Zu-Hang Sheng - US grants

Specific Aims: Syntaphilin (SNPH) is a neuron-specific and axon-targeted protein initially identified in our lab as a candidate inhibitor of presynaptic function (1). 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 (2). Our study reveals that SNPH is required for maintaining a large number of axonal mitochondria in a stationary state through an interaction with the microtubule (MT)-based cytoskeleton. Three lines of evidence support this view. First, the mitochondria that associated with exogenously expressed GFP-SNPH were almost entirely immobile. This occurs through interactions with the cytoskeleton because SNPH contains a MT-binding domain, which is necessary and sufficient for SNPH-mediated immobilization of axonal mitochondria. Second, by recording mitochondrial movement in living neurons followed by retrospective immunostaining for endogenous SNPH, we demonstrate that the immobility of axonal mitochondria depends on their association with endogenous SNPH, and further reveal a binomial distribution with a strong correlation between the endogenous SNPH-tagged mitochondria (62%) and stationary mitochondria (65%). Finally, deletion of the snph gene in mice resulted in a substantially higher proportion of axonal mitochondria in the mobile state than that found in wild-type neurons, and reduced the densities of total and inter-bouton mitochondria in axons. The snph mutant neurons exhibit enhanced short-term facilitation during prolonged stimulation, by affecting calcium signaling at presynaptic boutons. This phenotype is fully rescued by reintroducing the snph gene into the mutant neurons. Thus, SNPH acts as a static anchor for docking/retaining mitochondria in axons and at synapses. These findings reveal for the first time a neuron-specific protein capable of docking axonal mitochondria and regulating their densities within axons. Mitochondrial balance between the motile and stationary phases is a target for regulating mitochondrial redistribution. How are motile mitochondria recruited to the stationary pool? In particular, the mechanisms regulating SNPH-mediated mitochondrial anchoring at axons remain elusive. By applying a proteomic approach combined with time-lapse imaging in live snph (+/+) and (-/-) neurons, we revealed that dynein light chain LC8 enhances the docking efficiency by binding to SNPH (3). Four lines of evidence support this view. First, SNPH interacts with LC8 via its 7-residue motif (ERAIQTD);the SNPH-LC8 complex is detected by immunoprecipitation of brain homogenates;the interaction is independent of the dynein motor complex. Second, SNPH recruits LC8 to axonal mitochondria via its 7-residue LC8-binding motif. Deleting this motif reduces the SNPH capacity in docking axonal mitochondria. Third, elevated LC8 expression in snph (+/+) neurons inhibits the mobility of axonal mitochondria. In contrast, this effect is not observed in snph null neurons, suggesting that the role of LC8 is depending on its interaction with SNPH. Forthermore, CD spectrum analysis revealed that LC8 enhances docking by stabilizing an α−helical coiled-coil within the MT-binding domain of SNPH against thermal unfolding. Altogether, our studies provide new mechanistic insights into how SNPH and LC8 coordinately immobilize mitochondria through enhanced interaction of SNPH and MTs. In summary, using genetic mouse models combined with time-lapse imaging in live neurons, we elucidate molecular mechanism underlying the complex mobility patterns of axonal mitochondria. Such a mechanism enables neurons to maintain proper mitochondrial densities within axons and near synapses. We further provide the physiological evidence that the mobility and density of axonal mitochondria play a critical role in short-term synaptic plasticity. It is expected that defective mitochondrial docking/anchoring could affect neuronal functions. Dysfunction and defective trafficking of axonal mitochondria have been implicated in the pathologic processes of neurodegenerative diseases such as Alzheimers and Huntingtons and amyotrophic lateral sclerosis. The continued application of live cell imaging in combination with a multi-disciplinary analysis of genetically crossed mouse models will improve our understanding as how the changes in mitochondrial mobility affect axonal neurodegeneration. Papers published from the lab related to the project: 1. Guifang Lao, Volker Scheuss, Claudia M. Gerwin, Qingning Su, Sumiko Mochida, Jens Rettig, and Zu-Hang Sheng (2000). Neuron 25, 191-201. 2. Jian-Sheng Kang,Jin-Hua Tian, Philip Zald, Ping-Yue Pan, Cuiling Li, Chuxia Deng, and Zu-Hang Sheng. (2008). Cell 132, 137-148. 3. Yan-Min Chen, Claudia Gerwin, and Zu-Hang Sheng. (2009). Journal of Neuroscience 29, 9428-9437.

Specific Aim 1: Discovery of A New Pathway for the Activity-Dependent Plasticity through Axonal Transport Neuronal transport includes the intracellular trafficking route for membranous protein carriers from the soma to nerve terminals where they deliver cargos for synapse formation. The contents of these transport packets include protein components of SV and AZs, exocytotic machinery, channels, and adhesion molecules. Cargos must be attached to their transport motors with a high degree of specificity to preserve cargo identity and targeted trafficking. However, the mechanism underlying motor-cargo interactions remains unresolved. The SNARE protein syntaxin-1, a key component of SV fusion machinery, is transported to the plasma membrane in cargos. We identified a novel syntaxin-binding and kinesin-1 motor (KIF5)-associated protein named syntabulin (1, 2). Our studies with loss-of-function analysis established that syntabulin is an adaptor capable of conjoining syntaxin-1 and KIF5 motors, thereby mediating transport of syntaxin-1 to neuronal processes. Remodeling of pre-existing synapses and the formation of new synapses play an important role in the various forms of synaptic plasticity of complex neuronal networks. Previously identified mechanisms underlying activity-dependent synaptic plasticity include activation of transcriptional factors, new protein synthesis, and reorganization of the actin filaments at synapses. Thus, efficient and targeted axonal transport of newly synthesized synaptic components to presynaptic boutons would be critical in response to neuronal activity. However, the contribution of the microtubule-based axonal transport to the activity-induced formation of new synapses are unknown. Since syntaxin-1 is a component of the AZ precursor cargos, further characterization of syntabulins role in neuronal trafficking will contribute to understanding the molecular mechanisms of the axonal delivery of presynaptic components. Our ongoing research reveals that syntaxin-1, syntabulin, and KIF5 comprise the transport machinery critical for anterograde axonal transport of the AZ precursors and contributes to presynaptic assembly (3). Knockdown of syntabulin or disruption of the syntaxin-1-syntabulin-KIF5B complex impairs the anterograde transport of AZ components out of the soma and reduces the axonal densities of SV clusters and FM4-64 loading. Furthermore, syntabulin loss-of-function results in a reduction in both the amplitude of postsynaptic currents and the frequency of asynchronous quantal events, and abolishes the activity-induced recruitment of new AZ components into the axons and subsequent co-clustering with SVs. Consequently, syntabulin loss-of-function blocks the formation of new presynaptic boutons during activity-dependent synaptic plasticity. These studies establish for the first time that a kinesin motor-adaptor complex is critical for the anterograde axonal transport of AZ components, thus contributing to activity-dependent presynaptic assembly during neuronal development. To explore the function of syntabulin in vivo, we will generate the conditional KO mice with functional disruption of the syntabulin gene. Phenotype analysis of the KO mice and the mutant neurons will clarify the molecular details of how this protein mediates the trafficking of the AZ precursor vesicles essential for presynapse assembly and synaptic plasticity. Specific Aim 2: Identification of An Essential Role of Snapin in Synchronizing Fast Neurotransmitter Release Information coding in the brain depends on the timing of action potentials, which is influenced by integration of unitary excitatory inputs. The size and shape of excitatory postsynaptic currents (EPSCs) are two decisive factors in tuning the temporal and spatial precision of spiking and can be modulated by synaptic vesicle (SV) fusion process. Ca2+-triggered neurotransmitter release depends on the presence of a pool of primed release-ready SVs, which determines the release probability of a synapse. The priming step corresponds to assembly of the SNARE complex in which synaptobrevin interacts with SNAP-25 and syntaxin-1 to form a metastable structure before fusion. Maturation of SVs into a release-ready state requires synaptotagmin I (Syt I), a Ca2+ sensor of fast neurotransmission. Accurate assembly of Syt I-SNARE fusion machineries is critical for the precise timing of fast release. Ca2+-dependent and independent interactions between Syt I and SNAREs suggest that before the Ca2+ trigger, a loose pre-fusion Syt I-SNARE complex is assembled during the priming process. Ca2+ influx sensitizes the Ca2+ sensor Syt I and induces its subsequent tight coupling to the SNARE complex. While much attention in the past decade has been given to the SNARE-regulatory proteins in studying SV release probability and short-term plasticity, our understanding of the molecular mechanisms that govern the tuning of EPSC shape is largely lacking. We initially identified Snapin as a SNAP-25-binding protein that enhances the association of Syt I with the SNAREs (4-6). Using snapin knockout mice, we demonstrated that Snapin modulates fast exocytosis of large dense-core vesicles in chromaffin cells (7). Deletion of snapin leads to a reduced amount of Syt I-SNARE complex in mouse brain. We recently characterized the function of Snapin in synchronizing SV fusion at central synapses (8). By recording synaptic transmission between cultured cortical neurons from snapin-deficient mice, we found that snapin mutant neurons exhibit EPSCs with multiple peaks and fail to follow sustained firing under high-frequency stimulations. Re-introducing snapin into the mutant presynaptic neurons effectively accelerates EPSC kinetics to the greater extent found in (+/+) neurons by boosting the synchronicity of SV fusion. The marked increase in rise/decay time and synaptic delay time observed in snapin-deficient neurons changes the shape of the EPSC and impairs both synaptic efficacy and precision. At snapin-deficient nerve terminals, SVs are likely heterogeneously primed due to the unfavorable or unstable association of Syt I with the metastable SNARE complex before the Ca2+ sensing. It leads to two defects: (1) fewer fusion competent vesicles, and hence decreased size of EPSCs;and (2) fewer vesicles undergoing synchronized fusion within a narrow time window during excitation-secretion coupling. Thus, our studies reveal the role of Snapin as a unique synchronizer of calcium-triggered SV fusion at central synapses. Papers published from the lab related to the projects: 1. Qingning Su*, Qian Cai*, Claudia Gerwin, Carolyn L. Smith, Zu-Hang Sheng (2004). Nature Cell Biology 6, 941-953. 2. Qian Cai, Claudia Gerwin, and Zu-Hang Sheng. (2005). Journal of Cell Biology 170, 959-969. 3. Qian Cai, Pingyue Pan, and Zu-Hang Sheng. (2007). Journal of Neuroscience 27, 7284-7296. 4. Jeffrey M. Ilardi, Sumiko Mochida, and Zu-Hang Sheng (1999). Nature Neuroscience 2, 119-124. 5. Milan G. Chheda, Uri Ashery, Pratima Thakur, Jens Rettig, and Zu-Hang Sheng (2001). Nature Cell Biology 3, 331-338. 6. Pratima Thakur, David R. Stevens, Zu-Hang Sheng and Jens Rettig (2004), Journal of Neuroscience 24, 6476-6481. 7. Jin-Hua Tian, et al (2005). Journal of Neuroscience 25, 10546-10555. 8. Ping-Yue Pan, Jin-Hua Tian and Zu-Hang Sheng (2009). Neuron 61, 412-424.

Specific Aims: Syntaphilin (SNPH) is a neuron-specific and axon-targeted protein initially identified in our lab as a candidate inhibitor of presynaptic function (Lao et al., Neuron 2000). Our effort in generating SNPH KO mice has led to the discovery of a role for SNPH in the control of axonal mitochondrial motility (Kang et al., Cell 2008). Our study reveals that SNPH is required for maintaining a large number of axonal mitochondria in a stationary state through an interaction with the microtubule (MT)-based cytoskeleton. Deletion of the snph gene in mice results in a substantially higher proportion of axonal mitochondria in mobile state than that found in wild-type neurons, and reduced total and inter-bouton mitochondria density within axons. The snph mutant neurons exhibit enhanced short-term synaptic facilitation during prolonged stimulation by affecting calcium signaling at presynaptic boutons. This phenotype is fully rescued by reintroducing the snph gene into the mutant neurons. Thus, SNPH acts as a static anchor for docking/retaining mitochondria in axons and at synapses. These findings reveal for the first time a neuron-specific protein capable of docking axonal mitochondria within axons and near synapses. By applying a proteomic approach combined with time-lapse imaging, we further revealed that dynein light chain LC8 enhances the mitochondrial docking through its binding to SNPH (Chen et al., J Neuroscience 2009). SNPH recruits LC8 to axonal mitochondria via the 7-residue (ERAIQTD) LC8-binding motif and this interaction is independent of the dynein motor complex. Elevated LC8 expression in wild-type neurons inhibits axonal mitochondrial mobility. In contrast, this effect is not observed in snph-null neurons, suggesting the role of LC8 via its interaction with SNPH. CD spectrum analysis revealed that LC8 enhances mitochondrial docking by stabilizing an helix coiled-coil within the MT-binding domain of SNPH, thus providing new mechanistic insight into how SNPH and LC8 coordinately immobilize axonal mitochondria. Synaptic structure and function are highly plastic and undergo activity-dependent remodeling, thereby altering mitochondrial mobility and distribution. Axonal mitochondria exhibit the complex mobility pattern by coupling two opposing molecular motors kinesins and dynein and by attaching to docking/anchoring receptor SNPH. Identification of SNPH as a docking protein provides the molecular target for regulating the mobility and distribution of axonal mitochondria in response to neuronal activity. Our ongoing studies using the snph KO mouse will provide molecular details on how SNPH regulates mitochondrial transport and presynaptic function. In particular, our research will provide a molecular basis for addressing whether the motors and docking receptor share a single system of regulation or are modulated through distinct signal pathways. Mitochondrial dysfunction, altered mitochondrial dynamics and mobility, and perturbation of their turnover are involved in the pathology of several major neurodegenerative diseases including Parkinsons, Alzheimers, Huntingtons disease and amyotrophic lateral sclerosis (ALS). For example, ALS associated SOD1G93A and Huntingtons-linked Htt72Q transgenic mice display abnormal mitochondrial movement in neurons where mitochondria move more slowly, stop more frequently and travel shorter distances. However, whether defective mitochondrial transport plays a role in axonal degeneration remains largely unknown. Deleting the snph gene in mice recruits a majority of axonal mitochondria into an actively motile state. Our ongoing research is addressing (1) whether enhanced mobility of axonal mitochondria by crossing the snph (-/-) mice with the aforementioned diseased mouse lines contributes to efficient removal of dysfunctional mitochondria from synapses and distal axons and (2) whether enhanced mitochondrial mobility benefits the turnover of dysfunctional mitochondria via the mitophagy pathway or membrane fusion/fission dynamics. These studies will provide cellular and genetic clues as to whether manipulating mitochondrial transport and turnover may leads 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. Papers published from the lab related to the project: Guifang Lao, Volker Scheuss, Claudia M. Gerwin, Qingning Su, Sumiko Mochida, Jens Rettig, and Zu-Hang Sheng (2000). Neuron 25, 191-201. Qian Cai, Claudia Gerwin, and Zu-Hang Sheng (2005). Journal of Cell Biology 170, 959-969. Jian-Sheng Kang,Jin-Hua Tian, Philip Zald, Ping-Yue Pan, Cuiling Li, Chuxia Deng, and Zu-Hang Sheng (2008). Cell 132, 137-148. Yan-Min Chen, Claudia Gerwin, and Zu-Hang Sheng. (2009). Journal of Neuroscience 29, 9428-9437. Qian Cai, Zu-Hang Sheng (2009). Experimental Neurology 218, 257-267. Huan Ma, Qian Cai, Wenbo Lu, Zu-Hang Sheng (co-corresponding author), and Sumiko Mochida (2009). Journal of Neuroscience 29, 13019-13029. Qian Cai, Zu-Hang Sheng (2009). Neuron 61, 493-496.

Specific Aim 1. Mechanisms of Synchronized Synaptic Vesicle Release. Information coding in the brain depends on the timing of action potentials, which is influenced by integration of unitary excitatory inputs. The size and shape of excitatory postsynaptic currents (EPSCs) are two decisive factors in tuning the temporal and spatial precision of spiking and can be modulated by the SV fusion process. Our recent studies using the snapin-deficient cortical neurons combined with gene rescue experiments revealed a crucial role for Snapin in enhancing the efficacy of SV priming and in fine-tuning the precision of synchronous release by directly binding to synaptotagmin I (Pan et al., Neuron 2009). Snapin mutant neurons exhibit EPSCs with multiple peaks and fail to follow sustained firing under high-frequency stimulation. Re-introducing snapin into the mutant presynaptic neurons effectively accelerates EPSC kinetics by boosting the synchronicity of SV fusion. Thus, our studies reveal the role of Snapin as a unique synchronizer of SV fusion at central synapses. Several groups have independently reported an interaction between Snapin and dysbindin (BTNBP1)the product of a susceptibility gene found among the common genetic variations associated with schizophrenia. Our future studies aimed at (1) elucidating mechanisms of synchronized synaptic transmission;(2) determining whether Snapin acts in parallel or in a coordinated manner with other known priming proteins;and (3) evaluating Snapins role in the cognitive impairment prominent in schizophrenia. Specific Aim 2. Axonal Transport of Presynaptic Cargoes Essential for Synaptic 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 trans-Golgi network (TGN) in the soma to the synaptic terminals. Thus, efficient axonal transport of these newly synthesized components to nascent presynaptic boutons is critical in response to neuronal activity. Substantial evidence suggests that AZ precursor carriers are generated from TGN and traverse the developing axon to nascent synapses. Cargo vesicles must attach to their transport motors with a high degree of specificity to preserve cargo identity and targeted trafficking. 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 a motor adaptor capable of joining KIF5B and syntaxin-1 and enables syntaxin-1 transport to neuronal processes (Su et al., Nature Cell Biology, 2004). Using time-lapse imaging in live hippocampal neurons, we further demonstrate that the transport complex of syntaxin-1-syntabulin-KIF5B mediates axonal transport of the AZ components essential for presynaptic assembly. Syntabulin loss-of-function blocks formation of new presynaptic boutons during activity-dependent synaptic plasticity in developing neurons (Cai et al., J Neuroscience 2007). Our studies establish that kinesin-mediated and MT-based anterograde axonal transport is another critical factor in the cellular mechanism underlying activity-dependent presynaptic plasticity. Our recent study further demonstrated the critical role of syntabulin-mediated axonal transport in the maintenance of presynaptic function and regulation of synaptic plasticity in well-matured sympathetic SCG neurons in culture (Ma et al., J Neuroscience 2009). Our findings provide a molecular basis for future studies aimed at (1) determining whether the motor-adaptor complex regulates the transport rate in response to synaptic activity;(2) identifying the sorting signals for the axon-targeted delivery of the AZ cargo. Specific Aim 3. Regulation of Retrograde Transport and 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. An impaired autophagy-lysosomal system has been associated with the pathogenesis of neurodegenerative diseases. However, the mechanisms regulating the autophagy-lysosomal system in neurons remain incompletely understood. Dynein-mediated retrograde transport can enhance late endocytic trafficking by driving late endosomes and lysosomes close enough to fuse with higher efficiency, thus ensuring proper autophagy-lysosomal function. However, the mechanisms of coordinating these dynamic cellular processes remain unclear. In addition to its association with SVs, Snapin is present in cytosol and membrane-associated fractions in neuronal and non-neuronal cells and is co-purified with late endocytic organelles. Our recent study uncovered a critical role for Snapin in regulating late endocytic transport and membrane trafficking (Cai et al., Neuron in press). 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, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene. Thus, our study highlights new mechanistic insights into how Snapin-dynein coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function. Autophagy-lysosomal system is essential for quality control of intracellular components and maintenance of cellular homeostasis. Lysosomal dysfunction is one of the main cellular defects contributing to the pathogenesis of a range of neurodegenerative diseases associated with aggregation-prone intracytosolic proteins. The snapin KO mouse provides us with a unique genetic tool for characterizing the role of late endocytic transport in neurodegeneration. Papers published from the lab related to the projects: Qingning Su, Qian Cai, Claudia Gerwin, Carolyn L. Smith, Zu-Hang Sheng (2004). Nature Cell Biology 6, 941-953. Qian Cai, Claudia Gerwin, and Zu-Hang Sheng. (2005). Journal of Cell Biology 170, 959-969. Qian Cai, Pingyue Pan, and Zu-Hang Sheng. (2007). Journal of Neuroscience 27, 7284-7296. Jin-Hua Tian, Zheng-Xing Wu, Michael Unzicker, Li Lu, Qian Cai, Cuiling Li, Claudia Schirra, Ulf Matti, David Stevens, Chuxia Deng, Jens Rettig, and Zu-Hang Sheng (2005). Journal of Neuroscience 25, 10546-10555. Ping-Yue Pan, Jin-Hua Tian and Zu-Hang Sheng (2009). Neuron 61, 412-424. Qian Cai, Zu-Hang Sheng (2009). Neuroscientist 15, 78-89. Qian Cai, Li Lu, Jin-Hua Tian, Yi-Bing Zhu, Haifa Qiao, and Zu-Hang Sheng (2010). Neuron (in press).

Specific Aim 1. Regulation of Synaptic Vesicle Dynamics and Release. Information coding in the brain depends on the timing of action potentials, which is influenced by integration of unitary excitatory inputs. The size and shape of excitatory postsynaptic currents (EPSCs) are two decisive factors in tuning the temporal and spatial precision of spiking and can be modulated by the SV fusion process. Our recent studies using the snapin-deficient cortical neurons combined with gene rescue experiments revealed a crucial role for Snapin in enhancing the efficacy of SV priming and in fine-tuning the precision of synchronous release (Pan et al., Neuron 2009). Snapin mutant neurons exhibit EPSCs with multiple peaks and fail to follow sustained firing under high-frequency stimulation. Re-introducing snapin into the mutant presynaptic neurons effectively accelerates EPSC kinetics by boosting the synchronicity of SV fusion. Thus, our studies reveal the role of Snapin as a unique synchronizer of SV fusion at central synapses. Several groups have independently reported an interaction between Snapin and dysbindin (BTNBP1)the product of a susceptibility gene found among the common genetic variations associated with schizophrenia. Our recent study also identified that Snapin acts as dynein motor adaptor for late endosomes (Cai et al., Neuron 2010). This raises an interesting question whether Snapin regulates synaptic vesicle transport and dynamics at release sites, thus contributing to regulation of synaptic transmission. Our current studies aim at (1) elucidating mechanisms by which Snapin regulates synchronized synaptic transmission;(2) determining whether Snapin-dynein transport complex regulates synaptic vesicle density and dynamics at presynaptic terminals;and (3) evaluating Snapins role in the cognitive impairment prominent in schizophrenia. Specific Aim 2. Regulation of Presynaptic Cargo Transport and its Impact on Synaptic 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 trans-Golgi network (TGN) in the soma to the synaptic terminals. Thus, efficient axonal transport of these newly synthesized components to nascent presynaptic boutons is critical in response to neuronal activity. Substantial evidence suggests that AZ precursor carriers are generated from TGN and traverse the developing axon to nascent synapses. Cargo vesicles must attach to their transport motors with a high degree of specificity to preserve cargo identity and targeted trafficking. 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 a motor adaptor capable of joining KIF5B and syntaxin-1 and enables syntaxin-1 transport to neuronal processes (Su et al., Nature Cell Biology, 2004). Using time-lapse imaging in live hippocampal neurons, we further demonstrate that the transport complex of syntaxin-1-syntabulin-KIF5B mediates axonal transport of the AZ components essential for presynaptic assembly. Syntabulin loss-of-function blocks formation of new presynaptic boutons during activity-dependent synaptic plasticity in developing neurons (Cai et al., J Neuroscience 2007). Our studies establish that kinesin-mediated and MT-based anterograde axonal transport is another critical factor in the cellular mechanism underlying activity-dependent presynaptic plasticity. Our recent study further demonstrated the critical role of syntabulin-mediated axonal transport in the maintenance of presynaptic function and regulation of synaptic plasticity in well-matured sympathetic SCG neurons in culture (Ma et al., J Neuroscience 2009). Our findings provide a molecular basis for future studies. Conditional syntabulin knockout mice are in generation and we will use this genetic mouse line to (1) determine whether deficiency in syntabulin/KIF5-mediated anterograde axonal transport has any impact on synapse formation and maintenance, and synaptic plasticity;(2) determine whether the motor-adaptor complex regulates the transport rate in response to synaptic activity;(3) identify the sorting signals for the axon-targeted delivery of the AZ cargo. Specific Aim 3. Retrograde Transport and Impact on Neuronal 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. Autophagy-lysosomal system is essential for quality control of intracellular components and mitochondria, and maintenance of cellular homeostasis. An impaired autophagy-lysosomal system has been associated with the pathogenesis of several major neurodegenerative diseases. However, the mechanisms regulating the autophagy-lysosomal system in neurons remain incompletely understood. Dynein-mediated retrograde transport can enhance late endocytic trafficking to some, where lysosomes are predominantly localized, and drive late endosomes and lysosomes close enough to fuse with higher efficiency, thus ensuring proper autophagy-lysosomal function. In addition to its association with SVs, Snapin is present in cytosol and membrane-associated fractions in neuronal and non-neuronal cells and is co-purified with late endocytic organelles. 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, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene. Thus, our study highlights new mechanistic insights into how Snapin-dynein coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function. Our research goal is to identify the cellular pathways for clearance of aggregation-prone proteins by regulating the autophagy-lysosomal system. The snapin KO mouse provides us with a unique genetic tool for characterizing the role of late endocytic transport in neurodegeneration. The conditional snapin KO mice are generated and are being crossing with several disease mouse lines including mutant SOD1-linked ALS disease model. These studies will provide genetic evidence as to whether manipulating the late endocytic pathway will ultimately lead 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. Related publications from the lab: Qingning Su, Qian Cai, Claudia Gerwin, Carolyn L. Smith, Zu-Hang Sheng (2004) Syntabulin: a microtubule-associated protein implicated in syntaxin transport in neurons, Nature Cell Biology 6, 941-953. Qian Cai, Pingyue Pan, and Zu-Hang Sheng. (2007). Syntabulin-kinesin-1 family 5B-mediated axonal transport contributes to activity-dependent presynaptic assembly. Journal of Neuroscience 27, 7284-7296. Ping-Yue Pan, Jin-Hua Tian and Zu-Hang Sheng (2009). Snapin Facilitates the Synchronization of Synaptic Vesicle Fusion. Neuron 61, 412-424.

Our current hypothesis is that mitochondrial trafficking and distribution is regulated in order to sense, integrate, and respond to changes in metabolic and growth status, synaptic activity, and pathological stress. Specific Aims 1-3 are formulated to address three fundamental questions: (Aim 1) how axonal mitochondria are recruited to and captured at active synapses; (Aim 2) how SNPH turns its anchoring on or off by sensing local ATP levels, and how the energy signaling enables neurons distribute axonal mitochondria into areas where energy consumption is high during development and regeneration; and (Aim 3) how neurons maintain and recover stressed mitochondria prior to the activation of Parkin-mediated mitophagy under physiological and pathological conditions. Specific Aim 1. Elucidate mechanisms anchoring mitochondria at presynaptic terminals. Presynaptic activity imposes large energetic demands that are met by local ATP synthesis via glycolysis and oxidative phosphorylation. We propose that mitochondrial anchoring at presynaptic terminals is regulated to sense synaptic activity and is required to sustain synaptic transmission. Our previous study revealed SNPH as a static anchor that holds axonal mitochondria stationary via microtubule interactions (Kang et al., Cell 2008). Deleting snph dramatically increases axonal mitochondrial motility in vitro and in vivo. Using the snph knockout (KO) mouse model, we further revealed an engine-brake switch mechanism, by which KIF5-SNPH interaction is regulated in order to move or stop axonal mitochondria in response to synaptic activity (Chen and Sheng JCB 2013). We also provided mechanistic insights into how motile axonal mitochondria contribute to the pulse-to-pulse variability of presynaptic strength, the most notable feature of synaptic transmission in response to repeated stimulations (Sun et al., Cell Reports 2013). ATP production from presynaptic mitochondria is the main energy source to sustain synaptic transmission. However, the mechanisms anchoring mitochondria at presynaptic terminals remain elusive. There is a critical need to understand how mitochondria are recruited to and retained at active synapses. We are testing our hypothesis that mobile axonal mitochondria can be captured at presynaptic terminals via SNPH-mediated anchoring to presynaptic actin filaments. This anchoring may ensure mitochondrial docking in response to change in synaptic activity and energy requirement by triggering energy sensing pathway to turn on and off the SNPH-actin anchoring mechanism. Specific Aim 2. Elucidate mechanisms linking energy sensing and mitochondrial transport to support neuronal growth and regeneration. Mitochondria trafficking to and anchoring at metabolically active regions provides local energy stations that constantly supply ATP. Regulation of mitochondrial transport and distribution is therefore a central issue concerning the maintenance of energy homeostasis throughout nerve cells. Neuronal growth and regrowth require high energy consumption to drive the synthesis of raw building materials and deliver these materials to growing tips. Proper mitochondrial transport into growth cones and injured axons ensures adequate ATP supply. Recent studies have established a correlation between polarized mitochondrial transport and axonal and dendritic morphology, thus neurons may have a unique mechanism for delivering mitochondria to distal axons by sensing energy requirements. AMPK serves as a master regulator of cellular energy homeostasis as it becomes activated when intracellular ATP supplies become depleted. We are testing our hypothesis that SNPH turns on and off its mitochondrial anchoring by sensing local ATP/ADP ratio through phosphorylation and dephosphorylation of SNPH, thus enabling neurons to re-mobilize and re-distribute axonal mitochondria into areas where energy consumption is in high demand. Specific Aim 3. Elucidate mechanisms maintaining and recovering stressed mitochondria. Parkin-mediated mitophagy is a key cellular pathway to eliminate damaged mitochondria in many non-neuronal cells. Our previous studies revealed that Parkin-mediated mitophagy is observed only in a small portion of mature neurons and occurs much more slowly than in non-neuronal cells (Cai et al., Current Biology 2012; Lin et al., Neuron 2017). In Parkin and Pink mutant flies, density and integrity of axonal mitochondria in motor neurons is comparable to WT. These findings argue for unique mechanisms that maintain and recover neuronal mitochondrial integrity and thus energy homeostasis in the early stages of mitochondrial stress, rather than acute global elimination of stressed mitochondria by activating Parkin-mediated mitophagy. In order to support this assumption, we are addressing two fundamental questions: (1) Can neurons recover chronically stressed mitochondria before Parkin-mediated mitophagy is activated? (2) Is mitophagy the last resort for mitochondrial quality control after recovery mechanisms have failed? Addressing these two questions is relevant to several major neurodegenerative diseases that associate with chronic mitochondrial stress. Our working model is that neurons have an intrinsic checkpoint mechanism for recovering stressed mitochondria through regulation of mitochondrial dynamics and ER-Mito contacts. If this pathway fails, Parkin-mediated mitophagy is subsequently activated to degrade stressed mitochondria. We propose that mitochondria-resident Mul1-Mfn2 pathway is critical to recovering stressed mitochondria, thus limiting neuronal mitophagy and maintaining energy homeostasis under chronic stress conditions. Publications: Kang, J.-S., J.-H. Tian, P. Zald, P.-Y. Pan, C. Li, C. Deng, and Z.-H. Sheng (2008). Docking of axonal mitochondria by syntaphilin controls their mobility and affects short-term facilitation. Cell 132, 137-148. Cai, Q., H. M. Zakaria, A. Simone, and Z.-H. Sheng (2012). Spatial parkin translocation and degradation of depolarized mitochondria via mitophagy in live cortical neurons. Current Biology 22, 545-552. Chen, Y. and Z.-H. Sheng (2013). Kinesin1-syntaphilin coupling mediates activity-dependent regulation of axonal mitochondrial transport. Journal of Cell Biology 202, 351-364. Sun, T., H. Qiao, P.-Y. Pan, Y. Chen, and Z.-H. Sheng (2013). Mobile axonal mitochondria contribute to the variability of presynaptic strength. Cell Reports 4, 413-419. Yuxiang Xie, Bing Zhou, Mei-Yao Lin, Shiwei Wang, Kevin D. Foust, and Zu-Hang Sheng. (2015) Endolysosome deficits augment mitochondria pathology in spinal motor neurons of asymptomatic fALS-linked mice. Neuron 87, 355-370. Morsci, N. S., D. H. Hall, M. Driscoll, and Z.-H. Sheng (2016). Age-related phasic patterns of mitochondrial maintenance in adult C. elegans neurons. Journal of Neuroscience 36, 1373-1385. Zhou, B., P. Yu, MY. Lin, T. Sun, Y. Chen, and Z.-H. Sheng (2016). Facilitation of axon regeneration by enhancing mitochondrial transport and rescuing energy deficits. Journal of Cell Biology 214, 203-119. Lin*, M-Y., X-T. Cheng* (equal contributions), P Tammineni, Y. Xie, B. Zhou, Q. Cai, and Z-H. Sheng (2017). Releasing syntaphilin removes stressed mitochondria from axons independent of mitophagy under pathophysiological conditions. Neuron 94, 595-610. Lin* M-Y, Cheng* X-T, Xie Y, Cai Q & Sheng Z-H (2017). Removing dysfunctional mitochondria from axons independent of mitophagy under pathophysiological conditions. Autophagy 13, 1792-1794. Sheng Z-H (2017). The interplay of axonal energy homeostasis and mitochondrial trafficking and anchoring. Trends in Cell Biology 27, 403-416 (Invited review).

Specific Aim 1. Mechanisms regulating neurotrophin retrograde signaling and neuronal growth and survival. 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. Using snapin deficient mice and gene rescue experiments combined with compartmentalized cultures of live cortical neurons, we recently revealed that Snapin, as a dynein adaptor, mediates retrograde axonal transport of TrkB signaling endosomes. Such a role is essential for dendritic growth of cortical neurons. Deleting snapin or disrupting Snapin-dynein interaction abolishes TrkB retrograde transport, impairs BDNF-induced retrograde signaling from axonal terminals to the nucleus, and decreases dendritic growth. Such defects were rescued by reintroducing snapin gene. Our study indicates that Snapin-dynein coupling is one of the primary mechanisms driving BDNF-TrkB retrograde transport, thus providing new mechanistic insights into the regulation of neuronal growth and survival (Zhou et al., Cell Reports, 2012). Specific Aim 2. 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 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, combined with reduced neuron viability and neurodegeneration (Cai and Sheng, Autophagy 2011; Zhou et al., 2011). 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 summay, our ongoing study provides new mechanistic insights into (1) how Snapin regulates retrograde axonal transport of neurotrophin signaling endosomes and late endosomal-lysosomal organelles; (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. Related publications from the lab: Qingning Su*, Qian Cai*(equal contributions), Claudia Gerwin, Carolyn L. Smith, Zu-Hang Sheng (2004) Syntabulin: a microtubule-associated protein implicated in syntaxin transport in neurons, Nature Cell Biology 6, 941-953. Qian Cai, Pingyue Pan, and Zu-Hang Sheng. (2007). Syntabulin-kinesin-1 family 5B-mediated axonal transport contributes to activity-dependent presynaptic assembly. Journal of Neuroscience 27, 7284-7296. Huan Ma, Qian Cai, Wenbo Lu, Zu-Hang Sheng (co-corresponding author), and Sumiko Mochida. (2009). KIF5 motor adaptor syntabulin maintains synaptic transmission in sympathetic neurons. Journal of Neuroscience 29, 13019-13029. Qian Cai, Li Lu, Jin-Hua Tian, Yi-Bing Zhu, Haifa Qiao, Zu-Hang Sheng. (2010). Snapin-regulated late endosomal transport is critical for efficient autophagy-lysosomal function in neurons. Neuron 68, 73-86. Qian Cai and Zu-Hang Sheng. (2011). Uncovering the role of Snapin in regulating autophagy-lysosomal Function. Autophage 7, 445-447. Bing Zhou, Yi-Bing Zhu, Lin Lin, Qian Cai, and Zu-Hang Sheng. (2011). Snapin deficiency is associated with developmental defects of the central nervous system Bioscience Report 31, 151-158. Bing Zhou, Qian Cai, Yuxiang Xie, and Zu-Hang Sheng (2012). Snapin recruits dynein to BDNF-TrkB signaling endosomes for retrograde axonal transport and is essential for dendrite growth of cortical neurons, Cell Reports 2, 42-51.