The role of LRRK2 in regulating manganese toxicity
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Manganese (Mn) is essential for normal metabolic function and nutrition but also one of the most instrumental metals that correlates with increased susceptibility in the development of Parkinson's disease. The disorder known as manganism is caused by excess exposure to manganese and in the later stages of the disorder the symptoms become similar to those seen with Parkinson's disease which implies there is a possible common link between the two disorders. There is evidence that susceptibility to develop manganism may be associated with genes that induce early and late-onset of Parkinson's Disease (PD). There are significant differences in susceptibility and symptoms associated with manganism which could imply an underlying genetic variability. Mutations in the gene LRRK2 (leucine-rich repeat kinase-2) are the most prominent causes of late-onset Parkinson's disease; the most common being the G2019S LRRK2 mutation. Whether or not a change in LRRK2 activity also increases susceptibility to acquire manganism needs to be examined and could provide further explanation concerning the link between the two disorders. Although chronic exposure to Mn does not directly cause dopaminergic cell loss characteristic of Parkinson's disease there is evidence indicating excess exposure to Mn can increase vulnerability to developing PD. To determine the role of LRRK2 in the development of Mn toxicity I examined the effect of knocking down LRRK2 expression in mammalian cell culture models and performed toxicity tests with Mn and dopamine (DA) to determine the dose-response relationships. Mn promotes disruption of mitochondrial function by activating oxidative stress-related processes and promoting apoptotic signals such as phosphorylated p38 and JNK which ultimately lead to cell death. For this project I have examined the specific cellular processes by which manganese functions in relation to down-regulated LRRK2. It was hypothesized that the down-regulation of LRRK2 would potentiate Mn toxicity and thus would show an increase in susceptibility to develop manganism. HEK293 and HEK-YFP-DAT (HEK cells containing dopamine transporter) cells were stably transfected using nucleofection with shRNA to silence LRRK2 expression. Mn and DA toxicity were assessed by performing MTT assays and ROS generation which is a marker for cell death. To assess the effects of Mn on changes in apoptotic signaling P-JNK and P-p38 assays were performed by Western blotting. Results of these studies demonstrated that LRRK2 down-regulation potentiated Mn toxicity in both HEK and DAT cell lines. LRRK2 knock-down (KD) also potentiated DA toxicity in transfected DAT cells. Treatment of Mn and DA produced an increase in toxicity as measured by the MTT assays as well as increases in apoptotic signal phosphorylated JNK and ROS in LRRK2 KD cells compared to control HEK cells. Due to the fact that LRRK2 is a kinase that plays a role in the phosphorylation of p38 surprisingly there was a decrease in phosphorylated p38 apoptotic signal in the transfected LRRK2 KD cells due to the lack of LRRK2 activity. Based on the results found in this study it can be concluded that down-regulation of LRRK2 can indeed potentiate Mn and DA toxicity, and therefore can increase susceptibility to developing manganese toxicity as well as PD. The down-regulation of LRRK2 caused an increase in cell death, apoptotic marker expressions, and oxidative damage due to formation of reactive oxygen species (ROS) in the presence of Mn and DA.