RIP3 facilitates necroptosis through CaMKII and AIF after intracerebral hemorrhage in mice

• Published in: Neuroscience letters 2021-04, Vol.749, p.135699-135699, Article 135699
• Main Authors: Xu, Yang, Wu, Xiaodong, Hu, Wenjie, Yu, Dijing, Shao, Zhiding, Li, Weifei, Huang, Tingting, Zhang, Ji, Zhu, Xiaolong, Li, Xueqin, Yang, Hui, Chu, Zhaohu, Lv, Kun
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis-inducing factor (AIF); Brain Edema - metabolism; Ca2+/calmodulin-dependent protein kinase II (CaMKII); Calcium-Calmodulin-Dependent Protein Kinase Type 2 - metabolism; Cerebral Hemorrhage - metabolism; Intracerebral hemorrhage (ICH); Male; Mice; Mice, Inbred C57BL; Necroptosis; Neurons - metabolism; Protein Kinases - metabolism; Receptor-interacting protein 3 (RIP3); Receptor-Interacting Protein Serine-Threonine Kinases - metabolism; Signal Transduction - physiology
• ISSN: 0304-3940; 1872-7972
• DOI: 10.1016/j.neulet.2021.135699

•The expressions of RIP3, p-MLKL, AIF and CaMKII increased in a time-dependent manner after ICH.•AIF leaks through the mPTP channel and forms a complex with RIP3 in the cytoplasm.•RIP3-AIF nucleus translocation is primary step of necroptosis.•Inhibition of RIP3/AIF/CaMKII signaling improved neurological functions after ICH. Necroptosis-induced neuronal damage after intracerebral hemorrhage (ICH) has been documented recently. Previous studies have reported that RIP3 and its complex are recognized as central mediators of necroptosis. In this study, the role of RIP3 in the activation of CaMKII and AIF was investigated. We induced ICH in C57BL/6 mice by injecting collagenase IV into the basal ganglia. ICH mice were pretreated with the mPTP inhibitor CsA and the CAMKII inhibitor Kn-93, RIP3 siRNA or RIP3 rAAV. Brain edema and neurobehavior were evaluated. The expression of RIP3, p-MLKL, AIF, and CaMKII proteins was evaluated by western blotting, immunofluorescence (IF) and immunoprecipitation (IP). Significant increases in RIP3, p-MLKL, CaMKII and AIF expression were observed in ICH mice, and RIP3-AIF colocalized in the nucleus. Overexpression of RIP3 by rAAV upregulated AIF expression in both the cytoplasm and nucleus, while CaMKII expression was increased in the cytoplasm. The interaction of RIP3-AIF and RIP3-CaMKII was detected after ICH injury. These complexes were inhibited by CsA with Kn-93 or RIP3 siRNA pretreatment, which reduced brain edema and neurological deficits. Our findings revealed that ICH induced necroptotic neuronal death through the RIP3-CaMKII complex and the RIP3-AIF signaling pathway. Moreover, blockade of mPTP opening could suppress the pathogenesis of necroptosis.