Mitochrondrial Etiologies of Parkinsons Disease

University Of Virginia Charlottesville, Charlottesville VA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] This is an application to renew the University of Virginia Udall Parkinson's Disease Research Center of Excellence. The focus of this Center is to define the contributions of mitochondrial genes to the pathogenesis and progression of Parkinson's disease (PD), a major neurodegenerative disease of adults that afflicts ~1 million Americans. Although most PD primary motor symptoms arise from loss of nigral dopamine neurons, brain involvement with PD is more extensive. Understanding PD pathogenesis requires defining mechanisms of both dopamine neuron vulnerability and processes that degrade synaptic function in other areas. One appealing unifying mechanism is excessive protein damage and aggregation, leading to increased neuronal stress and formation of Lewy neuritis and Lewy bodies. Because the mitochondrial transgenic cybrid cell model recapitulates essential biochemical and neuropathological components of sporadic PD, including formation of Lewy bodies and aggregates resembling Lewy neurites, PD mitochondrial gene expression appears to contribute to pathogenesis. However, traditional mitochondrial DNA (mtDNA) sequencing has not revealed PD-associated mtDNA mutations, signifying that a more intensive approach is needed to uncover this relationship. This proposal has four Projects and two Cores, a Consortium with the University of Oregon and ongoing collaboration with the Duke University Udall Center. Project 1utilizes an intensive mtDNA sequencing strategy to identify low level abundance mtDNA mutations in complex I genes and tRNA's. Our use of this sequencing approach has uncovered extensive, low-abundance, heteroplasmic mutations in PD brain homogenates and will be applied to analysis of mtDNA in laser-captured nigral neurons from PD and CTL brains and in PD/CTL cybrids. In Projects 2 and 3, complementary studies in PD/CTL cybrids differentiated into non dividing DA neurons will define how protein aggregation occurs, what the consequences are for neuronal function and survival, and how manipulation of endogenous and exogenous oxidative stresses affects these processes. Comparisons will be made to differentiated neurons expressing A30P and A53T a-synuclein mutations causal for some cases of familial PD. In Project 4, central and peripheral oxidative stress will be measured in PD subjects and correlated with cybrid oxidative stress and alterations in complex subunit assembly and post-translational modifications. The in vivo antioxidative actions of a novel neuroprotectant, R(+) PPX, will be tested in PD subjects. The outcome of this Program will be a comprehensive understanding of mitochondrial genetics in vulnerable PD nigral neurons and cybrid neurons, greater insights into the relationships among protein aggregation, neuronal function and cell survival, increased molecular insights into oxidative stress and complex I dysfunction in PD, and early clinical development of a novel neuroprotective therapy for PD. [unreadable] [unreadable] PROJECT 1 [unreadable] [unreadable] Title: Mitochondrial DNA mutations in Parkinson's disease [unreadable] [unreadable] PI: W. Davis Parker, MD [unreadable] [unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] The two Specific Aims of Project 1 are focused on identifying mitochondrial DNA mutations associated with the pathogenesis of idiopathic Parkinson's disease (PD). Multiple PD tissues, including nigral and non-nigral brain, exhibit a loss of catalytic activity of complex I in the electron transport chain (ETC). This systemic abnormality of PD mitochondrial ETC function is linked to the pathogenesis of PD by the actions of MPTP and rotenone, neurotoxins that reproduce PD pathology through inhibition of complex I. The anatomically widespread presence of the PD complex I defect is consistent with a genetic origin, although other causes are possible. Transfer of mitochondrial DNA (mtDNA) from PD platelets into neural host cells depleted of their endogenous mtDNA, creating cytoplasmic hybrid (cybrid) cells, reproduced the loss of complex I activity. Cybrids reproduce many pathogenic features of PD including increased oxidative stress and Lewy body formation. These results provide compelling evidence that mitochondrial DNA is likely to play an important role in the pathogenesis of PD. However, studies to date have not identified a high correlation between specific mtDNA mutations and PD. An intensive sequencing of the seven mitochondrial complex I genes in PD and CTL brains revealed extensive low-level heteroplasmy. PD-associated mutations may be revealed by clustering algorithms. These sequencing studies were conducted on brain homogenates and reflect mutations averaged across millions of cells of multiple types, and the question of mtDNA mutations specific to PD will have to be answered by sequencing mtDNA from single neurons. Project 1 will sequence mtDNA in single dopaminergic neurons from PD and CTL substantia nigra and in single PD and CTL cybrid cells. The presence of Lewy bodies in PD nigral neurons and PD cybrids will identify impaired cells and their mtDNA sequences will be compared to control cells (both nigral neurons and cybrids) as well as to non-Lewy body PD nigral neurons. [unreadable] [unreadable]