Mitochondrial Mechanisms of Toxicant Induced Dopaminergic Neurodegeneration in Caenorhabditis elegans

Abstract

Parkinson’s Disease (PD) is the 2nd most prevalent neurodegenerative disease, and the most rapidly increasing. Characterized by the progressive loss of dopaminergic neurons in the substantia nigra region of the brain, PD patients demonstrate debilitating motor symptoms and a 75% chance of developing dementia within 10 years. Roughly 15% of PD cases can be attributed to genetic causes, leaving 85% of cases as idiopathic. Environmental exposures represent a significant contributor to the presence of idiopathic PD, and multiple toxicants ranging from pesticides to industrial solvents have been associated with increased risk for PD onset. One common intracellular target of both genetic mutations and environmental exposures associated with PD is mitochondria, the major energy producing organelle within eukaryotic cells. The exact mechanisms by which mitochondrial dysfunction induces dopaminergic neurodegeneration remain unclear, though previous research indicates bioenergetic deficits and aberrant cellular redox homeostasis may be main culprits. On top of thousands of recognized mitochondrial toxicants (mitotoxicants), few of which have been tested for dopaminergic neurotoxicity, new compounds continue to be produced each year creating a greater need for understanding the role mitochondrial dysfunction plays in PD onset. In this dissertation I developed a preliminary pipeline for the medium throughput assessment of mitotoxicant induction of dopaminergic neurodegeneration, and subsequently investigated how both direct mitochondrial toxicants, such as Complex I inhibitors, and broader mitochondrial stressors, such as a high sugar diet, induce or alter susceptibility to toxicant induced dopaminergic neurodegeneration. This work employs the small nematode Caenorhabditis elegans for its rapid life cycle, highly tractable genome, and well conserved mitochondrial and neuronal biology. Transgenic C. elegans strains were utilized for in vivo assessment of neuronal morphology, real-time whole-worm and neuronal bioenergetic and redox assessment, and quantification of behavior directly related to dopaminergic neuronal function. In Aim 1 this was coupled with exposure to multiple established mitochondrial toxicants to establish a systematic method for assessing mitotoxicant induction of dopaminergic neurodegeneration. In Aim 2, the roles of ATP depletion and superoxide anion production in Complex I inhibitor induced dopaminergic neurodegeneration were delineated using exposure to the Complex I inhibitors rotenone and pyridaben, a genetic model of Complex I superoxide anion production in which worms express NUO-1::SuperNova, and use of the suppressor of Comple I Q-site electron leak (S1QEL1.1). Finally, Aim 3 tested how a systemic stressor that increases mitochondrial oxidative stress, high sugar diets, would alter susceptibility to a dopaminergic neurotoxicant. Aim 1 work to develop a preliminary pipeline for evaluating mitotoxicant induction of dopaminergic neurodegeneration resulted in a 4-step method for assessment. Doses of mitotoxicants were selected by induction of organism-wide ATP depletion, with the EC10 and EC50 after 6 hours of exposure selected for further assessment. Both doses of each toxicant were assessed for lethality, impact on dopaminergic dendrite morphology, and neuronal function. Two systems, one automated and one manual, were developed for the quantification of morphological dendritic damage in the cephalic neurons. AUDDIT, the fully automated system, accurately replicates manual scoring of neurons after toxicant exposure but requires further optimization to reduce errors in dendrite tracking. A 7-point scoring scale was also supplied for manual evaluation and was employed throughout the subsequent analysis. Optimal detection of neuronal dysfunction occurred after 48-hours of continuous exposure in contrast to peak detection of morphological damage which occurred after 96-hours of continuous exposure. Taken together, these data indicate the Complex I inhibitor rotenone as the most potent toxicant tested resulting in both morphological damage and loss of neuronal function. The Complex II inhibitor wact-11 and Complex III inhibitor Antimycin A did not induce morphological damage, but caused loss of function suggesting they should be explored further. The uncoupler FCCP neither caused functional loss nor morphological damage, indicating that in this exposure paradigm it is not a dopaminergic neurotoxicant. Finally, the Complex II inhibitor fluopyram induced both morphological damage and functional loss, however this occurred alongside elevated lethality, a confounding effect that prevents drawing clear conclusions. This represents the initial development of a pipeline suitable for testing ATP-depleting mitochondrial toxicants for induction of dopaminergic neurodegeneration in C. elegans that can be employed together with other in vitro models and model organisms for screening toxicants and exploring mechanisms by which mitotoxicants may play a substantive role in PD cases. In the second aim of this dissertation focused on delineating the roles of superoxide anion production and ATP depletion by Complex I inhibitors in their induction of dopaminergic neurodegeneration. I observed induction of morphological damage by rotenone and neuronal loss of function by both rotenone and pyridaben, but assessment of neuronal redox and bioenergetic status failed to provide clarity on the relative contributions of each mechanism to neurodegeneration. I first attempted to sensitize the worms to mitochondrial superoxide stress by employing a mutant lacking the glyoxylate shunt, a pathway shown to protect from mitochondrial superoxide in worms. This caused elevated baseline morphological damage, but abrogated rotenone and pyridaben induced dysfunction and damage in contrast to the hypothesis. Second, I found that light activated production of superoxide anion local to complex I was sufficient to induce both dendritic damage and neuronal dysfunction, suggesting that enzymatic inhibition of Complex I may not be required for the effects of rotenone and pyridaben. Finally, coexposure to S1QEL1.1 further supported a causal role for superoxide anion production as blocking Complex I Q-site superoxide anion production fully rescued the morphological and function impacts of both rotenone and pyridaben exposure without rescuing organism-wide ATP depletion. Overall, these results indicate that Complex I superoxide production is not only singly capable of inducing dopaminergic neurodegeneration, but also potentially the main mechanism by which Complex I inhibitors do so. The final aim of this work sought to address how a mitochondrial stressor not directly targeting the mitochondria may alter susceptibility to toxicant induced dopaminergic neurodegeneration. High sugar consumption, a key component of western diets, induced elevated oxidative stress in both worm and mouse models. I hypothesized that chronic elevated oxidative stress would render the worms more susceptible to 6-hydroxydopamine, a dopaminergic neurotoxicant that acts by Complex I and IV inhibition and auto-oxidation. Despite replicating lifespan and reproductive losses, chronic high glucose or fructose diets throughout adulthood did not result in a more oxidized baseline redox tone. Furthermore, high sugar diets modulated DAT-1 dopamine reuptake transporter uptake of 6-OHDA resulting in a smaller increase in glutathione oxidation after exposure and protection from neurodegeneration. This occurred despite identical decreases in the ATP:ADP ratio within dopaminergic neurons, supporting the finding of Aim 2 that redox, not bioenergetic, dysfunction is the key component of neurodegeneration inducing mitochondrial toxicants. Use of high sugar diets did not allow for testing of the hypothesis that lifelong enhanced oxidative stress would increase susceptibility to neurodegeneration; However, these data underscore the value of assessing lifestyle and other external factors that interact modulate susceptibility to mitochondrial toxicants. In combination these Aims provide a system to improve future evaluations of mitotoxicant induced dopaminergic neurodegeneration and suggest increased oxidative stress within the mitochondria is a key event in the pathway by which mitotoxicants induce dopaminergic neurodegeneration. Demonstrated in this work through an exploration of Complex I inhibitors and high sugar diets, both alternative mitochondrial toxicants and external stressors should be evaluated in the future to comprehensively understand how we can mitigate risk for PD moving forward.