Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Parkinson’s disease (PD) is a complex and multifaceted illness characterized by progressive dopaminergic neuronal loss and debilitating motor, cognitive, and behavioral symptoms. A plethora of studies demonstrate a link between pesticide exposure and increased risk of PD. In this context, mounting experimental evidence indicate that dichlorvos (DCV), an organophosphate increases the risk of developing PD; however, the exact mechanism(s) of neurotoxicity remains elusive. Hence, understanding the cellular mechanisms that increase the vulnerability of dopaminergic neurons to pesticide-induced neuronal injury is critical. In recent years, a redox-sensitive non-receptor c-Abl tyrosine kinase has been shown to promote dopaminergic neurotoxicity in experimental models of PD via impaired protein clearance and oxidative stress mechanisms. Given that DCV causes dopaminergic neurotoxicity via an oxidative stress mechanism, we hypothesized that exposure of dopaminergic neuronal cells to DCV would induce mitochondria-mediated cell death signaling events while triggering impairment in cellular proteastasis via a c-Abl-dependent mechanism. We demonstrate that DCV-treated SH-SY5Y cells exhibit increased c-Abl activation, mitochondrial dysfunction, increased Bax/Bcl2 ratio, cytochrome C release, disruption of autophagy and proteasomal and delayed apoptotic cell death. Intriguingly, inhibition of c-Abl via pharmacological and genetic modulation restored cellular proteastasis and ameliorated mitochondria-mediated cell death signaling events, while over expression of c-Abl WT construct exacerbated cellular proteastasis and mitochondria-dependent cell death signaling events. Together, these findings indicate that DCV-induced c-Abl activation contributes to disruption of protein degradation machinery and associated neuronal apoptosis via the induction of mitochondria-mediated cell death signaling events.
In next set of studies, we investigated the role of c-Abl kinase in microglial activation in response to tandem exposure to pathogen-associated molecular pattern (PAMP) activator Lipopolysaccharide (LPS), an inflammagen, and danger-associated molecular pattern (DAMP) inducer rotenone (ROT), a mitochondrial complex I inhibitor and a Parkinsonian toxicant. Increasing evidence from human and experimental studies demonstrates a positive association between hyperactivated microglia and PD-associated dopaminergic neuropathology in the substantia nigra. The signaling pathways that lead to chronic activation of microglia remain poorly understood. Emerging evidence indicates that microglial NLRP3 inflammasome activation may cause neuronal injury in numerous neurodegernerative diseases, including Alzheimer’s disease via the induction of mitochondrial oxidative stress-dependent mechanisms. Given that c-Abl is a redox sensitive kinase, we hypothesized that c-Abl kinase may act as an upstream regulator of NLRP3 inflammasome. To investigate our hypothesis, we used the two well-characterized mouse microglial cell culture systems namely BV2 cells and primary mouse microglia. Upon sequential exposure of microglial cells to LPS and ROT, c-Abl tyrosine kinase activity was upregulated as evidenced by hyperphosphorylation at Y412 and Y245 sites, respectively. The activated c-Abl kinase subsequently tyrosine phosphorylated pro-inflammatory kinase PKC at Tyr311 site, with concomitant activation of the NF-B pathway, induction of mitochondrial oxidative stress, autophagolysosomal system (ALS) dysfunction, and increased NLRP3 inflammasome activation as evidenced by increased caspase 1 cleavage; and IL-1 and IL-18 release. The importance of c-Abl in the activation of NLRP3 inflammasome was further confirmed in an in vivo neuroinflammation model of PD, namely the LPS mouse model. In conclusion, we report that LPS/ROT-induces an early oxidative stress response that positively correlated with c-Abl activation, which, in turn accelerated mitochondrial oxidative stress that eventually contribute to ALS dysfunction and associated activation of the NLRP3 inflammasome in the microglia.
Interestingly, systemic infection has been shown to increase the risk of PD. To further characterize the impact of infection on PD-related neuroinflammation and dopaminergic neuronal loss, we hypothesized that microglia exposed to Parkinsonian toxicant ROT may exhibit increased sensitivity to LPS. First, we demonstrated the critical contribution of lysosomal cathepsin B in ROT/LPS-induced activation of NLRP3 inflammasome using genetic modulation techniques and pharmacological inhibitor (CA074) studies in both BV2 cells and primary microglia. In the next set of studies, we showed that ROT/LPS treatment upregulates microglial ER stress response, thereby leading to the activation of NLRP3 inflammasome via upregulation of thioredoxin-interacting protein (TXNIP) and downregulation of thioredoxin (Trx). Furthermore, using salubrinal, an ER stress inhibitor, TXNIP gene silencing technology, and c-Abl inhibition, we further demonstrated that c-Abl mediated activation of the ROS/Trx/ TXNIP signaling axis is involved in ROT/LPS-induced NLRP3 inflammasome activation. Notably, we validated our in vitro findings in aged rats that were sequentially exposed to ROT and LPS, in which NLRP3 inflammasome activation positively correlated with ER stress response and TH neuronal loss. Taken together, these results indicate that ROT/LPS-induced activation of c-Abl via an ER stress mechanism may lead to the activation of TXNIP-associated NLRP3 inflammasome, thereby leading to a heightened inflammatory response and associated dopaminergic neuronal loss.
Collectively, our studies highlight the pivotal role of c-Abl in dopaminergic neurodegeneration subsequent to exposure to a pesticide such as DCV. Additionally, we showed that c-Abl serves as a critical upstream regulator of ROT/LPS-induced microglial activation response via cathepsin B activation and ER stress-associated TXNIP activation and resultant NLRP3 inflammasome activation. Importantly, for the first time, we demonstrated that LPS augments ROT-induced dopaminergic neurotoxicity via the induction of c-Abl-mediated mitochondrial oxidative stress and ER stress-dependent signaling mechanisms in microglial cells.
Vivek Jitendrabhai Lawana
Lawana, Vivek Jitendrabhai, "Investigations into the molecular mechanisms of c-Abl in pesticide-induced dopaminergic neurodegeneration and microglial activation: Implications for Parkinson’s Disease" (2018). Graduate Theses and Dissertations. 17235.
Available for download on Sunday, December 06, 2020