Parkinsons disease (PD) neurodegeneration is characterized by loss of the dopaminergic cells of the substantia nigra, and has been linked to oxidative stress and mitochondrial dysfunction. The reactive neurotransmitter dopamine (DA) may play a role in neuronal vulnerability. DA oxidation has been shown to elicit dopaminergic toxicity in animal models, covalently modify proteins, and affect mitochondrial function. However, mitochondrial protein targets of DA modification are unknown. In this study, I utilized proteomic techniques to identify and characterize mitochondrial proteins altered following in vitro exposure to DA oxidation. Using two-dimensional difference in-gel electrophoresis and mass spectrometry analyses, I identified a subset of mitochondrial proteins that exhibited decreased abundance following exposure of isolated rat brain mitochondria to DA quinone (DAQ). Losses of two of these proteins, mitochondrial creatine kinase (MtCK) and mitofilin were further confirmed by Western blot analyses. Western blot also confirmed significant decreases of these two proteins in differentiated PC12 cells exposed to DA. I next utilized two-dimensional gel electrophoresis with autoradiography to identify proteins covalently modified by DAQ. I identified a subset of proteins covalently modified by ¹⁴C-DA from rat brain mitochondria exposed to ¹⁴C-DAQ and from differentiated SH-SY5Y cells exposed to ¹⁴C-DA. Proteins including mortalin/GRP75/mtHSP70, subunits of Complex I, MtCK, and mitofilin, amongst other proteins, were found to be covalently modified. We chose to further examine mitofilin, a protein implicated in maintaining mitochondrial structure. To characterize the effect of altered mitofilin levels on cell viability, I utilized overexpression and knockdown techniques to modulate mitofilin expression in dopaminergic cell lines, differentiated PC12 and SH-SY5Y cells, and examined their response to dopaminergic toxins, DA and rotenone. I found that increased mitofilin expression was protective against both DA- and rotenone-induced toxicity in both cell lines, and decreased mitofilin enhanced DA-induced toxicity in differentiated SH-SY5Y cells. Therefore, in this thesis, I identified a subset of mitochondrial and cellular proteins that are potential targets of DA-induced modification, and may have roles in PD pathogenesis. Modulating the expression level of one of these proteins, mitofilin, affected the cellular response to toxins, and may play a role in dopaminergic cell vulnerability.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-08012008-151519 |
| Date | 04 November 2008 |
| Creators | Van Laar, Victor Steven |
| Contributors | Donald B. DeFranco, Ph.D., David Allan Butterfield, Ph.D., Teresa G. Hastings, Ph.D., Michael Cascio, Ph.D., John Timothy Greenamyre, M.D., Ph.D., John Patrick Card, Ph.D. |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-08012008-151519/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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