A unifying pathology of age-related neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS) is the accumulation of misfolded proteins and protein aggregates in the nervous system. Paradoxically, aggregation- prone proteins are expressed in both neurons and astrocytes, yet neurons appear more vulnerable to the accumulation of protein aggregates. These observations suggest cell-type-specific differences in how quality control pathways are managed in neurons and astrocytes to regulate the integrity of the proteome. In fact, we have found striking differences in how the autophagy-lysosomal pathway and the ubiquitin-proteasome system are managed between neurons and astrocytes. Despite this knowledge, a comprehensive understanding of the cell-type-specific pathways for regulating proteostasis in neurons versus astrocytes has not been achieved. Moreover, the relationship between neurons and astrocytes in the context of proteotoxic stress is largely unknown. It is well established that astrocytes play critical roles in neuronal homeostasis, function, and neuroprotection. By contrast, astrocytes are also emerging as key players in disease pathogenesis. However, we are only at the inception of understanding the molecular and functional properties of neuron-astrocyte interactions in health and disease. Thus, this proposal will define the regulation of lysosome-mediated pathways (autophagy and endolysosomal) within (Aim 1) and between (Aim 2) astrocytes and neurons in response to proteotoxic stress elicited by fibrillar forms of a-synuclein, a model for a-synucleinopathies including PD. To elucidate cell-type-specific responses, we developed a robust system to coculture primary neurons and astrocytes that recapitulates morphological, proteomic, and functional signatures of astrocytes in vivo. Based on our preliminary data, we hypothesize that autophagy and lysosomal pathways are adapted differently in astrocytes versus neurons, which may render neurons preferentially vulnerable to proteotoxic stress associated with a-synuclein aggregation. To test this hypothesis, we will (1) define the sequence of degradation of a-synuclein fibrils via lysosome- mediated pathways within astrocytes as compared with neurons, and (2) define the role of the autophagy receptor TAX1BP1 in the coupling of neuron-astrocyte stress responses to a-synuclein proteotoxins. We employ live-cell imaging combined with quantitative cell biology and biochemistry to investigate these processes at high spatiotemporal resolution in a compartment-specific manner. These studies will reveal new concepts in how quality control pathways are managed in the neuron-astrocyte unit in response to proteotoxic stress. This information will also inform cell-type-specific vulnerabilities in neurodegenerative disease and enable more specific strategies to mitigate neuronal dysfunction and death.