br Materials and methods br Results br Discussion Because

Materials and methods
Results
Discussion Because the Ser/Thr kinase domain of LRRK2 is homologous to that of other MLK family proteins belonging to the MAP3K superfamily, LRRK2 is suggested to be involved in MAPK signaling and activation of neuronal cell death by phosphorylating its downstream targets [1], [2]. LRRK2 enhanced p38 MAPK activation in a model of PD and stimulated microglial ONO-5046 [7], [10]. Increasing evidence supports the putative association between LRRK2 function and p38 MAPK signaling cascade [6], [7], [8], [9], [10], [45], [46]. In this study, we found that an axis in the p38 MAPK pathway was regulated by LRRK2 through the activation of ASK1. Activation of ASK1 by LRRK2 triggered downstream cascades that promoted ASK1-mediated neuronal cell death and development of neurodegenerative diseases (Fig. 8). A recent study showed that LRRK2 possibly functions as a scaffolding protein and pre-assembles signaling complexes at specific subcellular locations [2]. Previous studies have shown that recombinant LRRK2 phosphorylates MKK3, MKK4, MKK6, or MKK7 and that LRRK2 G2019S binds to and phosphorylates these proteins more effectively [31], [32]. In line with these reports, we observed that LRRK2 served as a scaffolding protein through its different interaction domains. Because several MAPK proteins bind to different domains of LRRK2, it is possible that LRRK2 acts as a scaffolding protein in the MAPK pathway. Moreover, we expected that these approaches would ultimately lead us to identify specific molecular mechanisms underlying MAPK activation by LRRK2. We underscored the importance of LRRK2 as a scaffolding protein in the p38 MAPK pathway by determining that LRRK2 interacted not only with ASK1 but also with its downstream kinases MKK3/6 and p38 MAPK through its different domains, including WD40, LRR, and GTPase domains, in an ASK1-independent manner. These results suggested that LRRK2 enhanced the p38 MAPK pathway by serving as a scaffolding protein and the JNK pathway by serving as a JIP1-scaffolding protein to tether the sequential signaling complexes after stimulation by oxidative stress. However, the mechanisms through which LRRK2 controls ERK activity remain to be determined. By performing in silico and kinase assays, we showed that phosphorylation of ASK1 at Thr832 by LRRK2 resulted in the autophosphorylation of Thr845 and facilitated the complete activation of ASK1 and vice versa. Our finding suggested a unique primed phosphorylation mechanism in ASK1 and its complete activation under oxidative stress. Thus, these results indicated that LRRK2 serves as a pivotal kinase of ASK1 by regulating multi-stimulatory signaling pathways in a sophisticated manner. ASK1 is a major kinase that is activated by stress and plays a crucial role in neuronal apoptosis. Oxidative stress promotes neuronal apoptosis by activating ASK1 through the p38 MAPK pathway in vivo [47]. LRRK2 G2019S decreases nuclear envelope organization, clonal expansion, and neuronal differentiation of iPSCs obtained from patients with PD [39], [42]. NSCs expressing LRRK2 G2019S were more sensitive to autophagic stress that induced neuronal cell death than gene-collected NSCs. Neurotoxicity increased by LRRK2 G2019S was reduced by a selective inhibitor of ASK1. Interestingly, phosphorylation of ASK1 at both Thr832 and Thr845 was stimulated in NSCs expressing LRRK2 G2019S. This coordinated phosphorylation of ASK1 suggested that LRRK2 exerted its neurotoxic effects by phosphorylating ASK1 and by regulating the activation of the ASK1–p38 MAPK pathway to induce apoptosis of NSCs.
Transparency document
The apoptosis signal-regulating kinase 1 (ASK1) is a serine/threonine protein kinase in the triple mitogen activated protein kinase (MAP3K) family that plays a critical role in the cellular response to a wide variety of environmental and biological stressors. Specifically, ASK1 is activated in response to increases in reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, cytotoxic cytokines, and the activation of certain G protein-coupled receptor (GPCR) agonists. ASK1 activates both the p38 MAP and the c-jun-N-terminal (JNK) kinase pathways, through activation of the MAP2Ks, MKK3/MKK6 and MKK4/MKK7 respectively. The MAPK signaling pathway is involved in a variety of fundamental cellular processes such as cell survival, growth, proliferation, differentiation, stress response, and apoptosis. As a result, ASK1 is activated in a number of human pathological conditions including cancer, autoimmune disease, diabetes, cardiac, inflammatory and several neurodegenerative diseases. Consequently, inhibition of ASK1 represents an attractive strategy to provide significant benefit in multiple stress-induced diseased states.