Dynamics of Axonal Autophagy in Neurons

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Project Summary Autophagy is an essential cellular degradative pathway triggered by environmental stress in many cell types. In neurons, autophagy has a further role as a constitutively active mechanism that maintains axonal homeostasis. In vitro and in vivo, autophagosomes are generated de novo at axon terminals and synaptic sites. Once formed, axonal autophagosomes are trafficked back to the soma by the retrograde microtubule motor protein cytoplasmic dynein. Autophagosomes mature en route through fusion with late endosomes and lysosomes. Cargo degradation also occurs during transport along the axon, leading to the somal delivery of digested contents for recycling in new biosynthetic pathways. Axonal autophagy degrades mitochondrial fragments and disease- associated protein aggregates, suggesting a key role in the maintenance of axonal homeostasis. Consistent with this hypothesis, neuron-specific ablation of autophagy is sufficient to cause neurodegeneration. However, many outstanding questions remain that must be addressed: How is autophagy regulated in neurons? What controls the localization and timing of autophagosome formation and cargo engulfment? What is the function of axonal autophagy – what cargos are targeted for degradation, and by what mechanisms? And how does the axonal autophagy pathway intersect with the endolysosomal pathway to effectively degrade cargos such as dysfunctional organelles and aggregated proteins? To address these questions, we will use live cell imaging in primary neurons and gene-edited iPSC-derived human neurons, in concert with biochemical and biophysical approaches including proteomic analysis and computational modeling, to query the basic mechanisms of axonal autophagy and how these mechanisms are perturbed by neuronal stressors including mitochondrial dysfunction, protein aggregation, and lysosomal damage. We will address the following specific aims: Aim 1: How is autophagy spatially and temporally regulated in neurons? What controls the initiation of autophagy at the axon terminal or presynaptic sites? Aim 2: What cargos are degraded by axonal autophagy? Is cargo engulfment a selective process, or nonspecific? Is there preferential uptake of some cargos, and if so, what are these cargos? What mechanisms control cargo uptake? And Aim 3: How does the autophagy pathway intersect with the lysosomal pathway? How is autophagosome-lysosome fusion regulated? Why is axonal autophagy so dependent on retrograde axonal transport? And what mechanisms regulate lysosomal health along the axon, as lysosomes are required for the effective clearance of engulfed cargos by autophagy. Given the essential and conserved role that autophagy plays in neurons, we anticipate that these studies will significantly advance our understanding of neuronal cell biology, providing important insights into the mechanisms maintaining axonal. homeostasis and how the perturbation of these mechanisms may lead to neurodegeneration. We hope that these advances will provide new ideas on how to best intervene therapeutically to treat diseases such as ALS, Huntington's, and Parkinson's disease.