Altered expression of circular RNA in patients with cervical artery dissection.

Cervical artery dissection (CeAD), a special cerebrovascular disease and the main cause of stroke in young people, can present with ischemic stroke, headache, subarachnoid hemorrhage, and other symptoms, increasing the possibility of misdiagnosis. As a special class of non-coding RNAs, circRNAs are commonly found in organisms and can play regulatory roles in transcription and post-transcription processes, affecting gene expression.CircRNAs have reported to be associated with neurological diseases; however, their role in CeAD has not been discerned. In this study, we aimed to elucidate the pathophysiological changes in patients with CeAD and identify biomarkers. Peripheral blood mononuclear cells from patients with CeAD and healthy controls were sequenced using high-throughput sequencing. We detected 460 differently expressed circRNAs in patients with CeAD (p < 0.5, fold difference ≥ 2), of which 240 were upregulated and 220 were downregulated. Four circRNAs showed significant differences in expression, which were validated using qRT-PCR. These results suggested that three circRNAs were consistent with high-throughput sequencing results. Bioinformatics analysis demonstrated that these differentially expressed circRNAs were involved in protein metabolism, regulation, synapses, and other pathophysiological processes during CeAD-induced stroke. Additionally, various pathways related to inflammation were closely associated with circRNAs. Based on our results, we suggest that the aberrant expression of circRNAs in CeAD may serve as a biomarker for its diagnosis and as a potential therapeutic target.

EGR1-Driven METTL3 Activation Curtails VIM-Mediated Neuron Injury in Epilepsy.

Uncovering mechanisms underlying epileptogenesis aids in preventing further epilepsy progression and to lessen seizure severity and frequency. The purpose of this study is to explore the antiepileptogenic and neuroprotective mechanisms of EGR1 in neuron injuries encountered in epilepsy. Bioinformatics analysis was conducted to identify the key genes related to epilepsy. The mice were rendered epileptic using the kainic acid protocol, followed by measurement of seizure severity, high amplitude and frequency, pathological changes of hippocampal tissues and neuron apoptosis. Furthermore, an in vitro epilepsy model was constructed in the neurons isolated from newborn mice, which was then subjected to loss- and gain-of-function investigations, followed by neuron injury and apoptosis assessment. Interactions among EGR1, METTL3, and VIM were analyzed by a series of mechanistic experiments. In the mouse and cell models of epilepsy, VIM was robustly induced. However, its knockdown reduced hippocampal neuron injury and apoptosis. Meanwhile, VIM knockdown decreased inflammatory response and neuron apoptosis in vivo. Mechanistic investigations indicated that EGR1 transcriptionally activated METTL3, which in turn downregulated VIM expression through m6A modification. EGR1 activated METTL3 and reduced VIM expression, thereby impairing hippocampal neuron injury and apoptosis, preventing epilepsy progression. Taken together, this study demonstrates that EGR1 alleviates neuron injuries in epilepsy by inducing METTL3-mediated inhibition of VIM, which provides clues for the development of novel antiepileptic treatments.

Temporal patterns and distribution of pyroptosis-related molecules and effects of human mesenchymal stem cells on pyroptosis following cerebral ischemia/reperfusion in rats.

OBJECTIVES: Pyroptosis is a new type of programmed cell death that has a strong proinflammatory effect. The present study investigated the dynamic changes of pyroptosis-related molecules and the effect of mesenchymal stem cells (MSCs) on pyroptosis following cerebral ischemia/reperfusion (I/R). MATERIALS AND METHODS: The temporal pattern and cellular distribution of caspase-1, Gasdermin D and E (GSDMD and GSDME) in the peri-infarct area, and the effect of human MSCs on GSDMD, IL-1ß, IL-18, Lactate dehydrogenase (LDH) and neurological function were studied in a rat model of transient focal cerebral ischemia. RESULTS: The expression of caspase-1 mRNA increased with time, with a protein level of pro-caspase-1 comparable to its mRNA level, while the level of cleaved-caspase-1 protein peaked at 48 h following I/R. Increased levels of GSDMD mRNA and protein were also observed, with a peak level at 24 h. There were no significant changes in GSDME mRNA or protein expression after I/R. In regards to changes in the number of cells expressing GSDMD after I/R, that for neurons was more significant than those for microglia and astrocytes. The modified neurological severity score discrepancy and the expression of GSDMD showed no significant differences within 24 h following I/R between the MSC- and NS-treated groups, but MSCs treatment promoted the secretion of IL-1ß, IL-18 and LDH. CONCLUSIONS: In the early stage of cerebral infarction in rats, there were dynamic changes in pyroptosis-related molecules (caspase-1 and GSDMD), but MSCs showed no effect on the levels of GSDMD or neurological function.

Microglial and Neuronal Cell Pyroptosis Induced by Oxygen-Glucose Deprivation/Reoxygenation Aggravates Cell Injury via Activation of the Caspase-1/GSDMD Signaling Pathway.

Pyroptosis is a new type of programmed cell death, which induces a strong pro-inflammatory reaction. However, the mechanism of pyroptosis after brain ischemia/reperfusion (I/R) and the interaction between different neural cell types are still unclear. This study comprehensively explored the mechanisms and interactions of microglial and neuronal pyroptosisin the simulated I/R environment in vitro. The BV2 (as microglial) and HT22(as neuronal) cells were treated by oxygen-glucose deprivation/reoxygenation (OGD/R). Both BV2 and HT22 cells underwent pyroptosis after OGD/R, and the pyroptosis occurred at earlier time point in HT22than that of BV2. Caspase-11 and Gasdermin E expression in BV2 and HT22 cells did not change significantly after OGD/R. Inhibition of caspase-1 or GSDMD activity, or down-regulation of GSDMD expression, alleviated pyroptosis in both BV2 and HT22 cells after OGD/R. Transwell studies further showed that OGD/R-treated HT22 or BV2 cells aggravated pyroptosis of adjacent non-OGD/R-treated cells, which could be relieved by inhibition of caspase-1 or GSDMD. These results suggested that OGD/R induces pyroptosis of microglia and neuronal cells and aggravates cell injury via activation of caspase-1/GSDMD signaling pathway. Our findings indicated that caspase-1 and GSDMD may be therapeutic targets after cerebral I/R.

CircRNA expression profiles and function prediction in peripheral blood mononuclear cells of patients with acute ischemic stroke.

Most circular RNAs (circRNAs) belong to a novel class of noncoding RNAs that are produced by back-splicing reactions, and they regulate physiological and pathophysiological processes in human disease. Although circRNA expression has been shown to be altered in the ischemic cerebral tissue in animal studies, the expression profile of circRNA in the patients with acute ischemic stroke (AIS) has not been investigated to date. In this investigation, high-throughput sequencing was carried out to compare the circRNA expression of peripheral blood mononuclear cells (PBMCs) from five patients with AIS and five healthy subjects. A total of 521 circRNAs were expressed differentially between the patients with AIS and healthy controls (p < .05, fold difference ≥2) including 373 upregulated circRNAs and 148 downregulated circRNAs in patients with AIS compared to controls. Thirteen candidate circRNAs were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics analyses showed that these differentially expressed circRNAs were highly conserved, as well as eight circRNAs that were confirmed by qRT-PCR containing binding sites to multiple microRNAs. Kyoto Encyclopedia of Genes and Genomes pathway enrichment and gene ontology analyses indicated that the aberrantly expressed circRNAs participated in many pathophysiological processes of AIS, especially regarding inflammation and immunity. In conclusion, patients with AIS differentially express certain circRNAs in PBMCs, which may be diagnostic biomarkers or potential therapeutic targets.

The TLR9 Antagonist iCpG-ODN at Different Dosages Inhibits Cerebral Ischemia/Reperfusion Injury in Mice.

BACKGROUND: Inflammatory responses are important mechanisms that are involved in cerebral ischemia/reperfusion(I/R) injury. Whether toll-like receptor 9(TLR9), which belongs to the innate immune system, takes part in the inflammatory responses following cerebral I/R remains unclear. METHOD: This study examined the effect of different dosages of the TLR9 antagonist inhibitory oligodeoxynucleotide (iCpG-ODN) on cerebral I/R injury by using a mouse model of transient middle cerebral artery occlusion. Neurological function, infarct size, splenocytes and the expression of TLR9 and the downstream products of the TLR9 pathways were determined after cerebral I/R for up to 72 hours. RESULTS: The Clark's focal symptom scoring showed iCpG-ODN improved neurological deficits following focal cerebral I/R. The iCpG-ODN administration significantly decreased the infarct size in a dose-dependent manner. RT-PCR showed that iCpG-ODN attenuated the I/R-induced RNA expression of TLR9. Immunoblot showed that iCpG-ODN prevented I/R-induced increases in NFκB and IRF7 levels and that it further downregulated the levels of IL-1ß, TNF-α, and INF-ß in the brain. iCpG-ODN did not alter the levels of TNF-α or INF-ß in the peripheral blood or affect stroke-induced changes in the number of splenocytes. CONCLUSION: These findings suggest that iCpG-ODN induced protection against cerebral I/R via inhibiting inflammatory responses in a dose-dependent manner and may be useful in therapy for stroke patients.

Toll-Like Receptor 3 and Interferon ß mRNA Expressions Were Increased in Peripheral Blood of Ischemic Stroke Patients with Good Outcome.

BACKGROUND: Innate immunity plays an important role in brain ischemic injury, but there are only few studies on the effects of toll-like receptors (TLRs) on cerebral infarction patients up to now. We aimed to evaluate the TLR mRNA expression of patients with different outcomes. METHODS: Eighty-six cases suffering from cerebral infarction within 14 days were assigned into the good outcome group (n = 47) and the bad outcome group (n = 39) depending on the modified Rankin Scale scores (mRS ≤2 at 90 days following stroke onset was good outcome). We measured the mRNA expression of TLRs in peripheral blood mononuclear cells of patients at 24 hours, 3 days, 4 days, 7 days, and 14 days from onset. The National Institutes of Health Stroke Scale score and infarction volume were assessed on admission and at 7-14 days, respectively. RESULTS: Only TLR3 mRNA expression of the good outcome group was higher than that of the bad outcome group at acute and subacute phases. TLR7 expressions of the good outcome group increased within 3 days following stroke onset. Moreover, the two groups had no significant differences in terms of mRNA expressions of TLR2, TLR4, TLR8, and TLR9. The expression of interferon ß of the good outcome group was higher than that of the bad outcome group, and it had a positive correlation with the expressions of TLR3 and interferon regulatory factor 3. CONCLUSIONS: TLR3 and interferon ß mRNA expressions were increased in the peripheral blood of ischemic stroke patients with good outcome, which may imply their neuroprotection.

Transcription of the human sodium channel SCN1A gene is repressed by a scaffolding protein RACK1.

Voltage-gated sodium channel α subunit type I (Nav1.1, encoded by SCN1A gene) plays a critical role in the initiation of action potential in the central nervous system. Downregulated expression of SCN1A is believed to be associated with epilepsy. Here, we found that the SCN1A promoter (P1c), located at the 5' untranslated exon 1c, drove the reporter gene expression in human NT2 cells, and a region between nt +53 and +62 downstream of the P1c promoter repressed the promoter activity. Further analyses showed that a scaffolding protein RACK1 (receptor for activated C kinase 1) was involved in binding to this silencer. Knockdown of RACK1 expression in NT2 cells deprived the repressive role of the silencer on the P1c promoter and increased SCN1A transcription, suggesting the potential involvement of RACK1 in negatively regulating SCN1A transcription via interaction with the silencer. Furthermore, we demonstrated that the binding of the protein complex including RACK1 to the SCN1A promoter motif was decreased in neuron-like differentiation of the NT2 cells induced by retinoic acid and resulted in the upregulation of SCN1A transcription. Taken together, this study reports a novel role of RACK1 in regulating SCN1A expression that participates in retinoic acid-induced neuronal differentiation of NT2 cells.

A novel variant in the 3' UTR of human SCN1A gene from a patient with Dravet syndrome decreases mRNA stability mediated by GAPDH's binding.

Mutations in the SCN1A gene-encoding voltage-gated sodium channel α-I subunit (Nav1.1) cause various spectrum of epilepsies including Dravet syndrome (DS), a severe and intractable form. A large number of SCN1A mutations identified from the DS patients lead to the loss of function or truncation of Nav1.1 that result in a haploinsufficiency effects, indicating that the exact expression level of SCN1A should be essential to maintain normal brain function. In this study, we have identified five variants c.*1025T>C, c.*1031A>T, c.*1739C>T, c.*1794C>T and c.*1961C>T in the SCN1A 3' UTR in the patients with DS. The c.*1025T>C, c.*1031A>T and c.*1794C>T are conserved among different species. Of all the five variants, only c.*1794C>T is a novel variant and alters the predicted secondary structure of the 3' UTR. We also show that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) only binds to the 3' UTR sequence containing the mutation allele 1794U but not the wild-type allele 1794C, indicating that the mutation allele forms a new GAPDH-binding site. Functional analyses show that the variant negatively regulates the reporter gene expression by affecting the mRNA stability that is mediated by GAPDH's binding, and this phenomenon could be reversed by shRNA-induced GAPDH knockdown. These findings suggest that GAPDH and the 3'-UTR variant are involved in regulating SCN1A expression at post-transcriptional level, which may provide an important clue for further investigating on the relationship between 3'-UTR variants and SCN1A-related diseases.