Demonstrating or verifying a current or past exposure to an environmental mitochondrial toxin is extraordinarily difficult. For any given toxin, tissue distribution and pharmacokinetics of the toxin may be unknown. Low-level exposure may not produce a clinical phenotype, and depending on the assay chosen, functional mitochondrial impairment or enzyme inhibition may not persist beyond the acute exposure (or metabolism/excretion of the toxin). Thus, there is a pressing need to develop a biomarker for exposure to environmental mitochondrial inhibitors that is (i) sensitive, (i) at least semi- quantitative, (iii) enduring after toxin exposure has ceased, (iv) stable after specimen collection, and (v) highly reproducible. Another problem is that after exposure to certain mitochondrial toxins, some tissues are selectively vulnerable to damage while others are resistant. For example, when rats are exposed to rotenone chronically, they develop selective degeneration of the nigrostriatal dopamine system similar to Parkinson disease. The molecular and physiological basis for such heterogeneity in biological response is obscure and no biomarker of selective vulnerability exists. Our preliminary data suggest that mtDNA damage in blood or skeletal muscle may provide a biomarker of past or ongoing toxin exposure, and nuclear DNA (nDNA) damage may be a preclinical biomarker of selective vulnerability. For these studies, we will use an extremely sensitive PCR-based assay of DNA damage (both mtDNA &nDNA) that simultaneously allows assessment of multiple forms of damage, and further allows assessment of mtDNA and nDNA damage in the same samples, without a need for mitochondrial isolation.
The Specific Aims of this proposal are: 1. (a) Determine how soon after rotenone exposure mtDNA damage can be detected in blood and muscle. (b) Determine the duration, after a single exposure, that mtDNA damage can be detected. 2. Determine the minimal level of complex I inhibition that is required to cause detectable mtDNA damage. 3. Determine whether nuclear DNA (nDNA) damage is a marker of tissues that are selectively vulnerable to mitochondrial toxin-induced degeneration.!! 4. (a) Determine whether peripheral mtDNA damage is a common feature of systemically active complex I inhibitors. (b) Determine whether mtDNA damage is a common feature of other ETC inhibitors, including those acting at complexes II-IV. Preliminary results suggest our assay will provide a biomarker that is relatively simple, extremely sensitive, quantitative, enduring after exposure has ceased, stable after collection, and highly reproducible.
Exposures to environmental mitochondrial toxins are extremely difficult to demonstrate or verify. We have employed an extremely sensitive assay for mitochondrial DNA damage and have shown that it can detect subclinical exposures, and can detect an exposure even after the primary affect of the toxin has ended. We now propose to further characterize the sensitivity and utility of this biomarker.
|Sanders, Laurie H; Paul, Kimberly C; Howlett, Evan H et al. (2017) Editor's Highlight: Base Excision Repair Variants and Pesticide Exposure Increase Parkinson's Disease Risk. Toxicol Sci 158:188-198|
|Tapias, Victor; Hu, Xiaoping; Luk, Kelvin C et al. (2017) Synthetic alpha-synuclein fibrils cause mitochondrial impairment and selective dopamine neurodegeneration in part via iNOS-mediated nitric oxide production. Cell Mol Life Sci 74:2851-2874|
|Di Maio, Roberto; Barrett, Paul J; Hoffman, Eric K et al. (2016) ?-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson's disease. Sci Transl Med 8:342ra78|
|Tyurina, Y Y; Polimova, A M; Maciel, E et al. (2015) LC/MS analysis of cardiolipins in substantia nigra and plasma of rotenone-treated rats: Implication for mitochondrial dysfunction in Parkinson's disease. Free Radic Res 49:681-91|
|Rooney, John P; Ryde, Ian T; Sanders, Laurie H et al. (2015) PCR based determination of mitochondrial DNA copy number in multiple species. Methods Mol Biol 1241:23-38|
|Greenamyre, J Timothy; Sanders, Laurie H; Gasser, Thomas (2015) Fruit flies, bile acids, and Parkinson disease: a mitochondrial connection? Neurology 85:838-9|
|Zharikov, Alevtina D; Cannon, Jason R; Tapias, Victor et al. (2015) shRNA targeting ?-synuclein prevents neurodegeneration in a Parkinson's disease model. J Clin Invest 125:2721-35|
|Sanders, Laurie H; Howlett, Evan H; McCoy, Jennifer et al. (2014) Mitochondrial DNA damage as a peripheral biomarker for mitochondrial toxin exposure in rats. Toxicol Sci 142:395-402|
|Sanders, Laurie H; McCoy, Jennifer; Hu, Xiaoping et al. (2014) Mitochondrial DNA damage: molecular marker of vulnerable nigral neurons in Parkinson's disease. Neurobiol Dis 70:214-23|
|Sanders, Laurie H; Laganière, Josée; Cooper, Oliver et al. (2014) LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction. Neurobiol Dis 62:381-6|
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