Abstract
BackgroundOur previous study indicated that hypoxic preconditioning reduced receptor interacting protein (RIP) 3-mediated necroptotic neuronal death in hippocampal CA1 of adult rats after transient global cerebral ischemia (tGCI). Although mixed lineage kinase domain-like (MLKL) has emerged as a crucial molecule for necroptosis induction downstream of RIP3, how MLKL executes necroptosis is not yet well understood. In this study, we aim to elucidate the molecular mechanism underlying hypoxic preconditioning that inactivates MLKL-dependent neuronal necroptosis after tGCI.MethodsTransient global cerebral ischemia was induced by the four-vessel occlusion method. Twenty-four hours before ischemia, rats were exposed to systemic hypoxia with 8% O2 for 30 min. Western blotting was used to detect the expression of MLKL and interleukin-1 type 1 receptor (IL-1R1) in CA1. Immunoprecipitation was used to assess the interactions among IL-1R1, RIP3, and phosphorylated MLKL (p-MLKL). The concentration of intracellular free calcium ion (Ca2+) was measured using Fluo-4 AM. Silencing and overexpression studies were used to study the role of p-MLKL in tGCI-induced neuronal death.ResultsHypoxic preconditioning decreased the phosphorylation of MLKL both in neurons and microglia of CA1 after tGCI. The knockdown of MLKL with siRNA decreased the expression of p-MLKL and exerted neuroprotective effects after tGCI, whereas treatment with lentiviral delivery of MLKL showed opposite results. Mechanistically, hypoxic preconditioning or MLKL siRNA attenuated the RIP3-p-MLKL interaction, reduced the plasma membrane translocation of p-MLKL, and blocked Ca2+ influx after tGCI. Furthermore, hypoxic preconditioning downregulated the expression of IL-1R1 in CA1 after tGCI. Additionally, neutralizing IL-1R1 with its antagonist disrupted the interaction between IL-1R1 and the necrosome, attenuated the expression and the plasma membrane translocation of p-MLKL, thus alleviating neuronal death after tGCI.ConclusionsThese data support that the inhibition of MLKL-dependent neuronal necroptosis through downregulating IL-1R1 contributes to neuroprotection of hypoxic preconditioning against tGCI.
Highlights
Ischemic stroke, often accompanied by high morbidity, mortality, and disability, is considered to be one of major causes of death and disability worldwide
Hypoxic preconditioning or mixed lineage kinase domain-like (MLKL) siRNA attenuated the RIP3-phosphorylated MLKL (p-MLKL) interaction, reduced the plasma membrane translocation of p-MLKL, and blocked Ca2+ influx after transient global cerebral ischemia (tGCI)
Neutralizing Interleukin-1 type 1 receptor (IL-1R1) with its antagonist disrupted the interaction between IL-1R1 and the necrosome, attenuated the expression and the plasma membrane translocation of p-MLKL, alleviating neuronal death after tGCI. These data support that the inhibition of MLKL-dependent neuronal necroptosis through downregulating IL-1R1 contributes to neuroprotection of hypoxic preconditioning against tGCI
Summary
Often accompanied by high morbidity, mortality, and disability, is considered to be one of major causes of death and disability worldwide. Our previous studies demonstrated that both apoptosis and autophagy are involved in delayed neuronal death in CA1 after tGCI [1,2,3]. We reported that necroptosis contributes to hippocampal neuronal death after tGCI in adult rats. Our study further revealed that 30 min of hypoxic preconditioning applied 1 day before 10 min of tGCI-reduced necroptotic neuronal death in CA1 [6]. The precise mechanisms by which hypoxic preconditioning attenuates necroptotic neuronal death in CA1 region after tGCI remain poorly understood. Our previous study indicated that hypoxic preconditioning reduced receptor interacting protein (RIP) 3-mediated necroptotic neuronal death in hippocampal CA1 of adult rats after transient global cerebral ischemia (tGCI). We aim to elucidate the molecular mechanism underlying hypoxic preconditioning that inactivates MLKL-dependent neuronal necroptosis after tGCI
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