MiR-342-5p protects neurons from cerebral ischemia induced-apoptosis through regulation of Akt/NF-κB pathways by targeting CCAR2

ObjectivesIschemic stroke causes high morbidity, mortality and health burden in the world. MiR-342-5p was associated with Alzheimer's disease and cardio-protection. Herein, we aimed to reveal effects of miR-342-5p on cerebral ischemia injury as well as novel targets for stroke.Materials and methodsAgomiR-342-5p was intracerebroventricularly injected into the middle cerebral artery occlusion (MCAO) mouse models to evaluate functions of miR-342-5p on cerebral ischemia. RT-qPCR and western blot assays were used to evaluate genes expression. Oxygen-glucose deprivation (OGD) was used as an in vitro model for ischemia. Viability and apoptosis ratio of neurons was evaluated by CCK-8, LDH release detection, and flow cytometry. The potential targets of miR-342-5p were predicted by Targetscan, and their interaction was confirmed by luciferase assay.ResultsThe intervention of miR-342-5p effectively attenuated ischemic injury in MCAO mice. MiR-342-5p overexpression could protect neurons against OGD-induced injury, as revealed by increased cell viability and BCL2 expression, and decreased LDH release, apoptosis ratio, and BAX expression in OGD-induced neurons. Mechanically, miR-342-5p could directly bound with CCAR2 to inhibit its expression. Overexpressing CARR2 aggravated the OGD-induced injury of neurons, which was partly restrained by overexpressing miR-342-5p reversed. Furthermore, miR-342-5p/CARR2 axis regulates Akt/NF-κB signaling pathway in vitro as well as in vivo cerebral ischemia models.ConclusionsMiR-342-5p inhibited neuron apoptosis by regulating Akt/NF-kB signaling pathway via CCAR2 suppression. Our findings revealed the neuroprotection of miR-342-5p in cerebral ischemia.     

Possible neuroprotective property of nicotinic acetylcholine receptors in association with predominant upregulation of glial cell line-derived neurotrophic factor in astrocytes

The underlying mechanisms are still unclear for the neuroprotective properties of nicotine to date, whereas we have shown functional expression of nicotinic acetylcholine receptors (nAChRs) responsible for the influx of extracellular Ca²⁺ in cultured rat cortical astrocytes. In this study, we investigated the possible involvement of astrocytic nAChRs in the neuroprotection by this agonist. Exposure to nicotine predominantly induced mRNA expression of glial cell line‐derived neurotrophic factor (GDNF) among the different neurotrophic factors examined in cultured astrocytes, in a manner sensitive to nAChR antagonists, nifedipine, and aCa²⁺ chelator. Nicotine significantly increased GDNF in a concentration‐dependent manner in cultured astrocytes but not in neurons or neural progenitors even at the highest concentration used. In cultured astrocytes, a transient increase was seen in the expression of mRNA and corresponding protein for GDNF during sustained exposure to nicotine for 24 hr. Cytotoxicity mediated by oxidative, calcium, mitochondrial, or endoplasmic reticulum stress was invariably protected against in cortical neurons cultured with conditioned medium from astrocytes previously exposed to nicotine, and preincubation with the anti‐GDNF antibody reduced the neuroprotection by conditioned medium from astrocytes exposed to nicotine. Intraperitoneal administration of nicotine transiently increased the number of cells immunoreactive for both GDNF and glial fibrillary acidic protein in rat cerebral cortex. These results suggest that astrocytic nAChRs play a role in the neuroprotection against different cytotoxins after predominant upregulation of GDNF expression through a mechanism relevant to the acceleration of extracellular Ca²⁺ influx in rat brain in a particular situation. © 2012 Wiley Periodicals, Inc.     

miR-384-5p ameliorates neuropathic pain by targeting SCN3A in a rat model of chronic constriction injury

ABSTRACT

Objective: To explore the potential regulation mechanisms of miR-384-5p in Neuropathic pain (NP).

Methods: Rat model of chronic constriction injury (CCI) was established to induce NP in vivo. NP levels were assessed using Withdrawal Threshold (PWT) and Paw Withdrawal Latency (PWL). qPCR and Western blotting were used to determine the relative expression of miR-384-5p and SCN3A. The inflammation response in spinal microglia cells was determined by ELISA assay. Immunofluorescence assay was used to demonstrate the co-localization of miR-384-5p with SCN3A in rat dorsal root ganglions (DRGs). The target genes of miR-384-5p were verified by dual-luciferase report assays.

Results: In the current study, the miR-384-5p expression level was significantly downregulated in CCI rats when comparing to the sham group. In addition, miR-384-5p agomir significantly repressed mechanical allodynia and heat hyperalgesia in CCI rats. Meanwhile, the current study indicated miR‐384‐5p could decrease inflammation progress in spinal microglia cells incubated in lipopolysaccharide. Consistently, overexpression of miR-384-5p obviously depressed inflammation cytokine levels in CCI rats. Dual-luciferase reporter assays indicated that SCN3A is a target gene of miR-384-5p.

Conclusion: miR-384-5p is a negative regulator in the development of neuropathic pain by regulating SCN3A, indicating that miR-384-5p might be a promising therapeutic target in the treatment of neuropathic pain.

Abbreviations: CCI: Chronic constriction injury; ZEB1: Zinc finger E box binding protein-1; MAPK6: Mitogen-activated protein kinase 6; COX-2: cyclooxygenase-2.     

Bioinformatics and microarray analysis of microRNA expression profiles of murine embryonic stem cells,neural stem cells induced from ESCs and isolated from E8·5 mouse neural tube

Abstract

To better understand whether microRNAs (miRNAs) are involved in the self-renewal of stem cells and fate determination of neural stem cells and to identify the miRNA expression patterns of different neural stem cells (NSC) in vitro and in vivo, we examined miRNA expression profiles of murine embryonic stem cells (ESC), NSC induced from ESC and isolated from E8·5 mouse neural tube (E8·5-NSC) using microarray technique. It was found that a total of 40 miRNAs had similar expression level in all the three cells [false discovery rate (FDR)=0, fold change <3·0]. Moreover, q-PCR showed that some members of miR-106b and miR-17–92 families were expressed in the ESC, NSC induced from ESC (ESC-NSC) and hematopoietic stem cells (HSC). Bioinformatical analysis showed that 'stemness genes' (p21/CDKN1A, p57/CDKN1C and PTEN) were putative targets of miR-106b and miR-17–92 families. A total of 95 miRNAs were found to experience significant change (FDR=0, fold change >5·0) when the ESC differentiated into NSC. On the basis of miRNA, mRNA expression variance and predicted target genes of miRNA, we formulated a bioinformatical model for miRNA control of ESC-NSC differentiation. Then, the miRNA expression pattern was compared between NSC obtained in vitro and in vivo, and it was found that only 8% of miRNAs were different between the two NSCs. This study suggested that miR-106b and miR-17–92 families may promote the renewal of stem cells by targeting PTEN, p21/CDKN1A and p57/CDKN1C. Some miRNAs may play a key role in gene re-programming during ESC-NSC differentiation, and a substantial homogeneity exists between NSCs derived in vitro and those in vivo.     

Ketamine up-regulates a cluster of intronic miRNAs within the serotonin receptor 2C gene by inhibiting glycogen synthase kinase-3

Objectives: We examined mechanisms that contribute to the rapid antidepressant effect of ketamine in mice that is dependent on glycogen synthase kinase-3 (GSK3) inhibition.

Methods: We measured serotonergic (5HT)-2C-receptor (5HTR2C) cluster microRNA (miRNA) levels in mouse hippocampus after administering an antidepressant dose of ketamine (10?mg/kg) in wild-type and GSK3 knockin mice, after GSK3 inhibition with L803-mts, and in learned helpless mice.

Results: Ketamine up-regulated cluster miRNAs 448-3p, 764-5p, 1264-3p, 1298-5p and 1912-3p (2- to 11-fold). This up-regulation was abolished in GSK3 knockin mice that express mutant constitutively active GSK3. The GSK3 specific inhibitor L803-mts was antidepressant in the learned helplessness and novelty suppressed feeding depression-like behaviours and up-regulated the 5HTR2C miRNA cluster in mouse hippocampus. After administration of the learned helplessness paradigm mice were divided into cohorts that were resilient (non-depressed) or were susceptible (depressed) to learned helplessness. The resilient, but not depressed, mice displayed increased hippocampal levels of miRNAs 448-3p and 1264-3p. Administration of an antagonist to miRNA 448-3p diminished the antidepressant effect of ketamine in the learned helplessness paradigm, indicating that up-regulation of miRNA 448-3p provides an antidepressant action.

Conclusions: These findings identify a new outcome of GSK3 inhibition by ketamine that may contribute to antidepressant effects.     

Neuroprotective effect of ischemic preconditioning via modulating the expression of cerebral miRNAs against transient cerebral ischemia in diabetic rats

Objectives: In this study, we aimed to evaluate the effect of the Ischemic preconditioning (IPreC) on the expression profile of cerebral miRNAs against stroke by induced transient middle cerebral artery occlusion (MCAo) in diabetic rats.

Methods: Eighty male Spraque Dawley rats were allocated to eight groups. In order to evaluate the expression profile of miRNAs, we induced transient MCAo seven days after STZ-induced diabetes (DM). Also we performed IPreC 72 h before transient MCAo to assess whether IPreC could have a neuroprotective effect against ischemia-reperfusion injury.

Results: The general characteristics of STZ-treated rats included reduced body weight and elevated blood glucose levels compared to non-diabetic ones. We demonstrated that miRNA expression profiles, which are determined for biological functions such as aquaporin 4 formation (miR-29b-2, miR-124a-3p, miR-130a, miR-223 and miR-320a), glutamate toxicity (miR107, miR-145, miR-223), salvageable ischemic area (miR-9a, miR-19b, miR-29b-2, miR-341, miR-339–5p, miR-15–5p, miR-99b-5p), and neoangiogenesis (let-7f-5p, miR-126a and miR-322–3p), were regulated following IPreC. Ischemic preconditioning before cerebral ischemia significantly reduced infarction size compared with the other groups [IPreC + MCAo (27 ± 11 mm³) vs. MCAo (109 ± 15 mm³) p < 0.001; DM + IPreC + MCAo (38 ± 9 mm³) vs. DM + MCAo (165 ± 41 mm³) p < 0.001, respectively].

Discussion: The study results revealed the neuroprotective effects of ischemic preconditioning, supported with the upregulated pro-survival miRNAs in MCA infarcts.     

帕金森病患者血清胞外囊泡中miRNAs表达水平变化及其对疾病进展的影响

目的 探讨帕金森病患者血清胞外囊泡中微小RNAs(miRNAs)表达水平变化及其对疾病进展的影响。方法 采用回顾性研究,收集本院85例帕金森病患者的临床资料,根据发病年龄段的不同分为早发组(早发性帕金森病,发病年龄不超过50岁)37例和晚发组(晚发性帕金森病,发病年龄超过50岁)48例,另以本院30例体检健康人员为对照组; 采集3组血清标本,并提取胞外囊泡,采用miRNA表达谱芯片和实时荧光定量聚合酶链反应对miRNAs表达水平进行检测; 通过Cytoscape分析miRNAs共同调控的靶基因,并通过String数据库探讨靶基因的相互作用和功能; 采用实时荧光定量聚合酶链反应检测早发组与晚发组血清胞外囊泡中靶基因的相对表达水平。结果 晚发组血清胞外囊泡中miR-500a-5p和miR-451表达水平较对照组均显著降低,而miR-1180-3p和miR-143-3p表达水平较对照组显著升高(P<0.01)。与早发组比较,晚发组血清胞外囊泡中miR-143-3p表达水平明显降低(P<0.01); 2组血清胞外囊泡中miR-451,miR-500a-5p及miR-1180-3p表达水平比较均无明显差异(P>0.05)。经Cytoscape分析发现,Akt1和Bcl-2是miR-143-3p的关键靶基因。与早发组比较,晚发组血清胞外囊泡中Bcl-2表达水平明显升高(P<0.01); 2组血清胞外囊泡中Akt1表达水平比较并无明显差异(P>0.05)。结论 miR-143-3p与其靶基因Bcl-2均异常表达于帕金森病患者,其中miR-143-3p在早发性帕金森病患者血清胞外囊泡中的表达水平明显提高,Bcl-2的表达水平明显降低,提示二者表达水平的异常改变可能与帕金森病的疾病进展密切相关。     

Computational investigation of mechanistic insights of Aβ42 interactions against extracellular domain of nAChRα7 in Alzheimer’s disease

Aim: Amyloid beta (Aβ) 1-42, which is a basic constituent of amyloid plaques, binds with extracellular transmembrane receptor nicotine acetylcholine receptor α7 (nAChRα7) in Alzheimer’s disease.

Materials and Methods: In the current study, a computational approach was employed to explore the active binding sites of nAChRα7 through Aβ 1–42 interactions and their involvement in the activation of downstream signalling pathways. Sequential and structural analyses were performed on the extracellular part of nAChRα7 to identify its core active binding site.

Results: Results showed that a conserved residual pattern and well superimposed structures were observed in all nAChRs proteins. Molecular docking servers were used to predict the common interactive residues in nAChRα7 and Aβ1–42 proteins. The docking profile results showed some common interactive residues such as Glu22, Ala42 and Trp171 may consider as the active key player in the activation of downstream signalling pathways. Moreover, the signal communication and receiving efficacy of best-docked complexes was checked through DynOmic online server. Furthermore, the results from molecular dynamic simulation experiment showed the stability of nAChRα7. The generated root mean square deviations and fluctuations (RMSD/F), solvent accessible surface area (SASA) and radius of gyration (Rg) graphs of nAChRα7 also showed its backbone stability and compactness, respectively.

Conclusion: Taken together, our predicted results intimated the structural insight on the molecular interactions of beta amyloid protein involved in the activation of nAChRα7 receptor. In future, a better understanding of nAChRα7 and their interconnected proteins signalling cascade may be consider as target to cure Alzheimer’s disease.     

Activation of α2A‐containing nicotinic acetylcholine receptors mediates nicotine‐induced motor output in embryonic zebrafish

It is well established that cholinergic signaling has critical roles during central nervous system development. In physiological and behavioral studies, activation of nicotinic acetylcholine receptors (nAChRs) has been implicated in mediating cholinergic signaling. In developing spinal cord, cholinergic transmission is associated with neural circuits responsible for producing locomotor behaviors. In this study, we investigated the expression pattern of the α2A nAChR subunit as previous evidence suggested it could be expressed by spinal neurons. In situ hybridization and immunohistochemistry revealed that the α2A nAChR subunits are expressed in spinal Rohon–Beard (RB) neurons and olfactory sensory neurons in young embryos. To examine the functional role of the α2A nAChR subunit during embryogenesis, we blocked its expression using antisense modified oligonucleotides. Blocking the expression of α2A nAChR subunits had no effect on spontaneous motor activity. However, it did alter the embryonic nicotine‐induced motor output. This reduction in motor activity was not accompanied by defects in neuronal and muscle elements associated with the motor output. Moreover, the anatomy and functionality of RB neurons was normal even in the absence of the α2A nAChR subunit. Thus, we propose that α2A‐containing nAChRs are dispensable for normal RB development. However, in the context of nicotine‐induced motor output, α2A‐containing nAChRs on RB neurons provide the substrate that nicotine acts upon to induce the motor output. These findings also indicate that functional neuronal nAChRs are present within spinal cord at the time when locomotor output in zebrafish first begins to manifest itself.     

Neuronal Ca2+ sensor VILIP-1 leads to the upregulation of functional α4β2 nicotinic acetylcholine receptors in hippocampal neurons

The neuronal Ca²⁺-sensor protein VILIP-1, known to affect clathrin-dependent receptor trafficking, has been shown to interact with the cytoplasmic loop of the α4-subunit of the α4β2 nicotinic acetylcholine receptor (nAChR), which is the most abundant nAChR subtype with high-affinity for nicotine in the brain. The α4β2 nAChR is crucial for nicotine addiction and the beneficial effects of nicotine on cognition. Its dysfunction has been implicated in frontal lobe epilepsy, Alzheimer's disease and schizophrenia. Here we report that overexpression of VILIP-1 enhances ACh responsiveness, whereas siRNA against VILIP-1 reduces α4β2 nAChR currents of hippocampal neurons. The underlying molecular mechanism likely involves enhanced constitutive exocytosis of α4β2 nAChRs mediated by VILIP-1. The two interaction partners co-localize in a Ca²⁺-dependent manner with syntaxin-6, a Golgi-SNARE protein involved in trans-Golgi membrane trafficking. Thus, we speculate that regulation of VILIP-1-expression might modulate surface expression of ligand-gated ion channels, such as the α4β2 nAChRs, possibly comprising a novel form of physiological up-regulation of ligand-gated ion channels.     

Association of miR-122, miR-124 miR-126 and miR-143 gene polymorphisms with ischemic stroke in the northern Chinese Han population

Abstract

Objectives: Single nucleotide polymorphisms (SNPs) in microRNA (miRNA) genes can have diverse functional consequence by affecting the processing and target selection of miRNA. Recent evidence indicates that miRNA play important roles in the pathogenesis of atherosclerosis, which is a major cause of ischemic stroke (IS). The aim of this study was to clarify whether genetic variations in four miRNA genes (miR-143 rs4705342, miR-122 rs17669, miR-126 rs4636297, and miR-124 rs531564) contribute to IS susceptibility.

Methods: A case-control study was used to explore miRNA genetic polymorphisms in 567 IS patients and 552 control subjects that were frequency matched by age and gender. We genotyped four SNPs using polymerase chain reaction/ligation detection reaction.

Results: The miR-126 gene rs4636297 polymorphism was associated with decreased small vessel stroke risk (GA vs. GG: odds ratio (OR)?=?0.62, p?=?.015; GA?+?AA vs. GG: OR?=?0.637, p?=?.018; A vs. G: OR?=?0.696, p?=?.033). Using logistic regression analysis, this significant association remained after adjusting for confounding risk factors (adjusted OR?=?0.626, 95% confidence interval (CI)?=?0.426–0.921). However, the three other miRNA (miR-143 rs4705342, miR-122 rs17669, and miR-124 r531564) were not associated with IS risk under allele or genotype, nor in different inheritance models. In addition, there were no significant associations with stroke subtypes for these three miRNA SNPs.

Conclusion: Our study suggests that the miR-126 gene rs4636297 polymorphism may play an important role in the pathogenesis of small vessel occlusive stroke in the northern Chinese Han population.     

Chronic neonatal nicotine upregulates heteromeric nicotinic acetylcholine receptor binding without change in subunit mRNA expression

Smoking during pregnancy chronically exposes the fetus to nicotine resulting in long-term behavioral and cognitive deficits. Nicotine binds to neuronal nicotinic acetylcholine receptors (nAChRs), pentameric ligand-gated ion channels widely expressed in the nervous system. Chronic nicotine upregulates high-affinity nAChRs in animals and smokers. Here we determined if chronic nicotine treatment during a developmental period corresponding to the human third trimester regulates nAChR expression. Rat pups were intubated orally three times per day with or without nicotine (6 mg/kg/day) from postnatal day 1 to 8. Subunit mRNA expression was assessed by in situ hybridization. Expression of heteromeric and homomeric nAChR receptor was evaluated by autoradiography using (125)I-epibatidine and (125)I-alphabungarotoxin, respectively. nAChR expression was analyzed in cortex, hippocampus, thalamus and medial habenula from autoradiograms using computer assisted image analysis. Nicotine induced significant upregulation of heteromeric but not homomeric nAChRs in hippocampus, cortex and thalamus without changes in subunit mRNA expression. No effect of chronic nicotine on receptor expression was detected in the medial habenula, suggesting that nicotine's effect was mainly on alpha4beta2-type heteromeric nAChRs. The nicotine-induced upregulation was reversed after nicotine withdrawal. Receptor blockade by DHbetaE, an antagonist for heteromeric alpha4/beta2 nAChRs, did not prevent upregulation but increased expression to a similar degree as nicotine. Combination of both drugs had a cumulative effect. Thus, although transient, intermittent nicotine exposure as seen in smoking mothers is sufficient to upregulate heteromeric nAChRs during a critical period of brain development and could contribute to the behavioral deficits found in children whose mother smoked.     

Dexmedetomidine Alleviates Hypoxic-Ischemic Brain Damage in Neonatal Rats Through Reducing MicroRNA-134-5p-Mediated NLRX1 Downregulation

BackgroundDexmedetomidine (Dex) is a safe and effective anesthetic adjunct which also has neuroprotective roles. This study aimed to validate the role of Dex in hypoxic-ischemic brain damage (HIBD) in neonatal rats and the functional molecules.MethodsA neonatal rat model of HIBD was established and treated with Dex. The learning and memory abilities of rats were determined by Morris water maze tests. The left-hemisphere encephalatrophy, pathological changes, neuronal apoptosis, and inflammation in rat hippocampal tissues were examined to evaluate the treating effects of Dex on HIBD. Differentially expressed microRNAs (miRNAs) in rats with HIBD were screened using microarray analysis. Potential downstream molecules mediated by miR-134-5p were predicted using bioinformatics analysis. Altered expression of miR-134-5p and NLR family member X1 (NLRX1) was induced in rats after Dex treatment for rescue experiments.ResultsDex treatment significantly enhanced the learning and memory abilities of rats and reduced encephalatrophy in rats. It also alleviated pathological changes, neuronal apoptosis, and the production of pro-inflammatory cytokines in rat hippocampal tissues. miR-134-5p was significantly upregulated in rats with HIBD. Dex treatment reduced the expression of miR-134-5p. NLRX1 was a target gene of miR-134-5p and it reduced the phosphorylation of IκBα and p65, namely the activation of NF-κB signaling. Overexpression of miR-134-5p blocked, whereas overexpression of NLRX1 strengthened the protective effects of Dex on neonatal rats.ConclusionThis study demonstrates that Dex treatment can alleviate HIBD in neonatal rats through restoring NLRX1 expression by suppressing miR-134-5p.     

Relationship between the increased cell surface alpha7 nicotinic receptor expression and neuroprotection induced by several nicotinic receptor agonists.

Nicotine and other nicotinic acetylcholine receptor agonists have been shown to exert neuroprotective actions in vivo and in vitro by an as yet unknown mechanism. Even the identification of the subtype of nicotinic receptor(s) mediating this action has not been determined. In neural cell lines, the induction of cytoprotection often requires exposure to nicotine for up to 24 hr to produce a full protective effect. One phenomenon associated with chronic exposure of neural cells to nAChR agonists is the increased expression of nAChRs (upregulation), possibly as a response to desensitization. Because nicotinic receptors desensitize rapidly in the continuous presence of agonist, we investigated whether the neuroprotective actions produced by different nicotinic receptor agonists was related to their ability to induce nicotinic receptor upregulation. Differentiated PC12 cells were preincubated for 24 hr with various nAChR ligands, and the cells were subsequently deprived of both NGF and serum to induce cytotoxicity. Under control conditions cell viability was reduced to 66.5 +/- 5.4% of control by trophic factor withdrawal. For those cells pretreated with nicotine (1 nM-100 microM) cell viability increased from 74.2 +/- 1.5 to 97.3 +/- 4%. The neuroprotective action of nicotine was blocked by co-treatment with either 5 microM mecamylamine or 10 nM methyllycaconitine (MLA). The high potency blockade by MLA suggested that neuroprotection was mediated through the alpha7 nicotinic receptor subtype. For the seven agonists examined for neuroprotective activity, only nicotine was capable of evoking a near maximal (near 100% cell viability) neuroprotective action. The next most effective group included epibatidine, 4OHGTS-21, methycarbamylcholine, and 1,1-dimethyl-4-phenyl-piperazinium iodide. These least effective group included cytisine and tetraethylammonium. Incubation of differentiated PC12 cells with 10 microM nicotine increased the number of [(125)I]alpha bungarotoxin ([(125)I]alphaBGTbinding sites by 41% from 82.6 +/- 3.67 to 117 +/- 10.3 fmol/mg protein). Under similar conditions of incubation, the nicotinic receptor agonist cytisine (that was least effective in terms of neuroprotection) failed to increase the number of [(125)I]alphaBGT binding sites. Cells expressing increased levels of cell surface [(125)I]alphaBGT binding sites received added neuroprotective benefit from nicotine. Thus the induced upregulation of the alpha7 subtype of nicotinic receptors during chronic exposure to nicotine may be responsible for the drug's neuroprotective action.     

Circ_0101874 overexpression strengthens PDE4D expression by targeting miR-335-5p to promote neuronal injury in ischemic stroke

BackgroundIschemic stroke has been a public concern, while its pathogenesis is not fully understood. Increasing evidence suggests that circular RNAs (circRNAs) are involved in this disorder. The purpose of this study was to explore the role of circ_0101874 in ischemic stroke.MethodsThe in vivo model of ischemic stroke was established in mice with middle cerebral artery occlusion (MCAO) treatment. The in vitro model of ischemic stroke was established in SK-N-SH cells with oxygen-glucose deprivation (OGD) treatment. The expression of circ_0101874, miR-335-5p and phosphodiesterase 4D (PDE4D) mRNA was measured by quantitative real-time PCR (qPCR). The release of inflammatory factors was checked by ELISA. Cell viability, cell proliferation and cell apoptosis were detected using CCK-8 assay, EdU assay and flow cytometry assay, respectively. The protein levels of cyclinD1, cleaved-caspase-3 and PDE4D were detected by western blot. The interaction between miR-335-5p and circ_0101874 or PDE4D was validated by dual-luciferase reporter assay and RIP assay.ResultsCirc_0101874 was highly expressed in MCAO animal models and OGD-induced SK-N-SH cells. Circ_0101874 knockdown suppressed OGD-enhanced inflammation, cell apoptosis and oxidative stress and promoted OGD-inhibited cell viability and cell proliferation in SK-N-SH cells. Circ_0101874 directly bound to miR-335-5p, and miR-335-5p inhibition reversed the effects of circ_0101874 knockdown. PDE4D was a target gene of miR-335-5p, and PDE4D overexpression recovered OGD-promoted SK-N-SH cell injuries that were blocked by miR-335-5p enrichment. Circ_0101874 bound to miR-335-5p to enhance the expression of PDE4D.ConclusionCirc_0101874 knockdown alleviated OGD-induced neuronal cell injury by suppressing PDE4D via regulating miR-335-5p.     

Effects of miR-199a on autophagy by targeting glycogen synthase kinase 3β to activate PTEN/AKT/mTOR signaling in an MPP+ in vitro model of Parkinson’s disease

ABSTRACT

Objectives: miR-199a can regulate autophagy, its underlying mechanisms remain unknown. The purpose of this study was to investigate the mechanisms of miR-199a involved in regulating autophagy in a 1-methyl-4-phenylpyridine (MPP⁺)-induced in vitro model of PD.

Methods: PC12 cells were incubated in MPP⁺, and the expression levels of miR-199a were bidirectionally regulated via either transfection of an miR-199a mimic or incubation in miR-199a inhibitors. The experimental manipulations were divided into four groups, including the control group, MPP⁺ group, MPP⁺ + miR-199a mimic group, and MPP⁺ + miR-199a inhibitor group. MTT, CCK-8, qRT-PCR, Western blotting and linear correlation analysis were performed to evaluate various experimental indicators.

Results: At increasing MPP⁺ concentrations, the following results were found: the expression levels of miR-199a, phosphorylated AKT and mTOR proteins expression decreased; the expression levels of phosphatase and tensin homologue (PTEN), GSK3β, Beclin1, and LC3II increased; PC12 autophagy increased; and cellular viability and survival rates decreased. Transfection of an miR-199a mimic increased miR-199a expression and induced all of the following: the expression levels of PTEN, GSK3β, Beclin1, and LC3II decreased; the expression levels of phosphorylated AKT and mTOR proteins expression increased; PC12 autophagy decreased; and cellular viability and survival rates increased.

Discussion: In this in vitro study, we found that increasing miR-199a expression in PC12 cells reduced protein levels of Beclin1 and LC3II, decreased autophagy, enhanced cellular viability, increased survival rate, and ameliorated MPP⁺-induced parkinsonian-like cellular pathologies by targeting pro-autophagic pathways and GSK3β to activate PTEN/AKT/mTOR signaling.     

Fructose-1,6-bisphosphate preserves intracellular glutathione and protects cortical neurons against oxidative stress

The use of alcohol and nicotine are highly correlated, suggesting an underlying biochemical interaction. Chronic nicotine exposure results in a deactivation and subsequent upregulation of the expression of nicotinic acetylcholine receptors (nAChRs). Upregulation is thought to represent certain aspects of physical dependence on nicotine. If alcohol also alters nAChR expression or modulates the nicotine-induced upregulation, it could partially explain the high rate of co-abuse of these two drugs. We examined the effects of ethanol on the expression and nicotine-induced upregulation of nAChRs in two cell lines expressing different receptor subtypes. As measured by ligand binding, ethanol initially decreased nAChR expression in M10 cells but increased expression with a more chronic exposure. In the presence of nicotine, the effect of ethanol was similar; initially acting to blunt the upregulation of receptor expression caused by nicotine but enhancing the upregulation with 96 h of exposure. The upregulation of nAChRs was long lasting, remaining above control levels for as long as 7 days following removal of nicotine and ethanol. In PC12 cells, ethanol increased expression at all time points examined. A protein phosphatase inhibitor reduced nicotine-induced upregulation and a PKC inhibitor blocked the ethanol-induced decrease in nAChR expression. These data suggest that ethanol and nicotine interact at the level of the PKC pathway to regulate expression of nAChRs.     

Effect of chronic nicotine treatment on localization of neuronal nicotinic acetylcholine receptors at cellular level

Chronic nicotine treatment increases the number of neuronal nicotinic acetylcholine receptors (nAChRs). Localization of nAChRs at a cellular level determines their functional role. However, changes in the localization of nAChRs caused by chronic nicotine treatment are not well known. In this study, we have examined the effects of chronic nicotine treatment on alpha7 and beta2 nAChR subunits in vitro in cell lines and in vivo in mouse striatum. In vitro, two different cell lines were used, SH-SY5Y cells endogenously expressing several nAChR subtypes and SH-EP1-halpha7 cells, transfected with the human alpha7 nAChR subunit gene. Effects of chronic nicotine treatment (10 microM, 3 days) were studied in vitro by using confocal and electron microscopy and calcium fluorometry. In vitro in SH-SY5Y cells, alpha7 and beta2 subunits formed groups, unlike alpha7 subunits in SH-EP1-halpha7 cells, which were partially localized on endoplastic reticulum. Chronic nicotine treatment did not change the localization of nAChRs in endosomes, but caused clustering of alpha7 subunits in SH-EP1-halpha7 cells. In vivo, nicotine was given to mice in their drinking water for 7 weeks. Results showed that alpha7 and beta2 subunits formed groups, and that chronic nicotine treatment increased the size of the clusters. As a conclusion, our data show that there are large intracellular pools of nAChR subunits, which are partially localized on endoplastic reticulum. Chronic nicotine treatment does not change endocytotic trafficking of nAChRs. Chronic nicotine treatment increased clustering of nAChRs, which could have a role in the release of dopamine (DA) evoked by nicotine.