Advisory Committee Chair
Jianhua Zhang
Advisory Committee Members
Scott Ballinger
Jim Collawn
Rakesh Patel
Talene Yacoubian
Document Type
Dissertation
Date of Award
2015
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease and Huntington's disease are all characterized by metabolic dysfunction, increased oxidative damage to proteins and organelles, formation of proteinaceous inclusions, decreased autophagic and proteasomal function, and eventual neuronal and glial cell death. While our understanding of the mechanisms that underlie many of these pathologies is constantly growing, their exact cause, onset, interplay and progression still remain unclear. The gap between the description of disease pathologies and understanding the fundamental mechanisms of disease pathogenesis, progression and potential therapeutics to mitigate disease progression is still large. Based on the observation that altered glucose utilization, increased oxidative damage, and accumulation of damaged mitochondria and proteins normally degraded in healthy cells by autophagy all occur during neurodegeneration, we proposed the hypothesis that redox and metabolic regulation of autophagy plays an important role in neuronal health and survival. To test this hypothesis, we used both differentiated SH- SY5Y cells and primary cortical neurons exposed to 2DG or glucose deprivation conditions. In differentiated SH-SY5Y cells, 2DG inhibition of hexokinase decreased HNE-induced autophagy, resulting in a shift to an apoptotic phenotype and enhanced cell death. These effects were reversible by inhibiting apoptosis with the pan-caspase inhibitor ZVAD-fmk, and were specific to diminished hexokinase function, as glucose deprivation did not result in the same effects. While the exact mechanism of 2DG inhibition of autophagy still remains uncertain, it does appear to relate to inhibiting glycolytic enzyme function, as inhibition of GAPDH had a similar effect. 2DG effects also appeared to be independent of improper glycosylation, or effects on the pentose phosphate pathway (PPP), as neither ER stress, nor GSH levels appeared to be altered following treatment. 2DG effects may not be purely through alterations to glycolysis, as Bcl-2 levels were increased, indicating possible increased binding of Beclin-1, and LAMP-1 levels were decreased, indicating a possible decrease in lysosome number. Both of these events, which occurred following treatment, could also play a role in autophagy inhibition. We also aimed to determine if the autophagy inhibition by 2DG is preceded by early metabolic dysfunction using the Seahorse Extracellular flux analyzer. Using rat E18 primary cortical neurons, we determined that 2DG significantly enhanced HNE- induced mitochondrial dysfunction, but glucose deprivation did not. This decrease in mitochondrial dysfunction was also coupled to decreased protein OGlcNAcylation and basal and stress induced autophagy, similar to the effects observed in differentiated SH- SY5Y cells. While glucose deprivation showed similar effects on mitochondrial function as control glucose conditions, the ability to induce autophagy remained intact, although measurements of flux indicated a possible decrease in autophagic flux over time. Overall these studies indicate that glucose metabolism, specifically proper function of the enzyme hexokinase, is a key regulator of neuronal autophagic function. Not only can improper hexokinase function alter mitochondrial function and autophagy induction in the presence of a secondary stressor, it can also alter glucose usage by other pathways in the cell, such as protein OGlcNAcylation. Understanding how decreased function of key glucose-processing enzymes contributes to autophagic dysfunction could provide new therapeutic possibilities for the treatment of neurodegenerative diseases.
Recommended Citation
Dodson, Matthew, "Metabolic and Oxidative Regulation of Neuronal Autophagy and Survival" (2015). All ETDs from UAB. 1537.
https://digitalcommons.library.uab.edu/etd-collection/1537