Neuronal HT22 Cells Research Articles

The cGAS/STING signaling pathway is involved in sevoflurane induced neuronal necroptosis via regulating microglia M1 polarization

ObjectiveThe specific mechanisms of sevoflurane-induced neurotoxicity are still undetermined. The aim of the current study was to investigate the role of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway in sevoflurane-induced neuronal necroptosis. MethodsBV2 microglial cells were divided into a control group and a 4% sevoflurane exposure group. Western blotting was used to detect expression of the M1 polarization marker inducible nitric oxide synthase (iNOS). RNA was collected for RNA sequencing analysis. After STING knockdown in microglia, western blotting was performed to examine expression of the pro-inflammatory markers CD16 and CD32. The tumor necrosis factor-α (TNF-α) level in media was detected using an enzyme-linked immunosorbent assay. BV2 microglia conditioned media was collected to incubate HT22 neuronal cells, and their cell activity was measured using a CCK8 assay. Calcium was observed by fluorescence. Western blotting was performed to evaluate receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) expression. Neuronal necroptosis rate were detected using flow cytometry. ResultsSevoflurane exposure promoted microglial M1 polarization. The cGAS/STING pathway was screened and identified by RNA sequencing analysis of sevoflurane-exposed microglia and the control group. Compared with the control group, STING knockdown in microglia rescued the amoeboid morphology, inhibited TNF-α release, and significantly decreased iNOS, CD16, and CD32 expression. Moreover, calcium ions and necroptosis within neurons were decreased, and RIPK1, RIPK3, and p-MLKL expression was markedly decreased in microglia media culture with STING knockdown. ConclusionThese results suggest that sevoflurane can regulate microglial M1 polarization by activating the cGAS/STING signaling pathway and increasing immune factor release, thus accelerating the neuronal necroptosis induced by calcium overload.

CB1 receptor mediates anesthetic drug ketamine‑induced neuroprotection against glutamate in HT22 cells.

The anesthetic drug, ketamine (KTM) has been shown to induce therapeutic effects against major depressive disorder (MDD), however the related underlying mechanisms remain unclear. In the present study, HT22 neuronal cells were treated with glutamate to imitate oxidative stress injury in MDD, and it was hypothesized that the cannabinoid type 1 (CB1) receptor mediates KTM-induced neuroprotection via ameliorating mitochondrial function in glutamate-treated neuronal cells. Compared with the control, glutamate decreased cell viability and intracellular antioxidants, including glutathione (GSH), catalase and superoxide dismutase 2 levels, and inhibited mitochondrial function simultaneously. Moreover, glutamate increased lactate dehydrogenase release, cellular apoptosis level, cleaved caspase-3 expression and intracellular oxidants, such as reactive oxygen species, oxidized GSH and mitochondrial superoxide in the cells. The presence of KTM, however, significantly decreased the glutamate-induced oxidative stress injury, ameliorated the antioxidant/oxidant levels in the cells, enhanced mitochondrial function and upregulated CB1 receptor expression (P<0.05). Co-administration of the CB1 receptor antagonist AM251 markedly abolished the KTM-induced cytoprotective effects and ameliorations of antioxidant/oxidant levels and mitochondrial function, and also reversed CB1 upregulation (P<0.05). These observations indicated that KTM decreases the oxidative stress injury caused by glutamate in HT22 neuronal cells, and the neuroprotective effects may be mediated by the CB1 receptor.

Tocotrienol-rich fraction enhances cell proliferation and memory formation in hippocampal HT22 neuronal cells through BDNF/TrkB pathway

Memory formation is influenced by neurogenesis and long-term potentiation (LTP) of neurons in the hippocampus. In this study, we aimed to elucidate the mechanism of action of tocotrienol-rich fraction (TRF) involved in memory formation. Two cell models were employed, differentiated and undifferentiated HT22 neuronal cells. Our findings demonstrated that treatment with TRF increased cell proliferation of undifferentiated HT22 cells in a time-dependent manner by activating the brain-derived neurotrophic factor/tropomyosin receptor kinase B (BDNF/TrkB) pathway. Elevated levels of cyclin D and E were also observed, indicating an increase in cell cycle progression and proliferation. In differentiated HT22 cells, TRF upregulated expressions of BDNF, phosphorylation of glutamate receptor 1 and Ca2+/calmodulin-dependent protein kinase II, potentially leading to stronger synaptic strength and contributing to LTP. Taken together, these findings suggest that TRF supplementation may enhance memory formation in HT22 cells through modulation of the BDNF/TrkB pathway.

A New Strategy for the Synthesis of (+)‐Crambescin B Decarboxylate

AbstractCrambescin B decarboxylate is an inhibitor of voltage‐gated sodium channels and a potential biochemical tool. Herein, we report the asymmetric total synthesis of crambescin B decarboxylate using a new synthetic strategy. The key aspects of this new protocol include: 1) the use of an optically pure epoxide, synthesized by the Jacobsen hydrolytic kinetic resolution, as the starting material for guanidino‐aziridine, and 2) the introduction of the THF ring through nucleophilic substitution using an acyl anion equivalent. The synthetic protocol reported herein is nine steps shorter than the previously reported shortest method. We determined the cytotoxicity and protective effects of crambescin B decarboxylate in mouse‐hippocampus‐derived neuronal HT22 cells with an MTS assay. Cell viability was observed to decrease in a concentration‐dependent manner without cell detachment or shrinkage. In addition, crambescin B decarboxylate protected glutamate‐induced cell death.

Circular RNA circEfnb2 promotes cell injury after cerebral infarction by sponging miR-202-5p and regulating TRAF3 expression

BackgroundExogenous neural cell transplantation may be therapeutic for stroke, cerebral ischemic injury. Among other mechanisms, increasing findings indicated circular RNAs (circRNAs) regulate the pathogenesis progression of cerebral ischemia. Mmu_circ_0015034 (circEfnb2) was upregulated in focal cortical infarction established by middle cerebral artery occlusion (MCAO) in mice. Our study was designed to probe the molecular mechanism of circEfnb2 in the oxygen-glucose deprivation/reperfusion (OGD/R)-induced neuronal damage in cerebral ischemia. MethodsWe established an in vitro OGD/R cell model. CircEfnb2 and microRNA-202-5p (miR-202-5p) levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR). Lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS) levels were assessed using specific kits. Tumor necrosis factor-α (TNF-α) and Interleukin-1β (IL-1β) levels were examined using an Enzyme-linked immunosorbent assay (ELISA). Flow cytometry analysis evaluated cell apoptosis. Protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), cleaved caspase 3, and Tumor necrosis factor receptor-associated factor 3 (TRAF3) were determined using Western blot assay. ResultsOverall, circEfnb2 was highly expressed whereas miR-202-5p was decreased in OGD/R-treated mouse hippocampal neuronal HT22 cells compared to normal controls (both p > 0.05). From an in vitro functional perspective, circEfnb2 knockdown attenuated an OGD/R-triggered neuronal injury compared to controls (p > 0.05). Mechanically, circEfnb2 acted as a sponge of miR-202-5p; downregulation of miR-202-5p annulled the inhibitory roles of circEfnb2 silencing in an OGD/R-caused neuronal injury model. Our analysis showed that miR-202-5p directly targeted TRAF3 as enhanced TRAF3 abolished the effects of miR-202-5p in the OGD/R-induced neuronal injury. In vivo, lentivirus with a short hairpin (sh)-circEfnb2 inhibited cerebral injury, when injected into cerebral cortex in MCAO mice (p > 0.05). ConclusionOur results suggest that circEfnb2 deficiency may decrease OGD/R-induced HT22 cell damage by modulating the miR-202-5p/TRAF3 axis. This explanation may provide a new direction for cerebral infarction potential therapeutic targets.

AMPK Activation by Cimicifuga racemosa Extract Ze 450 Is Associated with Metabolic Effects and Cellular Resilience against Age-Related Pathologies in Different Tissue Cell Types.

Cimicifuga racemosa extracts (CREs) have gained well-established use for the treatment of menopausal symptoms such as hot flushes and excessive sweating, and weight gain. While the clinical effects of CREs have been well documented, the mechanisms underlying these effects are largely unknown. More recently, the metabolic effects of the CRE Ze 450 were demonstrated in cultured cells in vitro and in mouse models of obesity in vivo. At the molecular level, metabolic regulation, enhanced insulin sensitivity, and increased glucose uptake were linked to the activation of AMP-activated protein kinase (AMPK). Therefore, we tested the effects of Ze 450 on AMPK phosphorylation and thus activation in cells from different tissues, i.e., murine C2C12 myoblast cells, human HEPG2 liver cells, mouse HT22 neuronal cells, and in murine 3T3L1 adipocytes. Using a FRET-based HTRF-assay, we found that Ze 450 induced AMPK phosphorylation and the activation of this key enzyme of metabolic regulation in cells from various different tissues including C2C12 (muscle), HEPG2 (liver), HT22 (hippocampal), and 3T3-L1 (adipocyte) cells. In C2C12 muscle cells, enhanced AMPK activation was accompanied by reduced mitochondrial respiration and enhanced glucose uptake. Further, Ze 450 enhanced the resilience of the cells against oxidative death induced by ferroptosis inducers erastin or RSL3. Our findings suggest a general effect of Cimicifuga racemosa on AMPK activation in different tissues and across species. This may have a significant impact on expanded therapeutic applications of Ze 450, since AMPK activation and the related metabolic effects have been previously associated with anti-aging effects and the prevention of the metabolic syndrome.

4-Hydroxysesamin, a Modified Natural Compound, Attenuates Neuronal Apoptosis After Ischemic Stroke via Inhibiting MAPK Pathway.

The 4-hydroxysesamin (4-HS, a di-tetrahydrofuran lignin) is a modified sesamin that was prepared in the laboratory. This preclinical study was designed to preliminarily investigate the neuroprotective properties of 4-HS. In vitro, neuronal injury and inflammation were simulated by oxygen-glucose deprivation and lipopolysaccharide (LPS) exposure in mouse hippocampal neuronal HT22 cell line, and treated with 4-HS and/or metformin (MET, MAPK pathway activator for exploring mechanism). CCK-8, flow cytometry, and enzyme-linked immunosorbent assay were performed to evaluate cell viability, apoptosis, and inflammation. Apoptosis- and pathway-related proteins were detected by Western blotting. Middle cerebral artery occlusion (MCAO) was constructed as a stroke model and treated with 4-HS for in vivo confirmation. Histological staining was used for in vivo evaluation of 4-HS properties. The 4-HS showed similar anti-inflammatory activity to sesamin but did not affect the cell viability of HT22 cells. In vitro, 4-HS improved the cell viability, ameliorated neuronal apoptosis, along with the reversion of apoptotic proteins (Bax, cleaved-caspase 3/9, Bcl-2) expression and inflammatory cytokines (IL-6, TNF-α, IL-10) in LPS-treated HT22 cells. The 4-HS suppressed the phosphorylation of ERK, JNK, and p38 but the addition of MET reversed 4-HS-induced changes of phenotype and protein expression in LPS-treated cells. In vivo, 4-HS showed apparent improvement in cerebral infarction, brain tissue morphology, neuronal architecture, apoptosis, and inflammation of MCAO mice, and also showed inhibiting effects on the phosphorylation of ERK, JNK, and p38, confirming in vivo results. In this first pre-clinical study on 4-HS, we preliminarily demonstrated the neuroprotective properties of 4-HS both in cell and animal models, and proposed that the underlying mechanism might be associated with the MAPK pathway.

TREM2 Alleviates Subarachnoid Hemorrhage-Induced Brain Injury through Attenuating Neuroinflammation and Programmed Cell Death in Vivo and in Vitro.

Apoptosis and pyroptosis are two types of programmed cell death related to the neuroinflammatory reaction after subarachnoid hemorrhage (SAH). Research indicates that triggering receptor expressed on myeloid cells 2 (TREM2) can regulate the SAH-induced inflammatory response. However, whether TREM2 regulates programmed cell death (apoptosis and pyroptosis) remains to be clarified. The purpose of the present study was to investigate the effects of TREM2 on cell death in SAH. SAH was induced in adult male C57BL/6J mice by endovascular perforation. An in-vitro cellular model of SAH was established by treating cocultured BV2 microglia and HT22 neuronal cells with oxyhemoglobin. TREM2 overexpression or knockdown was carried out by intraventricular lentivirus injection at 7 d before SAH induction in mice or lentiviral transfection, respectively. Neurobehavioral tests as well as western blot, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, Evans blue (EB) staining, Nissl staining, and flow cytometry assays were performed to investigate the neuroprotective role of TREM2 after SAH. After SAH, the TREM2 mRNA and protein levels were elevated in SAH mice, exhibiting a peak at 72 h. TREM2 overexpression improved the SAH-induced neurological deficits in mice, while TREM2 knockdown worsened them. In the brains of mice with TREM2 overexpression, less neuronal death and more neuronal survival were detected at 72 h post SAH. Meanwhile, TREM2 overexpression showed an inhibitory effect on microglial activation, neutrophil infiltration, and the expression of cell death marker proteins. Consistent results were obtained in vitro. Our research indicates the important role of TREM2 on cell death after SAH, suggesting that targeting TREM2 might be an effective approach for treating SAH.

DDQ anti-aging properties expressed with improved mitophagy in mutant tau HT22 neuronal cells

The purpose of our study is to develop age-related phosphorylated tau (p-tau) inhibitors, for Alzheimer’s disease (AD). There are wide-ranging therapeutic molecules available in the market and tested for age-related p-tau inhibition to enhance phosphatase activity and microtubule stability in AD neurons. Until now there are no such small molecules claimed to show promising results to delay the disease process of AD. However, a recently developed molecule, DDQ, has been shown to reduce abnormal protein–protein interactions and protect neurons from mutant protein-induced toxicities in the disease process. In addition, DDQ reduced age- and Aβ-induced oxidative stress, mitochondrial dysfunction, and synaptic toxicity. To date, there are no published reports on the p-tau interaction of DDQ and Sirt3 upregulation with CREB-mediated mitophagy activation in AD neurons. In the current study, HT22 cells were transfected with mutant Tau (mTau) cDNA and treated with the novel molecule DDQ. Cell survival, immunoblotting, and immunofluorescence analysis were conducted to assess cell viability and synaptic and mitophagy proteins in treated and untreated cell groups. As expected, we found cell survival was decreased in mTau-HT22 cells when compared with control HT22 cells. However, cell survival was increased in DDQ-treated mTau-HT22 cells when compared with mTau HT22 cells. P-tau and total tau proteins were significantly reduced in DDQ-treated mTau-HT22 cells, and MAP2 levels were increased. Anti-aging proteins like Sirt3, and CREB levels were increased in DDQ-treated HT22 cells and also in mTau-HT22 cells treated DDQ. Mitophagy proteins were decreased in mTau-HT22 cells and these were increased in DDQ-treated mTau-HT22 cells. These observations strongly suggest that DDQ has anti-p-tau and anti-aging properties, via Sirt3 overexpression and increased mitophagy proteins. Our study findings may have implications for healthy aging to the development of p-tau targeted therapeutics in AD and tauopathies.

Manganese disrupts the maturation and degradation of axonal autophagosome leading to hippocampal synaptic toxicity in mice via the activation of LRRK2 on phosphorylation of Rab10

Manganese (Mn) overexposure induces hippocampal synaptotoxicity by the accumulation of dysfunctional synaptic vesicles (SVs). Leucine-rich repeat kinase 2 (LRRK2) kinase activity is involved in regulating axonal transport (autophagosomal maturation) and lysosomal function. Nevertheless, it remains unclear whether Mn-induced synaptotoxicity is associated with the LRRK2-mediated disruption of autophagosomal maturation in axonal transport and the impairment of lysosomes in hippocampal neurons. Here, we established models of manganism in C57BL/6 mice and hippocampal neuronal HT22 cells to verify the role of LRRK2-mediated Rab10 phosphorylation in the Mn-induced dysfunction of autophagy- lysosomal fusion. Our results proved that Mn-induced the disorder of axonal transport and that lysosome impairments were associated with the increased recruitment of phospho-Rab10 at the axon and lysosomes. Next, we established Lrrk2-KD and LRRK2 kinase- specific inhibitor (GNE-0877, GNE) pre-treated HT22 cells to inhibit Lrrk2 gene expression and kinase activity, respectively. In Mn-treated Lrrk2-KD or GNE-pretreated normal neurons, our results indicated that lysosomal pH and integrity and autophagic flow were restored, indicating by decreased levels of phospho-Rab10 on lysosomes and JNK-interacting proteins (JIP4). In addition, GNE pretreatment could provide protection against Mn-induced synaptotoxicity in vivo, which was evidenced by the partial recovery in synaptic plasticity and synaptic damage. Thus, the Mn-induced abnormal activation of LRRK2 affected lysosomes and the recruitment of phospho-Rab10 by JIP4, which disrupted autophagosomal maturation in proximal axons and resulted in the hippocampal synaptic toxicity of mice.

Modulating the aggregation of human prion protein PrP106-126 by an indole-based cyclometallated palladium complex.

The spontaneous aggregation of infectious or misfolded forms of prion protein is known to be responsible for neurotoxicity in brain cells, which ultimately leads to the progression of prion disorders. Bovine spongiform encephalopathy (BSE) in animals and Creutzfeldt-Jakob disease (CJD) in humans are glaring examples in this regard. Square-planar complexes with labile ligands and indole-based compounds are found to be efficiently inhibitory against protein aggregation. Herein, we report the synthesis of an indole-based cyclometallated palladium complex. The ligand and complex were characterized by various spectroscopic techniques such as UV-visible, NMR, IR, and HRMS. The molecular structure of the complex was confirmed by single-crystal X-ray crystallography. The interaction of the complex with PrP106-126 was studied using UV-visible spectroscopy, CD spectroscopy, MALDI-TOF MS, and molecular docking. The inhibition effects of the complex on the PrP106-126 aggregation, fibrillization and amyloid formation phenomena were analysed through the ThT assay, CD, TEM and AFM. The effect of the complex on the aggregation process of PrP106-126 was determined kinetically through the ThT assay. The complex presented high binding affinity with the peptide and influenced the peptide's conformation and aggregation in different modes of binding. Furthermore, the MTT assay on neuronal HT-22 cells showed considerable protective properties of the complex against PrP106-126-mediated cytotoxicity. These findings suggest that the compound influences peptide aggregation in different ways, and the anti-aggregation action is primarily associated with the metal's physicochemical properties and the reactivity rather than the ligand. As a result, we propose that this compound be investigated as a potential therapeutic molecule in metallopharmaceutical research to treat prion disease (PD).

Role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in autophagy activation following subarachnoid hemorrhage

BackgroundEarly brain injury (EBI) refers to a severe brain injury that occurs within hours to days after subarachnoid hemorrhage (SAH). Neuronal damage in EBI is considered a key factor leading to poor prognosis. Currently, our understanding of the mechanisms of neuronal damage, such as neuronal autophagy, is still incomplete. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in metabolism and plays an important role in autophagy. Based on this, this study will further explore the regulation of autophagy by GAPDH after SAH, which may provide a new treatment strategy for improving the prognosis of SAH patients. MethodsThe rat SAH model was established by endovascular puncturing, and the trend of autophagy in hippocampal neurons at different time points was discussed. Additionally, an in vitro SAH model was created using the oxygenated hemoglobin and hippocampal neuronal HT22 cell line. Through siRNA and overexpression adenovirus techniques, we further investigated the relationship between the key enzyme GAPDH and autophagy in the in vitro SAH model. ResultsWe observed significant neuronal damage in the hippocampus 24 h after SAH, and the proteomics showed significant enrichment of autophagy-related pathways at this time point. Further studies showed that the expression of LC3 and Beclin1 peaked at 24 h, and the nuclear translocation of GAPDH occurred simultaneously with SAH-induced neuronal autophagy. Our in vitro SAH model confirmed the role of GAPDH in regulating the level of autophagy in HT22 cells. Knockdown of GAPDH significantly reduced the level of autophagy, while overexpression of GAPDH increased the level of autophagy. ConclusionThis study shows the trend of autophagy in hippocampal neurons after SAH, and reveals the regulatory role of GAPDH in SAH-induced autophagy. However, further studies are needed to reveal the exact mechanism of GAPDH in the nuclear translocation regulation of autophagy and validate in animal models.

BANF1 promotes glutamate-induced apoptosis of HT-22 hippocampal neurons.

Glutamate exposure was fatal to HT-22 neuronal cells that derived from mouse hippocampus. This is often used as a model for hippocampus neurodegeneration in vitro. The targets relevant to glutamate-induced neuronal toxicity is not fully understood. In this study, we aimed to identify crucial factors associated with glutamate-induced cytotoxicity in HT-22 cells. HT-22 cells were treated with 7.5 mM glutamate for 24 h and isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis conducted to identify the differentially expressed proteins. Differential proteins were subjected to Gene Ontology analyses. Upregulation of barrier to autointegration factor (BANF1/BANF1) protein was confirmed by RT-qPCR and western blotting. Cell viability was measured by CKK-8 and MTT assays. Cell apoptosis rates and intracellular reactive oxygen species (ROS) levels were detected using flow cytometry. A total of 5811 proteins were quantified by iTRAQ, 50 of which were recognized as significantly differential proteins (fold change ≥ 1.5 and P ≤ 0.05); 26 proteins were up-regulated and 24 were down-regulated after exposure to glutamate. GO enrichment analysis showed that the apoptotic signaling pathway was involved in cell death induced by glutamate. BANF1 expression level was markedly increased in HT-22 cells after glutamate treatment. Further, knockdown of BANF1 alleviated glutamate-mediated cell death with lower ROS levels. In conclusion, we successfully filtered out differential proteins relevant to glutamate-mediated cytotoxicity. BANF1 upregulation promoted glutamate-induced apoptosis of HT-22 cells by enhancing ROS generation.

Neuron-derived exosomes mediate sevoflurane-induced neurotoxicity in neonatal mice via transferring lncRNA Gas5 and promoting M1 polarization of microglia.

Sevoflurane exposure during rapid brain development induces neuronal apoptosis and causes memory and cognitive deficits in neonatal mice. Exosomes that transfer genetic materials including long non-coding RNAs (lncRNAs) between cells play a critical role in intercellular communication. However, the lncRNAs found in exosomes derived from neurons treated with sevoflurane and their potential role in promoting neurotoxicity remain unknown. In this study, we investigated the role of cross-talk of newborn mouse neurons with microglial cells in sevoflurane-induced neurotoxicity. Mouse hippocampal neuronal HT22 cells were exposed to sevoflurane, and then co-cultured with BV2 microglial cells. We showed that sevoflurane treatment markedly increased the expression of the lncRNA growth arrest-specific 5 (Gas5) in neuron-derived extracellular vesicles, which inhibited neuronal proliferation and induced neuronal apoptosis by promoting M1 polarization of microglia and the release of inflammatory cytokines. We further revealed that the exosomal lncRNA Gas5 significantly upregulated Foxo3 as a competitive endogenous RNA of miR-212-3p in BV2 cells, and activated the NF-κB pathway to promote M1 microglial polarization and the secretion of inflammatory cytokines, thereby exacerbating neuronal damage. In neonatal mice, intracranial injection of the exosomes derived from sevoflurane-treated neurons into the bilateral hippocampi significantly increased the proportion of M1 microglia, inhibited neuronal proliferation and promoted apoptosis, ultimately leading to neurotoxicity. Similar results were observed in vitro in BV2 cells treated with the CM from HT22 cells after sevoflurane exposure. We conclude that sevoflurane induces the transfer of lncRNA Gas5-containing exosomes from neurons, which in turn regulates the M1 polarization of microglia and contributes to neurotoxicity. Thus, modulating the expression of lncRNA Gas5 or the secretion of exosomes could be a strategy for addressing sevoflurane-induced neurotoxicity.

Regulation of microglia-mediated inflammation by host lncRNA Gm20559 upon flaviviral infection

BackgroundJapanese Encephalitis Virus (JEV) and West Nile Viruses (WNV) are neurotropic flaviviruses which cause neuronal death and exaggerated glial activation in the central nervous system. Role of host long non coding RNAs in shaping microglial inflammation upon flavivirus infections has been unexplored. This study attempted to decipher the role of lncRNA Gm20559 in regulating microglial inflammatory response in context of flaviviruses. MethodsAntisense oligonucleotide LNA Gapmers designed against lncRNA Gm20559 and non-specific site (negative control) were used for Gm20559 knockdown in JEV and WNV-infected N9 microglial cells. Upon establishing successful Gm20559 knockdown, expression of various proinflammatory cytokines, chemokines, interferon-stimulated genes (ISGs) and RIG-I were checked by qRT-PCR and cytometric bead array. Western Blotting was done to analyse the phosphorylation level of various inflammatory markers and viral non-structural protein expression. Plaque Assays were employed to quantify viral titres in microglial supernatant upon knocking down Gm20559. Effect of microglial supernatant on HT22 neuronal cells was assessed by checking expression of apoptotic protein and viral non-structural protein by Western Blotting. ResultsUpregulation in Gm20559 expression was observed in BALB/c pup brains, primary microglia as well as N9 microglia cell line upon both JEV and WNV infection. Knockdown of Gm20559 in JEV and WNV-infected N9 cell led to the reduction of major proinflammatory cytokines – IL-1β, IL-6, IP-10 and IFN-β. Inhibition of Gm20559 upon JEV infection in N9 microglia also led to downregulation of RIG-I and OAS-2, which was not the case in WNV-infected N9 microglia. Phosphorylation level of P38 MAPK was reduced in case of JEV-infected N9 microglia and not WNV-infected N9 microglia. Whereas phosphorylation of NF-κB pathway was unchanged upon Gm20559 knockdown in both JEV and WNV-infected N9 microglia. However, treating HT22 cells with JEV and WNV-infected microglial supernatant with and without Gm20559 could not trigger cell death or influence viral replication. ConclusionKnockdown studies on lncRNA Gm20559 suggests its pivotal role in maintaining the inflammatory milieu of microglia in flaviviral infection by modulating the expression of various pro-inflammatory cytokines. However, Gm20559-induced increased microglial proinflammatory response upon flavivirus infection fails to trigger neuronal death.

Deubiquitylating Enzyme OTUB1 Facilitates Neuronal Survival After Intracerebral Hemorrhage Via Inhibiting NF-κB-triggered Apoptotic Cascades.

The deubiquitylase OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) has been implicated in the pathogenesis of various human diseases. However, the molecular mechanism by which OTUB1 participates in the pathogenesis of intracerebral hemorrhage (ICH) remains elusive. In the present study, we established an autologous whole blood fusion-induced ICH model in C57BL/6J mice. We showed that the upregulation of OTUB1 contributes to the attenuation of Nuclear factor kappa B (NF-κB) and its downstream apoptotic signaling after ICH. OTUB1 directly associates with NF-κB precursors p105 and p100 after ICH, leading to attenuated polyubiquitylation of p105 and p100. Moreover, we revealed that NF-κB signaling was modestly activated both in ICH tissues and hemin-exposed HT-22 neuronal cells, accompanied with the activation of NF-κB downstream pro-apoptotic signaling. Notably, overexpression of OTUB1 strongly inhibited hemin-induced NF-κB activation, whereas interference of OTUB1 led to the opposite effect. Finally, we revealed that lentiviral transduction of OTUB1 markedly ameliorated hemin-induced apoptotic signaling and HT-22 neuronal death. Collectively, these findings suggest that the upregulation of OTUB1 serves as a neuroprotective mechanism in antagonizing neuroinflammation-induced NF-κB signaling and neuronal death, shed new light on manipulating intracellular deubiquitylating pathways as novel interventive approaches against ICH-induced secondary neuronal damage and death.

Effect of cycloastragenol and punicalagin on Prp(106–126) and Aβ(25–35) oligomerization and fibrillizaton

Numerous neurological disorders, including prion, Parkinson's, and Alzheimer's disease (AD), are identified as being caused by alterations in protein conformation, aggregation, and metal ion dyshomeostasis. Recent years have seen a significant increase in the exploration and study of natural products (NPs) from plant and microbial sources for their therapeutic potential against several diseases, including cancer, diabetes, cardiovascular disease, and neurodegenerative diseases. In this study, we have examined the effect of two NPs, cycloastragenol (CAG) and punicalagin (PCG), on the metal-induced oligomerization and aggregation of Aβ25–35 and PrP106–126 peptides. The peptide aggregation and inhibitory properties of both NPs were examined by the thioflavin-T (ThT) assay, MALDI-TOF, circular dichroism (CD) spectroscopy, and transmission electron microscopy (TEM). Among the two NPs, PCG significantly binds to the peptides, chelates metal ions (Cu2+ and Zn2+), inhibits peptide aggregation, substantially reduces oxidative stress, and controls the production of reactive oxygen species (ROS). Both NPs exhibited low cytotoxicity and prominently mitigated peptide-mediated cell cytotoxicity in hippocampal neuronal HT-22 cells by covalent bonding and hydrophobic interactions.

Lactobacillus-derived extracellular vesicles counteract Aβ42-induced abnormal transcriptional changes through the upregulation of MeCP2 and Sirt1 and improve Aβ pathology in Tg-APP/PS1 mice.

Mounting evidence suggests that probiotics are beneficial for treating Alzheimer's disease (AD). However, the mechanisms by which specific probiotics modify AD pathophysiology are not clearly understood. In this study, we investigated whether Lactobacillus paracasei-derived extracellular vesicles (Lpc-EV) can directly act on neuronal cells to modify amyloid-beta (Aβ)-induced transcriptional changes and Aβ pathology in the brains of Tg-APP/PS1 mice. Lpc-EV treatment in HT22 neuronal cells counteracts Aβ-induced downregulation of Brain-derived neurotrophic factor (Bdnf), Neurotrophin 3 (Nt3), Nt4/5, and TrkB receptor, and reverses Aβ-induced altered expression of diverse nuclear factors, including the downregulation of Methyl-CpG binding protein 2 (Mecp2) and Sirtuin 1 (Sirt1). Systematic siRNA-mediated knockdown experiments indicate that the upregulation of Bdnf, Nt3, Nt4/5, and TrkB by Lpc-EV is mediated via multiple epigenetic factors whose activation converges on Mecp2 and Sirt1. In addition, Lpc-EV reverses Aβ-induced downregulation of the Aβ-degrading proteases Matrix metalloproteinase 2 (Mmp-2), Mmp-9, and Neprilysin (Nep), whose upregulation is also controlled by MeCP2 and Sirt1. Lpc-EV treatment restores the downregulated expression of Bdnf, Nt4/5, TrkB, Mmp-2, Mmp-9, and Nep; induces the upregulation of MeCP2 and Sirt1 in the hippocampus; alleviates Aβ accumulation and neuroinflammatory responses in the brain; and mitigates cognitive decline in Tg-APP/PS1 mice. These results suggest that Lpc-EV cargo contains a neuroactive component that upregulates the expression of neurotrophic factors and Aβ-degrading proteases (Mmp-2, Mmp-9, and Nep) through the upregulation of MeCP2 and Sirt1, and ameliorates Aβ pathology and cognitive deficits in Tg-APP/PS1 mice.

Idebenone protects against ethanol toxicity in HT-22 cells through strengthening neuroimmune response

Idebenone, an analogue of coenzyme Q10, may function as a neuroprotective agent with its antioxidant and anti-inflammatory properties. The current report was designed to examine the beneficial effects of idebenone on ethanol-related neurotoxicity in hippocampal neuronal HT-22 cells in vitro and annotate the neuroprotective mechanism of idebenone. 75 mM ethanol was applied to the cells for 24h to develop ethanol toxicity. Then, different concentrations of idebenone (final concentration in the well to be 1, 2.5, and 5 μM) were applied to HT-22 cells for 24 h to explore the protective impact against ethanol-induced hippocampal damage. Cell viability was evaluated with MTT test. MDA, SOD, and GSH concentrations were examined to interpret oxidative damage. Moreover, the effects of idebenone on IL-1β, IL-6, and IL-23 neuroimmune-related genes expression levels were assigned by the RT-PCR analysis. In our study, 75 mM ethanol decreased neuronal cell viability by approximately 61%. All concentrations of idebenone were not toxic to neurons. In addition, idebenone increased cell viability by reducing the damage caused by alcohol. Idebenone reversed the reduction in antioxidant capacity caused by ethanol through decreasing MDA and increasing SOD and GSH levels. In addition, idebenone attenuated ethanol-induced impairment in neuroimmune and neuroinflammatory responses by reducing IL-1β, IL-6, and IL-23 mRNA expression levels. Treatment with idebenone increased antioxidant capacity and a significant improvement was achieved in neuroimmune and neuroinflammatory parameters. Possible mechanisms underlying these beneficial effects cover the down-regulation of IL-1β, IL-6, and IL-23 receptors, and antioxidant restoration of idebenone.

Electro-conductive chitosan/graphene bio-nanocomposite scaffold for tissue engineering of the central nervous system

Degenerative central nervous system (CNS) disorders and traumatic brain injuries are common nowadays. These may induce the loss of neuronal cells and delicate connections essential for optimal CNS function. The CNS tissue has restricted regeneration ability, hindering the development of effective therapies. Developing cell and tissue instructive materials may bring up new treatment possibilities. In this study, chitosan-graphene nano platelets (GNPs) composite films were developed to regenerate brain cells. This study evaluates the effects of GNP concentration (0.5, 1 and 2 wt%) and their alignment on mechanical, electrical, surface, protein adsorption and biological properties of the regenerative scaffolds. Incorporating and aligning GNPs into chitosan matrix improved all the physical and biological properties. On reinforced scaffolds, HT22 cell morphology mimics pyramidal brain cells, which are responsible for the brain's highly branched neural network. Additionally, the reinforced scaffolds supported Mesenchymal Stem like Cells growth and were biocompatible in vivo. The alignment of GNPs in the chitosan matrix offered the appropriate physicochemical and biological properties to promote adhesion, proliferation and shape morphogenesis of hippocampal HT22 neuronal cells. Overall, this study delineates the enormous potential offered by the GNP-reinforced scaffolds for regeneration of central nervous system, especially the brain.