The autophagy core complexes in neuronal quality control

PROJECT SUMMARY Autophagy is universal in eukaryotes as a cytoprotective mechanism for the clearance of inclusions, damaged organelles, and other harmful materials. Autophagy has major roles in aging and neurodegeneration, including in Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal degeneration, and Huntington's disease. Autophagy involves the formation of a unique cup-shaped double membrane, the phagophore, which expands, engulfs cargo, and finally closes and fuses with lysosomes. The importance of autophagy as a neuroprotective mechanism has motivated attempts to therapeutically enhance autophagy in the brain. In contrast to nearly all other tissues, bulk autophagy in neurons is decoupled from amino acid levels and mTORC1 signaling. Growing evidence points to levels of the lipid phosphatidylinositol 3-phosphate (PI(3)P) as rate-limiting in autophagy in neurons. Our laboratory has done much of the paradigm shaping work on the molecular, structural, and biochemical mechanisms of regulation of the class III PI 3-kinase complexes that generate PI(3)P, positive and negative regulators of PI 3-kinase complexes, and their PI(3)P-dependent downstream effectors in autophagy. Here, we will combine cryo-EM, structural modeling, biochemical reconstitution, and functional assays and cell imaging in i3Neurons to determine how PI(3)P levels are controlled, and in turn control neuronal autophagy. Assays of i3Neurons will differentiate between bulk autophagy and different forms of mitophagy, and further differentiate between autophagy events in axons, axonal boutons, and soma. Imaging tools will include mitoKeima and HALO-based assays and CLEM and FIB- SEM imaging of autophagy in axons and axonal boutons in i3Neurons bearing a range of knockouts, autophagy probes, and fluorescently tagged subunits of autophagy core complexes. We will determine how the key lipid kinase complex of autophagy, the class III PI 3-kinase complexes I (PI3KC3-C1) is regulated by the key autophagy-initiating ULK1 complex. We will determine the high-resolution structure of the PI3KC3- C1:ULK1C supercomplex and analyze its function and regulation in distinct compartments within neurons.