The goals of this proposal are to define the molecular mechanisms by which HtrA2-mediated cleavage of RIP1 regulates inflammation and cell death in striatal neurons in response to TNF? and oxidative stress, and to assess the pathological relevance of upstream and downstream effectors of RIP1 (i.e. TNF? and RIP3) in the onset and progression of basal ganglia disorder in HtrA2-mutant (Mnd2; motor neuron degeneration 2) mice. TNF? and oxidative stress are significant underlying factors for many neurodegenerative diseases, and identification of the key molecular events that control neuronal response to TNF? and oxidative stress are an important and urgent task for the development of novel diagnostic approaches and therapeutic strategies. RIP1 is a dual-function (adaptor and kinase) protein that plays a key role in TNF?- and oxidative stress-induced cell survival, apoptosis and necroptosis. The ability of RIP1 to modulate cell survival is largely controlled by its K63- linked ubiquitination and NF-?B activation, and the prodeath function of it is dependent on its kinase activity. It is known that RIP1 overexpression promotes both NF-?B activation and cell death; however, the post-translational regulation of RIP1 protein abundance under basal and chronic stress conditions is largely unknown. HtrA2 is a serine protease and has been shown to promote cell death by degrading anti-apoptotic XIAP and cIAP1/2 proteins; however, gene knockout (KO) studies reveal that HtrA2 KO mice exhibit neurodegenerative disorder characteristic of Parkinson?s disease (PD), and that HtrA2 KO cells display significantly increased susceptibility to stress-induced apoptosis and necrosis. Notably, Mnd2 mice exhibit the same phenotype with HtrA2 KO mice, and were found to carry a missense mutation in the protease domain of HtrA2 gene. Mutations in human HTRA2 gene have also been found in sporadic PD patients, suggesting that HtrA2 enzymatic activity is required for the suppression of inflammation and neuronal death. However, the substrate(s) responsible for PD-like phenotype in Mnd2 mice have not yet been identified. We found that RIP1 is cleaved by HtrA2 in hematopoietic and brain tissues of wild-type (WT) but not of Mnd2 mice. Importantly, knockdown of RIP1 in Mnd2 and HtrA2 KO cells significantly suppressed the susceptibility of these cells to cellular stresses. Given that RIP1 overexpression is known to cause NF-?B activation and cell death, we hypothesize that HtrA2-mediated cleavage of RIP1 limits its proinflammatory and prodeath functions to maintain the homeostasis of organ/tissues that are sensitive to RIP1 protein abundance, and that altered regulation of this process under conditions of chronic stresses could lead to RIP1-mediated inflammation and neurodegeneration. To test these hypotheses, we propose to carry out the following specific aims:
Aim -1. Define the mechanisms by which RIP1 triggers inflammation and neuronal death in the striatum of Mnd2 mice;
Aim -2. Assess the pathological relevance of TNF? and RIP3 in the development of PD-like disease in Mnd2 mice. Thus the completion of this project has the potential to guide the development of novel approaches for the therapy of neurodegenerative disease through the intervention of RIP1 cleavage.
The goals of this proposal are to define the molecular mechanisms by which HtrA2-mediated RIP1 cleavage regulates inflammation and cell death; both are underlying causes of many neurodegenerative diseases. Thus our study has the potential to guide the development of novel approaches for the therapy of neurodegeneration.