Mitochondrial DNA (mtDNA) mutations and mitochondrial dysfunction

Activity of mitochondrial complex I is decreased in Parkinson’s disease (PD). Key features of PD are reproduced by complex I inhibitors (MPTP or rotenone). "Cybrid" cell lines expressing mtDNA from PD patients also show complex I deficiency, implicating a role for mtDNA mutations. We have screened large numbers of PD patients and controls for mtDNA mutations by comprehensive sequencing of the mtDNA encoded complex I and tRNA genes followed by additional screening by restriction fragment length polymorphism (RFLP) analyses. Several PD patients with potentially pathogenic mutations were identified. However, significant mtDNA mutations were absent in the majority of patients. This led to our focus on somatic mtDNA mutations.

Somatic Mitochondrial DNA Mutations and Substantia Nigra Neuronal Degeneration

One explanation for the failure to identify mutations in the majority of PD patients may be that acquired mtDNA mutations, which may not reach levels detectable by sequencing at any individual site, cumulatively could cause mitochondrial dysfunction. Neurons may be particularly susceptible to oxidative damage, which can induce acquired (somatic) mutations, particularly in mtDNA. We are now using laser capture microdissection (LCM) in order to isolate specific cell types from different brain regions from postmortem human brain from early PD, late PD, and control cases to assess the potential role of somatic mtDNA mutations in the loss of substantia nigra neurons in PD.

A related set of studies involve assessing the potential functional impact of somatic mtDNA mutations using the Polg "mutator mice." These transgenic mice express proofreading deficient mtDNA polymerase gamma, resulting in an accelerated accumulation of somatic mtDNA mutations. Though they appear normal at birth, they develop a premature aging phenotype. We are investigating the hypothesis that the increase in somatic mtDNA mutations in these mice will enhance the susceptibility of dopaminergic neurons in the substantia nigra to degeneration.

Strategies for Neuroprotection in PD

Mutations, or duplication or triplication of the normal in alpha-synuclein gene, lead to autosomal dominant PD. Increased levels of expression of alpha-synuclein in dopaminergic cells increases susceptibility to oxidative stress. We are investigating transcriptional regulation of antioxidant defense mechanisms, both in terms of the potential impact of alpha-synuclein on these mechanisms, and as potential neuroprotective targets. Ongoing translational work in the lab involves testing potential small molecule and gene therapy neuroprotective strategies in transgenic mice overexpressing wild-type or mutant alpha-synuclein, as well as in the MPTP toxin model of PD.

PGC-1α is a transcriptional coactivator that coordinately upregulates genes required for mitochondrial biogenesis, and also upregulates multiple antioxidant genes. Prior studies suggest an important role for PGC-1α in protection against oxidative stress in the brain, and implicate low PGC-1α as playing an important role in PD. We are now using stereotaxic injection of viral vectors to overexpress PGC-1α in the substantia nigra in mice to test the prediction that it will protect against MPTP toxicity and against the chronic degeneration that occurs in transgenic alpha-synuclein overexpressing mouse models of PD. Our initial studies demonstrate an unexpected downregulation of Pitx3 when PGC-1α is overexpressed at high levels. Pitx3 is a transcription factor that is important for the maintenance and survival of dopaminergic neurons. We are now investigating strategies to achieve the neuroprotective potential of PGC-1α as a therapeutic target while avoiding this potentially deleterious effect on Pitx3.

In relation to our work on the role of somatic mtDNA mutations in aging and neurodegeneration, we also are studying strategies to drive selection against a heteroplasmic pathogenic mtDNA mutation. Using in vitro and in vivo models, we are using different strategies to inhibit mTOR kinase activity to upregulate mitophagy, which is a natural cellular mechanism for preferentially degrading dysfunctional mitochondria. We find that this process holds promise as a therapeutic strategy to reduce levels of heteroplasmic mtDNA mutations.