Melatonin alleviates early brain injury by inhibiting the NRF2-mediated ferroptosis pathway after subarachnoid hemorrhage.

Ferroptosis is a novel form of cell death that plays a critical role in the pathological and physiological processes of early brain injury following subarachnoid hemorrhage. Melatonin, as the most potent endogenous antioxidant, has shown strong protective effects against pathological changes following subarachnoid hemorrhage, but its impact on ferroptosis induced by subarachnoid hemorrhage remains unexplored. In our study, we established a subarachnoid hemorrhage model in male SD rats. We found that subarachnoid hemorrhage induced changes in ferroptosis-related indicators such as lipid peroxidation and iron metabolism, while intraperitoneal injection of melatonin (40 mg/kg) effectively ameliorated these changes to a certain degree. Moreover, in a subset of rats with subarachnoid hemorrhage who received pre-treatment via intravenous injection of the melatonin receptor antagonist Luzindole (1 mg/kg) and 4P-PDOT (1 mg/kg), we found that the protective effect of melatonin against subarachnoid hemorrhage includes inhibition of lipid peroxidation and reduction of iron accumulation depended on melatonin receptor 1B (MT2). Furthermore, our study demonstrated that melatonin inhibited neuronal ferroptosis by activating the NRF2 signaling pathway, as evidenced by in vivo inhibition of NRF2. In summary, melatonin acts through MT2 and activates NRF2 and downstream genes such as HO-1/NQO1 to inhibit ferroptosis in subarachnoid hemorrhage-induced neuronal injury, thereby improving neurological function in rats. These results suggest that melatonin is a promising therapeutic target for subarachnoid hemorrhage.

P68 RNA helicase promotes invasion of glioma cells through negatively regulating DUSP5.

Gliomas are the most common central nervous system tumors. They show malignant characteristics indicating rapid proliferation and a high invasive capacity and are associated with a poor prognosis. In our previous study, p68 was overexpressed in glioma cells and correlated with both the degree of glioma differentiation and poor overall survival. Downregulating p68 significantly suppressed proliferation in glioma cells. Moreover, we found that the p68 gene promoted glioma cell growth by activating the nuclear factor-κB signaling pathway by a downstream molecular mechanism that remains incompletely understood. In this study, we found that dual specificity phosphatase 5 (DUSP5) is a downstream target of p68, using microarray analysis, and that p68 negatively regulates DUSP5. Upregulating DUSP5 in stably expressing cell lines (U87 and LN-229) suppressed proliferation, invasion, and migration in glioma cells in vitro, consistent with the downregulation of p68. Furthermore, upregulating DUSP5 inhibited ERK phosphorylation, whereas downregulating DUSP5 rescued the level of ERK phosphorylation, indicating that DUSP5 might negatively regulate ERK signaling. Additionally, we show that DUSP5 levels were lower in high-grade glioma than in low-grade glioma. These results suggest that the p68-induced negative regulation of DUSP5 promoted invasion by glioma cells and mediated the activation of the ERK signaling pathway.

MicroRNA-27a Promotes Inefficient Lysosomal Clearance in the Hippocampi of Rats Following Chronic Brain Hypoperfusion.

Chronic brain hypoperfusion (CBH) induces the accumulation of abnormal cellular proteins, accompanied by cognitive decline, and the autophagic-lysosomal system is abnormal in dementia. Whether CBH accounts for autophagic-lysosomal neuropathology remains unknown. Here, we show that CBH significantly increased the number of autophagic vacuoles (AVs) with high LC3-II levels, but decreased SQSTM1 and cathepsin D levels in the hippocampi of rats following bilateral common carotid artery occlusion (2VO) for 2 weeks. Further studies showed that microRNA-27a (Mir27a) was upregulated at 2 weeks compared with the sham group. Additionally, LAMP-2 proteins were downregulated by Mir27a overexpression, upregulated by Mir27a inhibition, and unchanged by binding-site mutations or miR-masks, indicating that lamp-2 is the target of Mir27a. Knockdown of endogenous Mir27a prevented the reduction of LAMP-2 protein expression as well as the accumulation of AVs in the hippocampi of 2VO rats. Overexpression of Mir27a induced, while the knockdown of Mir27a reduced, the accumulation of AVs and the LC3-II level in cultured neonatal rat neurons. The results revealed that CBH in rats at 2 weeks could induce inefficient lysosomal clearance, which is regulated by the Mir27a-mediated downregulation of LAMP-2 protein expression. These findings provide an insight into a novel molecular mechanism of autophagy at the miRNA level.

MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navß2 protein coding region in rat with chronic brain hypoperfusion.

BACKGROUND: Previous studies have demonstrated that the trafficking defects of Nav1.1/Nav1.2 are involved in the dementia pathophysiology. However, the detailed mechanisms are not fully understood. Moreover, whether the impaired miRNAs regulation linked to dementia is a key player in sodium channel trafficking disturbance remains unclear. The cognitive impairment induced by chronic cerebral ischemia through chronic brain hypoperfusion (CBH) is likely reason to precede dementia. Therefore, our goal in the present study was to examine the role of microRNA-9 (miR-9) in regulating Nav1.1/Nav1.2 trafficking under CBH generated by bilateral common carotid artery occlusion (2VO). RESULTS: The impairment of Nav1.1/Nav1.2 trafficking and decreased expression of Navß2 were found in the hippocampi and cortices of rats following CBH generated by bilateral 2VO. MiR-9 was increased in both the hippocampi and cortices of rats following CBH by qRT-PCR. Intriguingly, miR-9 suppressed, while AMO-miR-9 enhanced, the trafficking of Nav1.1/Nav1.2 from cytoplasm to cell membrane. Further study showed that overexpression of miR-9 inhibited the Navß2 expression by targeting on its coding sequence (CDS) domain by dual luciferase assay. However, binding-site mutation or miR-masks failed to influence Navß2 expression as well as Nav1.1/Nav1.2 trafficking process, indicating that Navß2 is a potential target for miR-9. Lentivirus-mediated miR-9 overexpression also inhibited Navß2 expression and elicited translocation deficits to cell membrane of Nav1.1/Nav1.2 in rats, whereas injection of lentivirus-mediated miR-9 knockdown could reverse the impaired trafficking of Nav1.1/Nav1.2 triggered by 2VO. CONCLUSIONS: We conclude that miR-9 may play a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Navß2 protein in 2VO rats at post-transcriptional level, and inhibition of miR-9 may be a potentially valuable approach to prevent Nav1.1/Nav1.2 trafficking disturbance induced by CBH.

Expression of amyloid-associated miRNAs in both the forebrain cortex and hippocampus of middle-aged rat.

BACKGROUND: Aging is associated with the gradual cognitive decline and shows the typical senile plaque formation in the brain, which results from the aggregation of beta amyloid (Aß) peptide following the abnormal proteolytic processing of amyloid precursor protein (APP) by ß-secretase (BACE1) and γ-secretase. Accumulating evidence indicates that several microRNAs (miRNAs) are involved in the Alzheimer's disease (AD) by regulating the expression of APP and BACE1 proteins. However, the cognitive ability and the expression profile of the APP- and BACE1-associated miRNAs in the middle-aged population are largely unknown. METHODS: The learning and memory ability in rats were determined by Morris Water Maze test. The protein levels of APP and BACE1 were detected by western blotting. The quantitative polymerase chain reaction was used to identify the miRNAs levels in forebrain cortex and the hippocampus. RESULTS: Middle-aged rats have declined learning ability without changes in the memory ability, and increased APP and BACE1 protein expression in the forebrain cortex. Computational analysis using Targetscan and Pictar databases reveals that totally 4 predicted miRNAs have conserved binding site with APP, namely miR-106b, -17-5p, -153, -101. All of them showed decreased expression in both the forebrain cortex and hippocampus. Among the 10 predicted miRNAs targeting BACE1, different expression profiles were identified in the forebrain cortex (decreased: miR-9, -19a, -135a, -15b, -16, -195, -29c, -214; increased: miR-124; no change: miR-141) and the hippocampus (decreased: miR-9, -15b, -16, -195, -29c, -124; increased: miR-19a, -135a, -214, -141) in the middle-aged rats compared with the young rats. CONCLUSION: Our results provided the first evidence that middle-aged rats have begun displaying cognitive disability with abnormal expression of APP- and BACE1-related miRNAs in the hippocampus and forebrain cortex.