title: RIPK3 interactions with MLKL and CaMKII mediate oligodendrocytes death in the developing brain.
abstract: Oligodendrocyte progenitor cells (OPCs) death is a key contributor to cerebral white matter injury (WMI) in the developing brain. A previous study by our group indicated that receptor-interacting proteins (RIPs) are crucial in mediating necroptosis in developing neurons. However, whether this mechanism is involved in OPCs death is unclear. We aimed to explore the mechanisms of RIP-mediated oligodendrocytes (OLs) death in the developing brain. Oligodendrocytes necroptosis was induced by oxygen-glucose deprivation plus caspase inhibitor zVAD treatment (OGD/zVAD) in vitro. Western blotting and immunofluorescence were used to detect RIPK1, RIPK3, mixed lineage kinase domain-like protein (MLKL), and Ca2+and calmodulin-dependent protein kinase IIδ (CaMKIIδ). Immunoprecipitation was used to assess the interactions between RIPK3 and RIPK1, MLKL, and CaMKIIδ. Necrostatin-1 was used to disturb the RIPK3-RIPK1 interaction, and siRNA was used to inhibit RIPK3 or MLKL expression. Oligodendrocytes death was examined using PI staining, EM, and cell membrane leakage assays. In vivo, brain damage in neonatal rats was induced by hypoxia-ischemia (HI). This was followed by an examination of myelin development. We found that OGD/zVAD treatment upregulates the expression of RIPK3 and the interaction of RIPK3 with RIPK1, MLKL, and CaMKIIδ. Inhibition of the RIPK3-MLKL or RIPK3-CaMKIIδ interaction attenuates OLs death induced by OGD/zVAD. These protective mechanisms involve the translocation of MLKL to the OLs membrane, and the phosphorylation of CaMKIIδ. However, inhibition of the RIPK3-RIPK1 interaction did not protect OLs death induced by OGD/zVAD. In vivo studies indicated that the disrupted development of myelin was attenuated after the inhibition of RIPK3-MLKL or RIPK3-CaMKIIδ interaction. Taken together, our data indicate that RIPK3 is a key factor in protection against OLs death and abnormal myelin development via its interaction with MLKL and CaMKIIδ after HI. This suggests that RIPK3 may be a potential target for the treatment of WMI in neonates.
authors.affiliation: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, China.|Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Qu
authors.affiliation: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, China.|Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Tang
authors.affiliation: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, China.|Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, China.
authors.forename: Huiqing
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, China.|Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, China.
authors.forename: Shiping
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, China.|Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, China.
authors.forename: Fengyan
authors.initials: F
authors.lastname: Zhao
authors.affiliation: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, China.|Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, China.|Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, China.|Department of Pediatrics, Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati 45229, OH, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Richard Lu
authors.affiliation: Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, China.|Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu 610041, China.|Department of Pediatrics, University of California, San Francisco, CA 94143, USA.
authors.forename: Dezhi
authors.initials: D
authors.lastname: Mu
mesh_terms: D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism; D001921:Brain; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D061945:Caspase Inhibitors / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D009186:Myelin Sheath / Q000378:metabolism; D009836:Oligodendroglia / Q000378:metabolism* / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011494:Protein Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
chemical_list: D061945:Caspase Inhibitors; D034741:RNA, Small Interfering; C000610381:MLKL protein, rat; D011494:Protein Kinases; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2