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Advisory Committee Chair

Candace Floyd

Advisory Committee Members

Erik Roberson

John Chatham

Talene Yacoubian

Document Type

Dissertation

Date of Award

2014

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Recent studies have provided strong evidence that alterations in protein degradation pathways, such as the autophagy-lysosome pathway (ALP), may contribute to neuronal dysfunction and death which lead to clinical symptoms diagnosed with various neurodegenerative diseases. Parkinson's disease (PD) is characterized by neuromuscular abnormalities resulting from the pathological loss of substantia nigra dopaminergic neurons and widespread detection of Lewy bodies, intracellular protein inclusions composed primarily of α-synuclein. Additionally, altered gene function regulating the expression of α-synuclein has been directly linked to the PD pathogenesis. Autophagy is an intracellular degradation process that when altered can lead to the accumulation neurotoxic proteins such as α-synuclein. Experimental inhibition of the ALP in models of PD is effective at reproducing α-synuclein accumulation and cell death. Conversely, stimuli that enhance ALP function are protective in PD and other models of neurodegenerative disease. Macroautophagy is a subset of autophagy that regulates degradation by sequestering cytosolic material within double-membraned vesicles, or autophagosomes, that are ultimately degraded by the lysosome. The vacuolar type-ATPase (V-ATPase) regulates acidic vesicle pH, and its inhibition by bafilomycin A1 (BafA1) is mediated through its interaction the V-ATPase subunit ATP6V0C. BafA1-mediated inhibition of V-ATPase leads to elevated lysosome pH, altered autophagy, accumulation of α-synuclein, and cell death. While autophagy is known to be important in maintenance of neuronal cell function, its potential causative role in the progression of neurodegenerative diseases require further investigation. We aim to (1) assess rotenone-induced alterations to the ALP that contribute to neurodegenerative pathology; (2) characterize ALP function and neuronal toxicity after genetic inhibition ATP6V0C under basal and stressed conditions; and (3) further understand low-dose BafA1-mediated neuroprotection in models of neurodegeneration. Using an in vitro model of PD, we treated differentiated neuronal cells with the environmental pesticide rotenone, a known neurotoxin linked to PD. Although previous studies report rotenone cause autophagosome accumulation, whether this accumulation is due to increased autophagosome production or decreased degradation is not entirely clear. In our studies we observed that rotenone induced accumulation of autophagosomes and α-synuclein that resulted from decreased lysosome-mediated degradation. We provide evidence that rotenone-induced lysosome dysfunction is caused by an elevation of pH concomitant with a reduction in cellular energy levels prior to cell death. These studies conclude that rotenone has a pronounced effect on macroautophagy completion that may contribute to its neurotoxic potential. Lysosome dysfunction is linked to neuronal cell death in many neurodegenerative diseases. As V-ATPase is a critical regulator of lysosome function, we investigated whether knockdown of the V-ATPase subunit, ATP6V0C, could alter the ALP and cause PD-like cellular pathologies. We found that knockdown of ATP6V0C alone caused an elevation in acidic vesicle pH, increased autophagosome accumulation, and decreased autophagic turnover. These observed alterations in the ALP occurred concomitantly with increased detection of α-synuclein. Knockdown of ATP6V0C alone caused a decrease in neurite length which was further exacerbated with BafA1 treatment. Although ATP6V0C knockdown alone did not alter cell viability, it caused a shift in the dose-response sensitizing cells to BafA1 toxicity. Together these results indicate a role for ATP6V0C in maintaining constitutive and stress-induced ALP function, in particular the metabolism of substrates that accumulate in age-related neurodegenerative disease and may contribute to disease pathogenesis. Our lab has previously shown low concentrations of BafA1 that do not inhibit V-ATPase effectively attenuate ALP-related neuronal pathology by enhancing ALP function in in vitro models of neurodegeneration. Additionally, low-dose BafA1 can protect dopaminergic cells from α-synuclein toxicity. However, prior to these studies whether low-dose BafA1provided ALP-dependent protection was not known. Using the lysosome inhibitor chloroquine to induce ALP dysfunction and cell death, we investigated whether low-dose BafA1 protection could be maintained in the absence of important ALP-related proteins. siRNA-mediated knockdown of both ATP6V0C and the autophagy related protein Atg7 prevented the BafA1-mediated attenuation of neuronal cell death induced by chloroquine, which induces cell death due to lysosome dysfunction . We also provide evidence that low-dose BafA1 enhances autophagic flux during periods of lysosome dysfunction, an effect that is inhibited by knockdown of ATP6V0C. Our findings suggest that low-dose BafA1 confers neuroprotection directly through enhanced ALP function and provides support for the therapeutic potential of methods which may enhance autophagy to treat neurodegenerative pathologies. Together, our findings highlight the importance of the ALP in maintenance of neuronal function. As such the ALP may be a useful therapeutic target to attenuate or prevent neuron loss in neurodegenerative disease. Current strategies that are intended to maintain neuronal cell viability and in turn dopaminergic signaling by may prove to be extremely useful in slowing or halting disease progression either alone or in combination with current therapeutic methods.

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