This application tests the hypothesis that biochemically distinct oligomers of a-synuclein induce neuron degeneration in part by inhibiting the function of chaperone-mediated autophagy (CMA). a-Synuclein (a- syn) has been identified as one of the major components of the protein inclusions in the brains of subjects with sporadic and familial forms of Parkinson's disease (PD) and related neurodegenerative disorders. Deregulation in dopamine metabolism resulting in increased cytosolic dopamine that undergoes oxidation has been also considered as contributing pathogenic mechanism for the selective neuron dysfunction and death in PD. We have provided evidence that oxidized dopamine interacts with a-syn and propose that this interaction unifies two potential neurotoxic mechanisms responsible for PD and disorders characterized by a-syn inclusions. Despite the profound implications in the pathogenesis of disease the interaction of a-syn with dopamine in vivo has not been evaluated. Therefore we will elevate the levels of dopamine in the substantia nigra of the mice expressing human a-syn with the pathogenic A53T mutation driven by the mouse PrP promoter by injecting lentiviral vectors that deliver cDNA of human tyrosine hydroxylase (TH) with N-terminus R37E, R38E mutation (TH-RREE), which is not feed-back inhibited by dopamine. In these mice we will then: 1) Compare and contrast the effects of dopamine on regional formation and biochemical properties of a-syn oligomers. Experiments will define the regional distribution, biochemical and biophysical properties of the soluble a-syn oligomers. Experiments will also test the ex vivo effects of oligomers on neuron dysfunction, vesicular association and a-syn aggregation. 2) Evaluate the regional association of a-syn with CMA and determine the in vivo effects of increasing dopamine levels. Experiments will test the novel hypothesis that compromised function of CMA resulting from the interaction with oxidized dopamine-induced a-syn oligomers leads to neuron dysfunction by the accumulation of S-nitrosylated glyceraldehyde phosphate dehydrogenase. This hypothesis unites the effect of a-syn oligomers in blocking CMA with the previously established effects of nitric oxide-induced dopaminergic neuron death. 3) Investigate the effect of increasing the levels of dopamine on the phenotype of A53T a-syn expressing mice. Onset of phenotype will be determined followed by a comprehensive immunohistochemical and biochemical analysis of the brains for dopamine neuron viability, inclusion formation, and astrogliosis.
The conversion of soluble proteins into insoluble aggregates is a common pathological hallmark of age-related neurodegenerative disorders. In Parkinson's disease and related disorders the presence of intracellular inclusions composed mostly of aggregated a-synuclein represents an indispensable neuropathological diagnostic tool and suggests that the formation of insoluble a-synuclein is an important factor in pathogenesis. Parkinson's disease and related disorders are also characterized by the loss of neurons that synthesize the neurotransmitter dopamine. It has been postulated but not tested in a living organism that the aggregation and potential toxicity of a-synuclein is intimately influenced by dopamine. Experiments in this application offer a unique opportunity to explore the significance of the dopamine a-synuclein interaction in vivo and generated mice that may recapitulate additional features of the disease such as degeneration of dopamine-producing neurons improving our most basic understanding of the molecular mechanism of these neurodegenerative disorders.
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