Compartment-Specific Regulation Of Autophagy In Neurons

Vineet Vinay Kulkarni, University of Pennsylvania

Abstract

Macroautophagy (hereafter autophagy) is a fundamental catabolic process that delivers damaged cytoplasmic proteins and organelles to the lysosome for turnover. In this capacity, autophagy controls the composition and integrity of organelles and the cellular proteome. Autophagy is vital for the development, functionality, and survival of the nervous system. Autophagy is also essential for learning and memory, suggesting critical roles for autophagy at the neuronal synapse. The mechanisms and cellular processes that regulate autophagy in neurons and astrocytes, however, are poorly understood. In this thesis we have elucidated how synaptic activity, which is a fundamental aspect of neuronal physiology, regulates the dynamics and function of autophagic vacuoles in primary hippocampal neurons using live-cell confocal microscopy. We found that synaptic activity regulates the motility of autophagic vacuoles (AVs) in dendrites. Stimulation of synaptic activity dampened AV motility, whereas silencing synaptic activity induced AV motility. Activity-dependent effects on dendritic AV motility are local and reversible. Importantly, these effects are compartment-specific, occurring in dendrites and not in axons. Most strikingly, synaptic activity increases the presence of degradative autolysosomes in dendrites and not in axons. We have further defined the molecular underpinnings of autophagy in primary cortical neurons and astrocytes in response to metabolic stress, using a combination of confocal microscopy and immunoblotting. We found that inducing metabolic stress by nutrient deprivation or pharmacological inhibition of MTOR (mechanistic target of rapamycin kinase) robustly activates autophagy in astrocytes, but have less pronounced effects on autophagy in neurons. Combined, our studies indicate how different cellular processes regulate autophagy in two dominant cell types of the brain. These findings raise important implications for how neurons and glia manage cellular stress, and how they may collaborate to maintain homeostasis in the brain.