2a Cells Research Articles

The neuroprotective effect of short-chain fatty acids against hypoxia-reperfusion injury

Gut microbe-derived short-chain fatty acids (SCFAs) are known to have a profound impact on various brain functions, including cognition, mood, and overall neurological health. However, their role, if any, in protecting against hypoxic injury and ischemic stroke has not been extensively studied. In this study, we investigated the effects of two major SCFAs abundant in the gut, propionate (P) and butyrate (B), on hypoxia-reperfusion injury using a neuronal cell line and a zebrafish model. Neuro 2a (N2a) cells treated with P and B exhibited reduced levels of mitochondrial and cytosolic reactive oxygen species (ROS), diminished loss of mitochondrial membrane potential, suppressed caspase activation, and lower rates of cell death when exposed to CoCl2, a chemical commonly used to simulate hypoxia. Furthermore, adult zebrafish fed SCFA-supplemented feeds showed less susceptibility to hypoxic conditions compared to the control group, as indicated by multiple behavioral measures. Histological analysis of 2,3,5-Triphenyltetrazolium chloride (TTC) stained brain sections revealed less damage in the SCFA-fed group. We also found that Fatty Acid Binding Protein 7 (FABP7), also known as Brain Lipid Binding Protein (BLBP), a neuroprotective fatty acid binding protein, was upregulated in the brains of the SCFA-fed group. Additionally, when FABP7 was overexpressed in N2a cells, it protected the cells from injury caused by CoCl2 treatment. Overall, our data provide evidence for a neuroprotective role of P and B against hypoxic brain injury and suggest the potential of dietary supplementation with SCFAs to mitigate stroke-induced brain damage.

Polyoxometalate-Polymer Directed Macromolecular Architectonics of Silver Nanoparticles as Effective Antimicrobials.

A novel inorganic-organic-inorganic ternary bioactive material formulated on antimicrobial peptide-based polymer has been reported. Supramolecular approach has been employed to incorporate molecularly crowded tyrosine-based polymer stabilized silver nanoparticles into membrane bound vesicles exploiting polyoxometalate-triggered surface templating strategy. Utilizing the covalent reversible addition fragmentation chain transfer (RAFT) polymerization and exploiting templated supramolecular architectonics at biopolymer interface, the bioactive ternary polymeric nanohybrids have been designed against Shigellosis leveraging the antibacterial activities of silver nanoparticle, cationic amphiphilic tyrosine polymer and inorganic polyoxometalate. The detail investigation against Shigella flexneri 2a cell line demonstrates that the collaborative mechanism of the ternary hybrid composite enhances the bactericidal activity in comparison to only polyoxometalate and polymer stabilized silver nanoparticle with an altered mechanism of action which is established via detailed biological analysis.

Increased expression of SLC25A18 is associated with Alzheimer’s disease and is involved in Aβ42-induced mitochondrial dysfunction and apoptosis in neuronal cells

Alzheimer’s disease (AD) is currently one of the most serious public health concerns in the world. However, the best approach to treat AD has yet to be discovered, implying that we must continue to work hard to find new AD target genes. In this study, we further analysed Gene Expression Omnibus (GEO) data and discovered that the expression of the Mitochondria glutamate carrier SLC25A18 is associated with AD by screening the differentially expressed genes in different regions of the brains of Alzheimer’s disease patients. To verify the expression of SLC25A18 during Alzheimer’s disease development, we analysed animal models (5×FAD transgenic AD animal model, chemically induced AD animal model, natural ageing animal model), and the results showed that the expression of SLC25A18 was increased in animal models of AD. Further investigation of the different regions found that SLC25A18 expression was elevated in the EC, TeA, and CA3, and expressed in neurons. Next, We found that Aβ42 treatment elevated SLC25A18 expression in Neuro 2A cells. Reducing SLC25A18 expression attenuated mitochondrial dysfunction and neuronal apoptosis caused by Aβ42. Overexpression of SLC25A18 increased ATP and intracellular superoxide anions but decreased mitochondrial membrane potential. The results indicate that SLC25A18 affects mitochondrial function and neuronal apoptosis, and is related to AD, which makes it a potential target for treating brain dysfunction.

Toxicity and magnetometry evaluation of the uptake of core-shell maghemite-silica nanoparticles by neuroblastoma cells.

Nanoparticle uptake by cells is a key parameter in their performance in biomedical applications. However, the use of quantitative, non-destructive techniques to obtain the amount of nanoparticles internalized by cells is still uncommon. We have studied the cellular uptake and the toxicity of core-shell maghemite-silica magnetic nanoparticles (MNPs), with a core diameter of 9 nm and a shell thickness of 3 nm. The internalization of the nanoparticles by mouse neuroblastoma 2a cells was evaluated by sensitive and non-destructive Superconducting Quantum Interference Device (SQUID) magnetometry and corroborated by graphite furnace atomic absorption spectroscopy. We were thus able to study the toxicity of the nanoparticles for well-quantified MNP uptake in terms of nanoparticle density within the cell. No significant variation in cell viability or growth rate was detected for any tested exposure. Yet, an increase in both the amount of mitochondrial superoxide and in the lysosomal activity was detected for the highest concentration (100 μg ml-1) and incubation time (24 h), suggesting the onset of a disruption in ROS homeostasis, which may lead to an impairment in antioxidant responses. Our results validate SQUID magnetometry as a sensitive technique to quantify MNP uptake and demonstrate the non-toxic nature of these core-shell MNPs under our culture conditions.

Electropositive Citric Acid-Polyethyleneimine Carbon Dots Carrying the PINK1 Gene Regulate ATP-Related Metabolic Dysfunction in APP/PS1-N2a Cells.

(1) Background: Alzheimer's disease (AD) is characterized by β-amyloid (Aβ) peptide accumulation and mitochondrial dysfunction during the early stage of disease. PINK1 regulates the balance between mitochondrial homeostasis and bioenergy supply and demand via the PINK1/Parkin pathway, Na+/Ca2+ exchange, and other pathways. (2) Methods: In this study, we synthesized positively charged carbon dots (CA-PEI CDs) using citric acid (CA) and polyethyleneimine (PEI) and used them as vectors to express PINK1 genes in the APP/PS1-N2a cell line to determine mitochondrial function, electron transport chain (ETC) activity, and ATP-related metabolomics. (3) Results: Our findings showed that the CA-PEI CDs exhibit the characteristics of photoluminescence, low toxicity, and concentrated DNA. They are ideal biological carriers for gene delivery. PINK1 overexpression significantly increased the mitochondrial membrane potential in APP/PS1-N2a cells and reduced reactive-oxygen-species generation and Aβ1-40 and Aβ1-42 levels. An increase in the activity of NADH ubiquinone oxidoreductase (complex I, CI) and cytochrome C oxidase (complex IV, CIV) induces the oxidative phosphorylation of mitochondria, increasing ATP generation. (4) Conclusions: These findings indicate that the PINK gene can alleviate AD by increasing bioenergetic metabolism, reducing Aβ1-40 and Aβ1-42, and increasing ATP production.

Network Pharmacology and Molecular Docking Identify the Potential Mechanism and Therapeutic Role of Scutellaria baicalensis in Alzheimer's Disease.

Scutellaria baicalensis, a traditional Chinese medicinal herb renowned for its anti-inflammatory, antioxidant, and anti-tumor properties, has shown promise in alleviating cognitive impairment associated with Alzheimer's disease. Nonetheless, the exact neuroprotective mechanism of Scutellaria baicalensis against Alzheimer's disease remains unclear. In this study, network pharmacology was employed to explore the possible mechanisms by which Scutellaria baicalensis protects against Alzheimer's disease. The active compounds of Scutellaria baicalensis were retrieved from the TCMSP database, and their corresponding targets were identified. Alzheimer's disease-related targets were obtained through searches in the GeneCards and OMIM databases. Cytoscape 3.6.0 software was utilized to construct a regulatory network illustrating the "active ingredient-target" relationships. Subsequently, the target genes affected by Scutellaria baicalensis in the context of Alzheimer's disease were input into the String database to establish a PPI network. GO analysis and KEGG analysis were conducted using the DAVID database to predict the potential pathways associated with these key targets. Following this, the capacity of these active ingredients to bind to core targets was confirmed through molecular docking. In vitro experiments were then carried out for further validation. A total of 36 active ingredients from Scutellaria baicalensis were screened out, which corresponded to 365 targets. Molecular docking results demonstrated the robust binding abilities of Baicalein, Wogonin, and 5,2'-Dihydroxy-6,7,8-trimethoxyflavone to key target proteins (SRC, PIK3R1, and STAT3). In vitro experiments showed that the active components of Scutellaria baicalensis can inhibit STAT3 expression by downregulating the PIK3R1/SRC pathway in Neuro 2A cells. In summary, these findings collectively suggest that Scutellaria baicalensis holds promise as a viable treatment option for Alzheimer's disease.

Homocysteine-potentiated Kelch-like ECH-associated protein 1 promotes senescence of neuroblastoma 2a cells via inhibiting ubiquitination of β-catenin.

Elevated serum homocysteine (Hcy) level is a risk factor for Alzheimer's disease (AD) and accelerates cell aging. However, the mechanism by which Hcy induces neuronal senescence remains largely unknown. In this study, we observed that Hcy significantly promoted senescence in neuroblastoma 2a (N2a) cells with elevated β-catenin and Kelch-like ECH-associated protein 1 (KEAP1) levels. Intriguingly, Hcy promoted the interaction between KEAP1 and the Wilms tumor gene on the X chromosome (WTX) while hampering the β-catenin-WTX interaction. Mechanistically, Hcy attenuated the methylation level of the KEAP1 promoter CpG island and activated KEAP1 transcription. However, a slow degradation rate rather than transcriptional activation contributed to the high level of β-catenin. Hcy-upregulated KEAP1 competed with β-catenin to bind to WTX. Knockdown of both β-catenin and KEAP1 attenuated Hcy-induced senescence in N2a cells. Our data highlight a crucial role of the KEAP1-β-catenin pathway in Hcy-induced neuronal-like senescence and uncover a promising target for AD treatment.

Involvement of Spinal Neuroplastin 65 in Neuropathic Pain by GABAA Receptor α2 Subunit Regulation.

Neuropathic pain (NP) is a highly challenging condition with complex pathological mechanisms, and the spinal gamma aminobutyric acid A receptor receptor plays a crucial role in its progression. Recent studies have revealed a potential interaction between neuroplastin 65 (NP65) and gamma aminobutyric acid A receptor α2 subunit (GABAAR-α2) on the cell surface. We hypothesize that NP65 is involved in the pathogenesis of NP by regulating the level of GABAAR-α2. A chronic constrictive injury (CCI) pain model was established in male Sprague-Dawley rats to verify the change in spinal NP65 expression. Alterations in pain behavior and GABAAR-α2 protein expression were observed after intrathecal injection of NP65 overexpressing adeno-associated virus (AAV) in CCI rats. In vitro investigations on Neuroblastoma 2a cells, the effect of NP65 on GABAAR-α2 expression via the calcineurin-nuclear factor of activated T-cell 4 (CaN-NFATc4) signaling pathway was evaluated by manipulating NP65 expression. The expression level of NP65 protein and mRNA in the CCI group were significantly decreased (P < .05; analysis of variance [ANOVA]). After intrathecal injection of NP65, overexpression of AAV and pain behavior in CCI rats were significantly alleviated, and levels of GABAAR-α2 were upregulated. In vitro experiments verified alterations in the expression of GABAAR-α2, CaN, and phosphorylated NFATc4 on the application of NP65 with plasmid or small interfering RNA, respectively. After the application of the specific CaN inhibitor cyclosporine A (CsA), the changes in NP65 expression did not produce subsequent alterations in the expression of GABAAR-α2, CaN, or phosphorylated NFATc4 proteins. NP65 modulates the level of GABAAR-α2 through the CaN-NFATc4 signaling pathway, which may serve as the underlying mechanism of NP.

Inhibition of Kv2.1 potassium channels by the antidepressant drug sertraline

Sertraline is a commonly used antidepressant of the selective serotonin reuptake inhibitors (SSRIs) class. In this study, we have used the patch-clamp technique to assess the effects of sertraline on Kv2.1 channels heterologously expressed in HEK-293 cells and on the voltage-gated potassium currents (IKv) of Neuro 2a cells, which are predominantly mediated by Kv2.1 channels. Our results reveal that sertraline inhibits Kv2.1 channels in a concentration-dependent manner. The sertraline-induced inhibition was not voltage-dependent and did not require the channels to be open. The kinetics of activation and deactivation were accelerated and decelerated, respectively, by sertraline. Moreover, the inhibition by this drug was use-dependent. Notably, sertraline significantly modified the inactivation mechanism of Kv2.1 channels; the steady-state inactivation was shifted to hyperpolarized potentials, the closed-state inactivation was enhanced and accelerated, and the recovery from inactivation was slowed, suggesting that this is the main mechanism by which sertraline inhibits Kv2.1 channels. Overall, this study provides novel insights into the pharmacological actions of sertraline on Kv2.1 channels, shedding light on the intricate interaction between SSRIs and ion channel function.

Transcriptomic changes associated with oral immunotherapy for food allergy.

This review summarizes recent advances in characterizing the transcriptional pathways associated with outcomes following Oral Immunotherapy. Recent technological advances including single-cell sequencing are transforming the ways in which the transcriptional landscape is understood. The application of these technologies is still in its infancy in food allergy but here we summarize current understanding of gene expression changes following oral immunotherapy for food allergy and specific signatures underpinning the different clinical outcomes of desensitization and remission (sustained unresponsiveness). T helper 2A cells have been identified as a cell type which correlates with disease activity and is modified by treatment. Molecular features at study entry may differentiate individuals who achieve more positive outcomes during OIT. Recent findings point to T cell anergy and Type 1 interferon pathways as potential mechanisms supporting redirection of the allergen-specific immune response away from allergy towards remission. Despite these developments in our understanding of immune mechanisms following OIT, there are still significant gaps. Additional studies examining immune signatures associated with long term and well-defined clinical outcomes are required to gain a more complete understanding of the pathways leading to remission of allergy, in order to optimize treatments and gain improved outcomes for patients.

Hesperidin Methyl Chalcone reduces extracellular Aβ(25-35) peptide aggregation and fibrillation and also protects Neuro 2a cells from Aβ(25-35) induced neuronal dysfunction

In the present study, we evaluated the neuroprotective potential of Hesperidin Methyl Chalcone (HMC) against the neurotoxicity induced by Aβ(25-35) peptide. HMC demonstrated higher free-radical scavenging activity than Hesperidin in initial cell-free studies. Investigations using the fluorescent dye thioflavin T with Aβ(25-35) peptide showed that HMC has the ability to combat extracellular amyloid aggregation by possessing anti-aggregation property against oligomers and by disaggregating mature fibrils. Also, the results of the molecular simulation studies show that HMC ameliorated oligomer formation. Further, the anti-Alzheimer’s property of HMC was investigated in in vitro cell conditions by pre-treating the neuro 2a (N2a) cells with HMC before inducing Aβ(25-35) toxicity. The findings demonstrate that HMC increased cell viability, reduced oxidative stress, prevented macromolecular damage, allayed mitochondrial dysfunction, and exhibited anticholinesterase activity. HMC also reduced Aβ induced neuronal cell death by modulating caspase-3 activity, Bax expression and Bcl2 overexpression, demonstrating that HMC pre-treatment reduced mitochondrial damage and intrinsic apoptosis induced by Aβ(25-35).In silico evaluation against potential AD targets reveal that HMC could be a potent inhibitor of BACE-1, inhibiting the formation of toxic Aβ peptides. Overall, the findings imply that the neuroprotective efficacy of HMC has high prospects for addressing a variety of pathogenic consequences caused by amyloid beta in AD situations and alleviating cognitive impairments.

Microglia-dependent neuroprotective effects of 4-octyl itaconate against rotenone-and MPP+-induced neurotoxicity in Parkinson’s disease

Chronic neuroinflammation is implicated in the pathogenesis of Parkinson’s disease (PD), one of the most common neurodegenerative diseases. Itaconate, an endogenous metabolite derived from the tricarboxylic acid cycle via immune‐responsive gene 1 activity, may mediate anti-inflammatory responses by activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway. This study investigates the neuroprotective potential of 4-octyl itaconate (OI), a cell-permeable derivative of itaconate, in cellular models of PD. OI not only suppressed lipopolysaccharide-induced proinflammatory cascades of inducible nitric oxide synthase, cyclooxygenase-2, and cytokines release in mouse BV2 microglial cells but also activated the Nrf2 signaling pathway and its downstream targets in these cells. Conditioned medium derived from OI-treated BV2 cells protected against rotenone- and MPP+-induced neurotoxicity in Neuro 2A cells. Overall, our findings support the anti-inflammatory neuroprotective potential of OI in PD.

A Porphyrin-based NIR Fluorescent Probe for Bi3+ and Potential Applications.

Herein, we have prepared a 5,10,15,20-Tetrakis(4-hydroxyphenyl) porphyrin (P) which acts as a probe for selective and sensitive detection of Bi3+ ions. Probe P was obtained by reacting pyrrole with 4-hydroxyl benzaldehyde and characterized by NMR, IR, and ESI-MS. All photo-physical studies of P were tested in DMSO:H2O (8:2, v/v) media by spectrophotometry and spectrofluorometry respectively. The selectivity of P was tested with different metal ions in solution as well as in the solid phase, only Bi3+ showed red fluorescence quenching while with other metal ions, no such effect was observed. The Job's plot unveiled the 1:1 stoichiometric binding ratio of the probe with Bi3+ and anticipated association constant of 3.4 ×105 M-1, whereas the Stern-Volmer quenching constant was noticed to be 5.6 ×105 M-1. Probe P could detect Bi3+ down to 27 nM by spectrofluorometric. The binding mechanism of P with Bi3+ was well supported with NMR, mass, and DFT studies. Further, the P was applied for the quantitative determination of Bi3+ in various water samples and the biocompatibility of P was examined using neuro 2A (N2a) cells. Overall, probe P proves promising for the detection of Bi3+ in the semi-aqueous phase and it is the first report as a colorimetric and fluorogenic probe.

Increased IP3R-3 degradation induced by acrylamide promoted Ca2+-dependent calpain activation and axon damage in rats

Occupational and environmental exposure to acrylamide (ACR) can cause selective peripheral and central nerve fiber degeneration. IP3R-3 is an important transmembrane Ca2+ channel on the endoplasmic reticulum (ER), previous studies have found that ACR could induce Ca2+-dependent calpain activation and axon injury, but the exact role of IP3R-3 in ACR neuropathy is still unclear. Here we show that ACR exposure (40 mg/kg) markedly increased the ubiquitination of IP3R-3 in rat spinal cords, and promoted the degradation of IP3R-3 through the ubiquitin-proteasome pathway. Furthermore, the normal structure of ER, especially the mitochondrial associated membranes (MAMs) component, was significantly impaired in ACR neuropathy, and the ER stress pathway was activated, which indicated that the aberrant increase of cytoplasmic Ca2+ could be attributed the destruction of IP3R-3. Further investigation demonstrated that the proteasome inhibitor MG-132 effectively rescued the IP3R-3 loss, attenuated the intracellular Ca2+ increase, and reduced the axon loss of Neuron 2a (N2a) cells following ACR exposure. Moreover, the calpain inhibitor ALLN also reduced the loss of IP3R-3 and axon injury in N2a cells, but did not alleviate the Ca2+ increase in cytosol, supporting that the abnormal ubiquitination of IP3R-3 was the upstream of the cellular Ca2+ rise and axon damage in ACR neuropathy. Taken together, our results suggested that the aberrant IP3R-3 degradation played an important role in the disturbance of Ca2+ homeostasis and the downstream axon loss in ACR neuropathy, thus providing a potential therapeutic target for ACR neurotoxicity.

Analogs of 6-Bromohypaphorine with Increased Agonist Potency for α7 Nicotinic Receptor as Anti-Inflammatory Analgesic Agents.

Hypaphorines, tryptophan derivatives, have anti-inflammatory activity, but their mechanism of action was largely unknown. Marine alkaloid L-6-bromohypaphorine with EC50 of 80 μM acts as an agonist of α7 nicotinic acetylcholine receptor (nAChR) involved in anti-inflammatory regulation. We designed the 6-substituted hypaphorine analogs with increased potency using virtual screening of their binding to the α7 nAChR molecular model. Fourteen designed analogs were synthesized and tested in vitro by calcium fluorescence assay on the α7 nAChR expressed in neuro 2a cells, methoxy ester of D-6-iodohypaphorine (6ID) showing the highest potency (EC50 610 nM), being almost inactive toward α9α10 nAChR. The macrophages cytometry revealed an anti-inflammatory activity, decreasing the expression of TLR4 and increasing CD86, similarly to the action of PNU282987, a selective α7 nAChR agonist. 6ID administration in doses 0.1 and 0.5 mg/kg decreased carrageenan-induced allodynia and hyperalgesia in rodents, in accord with its anti-inflammatory action. Methoxy ester of D-6-nitrohypaphorine demonstrated anti-oedemic and analgesic effects in arthritis rat model at i.p. doses 0.05-0.26 mg/kg. Tested compounds showed excellent tolerability with no acute in vivo toxicity in dosages up to 100 mg/kg i.p. Thus, combining molecular modelling and natural product-inspired drug design improved the desired activity of the chosen nAChR ligand.

Aptamer Reduces Aggregation of Mutant Huntingtin and Rescues Proteostasis Network in Non-Neuronal and Neuronal Cells.

Aggregation of mutant huntingtin is a pathological hallmark of Huntington's disease (HD). Protein aggregation results in various cellular dysfunctions, such as increase in oxidative stress, mitochondrial damage, proteostasis imbalance, etc., which finally cause cell death. Previously, specific RNA aptamers with high affinity for mutant huntingtin were selected. In the current study, we show that the selected aptamer inhibits aggregation of mutant huntingtin (EGFP-74Q) in HEK293 and Neuro 2a cell models of HD. The presence of aptamer decreases sequestration of chaperones and increases their cellular levels. This is accompanied by improved mitochondrial membrane permeability, reduced oxidative stress, and increased cell survival. Thus, RNA aptamers can be explored further as inhibitors of protein aggregation in protein misfolding diseases.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

Direct targeting of DOCK4 by miRNA-181d in oxygen-glucose deprivation/reoxygenation-mediated neuronal injury

The miRNA-181 (miR-181) family regulates neuronal persistence during cerebral ischemia/reperfusion injury (CI/RI). Since the effect of miR-181d on CI/RI has never been studied, the current work sought to determine the involvement of miR-181d in neuronal apoptosis after brain I/R injury. To replicate in vivo and in vitro CI/RI, a transient middle cerebral artery occlusion (tMCAO) model in rats and an oxygen-glucose deficiency/reoxygenation (OGD/R) model in neuro 2A cells were developed. In both in vivo and in vitro stroke models, the expression of miR-181d was considerably higher. miR-181d suppression reduced apoptosis and oxidative stress in OGD/R-treated neuroblastoma cells, but miR-181d overexpression increased both. Furthermore, it was observed that miR-181d has a direct target in dedicator of cytokinesis 4 (DOCK4). The overexpression of DOCK4 partially overcame cell apoptosis and oxidative stress induced by miR-181d upregulation and OGD/R injury. Furthermore, the DOCK4 rs2074130 mutation was related to lower DOCK4 levels in ischemic stroke (IS) peripheral blood and higher susceptibility to IS. These findings suggest that downregulating miR-181d protects neurons from ischemic damage by targeting DOCK4, implying that the miR-181d/DOCK4 axis might be a novel therapeutic target for IS.Graphical

Extracellular CIRP Induces Calpain Activation in Neurons via PLC-IP3-Dependent Calcium Pathway.

Abnormal calcium homeostasis, activation of protease calpain, generation of p25 and hyperactivation of cyclin-dependent kinase 5 (Cdk5) have all been implicated in the pathogenesis of neurogenerative diseases including Alzheimer's disease. We have recently shown that extracellular cold-inducible RNA-binding protein (eCIRP) induces Cdk5 activation via p25. However, the precise molecular mechanism by which eCIRP regulates calcium signaling and calpain remains to be addressed. We hypothesized that eCIRP regulates p25 via Ca2+-dependent calpain activation. eCIRP increased calpain activity and decreased the endogenous calpain inhibitor calpastatin in Neuro 2a (N2a) cells. Calpain inhibition with calpeptin attenuated eCIRP-induced calpain activity and p25. eCIRP specifically upregulated cytosolic calpain 1, and calpain 1 silencing attenuated the eCIRP-induced increase in p25. eCIRP stimulation increased cytosolic free Ca2+, especially in hippocampal neuronal HT22 cells, which was attenuated by the eCIRP inhibitor Compound 23 (C23). Endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor (IP3R) inhibition using 2-aminoethoxy-diphenyl-borate or xestospongin-C (X-C), interleukin-6 receptor alpha (IL-6Rα)-neutralization, and phospholipase C (PLC) inhibition with U73122 attenuated eCIRP-induced Ca2+ increase, while Ca2+ influx across the plasma membrane remained unaffected by eCIRP. Finally, C23, IL-6Rα antibody, U73122 and X-C attenuated eCIRP-induced p25 in HT-22 cells. In conclusion, the current study uncovers eCIRP-triggered Ca2+ release from ER stores in an IL-6Rα/PLC/IP3-dependent manner as a novel molecular mechanism underlying eCIRP's induction of Cdk5 activity and potential involvement in neurodegeneration.

Exaggerated levels of some specific TLRs, cytokines and chemokines in Japanese encephalitis infected BV2 and neuro 2A cell lines associated with worst outcome

Japanese encephalitis (JE) disease, a viral brain fever is caused by Japanese encephalitis virus (JEV). Despite the availability of effective vaccines against this deadly infection, JE is the leading cause of epidemic viral encephalitis in children in South-east Asia. There is no treatment available for the JE disease which might be due to incomplete understanding of the pathogenesis of JE virus. The JEV infections lead to permanent neurological deficits even in those who survive from the infection. Activated microglia may play a potentially detrimental role by eliciting the expression of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) influencing the surrounding brain tissue. Microglial activation, proinflammatory cytokine release and leukocytes trafficking are associated following JEV infection in central nervous system (CNS). How the pattern recognition receptors sense the viral nucleic acid and how the microglial and neuronal cells behaves following JEV infection is still unelucidated. There is scarcity of data on the expression levels of toll like receptors (TLRs), cytokines and chemokines in JEV infection in invitro model. To explore the molecular mechanisms of JEV infection of microglial cells and neuronal cells, we studied the expression profile of TLRs, cytokines and chemokines in JEV infected microglial cell line BV2 and Neuronal cell line Neuro 2A. For the present study, we developed the mouse model of encephalitis by intracerebral (IC) injection of JE virus for virus propagation, disease progression and damage study. Our results demonstrate the exaggerated release of some specific TLRs, cytokines and chemokines in invitro cell culture of microglial and Neuro 2A cell line, which are associated with bad outcome in invivo study.