Mechanisms of Sensing and Responding to Lysosomal Stress in Neurons

所有细胞中的蛋白质稳态都是通过调节蛋白质合成和蛋白质之间的平衡来维持的

Protein homeostasis in all cells is maintained by regulating the balance between protein synthesis and protein degradation. Since neurons are extraordinarily large and extremely long-lived, maintenance of the neuronal proteome is unusually challenging. Disruption of normal protein turnover can lead to accumulation of toxic aggregates, neuronal dysfunction, and death. Not surprisingly then, genes linked to degradative pathways are frequently linked to diseases of the nervous system. There is a big knowledge gap for how neurons monitor and handle proteostatic stress, and how axons and dendrites might have adapted different mechanisms to do this effectively. One cause of proteostatic stress is lysosomal damage which occurs much more frequently than previously realized. Agents of lysosomal damage include lysosomotropic drugs (such as LLOMe, chloroquine, SSRIs), neurotoxic aggregates (such as tau), and oxidative stress (as occurs after ischemic stroke). Lysosomal damage results in lysosomal membrane permeabilization (LMP) with immediate collapse of pH and calcium gradients, progressing to holes in the lysosomal membrane. Even though lysosomes are critical to neuronal function and often causally linked to neurological pathologies, the response of neurons to lysosomal damage on a mechanistic cellular level is poorly understood. This proposal focuses on new a damage response we discovered (late endosome rapid response “LERR”) for responding to and recovering from LMP. Current work in non-neuronal cells has discovered that cells respond to LMP by first trying to repair the damaged lysosome (in minutes). If repair fails, the cell disposes of damaged lysosomes via lysophagy (in hours) and initiates new lysosome biogenesis (24 hours). Our unpublished work discovered that undamaged compartments (especially LEs) rapidly change their dynamic behavior (in ~10 minutes). We pose the novel hypothesis that LEs mount a rapid response to LMP to maintain moderately degradative compartments in the short term. We propose two specific aims. Aim 1: Discover how endosomes in soma, dendrites, and axons respond to LMP. We will use vital sensors and multiplexing by live imaging of cortical neurons to determine the response of LEs and lysosomes in the soma, the dendrites, and the axon in order to elucidate how LMP responses are adapted to the great expanse of dendritic and axonal arbors. We hypothesize that dendritic compartments maintain moderate degradative capacity by halting fusion with damaged lysosomes in the soma. Aim 2: Discover if the neuronal “LE rapid response” is protective. Rab7 is the master regulator of LE maturation. We hypothesize that Rab7 effector cascades are required for the LE response to LMP and for return to normal after LLMOe washout. We will use pharmacological inhibition of key nodes of endosome maturation and transport in combination with acute approaches of Rab7 interference, including photoactivatable Rab7-dominant negative (DN) and degron tagged-Rab7-DN for rapid interference. Carrying out the proposed work holds the promise of establishing new paradigms for how protein turnover is spatially regulated in neurons.