Microglia-mediated Neurotoxicity Research Articles

Anti-neuroinflammatory potential of hydroxybenzoic ester derivatives: In silico insight and in vitro validation

This study explores the anti-neuroinflammatory and antioxidant potential of 42 hydroxybenzoic acid esters and three parent acids. Molecular docking simulations targeted key proteins involved in the lipopolysaccharide (LPS) signaling pathway to identify lead compounds for synthesis and in vitro evaluation. Among the screened esters, the most promising candidates demonstrated antioxidant activity comparable to vitamin C in ABTS and DPPH assays. Additionally, these esters significantly reduced the production of reactive oxygen species (ROS) and nitric oxide (NO) in H2O2- and LPS-stimulated BV2 microglial cells, indicating their ability to attenuate neuroinflammation. Further testing in SH-SY5Y neuronal cells exposed to microglia-derived supernatants confirmed the neuroprotective effects of these esters, reducing microglia-mediated neurotoxicity. These results suggest that hydroxybenzoic acid ester derivatives are promising candidates for mitigating microglia-driven neuroinflammation and protecting neurons, offering potential therapeutic applications for neurodegenerative diseases.

Microglia orchestrate synaptic and neuronal stripping: Implication in neuropsychiatric lupus.

Systemic lupus erythematosus (SLE), a multifactorial autoimmune disease, can affect the brain and cause neuropsychiatric dysfunction, also named neuropsychiatric lupus (NPSLE). Microglial activation is observed in NPSLE patients. However, the mechanisms regulating microglia-mediated neurotoxicity in NPSLE remain elusive. Here, we showed that M1-like proinflammatory cytokine levels were increased in the cerebrospinal fluid (CSF) of SLE patients, especially those with neuropsychiatric symptoms. We also demonstrated that MRL/lpr lupus mice developed anxiety-like behaviours and cognitive deficits in the early and active phases of lupus, respectively. An increase in microglial number was associated with upregulation of proinflammatory cytokines in the MRL/lpr mouse brain. RNA sequencing revealed that genes associated with phagocytosis and M1 polarization were upregulated in microglia from lupus mice. Functionally, activated microglia induced synaptic stripping invivo and promoted neuronal death invitro. Finally, tofacitinib ameliorated neuropsychiatric disorders in MRL/lpr mice, as evidenced by reductions in microglial number and synaptic/neuronal loss and alleviation of behavioural abnormalities. Thus, our results indicated that classically activated (M1) microglia play a crucial role in NPSLE pathogenesis. Minocycline and tofacitinib were found to alleviate NPSLE by inhibiting micrglial activation, providing a promising therapeutic strategy.

Nanotechnology for microglial targeting and inhibition of neuroinflammation underlying Alzheimer’s pathology

BackgroundAlzheimer's disease (AD) is considered to have a multifactorial etiology. The hallmark of AD is progressive neurodegeneration, which is characterized by the deepening loss of memory and a high mortality rate in the elderly. The neurodegeneration in AD is believed to be exacerbated following the intercoupled cascades of extracellular amyloid beta (Aβ) plaques, uncontrolled microglial activation, and neuroinflammation. Current therapies for AD are mostly designed to target the symptoms, with limited ability to address the mechanistic triggers for the disease. In this study, we report a novel nanotechnology based on microglial scavenger receptor (SR)-targeting amphiphilic nanoparticles (NPs) for the convergent alleviation of fibril Aβ (fAβ) burden, microglial modulation, and neuroprotection.MethodsWe designed a nanotechnology approach to regulate the SR-mediated intracellular fAβ trafficking within microglia. We synthesized SR-targeting sugar-based amphiphilic macromolecules (AM) and used them as a bioactive shell to fabricate serum-stable AM–NPs via flash nanoprecipitation. Using electron microscopy, in vitro approaches, ELISA, and confocal microscopy, we investigated the effect of AM–NPs on Aβ fibrilization, fAβ-mediated microglial inflammation, and neurotoxicity in BV2 microglia and SH-SY5Y neuroblastoma cell lines.ResultsAM–NPs interrupted Aβ fibrilization, attenuated fAβ microglial internalization via targeting the fAβ-specific SRs, arrested the fAβ-mediated microglial activation and pro-inflammatory response, and accelerated lysosomal degradation of intracellular fAβ. Moreover, AM–NPs counteracted the microglial-mediated neurotoxicity after exposure to fAβ.ConclusionsThe AM–NP nanotechnology presents a multifactorial strategy to target pathological Aβ aggregation and arrest the fAβ-mediated pathological progression in microglia and neurons.Graphical

2,4-Dichlorophenoxyacetic Acid Induces Degeneration of mDA Neurons In Vitro.

Background: Parkinson's disease (PD) affects 1-2% of the population over the age of 60 and the majority of PD cases are sporadic, without any family history of the disease. Neuroinflammation driven by microglia has been shown to promote the progression of midbrain dopaminergic (mDA) neuron loss through the release of neurotoxic factors. Interestingly, the risk of developing PD is significantly higher in distinct occupations, such as farming and agriculture, and is linked to the use of pesticides and herbicides. Methods: The neurotoxic features of 2,4-Dichlorophenoxyacetic acid (2,4D) at concentrations of 10 µM and 1 mM were analyzed in two distinct E14 midbrain neuron culture systems and in primary microglia. Results: The application of 1 mM 2,4D resulted in mDA neuron loss in neuron-enriched cultures. Notably, 2,4D-induced neurotoxicity significantly increased in the presence of microglia in neuron-glia cultures, suggesting that microglia-mediated neurotoxicity could be one mechanism for progressive neuron loss in this in vitro setup. However, 2,4D alone was unable to trigger microglia reactivity. Conclusions: Taken together, we demonstrate that 2,4D is neurotoxic for mDA neurons and that the presence of glia cells enhances 2,4D-induced neuron death. These data support the role of 2,4D as a risk factor for the development and progression of PD and further suggest the involvement of microglia during 2,4D-induced mDA neuron loss.

Silencing of long noncoding RNA X-inactive specific transcript alleviates Aβ1-42-induced microglia-mediated neurotoxicity by shifting microglial M1/M2 polarization.

This experimental study aims to investigate the role of long noncoding RNA X-inactive specific transcript (lncRNA XIST) in the microglial polarization and microglia-mediated neurotoxicity in Alzheimer's disease (AD). The levels of XIST and microRNA-107 (miR-107) were detected by quantitative real-time polymerase chain reaction. The spatial learning and memory capability of APPswe/PS1dE9 (APP/PS1) mice were evaluated by the Morris water maze test. The morphology of mouse hippocampus cells was evaluated by hematoxylin and eosin staining. The Iba1-positive microglia were labeled by immunohistochemistry staining. The protein levels were determined by western blot and enzyme-linked immunosorbent assay. Neurotoxicity was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, caspase-3 activity, and Cell Counting Kit-8 assay. The XIST, miR-107, and AD targets were predicted by bioinformatics analysis. The level of XIST was increased in APP/PS1 mice, and XIST silencing ameliorated AD progression. XIST silencing suppressed microglia activation, microglial M1 polarization, and proinflammatory factor levels, but promoted microglial M2 polarization in APP/PS1 mice and Aβ1-42-treated BV-2 cells. XIST knockdown reduced Aβ1-42-induced microglia-mediated apoptosis and enhanced cell viability in HT22 cells. XIST silencing down-regulated miR-107 level and attenuated Aβ1-42-caused suppression of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Those effects of XIST silencing were attenuated by miR-107 inhibitor or LY294002. Downregulation of XIST lessened Aβ1-42-induced microglia-mediated neurotoxicity by modulating microglial M1/M2 polarization, which may be mediated by the miR-107/PI3K/Akt pathway.

Kurarinone alleviates hemin-induced neuroinflammation and microglia-mediated neurotoxicity by shifting microglial M1/M2 polarization via regulating the IGF1/PI3K/Akt signaling.

Cerebral hemorrhage is a fatal disease that causes severe damage to local nerve function. The purpose of this research is to analyze the effect of kurarinone on hemin-induced neuroinflammation and neurotoxicity. In our study, according to the results of bioinformatics analysis, we hypothesized that kurarinone might modulate cerebral hemorrhage advancement via the insulin-like growth factor 1/phosphoinositide 3-kinase/protein kinase B (IGF1/PI3K/Akt) signaling. Kurarinone promoted M2 microglia polarization, and curbed M1 polarization and inflammation in human microglial cells (HMC3) cells with hemin treatment. Besides, kurarinone upregulated IGF1 expression and activated the PI3K/Akt signaling pathway in hemin-treated HMC3 cells. In addition, downregulation of IGF1 or inhibition of the PI3K/Akt signaling weakened the effects of kurarinone on microglia polarization and inflammation in HMC3 cells with hemin treatment. Kurarinone alleviated apoptosis and oxidative damage of SH-SY5Y cells co-cultured with hemin-treated HMC3 cells. In conclusion, kurarinone lessened hemin-induced neuroinflammation and microglia-mediated neurotoxicity by regulating microglial polarization through modulating the IGF1/PI3K/Akt signaling. These results delivered a new prospective therapeutic drug for the treatment of cerebral hemorrhage.

Sesamin protects against neurotoxicity via inhibition of microglial activation under high glucose circumstances through modulating p38 and JNK signaling pathways

Diabetes mellitus (DM), one of the principal causes of morbidity and mortality worldwide, is implicated in the progression of age-related neurodegenerative diseases (NDDs), in which microglial activation is a crucial mediator. Sesamin, a kind of phytochemical, shows inhibitory effects on microglial activation. The present study studied whether sesamin protects against neurotoxicity triggered by high glucose-induced microglial activation. We firstly demonstrated that high doses of glucose, which mimics hyperglycemia in DM, did induce the activation of murine BV2 microglial cells, increasing inflammatory responses such as the production of ROS or inflammatory mediators like IL-1β, TNF-⍺, and nitric oxide, through activation of p38 and JNK signaling pathways. Next, conditioned medium (CM) collected from high glucose-activated BV2 cell culture was used to show aggravated neurotoxicity in differentiated PC12 cells, indicating that high glucose-activated microglia could induce neurotoxicity. Interestingly, pretreatment of BV2 cells with sesamin diminished high glucose-induced microglia activation and inflammatory responses. Moreover, neurotoxicity in PC12 cells was found to be decreased in the group treated with CM from the sesamin-pretreated BV2 cell culture, suggesting sesamin inhibited microglial activation, thereby protecting neurons from activated microglia-mediated neurotoxicity. Thus, sesamin might be a potential compound to use in the prevention of diabetic-induced NDDs.

Microglia-Mediated Inflammation and Neural Stem Cell Differentiation in Alzheimer's Disease: Possible Therapeutic Role of KV1.3 Channel Blockade.

Increase of deposits of amyloid β peptides in the extracellular matrix is landmark during Alzheimer’s Disease (AD) due to the imbalance in the production vs. clearance. This accumulation of amyloid β deposits triggers microglial activation. Microglia plays a dual role in AD, a protective role by clearing the deposits of amyloid β peptides increasing the phagocytic response (CD163, IGF-1 or BDNF) and a cytotoxic role, releasing free radicals (ROS or NO) and proinflammatory cytokines (TNF-α, IL-1β) in response to reactive gliosis activated by the amyloid β aggregates. Microglia activation correlated with an increase KV1.3 channels expression, protein levels and current density. Several studies highlight the importance of KV1.3 in the activation of inflammatory response and inhibition of neural progenitor cell proliferation and neuronal differentiation. However, little is known about the pathways of this activation in neural stem cells differentiation and proliferation and the role in amyloid β accumulation. In recent studies using in vitro cells derived from mice models, it has been demonstrated that KV1.3 blockers inhibit microglia-mediated neurotoxicity in culture reducing the expression and production of the pro-inflammatory cytokines IL-1β and TNF-α through the NF-kB and p38MAPK pathway. Overall, we conclude that KV1.3 blockers change the course of AD development, reducing microglial cytotoxic activation and increasing neural stem cell differentiation. However, further investigations are needed to establish the specific pathway and to validate the use of this blocker as therapeutic treatment in Alzheimer patients.

Maf1 mitigates sevoflurane-induced microglial inflammatory damage and attenuates microglia-mediated neurotoxicity in HT-22 cells by activating the AMPK/Nrf2 signaling

BackgroundMaf1 has been found to play protective function against neuroinflammation and neuroapoptosis. This study seeks to explore whether and how Maf1 is involved in sevoflurane (Sev)-induced neuroinflammation and microglia-mediated neurotoxicity. MethodsqRT-PCR and western blot were used to detect the gene expression. ELISA was used to detect inflammatory factors. Cell viability was evaluated by using the Cell Counting Kit-8 kit. Neuroapoptosis was assessed with trhe Caspase-3 Assay Kit and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) technique. ResultsMaf1 expression was downregulated in Sev-stimulated BV2 microglial cells. Maf1 overexpression down-regulates the expression of pro-inflammatory M1-type markers (CD86, iNOS, IFN-γ) and up-regulates the expression of anti-inflammatory M2-type markers (CD206, TGF-β, Arg-1), and Maf1 reduces the Sev-induced inflammatory response in BV2 cells. After Maf1 overexpression, the relative expression of p-AMPK/AMPK and nucleus-Nrf2 increased significantly in BV2 cells treated with Sev. Inhibition of AMPK/Nrf2 pathway by compound C reverses anti-inflammatory effect of Maf1 in Sev-stimulated BV2 cells. Compound C reverses the effect of Maf1 on microglia-mediated neurotoxicity in HT-22 hippocampal neuronal cells. ConclusionsMaf1 mitigates Sev-induced microglial inflammatory damage and attenuates microglia-mediated neurotoxicity by activating the AMPK/Nrf2 signaling.

Anti-Neuroinflammatory Effects of a Semi-Synthetic Isoorientin-Based Glycogen Synthase Kinase-3β Inhibitor in Lipopolysaccharide-Activated Microglial Cells.

Neuroinflammation contributes to the pathogenesis of several neurodegenerative disorders. Glycogen synthase kinase-3β (GSK-3β) regulates the release of proinflammatory cytokines and promotes inflammatory responses in immune cells. Microglia are the resident mononuclear immune cells of the central nervous system. Here, we investigated the anti-neuroinflammatory effects of (2S,3S,4R,5R,6S)-6-(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-6-yl)-3,4,5-trihydroxy-N-((S)-1,1,1-trifluoropropan-2-yl)tetrahydro-2H-pyran-2-carboxamide (TFGF-18), a semisynthetic GSK-3β inhibitor, in lipopolysaccharide (LPS) activation of spontaneously immortalized SIM-A9 microglial cells and of mouse cortical microglia. TFGF-18 at 2.5 μM concentration inhibited LPS-induced production of nitric oxide by 56.3% and the proinflammatory cytokines TNF-α and IL-1β by 28.3 and 59.2% in SIM-A9 cells, respectively, relative to the LPS treatment control group. Pretreatment of mouse primary microglial cells with TFGF-18 at 2.5 μM concentration led to a reduction of 58.7% in TNF-α+ microglial cells at 24 h post-LPS stimulation. The migration of LPS-activated SIM-A9 cells was also reduced by 26.7% with pretreatment of TFGF-18 in a scratch assay. Analyses of signaling pathways demonstrated that TFGF-18 led to the suppression of LPS-induced GSK-3β activation and p65/NF-κB activity. Furthermore, the co-culture of SIM-A9 with SH-SY5Y neuroblastoma cells showed the suppression of TFGF-18 to microglia-mediated neurotoxicity in vitro. The findings indicate strong inhibitory effects of TFGF-18 on LPS-induced microglia activation via regulation of GSK-3β and downstream p65/NF-κB signaling. The results suggest a potential role of TFGF-18 in neuroprotection via its anti-neuroinflammatory effect.

Gualou Guizhi Granule Suppresses LPS-Induced Inflammatory Response of Microglia and Protects against Microglia-Mediated Neurotoxicity in HT-22 via Akt/NF-κB Signaling Pathways.

Neuroinflammation plays a crucial part in the commencement and advancement of ischemic stroke. Gualou Guizhi granule (GLGZG) is known to well exhibit neuroprotective effect, but it is not known whether GLGZG can regulate the inflammatory process at the cellular level in BV2 microglia cells and protect against microglia-mediated neurotoxicity in neurons. Herein, we aimed to investigate the anti-inflammatory effects of GLGZG on BV2 microglia cells and protection against microglia-mediated neurotoxicity in neurons. Methods. The cell model of neuroinflammation was constructed by lipopolysaccharide (LPS) to observe the effect of GLGZG in the presence or absence of GLGZG. The production of nitric oxide (NO), inflammatory mediators, was detected. Moreover, potential mechanisms associated with the anti-inflammatory effect, such as inhibition of microglial activation and nuclear factor kappa B (NF-κB), were also investigated. In addition, to prove whether GLGZG protects against microglia-mediated neurotoxicity, neuronal HT-22 cells were cultured in the conditioned medium. And cell survivability and neuronal apoptosis of HT-22 were evaluated. Results. It was found that a main regulator of inflammation, NO, is suppressed by GLGZG in BV2 microglial cells. Moreover, GLGZG dose dependently decreased the mRNA and protein levels of inducible NO synthase (iNOS) in LPS-stimulated BV2 cells. Additionally, GLGZG inhibited the expression and secretion of proinflammatory cytokines in BV2 microglial cells. Also, GLGZG inhibited LPS-activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in BV2 microglial cells at the intracellular level. GLGZG significantly affected Akt phosphorylation: phosphorylated forms of Akt increased. To check whether GLGZG protects against microglia-mediated neurotoxicity, neuronal HT-22 cells were incubated in the conditioned medium. GLGZG showed a neuroprotective effect by promoting cell survivability and suppressing neuronal apoptosis. Conclusions. GLGZG exerted its potential effects on suppressing inflammatory responses in LPS-induced BV2 cells by regulating NF-κB and Akt pathways. In addition, GLGZG could protect against microglia-mediated neurotoxicity in HT-22.

Tetrandrine ameliorated Alzheimer's disease through suppressing microglial inflammatory activation and neurotoxicity in the 5XFAD mouse

BackgroundAlzheimer's disease (AD) is a neurodegenerative disorder prevalent in the aged population. Tetrandrine is a natural metabolite isolated from herbal medicine Stephania tetrandra with various activities. PurposeIn this study, we investigated the therapeutic role of tetrandrine in 5XFAD mouse, a transgenic model of AD. Methods5XFAD mice were intraperitoneally injected with saline or different doses of tetrandrine (10, 20, and 40 mg/kg per 2 days) from the age of 5 months to 7 months followed by the determination of cognitive ability, amyloid plaque load, cell apoptosis, and inflammation in the brain. In vitro, the protective roles of tetrandrine against inflammatory activation of microglia and the resulting neurotoxicity were studied in BV2 cells and differentiated PC12 cells, respectively. ResultsMorris water maze test showed that two months of tetrandrine treatment dose-dependently improved the cognitive ability of 5XFAD mice. Immunostaining against Aβ 1-42 demonstrated reduced amyloid plaque deposition in the brain of tetrandrine-treated 5XFAD mice. TUNEL assay revealed decreased cell apoptosis in the hippocampus after tetrandrine treatment. Further, RT-PCR showed that the ectopic transcription of inflammation-associated genes including TNFα, IL-1β, IL-6, COX-2, iNOS, and p65 was reversed in 5XFAD mice treated with tetrandrine. In vitro, Aβ 1-42 stimulated the secretion of inflammatory cytokines TNFα and IL-1β in microglial BV2 cells as determined by ELISA, which was suppressed by tetrandrine pre-treatment. Tetrandrine pre-treatment also inhibited the expression of TLR4, p65, iNOS, and COX-2 in BV2 cells induced by Aβ 1-42. Most importantly, treatment of PC12-derived neuron-like cells with conditional medium from Aβ 1-42-stimulated BV2 cells remarkably impaired cell viability and promoted cell apoptosis, which was attenuated by the conditional medium from BV2 cells with tetrandrine pre-treatment. ConclusionCollectively, findings in this study demonstrated that tetrandrine ameliorates AD by suppressing microglia-mediated inflammation and neurotoxicity.

Atg7 deficiency in microglia drives an altered transcriptomic profile associated with an impaired neuroinflammatory response

Microglia, resident immunocompetent cells of the central nervous system, can display a range of reaction states and thereby exhibit distinct biological functions across development, adulthood and under disease conditions. Distinct gene expression profiles are reported to define each of these microglial reaction states. Hence, the identification of modulators of selective microglial transcriptomic signature, which have the potential to regulate unique microglial function has gained interest. Here, we report the identification of ATG7 (Autophagy-related 7) as a selective modulator of an NF-κB-dependent transcriptional program controlling the pro-inflammatory response of microglia. We also uncover that microglial Atg7-deficiency was associated with reduced microglia-mediated neurotoxicity, and thus a loss of biological function associated with the pro-inflammatory microglial reactive state. Further, we show that Atg7-deficiency in microglia did not impact on their ability to respond to alternative stimulus, such as one driving them towards an anti-inflammatory/tumor supportive phenotype. The identification of distinct regulators, such as Atg7, controlling specific microglial transcriptional programs could lead to developing novel therapeutic strategies aiming to manipulate selected microglial phenotypes, instead of the whole microglial population with is associated with several pitfalls.

GPR18 drives FAAH inhibition-induced neuroprotection against HIV-1 Tat-induced neurodegeneration

Human immunodeficiency virus type 1 (HIV-1) is known to provoke microglial immune responses which likely play a paramount role in the development of chronic neuroinflammatory conditions and neuronal damage related to HIV-1 associated neurocognitive disorders (HAND). In particular, HIV-1 Tat protein is a proinflammatory neurotoxin which predisposes neurons to synaptodendritic injury. Drugs targeting the degradative enzymes of endogenous cannabinoids have shown promise in reducing inflammation with minimal side effects in rodent models. Considering that markers of neuroinflammation can predict the extent of neuronal injury in HAND patients, we evaluated the neurotoxic effect of HIV-1 Tat-exposed microglia following blockade of fatty acid amid hydrolyze (FAAH), a catabolic enzyme responsible for degradation of endocannabinoids, e.g. anandamide (AEA). In the present study, cultured murine microglia were incubated with Tat and/or a FAAH inhibitor (PF3845). After 24 h, cells were imaged for morphological analysis and microglial conditioned media (MCM) was collected. Frontal cortex neuron cultures (DIV 7–11) were then exposed to MCM, and neurotoxicity was assessed via live cell calcium imaging and staining of actin positive dendritic structures. Results demonstrate a strong attenuation of microglial responses to Tat by PF3845 pretreatment, which is indicated by 1) microglial changes in morphology to a less proinflammatory phenotype using fractal analysis, 2) a decrease in release of neurotoxic cytokines/chemokines (MCP-1/CCL2) and matrix metalloproteinases (MMPs; MMP-9) using ELISA/multiplex assays, and 3) enhanced production of endocannabinoids (AEA) using LC/MS/MS. Additionally, PF3845's effects on Tat-induced microglial-mediated neurotoxicity, decreased dysregulation of neuronal intracellular calcium and prevented the loss of actin-positive staining and punctate structure in frontal cortex neuron cultures. Interestingly, these observed neuroprotective effects appeared to be independent of cannabinoid receptor activity (CB1R & CB2R). We found that a purported GPR18 antagonist, CID-85469571, blocked the neuroprotective effects of PF3845 in all experiments. Collectively, these experiments increase understanding of the role of FAAH inhibition and Tat in mediating microglial neurotoxicity in the HAND condition.

RGS10 physically and functionally interacts with STIM2 and requires store-operated calcium entry to regulate pro-inflammatory gene expression in microglia

Chronic activation of microglia is a driving factor in the progression of neuroinflammatory diseases, and mechanisms that regulate microglial inflammatory signaling are potential targets for novel therapeutics. Regulator of G protein Signaling 10 is the most abundant RGS protein in microglia, where it suppresses inflammatory gene expression and reduces microglia-mediated neurotoxicity. In particular, microglial RGS10 downregulates the expression of pro-inflammatory mediators including cyclooxygenase 2 (COX-2) following stimulation with lipopolysaccharide (LPS). However, the mechanism by which RGS10 affects inflammatory signaling is unknown and is independent of its canonical G protein targeted mechanism. Here, we sought to identify non-canonical RGS10 interacting partners that mediate its anti-inflammatory mechanism. Through RGS10 co-immunoprecipitation coupled with mass spectrometry, we identified STIM2, an endoplasmic reticulum (ER) localized calcium sensor and a component of the store-operated calcium entry (SOCE) machinery, as a novel RGS10 interacting protein in microglia. Direct immunoprecipitation experiments confirmed RGS10-STIM2 interaction in multiple microglia and macrophage cell lines, as well as in primary cells, with no interaction observed with the homologue STIM1. We further determined that STIM2, Orai channels, and the calcium-dependent phosphatase calcineurin are essential for LPS-induced COX-2 production in microglia, and this pathway is required for the inhibitory effect of RGS10 on COX-2. Additionally, our data demonstrated that RGS10 suppresses SOCE triggered by ER calcium depletion and that ER calcium depletion, which induces SOCE, amplifies pro-inflammatory genes. In addition to COX-2, we also show that RGS10 suppresses the expression of pro-inflammatory cytokines in microglia in response to thrombin and LPS stimulation, and all of these effects require SOCE. Collectively, the physical and functional links between RGS10 and STIM2 suggest a complex regulatory network connecting RGS10, SOCE, and pro-inflammatory gene expression in microglia, with broad implications in the pathogenesis and treatment of chronic neuroinflammation.

Velvet Antler Methanol Extracts Ameliorate Parkinson's Disease by Inhibiting Oxidative Stress and Neuroinflammation: From C. elegans to Mice.

Velvet antler is the traditional tonic food or medicine used in East Asia for treating aging-related diseases. Herein, we try to dissect the pharmacology of methanol extracts (MEs) of velvet antler on Parkinson's disease (PD). Caenorhabditis elegans studies showed that MEs decreased the aggregation of α-synuclein and protected oxidative stress-induced DAergic neuron degeneration. In vitro cellular data indicated that MEs suppressed the LPS-induced MAPKs and NF-κB activation, therefore inhibiting overproduction of reactive oxygen species, nitric oxide, tumor necrosis factor-α, and interleukin-6; blocking microglia activation; and protecting DAergic neurons from the microglia-mediated neurotoxicity. In vivo MPTP-induced PD mouse investigations found that MEs prevented MPTP-induced neuron loss in the substantia nigra and improved the behavioral rotating rod performance in MPTP-treated mice by increasing the expression level of tyrosine hydroxylase (TH) and downregulating α-synuclein protein expression. In all, these results demonstrate that MEs ameliorate PD by inhibiting oxidative stress and neuroinflammation.

Zanthoxylum zanthoxyloides inhibits lipopolysaccharide- and synthetic hemozoin-induced neuroinflammation in BV-2 microglia: roles of NF-κB transcription factor and NLRP3 inflammasome activation.

The effects of a root extract of Zanthoxylum zanthoxyloides on neuroinflammation in BV-2 microglia stimulated with LPS and hemozoin were investigated. ELISA, enzyme immunoassay and Griess assay were used to evaluate levels of cytokines, PGE2 and NO in culture supernatants, respectively. Microglia-mediated neurotoxicity was evaluated using a BV-2 microglia-HT-22 neuron transwell co-culture. Treatment with Z. zanthoxyloides caused reduced elevated levels of TNFα, IL-6, IL-1β, NO and PGE2, while increasing the levels of IL-10. In addition, there were reduced levels of iNOS and COX-2 proteins. This was accompanied by a prevention of microglia-mediated damage to HT-22 mouse hippocampal neurons. Z. zanthoxyloides reduced elevated levels of phospho-IκB and phospho-p65, while preventing degradation of IκB protein and DNA binding of p65. Further mechanistic studies revealed that Z. zanthoxyloides reduced the levels of pro-IL-1β and IL-1β in hemozoin-activated BV-2 microglia. This was accompanied by a reduction in caspase-1 activity and NLRP3 protein expression. Bioassay-guided fractionation resulted in the isolation of skimmianine as an anti-inflammatory compound in Z. zanthoxyloides. This is the first report showing the inhibition of neuroinflammation in LPS- and hemozoin-activated BV-2 microglia by the root extract of Z. zanthoxyloides by targeting the activation of both NF-κB and NLRP3 inflammasome.

Targeting chronic and evolving neuroinflammation following traumatic brain injury to improve long-term outcomes: insights from microglial-depletion models.

Targeting chronic and evolving neuroinflammation following traumatic brain injury to improve long-term outcomes: insights from microglial-depletion models.

Urban airborne PM2.5-activated microglia mediate neurotoxicity through glutaminase-containing extracellular vesicles in olfactory bulb

Emerging evidence has showed that exposure to airborne particulate matter (PM) with an aerodynamic diameter less than 2.5 μm (PM2.5) is associated with neurodegeneration. Our previous studies in vitro found that PM2.5 exposure causes primary neurons damage through activating microglia. However, the molecular mechanism of microglia-mediated neurotoxicity remains to elucidate. In this study, five groups (N = 13 or 10) of six-week-old male C57BL/6 mice were daily exposed to PM2.5 (0.1 or 1 mg/kg/day body weight), Chelex-treated PM2.5 (1 mg/kg/day body weight), PM2.5 (1 mg/kg/day body weight) plus CB-839 (glutaminase inhibitor), or deionized water by intranasal instillation for 28 days, respectively. Compared with the control groups, We found that PM2.5 triggered reactive oxygen species (ROS) generation and microglia activation evidenced by significant increase of ionized calcium binding adaptor molecule-1 (IBa-1) staining in the mouse olfactory bulbs (OB). Data from transmission electron microscope (TEM) images and Western blot analysis showed that PM2.5 significantly increased extracellular vesicles (EVs) release from OB or murine microglial line BV2 cells, and glutaminase C (GAC) expression and glutamate generation in isolated OB and BV2 cells. However, treatment with N-acetylcysteine (NAC) or CB-839 significantly diminished the number of EVs and the expression of GAC and abolished PM2.5-induced neurotoxicity. These findings provide new insights that PM2.5 induces oxidative stress and microglia activation through its metal contents and glutaminase-containing EVs in OBs, which may serve as a potential pathway/mechanism of excessive glutamate generation in PM2.5-induced neurotoxicity.

PHLDA1 promotes microglia-mediated neuroinflammation via regulating K63-linked ubiquitination of TRAF6

Microglia-mediated neuroinflammation plays an important role in the progression of neurodegenerative diseases including Parkinson's disease (PD). Pleckstrin homology-like domain family A member 1 (PHLDA1) plays an important role in immunological regulation, particularly in the Toll-like receptor-mediated immune response. Here, we explored the potential roles of PHLDA1 in microglia-mediated inflammation and neuronal protection. We found that PHLDA1 expression was rapidly increased in response to inflammatory stimuli in microglia cells in vivo or in vitro. Knockdown of PHLDA1 using adeno-associated virus serotype (AAV) ameliorated MPTP-induced motor deficits and inhibited neuroinflammation in mice. In support of this observation in vivo, we found that LPS-induced proinflammatory gene expression, including TNF-α, IL-1β, iNOS, and COX-2, was decreased in PHLDA1-deficient microglial cells. Mechanistic studies demonstrated that increased expression of PHLDA1, upon LPS stimulation in microglia, led to direct interaction with TRAF6 and enhanced its K63-linked ubiquitination-mediated NF-κB signaling activation. PHLDA1 deficiency interfered with TRAF6 K63-linked ubiquitination and inhibited microglial inflammatory responses. These findings reveal the first evidence that PHLDA1 is an important modulator of microglial function that is associated with microglia-mediated dopaminergic neurotoxicity. The data therefore provided the first evidence that PHLDA1 may be a potent modulator for neuroinflammation, and PHLDA1 may be a novel drug target for treatment of neuroinflammation-related diseases such as PD.