Van Laar, Victor Steven
(2008)
PROTEOMIC ANALYSIS OF DOPAMINE OXIDATION INDUCED MODIFICATIONS TO MITOCHONDRIAL PROTEINS: IMPLICATIONS FOR PARKINSON'S DISEASE.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Parkinson's 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.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
| Creators | Email | Pitt Username | ORCID  |
|---|
| Van Laar, Victor Steven | viv2@pitt.edu | VIV2 | |
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| ETD Committee: |
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| Date: |
4 November 2008 |
| Date Type: |
Completion |
| Defense Date: |
23 July 2008 |
| Approval Date: |
4 November 2008 |
| Submission Date: |
1 August 2008 |
| Access Restriction: |
5 year -- Restrict access to University of Pittsburgh for a period of 5 years. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Neuroscience |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
dopamine oxidation; dopamine quinone; mitochondria; mitofilin; Parkinson's disease; proteomics |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-08012008-151519/, etd-08012008-151519 |
| Date Deposited: |
10 Nov 2011 19:56 |
| Last Modified: |
15 Nov 2016 13:47 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/8803 |
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