Single-cell genetic analysis of the pathogenic role of neuronal genomic instability in environmental toxicant exposure

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Project Summary/Abstract The majority of all Parkinson’s disease (PD) cases have no identifiable inheritance and occur in a sporadic form. Epidemiologic studies have found an increased risk of PD associated with exposure to environmental, while the reduced risk of PD associated with coffee consumption. The recent single-neuron sequencing studies offer direct evidence there is an age-dependent increase of somatic brain mutations, suggesting adult terminally differentiated neurons can acquire somatic mutations, possibly precipitated by environmental factors. Given the plethora of correlational evidence describing pathological conditions in which levels of DNA damage/response and mutations were identified, the most critical question is the extent to which the significance of such DNA instability has relevance for the pathogenesis of neurodegeneration. One clue comes from a protective environmental factor of PD, caffeine. Its antiparkinsonian effects have been attributed to the potent inhibition of the Ataxia-Telangiectasia Mutated (ATM) pathway. ATM is the key orchestrator of oxidative stress and DNA damage response (DDR). Robust DDR has been closely connected to multiple neurodegenerative disorders. We hypothesize that the exposure to PD associated environmental toxicants compromises genomic stability to abnormally activate the DNA damage response (converging on ATM activation), which plays a central pathogenic role to lead to the final demise of neurodegeneration. Attenuating abnormally activated ATM signaling is neuroprotective. Employing our newly developed Mosaicism with Repeat Frameshift (MORF) strategy, we demonstrated the instability of a hypermutable repeat sequence (accounting for 3% of human genome) could be harnessed for sparse and stochastic visualize neurodegeneration and genomic instability in aging, stroke, and neurodegeneration. In aim 1, we will develop an AAV mediated quantitative ratiometric sensor of genomic instability that integrates the varying lengths of mononucleotide repeats, incompatible LoxP sites, and membrane-tethered multi-color fluorescence proteins. We will use both AAV and transgenic MORF strategy to track single-neuron with DNA instability after environmental toxicant exposure to determine the pathologic consequence. In aim 2, we will determine if the genetic reduction of DNA damage response in somatic cells with DNA instability can rescue DA neuron degeneration. Our project will develop a novel genetic sensor and actuator to study somatic brain mosaicism. The results will reveal the pathogenic significance of environment-driven neuronal genomic instability and the crucial role of the DDR pathway, thus transforming the current paradigm of the pathogenic role of environmental toxicant exposure in sporadic PD. 1