Axonal transport of endolysosomal organelles and presynaptic cargos for the maintenance of axon cellular homeostasis and presynaptic function

National Institute Of Neurological Disorders And Stroke

Investigators

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Abstract

By combining live imaging of neurons from genetic mouse models with gene-rescue approaches, we have made several key discoveries over the past decade: (1) Dynein adaptor for lysosomal transport: We identified snapin as a dynein motor adaptor that drives retrograde transport of endo-lysosomes from distal axons to the soma, where mature lysosomes are enriched, thereby maintaining neuronal degradative capacity (Cai et al., Neuron 2010). (2) Motor-adaptor sharing in autophagy clearance: We proposed a motor-adaptor sharing model in which late endosome-loaded dynein-snapin complexes also transport autophagosomes after their fusion with late endosomes to form amphisomes, ensuring effective autophagic clearance from distal axons (Cheng et al., JCB 2015). (3) Endo-lysosomal regulation of synaptic activity: We demonstrated that the axonal endo-lysosomal pathway regulates presynaptic activity by shuttling recycled synaptic vesicles towards degradation, ensuring presynaptic homeostasis (Di Giovanni and Sheng, EMBO J, 2015). (4) Early lysosomal pathology in ALS: Using a familial ALS (fALS) mouse model, we provided in vitro and in vivo evidence that progressive lysosomal deficits represent early pathological events in motor neurons (Xie and Zhou et al., Neuron 2015). (5) Defining degradative lysosomes in neurons: We established experimental guidelines for labeling mature degradative lysosomes in vitro and in vivo, characterizing how their distribution, trafficking, and functionality contribute to neuronal health and disease (Cheng et al., JCB 2018). (6) Soma-derived delivery of degradative lysosomes: We discovered that soma-derived transport of mature lysosomes into axons is critical to maintain local degradative capacity for disease-linked protein aggregates and damaged organelles (Farfel-Becker et al., Cell Reports 2019). (7) Presynaptic cargo transport: We identified syntabulin as a kinesin-1 motor adaptor that drives transport of presynaptic cargos into axons, thereby supporting presynaptic assembly, maintenance, and remodeling (Su and Cai et al., Nature Cell Biology, 2004; Cai et al., JCB, 2005; Cai et al., Journal of Neuroscience, 2007). Together, these studies have conceptually advanced our understanding of bidirectional axonal transport as central mechanisms for maintaining axonal and presynaptic homeostasis, providing a strong foundation for the recent accomplishments described below: Accomplishment 1: Lipid-driven impairment of axonal lysosome delivery in NPC neurons (Roney et al., Developmental Cell 2021; Roney et al., JCB 2022) Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder characterized by lipid accumulation in endolysosomes. A hallmark pathologic feature, observed in both NPC patients and mouse models, is pronounced axonal dystrophy—bulbous swellings filled with degradative organelles. Notably, this phenotype arises before symptom onset and neuronal degeneration, underscoring the importance of understanding early cellular events that trigger axonal degeneration. Using STED super-resolution live imaging, combined with genetic and pharmacological approaches, we uncovered a lipid-dependent mechanism driving autophagic stress and axon dystrophy. We found that chronic lysosomal dysfunction in NPC neurons disrupts axonal trafficking and positioning of mature lysosomes, thereby impairing lysosomal clearance and causing autophagic organelle buildup within axons. Normally, axonal lysosome delivery relies on kinesin-1–SKIP–Arl8 motor-adaptor complexes, where the small GTPase Arl8b couples lysosomes to kinesin-1 motors via its effector SKIP. We discovered that elevated cholesterol on NPC lysosomal membranes aberrantly sequesters kinesin-1 and Arl8, blocking lysosome entry into axons and driving autophagosome accumulation. Importantly, pharmacological reduction of lysosomal cholesterol with HPβCD restored lysosome transport, alleviated axonal autophagic stress, and reduced neuronal death. These findings establish a mechanistic link between altered lysosomal membrane lipid composition and defective axonal lysosome delivery, providing key biological insights into axonopathy in NPC and supporting the translational potential of HPβCD in restoring axonal degradative capacity at early disease stages. Accomplishment 2: Presynaptic mechanism underlying autism-like synaptic dysfunction and social behavioral traits (Xiong et al., Molecular Psychiatry 2021; Xiong and Sheng, JCB 2024) Synapse formation and maintenance rely on axonal trafficking of presynaptic proteins from the soma to presynaptic terminals. Disruption of these processes is a hallmark of neurodevelopmental disorders. We previously identified syntabulin (STB) as a kinesin-1 motor adaptor essential for presynaptic cargo transport during synapse assembly and maintenance (Su, Cai et al., Nat Cell Biol. 2004). STB expression peaks during early postnatal brain development and declines with maturation. The human STB gene resides within the autism susceptibility locus 8q22–24, and whole-exome sequencing identified a de novo autism-linked missense variant (R178Q). Based on these findings, we hypothesized that defective axonal transport of presynaptic cargos is a key causative mechanism for autism-associated synaptic dysfunction and behavioral abnormalities. Our recent work supports this hypothesis. Conditional knockout (Stb cKO) mice display impaired presynaptic cargo transport, reduced presynaptic terminals and active zone density, altered synaptic transmission/plasticity, and core autism-like traits including deficits in social recognition/communication, increased stereotypy, and impaired spatial learning and memory (Xiong et al., Molecular Psychiatry 2021). To directly test the genetic link, we recently used CRISPR/Cas9 to generate a human autism-linked Stb-R178Q knock-in (Stb-Kn) mouse line. This missense mutation is a loss-of-function variant that fails to function as a kinesin-1 adaptor. Strikingly, Stb-Kn mice phenocopy transport and synaptic deficits, along with hallmark autistic behaviors (Xiong et al., in preparation). Together, these genetic models provide the first direct evidence that impaired axonal transport and presynaptic mechanisms underly autism-linked synaptic dysfunction and behavioral abnormalities (Xiong and Sheng, JCB 2024). They also offer a unique platform for reprogramming axonal transport in defined brain circuits. This research aligns closely with recent NIH priorities aimed at advancing mechanistic understanding of autism etiology and informing therapeutic strategies. Accomplishment 3: Impaired axonal endo-lysosomal (E-L) transport contributes to dopaminergic neuron loss and Parkinson’s disease (PD) phenotypes (Xie et al., in preparation). In PD, axonopathy is one of the earliest pathological events that precedes dopaminergic (DA) neuron loss. Defective E-L transport has merged as a key contributor to PD-associated axonopathy by impairing clearance of damaged organelles and α-synuclein aggregates. However, the mechanisms by which PD-linked gene mutations disturb E-L transport in DA neurons remains elusive. To address this, we generated an SNpc DA-targeted snapin cKO mouse model. These mice exhibit striking PD-like features, including (1) progressive loss of SNpc DA neurons by 60.5% at 8 months of age; (2) substantial loss of TH-positive axons in the SNc region, with surviving axons showing fragmentation and swollen profiles filled with degradative multivesicular bodies (MVBs) and autophagic vacuoles (AVs); (3) impaired motor coordination, along with hallmark PD motor symptoms such as resting tremor, muscle rigidity, difficulty walking, slower body movement, and shuffling gait; and (4) body weight loss beginning after 8 months of age. Based on these findings, we hypothesize that pathogenic LRRK2-snapin signaling perturbs E-L axonal transport, leading to DA neuron degeneration. Ongoing studies are focused on defining the role of pathogenic LRRK2-snapin signaling in PD pathogenesis, including LRRK2-mediated phase transitions of snapin-motor complexes that regulate bidirectional autophagosomal and lysosomal transport to maintain axonal and presynaptic homeostasis.