Ultrastructural alterations to neuronal autophagy during aging delineated with Cryo-EM tomography

PROJECT SUMMARY/ABSTRACT Misregulation of autophagy has been implicated in aging and age-related neurodegenerative disorders. Despite its importance, relatively little is known about autophagy in neurons, especially in the context of aging. This gap in knowledge has prevented understanding how neuronal autophagy helps maintain neuronal homeostasis and function during aging. The long-term goal is to elucidate the molecular mechanisms that regulate neuronal autophagy during aging to identify potential therapeutic targets for neurodegenerative diseases and age-related cognitive decline. The overall objective of this application is to interrogate how age produces ultrastructural alterations to neuronal autophagosome formation and maturation that ultimately disrupt nervous system function. The central hypothesis is that in neurons, aging results in defects in autophagic vesicle ultrastructure and disrupts spatial relationships with neighboring subcellular structures. Further, failure to form productive autophagosomes leads to an accumulation of unsequestered and undegraded cargo in the axons, ultimately resulting in loss of neuronal homeostasis and function. The rationale is that elucidating age-related ultrastructural changes to autophagic vesicles in neurons will provide a foundation to interrogate the intervention point in the neuronal autophagy pathway to halt or reverse age- induced loss of neuronal health and function. The central hypothesis will be tested by accomplishing two specific aims: 1) compare the ultrastructural features of axonal autophagic vesicles during aging using correlative light and electron microscopy (CLEM); and 2) determine the ultrastructural basis for WIPI2 rescue of autophagy defects in aged neurons. In the first aim, cryo-electron tomography (cryo-ET) will be used to elucidate how aging induces ultrastructural changes to autophagic vesicles in mammalian neurons. Additionally, how aging modulates the architecture of the axonal compartment and the spatial relationships between autophagic vesicles and neighboring organelles will be quantified using cryo-ET. For the second aim, the complementary approaches of multi-color live-cell microscopy and cryo-ET will be leveraged to quantify how ectopic expression of a single autophagy component, WIPI2, rejuvenates the axonal compartment and restores autophagosome biogenesis in aged mice. The research proposed in this application is innovative because it utilizes live-cell imaging combined with CLEM to ask how aging modulates the autophagic pathway and alters the sub-cellular ultrastructure in primary murine neurons. The proposed research is significant because it is expected to identify stages of neuronal autophagy that are potential targets for intervention to prevent neuronal decline. Ultimately, the knowledge generated by this proposal has the potential to provide new opportunities for the development of innovative therapies to delay age-related cognitive decline and neurodegeneration.