Mechanism of Manganese-induced neurotoxicity via microglial Leucine Rich Repeat Kinase 2 (LRRK2) - Project Summary Chronic exposure to elevated manganese (Mn) levels in occupational/environmental settings causes a neurological disease referred to as manganism, a Parkinson's disease (PD)-like symptoms, but its toxicity mechanisms are not completely understood. Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein that includes a kinase domain. Although the LRRK2 function is not fully understood, its kinase activity has been implicated in vesicle trafficking and autophagy. Intriguingly, certain environmental toxicants, such as the pesticide rotenone, increase LRRK2 kinase activity. Furthermore, the LRRK2 mutation in G2019S, which is most frequently linked to familial PD, exerts LRRK2 hyper-kinase activity. These indicate that elevated LRRK2 kinase activity is common in both environmental toxicants-induced wild-type (WT) and G2019S mutation. There is strong evidence that LRRK2 kinase is increased by certain environmental toxicants, such as rotenone. Importantly, we found that Mn increased LRRK2 expression and its kinase activity in both WT and G2019S mouse brains as well as microglial culture. Given that both Mn and LRRK2 modulate microglial proinflammatory responses, microglial LRRK2 may mediate Mn-induced neurotoxicity. 14-3-3 proteins, which are highly conserved regulatory proteins, are known to interact with LRRK2 and decrease its kinase activity. Notably, Mn decreased 14-3-3 expression and its interaction with LRRK2, which was further pronounced in G2019S. The downstream pathological mechanism(s) of this Mn-increased LRRK2 may include impairment of mitochondrial fission-fusion, lysosomal defects, and mito/autophagy as both Mn and LRRK2 are involved in these processes. Our central hypothesis is that Mn-induced LRRK2 hyper-expression/kinase activity in microglia leads to impairment of mitochondrial fission-fusion, lysosomal function, and mitophagy, with subsequent increase of inflammasome and cytokines, resulting in neuronal injury by exosome and paracrine modes. The hypothesis will be tested by 3 Aims. Aim 1 will investigate the mechanisms of Mn-induced LRRK2 upregulation and kinase activity via YY1 and Sp1, as well as dysregulation of 14-3-3s and their binding to LRRK2 in microglia. Aim 2 will investigate the downstream toxicity mechanisms of Mn-increased microglial LRRK2 kinase and subsequent neuronal injury in a microglia- neuron coculture system. Whether all these effects are exacerbated in G2019S due to LRRK2 hyper-kinase activity will also be tested. Aim 3 will test if microglial LRRK2 kinase activity is critical for Mn-induced neurotoxicity in in vivo mouse models. We will test the role of microglial LRRK2 in Mn-induced motor deficits in mice using pharmacological and genetic approaches. The outcome of the proposed studies will be highly impactful as we will identify cellular and molecular targets of Mn toxicity mechanisms via microglial LRRK2, thus contributing to the development of therapeutic strategies for Mn toxicity. Moreover, our findings will also provide invaluable insights into how Mn and other environmental neurotoxins that increase LRRK2 kinase activity could be a potential risk factor for the development of LRRK2-associated PD.
