Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Anumantha G. Kanthasamy
Parkinson's disease (PD) is a chronic age progressive neurodegenerative disease that affects about 1% of people over the age of 60. The hallmark pathological features of PD is characterized by the loss of 60-70% dopaminergic neurons, formation of Lewy bodies and aggregation of α-synuclein in the substantia nigra of the midbrain region. There are multiple factors that contribute to the origin of the disease and also these factors further trigger an avalanche of pathways, thus prominently increasing the risk of incurring PD. Although the etiopathology of PD is not very well understood, growing evidences on mitochondrial dysfunction and oxidative stress suggests to serve as one of the critical cause of PD. Many years of research has shown that incessant chronic exposure to several classes of pesticides lead to mitochondria dysfunction and degeneration of dopamingeric neuronal cells leading to PD. The primary target and goal of my Ph.D thesis was to particularly understand the specific role of mitochondrial complex I class of pesticides in causing apoptotic death of dopaminergic neurons. Recently we have shown the green house pesticides, tebufenpyrad and pyridaben mediate noteworthy effect on causing mitochondrial toxicity in the N27 dopaminergic neuronal cells. These pesticides are classified as mitochondrial complex I inhibitors and are functionally analogous to the Parkinsonian toxicant rotenone, in terms of target and mode of action. Herein, we demonstrate that acute exposure of tebufenpyrad or pyridaben to the N27 neuronal cells, induced significant mitochondrial structural and functional dysfunction by significant impairment of mitochondrial respiration and ATP production. Suggesting that the pesticides toxicity is associated with oxidative damage. Now on a novel direction of trying to understand the dopamingeric neuronal cell death post complex I inhibitor exposure, we looked into PKCδ a pro-apoptotic signaling kinase and its downstream target of phosphorylation. In this study, we show that tebufenpyrad exposure to the dopaminergic neuronal cells, lead to the activation of PKCδ in a caspase-3 dependent manner, which was marked by the cleavage of PKCδ and phosphorylation of PKCδ at T505. Importantly, we identified that activated PKCδ potentiated the phosphorylation of laminB1 at T575, leading to damage of the nuclear membrane integrity. Moreover the activation of PKCδ, phosphorylation of laminB1 at T575 and laminB1 loss was also observed in the PD substantia nigra brain tissues, but not in the control brains. Thereby noticeably extrapolating translational evidence and importance of our findings. Next we had ventured onto a complete novel pathway that involves the interplay between mitochondrial dysfunction and epigenetic modification such as hyperacetylation of histones H3 and H4 post mitochondrial complex 1 targeting pesticides in the dopamingeric neuronal cells. Herein, post exposure of N27 cells to pyridaben we demonstrate a dose and time-dependent increase in acetylation of histones H3 and H4. Also, further analysis performed using immuno-fluorescence staining and confocal microscopy, confirmed the site specific AcH3(K23) and AcH4(K5) up-regulation prior to rotenone or pyridaben exposure in the N27 neuronal cells. Interestingly, pyridaben or rotenone treated nuclei also demonstrate a nuclear structural defect in the form of topological alteration or chromatin declustering. Adding on, this suspected nuclear morphological change was marked by a time dependent loss of HP-1α, a very well studied marker of heterochromatin, suggesting the involvement of chromatin remodeling like process post the mitochondrial inhibiting pesticides exposure in the N27 cells. Transgenic mitochondrial defective in vitro model of TFAM CRISPR/Cas9 knockdown N27 cells also show hyperacetylation of histones H3 and H4 compared to the control CRISPR/Cas9. Furthermore, the Mitopark transgenic animal model of PD in addition demonstrated an age progressive acetylation of histones H3 and H4, and also AcH3(K23) and AcH4(K5) up-regulation and nuclear declustering morphology. Finally, our data was conclusively confirmed by the analysis of post-mortem human substantia nigra tissues, which demonstrated hyperacetylation of H3 and H4 and formation of vivid nuclear declustered structures in the PD brains. The amalgamative interpretation of these results suggests that histone hyperacetylation is a key epigenetic mechanism in the nigral dopaminergic neuronal cells following mitochondrial dysfunction and neurotoxicity in the neuronal models of PD. Overall, our results suggests that exposure of dopaminergic neuronal cells to mitochondrial complex I inhibiting pesticides leads to accentuated PKCδ kinase activity, nuclear membrane damage and histone hyperacetylation, resulting in apoptotic dopaminergic neuronal cell death. Thus intervening such mechanisms, merits the development of strategies for diagnosis and drug based treatments for PD.
Adhithiya Charli Manohar Charle
Manohar Charle, Adhithiya Charli, "Interplay between mitochondrial dysfunction and epigenetic alterations in environmental linked Parkinson's Disease" (2018). Graduate Theses and Dissertations. 16631.