Mouse Microglia Research Articles

Pyruvate maintains and enhances the pro-inflammatory response of microglia caused by glucose deficiency in early stroke.

Pro-inflammatory microglia mainly rely on glycolysis to maintain cytokine production during ischemia, accompanied by an increase in inducible nitric oxide synthase (iNOS) and monocarboxylate transporter 1 (MCT1). The role of energy metabolism in the pro-inflammatory response of microglia is currently unclear. In this study, we tested the response of microglia in mice after cerebral ischemia and simulated an energy environment in vitro using low glucose culture medium. The research results indicate that the expression levels of iNOS and arginase 1 (ARG1) increase in the ischemic mouse brain, but the upregulation of MCT1 expression is mainly present in iNOS positive microglia. In microglia exposed to low glucose conditions, iNOS and MCT1 levels increased, while ARG1 levels decreased. Under the same conditions, knocking down MCT1 in microglia leads to a decrease in iNOS levels, while overexpression of MCT1 leads to the opposite result. The use of NF-κB inhibitors reduced the expression levels of iNOS and MCT1 in microglia. In summary, our data indicate that pyruvate maintains and enhances the NF-κB regulated pro-inflammatory response of microglia induced by low glucose.

Cofilin Inhibitor Improves Neurological and Cognitive Functions after Intracerebral Hemorrhage by Suppressing Endoplasmic Reticulum Stress Related-Neuroinflammation.

Neuroinflammation after intracerebral hemorrhage (ICH) is a crucial factor that determines the extent of the injury. Cofilin is a cytoskeleton-associated protein that drives neuroinflammation and microglia activation. A novel cofilin inhibitor (CI) synthesized and developed in our lab has turned out to be a potential therapeutic agent for targeting cofilin-mediated neuroinflammation in an in vitro model of ICH and traumatic brain injury. The current study aims to examine the therapeutic potential of CI in a mouse collagenase model of ICH and examine the neurobehavioral outcomes and its mechanism of action. Male mice were subjected to intrastriatal collagenase injection to induce ICH, and sham mice received needle insertion. Various concentrations (25, 50, and 100 mg/kg) of CI were administered to different cohorts of the animals as a single intravenous injection 3 h following ICH and intraperitoneally every 12 h for 3 days. The animals were tested for neurobehavioral parameters for up to 7 days and sacrificed to collect brains for hematoma volume measurement, Western blotting, and immunohistochemistry. Blood was collected for cofilin, TNF-α, and IL-1β assessments. The results indicated that 50 mg/kg CI improved neurological outcomes, reversed post-stroke cognitive impairment, accelerated hematoma resolution, mitigated cofilin rods/aggregates, and reduced microglial and astrocyte activation in mice with ICH. Microglia morphological analysis demonstrated that CI restored the homeostasis ramification pattern of microglia in mice treated with CI. CI suppressed endoplasmic reticulum stress-related neuroinflammation by inhibiting inflammasomes and cell death signaling pathways. We also showed that CI prevented synaptic loss by reviving the pre- and post-synaptic markers. Our results unveil a novel therapeutic approach to treating ICH and open a window for using CI in clinical practice.

Conditioned culture medium of bone marrow mesenchymal stem cells promotes phenotypic transformation of microglia by regulating mitochondrial autophagy.

To study the mechanism by which conditioned medium of bone marrow mesenchymal stem cells (BMSCs-CM) facilitates the transition of pro-inflammatory polarized microglia to an anti-inflammatory phenotype. BV2 cells, a mouse microglia cell line, were transformed into a pro-inflammatory phenotype using lipopolysaccharide. The expression of phenotypic genes in BV2 cells was detected using real-time quantitative PCR (RT-qPCR). Enzyme-linked immunosorbent assay was used to measure inflammatory cytokine levels in BV2 cells co-cultured with BMSCs-CM. The expressions of mitophagy-associated proteins were determined using western blot. The mitochondrial membrane potential and ATP levels in BV2 cells were measured using JC-1 staining and an ATP assay kit, respectively. Additionally, we examined the proliferation, apoptosis, and migration of C8-D1A cells, a mouse astrocyte cell line, co-cultured with BV2 cells. After co- culture with BMSCs -CM, the mRNA expression of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase significantly decreased in pro-inflammatory BV2 cells, whereas the expression of CD206 and arginase-1 significantly increased. Moreover, TNF-α and interleukin-6 levels significantly decreased, whereas transforming growth factor-β and interleukin-10 levels significantly increased. Furthermore, co-culture with BMSCs-CM increased mitophagy-associated protein expression, ATP levels, mitochondrial and lysosomal co-localization in these cells and decreased reactive oxygen species levels. Importantly, BMSCs-CM reversed the decrease in the proliferation and migration of C8-D1A cells co-cultured with pro-inflammatory BV2 cells and inhibited the apoptosis of C8-D1A cells. BMSCs-CM may promote the transition of polarized microglia from a pro-inflammatory to an anti-inflammatory phenotype by regulating mitophagy and influences the functional state of astrocytes.

Differential LRRK2 Signalling and Gene Expression in WT-LRRK2 and G2019S-LRRK2 Mouse Microglia Treated with Zymosan and MLi2.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause autosomal dominant Parkinson's disease (PD), with the most common causative mutation being the LRRK2 p.G2019S within the kinase domain. LRRK2 protein is highly expressed in the human brain and also in the periphery, and high expression of dominant PD genes in immune cells suggests involvement of microglia and macrophages in inflammation related to PD. LRRK2 is known to respond to extracellular signalling including TLR4, resulting in alterations in gene expression, with the response to TLR2 signalling through zymosan being less known. Here, we investigated the effects of zymosan, a TLR2 agonist and the potent and specific LRRK2 kinase inhibitor MLi-2 on gene expression in microglia from LRRK2-WT and LRRK2 p.G2019S knock-in mice by RNA-sequencing analysis. We observed both overlapping and distinct zymosan and MLi-2 mediated gene expression profiles in microglia. At least two candidate genome-wide association (GWAS) hits for PD, CathepsinB (Ctsb) and Glycoprotein-nmb (Gpnmb), were notably downregulated by zymosan treatment. Genes involved in inflammatory response and nervous system development were up and downregulated, respectively, with zymosan treatment, while MLi-2 treatment particularly exhibited upregulated genes for ion transmembrane transport regulation. Furthermore, we observed that the top twenty most significantly differentially expressed genes in LRRK2 p.G2019S microglia show enriched biological processes in iron transport and response to oxidative stress. Overall, these results suggest that microglial LRRK2 may contribute to PD pathogenesis through altered inflammatory pathways. Our findings should encourage future investigations of these putative avenues in the context of PD pathogenesis.

Xenografted human microglia in response to Alzheimer’s disease Aβ pathology

AbstractBackgroundNeuroinflammation is a pathological hallmark of Alzheimer’s disease (AD). Murine studies have revealed that mouse microglia can adopt several cell states in response to disease insults. However, single‐nuclei RNA sequencing studies have found divergent results when recalling those cell states in human post‐mortem tissue.MethodTo overcome this limitation, we have sequenced 127,000 single cell human microglia isolated from an AD xenotransplantation model.ResultIn response to plaques, human microglia can adopt several cell states including a disease‐associated microglia (DAM) and an antigen‐presenting response (HLA). When reacting to Aβ soluble aggregates, human microglia produces a pro‐inflammatory response cytokine response (CRM). TREM2 and APOE polymorphisms affect the development of the DAM‐HLA response, suggesting an important role of this response in AD.ConclusionOverall, this multi‐faceted response to amyloid pathologies highlights a complex scenario where these distinct phenotypes might influence the outcome on the disease.

A novel anti‐amyloid‐beta oligomer antibody reduces plaque burden and slows cognitive decline in 5xFAD mice

AbstractBackgroundThe soluble amyloid‐beta (Aβ) oligomers are the more toxic form of Aβ as compared with monomers and insoluble fibrils. Lecanemab, a humanized monoclonal antibody (mAb) with high affinity for Aβ oligomers, reduced cognitive decline in patients with early Alzheimer’s disease in a phase 3 clinical trial. Here we report a novel fully‐human anti‐Aβ oligomer mAb with profiles similar to Lecanemab.MethodAlivaMab human F(ab’)2 transgenic mice were immunized with Aβ oligomers to generate Aβ oligomer selective mAbs. The antibodies were screened for, and ranked, based on their selectivity for Aβ oligomer over monomer and ability in mediating the uptake of Aβ oligomers by microglia. Pharmacokinetics (PK) and brain penetration of lead mAb candidates was determined in mice. In vivo efficacy was evaluated in 7‐month old 5xFAD mice with weekly intraperitoneal injection of antibody lead at 1, 3, or 10 mg/kg dose levels for 4 months. After completion of dosing, open field and novel object recognition tests were performed on the treated mice and amyloid plaque burden in the brain was measured by Campbell‐Switzer stain and ELISA.ResultLead antibody 17P04 exhibited higher oligomer selectivity than that of mAb158 (mouse parental antibody of Lecanemab which served as reference antibody control) and induced antibody‐dependent cell‐mediated phagocytosis of Aβ oligomers in mouse microglia cells. PK study in mice showed that 17P04 had longer half‐life in the plasma and the brain, and demonstrated higher brain penetrance in comparison with mAb158. Subchronic treatment of 5xFAD mice with 17P04 reduced Aβ plaque burden in the brain in a dose dependent manner, reversed the abnormal activity of 5xFAD mice in open field test, and reduced cognitive decline as evaluated by novel object recognition.ConclusionWe identified a novel fully‐human anti‐Aβ oligomer antibody with superior Aβ oligomer selectivity, half‐life and brain exposure. Lead antibody 17P04 demonstrated high efficacy in plaque clearance and cognitive protection in 5xFAD mice.

Cortical microglia dynamics are conserved during voluntary wheel running.

We present the first demonstration of chronic in vivo imaging of microglia in mice undergoing voluntary wheel running. We find that healthy mice undergoing voluntary wheel running have similar microglia dynamics, morphologies, and responses to injury when compared to sedentary mice. This suggests that exercise over a period of 1 mo does not grossly alter cortical microglial phenotypes and that exercise may exert its beneficial effects on the brain through other mechanisms. Future work examining how microglia dynamics may be altered during exercise in disease or injury models could provide further insights into the therapeutic benefit of exercise.NEW & NOTEWORTHY We demonstrate the first use of chronic in vivo imaging of microglia over time during physical exercise. We found that microglia movement, morphology, and process motility were remarkably stable during voluntary wheel running (VWR). Additionally, microglia in running mice respond similarly to laser ablation injury compared to sedentary mice. These findings indicate that VWR does not induce changes in microglia dynamics in healthy adults. Exercise may elicit positive effects on the brain through other mechanisms.

GPNMB Ameliorates Neuroinflammation Via the Modulation of AMPK/NFκB Signaling Pathway After SAH in Mice

Glycoprotein non-metastatic melanoma protein B (GPNMB) got its name from the first discovery in a cell line of non-metastatic melanoma. Later studies found that GPNMB is widely expressed in various tissues and cells of the human body, most abundant in neural tissue, epithelial tissue, bone tissue, and monocyte-macrophage system. GPNMB has been shown to have anti-inflammatory effects in a variety of neurological diseases, however, it has not been reported in subarachnoid hemorrhage (SAH). Male CD-1 mice were used and intra-arterial puncture method was applied to establish the SAH model. Exogenous recombinant GPNMB (rGPNMB) was injected intracerebroventricularly 1 h after SAH. SAH grading, brain edema and blood–brain barrier (BBB) integrity were quantified, and neurobehavioral tests were performed to evaluate the effect of GPNMB on the outcome. Dorsomorphin, the selective inhibitor on AMPK was introduced to study the downstream signaling through which the GPNMB works. Furthermore, western blot, immunofluorescence staining and ELISA were utilized to confirm the signaling. After SAH, GPNMB expression increased significantly as a result of the inflammatory response. GPNMB was expressed extensively in mouse microglia, astrocytes and neurons. The administration of rGPNMB could alleviate brain edema, restore BBB integrity and improve the neurological outcome of mice with SAH. GPNMB treatment significantly magnified the expression of p-AMPK while p-NFκB, IL-1β, IL-6 and TNF-α were suppressed; in the meantime, the combined administration of GPNMB and AMPK inhibitor could decrease the intensity of p-AMPK and reverse the quantity of p-NFκB and the above inflammatory cytokines. GPNMB has the potential of ameliorating the brain edema and neuroinflammation, protecting the BBB and improving the neurological outcome, possibly via the AMPK/NFκB signaling pathway.Graphical

Microglial FoxO3a deficiency ameliorates ferroptosis-induced brain injury of intracerebral haemorrhage via regulating autophagy and heme oxygenase-1.

Microglial HO-1 regulates iron metabolism in the brain. Intracerebral haemorrhage (ICH) shares features of ferroptosis and necroptosis; hemin is an oxidized product of haemoglobin from lysed red blood cells, leading to secondary injury. However, little is known about the underlying molecular mechanisms attributable to secondary injury by hemin or ICH. In this study, we first show that FoxO3a was highly co-located with neurons and microglia but not astrocytes area of ICH model mice. Hemin activated FoxO3a/ATG-mediated autophagy and HO-1 signalling resulting in ferroptosis invitro and in a mice model of brain haemorrhage. Accordingly, autophagy inhibitor Baf-A1 or HO-1 inhibitor ZnPP protected against hemin-induced ferroptosis. Hemin promoted ferroptosis of neuronal cells via FoxO3a/ATG-mediated autophagy and HO-1 signalling pathway. Knock-down of FoxO3a inhibited autophagy and prevented hemin-induced ferroptosis dependent of HO-1 signalling. We first showed that hemin stimulated microglial FoxO3a/HO-1 expression and enhanced the microglial polarisation towards the M1 phenotype, while knockdown of microglial FoxO3a inhibited pro-inflammatory cytokine production in microglia. Furthermore, the microglia activation in the striatum showed significant along with a high expression level of FoxO3a in the ICH mice. We found that conditional knockout of FoxO3a in microglia in mice alleviated neurological deficits and microglia activation as well as ferroptosis-induced striatum injury in the autologous blood-induced ICH model. We demonstrate, for the first time, that FoxO3a/ATG-mediated autophagy and HO-1 play an important role in microglial activation and ferroptosis-induced striatum injury of ICH, identifying a new therapeutic avenue for the treatment of ICH.

Nanoplastics activate a TLR4/p38-mediated pro-inflammatory response in human intestinal and mouse microglia cells

The crescent presence of nanoplastics in the environment raises concerns regarding their potential impact on health. This study exposed human colon adenocarcinoma cells (HT29) and microglia cells (N9) to nanoplastics (25 nm, 50 nm, and 100 nm Polystyrene) to investigate their inflammatory responses, which are vital for body's defence. Although cytotoxicity remained generally low, HT29 cells exhibited a notable upregulation of p50 and p38 expression, concomitant with elevated TLR4 expression, in contrast with N9 cells that showed a less pronounced upregulation of these proteins. Additionally, nanoplastic exposure increased IL-1ß levels, partially attenuated by pre-exposure to TLR4 or p38 inhibitors. Intriguingly, N9 cells exposed to nanoplastics exhibited substantial increases in iNOS mRNA. This effect was entirely prevented by pre-exposure to TLR4 or p38 inhibitors, while TNF-α mRNA levels remained relatively stable. These findings underscore the potential of nanoplastics to activate inflammatory pathways, with response kinetics varying depending on the cell type.

Long non-coding RNA MIR17HG impedes FOSL2-mediated transcription activation of HIC1 to maintain a pro-inflammatory phenotype of microglia during intracerebral haemorrhage.

Activation and polarization of microglia play decisive roles in the progression of intracerebral haemorrhage (ICH), and lactate exposure correlates with microglia polarization. This study explores molecules influencing lactate production and microglia phenotype alteration following ICH. A murine model of ICH was induced by intracerebral injection of collagenase. The mice experienced autonomous neurological function recovery, haematoma resolution and rapid lactate production, along with a gradual increase in angiogenesis activity, neuronal recovery and an M1-to-M2 phenotype change of microglia. Galloflavin, a lactate dehydrogenase antagonist, suppressed this phenotype change and the functional recovery in mice. FOS like 2 (FOSL2) was significantly upregulated in the brain tissues from day 7 post-ICH. Overexpression of FOSL2 induced an M1-to-M2 phenotype shift in microglia and accelerated lactate production in vivo and in haemoglobin-treated microglia in vitro. Long non-coding RNA MIR17HG impeded FOSL2-mediated transcription activation of hypermethylated in cancer 1 (HIC1). MIR17HG overexpression induced pro-inflammatory activation of microglia in mice, which was blocked by further HIC1 overexpression. Overall, this study demonstrates that MIR17HG maintains a pro-inflammatory phenotype of microglia during ICH progression by negating FOSL2-mediated transcription activation of HIC1. Specific inhibition of MIR17HG or upregulation of FOSL2 or HIC1 may favour inflammation inhibition and haematoma resolution in ICH.

Species-specific metabolic reprogramming in human and mouse microglia during inflammatory pathway induction

Metabolic reprogramming is a hallmark of the immune cells in response to inflammatory stimuli. This metabolic process involves a switch from oxidative phosphorylation (OXPHOS) to glycolysis or alterations in other metabolic pathways. However, most of the experimental findings have been acquired in murine immune cells, and little is known about the metabolic reprogramming of human microglia. In this study, we investigate the transcriptomic, proteomic, and metabolic profiles of mouse and iPSC-derived human microglia challenged with the TLR4 agonist LPS. We demonstrate that both species display a metabolic shift and an overall increased glycolytic gene signature in response to LPS treatment. The metabolic reprogramming is characterized by the upregulation of hexokinases in mouse microglia and phosphofructokinases in human microglia. This study provides a direct comparison of metabolism between mouse and human microglia, highlighting the species-specific pathways involved in immunometabolism and the importance of considering these differences in translational research.

A microglial activity state biomarker panel differentiates FTD-granulin and Alzheimer’s disease patients from controls

BackgroundWith the emergence of microglia-modulating therapies there is an urgent need for reliable biomarkers to evaluate microglial activation states.MethodsUsing mouse models and human induced pluripotent stem cell-derived microglia (hiMGL), genetically modified to yield the most opposite homeostatic (TREM2-knockout) and disease-associated (GRN-knockout) states, we identified microglia activity-dependent markers. Non-targeted mass spectrometry was used to identify proteomic changes in microglia and cerebrospinal fluid (CSF) of Grn- and Trem2-knockout mice. Additionally, we analyzed the proteome of GRN- and TREM2-knockout hiMGL and their conditioned media. Candidate marker proteins were tested in two independent patient cohorts, the ALLFTD cohort (GRN mutation carriers versus non-carriers), as well as the proteomic data set available from the EMIF-AD MBD study.ResultsWe identified proteomic changes between the opposite activation states in mouse microglia and CSF, as well as in hiMGL cell lysates and conditioned media. For further verification, we analyzed the CSF proteome of heterozygous GRN mutation carriers suffering from frontotemporal dementia (FTD). We identified a panel of six proteins (FABP3, MDH1, GDI1, CAPG, CD44, GPNMB) as potential indicators for microglial activation. Moreover, we confirmed three of these proteins (FABP3, GDI1, MDH1) to be significantly elevated in the CSF of Alzheimer’s (AD) patients. Remarkably, each of these markers differentiated amyloid-positive cases with mild cognitive impairment (MCI) from amyloid-negative individuals.ConclusionsThe identified candidate proteins reflect microglia activity and may be relevant for monitoring the microglial response in clinical practice and clinical trials modulating microglial activity and amyloid deposition. Moreover, the finding that three of these markers differentiate amyloid-positive from amyloid-negative MCI cases in the AD cohort suggests that these proteins associate with a very early immune response to seeded amyloid. This is consistent with our previous findings in the Dominantly Inherited Alzheimer’s Disease Network (DIAN) cohort, where soluble TREM2 increases as early as 21 years before symptom onset. Moreover, in mouse models for amyloidogenesis, seeding of amyloid is limited by physiologically active microglia further supporting their early protective role. The biological functions of some of our main candidates (FABP3, CD44, GPNMB) also further emphasize that lipid dysmetabolism may be a common feature of neurodegenerative disorders.

Senescent Microglia Represent a Subset of Disease-Associated Microglia in P301S Mice.

The existence and contribution of microglia with senescent-like alterations in the pathogenesis of age-related neurodegenerative diseases like Alzheimer's disease (AD) have been suggested in recent years. However, the identification of this distinct microglial population in vivo has proven challenging, largely due to overlaps in the inflammatory phenotype of activated and senescent microglia. Furthermore, attempts at recapitulating senescence in microglia in vitro are limited. To identify and characterize senescent microglia that occur in vivo in an animal model of neurodegeneration driven by pathologic tau. We analyzed the RNA expression patterns of individual microglia from normal mice and the pathogenic tau P301 S PS19 mouse model. We have previously demonstrated that p16-expressing senescent microglia occur in these mice when neurodegeneration has occurred. Here we identify a subset of disease-associated microglia with senescent features, notably characterized by the expression of Ccl4. This signature overlaps with established markers of senescence from other cell types. Our characterization of senescent microglia can be used to better understand the role of senescent microglia in various age-related contexts, including whether clearance of senescent microglia represents a viable therapeutic option.

Atorvastatin rescues hyperhomocysteinemia-induced cognitive deficits and neuroinflammatory gene changes

BackgroundEpidemiological data suggests statins could reduce the risk of dementia, and more specifically, Alzheimer’s disease (AD). Pre-clinical data suggests statins reduce the risk of dementia through their pleiotropic effects rather than their cholesterol lowering effects. While AD is a leading cause of dementia, it is frequently found co-morbidly with cerebral small vessel disease and other vascular contributions to cognitive impairment and dementia (VCID), which are another leading cause of dementia. In this study, we determined if atorvastatin ameliorated hyperhomocysteinemia (HHcy)-induced VCID.MethodsWild-type (C57Bl6/J) mice were placed on a diet to induce HHcy or a control diet each with or without atorvastatin for 14 weeks. Mice underwent novel object recognition testing before tissue collection. Plasma total cholesterol and total homocysteine as well as related metabolites were measured. Using qPCR and NanoString technology, we profiled glial cell-associated gene expression changes. Finally, microglial morphology, astrocyte end feet, and microhemorrhages were analyzed using histological methods.ResultsAtorvastatin treatment of HHcy in mice led to no changes in total cholesterol but decreases in total homocysteine in plasma. While HHcy decreased expression of many glial genes, atorvastatin rescued these gene changes, which mostly occurred in oligodendrocytes and microglia. Microglia in HHcy mice with atorvastatin were trending towards fewer processes compared to control with atorvastatin, but there were no atorvastatin effects on astrocyte end feet. While atorvastatin treatment was trending towards increasing the area of microhemorrhages in HHcy mice in the frontal cortex, it only slightly (non-significantly) reduced the number of microhemorrhages. Finally, atorvastatin treatment in HHcy mice led to improved cognition on the novel object recognition task.ConclusionsThese data suggest that atorvastatin rescued cognitive changes induced by HHcy most likely through lowering plasma total homocysteine and rescuing gene expression changes rather than impacts on vascular integrity or microglial changes.

Microglial depletion exacerbates axonal damage and motor dysfunction in mice with cuprizone-induced demyelination

The cuprizone (CPZ)-induced demyelination model, an animal model of Multiple sclerosis (MS), is characterized by demyelination and motor dysfunction due to microglial-mediated neuroinflammation. To determine the contribution of microglia to motor function during CPZ-induced demyelination, the microglia of mice in the CPZ-model were depleted using PLX3397 (PLX), an orally bioavailable selective colony stimulating factor 1 receptor inhibitor. PLX treatment aggravated motor dysfunction as shown by the pole, beam walk, ladder walk, and rotarod tests. PLX treatment removed microglia from the superior cerebellar peduncle (SCP), but not from the corpus callosum (CC). Although PLX treatment did not affect the degree of demyelination in both of CC and SCP, the expression of axonal damage marker APP (amyloid precursor protein) was increased. Increased TNF-α, IL-1β, and iNOS expressions were observed in PLX-treated mice. These results suggest that microglial depletion exacerbates axonal damage and motor dysfunction in CPZ model mice. In this study, we found that microglia contribute to motor function and axon-protective effects in CPZ-induced demyelination.

Electroacupuncture attenuates myocardial ischemia-reperfusion injury by inhibiting microglial engulfment of dendritic spines

A major side effect of reperfusion therapy following myocardial infarction is myocardial ischemia-reperfusion injury (MIRI). Electroacupuncture preconditioning (EA-pre) has a long history in the treatment of cardiovascular diseases. Here, we demonstrate how EA-pre attenuates MIRI by affecting the phagocytosis of neuronal dendritic spines of microglia of the fastigial nucleus (FNmicroglia). We observed that EA-pre increased activity in FNGABA and then improved myocardial injury by inhibiting abnormal activities of glutaminergic neurons of the FN (FNGlu) during MIRI. Interestingly, we observed changes in the quantity and shape of FN microglia in mice treated with EA-pre and a decrease in the phagocytosis of FNGABA neuronal dendritic spines by microglia. Furthermore, the effects of improving MIRI were reversed when EA-pre mice were chemically activated by intra-FN lipopolysaccharide injection. Overall, our results provide new insight indicating that EA-pre regulates microglial engulfment capacity, thus promoting the improvement of cardiac sympathetic nervous disorder during MIRI.

Microglia Sirt6 modulates the transcriptional activity of NRF2 to ameliorate high-fat diet-induced obesity.

Microglia play a pivotal role in neuroinflammation, while obesity triggers hypothalamic microglia activation and inflammation. Sirt6 is an important regulator of energy metabolism in many peripheral tissues and hypothalamic anorexic neurons. However, the exact mechanism for microglia Sirt6 in controlling high-fat diet-induced obesity remain unknown. Microglia Sirt6 expression levels under various nutritional conditions were measured in the hypothalamus of mice. Also, microglia Sirt6-deficient mice were provided various diets to monitor metabolic changes and hypothalamic inflammatory response. Besides, RNA-seq and Co-IP of microglia with Sirt6 alterations were conducted to further investigate the detailed mechanism by which Sirt6 modulated microglia activity. We found that Sirt6 was downregulated in hypothalamic microglia in mice given a high-fat diet (HFD). Additionally, knockout of microglia Sirt6 exacerbated high-fat diet-induced hypothalamic microglial activation and inflammation. As a result, mice were more prone to obesity, exhibiting a decrease in energy expenditure, impaired glucose tolerance, insulin and leptin resistance, and increased food intake. In vitro, Sirt6 overexpression in BV2 cells displayed protective effects against oleic acid and palmitic acid treatment-derived inflammatory response. Mechanically, Sirt6 deacetylated and stabilised NRF2 to increase the expression of anti-oxidative genes and defend against reactive oxygen species overload. Pharmacological inhibition of NRF2 eliminated the beneficial modulating effects of Sirt6 on microglial activity. Collectively, our results revealed that microglial Sirt6 was a primary contributor of microglial activation in the central regulation of obesity. Thus, microglial Sirt6 may be an important therapeutic target for obesity.

EHMT2 affects microglia polarization and aggravates neuronal damage and inflammatory response via regulating HMOX1.

This research was designed to ascertain the function of euchromatic histone lysine methyltransferase 2 (EHMT2) in ischemic stroke-induced neuronal damage and inflammatory response and its regulatory mechanism. Mouse microglia (BV-2 cells) were induced by oxygen glucose deprivation/reoxygenation (OGD/R) to establish a cellular model, and then co-cultured with HT22 hippocampal neurons. After that, HT22 cell viability and apoptosis were evaluated, followed by the measurement of apoptosis-related factors (B-cell lymphoma-2, Bcl-2 associated X, and cleaved-Caspase 3). Meanwhile, the expression of inducible nitric oxide synthase (M1 microglia polarization marker) and arginase 1 (M2 microglia polarization marker) in BV-2 cells was detected, as well as the levels of inflammatory factors (tumor necrosis factor-α, interleukin [IL]-6, IL-10, IL-1β, and IL-4). Additionally, the expression of EHMT2 and heme oxygenase 1 (HMOX1) in BV-2 cells was assessed by quantitative reverse transcription polymerase chain reaction and western blot, and the binding between EHMT2 and HMOX1 was predicted and verified. OGD/R treatment led to decreased cell viability and increased cell apoptosis in HT22 cells, and aggravated inflammatory response in BV-2 cells. In OGD/R-induced BV-2 cells, EHMT2 and HMOX1 were increasingly expressed, and knockdown of EHMT2 or HMOX1 in BV-2 cells could inhibit neuronal damage and inflammatory response. Moreover, EHMT2 promoted HMOX1 transcription level by histone methylation. Collected evidence showed that down-regulation of EHMT2 relieved neuronal damage and inflammatory response by inhibiting HMOX1 expression.

Cyclin-G-associated kinase GAK/dAux regulates autophagy initiation via ULK1/Atg1 in glia

Autophagy is a major means for the elimination of protein inclusions in neurons in neurodegenerative diseases such as Parkinson's disease (PD). Yet, the mechanism of autophagy in the other brain cell type, glia, is less well characterized and remains largely unknown. Here, we present evidence that the PD risk factor, Cyclin-G-associated kinase (GAK)/Drosophila homolog Auxilin (dAux), is a component in glial autophagy. The lack of GAK/dAux increases the autophagosome number and size in adult fly glia and mouse microglia, and generally up-regulates levels of components in the initiation and PI3K class III complexes. GAK/dAux interacts with the master initiation regulator UNC-51like autophagy activating kinase 1/Atg1 via its uncoating domain and regulates the trafficking of Atg1 and Atg9 to autophagosomes, hence controlling the onset of glial autophagy. On the other hand, lack of GAK/dAux impairs the autophagic flux and blocks substrate degradation, suggesting that GAK/dAux might play additional roles. Importantly, dAux contributes to PD-like symptoms including dopaminergic neurodegeneration and locomotor function in flies. Our findings identify an autophagy factor in glia; considering the pivotal role of glia under pathological conditions, targeting glial autophagy is potentially a therapeutic strategy for PD.