) determine whether the motor-adaptor complex regulates the transport in response to synaptic activity;(3) identify the sorting signals for the axon-targeted delivery of presynaptic cargo. In summary, our study provides new mechanistic insights into (1) how Snapin regulates retrograde axonal transport of late endosomal-lysosomal organelles and neurotrophin signaling endosomes;(2) how syntabulin mediates anterograde transport of presynaptic proteins for synaptic maintenance and plasticity. Our snapin and syntabulin mouse models provide us with unique genetic tools for characterizing the roles of both anterograde and retrograde axonal transport in neurodevelopment and neurodegeneration. These studies will provide genetic evidence as to whether manipulating axonal transport will reduce axonal degeneration, thereby ultimately leading to new therapeutic approaches. Pursuing these investigations will advance our knowledge of fundamental processes that may affect human neurological disorders and is thus the very essence of the mission of the National Institute of Neurological Disorders and Stroke.

Project Start
Project End
Budget Start
Budget End
Support Year
17
Fiscal Year
2013
Total Cost
$1,208,886
Indirect Cost
City
State
Country
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Sheng, Zu-Hang (2014) Mitochondrial trafficking and anchoring in neurons: New insight and implications. J Cell Biol 204:1087-98
Ohno, Nobuhiko; Chiang, Hao; Mahad, Don J et al. (2014) Mitochondrial immobilization mediated by syntaphilin facilitates survival of demyelinated axons. Proc Natl Acad Sci U S A 111:9953-8
Sun, Tao; Qiao, Haifa; Pan, Ping-Yue et al. (2013) Motile axonal mitochondria contribute to the variability of presynaptic strength. Cell Rep 4:413-9
Lu, Li; Cai, Qian; Tian, Jin-Hua et al. (2009) Snapin associates with late endocytic compartments and interacts with late endosomal SNAREs. Biosci Rep 29:261-9
Pan, Ping-Yue; Tian, Jin-Hua; Sheng, Zu-Hang (2009) Snapin facilitates the synchronization of synaptic vesicle fusion. Neuron 61:412-24
Lu, Wenbo; Ma, Huan; Sheng, Zu-Hang et al. (2009) Dynamin and activity regulate synaptic vesicle recycling in sympathetic neurons. J Biol Chem 284:1930-7
Cai, Qian; Sheng, Zu-Hang (2009) Molecular motors and synaptic assembly. Neuroscientist 15:78-89
Leenders, A G Miriam; Lin, Lin; Huang, Li-Dong et al. (2008) The role of MAP1A light chain 2 in synaptic surface retention of Cav2.2 channels in hippocampal neurons. J Neurosci 28:11333-46