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Defective mitochondrial DNA homeostasis in the substantia nigra in Parkinson disease

View Article: PubMed Central - PubMed

ABSTRACT

Increased somatic mitochondrial DNA (mtDNA) mutagenesis causes premature aging in mice, and mtDNA damage accumulates in the human brain with aging and neurodegenerative disorders such as Parkinson disease (PD). Here, we study the complete spectrum of mtDNA changes, including deletions, copy-number variation and point mutations, in single neurons from the dopaminergic substantia nigra and other brain areas of individuals with Parkinson disease and neurologically healthy controls. We show that in dopaminergic substantia nigra neurons of healthy individuals, mtDNA copy number increases with age, maintaining the pool of wild-type mtDNA population in spite of accumulating deletions. This upregulation fails to occur in individuals with Parkinson disease, however, resulting in depletion of the wild-type mtDNA population. By contrast, neuronal mtDNA point mutational load is not increased in Parkinson disease. Our findings suggest that dysregulation of mtDNA homeostasis is a key process in the pathogenesis of neuronal loss in Parkinson disease.

No MeSH data available.


Amplification and deep-sequencing of mtDNA from single dopaminergic substantia nigra neurons of individuals with PD and controls.(a) Schematic representation of human mtDNA, indicating the major arc (grey arc), where the vast majority of mtDNA deletions occur, and the position of the 4,767 bp fragment used for ultra-deep sequencing (magenta arc). mtDNA genes are coloured by type of transcript and designated by standard nomenclature. Genes encoding tRNAs are coloured green and designated by the one-letter code of their corresponding amino acid. (b) PCR products were amplified from single-cell lysates and analysed by agarose gel electrophoresis before sequencing. Samples with low (*) or no (#) amplification were omitted. (c) Mean sequencing depth and coverage of the mtDNA fragment after completed quality control. The solid black line shows the mean depth for all samples per mtDNA position and the shaded grey line corresponds to the 95% confidence interval (CI). Indicatively, 95% of the sequence was covered at >12,000 × depth and 80% at >50,000 × depth. (d) Total burden of mtDNA SNVs in single dopaminergic neurons of the substantia nigra plotted against the base 10 logarithm of heteroplasmy frequency (HF). Bars show 95% CIs. Point mtDNA variation is found at generally low levels in nigral neurons and the majority of variants cluster at heteroplasmic frequencies below 1%. There is no difference in point mutational burden between individuals with PD (blue) and controls (red).
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f7: Amplification and deep-sequencing of mtDNA from single dopaminergic substantia nigra neurons of individuals with PD and controls.(a) Schematic representation of human mtDNA, indicating the major arc (grey arc), where the vast majority of mtDNA deletions occur, and the position of the 4,767 bp fragment used for ultra-deep sequencing (magenta arc). mtDNA genes are coloured by type of transcript and designated by standard nomenclature. Genes encoding tRNAs are coloured green and designated by the one-letter code of their corresponding amino acid. (b) PCR products were amplified from single-cell lysates and analysed by agarose gel electrophoresis before sequencing. Samples with low (*) or no (#) amplification were omitted. (c) Mean sequencing depth and coverage of the mtDNA fragment after completed quality control. The solid black line shows the mean depth for all samples per mtDNA position and the shaded grey line corresponds to the 95% confidence interval (CI). Indicatively, 95% of the sequence was covered at >12,000 × depth and 80% at >50,000 × depth. (d) Total burden of mtDNA SNVs in single dopaminergic neurons of the substantia nigra plotted against the base 10 logarithm of heteroplasmy frequency (HF). Bars show 95% CIs. Point mtDNA variation is found at generally low levels in nigral neurons and the majority of variants cluster at heteroplasmic frequencies below 1%. There is no difference in point mutational burden between individuals with PD (blue) and controls (red).

Mentions: To assess whether point mutations also contribute to neuronal mtDNA pathology in PD, we performed ultra-deep sequencing of a ∼5 kb fragment of mtDNA (rCRS 1157–5924) in a total of 184 single dopaminergic substantia nigra neurons from the 10 individuals with PD and 10 matched controls. The sequenced fragment was localized away from the deletion region to ensure it represented the total mtDNA population of each neuron (for details see the ‘Methods' section and Fig. 7a,b). Sequence data from 144 neurons successfully passed quality control and were used in the analyses. High-sequence coverage (Fig. 7c) allowed for detection of single-nucleotide variants (SNVs) as low as at a heteroplasmic frequency (HF) of 0.1% (Fig. 7d). The point mutation frequency was estimated as number of SNVs per 1,000 bp mtDNA.


Defective mitochondrial DNA homeostasis in the substantia nigra in Parkinson disease
Amplification and deep-sequencing of mtDNA from single dopaminergic substantia nigra neurons of individuals with PD and controls.(a) Schematic representation of human mtDNA, indicating the major arc (grey arc), where the vast majority of mtDNA deletions occur, and the position of the 4,767 bp fragment used for ultra-deep sequencing (magenta arc). mtDNA genes are coloured by type of transcript and designated by standard nomenclature. Genes encoding tRNAs are coloured green and designated by the one-letter code of their corresponding amino acid. (b) PCR products were amplified from single-cell lysates and analysed by agarose gel electrophoresis before sequencing. Samples with low (*) or no (#) amplification were omitted. (c) Mean sequencing depth and coverage of the mtDNA fragment after completed quality control. The solid black line shows the mean depth for all samples per mtDNA position and the shaded grey line corresponds to the 95% confidence interval (CI). Indicatively, 95% of the sequence was covered at >12,000 × depth and 80% at >50,000 × depth. (d) Total burden of mtDNA SNVs in single dopaminergic neurons of the substantia nigra plotted against the base 10 logarithm of heteroplasmy frequency (HF). Bars show 95% CIs. Point mtDNA variation is found at generally low levels in nigral neurons and the majority of variants cluster at heteroplasmic frequencies below 1%. There is no difference in point mutational burden between individuals with PD (blue) and controls (red).
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f7: Amplification and deep-sequencing of mtDNA from single dopaminergic substantia nigra neurons of individuals with PD and controls.(a) Schematic representation of human mtDNA, indicating the major arc (grey arc), where the vast majority of mtDNA deletions occur, and the position of the 4,767 bp fragment used for ultra-deep sequencing (magenta arc). mtDNA genes are coloured by type of transcript and designated by standard nomenclature. Genes encoding tRNAs are coloured green and designated by the one-letter code of their corresponding amino acid. (b) PCR products were amplified from single-cell lysates and analysed by agarose gel electrophoresis before sequencing. Samples with low (*) or no (#) amplification were omitted. (c) Mean sequencing depth and coverage of the mtDNA fragment after completed quality control. The solid black line shows the mean depth for all samples per mtDNA position and the shaded grey line corresponds to the 95% confidence interval (CI). Indicatively, 95% of the sequence was covered at >12,000 × depth and 80% at >50,000 × depth. (d) Total burden of mtDNA SNVs in single dopaminergic neurons of the substantia nigra plotted against the base 10 logarithm of heteroplasmy frequency (HF). Bars show 95% CIs. Point mtDNA variation is found at generally low levels in nigral neurons and the majority of variants cluster at heteroplasmic frequencies below 1%. There is no difference in point mutational burden between individuals with PD (blue) and controls (red).
Mentions: To assess whether point mutations also contribute to neuronal mtDNA pathology in PD, we performed ultra-deep sequencing of a ∼5 kb fragment of mtDNA (rCRS 1157–5924) in a total of 184 single dopaminergic substantia nigra neurons from the 10 individuals with PD and 10 matched controls. The sequenced fragment was localized away from the deletion region to ensure it represented the total mtDNA population of each neuron (for details see the ‘Methods' section and Fig. 7a,b). Sequence data from 144 neurons successfully passed quality control and were used in the analyses. High-sequence coverage (Fig. 7c) allowed for detection of single-nucleotide variants (SNVs) as low as at a heteroplasmic frequency (HF) of 0.1% (Fig. 7d). The point mutation frequency was estimated as number of SNVs per 1,000 bp mtDNA.

View Article: PubMed Central - PubMed

ABSTRACT

Increased somatic mitochondrial DNA (mtDNA) mutagenesis causes premature aging in mice, and mtDNA damage accumulates in the human brain with aging and neurodegenerative disorders such as Parkinson disease (PD). Here, we study the complete spectrum of mtDNA changes, including deletions, copy-number variation and point mutations, in single neurons from the dopaminergic substantia nigra and other brain areas of individuals with Parkinson disease and neurologically healthy controls. We show that in dopaminergic substantia nigra neurons of healthy individuals, mtDNA copy number increases with age, maintaining the pool of wild-type mtDNA population in spite of accumulating deletions. This upregulation fails to occur in individuals with Parkinson disease, however, resulting in depletion of the wild-type mtDNA population. By contrast, neuronal mtDNA point mutational load is not increased in Parkinson disease. Our findings suggest that dysregulation of mtDNA homeostasis is a key process in the pathogenesis of neuronal loss in Parkinson disease.

No MeSH data available.