The following Specific Aims are formulated to address above fundamental questions:
Specific Aim 1. Retrograde Transport of LEs Regulates Neuronal Autophagy-Lysosomal Function. (Cai et al., Neuron 2010; Xie et al., Neuron 2015). Dynein motors drive retrograde transport of LEs, thus enhancing late endocytic trafficking from distal processes to the soma, where mature lysosomes are mainly localized. Our recent study uncovered a critical role for snapin in regulating late endocytic transport and endo-lysosomal trafficking. Snapin acts as a dynein motor adaptor by attaching dynein to late endosomes. The snapin KO neurons exhibit impaired retrograde transport of late endosomes, reduced lysosomal proteolysis, aberrant accumulation of immature lysosomes, and impaired clearance capacity of autolysosomes. Snapin deficiency leads to axonal degeneration and developmental defects in the central nervous system. Reintroducing the snapin transgene rescues these phenotypes. Our studies elucidate a new mechanism coordinating dynein-mediated late endocytic transport and endosomal-lysosomal trafficking. Such a mechanism is critical for maintaining cellular homeostasis essential for neuronal survival. Autophagy-lysosomal dysfunction is one of the cellular defects contributing to the pathogenesis of major neurodegenerative diseases associated with accumulation of aggregation-prone proteins and damaged organelles.
Specific Aim 2. Endo-lysosomal Trafficking and Sorting Regulates Synaptic Activity (Di Giovanni and Sheng, EMBO J 2015). Proper regulation of synaptic vesicle (SV) pool size is critical to maintain synaptic activity. Because early endosomes represent crossroads between local SV recycling and the endo-lysosomal degradation, this raises a fundamental question: are SVs sorted toward endo-lysosomal pathway for degradation? To address this question, we applied snapin dominant-negative mutants combined with dual-channel time-lapse imaging in live cortical neurons. Our study reveals that dynein-driven LE transport regulates the SV pool size. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin-/- phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endo-lysosomal pathway. SV components are co-transported with LEs along axons. Therefore, our study provides new mechanistic insights into maintaining and regulating SV pool size through snapin-mediated endosomal trafficking and sorting.
Specific Aim 3. Mechanism Removing Autophagosomes from Axons (Cheng et al., JCB 2015). Degradation of autophagic vacuoles (AVs) via lysosomes is an important homeostatic process over a neurons lifetime. Autophagy undergoes stepwise maturation: bulk cytoplasmic components and organelles are engulfed within double-membrane organelles termed autophagosomes, followed by fusion with LEs into amphisomes, or fusion with lysosomes into autolysosomes for degradation in the soma. However, it is unknown how autophagosomes in distal axons acquire their retrograde motility. We reveal a new motor-adaptor sharing mechanism driving autophagosome transport to the soma. LE-loaded dynein-snapin complexes mediate the retrograde transport of autophagosomes upon their fusion with LEs in distal axons. This motor-adaptor-sharing mechanism enables neurons to maintain effective autophagic clearance, thus reducing autophagic stress in axons. Disrupting dynein-snapin coupling blocks dynein recruitment to LEs and thus impairs retrograde transport of amphisomes toward the soma. Therefore, our study reveals a new cellular mechanism underlying the removal of distal AVs engulfing aggregated misfolded proteins and dysfunctional organelles associated with several major neurodegenerative diseases.
Specific Aim 4. Autophagy-Lysosomal Deficits Contributes to ALS-linked Early Pathology. (Xie et al., Neuron 2015). One pathological hallmark in fALS-linked motor neurons (MNs) is axonal accumulation of AVs, thus raising a fundamental question as to whether reduced autophagic clearance due to an impaired lysosomal system contributes to autophagic stress and axonal degeneration. We recently reveal progressive lysosomal deficits in spinal MNs beginning at early asymptomatic stages in fALS-linked mice expressing the human mutant SOD1G93A protein. Such deficits impair the degradation of AVs engulfing damaged mitochondria from distal axons. These early pathological changes are attributable to mutant hSOD1, which interferes with dynein-driven endo-lysosomal trafficking. Elucidation of this pathological mechanism is broadly relevant, because autophagy-lysosomal deficits are associated with several major neurodegenerative diseases. Therefore, enhancing lysosome function, rather than autophagy induction, is an alternative therapeutic strategy for ALS-linked clinical trials.
Specific Aim 5. Synaptic Cargo 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; this process requires the targeted delivery of newly synthesized synaptic cargoes from the soma to synapses. Our previous studies established that syntabulin is an adaptor capable of linking KIF5 motor and synaptic protein cargoes. Syntabulin-KIF5 coupling mediates axonal transport of synaptic components essential for presynaptic assembly and maintenance (Su et al., Nature Cell Biology 2004; Cai et al., Journal of Neuroscience 2007). Syntabulin loss-of-function blocks formation of new presynaptic terminals in developing neurons. Our studies establish that kinesin-mediated axonal transport is one mechanism underlying activity-dependent presynaptic plasticity. Conditional syntabulin knockout mice have been recently generated in the lab. We will use this mouse line to determine whether deficiency in syntabulin-mediated transport has any impact on synapse formation, maintenance and plasticity during health brain development and in neurological and mental disorders.
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