Project Summary. Neurons and astrocytes have unique demands in regulating the quality of their proteome. A key regulator of the proteome is autophagy, a lysosomal degradation pathway. During autophagy, cytoplasmic components are packaged into autophagosomes and delivered to lysosomes for cargo degradation. Autophagy is neuroprotective, as mutations in key autophagy genes cause neurodegeneration. Preliminary studies show that autophagy is regulated differently in neurons and astrocytes in multiple paradigms of stress. Despite the importance of autophagy, how it is regulated in neurons and astrocytes to facilitate cell- type specific functions and stress responses is largely unknown. Thus, the goal for this proposal is to define cell-type specific functions for the autophagy receptor p62 in neurons and astrocytes. P62 facilitates selective forms of autophagy by binding to ubiquitinated substrates and the autophagy marker LC3, thereby incorporating cargo into a forming autophagosome. P62 mitigates proteotoxic stress by targeting protein aggregates to the autophagy pathway. Additionally, p62 mitigates oxidative stress by targeting Keap1, a negative regulator of the antioxidant transcription factor NRF2, for degradation by autophagy. To examine functions of p62 in each cell type, we established a robust system to coculture neuron and astrocytes. This system recapitulates intercellular interactions found in vivo, and provides an easily manipulatable system for studying cell-type specific p62 function with high resolution. Using the coculture, I found by immunostain that metabolic stress (autophagy activator) induces formation of p62-ubiquitin positive structures (i.e., ubiquitinated cargo) only in neurons. Moreover, blocking ubiquitination significantly reduces p62 puncta formation and degradation in neurons as compared to astrocytes in basal and stress conditions. Astrocytes are crucial to combating oxidative stress, but the role of p62 in this pathway in astrocytes is largely unknown. I found that oxidative stress induces p62 levels selectively in astrocytes. Thus, I hypothesize that p62 functions primarily in selective autophagy in neurons, and primarily in the antioxidant pathway in astrocytes. Importantly, ALS-linked mutations in p62 fall in domains important for each function. But how p62 protects against neurodegeneration in each cell type is not understood. I hypothesize that ALS-linked mutations in p62 domains that are important for selective autophagy and antioxidant function will impair p62 function in neurons and astrocytes, respectively. To test these hypotheses, I will (Aim 1) define cell-type specific functions of p62 in neurons and astrocytes, and (Aim 2) determine the effects of ALS-linked mutations on p62 function in neurons and astrocytes. This study will elucidate cell-type specific contributions of neurons and astrocytes to neurodegeneration. In turn, understanding cell-type specific contributions to ALS will enable opportunities f...