Date of Award

1-1-2013

Document Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Daniel A. Linseman

Keywords

Amyotrophic Lateral Sclerosis, Glutathione, Mitochondria, Oxidative Stress

Abstract

Oxidative stress is a contributing factor to many neurodegenerative diseases. In particular, mitochondria are a key source of oxidative stress due to electron leakage at the level of the electron transport chain. To combat the endogenous production of reactive oxygen and reactive nitrogen species mitochondria are equipped with several redox-cycling systems, such as glutathione (GSH). Mitochondrial GSH has been shown to be a critical reservoir of this antioxidant, where selective depletion of mitochondrial GSH can induce apoptosis in several systems. Many studies have intricately linked Bcl-2 to cellular GSH status and it has been previously shown that Bcl-2 is a GSH binding protein. Therefore, we hypothesize that Bcl-2 may play an important role in regulating mitochondrial GSH transport.

Here, we show that inhibition of Bcl-2 in primary cerebellar granule neurons (CGNs) induced apoptosis, mitochondrial oxidative stress, selective depletion of the mitochondrial GSH pool, and led to inhibition of mitochondrial GSH transport. Furthermore, we found that Bcl-2 is an interacting partner with a previously identified mitochondrial GSH transporter, the 2-oxoglutarate carrier (OGC), and this interaction is modulated by GSH. To further support a role for Bcl-2 in regulating mitochondrial GSH transport, we show that Bcl-2 requires OGC for protection against apoptosis induced by oxidative stress and to increase the mitochondrial GSH pool. These data suggest that Bcl-2 plays a key role in regulating mitochondrial GSH transport and that the enhancement of the interaction between Bcl-2 and OGC by GSH may increase mitochondrial GSH transport.

The mechanisms of mitochondrial GSH transport thus far have only been extensively studied in liver and kidney, where the OGC and the dicarboxylate (DIC) carrier have been identified as inner membrane mitochondrial GSH transporters. Most studies examining mitochondrial GSH transport mechanisms in brain have not allowed for the distinction between neuronal and glial cell mitochondrial GSH transport mechanisms. Therefore, we employed primary cerebellar astrocytes and primary CGNs as a system to study mitochondrial GSH transport mechanisms and differences between neuronal and glial cells. It was found that cerebellar astrocytes use both the OGC and DIC to transport GSH into mitochondria, while CGNs preferentially use the DIC. In addition, discrete inhibition of one mitochondrial GSH transporter (DIC) using chemical inhibition and genetic knockdown, in CGNs, led to increased susceptibility to both oxidative and nitrosative stress. Overall, this suggests that inhibition of a single mitochondrial GSH transporter is sufficient to predispose neurons to oxidative stress, such as the conditions observed in neurodegenerative diseases.

Finally, the consequences of overexpressing a mitochondrial GSH transporter were examined. Stable NSC34 motor neuron- like cell lines were produced which overexpress OGC. The stable OGC cell lines had significantly increased mitochondrial GSH levels and were markedly resistant to both oxidative and nitrosative stress. More importantly, the stable OGC cell lines up-regulated Bcl-2 expression (a protein we have shown previously to interact with OGC) and this phenomenon was due to increased mitochondrial GSH levels. The up-regulation of Bcl-2 was required to sustain increased mitochondrial GSH levels and resistance to oxidative stress. Overall, this study implicates the importance of not only the mitochondrial GSH pool, but mitochondrial GSH transport for neuronal survival. These findings suggest that modulation of

mitochondrial GSH transport could be a novel therapeutic approach for neurodegenerative diseases.

Provenance

Recieved from ProQuest

Rights holder

Heather Marie Wilkins

File size

204 p.

File format

application/pdf

Language

en

Discipline

Neurosciences, Biochemistry

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