Mitochondrial DNA (mtDNA) mutations and mitochondrial dysfunction
Activity of mitochondrial complex I is decreased in 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 mutaions.
Somatic Mitcochondrial 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.
I. NAC: We are studying the impact of oral n-aceytlcysteine supplementation on the chronic progressive degeneration of dopaminergic neurons and terminals in these mouse models of PD.
II. PGC-1a and NRF2: 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. 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. Other studies focus on another potential neuroprotective target, NRF2, a transcription factor that is responsible for the upregulation of many antioxidant genes in response to oxidative stress.