Elsevier

Neurobiology of Aging

Volume 26, Issue 10, November–December 2005, Pages 1343-1355
Neurobiology of Aging

Somatic mitochondrial DNA mutations in single neurons and glia

https://doi.org/10.1016/j.neurobiolaging.2004.11.008Get rights and content

Abstract

Somatic mitochondrial DNA (mtDNA) point mutations reach high levels in the brain. However, the cell types that accumulate mutations and the patterns of mutations within individual cells are not known. We have quantified somatic mtDNA mutations in 28 single neurons and in 18 single glia from post-mortem human substantia nigra of six control subjects. Both neurons and glia contain mtDNA with somatic mutations. Single neurons harbor a geometric mean (95% CI) of 200.3 (152.9–262.4) somatic mtDNA point mutations per million base pairs, compared to 133.8 (97.5–184.9) for single glia (p = 0.0251). If mutations detected multiple times in the same cell are counted only once, the mean mutation level per million base pairs remains elevated in single neurons (146.9; 124.0–174.2) compared to single glia (100.5; 81.5–126.5; p = 0.009). Multiple distinct somatic point mutations are present in different cells from the same subject. Most of these mutations are individually present at low levels (less than 10–20% of mtDNA molecules), but with high aggregate mutation levels, particularly in neurons. These mutations may contribute to changes in brain function during normal aging and neurodegenerative disorders.

Introduction

Recent data indicate that somatic mtDNA mutations accumulate with age, and suggest that such mutations may have significant functional consequences. Single cytochrome c oxidase deficient muscle fibers from elderly subjects have high aggregate levels of individually rare mtDNA mutations [5], [17]. In the human brain, cytochrome c oxidase deficient hippocampal neurons have been shown to increase in prevalence with age [14], but the levels of mtDNA mutations in these cells is not known. We have previously reported that in brain homogenate somatic mtDNA mutations accumulate with age. Each specific somatic mutation is extremely rare, yet the aggregate level of all such mutations considered together is high, reaching an average of about 3 per mitochondrial genome in the cortex and substantia nigra of elderly subjects [34], [47]. The aggregate burden of these mutations is negatively correlated with mitochondrial electron transport chain function [34]. Others have reported that transgenic mice expressing a proofreading-deficient mtDNA polymerase exhibit accelerated aging and premature death [53], providing experimental evidence that the accumulation of somatic mtDNA mutations can be causative of age-related disorders. But a better understanding of the role of somatic mtDNA mutations in the human brain is hampered by lack of data on key issues, such the levels of somatic mtDNA mutations in different cell types in the brain and the patterns of accumulation of individual mutations within single cells.

Two different mechanisms could account for the pattern of multiple individually rare somatic mtDNA mutations that we have observed previously in brain homogenates (Fig. 1). In one scenario, single specific mtDNA point mutations might be present at high levels (present in most mtDNA molecules) within an individual cell, but with different mutations in different cells of the same subject. Any single specific mutation would therefore be rare in brain homogenate, even though it might be very frequent or even homoplasmic within an individual cell, because only one cell would contain that particular mutation. An alternative scenario is that each cell contains mtDNA with many different somatic mtDNA mutations, with each specific mutation present at low levels (present in only a small fraction of mtDNA molecules). In this second scenario, multiple distinct individually rare mutations result in a high aggregate mutational burden within a single cell. Although these two scenarios are indistinguishable at the level of tissue homogenate, they may differ in functional consequences. A mutation that is present at a high level within a single cell is more likely to be deleterious as it is less likely to be complemented by wild-type mtDNA molecules within the same cell. In contrast, functional complementation is more likely to occur within a cell with multiple different mutations, each present at a low level [2], [23], [43]. Analyses of brain homogenate DNA cannot distinguish between these two possible mechanisms. In addition, studies of brain homogenate cannot identify the cell types that are accumulating mutations.

We have applied the technology of Laser Capture Microdissection (LCM) to study mtDNA mutations in single cells from human brain. We have focused our attention on the neurons and glia found in the substantia nigra. Dopaminergic neurons in the substantia nigra are lost during normal aging [11], [24], [37], and exhibit selective vulnerability in PD [15], [36]. There is clear evidence for a deficit of mitochondrial electron transport chain function in the substantia nigra in PD [45]. The cellular origin of this deficit is less certain. While loss of dopaminergic neurons is the most characteristic feature of this disease, there is also evidence suggesting a failure of activation of astrocytes [40], which may play a role in the degenerative process [20], [51]. We have quantified somatic mtDNA mutations in isolated single neurons and single glia from post-mortem human substantia nigra using allele-specific PCR to preferentially amplify the targeted DNA, followed by a highly sensitive cloning-sequencing strategy.

Section snippets

Subjects

Brain tissue was obtained from the Massachusetts Alzheimer's Disease Research Center at Massachusetts General Hospital and from the Harvard Brain Tissue Resource Center at McLean Hospital, Boston, MA. Subjects were Caucasians without a clinical history of neurological disease and with normal post-mortem examinations of the brain. Tissue from six normal control subjects was studied, with ages at the time of death of 40, 57, 58, 58, 62, and 69 years. Post-mortem intervals ranged from 16.6 to 21.2 

Aggregate somatic point mutation levels in single neurons and single glia

For the primary analyses, if the same mutation appeared in multiple clones, it was counted once for each clone in which it appeared. We found that the geometric mean of somatic mtDNA point mutation levels per million base pairs in single neurons (200.3; 95% CI: 152.9, 262.4) was significantly higher than the geometric mean of mutation levels in single glia (133.8; 95% CI: 97.5, 184.9; p = 0.025, mixed effects model; Fig. 4A). This difference was highlighted at the extremes, with neurons

Discussion

The data presented here reveal that in post-mortem human substantia nigra there are high aggregate levels of somatic mtDNA mutations present in both neurons and glia. The mean level of somatic mtDNA point mutations in single neurons is significantly higher than the mean level in single glia. When recurrent mutations are counted once for each clone in which they occur, the increase was 1.5-fold. Extrapolating from our data suggests mean numbers of somatic point mutations per mitochondrial genome

Acknowledgements

We thank Dr. Matthew P. Frosch for his valuable guidance. We thank Drs. Guang-Jun Liu and Victor Lee for technical assistance. This work was supported by the APDA Advanced Center for Parkinson Research at MGH (I.C.C.), the American Federation for Aging Research (M.T.L.: Beeson Award), the NINDS (D.K.S.: K02 NS044482), the NIA (M.F.B.: AG20729), a Cotzias Fellowship Award from the American Parkinson Disease Association (D.K.S.), the Harvard Center for Neurodegeneration and Repair (R.A.B.), and

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