Inhibition of NLRP3 inflammasome alleviates cognitive deficits in a mouse model of anti-NMDAR encephalitis induced by active immunization.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a neurological disorder, characterized by cognitive deficits as one of its vital features. The nucleotide-binding oligomerization domain-like receptor (NLRP3) inflammasome is a key contributor to neuroinflammation and cognitive deficits in neurological diseases. However, the underlying mechanism of anti-NMDAR encephalitis remains unclear, and the biological function of the NLRP3 inflammasome in this condition has not been elucidated. In this study, a mouse model of anti-NMDAR encephalitis was induced by active immunization with the GluN1356-385 peptide (NEA model). The NLRP3 inflammasome in the hippocampus and temporal cortex was investigated using real-time quantitative PCR (RT-qPCR), western blotting, and immunofluorescence staining. The impact of MCC950 on cognitive function and NLRP3 inflammation was assessed. Confocal immunofluorescence staining and Sholl analysis were employed to examine the function and morphology of microglia. In the current study, we discovered overactivation of the NLRP3 inflammasome and an enhanced inflammatory response in the NEA model, particularly in the hippocampus and temporal cortex. Furthermore, significant cognitive dysfunction was observed in the NEA model. While, MCC950, a selective inhibitor of the NLRP3 inflammasome, sharply attenuated the inflammatory response in mice, leading to mitigated cognitive deficits of mice and more regular arrangements of neurons and reduced number of hyperchromatic cells were also observed in the hippocampus area. In addition, we found that the excess elevation of NLRP3 inflammasome was mainly expressed in microglia accompanied with the overactivation of microglia, while MCC950 treatment significantly inhibited the increased number and activated morphological changes of microglia in the NEA model. Altogether, our study reveals the vital role of overactivated NLRP3 signaling pathway in aggravating the inflammatory response and cognitive deficits and the potential protective effect of MCC950 in anti-NMDAR encephalitis. Thus, MCC950 represents a promising strategy for anti-inflammation in anti-NMDAR encephalitis and our study lays a theoretical foundation for it to become a clinically targeted drug.

The miR-23b-3p from adipose-derived stem cell exosomes alleviate inflammation in mice experiencing kainic acid-induced epileptic seizures.

Epilepsy is a common neurologic disorder. While a good clinical solution is still missing, studies have confirmed that exosomes (Exos) derived from adipose-derived stem cells (ADSCs) had a therapeutic effect on various diseases, including neurological diseases. Therefore, this study aimed to reveal whether ADSC-Exo treatment could improve kainic acid (KA)-induced seizures in epileptic mice. ADSCs and Exos were isolated. Mice were generated with KA-induced epileptic seizures. ELISA was used to detect inflammatory factor expression. Luciferase reporter analysis detection showed a relationship among miR-23b-3p, STAT1, and glyoxylate reductase 1 (GlyR1). ADSC-Exos had a protective effect on KA-induced seizures by inhibiting inflammatory factor expression and the M1 microglia phenotype. The result showed that miR-23b-3p played an important role in the Exo-mediated protective effect in KA-induced seizures in epileptic mice by regulating STAT1 and GlyR1. Luciferase reporter analysis confirmed that miR-23b-3p interacted with the 3'-UTR of STAT1 and GlyR1. The miR-23b-3p inhibited M1 microglia-mediated inflammatory factor expression in microglial cells by regulating STAT1 and GlyR1. The downregulation of miR-23b-3p decreased the protective effect of ADSC-Exos on KA-induced seizures in epileptic mice. The miR-23b-3p from ADSC-Exos alleviated inflammation in mice with KA-induced epileptic seizures.

Rapamycin alleviates mitochondrial dysfunction in anti-NMDAR encephalitis mice.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is one of the most prevalent forms of autoimmune encephalitis, characterized by a series of neurological and psychiatric symptoms, including cognitive impairment, seizures and psychosis. The underlying mechanism of anti-NMDAR encephalitis remains unclear. In the current study, the mouse model of anti-NMDAR encephalitis with active immunization was performed. We first uncovered excessive mitochondrial fission in the hippocampus and temporal cortex of anti-NMDAR encephalitis mice, indicated by elevated level of Phospho-DRP1 (Ser616) (p-Drp1-S616). Moreover, blockade of the autophagic flux was also demonstrated, leading to the accumulation of fragmented mitochondria, and elevated levels of mitochondrial reactive oxygen species (mtROS) and mitochondrial DNA (mtDNA) in anti-NMDAR encephalitis. More importantly, we found that the mTOR signaling pathway was overactivated, which could aggravate mitochondrial fission and inhibit autophagy, resulting in mitochondrial dysfunction. While rapamycin, the specific inhibitor of the mTOR signaling pathway, significantly alleviated mitochondrial dysfunction by inhibiting mitochondrial fission and enhancing autophagy. Levels of mtROS and mtDNA were markedly reduced after the treatment of rapamycin. In addition, rapamycin also significantly alleviated cognitive dysfunction and anxious behaviors found in anti-NMDAR encephalitis mice. Thus, our study reveals the vital role of mitochondrial dysfunction in pathological mechanism of anti-NMDAR encephalitis and lays a theoretical foundation for rapamycin to become a clinically targeted drug for anti-NMDAR encephalitis.

Systematic analysis identifies REST as an oncogenic and immunological biomarker in glioma.

The repressor element 1 silencing transcription factor (REST) has been proposed to function as a transcription factor to silence gene transcription by binding to repressor element 1 (RE1), a highly conserved DNA motif. The functions of REST in various tumors have been studied, but its role and correlation with immune cell infiltration remains uncertain in gliomas. REST expression was analyzed in datasets of The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) and validated by the Gene Expression Omnibus and Human Protein Atlas databases. The clinical prognosis of REST was evaluated by clinical survival data of TCGA cohort and validated by Chinese Glioma Genome Atlas cohort. MicroRNAs (miRNAs) contributing to REST overexpression in glioma were identified by a combination of a series of in silico analyses, including expression analysis, correlation analysis, and survival analysis. The correlations between immune cell infiltration level and REST expression were analyzed by TIMER2 and GEPIA2 tools. Enrichment analysis of REST was performed using STRING and Metascape tools. The expression and function of predicted upstream miRNAs at REST and their association with glioma malignancy and migration were also confirmed in glioma cell lines. REST was highly expressed and associated with poorer overall survival and disease-specific survival in glioma and some other tumors. MiR-105-5p and miR-9-5p were identified as the most potential upstream miRNAs of REST in glioma patient cohort and experiments in vitro. REST expression was positively correlated with infiltration of immune cells and the expression of immune checkpoints such as PD1/PD-L1 and CTLA-4 in glioma. Furthermore, histone deacetylase 1 (HDAC1) was a potential REST-related gene in glioma. Enrichment analysis of REST found chromatin organization and histone modification were the most significant enriched terms, and Hedgehog-Gli pathway might be involved in the effect of REST on the pathogenesis of glioma. Our study suggests REST to be an oncogenic gene and the biomarker of poor prognosis in glioma. High REST expression might affect the tumor microenvironment of glioma. More basic experiments and large clinical trials aimed at the carcinogenetic study of REST in glioma will be needed in the future.

Clinical features of epileptic seizures in patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes.

BACKGROUND AND PURPOSE: This study aimed to characterize the clinical features of epilepsy in mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) and analyze the clinical determinants for drug-resistant epilepsy in MELAS. METHODS: A single-center, retrospective study was conducted to investigate the clinical features of epilepsy in patients with MELAS. Collected variables included seizure semiology, electroencephalography (EEG), muscle biopsy, genetic testing, neuroimaging findings, resting serum lactic value and modified Rankin scale (mRS) of patients with MELAS. We also investigated the differences between the adult-onset group and the child-onset group and analyzed the risk factors for drug-resistant epilepsy in MELAS. RESULTS: We studied 97 patients (56 males: 41 females) with confirmed MELAS. Epileptic seizure occurred in 100.0% of patients and the initial symptom of 69.1% patients was epileptic seizure. The average age of disease onset was 21.0 years, ranging from 2 to 60 years. The seizure types of these patients with MELAS were variable, with generalized onset (51.5%) to be the most common type. The EEG changes in the patients with MELAS were mainly slow wave (90.9%) and epileptiform discharge (68.2%). The child-onset group with earlier seizure onset presented significantly higher resting serum lactic value (p = 0.0048) and lower incidence of stroke-like lesion in the brain (p = 0.003), especially in the temporal lobe (p < 0.001), compared with the adult-onset group. Importantly, drug-resistant epilepsy in MELAS was demonstrated to be closely related to the earlier age of seizure onset (p = 0.013), as well as the higher mRS score (p < 0.001) and higher resting serum lactic value (p = 0.009). CONCLUSION: Early identification of MELAS should be considered among individuals with recurrent epilepsy through clinical screening. Age of seizure onset and resting serum lactic value may predict the development of drug-resistant epilepsy in MELAS. Close observation and appropriate anti-epileptic treatment are indispensable for individuals with MELAS to improve the prognosis. Further studies with larger sample size are required to further evaluate the risk factors of drug-resistant epilepsy in MELAS and provide guidance on treatment of MELAS.

Vav2 is a novel APP-interacting protein that regulates APP protein level.

Amyloid precursor protein (APP) is a transmembrane protein that plays critical role in the pathogenesis of Alzheimer's disease (AD). It is also involved in many types of cancers. Increasing evidence has shown that the tyrosine phosphorylation site Y682 in the intracellular tail of APP is crucial for APP function. Here, we report that Vav2, a guanine nucleotide exchange factor (GEF) for Rho family GTPase, is a novel interaction partner of APP. We found that Vav2-SH2 domain was able to bind directly to the Y682-phosphorylated intracellular tail of APP through isothermal titration calorimetry and NMR titrating experiments. The crystal structure of Vav2-SH2 in complex with an APP-derived phosphopeptide was determined to understand the structural basis of this recognition specificity. The interaction of APP and Vav2 in a full-length manner was further confirmed in cells by GST pull-down, co-immunoprecipitation and immunofluorescence staining experiments. In addition, we found overexpression of Vav2 could inhibit APP degradation and markedly increase the protein levels of APP and its cleavage productions in 20E2 cells, and this function of Vav2 required a functional SH2 domain.

Upregulation of RCAN1.4 by HIF1α alleviates OGD-induced inflammatory response in astrocytes.

OBJECTIVE: Ischemic stroke is a leading cause of human mortality and long-term disability worldwide. As one of the main forms of regulator of calcineurin 1 (RCAN1), the contribution of RCAN1.4 in diverse biological and pathological conditions has been implicated. But the role of RCAN1.4 in ischemic stroke progression remains elusive. This study is to explore the expression changes and roles of RCAN1.4 in ischemic stroke as well as the underlying mechanisms for these changes and effects of RCAN1.4 in ischemic stroke. METHODS: Middle cerebral artery occlusion model in C57BL/6J mice and oxygen-glucose deprivation (OGD) model in primary astrocytes were performed to induce the cerebral ischemic stroke. The expression pattern of RCAN1.4 was assessed using real-time quantitative PCR and western blotting in vivo and in vitro. Mechanistically, the underlying mechanism for the elevation of RCAN1.4 in the upstream was investigated. Lentiviruses were administrated, and the effect of RCAN1.4 in postischemic inflammation was clearly clarified. RESULTS: Here we uncovered that RCAN1.4 was dramatically increased in mouse ischemic brains and OGD-induced primary astrocytes. HIF1α, activated upon OGD, significantly upregulated RCAN1.4 gene expression through specifically binding to the RCAN1.4 promoter region and activating its promoter activity. The functional hypoxia-responsive element (HRE) was located between -254 and -245 bp in the RCAN1.4 promoter region. Moreover, elevated RCAN1.4 alleviated the release of pro-inflammatory cytokines TNFα, IL1ß, IL6 and reduced expression of iNOS, COX2 in primary astrocytes upon OGD, whereas RCAN1.4 silencing has the opposite effect. Of note, RCAN1.4 overexpression inhibited OGD-induced NF-κB activation in primary astrocytes, leading to decreased degradation of IκBα and reduced nuclear translocation of NF-κB/p65. INTERPRETATION: Our results reveal a novel mechanism underscoring the upregulation of RCAN1.4 by HIF1α and the protective effect of RCAN1.4 against postischemic inflammation, suggesting its significance as a promising therapeutic target for ischemic stroke treatment.

Targeting upregulated RNA binding protein RCAN1.1: a promising strategy for neuroprotection in acute ischemic stroke.

AIMS: To explore the expression changes and roles of the RNA-binding protein RCAN1.1 in acute ischemic stroke (AIS), and to preliminarily confirm the medicinal value of the RNA aptamer R1SR13 in AIS by targeting RCAN1.1. METHODS: Two mouse AIS models of middle cerebral artery occlusion (MCAO) and right common carotid artery ligation (R-CCAL) and oxygen glucose deprivation (OGD) model of AIS in primary neurons and SH-SY5Y were performed. The expression pattern of RCAN1.1 was assessed using real-time quantitative PCR (RT-qPCR) and western blotting (WB) in vivo and in vitro. The underlying mechanism for the elevation of RCAN1.1 in the upstream was investigated. Lentiviruses were administrated and the effect of RCAN1.1 in AIS was assessed by ATP level, caspase 3/7 assay, TUNEL and WB. The protective function of R1SR13 in AIS was evaluated both in vivo and in vitro. RESULTS: In two mouse models of AIS, RCAN1.1 mRNA and RCAN1.1 L protein were significantly upregulated in the ischemic brain tissue. The same results were detected in the OGD model of primary neurons and SH-SY5Y. The mechanistic analysis proved that hypoxia-inducible factor-1α (HIF1α) could specifically activate the RCAN1.1 gene promoter through combining with the functional hypoxia-responsive element (HRE) site (-325 to -322 bp). The increased expression of RCAN1.1 L markedly depleted ATP production and aggravated neuronal apoptosis under OGD condition. R1SR13, an antagonizing RNA aptamer of RCAN1.1, was demonstrated to reduce neuronal apoptosis caused by the elevated RCAN1.1 L in the cellular and animal models of AIS. CONCLUSION: RCAN1.1 is a novel target gene of HIF1α and the functional HRE in the RCAN1.1 promoter region is -325 to -322 bp. The marked upregulation of RCAN1.1 in AIS promoted neuronal apoptosis, an effect that could be reversed by its RNA aptamer R1SR13 in vivo and in vitro. Thus, R1SR13 represents a promising strategy for neuroprotection in AIS and our study lays a theoretical foundation for it to become a clinically targeted drug.

Risk factors for postictal generalized EEG suppression in generalized convulsive seizure: A systematic review and meta-analysis.

BACKGROUND AND PURPOSE: Postictal generalized EEG suppression (PGES) has been suggested as a pathophysiological hallmark for sudden unexpected death in epilepsy (SUDEP). We aimed to characterize the clinical determinants for PGES occurrence after generalized convulsive seizures (GCS). METHODS: We systematically searched Pubmed, Embase and Medline databases up to 30 August 2021. Eligibility screening, data extraction, and quality assessment of the retrieved articles were conducted by two independent reviewers. Studies reporting potential risk factors of PGES occurrence in GCS were included for subsequent meta-analysis and PGES was defined as a generalized EEG attenuation of any duration >1s below 10µV, immediately or within 30s after an ictal EEG pattern has terminated. A fixed-effects model was applied when the heterogeneity is low (I2 values < 50%). Otherwise, a random-effects model was used (I2 values ≥ 50%). We assessed the odds ratio (OR) as outcome measure for dichotomous variables and the STD Mean Difference (SMD) for continuous variables. The Begg test and the Egger test was applied in the assessment of publication bias. RESULTS: A total of 15 relevant studies were identified, enrolling 2057 GCSs. The incidence of PGES in GCS from 15 studies varied from 23% to 86%. The longer tonic phase duration (SMD, 0.26; 95%CI, 0.13 to 0.39; p < 0.001), sleep state at GCS onset (OR,1.63; 95%CI, 1.24 to 2.16; p = 0.001), older age of epilepsy onset (SMD, 0.48; 95%CI, 0.21 to 0.75; p = 0.001), the presence of postictal immobility (OR, 78.05; 95%CI, 32.31 to 188.53; p < 0.001) and oxygen desaturation nadir (SMD, -0.54; 95%CI, -0.76 to -0.33; p < 0.001) showed significant association with the likelihood of having PGES in GCS, but not total seizure duration (SMD, -0.06; 95%CI, -0.20 to 0.08; p = 0.385), tonic-clonic duration (SMD, -0.12; 95%CI, -0.26 to 0.01; p = 0.071), clonic phase duration (SMD, -0.09; 95%CI, -0.27 to 0.08; p = 0.293), epilepsy duration of patients (SMD, -0.09; 95%CI, -0.27 to 0.08; p = 0.293) or lack of early O2 administration (OR, 1.59; 95%CI, 0.80 to 3.17; p = 0.184). CONCLUSION: The current study informed that PGES is common after GCS. Early identification should be considered among individuals with GCS at high risk of PGES through clinical screening. Further studies with larger sample size are required for individualized evaluation of the risk of PGES in GCS and more effort is needed to further evaluate the risk of SUDEP.