LRRK2: dropping (kinase) inhibitions and seeking an (immune) response.

Since the discovery in 2004 of mutations in LRRK2 that cause Parkinson’s disease (PD), extensive effort has been made to understand the cellular roles of the encoded protein. LRRK2 is a large (286kDa) multi-domain protein that harbours a kinase and GTPase domain. Most PD linked mutations are found in these two enzymatic domains while the most prevalent mutation, G2019S, enhances the kinase activity of the protein and induces neuronal cell death in cell and animal models. Because of the potential role of kinase activity in LRRK2 pathogenesis, a number of small pharmacological inhibitors against the kinase activity of LRRK2 have been developed that are now routine research tools. Recently, LRRK2 has been linked to inflammatory response signalling. Genome-wide association studies (GWAS) have identified common genetic variants around LRRK2 as risk factors for Crohn’s disease (Barrett et al., 2008), leprosy (Zhang et al., 2009) as well as PD (IPDGC and WTCCC2, 2011). LRRK2 is highly expressed in immune cells and upregulated in response to microbial and viral particles (Hakimi et al., 2011). Furthermore, Toll-Like Receptor (TLR) signalling has been shown to modulate LRRK2 function via changes in constitutive phosphorylation in macrophages (Dzamko et al., 2012). LRRK2 is also upregulated in peripheral blood mononuclear cells (PBMCs) upon INF-γ stimulation (Gardet et al., 2010), but the signalling events mediating this are largely unexplored. In this issue, Kuss et al. (2014) identify extracellular-signal-regulated kinase 5 (ERK5) as a modulator of LRRK2 expression. The authors demonstrate that induction of LRRK2 expression by IFN-γ is attenuated by knock down or molecular inhibition of ERK5 in human monocytic leukemia THP-1 cells or human peripheral blood monocytes differentiated to macrophages. ERK5 belongs to the mitogen-activated protein kinase (MAPK) family and is involved in regulation of cell proliferation and differentiation. Inhibition of ERK5 blocks expression of TLR2 induced by a TLR1/2 agonist and other cytokines in monocytes (Wilhelmsen et al., 2012), suggesting that ERK5 may play a role in IFN-γ and TLR2 signaling. The data from Kuss et al. (2014) provide an interesting concept for how LRRK2 might have different functions in different cell types. ERK5 and LRRK2 are both ubiquitously expressed in the human body but IFN-γ signalling is predominantly restricted to immune cells. This suggests that control of LRRK2 may vary between different cell types. Downstream of IFN-γ ERK5 may thus serve as a modular control for LRRK2 in different cells. ERK5 is activated by oxidative stress and plays a neuroprotective role in dopaminergic cells (Cavanaugh et al., 2006). Accumulation of reactive oxygen species and oxidative damage in the aging brain could therefore impair physiological ERK5 signalling and dysregulate LRRK2. Therefore, common signalling events in both microglia and neurons might be critical for pathogenesis. ERK5 is believed to mediate activation of Akt in response to growth factors to regulate cell survival but the signalling events involved are not fully defined. Kuss et al. (2014) suggest that LRRK2 is a downstream ERK5 effector whose levels are regulated in response to stimuli. As previous data has implicated LRRK2 in Akt phosphorylation (Ohta et al., 2011) a testable hypothesis is that activated ERK5 promotes upregulation of LRRK2 that in turn can drive downstream dysregulation of Akt signalling, thought to occur in degenerating neurons (Timmons et al., 2009). Again, these considerations support the idea that LRRK2 pathogenesis might include effects both on neuronal and non-neuronal cells, although this has yet to be tested in vivo. Part of the way in which Kuss et al. (2014) identified ERK5 was by considering some apparently difficult data. Although one kinase inhibitor, LRRK2-IN-1, could attenuate INF-γ-induced LRRK2 upregulation, this effect was not reproduced with four other chemically distinct LRRK2 kinase inhibitors. This therefore suggested that LRRK2 kinase inhibition is not the event mediating LRRK2 upregulation. A recent study by Luerman et al. (2013) used proteomics to show that LRRK2-IN-1 can inhibit ERK5, while also exhibiting a measurable effect on TNFα activation signalling. Kuss et al. (2014) therefore used two ERK5 selective inhibitors and knock down and observed similar effects to LRRK2-IN-1, indicating that ERK5 is involved in LRRK2 upregulation after IFN-γ stimulation. Although this underlines the limitations of any pharmacological approach, at the same time it presents a good example of how these non-specific cellular effects can be exploited to advance our understanding of LRRK2 biology. In summary, the study by Kuss et al. (2014) has two novel observations: a) ERK5 is involved in LRRK2 signalling after IFN-γ activation, and b) the exploitation of off-target effects of pharmacological inhibitors in deciphering LRRK2 function. It is important that all the experiments looked at endogenous proteins, suggesting relevance to physiological signalling events. Future directions might be to consider whether genetic variants found in GWAS, either for PD or Crohn’s disease, or familial mutations may further modulate the effects of ERK5 on LRRK2. It would also be important to investigate the nature of the apparent accumulation of mRNA; perhaps these are translational events which again would be important in understanding along with the effects of genetic variation around transcription factor binding sites. At the protein level, Kuss et al. (2014) propose that endogenous LRRK2 appears to accumulate in the cytoplasm of IFN-γ treated THP-1 cells. Following on from reports implicating ubiquiting-proteasome pathway in degradation of LRRK2 (Ko et al., 2009) it is possible that association with chaperone proteins mediates accumulation of the protein in the cytoplasm, which would be an additional mechanism for controlling activity of this protein. Some additional questions are whether LRRK2 is involved in ERK5/IFN-γ signalling in microglia, whether LRRK2 expression levels are important in inflammation response in the brain, and how could altered microglia function, caused by aberrant LRRK2, influence neurodegeneration in PD? As discussed above, LRRK2 can be upregulated by IFN-γ (Kuss et al., 2014) and TLR-mediated signalling can modulate LRRK2 phosphorylation. These events may be related and, potentially, co-ordinated during specific signalling situations (Figure 1). What is not known is how these events can modulate LRRK2 activity, which has been proposed to be important in autophagy and/or inflammation. Both these systems are believed to contribute to PD progression: dysregulation of macroautophagy signalling may partly mediate α-synuclein deposition, a key pathological marker of PD, while neuroinflammation can induce dopaminergic neuron loss (Qin et al., 2007). Autophagy is an integral part of immune response mediating antigen presentation, pathogen clearance and intracellular antigen sensing. What is becoming apparent is that PD progression may be a multifactorial sequence of signalling events that take place in both microglia and neurons, and data suggest that LRRK2 may function in both systems. Figure 1 LRRK2 is involved in inflammatory signalling. In response to IFN-γ and via ERK5 LRRK2 is upregulated in macrophage-like cells, while TLR activation mediates LRRK2 phosphorylation via IKK. Apparent involvement of LRRK2 in signalling events of inflammation ...