Investigation of Drp1 inhibition as potential therapy in pesticide-based models of Parkinson's disease

Abstract

In this study I explored the impact of Drp1 inhibition on pathology in two different pesticide-based models of Parkinson’s disease, and found that paraquat induced autophagy blockade in vitro, which was abrogated by inhibition of Drp1 function through siRNA knockdown or expression of the Drp1K38A dominant negative. Rotenone and paraquat are two pesticides implicated in the pathophysiology and progression of Parkinson’s disease, a hypokinetic neurodegenerative disorder which results in motor and cognitive changes in patients. Previous studies confirmed pesticide-induced loss of dopaminergic neurons in the substantia nigra pars compacta in animal models, however further research was necessary to explore the underlying mechanisms of this pathology. As both pesticides influence mitochondria, and mitochondrial dysfunction has been implicated in Parkinson’s disease, I investigated how modulation of the mitochondrial fission factor Drp1 influenced the pesticide-induced pathology. Exploration of rotenone in vivo recapitulated dopaminergic neuropathology, motor dysfunction and demonstrated induction of apoptosis, however the systemic toxicity and premature mortality proved it to be an inappropriate model for the exploration of potential therapeutic agents. Paraquat experiments in Oct3-/- mice demonstrated that pharmacological inhibition of Drp1 with mdivi-1 was protective against the paraquat-induced neuropathology, however investigation of genetic Drp1 modulation in the global heterozygous Drp1 knockout mouse model failed to reproduce dopaminergic neuropathology in paraquat-treated wild-type littermate controls, preventing exploration of any potential protective impact of the Drp1 reduction. Systemic toxicity was much more pronounced in male mice than females, suggestive of gender disparity in their sensitivity to paraquat, with relevance to the human disease as Parkinson’s disproportionately affects men. Further investigation of mechanisms of paraquat-induced toxicity in vitro implicated autophagy blockade as a mechanism of pathology, with Drp1 inhibition proving effective to abrogate the blockade. Drp1 inhibition in cells co-treated with paraquat and α-synuclein however was insufficient to restore function in tfLC3 HeLa cells. This implicates Drp1 in autophagy, where it was previously considered a mitochondrial fission protein. Whilst further exploration of how Drp1 influences autophagy is required, this work is the first to demonstrate a protective role of Drp1 inhibition against paraquat-induced pathology, including the partial resolution of autophagy blockade. Combined with previous work from our lab, this further supports the involvement of Drp1 in autophagy as a mechanism in Parkinson’s disease pathology, which validates the targeting of Drp1 as a potential therapeutic method to alter Parkinson’s disease progression.