Abstract
We previously reported that astrocyte-derived proinflammatory cytokine interleukin (IL)-17A could aggravate neuronal ischemic injuries and strength autophagy both in oxygen-glucose deprivation (OGD)/reoxygenation (R)-treated neurons and peri-infarct region of mice with middle cerebral artery occlusion (MCAO)/reperfusion (R)-simulated ischemic stroke. In this study, the role and molecular mechanism of IL-17A in autophagy were further explored under ischemic condition. We found that exogenous addition of rmIL-17A remarkably (P < 0.001) decreased cell viability, which companying with the increases of LC3 II accumulation (P < 0.05 or 0.01) and Beclin 1 levels (P < 0.05 or 0.001), and reduction of p62 levels (P < 0.01 or 0.001) in OGD/R-treated cortical neurons (n = 6). The levels of P-mTOR (Ser 2448) (P < 0.001) and P-S6 (Ser 240/244) (P < 0.01) significantly decreased without the involvement of Akt, ERK1/2 and AMPK in cortical neurons under rmIL-17A and OGD/R treatments (n = 6). Interestingly, the co-IP analysis exhibited that PP2B and mTOR could be reciprocally immunoprecipitated; and the addition of rmIL-17A increased their interactions, PP2B activities (P < 0.001), P-Src (P < 0.001), and P-PLCγ1 (P < 0.01) levels in OGD/R-treated neurons (n = 6 or 5). The PP2B inhibitor Cyclosporin A blocked the induction of excessive autophagy (P < 0.05 or <0.001) and increased cell viability (P < 0.001) after OGD/R and rmIL-17A treatments (n = 6). In addition, the ICV injection of IL-17A neutralizing mAb could attenuate autophagy levels (P < 0.01 or 0.001, n = 6) and improve neurological functions (P < 0.01 or 0.001, n = 10) of mice after 1 h MCAO/R 24 h or 7 d. These results suggested that IL-17A-mediated excessive autophagy aggravates neuronal ischemic injuries via Src-PP2B-mTOR pathway, and IL-17A neutralization may provide a potential therapeutic effect for ischemic stroke.
Highlights
As the leading cause of death, ischemic stroke (>80% of stroke) is a medical emergency with high morbidity and mortality [1, 2]
To further explore the role of IL-17A-enhanced autophagy levels in neuronal ischemic injuries, two autophagic inhibitors 3-methyladenine (3-MA) and Bafilomycin A1 (Baf A1) were applied. Both two inhibitors pretreatment could significantly improve the survival rates of neurons when compared with their corresponding oxygen-glucose deprivation (OGD)/R or OGD/R+IL17A, respectively (Figure 2A). 3-MA treatment, which blocked the nucleation stage of autophagy by inhibiting vacuolar protein sorting (VPS) 34 of class III phosphatidylinositol-3-kinase (PI3K) [24], could significantly decrease light chain 3-β (LC3) II accumulation in neurons exposed to OGD/R or OGD/R+IL-17A (Figure 2B)
These results suggested that IL-17A induces the elevated autophagy via Src-PP2B-mechanistic target of rapamycin (mTOR) pathway in neurons exposed to OGD/R
Summary
As the leading cause of death, ischemic stroke (>80% of stroke) is a medical emergency with high morbidity and mortality [1, 2] It causes multiple pathophysiological events including mitochondrial response, excitotoxicity, protein misfolding and immune response, which lead to the delayed neuronal loss [3]. The injection of IL-17A neutralizing monoclonal antibody (mAb) could reduce the infarct volume and improve neurological outcome of mice with ischemic stroke [6, 7]. How this intervention functions still remain unclear
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