Rab Proteins Are Mediators of Mitochondrial Quality Control in Parkinson’s Disease

Abstract

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, with more than 10 million global cases. PD is defined by degeneration of dopaminergic neurons in the substantia nigra pars compacta, promoting the manifestation of motor impairments such as tremor, postural instability, and bradykinesia, as well as a range of non-motor symptoms. Mutations in leucine-rich repeat kinase (LRRK2) are associated with late-onset autosomal dominant familial PD. One widely accepted pathogenic contributor to dopaminergic cell death in PD is mitochondrial dysfunction, with several abnormalities in the mitochondria having been reported in association with dopaminergic neurodegeneration in patients, animal models, and cellular systems representative of PD. We have observed mitochondrial defects in models of PD-causing LRRK2 mutation, including loss in integrity of mitochondrial DNA (mtDNA). PD-linked mutations in LRRK2 also lead to an increase in LRRK2 protein kinase activity and hyperphosphorylation of its substrates, including the Rab GTPase family of proteins. No disease-modifying therapies currently exist for PD due in part to an incomplete understanding of the disease’s etiologic mechanisms. In this dissertation, I discuss my work analyzing mechanisms of mitochondrial quality control relevant to PD. We explored the efficacy of a G2019S mutant selective LRRK2 inhibitor (EB-42168) to specifically target G2019S LRRK2 kinase activity while maintaining physiological LRRK2 function. We assessed the propensity of EB-42168 to reverse mtDNA damage in G2019S LRRK2 cell models relative to a non-selective LRRK2 inhibitor (MLi-2). Potency of LRRK2 kinase inhibition by EB-42168 or MLi-2 was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 through western immunoblot analysis. The Mito DNADX assay, a novel system that allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, was performed in parallel. We confirmed that EB-42168 selectively inhibits LRRK2 phosphorylation on G2019S LRRK2 relative to wild-type LRRK2, while MLi-2 was equipotent for wild-type and G2019S LRRK2. Acute treatment with EB-42168 effectively restored mtDNA damage to healthy control levels. To explore the potential mechanisms mediating mtDNA damage rescue, we analyzed the relationship between LRRK2 kinase inhibition, mtDNA damage, and the autophagic turnover of dysfunctional mitochondria, or mitophagy. We found that G2019S LRRK2 mitophagy defects were not alleviated with LRRK2 kinase inhibition, suggesting that mitophagy is not mechanistically regulating LRRK2 kinase-mediated reversal of mtDNA damage in this acute timeframe. Moving forward, we will further elucidate the mechanisms mediating LRRK2-dependent mitochondrial dysfunction, and how these mechanisms interplay to promote neuronal death. Next, I investigated the relationship between the Rab GTPase family and mitochondrial quality control. Overexpression of the phosphatase PPM1H, which has been shown to counteract LRRK2-mediated phosphorylation of select Rab protein substrates, rescued mtDNA damage in a model of G2019S LRRK2. This led us to hypothesize that select Rab protein family members are critical for mitochondrial quality control. To test this hypothesis, I performed a high-throughput siRNA-based knockdown screen of each of the Rab family proteins to identify novel effectors of mitophagy. A HeLa cell line expressing the mitophagy reporter mitochondrial mKeima was plated in 96-well format with pre-stamped siRNAs for each Rab family protein, followed by induction of mitophagy through treatment with the mitochondrial uncoupling agent FCCP. Confocal image-based analysis was then used to assess the involvement of each Rab family protein in depolarization-induced mitophagy, allowing us to identify several candidates for novel mitophagy effectors within the Rab family. Among these, I validated and further investigated Rab12 as a player in mitochondrial quality control. I demonstrated that Rab12 knockdown led to a significant enhancement of mitochondrial depolarization-induced mitophagy, and further showed that Rab12 knockdown led to an increase in mitochondrial content with a decreased average membrane potential. Together, this suggests that Rab12 knockdown leads to accumulation of poorly functioning mitochondria which show increased sensitivity to mitophagy-inducing stress conditions, suggesting Rab12 to have a positive role in maintaining mitochondrial quality. We suggest a hypothesized mechanism by which Rab12 promotes mitochondrial homeostasis through its roles in positively regulating macroautophagy, which allows the removal of aged or dysfunctional mitochondrial at baseline conditions. This work provides a framework through which future studies may investigate the role of the Rab family in mediating disease-relevant defects in mitochondrial quality control pathways, with the goal to identify targetable pathways to pursue rescue of mitochondrial quality control as a therapeutic avenue within PD and other disease contexts.