Project Summary/Abstract The endosomal-lysosomal system is responsible for cellular protein degradation, and thus for protein homeostasis (proteostasis) in all cells. Neurons are spatially complex, large cell types, and must survive for the lifetime of an organism. This makes maintenance of the neuronal proteome unusually challenging. Disruption of lysosomal protein degradation can lead to accumulation of toxic aggregates, neuronal dysfunction, and death. Normal protein degradation can be disrupted via direct damage to lysosomes, which leads to leakage of luminal lysosomal contents into the cytosol in a process known as lysosomal membrane permeabilization (LMP), which can facilitate cell death. LMP is often causally linked to neurological pathologies that disproportionately affect aging populations. Even though lysosomes are critical to neuronal proteostasis, the response of neurons to LMP on a mechanistic level is poorly understood. Further, despite the intimate relationship between endosomal and lysosomal function, the role of endosomes in LMP responses is unknown. The small GTPase Rab7 is a master regulator of late endosomes and lysosomes. It is also critical for autophagy. Mutations in Rab7 lead to Charcot Marie-Tooth 2B (CMT2B) disease, a progressive peripheral neuropathy. My preliminary data suggest that Rab7 is an acute responder to LMP. Following LMP, Rab7 accumulates on various enlarged vesicular compartments, including late endosomes (LEs) and lysosomes (Lys), in a hyperactive, GTP-bound state. Within neurons, this response is exclusively localized to the soma. In this proposal, I aim to discover (1) the requirement of Rab7 activation for downstream LMP responses, such as galectin-3 accumulation on Lys and enlargement of LEs, (2) the impact of CMT2B mutant alleles on known LMP responses and Rab7 activation post LMP induction, and (3) the impact of age on Rab7 mediated neuronal degradation and LMP responses. I posit that Rab7 activation is essential in coordinating early responses for endosomal damage control in LMP. I will test the hypothesis that Rab7 activation following LMP is an essential response that mediates a protective pathway in neurons, which is disrupted in CMT2B mutant alleles. In Aim 1, I will explore the necessity of Rab7 activation in (1) galectin-3 mediated lysophagy and (2) LE vacuolization in response to LMP. In Aim 2, I will test whether Lys/LE responses and Rab7 activation in response to LMP are impacted in CMT2B. In Aim 3, I will determine whether aging disrupts Rab7 mediated (1) cargo degradation and (2) LMP responses in wild- type and mutant CMT2B neurons. Together, these experiments will determine homeostatic mechanisms in neurons that address lysosomal insults and whether this is impacted in disease. My long-term goal is to discover and characterize compensatory endosomal response mechanisms to lysosomal disruptions in LMP that will be broadly applicable to neurological disorders for the ultimate design of be...