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Quantitative determination of deleted mitochondrial DNA relative to normal DNA in parkinsonian striatum by a kinetic
1Department of Biomedical Chemistry, Faculty of Medicine, University of Nagoya, Japan.
Abstract:
Deleted mitochondrial DNA (mtDNA) was accumulated in the parkinsonian striatum, but the same deleted mtDNA was also detectable in the control striatum when cycles of polymerase chain reaction were increased. To discriminate between these pathological and physiological conditions, we quantitatively analyzed the proportion of deleted mtDNA to normal mtDNA by measuring the incorporation of alpha-[32P]deoxycytosine triphosphate into mtDNA fragments by using a laser image analyzer. To estimate the molar ratio of the deleted mtDNA to normal mtDNA, the radioactivity was normalized by each fragment size. By plotting logarithms of normalized radioactivities against PCR amplification cycles, straight lines were obtained with different slopes. By extrapolation of the line to the zero amplification, the proportion of mutant mtDNA to normal mtDNA in the original sample from the parkinsonian striatum was estimated to be ca. 5%, which was at least ten times higher than the proportion of ca. 0.3% in the control striatum. These results indicate that phenotype of the mutant mtDNA as Parkinson's disease is expressed when the proportion of deleted mtDNA to normal mtDNA exceeds a threshold of ten times higher value than in the normal subject.
Insights
Parkinson's disease may be linked to a higher proportion of deleted mitochondrial DNA (mtDNA). This study quantifies deleted mtDNA, finding significantly higher levels in Parkinson's patients compared to controls.
Area of Science:
- Neuroscience
- Genetics
- Biochemistry
Background:
- Deleted mitochondrial DNA (mtDNA) is found in Parkinson's disease (PD) brains.
- Detecting deleted mtDNA in controls suggests a need for quantitative analysis to distinguish pathological from physiological levels.
Purpose of the Study:
- To quantitatively analyze the proportion of deleted mtDNA to normal mtDNA in parkinsonian and control striata.
- To establish a threshold for deleted mtDNA indicative of Parkinson's disease.
Main Methods:
- Quantitative analysis of deleted mtDNA proportion using alpha-[32P]deoxycytosine triphosphate incorporation and laser image analysis.
- Estimation of molar ratio of deleted to normal mtDNA by normalizing radioactivity by fragment size.
- Extrapolation of PCR amplification data to determine original mtDNA proportions.
Main Results:
- The proportion of deleted mtDNA to normal mtDNA in parkinsonian striatum was approximately 5%.
- This proportion was at least ten times higher than the approximately 0.3% found in control striata.
- A threshold for deleted mtDNA was identified, distinguishing PD from normal subjects.
Conclusions:
- Parkinson's disease phenotype expression correlates with deleted mtDNA levels exceeding a specific threshold.
- Quantitative analysis of deleted mtDNA is crucial for understanding its role in Parkinson's disease pathogenesis.
- Elevated deleted mtDNA proportion serves as a potential biomarker for Parkinson's disease.