Reelin Regulates Oxidative Stress-induced Neuronal Apoptosis By Inhibiting CaN-mediated DAPK1 Dephosphorylation

Objective:Ischemic stroke is one of the leading causes of death and disability worldwide.Understanding the mechanism of oxidative stress injury induced by ischemia/reperfusion?I/R?may provide a new treatment for ischemic stroke.Reelin protein is an extracellular matrix serine protease,which plays an important role in the migration and localization of newborn neurons.The downstream signaling pathway is closely related to cell survival,proliferation,differentiation and growth.Cerebral ischemia injury leads to intracellular Ca²⁺overload,which triggers the abnormal activation of calcineurin?CaN?,which leads to the activation of death-associated protein kinase 1?DAPK1?.CaN is a Ca²⁺/calmodulin-dependent serine/threonine phosphatase composed of catalytic and regulatory subunits that mediates neuronal apoptosis.DAPK1 is a specific"cell death signal"that acts as a positive mediator of apoptosis and promotes ischemia-induced neuronal apoptosis.The aim of this study was to evaluate the molecular mechanism of Reelin's regulation of neuronal apoptosis through CaN during oxidative stress injury,and to investigate the effect of CaN-DAPK1signaling on neuronal apoptosis after oxidative stress injury.Methods:Primary culture of neonatal SD rats?born within 0-24 hours?cerebral cortical neurons was performed.Hydrogen peroxide?H₂O₂?treatment was administered to cortical neurons to simulate oxidative stress injury during ischemia/reperfusion.Cellular immunofluorescence technique was used to observe the neuronal death,Reelin protein expression,colocalization of Disabled-1?Dab-1?with DAPK1,and colocalization of CaN with DAPK1.Co-Immunoprecipitation?Co-IP?technique was used to detect the interaction between CaN and DAPK1.Western-blot?WB?was used to detect the expression levels of cleaved caspase-3,Reelin protein,the cleavage of CaN and phosphorylated DAPK1.To investigate the regulation of Reelin on CaN and DAPK1 downstream of Phosphatidylinositol 3-kinase/protein kinase B/the mammalian target of rapamycin?PI3K/Akt/mTOR?pathway in rat cerebral cortical neurons.By administering exogenous recombinant protein,siRNA interference and pharmacological inhibition,the anti-apoptotic effect of Reelin was observed and its mechanism of action was explored.Results:?1?After the oxidative stress treatment of primary cultured cortical neurons with different concentrations of H₂O₂,the number of neuronal deaths increased and the expression of cleaved caspase-3 increased with the increase of H₂O₂ concentration.When the concentration of H₂O₂ was 100?mol/L,neuronal apoptosis was the most serious.?2?The expression level of Reelin protein was significantly increased after oxidative stress treatment of cortical neurons with 100?mol/L H₂O₂.The expression level of cleaved caspase-3 was further increased after siRNA was used to interfere with the expression of endogenous Reelin protein in oxidative stress-injured neurons.After administration of the exogenous recombinant protein mReelin,the number of neuronal deaths decreased,and the expression level of cleaved caspase-3 was relatively decreased.?3?Fluorescence results showed that Dab-1 colocalized with DAPK1.After administration of the exogenous recombinant protein mReelin,the PI3K/Akt pathway inhibitor Wortmannin and the mTOR pathway inhibitor Rapamycin were administered,the number of neuronal deaths increased,the expression level of cleaved caspase-3 was relatively increased,and the phosphorylation level of DAPK1 was relatively decreased.?4?After administration of mReelin,followed by treatment with the PI3K/Akt pathway inhibitor Wortmannin and the mTOR pathway inhibitor Rapamycin,the cleavage of CaN was relatively increased.After administration of CaN inhibitor FK506,the expression level of cleaved caspase-3 was decreased,and the phosphorylation level of DAPK1 was increased.Fluorescence results showed that CaN colocalized with DAPK1,and co-IP results confirmed the presence of binding between CaN and DAPK1.?5?After administration of caspase-3 inhibitor Z-DEVD-FMK,the cleavage of CaN is reduced.Conclusions:?1?H₂O₂ oxidative stress treatment can induce neuronal apoptosis in cerebral cortex.Reelin protein expression levels are significantly increased after neuronal damage by oxidative stress,and Reelin protein can reduce cortical neuron apoptosis by activating its downstream PI3K/Akt/mTOR signaling pathway.?2?After cerebral cortical neurons are damaged by oxidative stress,activation of CaN in the CaN-DAPK1 complex results in dephosphorylation of DAPK1,further leading to activation of caspase-3 and apoptosis of neurons.At the same time,the cleaved caspase-3 cleaves and activates CaN,thereby triggering the cell's apoptotic cycle.However,Reelin attenuates this process by activating the PI3K/Akt/mTOR signaling pathway.