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Morphine (Mor) has exhibited efficacy in safeguarding neurons against ischemic injuries by simulating ischemic/hypoxic preconditioning (I/HPC). Concurrently, autophagy plays a pivotal role in neuronal survival during IPC against ischemic stroke. However, the involvement of autophagy in Mor-induced neuroprotection and the potential mechanisms remain elusive. Our experiments further confirmed the effect of Mor in cellular and animal models of ischemic stroke and explored its potential mechanism. The findings revealed that Mor enhanced cell viability in a dose-dependent manner by augmenting autophagy levels and autophagic flux in neurons subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Pretreatment of Mor improved neurological outcome and reduced infarct size in mice with middle cerebral artery occlusion/reperfusion (MCAO/R) at 1, 7 and 14 days. Moreover, the use of autophagy inhibitors nullified the protective effects of Mor, leading to reactive oxygen species (ROS) accumulation, increased loss of mitochondrial membrane potential (MMP) and neuronal apoptosis in OGD/R neurons. Results further demonstrated that Mor-induced autophagy activation was regulated by mTOR-independent activation of the c-Jun NH2- terminal kinase (JNK)1/2 Pathway, both in vitro and in vivo. Overall, these findings suggested Mor-induced neuroprotection by activating autophagy, which were regulated by JNK1/2 pathway in ischemic stroke., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (Copyright © 2019 Liu, Han, Dai, Zheng, Liu, Li and Li.)

We have reported that neuron-specific conventional protein kinase C (cPKC)γ is involved in the development of cerebral hypoxic preconditioning (HPC) and the neuroprotection against ischemic injuries, but its molecular mechanism is unclear. In this study, the adult and postnatal 24 h C57BL/6J wild-type (cPKCγ +/+ ) and cPKCγ knockout (cPKCγ -/- ) mice were respectively used to establish the models of middle cerebral artery occlusion (MCAO)-induced ischemic stroke in vivo and oxygen-glucose deprivation (OGD)-treated primarily cultured cortical neurons as cell ischemia in vitro. The results showed that cPKCγ knockout could increase the infarct volume and neuronal cell loss in the peri-infarct region, and enhance the neurological deficits, the impaired coordination, and the reduced muscle strength of mice following 1 h MCAO/1-7 days reperfusion. Meanwhile, cPKCγ knockout significantly increased the conversion of LC3-I to LC3-II and beclin-1 protein expression, and resulted in more reductions in P-Akt, P-mTOR, and P-S6 phosphorylation levels in the peri-infarct region of mice with ischemic stroke. The autophagy inhibitor BafA1 could enhance or reduce neuronal cell loss in the peri-infarct region of cPKCγ +/+ and cPKCγ -/- mice after ischemic stroke. In addition, cPKCγ knockout and restoration could aggravate or alleviate OGD-induced neuronal ischemic injury in vitro through Akt-mTOR pathway-mediated autophagy. These results suggested that cPKCγ-modulated neuron-specific autophagy improves the neurological outcome of mice following ischemic stroke through the Akt-mTOR pathway, providing a potential therapeutic target for ischemic stroke.

The understanding of molecular mechanism underlying ischemia/reperfusion-induced neuronal death and neurological dysfunction may provide therapeutic targets for ischemic stroke. The up-regulated miRNA-30a among our previous identified 19 MicroRNAs (miRNAs) in mouse brain after 6 h middle cerebral artery occlusion (MCAO) could negatively regulate Beclin 1 messenger RNA (mRNA) resulting in decreased autophagic activity in tumor cells and cardiomyocytes, but its role in ischemic stroke is unclear. In this study, the effects of miRNA-30a on ischemic injury in N2A cells and cultured cortical neurons after oxygen glucose deprivation (OGD), and mouse brain with MCAO-induced ischemic stroke were evaluated. The results showed that miRNA-30a expression levels were up regulated in the brain of mice after 6 h MCAO without reperfusion, but significantly down regulated in the peri-infarct region of mice with 1 h MCAO/24 h reperfusion and in N2A cells after 1 h OGD/6-48 h reoxygenation. Both the conversion ratio of microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I and Beclin 1 protein level increased in N2A cells and cultured cortical neurons following 1 h OGD/24 h reoxygenation. The down-regulated miRNA-30a could attenuate 1 h OGD/24 h reoxygenation-induced ischemic injury in N2A cells and cultured cortical neurons through enhancing Beclin 1-mediated autophagy, as miRNA-30a recognized the 3'-untranslated region of beclin 1 mRNA to negatively regulate Beclin 1-protein level via promoting beclin 1 messenger RNA (mRNA) degradation, and Beclin 1 siRNA abolished anti-miR-30a-induced neuroprotection in 1 h OGD/24 h reoxygenation treated N2A cells. In addition, anti-miR-30a attenuated the neural cell loss and improved behavioral outcome of mice with ischemic stroke. These results suggested that down-regulation of miRNA-30a alleviates ischemic injury through enhancing beclin 1-mediated autophagy, providing a potential therapeutic target for ischemic stroke.

Activin A plays an essential role in ischemic stroke as a well-known neuroprotective factor. We previously reported that Activin A could promote white matter remyelination. However, the exact molecular mechanism of Activin A in neuronal protection post-stroke is still unclear. In this study, the middle cerebral artery occlusion/reperfusion (MCAO/R)-induced ischemic stroke mouse model and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated primary neurons were used to explore the molecular mechanism of Activin A-mediated neuroprotection against ischemic injuries. We found that Activin A significantly inhibits cGAS-STING-mediated excessive autophagy through the PI3K-PKB pathway, but not mTOR-dependent autophagy. Consequently, Activin A protected neurons against OGD/R-induced ischemic injury and improved cell survival in a dose-dependent manner. In addition, Activin A improved neurological functions and reduced infarct size of mice with MCAO/R-induced ischemic stroke by inhibiting autophagy. Furthermore, Activin A depended on ACVR1C receptor to exert neuroprotective effects in 1 h MCAO/R treated mice. Our findings showed that Activin A alleviated neuronal ischemic injury through inhibiting cGAS-STING-mediated excessive autophagy in mice with ischemic stroke, which may suggest a potential therapeutic target for ischemic stroke. [ABSTRACT FROM AUTHOR]

Stroke is one of the leading causes of death in the world, but its underlying mechanisms remain unclear. Both conventional protein kinase C ( cPKC)γ and ubiquitin C-terminal hydrolase L1 ( UCHL1) are neuron-specific proteins. In the models of 1-hr middle cerebral artery occlusion ( MCAO)/24-hr reperfusion in mice and 1-hr oxygen-glucose deprivation ( OGD)/24-hr reoxygenation in cortical neurons, we found that cPKCγ gene knockout remarkably aggravated ischaemic injuries and simultaneously increased the levels of cleaved (Cl)-caspase-3 and LC3-I proteolysis product LC3- II, and the ratio of TUNEL-positive cells to total neurons. Moreover, cPKCγ gene knockout could increase UCHL1 protein expression via elevating its mRNA level regulated by the nuclear factor κB inhibitor alpha (IκB-α)/nuclear factor κB ( NF-κB) pathway in cortical neurons. Both inhibitor and sh RNA of UCHL1 significantly reduced the ratio of LC3- II/total LC3, which contributed to neuronal survival after ischaemic stroke, but did not alter the level of Cl-caspase-3. In addition, UCHL1 sh RNA reversed the effect of cPKCγ on the phosphorylation levels of mTOR and ERK rather than that of AMPK and GSK-3β. In conclusion, our results suggest that cPKCγ activation alleviates ischaemic injuries of mice and cortical neurons through inhibiting UCHL1 expression, which may negatively regulate autophagy through ERK- mTOR pathway. [ABSTRACT FROM AUTHOR]

We have reported that conventional protein kinase Cγ (cPKCγ)-modulated neuron-specific autophagy improved the neurological outcome of mice following ischemic stroke through the Akt-mechanistic target of rapamycin (mTOR) pathway. However, its detailed molecular mechanism remains unclear. In this study, primary cortical neurons from postnatal one-day-old C57BL/6J cPKCγ wild-type (cPKCγ+/+) and knockout (cPKCγ−/−) mice suffering oxygen glucose deprivation/reperfusion (OGD/R) were used to simulate ischemia/reperfusion injury in vitro. A block of autophagic flux was observed in cPKCγ+/+ neurons under OGD/R exposure, characterized by accumulation of p62. Immunofluorescent results showed a decrease in colocalization between LC3 and Atg14 or Stx17 in cPKCγ+/+ neurons when compared with cPKCγ−/− neurons after OGD/R. However, the colocalization between LC3 and Lamp2 was barely decreased, indicating the presence of autolysosomes. The larger lysotracker-positive structures were also significantly increased. These results suggest that cPKCγ-induced inhibition of autophagy occurred at the stages of autophagosome formation, Stx17 anchoring, and the degradation of autolysosomes in particular. In addition, cPKCγ-modulated phosphorylation of mTOR at Ser 2481 was dependent on the site of Ser 2448, which may have blocked autophagic flux. cPKCγ-modulated sequential reactivation of mTOR inhibited autophagic flux in neurons exposed to OGD/R, which may provide endogenous interventional strategies for stroke, especially ischemia/reperfusion injury. [ABSTRACT FROM AUTHOR]