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

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Abstract

By employing live imaging of adult neurons from genetic mouse models combined with gene rescue experiments, we have made seven important discoveries in recent years: (1) We revealed that snapin acts as an adaptor of dynein motors driving endo-lysosome retrograde transport from distal axons to the soma, thus maintaining neuronal degradation capacity (Cai et al., Neuron 2010). (2) We revealed a motor-adaptor sharing model by which late endosome (LE)-loaded dynein-snapin complex drives the retrograde transport of autophagosomes upon their fusion with LEs into hybrid organelles, thus maintaining autophagic clearance in distal axons (Cheng et al., JCB 2015). (3) We demonstrated that endo-lysosomal pathway regulates presynaptic activity by shuttling protein components of synaptic vesicles (SVs) towards endo-lysosomal degradation pathways (Di Giovanni and Sheng, EMBO J, 2015). (4) We investigated a familial (f)ALS-linked mouse model and provided in vitro and in vivo evidence that progressive lysosomal deficits are early fALS-linked pathological events in motor neurons (Xie and Zhou et al., Neuron 2015). (5) We provided the guidelines for labeling degradative lysosomes in in vitro and in vivo nervous systems to characterize how lysosomal distribution, trafficking, and functionality contribute to neuronal health and disease progression (Cheng et al., JCB 2018). (6) We further characterized axonal transport of degradative lysosomes in developing and mature neurons and showed that axonal degradation capacity is maintained by the delivery of fresh degradative lysosomes from the soma (Farfel-Becker et al., Cell Reports 2019). (7) We identified syntabulin as a kinesin-1 motor adaptor that links and drives presynaptic Bassoon cargo transport in axons, thus contributing to 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). These studies conceptually advance our understanding of axonal transport in the maintenance of axonal and presynaptic homeostasis essential for neuronal function and survival, thus building a solid foundation for our research accomplishments in the current fiscal year. Accomplishment 1: Reveal lipid-mediated motor-adaptor sequestration that impairs axonal lysosome delivery leading to autophagic stress and dystrophy in NPC neurons (Roney et al., Developmental Cell 2021; Roney et al., Autophagy 2021) NPC is a neurodegenerative lysosomal storage disorder characterized by lipid accumulation in endo-lysosomes. One major pathologic feature observed in NPC patients and recapitulated in NPC mouse models is striking axonal dystrophy, which consists of bulbous swellings containing accumulated degradative organelles. This axonal phenotype precedes symptom onset and neurodegeneration, thus highlighting the importance of understanding the cellular events that precede axonal organelle accumulation and their contribution to neurodegeneration. We recently revealed a lipid-linked pathological mechanism underlying autophagic stress and axonal dystrophy. Using STED super-resolution and live imaging combined with genetic and pharmacologic interventions, we demonstrated that chronic lysosomal dysfunction in NPC neurons compromises the axonal trafficking and positioning of degradative lysosomes, which impairs autophagy-lysosomal clearance, leading to the accumulation of autophagic organelles within the axon. Axonal lysosome delivery is driven by the kinesin-1-SKIP-Arl8 motor-adaptor complex, where the small GTPase Arl8b links lysosomes to kinesin-1 motors through its effector SKIP. We discovered that elevated cholesterol levels on NPC lysosome membranes sequester kinesin-1 and Arl8, resulting in impaired lysosome transport into axons, thus contributing to axonal autophagosome accumulation. Pharmacologic reduction of lysosomal membrane cholesterol with HPCD rescues lysosome transport, thereby reducing axonal autophagic stress and neuron death in NPC. These findings demonstrate a new transport mechanism through which altered membrane lipid composition impairs lysosome delivery into axons and thus provide biological insights into the translational application of HPCD in restoring axonal degradation capacity at early stages of NPC. Accomplishment 2: Reveal axonal transport mechanism underlying autism-like synaptic dysfunction and social behavioral traits (Xiong et al., Molecular Psychiatry 2021) The formation and maintenance of synapses require long-distance axonal delivery of newly synthesized presynaptic proteins from the soma to distal synapses, raising the fundamental question of whether impaired axonal transport is associated with neurodevelopmental disorders such as autism. We previously revealed syntabulin (STB) as a kinesin-1 motor adaptor driving presynaptic cargo transport, thus contributing to presynaptic assembly and maintenance (Su, Cai et al., Nat Cell Biol. 2004). The STB gene locates within the autism susceptibility loci 8q22-24. A recent whole exome sequencing study identified an autism-linked de novo human STB (R178Q) missense variant. Thus, there is an urgent need to establish a mechanistic link between impaired axonal transport of presynaptic cargos and autism-like synaptic dysfunction and social behavioral abnormalities. Generating a conditional stb knockout (stb cKO) mouse provides us an in vivo model system to address this issue. STB expression in mouse brains peaks during the first two weeks of postnatal development and then progressively declines with brain maturation. The stb cKO neurons display impaired axonal transport of presynaptic cargos, reduced synapse density and active zones, and altered synaptic transmission and long-term plasticity. Intriguingly, stb cKO mice exhibit core autism-like traits, including defective social recognition and communication, increased stereotypic behavior, and impaired spatial learning and memory. These phenotypes are further confirmed by the human missense variant STB-R178Q, which loses its adaptor capacity for binding kinesin-1 motors. Expressing STB-R178Q fails to rescue reduced synapse formation and impaired synaptic transmission and plasticity in stb cKO neurons. Altogether, our study establishes that defective axonal transport impairs synapse formation and maintenance, thus providing one of the core presynaptic mechanisms underlying synaptic dysfunction and behavioral abnormalities that bear similarities to autism.