Mouse Hippocampal Neuronal Cell Line Research Articles

Momordica charantia L. Extract Protects Hippocampal Neuronal Cells against PAHs-Induced Neurotoxicity: Possible Active Constituents Include Stigmasterol and Vitamin E.

Polycyclic aromatic hydrocarbons (PAHs) have been recognized to cause neurobehavioral dysfunctions and disorder of cognition and behavioral patterns in childhood. Momordica charantia L. (MC) has been widely known for its nutraceutical and health-promoting properties. To date, the effect of MC for the prevention and handling of PAHs-induced neurotoxicity has not been reported. In the current study, the neuroprotective effects of MC and its underlying mechanisms were investigated in mouse hippocampal neuronal cell line (HT22); moreover, in silico analysis was performed with the phytochemicals MC to decipher their potential function as neuroprotectants. MC was demonstrated to possess neuroprotective effect by reducing reactive oxygen species’ (ROS’) production and down-regulating cyclin D1, p53, and p38 mitogen-activated protein kinase (MAPK) protein expressions, resulting in the inhibition of cell apoptosis and the normalization of cell cycle progression. Additionally, 28 phytochemicals of MC and their competence on inhibiting cytochrome P450 (CYP: CYP1A1, CYP1A2, and CYP1B1) functions were resolved. In silico analysis of vitamin E and stigmasterol revealed that their binding to either CYP1A1 or CYP1A2 was more efficient than the binding of each positive control (alizarin or purpurin). Together, MC is potentially an interesting neuroprotectant including vitamin E and stigmasterol as probable active components for the prevention for PAHs-induced neurotoxicity.

Protective Effect of Salidroside on Mitochondrial Disturbances via Reducing Mitophagy and Preserving Mitochondrial Morphology in OGD-induced Neuronal Injury.

Salidroside is the active ingredient extracted from Rhodiola rosea, and has been reported to show protective effects in cerebral ischemia, but the exact mechanisms of neuronal protective effects are still unrevealed. In this study, the protective effects of salidroside (1 µmol/L) in ameliorating neuronal injuries induced by oxygen-glucose deprivation (OGD), which is a classical model of cerebral ischemia, were clarified. The results showed that after 8 h of OGD, the mouse hippocampal neuronal cell line HT22 cells showed increased cell death, accompanied with mitochondrial fragmentation and augmented mitophagy. However, the cell viability of HT22 cells showed significant restoration after salidroside treatment. Mitochondrial morphology and mitochondrial function were effectively preserved by salidroside treatment. The protective effects of salidroside were further related to the prevention of mitochondrial over-fission. The results showed that mTOR could be recruited to the mitochondria after salidroside treatment, which might be responsible for inhibiting excessive mitophagy caused by OGD. Thus, salidroside was shown to play a protective role in reducing neuronal death under OGD by safeguarding mitochondrial function, which may provide evidence for further translational studies of salidroside in ischemic diseases.

Ischemia Injury induces mPTP opening by reducing Sirt3

Mitochondrial permeability transition pore (mPTP) opening is critical to mitochondrial apoptosis during ischemic injury. Sirtuin 3 (Sirt3) is a mitochondrial deacetylase known to play a major role in stress resistance and cell death. Our previous studies have shown that Sirt3 activates superoxide dismutase 2 and forkhead box O3a to reduce cellular reactive oxygen species. However, it is unclear the interaction between Sirt3 and mPTP and the roles they play in ischemic stroke. We used the middle cerebral artery occlusion (MCAO) model, a mouse model of stroke, to examine Sirt3 and mPTP-related protein levels. We then applied lentivirus packaged Sirt3 overexpression in HT22 cells, a mouse hippocampal neuronal cell line, to investigate the underlying mechanism. We found Sirt3 protein level was decreased in the penumbra area in MCAO mice, along with an increase in mPTP related proteins, namely voltage-dependent anion channel 1 (VDAC1) and adenine nucleotide translocator 1 (ANT1). Sirt3 overexpression suppressed the increase in VDAC1, ANT1 and cleaved caspase 3 that were induced by the serum and glucose deprivation (SGD) condition. Our studies suggest that ischemic injury induced mPTP opening and apoptosis by reducing Sirt3. It helps to identify new therapeutic targets for ischemic stroke.

Analysis of the role of Pur\u03b1 in the pathogenesis of Alzheimer's disease based on RNA-seq and ChIP-seq

Purine rich element binding protein A (Purα), encoded by the Purα gene, is an important transcriptional regulator that binds to DNA and RNA and is involved in processes such as DNA replication and RNA translation. Purα also plays an important role in the nervous system. To identify the function of Pura, we performed RNA sequence (RNA-seq) analysis of Purɑ-KO mouse hippocampal neuron cell line (HT22) to analyze the effect of Purα deletion on neuronal expression profiles. And combined with ChIP-seq analysis to explore the mechanism of Purα on gene regulation. In the end, totaly 656 differentially expressed genes between HT22 and Purα-KO HT22 cells have been found, which include 7 Alzheimer’s disease (AD)-related genes and 5 Aβ clearance related genes. 47 genes were regulated by Purα directly, the evidence based on CHIP-seq, which include Insr, Mapt, Vldlr, Jag1, etc. Our study provides the important informations of Purα in neuro-development. The possible regulative effects of Purα on AD-related genes consist inthe direct and indirect pathways of Purα in the pathogenesis of AD.

Kuwanon G protects HT22 cells from advanced glycation end product-induced damage.

The incidence of diabetic encephalopathy is increasing as the population ages. Evidence suggests that formation and accumulation of advanced glycation end products (AGEs) plays a pivotal role in disease progression, but limited research has been carried out in this area. A previous study demonstrated that Kuwanon G (KWG) had significant anti-oxidative stress and anti-inflammatory properties. As AGEs are oxidative products and inflammation is involved in their generation it is hypothesized that KWG may have effects against AGE-induced neuronal damage. In the present study, mouse hippocampal neuronal cell line HT22 was used. KWG was shown to significantly inhibit AGE-induced cell apoptosis in comparison with a control treatment, as determined by both MTT and flow cytometry. Compared with the AGEs group, expression of pro-apoptotic protein Bax was reduced and expression of anti-apoptotic protein Bcl-2 was increased in the AGEs + KWG group. Both intracellular and extracellular levels of acetylcholine and choline acetyltransferase were significantly elevated after KWG administration in comparison with controls whilethe level of acetylcholinesterase decreased. These changes in protein expression were accompanied by increased levels of superoxide dismutase and glutathione peroxidase synthesis and reduced production of malondialdehyde and reactive oxygen species. Intracellular signaling pathway protein levels were determined by western blot and immunocytochemistry. KWG administration was found to prevent AGE-induced changes to the phosphorylation levels of Akt, IκB-α, glycogen synthase kinase 3 (GSK3)-α and β, p38 MAPK and NF-κB p65 suggesting a potential neuroprotective effect of KWG against AGE-induced damage was via the PI3K/Akt/GSK3αβ signaling pathway. The findings of the present study suggest that KWG may be a potential treatment for diabetic encephalopathy.

Silver nanoparticles induced cytotoxicity in HT22 cells through autophagy and apoptosis via PI3K/AKT/mTOR signaling pathway

With the widespread application and inevitable environmental exposure, silver nanoparticles (AgNPs) can be accumulated in various organs. More serious concerns are raised on the biological safety and potential toxicity of AgNPs in the central nervous system (CNS), especially in the hippocampus. This study aimed to investigate the biological effects and the role of PI3K/AKT/mTOR signaling pathway in AgNPs mediated cytotoxicity using the mouse hippocampal neuronal cell line (HT22 cells). AgNPs reduced cell viability and induced membrane leakage in a dose-dependent manner, determined by the MTT and LDH assay. In doses of 25, 50, 100 μg mL-1 for 24 h, AgNPs promoted the excessive production of reactive oxygen species (ROS) and caused the oxidative stress in HT22 cells. AgNPs induced autophagy, determined by the transmission electron microscopy observation, upregulation of LC3 II/I and downregulation of p62 expression levels. The mechanistic investigation showed that the PI3K/AKT/mTOR signaling pathway was activated by phosphorylation, which was enrolled in an AgNP-induced autophagy process. AgNPs could further trigger the apoptosis by upregulation of caspase-3 and Bax and downregulation of Bcl-2 in HT22 cells. These results revealed AgNP-induced cytotoxicity in HT22 cells, which was mediated by autophagy and apoptosis via the PI3K/AKT/mTOR signaling pathway. The study could provide the experimental evidence and explanation for the potential neurotoxicity triggered by AgNPs in vitro.

Agomelatine confers neuroprotection against cisplatin-induced hippocampal neurotoxicity.

Neurotoxicity caused by cisplatin is a major obstacle during chemotherapy. Oxidative stress and inflammation are considered the primary mechanism behind neuronal damage which affects the continuing chemotherapy regimen. Agomelatine was recently described as a neuroprotective compound against toxic insults in the nervous systems. It is an analog of the well-known antioxidant and anti-inflammatory compound melatonin and currently used for depression and sleep disturbances. In the current study, we investigated the possible neuroprotective role of agomelatine against cisplatin-induced oxidative, inflammatory, and behavioral alterations in male rats. Our results show that agomelatine prevented cisplatin-induced neurotoxicity in the HT-22 mouse hippocampal neuronal cell line. Additionally, agomelatine treatment inhibited cisplatin-induced behavioral deficits and neuronal integrity in vivo. For the evaluation of the effect of agomelatine on oxidative stress and inflammation, GSH, MDA, TNF, and IL-6 levels were analyzed in HT-22 cells and hippocampal tissues. Agomelatine significantly attenuated oxidative stress and inflammation due to the cisplatin insult in vitro and in vivo. Also, agomelatine treatment ameliorated the neuronal pathology in the hippocampus, which is strongly related to cognition and memory. Taken together, our results indicate that in males, the neuroprotective effect of agomelatine is mediated through its antioxidant and anti-inflammatory actions abrogating functional deficits.

MiR-9-5p inhibits mitochondrial damage and oxidative stress in AD cell models by targeting GSK-3β.

This study aims to investigate the effects and underlying mechanisms of overexpression microRNA-9-5p (miR-9-5p) on the Aβ-induced mouse hippocampal neuron cell line HT22. Different concentrations of Aβ25-35 (10, 20, 40, 80, and 160μM) treatment were used to establish AD model in HT22 cells. The CCK-8 assay was used to measure the cell viability. The mRNA expression levels of miR-9-5p and glycogen synthase kinase-3β (GSK-3β) were determined by RT-qPCR. HT22 cell apoptosis was analyzed flow cytometry. MiR-9-5p was down-regulated in Aβ25-35-induced HT22 cells. GSK-3β is a functional target for miR-9-5p. MiR-9-5p overexpression inhibited Aβ25-35-induced mitochondrial dysfunction, cell apoptosis, and oxidative stress by regulating GSK-3β expression in HT22 cells. Furthermore, through targeting GSK-3β, overexpression of miR-9-5p partly activated nuclear factor Nrf2/Keap1 signaling, including part increases of Nrf2, HO-1, SOD-1, GCLC expression and slight decrease of Keap1 expression. Our results showed miR-9-5p may play a powerful role in the pathogenesis of AD.

MicroRNA-204-5p mediates sevoflurane-induced cytotoxicity in HT22 cells by targeting brain-derived neurotrophic factor.

Sevoflurane is widely used as an inhalational anesthetic in clinical practice. However, sevoflurane can cause cytotoxicity and induce learning capacity decline in patients. A previous publication indicated that miR-204-5p might have a close relationship with sevoflurane-induced neurotoxicity. When exposed to sevoflurane, the expression of miR-204-5p in neonatal hippocampus of rats was significantly increased. Hence, we aimed to investigate the role of miR-204-5p in sevoflurane-induced neurotoxicity using a mouse hippocampal neuronal cell line (HT22). The levels of miR-204-5p in HT22 cells were detected by RT-qPCR. In addition, the effects of miR-204-5p on cell viability, apoptosis and proliferation were evaluated by CCK-8, flow cytometric, and immunofluorescence assay, respectively. Western blotting was used to detect expressions of Bax, Bcl-2, active caspase 3, BDNF, TrkB, p-TrkB, Akt and p-Akt in HT22 cells. ELISA assay was used to examine the levels of total superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA) and reactive oxygen species (ROS) in cells. Meanwhile, the dual luciferase reporter system assay was employed to explore the interaction of miR-204-5p and BDNF in cells. The level of miR-204-5p was increased in sevoflurane-treated HT22 cells. Moreover, downregulation of miR-204-5p inhibited sevoflurane-induced apoptosis and promoted cell proliferation by upregulating the proteins of Bcl-2 and downregulating the expressions of Bax and active caspase-3 in HT22 cells. In addition, inhibition of miR-204-5p alleviated sevoflurane-induced oxidative injuries in HT22 cells via decline of ROS and MDA and upregulation of SOD and GSH. Furthermore, bioinformatics and dual luciferase assay demonstrated that miR-204-5p can inhibit the TrkB/Akt pathway by targeting BDNF. Our findings indicated that downregulation of miR-204-5p can decrease oxidative status in HT22 cells and alleviate sevoflurane-induced cytotoxicity through stimulating the BDNF/TrkB/Akt pathway. Therefore, miR-204-5p might be a potential biomarker and therapeutic target for the treatment of sevoflurane-induced neurotoxicity.

Excessive apoptosis and ROS induced by ethionine affect neural cell viability and differentiation.

The central nervous system (CNS) diseases are still a major cause of morbidity and mortality throughout the world, which imposes heavy burden on the development of society. Ethionine is a non-proteinogenic amino acid having similar chemical structure and activity to that of methionine, with which it competes. Previous studies have confirmed that ethionine affects various cellular functions by inhibiting the biosynthesis of proteins, RNA, DNA, and phospholipids, or all of them. The relationship of ethionine with some CNS diseases, including neural tube defects, has been investigated recently. However, the detailed effects of ethionine on the nerve cell bioactivities and the underlying mechanisms have not been fully explored. Herein, we systematically investigated the influences of ethionine on the proliferation, differentiation, and apoptosis of neural stem cells (NSCs) and post-mitotic nerve cells. We demonstrated that ethionine inhibited cell viability by disrupting the balance between proliferation and apoptosis, prevented NSCs from differentiating into neurons and astrocytes, and blocked cell progression from G1 to S phase via reducing cyclin D1 function in nerve cells including NSCs, a mouse hippocampal neuron cell line (HT-22), and a mouse brain neuroma cell line (Neuro-2a). We speculated that the inhibitory effect of ethionine on cell viability and differentiation are associated with increased reactive oxygen species production. Our results also supported the concept that ethionine may be an underlying cause of abnormal folate metabolism-induced CNS diseases. Our findings may provide important direction for the application of abnormal folate metabolism-induced CNS diseases in future NSC-based therapies.

Electrochemical property, antioxidant activities, water treatment and solar cell applications of titanium dioxide – zinc oxide hybrid nanocomposite based on graphene oxide nanosheet

Antioxidant activity has been detected with the HT22 mouse Hippocampal Neuronal cell line. The photocatalytic properties, electrochemical properties and solar cell applications tests of fabricated (Titanium dioxide (TiO2) nanoparticles-Zinc oxide (ZnO) nanoparticle) hybrid nanocomposite based on Graphene oxide nanosheet (GO) have been studied with different techniques. The electrochemical properties revealed by cyclic voltammetry analysis and the photocatalytic study have been evidenced by the interaction of synthesized nanoflower composite (GO@TiO2·ZnO) with mixed dyes (MB and RhB) and, also, with mixed pesticides (Tobaz and Metalxel). Absorbance intensities of examined organic pollution compounds were considerably quenched, this degradation was recorded by using the steady-state UV–Vis spectrophotometer under visible light source. Effect of fabricated GO@TiO2·ZnO hybrid nanocomposite on the HT22 mouse Hippocampal Neuronal cell line was measured and the reducing power activity showed the potential antioxidant property of the fabricate nanocomposite. The results showed that the fabricated hybrid nanoflower composite on graphene oxide (GO) surface has high photocatalytic efficiency for degradation of mixed organic compounds, this composite has been kept the viability of HT22 cells line. The electron-hole pair recombination was evidenced by photoluminescence (PL) analysis, where the fabricated nanocomposite has high electron transfer, so, the tests of solar cell applications have been revealed and confirmed that the fabricated hybrid nanocomposite has activity towards solar cell applications.

Beta-naphthoflavone inhibits LPS-induced inflammation in BV-2 cells via AKT/Nrf-2/HO-1-NF-κB signaling axis

Numerous studies have shown that over-activation of microglia could cause neuroinflammation and release pro-inflammatory mediators, which could result in neurodegenerative diseases, like Parkinson’s disease, Alzheimer’s disease etc. Beta-naphthoflavone (BNF) has anti-oxidant and anti-inflammatory effects in borderline tissues, but BNF has not been reported the effect associated with neuroinflammation. Therefore, the purpose of this experiment is to inquiry the impact and mechanism of BNF on neuroinflammation. The results indicated that BNF significantly inhibited the production of pro-inflammatory mediators (inducible nitric-oxide synthase (iNOS), Cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) andinterleukin-6 (IL-6)) in LPS-exposed BV-2 cells. Analysis of western blot results found that BNF accelerated the activation of AKT/Nrf-2/HO-1 signaling pathway and suppressed NF-κB pathway activation. Further study showed that BNF inhibited activation of NF-κB pathway via promoting HO-1, and SnPP IX (a HO-1 inhibitor) could inhibit anti-inflammatory function of BNF. We also found that BNF reduced the apoptosis rate of Human neuroblastoma cells (SHSY5Y) and mouse hippocampal neuron cell line (HT22) by inhibiting release of inflammatory mediators in LPS-exposed BV2 cells. In a word, our results suggested that BNF could inhibit inflammatory response via AKT/Nrf-2/HO-1-NF-κB signaling axis in BV-2 cells and exerts neuroprotective impact via inhibiting the activation of BV2 cells.

GABRG2 Deletion Linked to Genetic Epilepsy with Febrile Seizures Plus Affects the Expression of GABAA Receptor Subunits and Other Genes at Different Temperatures

Mutations in γ-aminobutyric acid A receptor (GABAA) subunits and sodium channel genes, especially GABRG2 and SCN1A, have been reported to be associated with febrile seizures (FS) and genetic epilepsy with febrile seizures plus (GEFS+). GEFS+ is a well-known family of epileptic syndrome with autosomal dominant inheritance in children. Its most common phenotypes are febrile seizures often with accessory afebrile generalized tonic-clonic seizures, febrile seizures plus (FS+), severe epileptic encephalopathy, as well as other types of generalized or localization-related seizures. However, the pathogenesis of febrile seizures remains largely unknown. Here, we generated a GABRG2 gene knockout cell line (HT22GABRG2KO) by applying the CRISPR/Cas9-mediated genomic deletion in HT-22 mouse hippocampal neuronal cell line to explore the function of GABRG2 in vitro. With mRNA-seq, we found significant changes in the expression profiles of several epilepsy-related genes when GABRG2 was knockout, some of them showing temperature-induced changes as well. Kyoto Encyclopedia Gene and Genomic (KEGG) analysis revealed a significant alteration in the MAPK and PI3K-Akt signaling pathways. We also observed an up-regulation of the matrix metalloproteinases (MMPs) family after GABRG2 knockout. Furthermore, the significant decrease in expression of GABRA1 and CACNA1A (but not others) with an increase in temperature is a novel finding. In summary, mutations in the GABAA receptor can lead to a decrease in numbers of receptors, which may cause the impairment of GABAergic pathway signaling. This data has been the first time to reveal that GABRG2 mutations would affect the function of other genes, and based on this finding we hope this work would also provide a new direction for the research of GABRG2 in GEFS+. It also may provide a molecular basis for the severity of epilepsy, and guide the clinical medication for the treatment of the epilepsy focused on the function on GABAA receptors, which, might be a new strategy for genetic diagnosis and targeted treatment of epilepsy.

The role of Interleukin-22 and its receptor Interleukin-22Rα on the regulation of inflammatory responses in the brain

Abstract Interleukin (IL)-22 is a member of IL-10 family, is a potent mediator of inflammatory responses. It is produced by activated CD4+ T cells and natural killer (NK) cells and takes effect on non-hematopoietic cells mainly stromal and epithelial cells. And, IL-22 receptor consists of IL-22Rα and IL-10Rβ. It is known that IL-22Rα expression is restricted to non-hematopoietic cells in the skin, pancreas, intestine, liver, lung and kidney. Even though IL-22 is mainly involved in the development of inflammatory responses, but there is a no report regarding their roles on inflammatory responses in the brain. In the present study, I investigated the role of IL-22 and its receptor on inflammatory responses in the brain using mouse microglia cell line, BV2 and mouse hippocampal neuronal cell line, HT22. When BV2 and HT22 were treated with rIL-22 (20 ng/ml), expression of cyclooxygenase (COX)-2, was increased and its expression is followed by the increased of PGE2 production. Next, I examined whether production of pro-inflammatory cytokines, such as such as IL-6 and TNF-α, and pro-inflammatory chemokines by IL-22 treatment. As a result, I confirmed that IL-6, TNF-α, and MIG/CXCL9 are remarkably increased. Taken together, IL-22α is spontaneously expressed on the cells, especially microglia and neuron and it is closely involved in the development of inflammatory responses in the brain.

Astrocyte-derived exosomes transfer miR-190b to inhibit oxygen and glucose deprivation-induced autophagy and neuronal apoptosis

ABSTRACT Our previous work has verified that astrocytes (AS)-derived exosomes (AS-Exo) inhibited autophagy and ameliorated neuronal damage in experimental ischemic stroke. However, the mechanism of AS-Exo regulation of autophagy remains unclear. The aim of this study was to investigate the regulatory mechanism of AS-Exo on neuronal autophagy. The mouse hippocampal neuronal cell line HT-22 was cultured in oxygen and glucose deprivation (OGD) condition to mimic ischemic injury. The primary astrocytes were used to isolate exosomes. Exosome labeling and uptake by HT-22 cells were observed by confocal laser microscopy. miR-190b expression was determined by qRT-PCR. HT-22 cell vitality and apoptosis were determined by CCK-8 assay and TUNEL staining, respectively. Levels of TNF-α, IL-6 and IL-1β were analyzed by ELISA. Protein levels of apoptosis-related cleaved caspase-3, Bax, Bcl-2 and autophagy-related Beclin-1, LC3-I/II, Atg7, P62 were determined by western blot. A dual-luciferase reporter assay was performed to confirm the direct interaction between miR-190b and Atg7. miR-190b expression in AS-Exo was found to be significantly higher than that in AS. AS-Exo-mediated transfer of miR-190b attenuated OGD-induced neuronal apoptosis via suppressing autophagy. Moreover, Atg7 was identified as a target of miR-190b. AS-Exo-mediated transfer of miR-190b regulated autophagy by targeting Atg7. Collectively, our data indicated that AS-Exo transferred miR-190b to inhibit OGD-induced autophagy and neuronal apoptosis.

Sodium tanshinone IIA sulfonate protects against Aβ1–42-induced cellular toxicity by modulating Aβ-degrading enzymes in HT22 cells

β-Amyloid (Aβ) plays an important role in the pathogenesis of Alzheimer's disease (AD). However, there is still no effective Aβ-targeting drugs for AD treatment. In this study, we explored the effect and mechanism of Sodium Tanshinone IIA Sulfonate (STS) on AD. Aβ-treated HT22 cells, an immortalized mouse hippocampal neuronal cell line, were employed. Different dosages of STS (0.1, 1 and 10 μM) were selected. STS improved cell viability and protected against Aβ-induced apoptosis in a dose-dependent manner. Furthermore, the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were decreased, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were significantly increased after STS treatment. STS decreased the levels of phosphorylate PKR-like (p-PERK), phosphorylate eukaryotic initiation factor 2 (p-eIF2α), phosphorylate inositol-requiring enzyme (p-IRE1α), X-box binding protein 1 (XBP1) and binding immunoglobulin heavy chain protein (Bip), while increased protein disulfide isomerase (PDI) levels in Aβ-treated HT22 cells. In addition, the levels of insulin degrading enzymes (IDE) and Nepterrilysin (NEP) (or call it CD10) were significantly increased after STS treatment. Taken together, these results indicated that STS might be effective in treating AD via increasing the levels of Aβ-degrading enzymes.

Lipidomics-based study on the neuroprotective effect of geissoschizine methyl ether against oxidative stress-induced cytotoxicity

Ethnopharmacological relevanceLipid homoeostasis is important for neurodevelopment, cell signaling and neurotransmission. Alteration of lipid metabolism has been demonstrated in many neurological disorders and neurodegenerative diseases. Geissoschizine methyl ether (GM) is an active alkaloid ingredient in the traditional Chinese medicine Uncaria hook. It has been shown that GM has strong potency in neuroprotective activity and GM reduces the production of reactive oxygen species by regulating glucose metabolism, which protects neurons against oxidative stress-induced cell death. However, it is unknown whether GM could regulate neuronal lipid metabolism during oxidative challenge. Aim of the studyThe current study aimed to explore whether GM regulates lipid metabolism in oxidative damaged neurons and to determine the underlying mechanism involved in this neuro-protection. Materials and methodsUsing a glutamate-induced oxidative toxicity model in mouse hippocampal neuronal cell line (HT-22 cells), we investigated the effect of GM on glutamate-induced lipid peroxidation, lipotoxicity and mitochondrial dysfunction. In order to clarify the mechanism underlying the neuroprotection by GM, lipid metabolomics was performed to investigate whether GM prevent oxidative stress-induced lipid metabolism disruption. Furthermore, the expression of lipid metabolism-related genes was measured. ResultsThe results show the protective effect of GM against oxidative stress through blocking glutamate-induced lipid peroxidation and lipotoxicity. Overall, lipidomics analysis revealed that glutamate treatment resulted in different extents of changes in a wide range of lipid classes such as fatty acids (FA), triacylglycerol (TG), sphingomyelin (SM), cardiolipin (CL), lysophosphatidylcholines (LPC). However, GM treatment can significantly reverse glutamate-induced lipids disorder to the homeostasis level. GM prevented the disruption of lipid metabolism by regulating the expression of lipid homeostasis related genes, which contributes to preserve mitochondrial function under oxidative damage. ConclusionThese findings clearly demonstrated a novel protective mechanism of GM against glutamate-induced oxidative toxicity in neurons via regulating lipid metabolism. GM may provide an effective approach for the prevention and treatment of oxidative damaged neurons.

Neuroprotective mechanisms of DNA methyltransferase in a mouse hippocampal neuronal cell line after hypoxic preconditioning.

Hypoxic preconditioning has been shown to improve hypoxic tolerance in mice, accompanied by the downregulation of DNA methyltransferases (DNMTs) in the brain. However, the roles played by DNMTs in the multiple neuroprotective mechanisms associated with hypoxic preconditioning remain poorly understood. This study aimed to establish an in vitro model of hypoxic preconditioning, using a cultured mouse hippocampal neuronal cell line (HT22 cells), to examine the effects of DNMTs on the endogenous neuroprotective mechanisms that occur during hypoxic preconditioning. HT22 cells were divided into a control group, which received no exposure to hypoxia, a hypoxia group, which was exposed to hypoxia once, and a hypoxic preconditioning group, which was exposed to four cycles of hypoxia. To test the ability of hypoxic preadaptation to induce hypoxic tolerance, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay. Cell viability improved in the hypoxic preconditioning group compared with that in the hypoxia group. The effects of hypoxic preconditioning on the cell cycle and apoptosis in HT22 cells were examined by western blot assay and flow cytometry. Compared with the hypoxia group, the expression levels of caspase-3 and spectrin, which are markers of early apoptosis and S-phase arrest, respectively, noticeably reduced in the hypoxic preconditioning group. Finally, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and western blot assay were used to investigate the changes in DNMT expression and activity during hypoxic preconditioning. The results showed that compared with the control group, hypoxic preconditioning downregulated the expression levels of DNMT3A and DNMT3B mRNA and protein in HT22 cells and decreased the activities of total DNMTs and DNMT3B. In conclusion, hypoxic preconditioning may exert anti-hypoxic neuroprotective effects, maintaining HT22 cell viability and inhibiting cell apoptosis. These neuroprotective mechanisms may be associated with the inhibition of DNMT3A and DNMT3B.

Effects of 5-Aza on p-Y1472 NR2B related to learning and memory in the mouse hippocampus

BackgroundWe have previously reported that 5-Aza-2-deoxycytidine (5-Aza-cdR) can repress protein serine/threonine phosphatase-1γ (PP1γ) expression and activity in the mouse hippocampus and affect the behaviour of mice in a water maze. It is well known that the phosphorylation of N-methyl-d-aspartate receptor 2B subunit (NR2B) plays a role in behaviour. In this study, we examined whether 5-Aza-cdR affects NR2B phosphorylation at tyrosine 1472 (p-Y1472 NR2B) and whether it affected the responses of the mice in a passive avoidance test. Methods5-Aza-cdR (10 μM) was administered to mice via intracerebroventricular injection (i.c.v). The learning and memory behaviour of the mice were evaluated by measuring their response in a step-down type passive avoidance test 24 h after the injection. The mRNA level of NR2B was measured by real-time PCR. NR2B and p-Y1472 NR2B protein expression in the mouse hippocampus was detected by western blot and immunofluorescence. CDK5 activity was detected by the ADP-Glo™ + CDK5/p35 Kinase Enzyme System. To further clarify whether the 5-Aza-cdR effects on behaviour were dependent on cellular proliferation or not, the effect of 5-Aza-cdR on the expression level of NR2B, the phosphorylation level of p-Y1472 NR2B, cell viability and the cell cycle were analysed using the immortalized mouse hippocampal neuronal cells neural cell line HT22 treated with 10 μM 5-Aza-cdR compared with an untreated control group. ResultsAfter injection with 5-Aza-cdR, the behaviour of the mice in the step-down test was improved, while their phosphorylation level of p-Y1472 NR2B was increased and their CDK5 activity was decreased in the hippocampus. In vitro experiments showed 10 μM 5-Aza-cdR increased the p-Y1472 NR2B phosphorylation level with inhibition of cell viability and cell cycle arrest. ConclusionsOur results suggested that the effect of 5-Aza-cdR on behaviour may be related to the increase in phosphorylation of p-Y1472 NR2B in the hippocampus.

Involvement of nerve growth factor in mouse hippocampal neuronal cell line (HT22) differentiation and underlying role of DNA methyltransferases

ABSTRACTDNA methylation is an epigenetic event involved in regulation of gene transcription during cell differentiation. DNA methyltransferases (DNMT) play a role in differentiation of neural stem cells into neurons. The aim of this study was to determine whether nerve growth factor (NGF) was involved in differentiation of mouse hippocampal neuronal cell line (HT22) as assessed by IncuCyte. Quantitative PCR and western blot were used to measure gene and protein expression of DNMT as well as the activity of DNMTs. Treatment with NGF was found to upregulate both gene and protein expressions as well as total activity of DNMTs in differentiating HT22 cells. Compared to undifferentiating cells, the percentage of differentiating cells at S phase increased significantly when incubated with NGF. In undifferentiated cells, NGF failed to induce gene and protein expressions and activity of DNMTs. Data demonstrate that differentiation of HT22 cells by exposure to NGF involve the activation of DNMTs pathway.