Neuroinflammatory mechanisms and translational approaches in environmental neurotoxicity models
Parkinson's disease (PD) is the most common neurodegenerative movement disorder first characterized in 1917 by James Parkinson, who called it the "shaking palsy". The pathogenesis of PD has not yet been elucidated, as it is a complex and multi‐factorial disease. PD is characterized by the progressive and selective loss of dopaminergic neurons within the substantia nigra. Both motor and non‐motor symptoms are seen in patients with PD, including bradykinesia, rigidity, postural instability, depression, olfactory deficits, and dementia. Idiopathic PD, which accounts for approximately 95% of known PD cases, is age‐related and affects approximately 3% of people over the age of 65. The pathology of PD was once believed to be simple, involving selective degeneration of the nigrostriatal pathway leading to the reduction of the nigrostriatal region's dopamine concentration. But the pathology of PD has been found to be much more complex, with evidence of degeneration of nondopaminergic transmitter systems as well as dopaminergic neurons. Over the years, new pathogenic mechanisms have been studied and implicated in the pathology of PD, including neuroinflammation, reactive microgliosis, oxidative stress, and mitochondrial dysfunction.
Recent studies evaluating exposure to transition metals have shown that manganese (Mn), by itself or in combination with an inflammatory stimulus, can activate pathways, leading to a neuroinflammatory response within the nigrostriatal dopaminergic system. Our lab has previously shown that the novel PKC isoform PKCδ contributes to manganese‐induced apoptosis. When catalytically cleaved, PKCδ activates caspase‐3, which consequently activates caspase‐9, leading to the activation of an apoptotic cascade. Previous studies by other laboratories showed that Mn can augment neuroinflammatory responses, including LPS‐induced neuroinflammatory responses. We sought to investigate if PKCδ plays a role in LPS/Mn‐induced inflammatory events using in vitro models of neuroinflammation. We showed increases in nitric oxide release, gp91phox protein expression, and the production of intracellular reactive oxygen species upon Mn/LPS treatment, which could be attenuated when cells were pretreated with the PKCδ inhibitor rottlerin. We also used primary microglia obtained from both wild‐type mice and PKCδ knockout mice as a cell culture model of neuroinflammation. The primary microglia were treated with Mn/LPS and we saw a marked decrease in the release of nitric oxide, production of intracellular reactive oxygen species, and the release of various cytokines (IL‐1β, IL‐6, IL‐10, IL‐12, and TNFα) in the PKCδ (‐/‐) microglia as compared to the microglia obtained from wild‐type mice. Collectively, our results suggest that the PKCδ signaling pathway may play a key role in regulating key proinflammatory events, including microglial activation, cytokine release, and NADPH oxidase complex activity induced by LPS/manganese treatment.
Our lab also studied the novel compound diapocynin, a metabolite and dimer of the naturally occurring NADPH oxidase inhibitor apocynin, and its ability to attenuate LPS/Mn‐induced neuroinflammatory events. Initially, we compared apocynin and diapocynin side‐by‐side to determine if diapocynin could effectively block neuroinflammatory events. We found that diapocynin had an EC50 about eight times lower than apocynin, and could more efficiently attenuate LPS‐induced neuroinflammatory events including nitric oxide production, production of intracellular reactive oxygen species, and the release of various cytokines. Thus, we sought to determine if diapocynin was also able to attenuate neuroinflammatory events induced upon treatment with a combination of LPS and manganese. Using microglial cell culture models of neuroinflammation, we showed that diapocynin pretreatment effectively attenuated the production of nitric oxide and intracellular reactive oxygen species, and was able to suppress the release of TNFα, IL‐1β, IL‐10, and IL‐12. Diapocynin pretreatment was also able to reduce the expression of the phox protein gp91phox, a major component of the NADPH oxidase complex. Collectively, these results suggest that diapocynin can effectively suppress Mn/LPS–induced neuroinflammatory events.
Collectively, we have discovered that PKCδ may be a potential therapeutic target in neuroinflammatory events. As determined using PKCδ knockout microglia, the kinase plays a key role in augmenting metal‐induced proinflammatory events in the brain. Additionally, we characterized the anti‐inflammatory effects of diapocynin, a metabolite of apocynin. After establishing diapocynin as a more effective anti‐inflammatory compound than apocynin, we characterized diapocynin as an effective suppressor of Mn/LPS‐induced microglia inflammatory responses. Overall, the work presented in this thesis has important implications for development of novel therapeutic strategies for Parkinson's disease.