Genetic studies unambiguously tie LRRK2 to several human diseases. Most notably, high frequencies of causative pathological mutations have been identified in late onset PD. Whole genome association studies pinpoint LRRK2 as one of a few genes where common genetic variability underlies susceptibility to leprosy and Crohn's disease, highlighting a potential immunologic function for LRRK2. Other evidence also suggests that immune function is involved in the pathogenesis of PD, since genetic variation in the HLA region associates with PD (Hamza et al., 2011
), and numerous pathologic studies describe microglia activation in PD. LRRK2 appears to be expressed in cells of the innate but not adaptive immune system. A recent study demonstrated that the expression of LRRK2 in cultured bone marrow- derived macrophages from mice is up regulated in response to LPS (Hakimi et al., 2011
). Another recent study demonstrated that LRRK2 expression is also stimulated by the IFN-γ response in peripheral blood mononuclear cells (Gardet et al., 2010
). Thus, it has been hypothesized that LRRK2 may mediate some aspect of signaling or differentiation in innate immune cells.
The recent development of highly specific and sensitive rabbit monoclonal LRRK2 antibodies and LRRK2 knockout mice enables immunohistochemistry studies with a higher degree of confidence. Our results show that in brain tissue challenged with the potent TLR4 agonist LPS, LRRK2 expression is induced in activated microglia. Immunoprecipitation of LRRK2 protein from brains treated with LPS revealed enhanced levels autophosphorylation, which implies greater LRRK2 activity. Direct demonstration of enhanced LRRK2 enzyme activity in neuroinflammation awaits the identification of bone fide LRRK2 kinase substrates. Interestingly, we found that the accumulation of LRRK2 protein which occurs during inflammatory signaling in primary microglia is not accompanied by significant changes in mRNA levels, suggesting important post-transcriptional regulation.
We find that inhibition of LRRK2, either by small molecule kinase inhibitors or RNAi knockdown, attenuates pro-inflammatory signaling in response to TLR4 activation. LRRK2 is hierarchically clustered in the tyrosine-kinase like superfamily nearby kinases important for inflammatory signaling in immune activation, such as the Interleukin-1 receptor associated kinase (IRAK) family and the mixed lineage kinase (MLK) family (West et al., 2007
). Thus, we hypothesized that LRRK2 may function as a stress response kinase during a neuroinflammatory stimulus in the brain by facilitating signal transduction pathways in affected cells. We utilized two approaches to dissect the role of LRRK2 in TLR4 mediated inflammatory responses in primary microglia: small molecule inhibition and RNAi knockdown of total protein. While a single inhibitory molecule may be confounded by off target effects, we find agreement with multiple effective molecules that are unlikely to have over lapping off targets. In cultured rat microglia, peak TNFα release was observed 6 hours post LPS exposure and reduced by 12 hours, and in the context of LRRK2 inhibition, peak induction was reduced by greater than 20% by either LRRK2 small molecule inhibition or RNAi. Likewise, reduced levels of the TNFα target iNOS were detected by western blot. Although this effect is modest in the overall pro-inflammatory response, a cumulative effect over time may link this modifying effect with susceptibility to neurodegeneration since both TNFα and iNOS have been implicated as critical determinants of neurotoxicity.
We also observed an effect of LRRK2 inhibition on the morphological response of microglia to LPS. Exposure of microglia to LPS in the context of inhibited LRRK2 prevents the normal morphological response of fine process extension and cytoskeleton remodeling. The functional role for microglial fine process extension during initial phases of inflammatory signaling are not clear, but may involve physical sequestration of infiltrating pathogens in vivo
in damaged areas by providing a network blockage (Davalos et al., 2005
). We hypothesized that inhibition of fine process extension during LPS stimulation may itself have an anti-inflammatory effect due to negative effects on receptor clustering and lipid raft shuttling. Although actin inhibition nearly abolished fine process extension, there were no anti-inflammatory effects observed. We therefore conclude that a full pro-inflammatory response mediates process extension, but process extension itself does not mediate pro-inflammatory signaling. Thus, LRRK2 inhibition may prevent a full inflammatory response required for fine-process extension, placing LRRK2 as an upstream stress-responsive kinase to TLR4 activation.
Although the current data would suggest an overall pro-inflammatory role for LRRK2, given the complexity of neuroinflammation and the limitations of interpreting microglial action in purified cultures in vitro, it is possible an overall anti-inflammatory role for LRRK2 may be likewise envisaged. Indeed, we find that LRRK2 inhibitors quell a chemotaxic response to the potent microglial chemoattractant ADP. However, we interpret these results with some caution since we were not able to perform comparable experiments under conditions of LRRK2 RNAi. In these experiments, it was not possible to remove microglia already transduced with lentiviral LRRK2 shRNA molecules, incubated for the time required to knockdown LRRK2 protein, without killing the microglia cells. Besides microglia, LRRK2 may also be expressed in anti-inflammatory IL-10 producing macrophages in the brain, and inhibition of LRRK2 in these cells may mitigate anti-inflammatory signaling. Transgenic and LRRK2 knockout mice can be utilized in future studies to clarify overall effects of LRRK2 in neuroinflammation.
In this study, we contribute to a growing body of evidence that suggests a possible modifying role for the immune system and inflammation in PD by demonstrating LRRK2 activation in microglia and critical function in pro-inflammatory responses. It seems hypothetically possible that activating mutations in LRRK2, such as the G2019S missense mutation in the kinase activation loop, may serve to exaggerate neuroinflammatory responses that predispose to neurodegeneration susceptibility in PD. Further studies with in vivo models of neuroinflammation and associated neurodegeneration are warranted and will be critical to address the pathophysiological function of LRRK2.