Parkinson's disease (PD) is a debilitating neurodegenerative disorder, affecting roughly 2% of those over the age of 80. Though most cases of PD are "sporadic", arising without a family history, a minority of cases have a clear autosomal dominant or recessive inheritance pattern. The most common known cause of autosomal recessive PD is homozygous inactivation of PARK2, which codes for the E3 ubiquitin ligase parkin. In addition, there is evidence that parkin inactivation may play a role in the pathogenesis of sporadic PD as well. As such, strategies aimed at increasing parkin activity hold therapeutic promise for sporadic PD.
Though much work has examined the functions of parkin, and, more recently, the mechanisms by which it is activated, substantially less is known about how parkin levels are regulated in the cell. This is particularly true for activated parkin. Understanding these regulatory mechanisms is critical for the effective development of therapeutic strategies based on upregulation of parkin activity. The work presented in this dissertation provides new insights into these regulatory mechanisms.
We show that relatively high doses of the dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) decrease parkin protein levels in vitro, analogously to previous findings using other cellular stressors. Characterizing this effect, we show that L-DOPA increases parkin degradation and that this occurs independently of L-DOPA's conversion to dopamine and of L-DOPA-induced cell death. Furthermore, we define two distinct pathways by which L-DOPA decreases parkin: an oxidative stress-dependent pathway and an oxidative stress-independent pathway. We show that the former overlaps with the previously defined mechanism of PINK1-mediated parkin activation. Specifically, parkin's association with PINK1-generated phosphorylated ubiquitin (phospho-Ub) leads to its proteasomal degradation downstream of oxidative stress, but not via autoubiquitination. Despite the involvement of PINK1 in parkin loss from L-DOPA treatment, we do not observe evidence of mitochondrial parkin activity after L-DOPA treatment, indicating that, surprisingly, parkin loss does not depend on this activity. Additionally, we provide evidence against the involvement of Trib3 and NADPH oxidases in parkin loss from L-DOPA. Finally, we provide preliminary evidence that parkin knockdown does not sensitize cells to L-DOPA-induced death.
Taken together, the findings in this dissertation contribute to the understanding of parkin regulation. Our observation that parkin's association with phospho-Ub leads to its degradation following L-DOPA treatment is of particular interest because it may represent the steady-state mechanism by which levels of activated parkin are kept in check. In light of this, an attractive therapeutic strategy may involve uncoupling parkin's activation by phospho-Ub from its phospho-Ub-dependent degradation.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D85X3SMM |
| Date | January 2018 |
| Creators | Kovalchuke, Lyudmila |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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