Biosynthesis of neuroprotective melatonin is dysregulated in Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative brain disorder associated with uncontrolled body movements, cognitive decline, and reduced circulating melatonin levels. Melatonin is a potent antioxidant and exogenous melatonin treatment is neuroprotective in experimental HD models. In neurons, melatonin is exclusively synthesized in the mitochondrial matrix. Thus, we investigated the integrity of melatonin biosynthesis pathways in pineal and extrapineal brain areas in human HD brain samples, in the R6/2 mouse model of HD and in full-length mutant huntingtin knock-in cells. Aralkylamine N-acetyltransferase (AANAT) is the rate-limiting step enzyme in the melatonin biosynthetic pathway. We found that AANAT expression is significantly decreased in the pineal gland and the striatum of HD patients compared to normal controls. In the R6/2 mouse forebrain, AANAT protein expression was decreased in synaptosomal, but not nonsynaptosomal, mitochondria and was associated with decreased synaptosomal melatonin levels compared to wild type mice. We also demonstrate sequestration of AANAT in mutant-huntingtin protein aggregates likely resulting in decreased AANAT bioavailability. Paradoxically, AANAT mRNA expression is increased in tissues where AANAT protein expression is decreased, suggesting a potential feedback loop that is, ultimately unsuccessful. In conclusion, we demonstrate that pineal, extrapineal, and synaptosomal melatonin levels are compromised in the brains of HD patients and R6/2 mice due, at least in part, to protein aggregation.

Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release.

G protein-coupled receptors (GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT1), its associated G protein, and ß-arrestins are on and within neuronal mitochondria. We discovered that the ligand melatonin is exclusively synthesized in the mitochondrial matrix and released by the organelle activating the mitochondrial MT1 signal-transduction pathway inhibiting stress-mediated cytochrome c release and caspase activation. These findings coupled with our observation that mitochondrial MT1 overexpression reduces ischemic brain injury in mice delineate a mitochondrial GPCR mechanism contributing to the neuroprotective action of melatonin. We propose a new term, "automitocrine," analogous to "autocrine" when a similar phenomenon occurs at the cellular level, to describe this unexpected intracellular organelle ligand-receptor pathway that opens a new research avenue investigating mitochondrial GPCR biology.

Long noncoding ribonucleic acid NKILA induces the endoplasmic reticulum stress/autophagy pathway and inhibits the nuclear factor-k-gene binding pathway in rats after intracerebral hemorrhage.

Long noncoding RNAs (lncRNAs) have emerged as an important class of molecules that have been associated with brain function and neurological disease, but the expression profiles of lncRNAs after intracerebral hemorrhage (ICH) remain to be elucidated. In this study, we determined the expression pattern of nuclear factor-k-gene binding (NF-kB) interacting lncRNA (NKILA) after ICH and examined its respective effects on the endoplasmic reticulum stress (ERS)/autophagy pathway, hippocampal neuron loss, and the NF-kB pathway after type VII collagenase-induced ICH in rats. The regulatory mechanisms of NKILA were investigated by an intraperitoneal injection of small interfering (siRNA) against NKILA into rats after ICH. NKILA inhibition mediated by siRNA against NKILA was shown to significantly reduce ERS and autophagy, activate the NF-kB pathway, decrease neurological deficits, brain edema, and injury, and induce blood-brain barrier breakdown, further leading to hippocampal neuron loss and the production of inflammation cytokines. Taken together, the demonstration that NKILA induces the ERS/autophagy pathway and inhibits the NF-kB pathway after ICH supports the concept that NKILA functions as a novel target that is required for the attenuation of brain injuries after ICH.

Oncogenic DIRAS3 promotes malignant phenotypes of glioma by activating EGFR-AKT signaling.

Epidermal growth factor receptor (EGFR)-Akt signaling cascade activation plays a pivotal role in gliomas malignant phenotype, especially in Classical and Mesenchymal subtype gliomas. However, the molecules and mechanisms underlying regulate and maintain the activation of EGFR-AKT signaling remains unclear. Previously reports showed that DIRAS3 inhibits cell proliferation and induces autophagy in ovarian, breast, lung and prostate cancers, which is heterozygosity loss or down-regulated in aforementioned cancers and functionally as a tumor suppressor, whereas the role of DIRAS3 in glioma is still veiled. Here, in this study, we investigated the biological function and role of DIRAS3 in gliomas, and found that DIRAS3 is up-regulated in gliomas and is positively correlated with poor prognosis of glioma patients, meanwhile, over-expressed DIRAS3 promotes glioma cells proliferation and invasion. Further mechanistic study showed that the expression level of DIRAS3 in Classical and Mesenchymal subtype GBMs is higher, and over-expression of DIRAS3 promotes EGFR-AKT signaling activation at the downstream of EGFR and increases AKT phosphorylation, meanwhile suppression of AKT by MK-2206 reverses the tumor promoting function of DIRAS3. Taken together, these findings reveal a novel oncogenic role of DIRAS3 in the development and progression of glioma, which suggest that DIRAS3 could serve as a potential diagnostic marker and a promising therapeutic target of gliomas.

Transcription Factor TFAP2B Exerts Neuroprotective Effects Targeting BNIP3-Mediated Mitophagy in Ischemia/Reperfusion Injury.

Cerebral ischemia-reperfusion injury (CIRI) leads to malignant brain edema, blood-brain barrier destruction, and neuronal apoptosis. N6-methyladenosine (m6A) RNA modification in CIRI was still limited explored. In this study, MeRIP- and RNA-sequencing were performed of middle cerebral artery occlusion and reperfusion (MCAO/R) rats to find novel potential molecular targets. Transcription factor TFAP2B stood out of which its m6A abundance decreased associated with a marked reduction of its mRNA based on cojoint interactive bioinformatics analysis of the MeRIP- and RNA-sequencing data. It was suggested TFAP2B could have a role in CIRI. Functionally, overexpression of TFAP2B in cultured primary neurons could effectively improve the cell survival and pro-survival autophagy in parallel with reduced cell apoptosis during OGD/R in vitro. Through the RNA-sequencing of TFAP2B overexpressed primary neurons and subsequent validation experiments, it was found that mitophagy receptor BNIP3 was one of the important targets of TFAP2B in OGD/R neurons through which TFAP2B could bind to its promoter region for transcriptional activation of BNIP3, thereby enhancing BNIP3-mediated mitophagy to protect against OGD/R injury of neurons. Lastly, TFAP2B was demonstrated to alleviate the MCAO/R damage to a certain extent in vivo. Although it failed to confirm TFAP2B dysregulation was m6A dependent in current research, this is the first research of TFAP2B in CIRI field with important guiding significance.

CD73-positive extracellular vesicles promote glioblastoma immunosuppression by inhibiting T-cell clonal expansion.

Extracellular vesicles are involved in the occurrence, progression and metastasis of glioblastoma (GBM). GBM can secrete a variety of tumour-derived extracellular vesicles (TDEVs) with high immunosuppressive activity that remotely suppress the systemic immune system, and therapy targeting TDEVs has potential efficacy. In this study, we detected a higher concentration of CD73+ TDEVs enriched in exosomes in central and peripheral body fluids of GBM patients than in those of patients with other brain tumours (low-grade glioma or brain metastases from melanoma or non-small-cell lung cancer). High CD73 expression was detected on the surface of T cells, and this CD73 was derived from TDEVs secreted by GBM cells. In vitro, we observed that CD73+ TDEVs released by GBM cell lines could be taken up by T cells. Moreover, excess adenosine was produced by AMP degradation around T cells and by adenosine receptor 2A (A2AR)-dependent inhibition of aerobic glycolysis and energy-related metabolic substrate production, thereby inhibiting the cell cycle entry and clonal proliferation of T cells. In vivo, defects in exosomal synthesis and CD73 expression significantly inhibited tumour growth in GBM tumour-bearing mice and restored the clonal proliferation of T cells in the central and peripheral regions. These data indicate that CD73+ TDEVs can be used as a potential target for GBM immunotherapy.

Molecular and Clinical Characterization of a Novel Prognostic and Immunologic Biomarker GPSM3 in Low-Grade Gliomas.

Background: as the most common malignancy of the central nervous system, low-grade glioma (LGG) patients suffered a poor prognosis. Tumor microenvironment, especially immune components, plays an important role in the progression of tumors. Thus, it is critical to explore the key immune-related genes, a comprehensive understanding of the TME in LGG helps us find novel cancer biomarkers and therapeutic targets. Methods: the GPSM3 expression level and the correlations between clinical characteristics and GPSM3 levels were analyzed with the data from CGGA and TCGA dataset. Univariate and multivariate cox regression model were built to predict the prognosis of LGG patients with multiple factors. Then the correlation between GPSM3 with immune cell infiltration was explored by ESTIMATE, CIBERSORT and TIMER2.0. At last, the correlation analyzed between GPSM3 expression and immune checkpoint related genes were also analyzed. Results: GPSM3 expression was overexpressed in LGG and negatively correlated to the GPSM3 DNA methylation. Univariate and multivariate Cox analysis demonstrated that GPSM3 expression was an independent prognostic factor in LGG patients. Functional characterization of GPSM3 revealed that it was associated with many immune processes to tumor cells. GPSM3 expression was positive related to the immune score, Stromal scores and ESTIMATE scores, but negative related to the Tumor purity. Immune features in the TME of GPSM3-high LGG group is characterized by a higher infiltrating of regulatory T cells, neutrophils, macrophages M2, and a lower proportion of monocytes than to the GPSM3-low group. Furthermore, GPSM3 expression exhibited significant correlations with the immune checkpoint-related genes, especially PD-1, PD-L1, PD-L2, CTLA4 and TIM3. Conclusions: these findings proved that GPSM3 could serve as a prognostic biomarker and potential immunotherapy target for LGG.

Transcriptional factor FoxM1-activated microRNA-335-3p maintains the self-renewal of neural stem cells by inhibiting p53 signaling pathway via Fmr1.

BACKGROUND: New mechanistic insights into the self-renewal ability and multipotent properties of neural stem cells (NSCs) are currently under active investigation for potential use in the treatment of neurological diseases. In this study, NSCs were isolated from the forebrain of fetal rats and cultured to induce NSC differentiation, which was associated with low expression of the non-coding RNA microRNA-335-3p (miR-335-3p). METHODS: Loss- and gain-of-function experiments were performed in NSCs after induction of differentiation. RESULTS: Overexpression of miR-335-3p or FoxM1 and inhibition of the Fmr1 or p53 signaling pathways facilitated neurosphere formation, enhanced proliferation and cell cycle entry of NSCs, but restricted NSC differentiation. Mechanistically, FoxM1 positively regulated miR-335-3p by binding to its promoter region, while miR-335-3p targeted and negatively regulated Fmr1. Additionally, the promotive effect of miR-335-3p on NSC self-renewal occurred via p53 signaling pathway inactivation. CONCLUSION: Taken together, miR-335-3p activated by FoxM1 could suppress NSC differentiation and promote NSC self-renewal by inactivating the p53 signaling pathway via Fmr1.

MiR-421 Binds to PINK1 and Enhances Neural Stem Cell Self-Renewal via HDAC3-Dependent FOXO3 Activation.

Dysfunctions of neural stem cells (NSCs) often lead to a variety of neurological diseases. Thus, therapies based on NSCs have gained increasing attention recently. It has been documented that microRNA (miR)-421 represses the autophagy and apoptosis of mouse hippocampal neurons and confers a role in the repair of ischemic brain injury (IBI). Herein, we aimed to illustrate the effects of miR-421 on NSC self-renewal. The downstream factors of miR-421 were predicted initially, followed by gain- and loss-of-function assays to examine their effects on NSC self-renewal. Immunoprecipitation and dual luciferase assays were conducted to validate the interaction among miR-421, PTEN-induced putative kinase 1 (PINK1), HDAC3, and forkhead box O3 (FOXO3). A mouse model with IBI was developed to substantiate the impact of the miR-421/PINK1/HDAC3/FOXO3 axis on NSC self-renewal. The expression of miR-421 was downregulated during differentiation of human embryonic NSCs, and miR-421 overexpression accelerated NSC self-renewal. Besides, miR-421 targeted PINK1 and restricted its expression in NSCs and further suppressed HDAC3 phosphorylation and enhanced FOXO3 acetylation. In conclusion, our data elucidated that miR-421 overexpression may facilitate NSC self-renewal through the PINK1/HDAC3/FOXO3 axis, which may provide potential therapeutic targets for the development of novel therapies for IBI.

MicroRNA-361-5p slows down gliomas development through regulating UBR5 to elevate ATMIN protein expression.

MicroRNA (miR)-361-5p has been studied to suppress gliomas development. Based on that, an insight into the regulatory mechanism of miR-361-5p in gliomas was supplemented from ubiquitin protein ligase E3 component N-recognin 5 (UBR5)-mediated ubiquitination of ataxia-telangiectasia mutated interactor (ATMIN). miR-361-5p, ATMIN, and UBR5 levels were clinically analyzed in gliomas tissues, which were further validated in gliomas cell lines. Loss/gain-of-function method was applied to determine the roles of miR-361-5p and UBR5 in gliomas, as to cell viability, migration, invasion, colony formation ability, and apoptosis in vitro and tumorigenesis in vivo. The relationship between miR-361-5p and UBR5 was verified and the interaction between UBR5 and ATMIN was explored. It was detected that reduced miR-361-5p and ATMIN and enhanced UBR5 levels showed in gliomas. Elevating miR-361-5p was repressive in gliomas progression. UBR5 was directly targeted by miR-361-5p. UBR5 can ubiquitinate ATMIN. miR-361-5p suppressed gliomas by regulating UBR5-mediated ubiquitination of ATMIN. Downregulating UBR5 impeded gliomas tumor growth in vivo. Upregulating miR-361-5p targets UBR5 to promote ATMIN protein expression, thus to recline the malignant phenotype of gliomas cells.

MicroRNA-579-3p Exerts Neuroprotective Effects Against Ischemic Stroke via Anti-Inflammation and Anti-Apoptosis.

BACKGROUND/AIMS: Multiple studies have found that microRNAs (miRNAs) are involved in the development of cerebral ischemia. MiR-579-3p can inhibit inflammatory responses and apoptosis, leading to ischemia/reperfusion (I/R) damage. However, the mechanism of how miR-579-3p actions in brain I/R injury remains unclear. This study aimed to investigate the mechanism of the role of miR-579-3p in brain I/R injury. METHODS: A rat model of cerebral ischemia-reperfusion injury was established by suture method. The effects of miR-579-3p on cerebral infarction size, brain water content, and neurological symptoms were evaluated. Flow cytometry was used to detect apoptosis. ELISA was used to detect the level of inflammatory factors. Western blot was used to detect the expression of P65, NCOA1, Bcl-2 and Bax. The relationship between miR-579-3p and NCOA1 was analyzed by bioinformatics analysis and luciferase assay. RESULTS: Overexpression of miR-579-3p reduced infarct volume, brain water content and neurological deficits. Overexpression of miR-579-3p inhibited the expression level of the inflammatory cytokines, such as TNF-α, IL-6, COX-2 and iNOS, and increased the expression level of IL-10. MiR-579-3p overexpression inhibited NF-кB activity by reducing NRIP1. In addition, miR-579-3p could reduce the apoptotic rate of cortical neurons. Overexpression of miR-579-3p inhibited the activity of caspase-3, increased the expression level of anti-apoptotic gene Bcl-2 in neurons, and decreased the expression level of apoptotic gene Bax. CONCLUSION: miR-579-3p can be used to treat brain I/R injury, and its neuroprotective effect may be ascribed to the reduction of inflammation and apoptosis.

MiR-150-3p targets SP1 and suppresses the growth of glioma cells.

Glioma has been considered as one of the most prevalent and common malignancy of the nervous system; however, the underlying mechanisms that are responsible for the occurrence and development of glioma still remain largely unknown. Amounting evidence highlights the critical regulatory function of miRNAs in carcinogenesis. Here, we showed that the expression of miR-150-3p was significantly decreased in glioma tissues and cell lines. Suppressed expression of miR-150-3p was associated with the lymph node metastasis of the glioma patients. Overexpression of miR-150-3p significantly inhibited the proliferation of glioma cells. Molecular study uncovered that the transcription factor specificity protein 1 (SP1) was identified as one of the targets of miR-150-3p Highly expressed miR-150-3p in glioma cells significantly decreased both the mRNA and protein levels of SP1. Consistently, the abundance of phosphatase and tension homolog deleted on chromosome ten (PTEN), a negative downstream target of SP1, was increased with the ectopic miR-150-3p Collectively, these results suggested that miR-150-3p suppressed the growth of glioma cells partially via regulating SP1 and possibly PTEN.

Apurinic/apyrimidinic endonuclease 1 (APE1) contributes to resveratrol­induced neuroprotection against oxygen­glucose deprivation and re­oxygenation injury in HT22 cells: Involvement in reducing oxidative DNA damage.

Resveratrol, a naturally occurring polyphenolic compound, exhibits a neuroprotective role in models of central nervous system diseases, including cerebral ischemia/reperfusion injury. Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme that contributes to base excision repair of oxidative DNA damage and redox activation of transcription factors, associated with neuronal survival against hypoxic­ischemic injury. It was hypothesized that resveratrol protects HT22 cells against oxygen­glucose deprivation and re­oxygenation (OGD/R)­induced injuries through upregulation of APE1. It was demonstrated that resveratrol pretreatment significantly increased the viability of HT22 cells and decreased the release of lactate dehydrogenase (LDH), accompanied by the upregulation of APE1 mRNA, and protein levels, as well as the activity of APE1 under OGD/R conditions. In addition, resveratrol reversed OGD/R­induced oxidative DNA damage as evidenced by the decreases in the levels of 8­hydroxy­2'­deoxyguanosine and APE sites. However, APE1 knockdown using short hairpin RNA sequence targeting APE1 abolished resveratrol­elicited beneficent effects against OGD/R­induced cytotoxicity and oxidative stress. This was indicated by decreased cell viability, superoxide dismutase activity and glutathione levels, and increased LDH release and reactive oxygen species levels. The results of the present study indicate that APE1 contributes to the protective effects of resveratrol against neonatal hypoxic­ischemic brain injuries, and suggest that DNA repair enzymes, including APE1, may be a unique strategy for neuroprotection against this disease.