Somatic mitochondrial DNA mutations in cortex and substantia nigra in aging and Parkinson’s disease
Introduction
Accumulation of mitochondrial DNA (mtDNA) mutations is hypothesized to play a role in aging and in age-related neurodegenerative diseases [36]. A potential mechanism that may contribute to the induction of these mutations is oxidative damage to mtDNA. 8-Hydroxy-2′-deoxyguanosine (OH8dG), a well-characterized marker of oxidative damage to DNA [28], is reported to be increased as much as 23-fold in mtDNA compared to nuclear DNA [23], [49], to increase with aging [2], [24], [42], and to increase further in neurodegenerative diseases [1], [19], [41], [51], [55], including Parkinson’s disease (PD) [1], [51], [55], [64], although these data are controversial due to potential technical artifacts [5]. OH8dG can induce mutations during DNA replication as it can pair with adenine as well as cytosine with almost equal efficiencies, resulting in G:C to T:A and T:A to G:C mutations [10], [32]. However, direct evidence for the accumulation with age of G:C to T:A and T:A to G:C mutations in mtDNA mutations is lacking.
Mitochondrial complex I (CI) activity in the substantia nigra (SN) declines with normal aging [8], [9]. This decline occurs to a greater extent in PD compared to age-matched controls [27], [40], [52], [53], [54]. Inhibitors of CI (MPP+ [33] or rotenone [6]) can induce parkinsonism and SN neuronal death in experimental animals, suggesting that CI deficiency may be central to the pathophysiology of PD. Cytoplasmic hybrid (“cybrid”) cell lines expressing mtDNA from PD patients also manifest CI deficiency, suggesting that mtDNA mutations account for the defect [22], [60]. However, extensive sequencing of the mtDNA-encoded CI and transfer RNA genes has failed to reveal significant mtDNA mutations in the majority of PD patients [57].
These data can be reconciled if acquired mtDNA mutations account for the CI defect. CI genes represent 7 of the 13 mtDNA protein-coding genes, which occupy 38% of the mitochondrial genome. As a result, CI may be preferentially affected by randomly positioned mutations compared to other products of the mitochondrial genome [13]. There is evidence that individual acquired mtDNA mutations do not achieve high mutational burdens in the brain, though the cumulative effect of multiple individually rare mutations may be significant [35], [56]. This situation of multiple different individually rare mtDNA species would be undetectable by standard sequencing methods, which detect mutations only when a specific mutation (at a particular base pair) is present in a large percentage of the mtDNA molecules. More sensitive techniques, such as two-dimensional denaturing gradient gel electrophoresis [61], still may lack the sensitivity required to detect this situation. However, these individually rare mutations can be detected by isolating and clonally expanding individual mtDNA species. Using a similar strategy, but with a lower fidelity PCR system than the one used for the current study, we have shown previously that levels of point mutations in mtDNA increase with age in the cortex and inversely correlate with mitochondrial electron transport chain activity [35]. We now report results using a highly sensitive cloning and sequencing strategy to estimate the frequency in a mitochondrial CI gene of acquired mtDNA mutations in the frontal cortex (FCtx) and SN in young and old control subjects as well as in PD. We also report analyses for the subset of G:C to T:A and T:A to G:C mutations that can be induced by oxidative stress.
Section snippets
Overview
The strategy to identify point mutations in single mtDNA molecules is as follows: (1) purification of DNA, (2) PCR amplification of a 1.2 kb section of the mtDNA using the Advantage HF-2 PCR Kit (Clontech, Palo Alto, CA, USA), which includes a polymerase with proofreading activity, (3) cloning of individual mtDNA fragments, (4) PCR amplification of at least 50 cloned mtDNA fragments, and (5) sequencing of the PCR-amplified cloned fragments.
Brain tissue
Frozen brain tissue from FCtx or SN was provided by the
Results
The basic strategy for identifying acquired mtDNA mutations was a modified version of a previously described protocol [35]. First, DNA was isolated from FCtx or SN. Next, a mtDNA fragment including part of the gene encoding the ND4 subunit of CI was amplified by PCR. This PCR product was then cloned and between 35 and 139 clones were selected. A “post-cloning” PCR reaction was then performed on DNA from each of these clones. Next, these PCR products each were sequenced in order to identify
Aggregate burden of mtDNA point mutations in elderly subjects
Though each individual point mutation analyzed here is present at an extremely low mutational burden and thus is unlikely to be of functional significance on its own, the aggregate burden of these mutations may reach significant levels. Somatic mtDNA mutations are predicted to accumulate in each of the seven mtDNA-encoded CI genes. If we consider a single gene, the ND5 gene (1812 base pairs, the longest of the CI genes), extrapolation from our results indicates that 39% of ND5 gene copies are
Acknowledgements
This work was supported by the NINDS (K08 NS01971 and K02 NS4311-01, DKS; and K24 NS02239, DRJ), a George C. Cotzias Fellowship Award from the American Parkinson Disease Foundation (DKS), an anonymous donor fund for Parkinson’s disease research (DKS), the NIA (K08 AG00798, MTL; R01 AG20729, MFB), and a Beeson Scholarship from the American Federation for Aging Research (MTL).
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