RTN1-C mediates cerebral ischemia/reperfusion injury via modulating autophagy.

It has been widely accepted that autophagic cell death exacerbates the progression of cerebral ischemia/reperfusion (I/R). Our previous study revealed that overexpression of reticulon protein 1-C (RTN1-C) is involved in cerebral I/R injury. However, the underlying mechanisms have not been studied intensively. This study was designed to evaluate the effect of RTN1-C on autophagy under cerebral I/R. Using an in vitro oxygen-glucose deprivation followed by reoxygenation and a transient middle cerebral artery occlusion model in rats, we found that the expression of RTN1-C protein was significantly upregulated. We also revealed that RTN1-C knockdown suppressed overactivated autophagy both in vivo and in vitro, as indicated by decreased expressions of autophagic proteins. The number of Beclin-1/propidium iodide-positive cells was significantly less in the LV-shRTN1-C group than in the LV-shNC group. In addition, rapamycin, an activator of autophagy, aggravated cerebral I/R injury. RTN1-C knockdown reduced brain infarct volume, improved neurological deficits, and attenuated cell vulnerability to cerebral I/R injury after rapamycin treatment. Taken together, our findings demonstrated that the modulation of autophagy from RTN1-C may play vital roles in cerebral I/R injury, providing a potential therapeutic treatment for ischemic brain injury.

RTN1-C is involved in high glucose-aggravated neuronal cell subjected to oxygen-glucose deprivation and reoxygenation injury via endoplasmic reticulum stress.

BACKGROUND: Patients suffering from diabetes mellitus experience poor outcomes after ischemic stroke. RTN1-C, ER-associated proteins localized in endoplasmic reticulum (ER) membrane, plays an important role in ER stress-induced apoptosis and regulates cellular susceptibility to different apoptosis pathways. Overexpression of RTN1-C can aggravate cerebral ischemia/reperfusion injury (IRI). ER stress plays a crucial role in hyperglycemia-aggravated cerebral IRI. In this study, we aimed to investigate the role of RTN1-C in high glucose-aggravated OGD/R-induced cell damage. MATERIALS AND METHODS: N2a cells and primary neuronal cells were cultured in normal glucose or high glucose conditions. We used a model of oxygen-glucose deprivation followed by reoxygenation (OGD/R). RTN1-C shRNA was used to knock down RTN1-C. The chemical chaperone 4-phenylbutyric acid (4-PBA) is a low molecular weight fatty acid that has the ability to stabilize mutant proteins and facilitate their folding, was used to inhibited ER stress. Cell viability and apoptosis were measured by CCK-8 and flow cytometry assays. The contents of ER stress-associated proteins, such as GRP78, cleaved caspase-12, CHOP and cleaved caspase-3, were detected by western blot. RESULTS: High glucose significantly decreased cell viability and increased cell apoptosis in OGD/R-treated neuronal cells. The contents of GRP78, cleaved caspase-12, CHOP and cleaved caspase-3 under high glucose conditions were higher than those under normal glucose conditions after OGD/R. Importantly, inhibition of ER stress by 4-PBA alleviated the high glucose-aggravated OGD/R-induced cell damage. Here, we demonstrated that high glucose increases RTN1-C expression in OGD/R-treated cells. More importantly, knockdown of RTN1-C expression dramatically reversed the high glucose-aggravated change in cell viability and apoptosis and relieved ER stress in OGD/R-treated cells. CONCLUSIONS: High glucose significantly increases RTN1-C expression in OGD/R-treated cells. RTN1-C affects high glucose-treated OGD/R cells by exacerbating ER stress.

RTN1-C mediates cerebral ischemia/reperfusion injury via ER stress and mitochondria-associated apoptosis pathways.

The reticulon family has been found to induce apoptosis, inhibit axon regeneration and regulate protein trafficking. However, little is known about the mechanisms of how reticulon proteins are involved in neuronal death-promoting processes during ischemia. Here, we report that the expression of Reticulon Protein 1-C (RTN1-C) was associated with the progression of cerebral ischemia/reperfusion (I/R) injury. Using a combination of rat middle cerebral artery occlusion (MCAO) stroke and oxygen-glucose deprivation followed by reoxygenation (OGD/R) models, we determined that the expression of RTN1-C was significantly increased during cerebral ischemic/reperfusion. RTN1-C overexpression induced apoptosis and increased the cell vulnerability to ischemic injury, whereas RTN1-C knockdown reversed ischemia-induced apoptosis and attenuated the vulnerability of OGD/R-treated neural cells. Mechanistically, we demonstrated that RTN1-C mediated OGD/R-induced apoptosis through ER stress and mitochondria-associated pathways. RTN1-C interacted with Bcl-xL and increased its localization in the ER, thus reducing the anti-apoptotic activity of Bcl-xL. Most importantly, knockdown of Rtn1-c expression in vivo attenuated apoptosis in MCAO rats and reduced the extent of I/R-induced brain injury, as assessed by infarct volume and neurological score. Collectively, these data support for the first time that RTN1-C may represent a novel candidate for therapies against cerebral ischemia/reperfusion injury.