Mitofusin 2 Integrates Mitochondrial Network Remodelling, Mitophagy and the Renewal of Respiratory Chain Proteins in Neurons After Oxygen and Glucose Deprivation.

In the efforts to develop effective therapeutic strategies limiting post-ischemic injury, mitochondria emerge as key element in determining the fate of the neurons. Mitochondrial damage can be alleviated by various mechanisms including mitochondrial network remodelling, mitochondrial elimination and mitochondrial protein biogenesis. However, the mechanisms regulating the relationship between these phenomena are poorly understood. Here we hypothesize that mitofusin 2 (Mfn2), a mitochondrial GTPase, involved in mitochondrial fusion, mitochondria trafficking and mitochondria and endoplasmic reticulum (ER) tethering, may act as a linking and regulatory factor in neurons following ischemic insult. To verify this assumption, we performed a temporal oxygen and glucose deprivation (OGD) on rat cortical primary culture to determine whether Mfn2 protein reduction may affect the onset of mitophagy, subsequent mitochondrial biogenesis and thus neuronal survival. In our study we found that Mfn2 knock-down increased the susceptibility of the neurons to the OGD. Mfn2 protein reduction prevented mitochondrial network remodelling and resulted in the prolonged mitophagosomes formation in response to the insult. Further on, Mfn2 protein reduction was accompanied by a reduced level of Parkin protein and an increased Parkin accumulation with mitochondria. As for Mfn2-expressing neurons, the OGD insult was followed by an elevated mtDNA content and an increase in the respiratory chain proteins. Neither of this phenomena were observed for Mfn2-reduced neurons. Collectively, our findings show that Mfn2 in neurons is involved in their response to mild and transient OGD stress, balancing the extent of elimination of defective mitochondria and positively influencing mitochondrial respiratory proteins levels. Our study confirms that Mfn2 is an essential element of the neuronal response to ischemic insult, necessary for the neuronal survival.

MiR-429 Inhibits the Angiogenesis of Human Brain Microvascular Endothelial Cells through SNAI2-Mediated GSK-3β/β-Catenin Pathway

MicroRNA (miRNA) dysfunction has been confirmed as a key event of ischemic stroke appearance. This study is aimed at revealing the role of miR-429 in the angiogenesis of HBMECs. The HBMECs were treated with oxygen and glucose deprivation (OGD) to establish the ischemic cell model. The qRT-PCR was used to measure the expression levels of the miR-429 in the serums of the patients or cells, and CCK-8, wound healing assay, and tube formation assay were used to observe the effects of miR-429 on the phenotype of HBMECs. Moreover, the Targetscan, dual-luciferase reporter assay, and Western blot were used to reveal the downstream target and regulation mechanism of miR-429 in OGD-induced HBMECs. The results showed that miR-429 was significantly upregulated in the serums of the patients, and overexpressed miR-429 could extremely inhibit the viability, migration, and tube formation of OGD-induced HBMECs. Furthermore, it was found that SNAI2 was a downstream factor of miR-429, and SNAI2 could rescue the effects of miR-429 on OGD-induced HBMECs. Besides, the Western blot showed that miR-429 could affect the activity of GSK-3β/β-catenin pathway via inhibiting the expression of SNAI2. In conclusion, this study suggests that miR-429 inhibits the angiogenesis of HBMECs through SNAI2-mediated GSK-3β/β-catenin pathway.

Zuogui Pill Attenuates Neuroinflammation and Improves Cognitive Function in Cerebral Ischemia Reperfusion-Injured Rats

<b><i>Introduction:</i></b> Cerebral ischemia and reperfusion (CI/R) injury is a devasting cerebrovascular disease, accompanied with ischemia stroke, cerebral infarction. Zuogui Pill (ZGP), as a Chinese traditional medicine, is proved to be effective in many diseases and cancers. Our study aimed to detect the roles of ZGP in CI/R injury. <b><i>Methods:</i></b> Neural stem cells were isolated from rats and induced by oxygen and glucose deprivation and recovery. CCK-8 and flow cytometry were applied to assess the function of ZGP on cell viability and apoptosis. Rat CI/R injury models were established by the middle cerebral artery occlusion and reperfusion. The function of ZGP on CI/R injury was identified via evaluating modified neurological severity score, infarct area, and cognitive impairment. <b><i>Results:</i></b> Compared to the control, the cell viability was obviously decreased in the oxygen and glucose deprivation and recovery (OGD/R) group, while the adverse influence on cells was reversed by cultured plus 10% ZGP serum. Consistently, ZGP attenuated the influence of OGD/R on cell apoptosis. More importantly, ZGP could alleviate CI/R injury of rats by reducing neurological damage and infarct area and promoting cognitive function. <b><i>Conclusion:</i></b> This study provided protective roles of ZGP on cell viability and apoptosis induced by OGD/R. In addition, ZGP played protective roles on neuroinflammation and cognitive function in rats.

Long noncoding RNA Meg3 mediates ferroptosis induced by oxygen and glucose deprivation combined with hyperglycemia in rat brain microvascular endothelial cells, through modulating the p53/GPX4 axis

Individuals with diabetes are exposed to a higher risk of perioperative stroke than non-diabetics mainly due to persistent hyperglycemia. LncRNA Meg3 has been considered as an important mediator in regulating ischemic stroke. However, the functional and regulatory roles of Meg3 in diabetic brain ischemic injury remain unclear. In this study, rat brain microvascular endothelial cells (RBMVECs) were exposed to 6 h of oxygen and glucose deprivation (OGD), and subsequent reperfusion via incubating cells with glucose of various high concentrations for 24 h to imitate in vitro diabetic brain ischemic injury. It was shown that the marker events of ferroptosis and increased Meg3 expression occurred after the injury induced by OGD combined with hyperglycemia. However, all ferroptotic events were reversed with the treatment of Meg3-siRNA. Moreover, in this in vitro model, p53 was also characterized as a downstream target of Meg3. Furthermore, p53 knockdown protected RBMVECs against OGD + hyperglycemic reperfusion-induced ferroptosis, while the overexpression of p53 exerted opposite effects, implying that p53 served as a positive regulator of ferroptosis. Additionally, the overexpression or knockdown of p53 significantly modulated GPX4 expression in RBMVECs exposed to the injury induced by OGD combined with hyperglycemic treatment. Furthermore, GPX4 expression was suppressed again after the reintroduction of p53 into cells silenced by Meg3. Finally, chromatin immunoprecipitation assay uncovered that p53 was bound to GPX4 promoter. Altogether, these data revealed that, by modulating GPX4 transcription and expression, the Meg3-p53 signaling pathway mediated the ferroptosis of RBMVECs upon injury induced by OGD combined with hyperglycemic reperfusion.

Transient Receptor Potential Ankyrin-1 (TRPA1) Block Protects against Loss of White Matter Function during Ischaemia in the Mouse Optic Nerve

Oligodendrocytes produce myelin, which provides insulation to axons and speeds up neuronal transmission. In ischaemic conditions, myelin is damaged, resulting in mental and physical disabilities. Recent evidence suggests that oligodendrocyte damage during ischaemia can be mediated by Transient Receptor Potential Ankyrin-1 (TRPA1), whose activation raises intracellular Ca2+ concentrations and damages compact myelin. Here, we show that TRPA1 is constitutively active in oligodendrocytes and the optic nerve, as the specific TRPA1 antagonist, A-967079, decreases basal oligodendrocyte Ca2+ concentrations and increases the size of the compound action potential (CAP). Conversely, TRPA1 agonists reduce the size of the optic nerve CAP in an A-967079-sensitive manner. These results indicate that glial TRPA1 regulates neuronal excitability in the white matter under physiological as well as pathological conditions. Importantly, we find that inhibition of TRPA1 prevents loss of CAPs during oxygen and glucose deprivation (OGD) and improves the recovery. TRPA1 block was effective when applied before, during, or after OGD, indicating that the TRPA1-mediated damage is occurring during both ischaemia and recovery, but importantly, that therapeutic intervention is possible after the ischaemic insult. These results indicate that TRPA1 has an important role in the brain, and that its block may be effective in treating many white matter diseases.