Project 458905

The molecular mechanisms underlying the pathogenesis of Parkinson's disease (PD) have been difficult to establish. However, studying alpha-synuclein (aSyn) - a known protein involved in PD pathology - has shed important light on some of these mechanisms. aSyn is primarily pre-synaptic and, at lower levels, nuclear. However, in PD, nuclear aSyn increases. I previously determined that increasing nuclear aSyn in mice, using the Snca-NLS mice which translocates aSyn to the nucleus, elicits motor dysfunction and cortical atrophy. The role of nuclear aSyn has not been well characterized, both in health and disease states. Therefore, I propose to better understand the molecular mechanisms of nuclear aSyn. First, I will determine the precise subnuclear localization of aSyn using two different microscopy techniques in primary cortical neurons, mouse brain, and PD patient brain tissue. Locating where aSyn exists in the nucleus will give clues to its nuclear function. We can then compare the localization in wildtype or healthy tissue to that of homozygous Snca-NLS mouse or PD patient tissue to determine how its localization changes in disease. Secondly, aSyn has been shown to bind to DNA and elicit changes in gene expression. To better understand this function of nuclear aSyn, I will determine where aSyn is binding to DNA and how aSyn is changing gene expression. From here, I will design dCas9-fusion proteins (dCas9, deactivated Cas9) to target the binding of aSyn to DNA using the specificity of Cas9 without its cleavage activity. Mapping where aSyn binds to DNA and changes gene expression will enable a more comprehensive understanding of the function of nuclear aSyn. Together, these two aims will be used to better elucidate the molecular mechanisms of nuclear aSyn natively and in disease.