Microglial Cultures Research Articles

Complement C3aR depletion reverses HIF-1α-induced metabolic impairment and enhances microglial response to Aβ pathology.

Microglia are the major cell type expressing complement C3a receptor (C3aR) in the brain. Using a knockin mouse line in which a Td-tomato reporter is incorporated into the endogenous C3ar1 locus, we identified 2 major subpopulations of microglia with differential C3aR expression. Expressing the Td-tomato reporter on the APPNL-G-F-knockin (APP-KI) background revealed a significant shift of microglia to a high-C3aR-expressing subpopulation and they were enriched around amyloid β (Aβ) plaques. Transcriptomic analysis of C3aR-positive microglia documented dysfunctional metabolic signatures, including upregulation of hypoxia-inducible factor 1 (HIF-1) signaling and abnormal lipid metabolism in APP-KI mice compared with wild-type controls. Using primary microglial cultures, we found that C3ar1-null microglia had lower HIF-1α expression and were resistant to hypoxia mimetic-induced metabolic changes and lipid droplet accumulation. These were associated with improved receptor recycling and Aβ phagocytosis. Crossing C3ar1-knockout mice with the APP-KI mice showed that C3aR ablation rescued the dysregulated lipid profiles and improved microglial phagocytic and clustering abilities. These were associated with ameliorated Aβ pathology and restored synaptic and cognitive function. Our studies identify a heightened C3aR/HIF-1α signaling axis that influences microglial metabolic and lipid homeostasis in Alzheimer disease, suggesting that targeting this pathway may offer therapeutic benefit.

Propofol inhibits neuroinflammation and metabolic reprogramming in microglia in vitro and in vivo.

Microglial activation-induced neuroinflammation is closely related to the development of sepsis-associated encephalopathy. Accumulating evidence suggests that changes in the metabolic profile of microglia is crucial for their response to inflammation. Propofol is widely used for sedation in mechanically ventilated patients with sepsis. Here, we investigate the effect of propofol on lipopolysaccharide-induced neuroinflammation, neuronal injuries, microglia metabolic reprogramming as well as the underlying molecular mechanisms. The neuroprotective effects of propofol (80mg/kg) in vivo were measured in the lipopolysaccharide (2mg/kg)-induced sepsis in mice through behavioral tests, Western blot analysis and immunofluorescent staining. The anti-inflammatory effects of propofol (50μM) in microglial cell cultures under lipopolysaccharide (10ng/ml) challenge were examined with Seahorse XF Glycolysis Stress test, ROS assay, Western blot, and immunofluorescent staining. We showed that propofol treatment reduced microglia activation and neuroinflammation, inhibited neuronal apoptosis and improved lipopolysaccharide-induced cognitive dysfunction. Propofol also attenuated lipopolysaccharide-stimulated increases of inducible nitric oxide synthase, nitric oxide, tumor necrosis factor-α, interlukin-1β and COX-2 in cultured BV-2 cells. Propofol-treated microglia showed a remarkable suppression of lipopolysaccharide-induced HIF-1α, PFKFB3, HK2 expression and along with downregulation of the ROS/PI3K/Akt/mTOR signaling pathway. Moreover, propofol attenuated the enhancement of mitochondrial respiration and glycolysis induced by lipopolysaccharide. Together, our data suggest that propofol attenuated inflammatory response by inhibiting metabolic reprogramming, at least in part, through downregulation of the ROS/PI3K/Akt/mTOR/HIF-1α signaling pathway.

Hydrogen peroxide signaling modulates neuronal differentiation via microglial polarization and Wnt/β-catenin pathway.

Reactive oxygen species (ROS) are generated within the cell and serve as second messengers in fundamental cellular processes under physiologic conditions. Although the deleterious effects of high-level ROS associated with oxidative stress are well established, it is unclear how the developing brain reacts to redox changes. Our aim is to investigate how redox alteration affects neurogenesis and the mechanism that underlies it.We investigated in vivo microglial polarization and neurogenesis in zebrafish after hydrogen peroxide (H2O2) incubation. To quantify intracellular H2O2 levels in vivo, a transgenic zebrafish line that expresses Hyper and termed Tg(actb2:hyper3)ka8 was used. Then, in vitro studies with N9 microglial cells, 3-dimensional neural stem cell (NSC)-microglia coculture, and conditioned medium experiments are carried out to comprehend the mechanism underlying the changes in neurogenesis upon redox modulation.In zebrafish, exposure to H2O2 altered embryonic neurogenesis, induced M1 polarization in microglia, and triggered the Wnt/β-catenin pathway. N9 microglial cell culture experiments revealed that exposure to H2O2 resulted in M1 polarization in microglial cells, and this polarization was mediated by the Wnt/β-catenin pathway. Redox modulation of microglia interfered with NSC differentiation in coculture experiments. Neuronal differentiation was significantly higher in NSCs cocultured with H2O2-treated microglia when compared to control microglia. Wnt inhibition prevented the effects of H2O2-treated microglia on NSCs. No significant alterations were observed in conditioned medium experiments.Our findings point to a robust interplay between microglia and neural progenitors influenced by the redox state. Intracellular H2O2 levels can interfere with neurogenesis by altering the phenotypic state of the microglia via the Wnt/β-catenin system.

Resistance to Degradation of Silk Fibroin Hydrogels Exposed to Neuroinflammatory Environments.

Central nervous system (CNS) diseases represent an extreme burden with significant social and economic costs. A common link in most brain pathologies is the appearance of inflammatory components that can jeopardize the stability of the implanted biomaterials and the effectiveness of therapies. Different silk fibroin scaffolds have been used in applications related to CNS disorders. Although some studies have analyzed the degradability of silk fibroin in non-cerebral tissues (almost exclusively upon non-inflammatory conditions), the stability of silk hydrogel scaffolds in the inflammatory nervous system has not been studied in depth. In this study, the stability of silk fibroin hydrogels exposed to different neuroinflammatory contexts has been explored using an in vitro microglial cell culture and two in vivo pathological models of cerebral stroke and Alzheimer's disease. This biomaterial was relatively stable and did not show signs of extensive degradation across time after implantation and during two weeks of in vivo analysis. This finding contrasted with the rapid degradation observed under the same in vivo conditions for other natural materials such as collagen. Our results support the suitability of silk fibroin hydrogels for intracerebral applications and highlight the potentiality of this vehicle for the release of molecules and cells for acute and chronic treatments in cerebral pathologies.

Chronic exposure to aflatoxin B1 increases hippocampal microglial pyroptosis and vulnerability to stress in mice

BackgroundChronic aflatoxin B1 (AFB1) exposure may increase the risk of multiple neuropsychiatric disorders. Stress is considered one of the main contributors to major depressive disorder. Whether and how chronic AFB1 exposure affects vulnerability to stress is unclear. MethodsMice were exposed for three weeks to AFB1 (100 µg/kg/d) and/or chronic mild stress (CMS). The vulnerability behaviors in response to stress were assessed in the forced swimming test (FST), sucrose preference test (SPT), and tail suspension test (TST). Microglial pyroptosis was investigated using immunofluorescence, enzyme-linked immunosorbent assays, and western blot assay in the hippocampus of mice. Hippocampal neurogenesis and the effects of AFB1-treated microglia on proliferation and differentiation of neural stem/precursor cells (NSPCs) were assessed via immunofluorescence in the hippocampus of mice. ResultsMice exposed to CMS in the presence of AFB1 exhibited markedly greater vulnerability to stress than mice treated with CMS or AFB1 alone, as indicated by reduced sucrose preference and longer immobility time in the forced swimming test. Chronic aflatoxin B1 exposure resulted in changes in the microglial morphology and increase in TUNEL+ microglia and GSDMD+ microglia in the hippocampal dentate gyrus. When mice were exposed to both CMS and AFB1, pyroptosis-related molecules (such as NLRP3, caspase-1, GSDMD-N, and interleukin-1β) were significantly upregulated in the hippocampus. These molecules were also significantly enhanced by AFB1 in primary microglial cultures. AFB1-treated mice showed decrease in the numbers of BrdU+, BrdU-DCX+, and BrdU-NeuN+ cells in the hippocampal dentate gyrus, as well as the percentages of BrdU+ cells that were NeuN+ in the presence or absence of CMS when compared with vehicle-treated mice. The combination of AFB1 and CMS exacerbated these effects to an even greater extent. The number of DCX+ cells correlated negatively with the percentage of ameboid microglia, TUNEL+ microglia and GSDMD+ microglia in the hippocampal dentate gyrus. AFB1-treated microglia suppressed the proliferation and neuronal differentiation of NSPCs in vitro. ConclusionChronic AFB1 exposure induces microglial pyroptosis, promoting an adverse neurogenic microenvironment that impairs hippocampal neurogenesis, which may render mice more vulnerable to stress.

Three-Dimensional Tracking of Intracellular Calcium and Redox State during Real-Time Control in a Hypoxic Gradient in Microglia Culture: Comparison of the Channel Blocker and Reoxygenation under Ischemic Shock.

Real-time three-dimensional (3-D) imaging is crucial for quantifying correlations among various molecules under acute ischemic stroke. Insights into such correlations may be decisive in selecting molecules capable of providing a protective effect within a shorter period. The major bottleneck is maintaining the cultures under severely hypoxic conditions while simultaneously 3-D imaging intracellular organelles with a microscope. Moreover, comparing the protective effect of drugs and reoxygenation remains challenging. To address this, we propose a novel workflow for the induction of gas-environment-based hypoxia in the HMC-3 cells along with 3-D imaging using laser-scanning-confocal microscopy. The imaging framework is complemented with a pipeline for quantifying time-lapse videos and cell-state classification. First, we show an imaging-based assessment of the in vitro model for hypoxia using a steep gradient in O2 with time. Second, we demonstrate the correlation between mitochondrial superoxide production and cytosolic calcium under acute hypoxia. We then test the efficacy of an L-type calcium channel blocker, compare the results with reoxygenation, and show that the blocker alleviates hypoxic conditions in terms of cytosolic calcium and viability within an acute window of one hour. Furthermore, we show that the drug reduces the expression of oxidative stress markers (HIF1A and OXR1) within the same time window. In the future, this model can also be used to investigate drug toxicity and efficacy under ischemic conditions.

MicroRNA-132 regulates gene expression programs involved in microglial homeostasis

microRNA-132 (miR-132), a known neuronal regulator, is one of the most robustly downregulated microRNAs (miRNAs) in the brain of Alzheimer's disease (AD) patients. Increasing miR-132 in AD mouse brain ameliorates amyloid and Tau pathologies, and also restores adult hippocampal neurogenesis and memory deficits. However, the functional pleiotropy of miRNAs requires in-depth analysis of the effects of miR-132 supplementation before it can be moved forward for AD therapy. We employ here miR-132 loss- and gain-of-function approaches using single-cell transcriptomics, proteomics, and in silico AGO-CLIP datasets to identify molecular pathways targeted by miR-132 in mouse hippocampus. We find that miR-132 modulation significantly affects the transition of microglia from a disease-associated to a homeostatic cell state. We confirm the regulatory role of miR-132 in shifting microglial cell states using human microglial cultures derived from induced pluripotent stem cells.

Extracellular vesicles released by hair follicle and adipose mesenchymal stromal cells induce comparable neuroprotective and anti-inflammatory effects in primary neuronal and microglial cultures

Background aimsDespite intensive research, to date, there is no effective treatment for neurodegenerative diseases. Among the different therapeutic approaches, recently, the use of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) has gained attention. MethodsIn the present work, we focused on medium/large extracellular vesicles (m/lEVs) derived from hair follicle–-derived (HF) MSCs, comparing their potential neuroprotective and anti-inflammatory effect against adipose tissue (AT)-MSC–derived m/lEVs. ResultsThe obtained m/lEVs were similar in size with comparable expression of surface protein markers. The neuroprotective effect of both HF-m/lEVs and AT-m/lEVs was statistically significant in dopaminergic primary cell cultures, increasing cell viability after the incubation with 6-hidroxydopamine neurotoxin. Moreover, the administration of HF-m/lEVs and AT-m/lEVs counteracted the lipopolysaccharide-induced inflammation in primary microglial cell cultures, decreasing the levels of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-1β. ConclusionsTaken together, HF-m/lEVs demonstrated comparable potential with that of AT-m/lEVs as multifaceted biopharmaceuticals for neurodegenerative disease treatment.

Microglial Priming in Bilirubin-Induced Neurotoxicity.

Neuroinflammation is a major contributor to bilirubin-induced neurotoxicity, which results in severe neurological deficits. Microglia are the primary immune cells in the brain, with M1 microglia promoting inflammatory injury and M2 microglia inhibiting neuroinflammation. Controlling microglial inflammation could be a promising therapeutic strategy for reducing bilirubin-induced neurotoxicity. Primary microglial cultures were prepared from 1-3-day-old rats. In the early stages of bilirubin treatment, pro-/anti-inflammatory (M1/M2) microglia mixed polarization was observed. In the late stages, bilirubin persistence induced dominant proinflammatory microglia, forming an inflammatory microenvironment and inducing iNOS expression as well as the release of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β. Simultaneously, nuclear factor-kappa B (NF-κB) was activated and translocated into the nucleus, upregulating inflammatory target genes. As well known, neuroinflammation can have an effect on N-methyl-D-aspartate receptor (NMDAR) expression or function, which is linked to cognition. Treatment with bilirubin-treated microglia-conditioned medium did affect the expression of IL-1β, NMDA receptor subunit 2A (NR2A), and NMDA receptor subunit 2B (NR2B) in neurons. However, VX-765 effectively reduces the levels of proinflammatory cytokines TNF-α, IL-6, and IL-1β, as well as the expressions of CD86, and increases the expressions of anti-inflammatory related Arg-1. A timely reduction in proinflammatory microglia could protect against bilirubin-induced neurotoxicity.

Aldo-Keto Reductase 1C15 Characterization and Protection in Ischemic Brain Injury.

Aldo-keto reductase (AKR) 1C15, a member of the AKR superfamily, was recently identified and cloned, and reported to alleviate oxidative stress in endothelial cells in rodent lungs. However, its expression and role in the brain and ischemic brain diseases have not been investigated. AKR1C15 expression was detected with real-time PCR. Mouse ischemic stroke and ischemic preconditioning (IPC) were established with middle cerebral artery occlusion (MCAO) for 1 h or 12 min, respectively. Recombinant AKR1C15 was administered intraperitoneally, and stroke outcome was evaluated with neurobehavioral tests and infarct volumes. Rat primary brain cell cultures were subjected to oxygen-glucose deprivation (OGD) to mimic ischemic injury. Cell survival or in vitro blood-brain barrier (BBB) permeability was measured, and nitric oxide (NO) release was detected. Immunostaining and Western blotting were used to evaluate oxidative-stress-related protein expression. AKR1C15 administration decreased the infarct volume and neurological deficits 2d post-stroke, and its early (1-h) administration after IPC abolished the protection of IPC against stroke. In rat primary brain cell cultures, AKR1C15 was most abundantly expressed in brain microvascular endothelial cells (BMVECs) and microglia. Its expression decreased upon OGD in most cell types except for BMVECs and microglia. In primary neuronal cultures, AKR1C15 treatment prevented OGD-induced cell death accompanied by decreased levels of 4-hydroxynonenal, 8-hydroxy-2'-deoxyguanosine, and heme oxygenase-1. In BMVEC cultures, AKR1C15 treatment protected against OGD-induced cell death and in vitro BBB leakage. In primary microglial cultures, AKR1C15 reduced the release of NO upon proinflammatory stimulation. Our results provide a characterization of the novel antioxidant AKR1C15 and demonstrate its protective role against ischemic injury, both in vivo and in vitro. AKR1C15 may be a promising agent for ischemic stroke treatment.

The human P2X7 receptor alters microglial morphology and cytokine secretion following immunomodulation.

Introduction: In recent years, purinergic signaling via the P2X7 receptor (P2X7R) on microglia has repeatedly been implicated in depression genesis. However, it remains unclear which role the human P2X7R (hP2X7R) plays in regulating both microglia morphology and cytokine secretion upon different environmental and immune stimuli, respectively. Methods: For this purpose, we used primary microglial cultures derived from a humanized microglia-specific conditional P2X7R knockout mouse line to emulate different gene-environment interactions between microglial hP2X7R and molecular proxies of psychosocial and pathogen-derived immune stimuli. Microglial cultures were subjected to treatments with the agonists 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP) and lipopolysaccharides (LPS) combined with specific P2X7R antagonists (JNJ-47965567, A-804598). Results: Morphotyping revealed overall high baseline activation due to the in vitro conditions. Both BzATP and LPS + BzATP treatment increased round/ameboid microglia and decreased polarized and ramified morphotypes. This effect was stronger in hP2X7R-proficient (CTRL) compared to knockout (KO) microglia. Aptly, we found antagonism with JNJ-4796556 and A-804598 to reduce round/ameboid microglia and increase complex morphologies only in CTRL but not KO microglia. Single cell shape descriptor analysis confirmed the morphotyping results. Compared to KO microglia, hP2X7R-targeted stimulation in CTRLs led to a more pronounced increase in microglial roundness and circularity along with an overall higher decrease in aspect ratio and shape complexity. JNJ-4796556 and A-804598, on the other hand, led to opposite dynamics. In KO microglia, similar trends were observed, yet the magnitude of responses was much smaller. Parallel assessment of 10 cytokines demonstrated the proinflammatory properties of hP2X7R. Following LPS + BzATP stimulation, IL-1β, IL-6, and TNFα levels were found to be higher and IL-4 levels lower in CTRL than in KO cultures. Vice versa, hP2X7R antagonists reduced proinflammatory cytokine levels and increased IL-4 secretion. Discussion: Taken together, our results help disentangle the complex function of microglial hP2X7R downstream of various immune stimuli. In addition, this is the first study in a humanized, microglia-specific in vitro model identifying a so far unknown potential link between microglial hP2X7R function and IL-27 levels.

Oxytocin attenuates microglial activation and restores social and non-social memory in APP/PS1 Alzheimer model mice

Alzheimer's disease (AD) is characterized by neurodegeneration, memory loss, and social withdrawal. Brain inflammation has emerged as a key pathogenic mechanism in AD. We hypothesized that oxytocin, a pro-social hypothalamic neuropeptide with anti-inflammatory properties, could have therapeutic actions in AD. Here, we investigated oxytocin expression in experimental models of AD, and evaluated the therapeutic potential of treatment with oxytocin. Amyloid-β peptide oligomers (AβOs) reduced oxytocin expression invitro and invivo, and treatment with oxytocin prevented microglial activation induced by AβOs in purified microglial cultures. Treatment of aged APP/PS1 mice, a mouse model of AD, with intranasal oxytocin attenuated microglial activation and favored deposition of Aβ in dense core plaques, a potentially neuroprotective mechanism. Remarkably, treatment withoxytocin alleviated social and non-social memory impairments in agedAPP/PS1 mice. Our findings point to oxytocin as a potential therapeutic target to reduce brain inflammation and correct memory deficits inAD.

Akebia saponin D acts via the PPAR-gamma pathway to reprogramme a pro-neurogenic microglia that can restore hippocampal neurogenesis in mice exposed to chronic mild stress.

Using drugs to modulate microglial function may be an effective way to treat disorders, such as depression, that involve impaired neurogenesis. Akebia saponin D (ASD) can cross the blood-brain barrier and exert anti-inflammatory and neuroprotective effects, so we wondered whether it might influence adult hippocampal neurogenesis to treat depression. We exposed C57BL/6 mice to chronic mild stress (CMS) as a model of depression and then gave them ASD intraperitoneally once daily for 3 weeks. We investigated the effects of ASD on microglial phenotype, hippocampal neurogenesis, and animal behavior. The potential role of the peroxisome proliferator-activated receptor-gamma (PPAR-γ) or BDNF-TrkB pathway in the pro-neurogenesis and anti-depressant of ASD was identified using there inhibitors GW9662 and K252a, respectively. The neurogenic effects of ASD-treated microglia were evaluated using conditioned culture methods. We found that CMS upregulated pro-inflammatory factors and inhibited hippocampal neurogenesis in dentate gyrus of mice, while inducing depressive-like behaviors. Dramatically, ASD (40 mg/kg) treatment reprogrammed an arginase (Arg)-1+ microglial phenotype in dentate gyrus, which increased brain-derived neurotrophic factor (BDNF) expression and restored the hippocampal neurogenesis, and partially ameliorated the depressive-like behaviors of the CMS-exposed mice. K252a or neurogenesis inhibitor blocked the pro-neurogenic, anti-depressant effects of ASD. Furthermore, ASD activated PPAR-γ in dentate gyrus of CMS mice as well as in primary microglial cultures treated with lipopolysaccharide. Blocking the PPAR-γ using GW9962 suppressed the ASD-reprogrammed Arg-1+ microglia and BDNF expression in dentate gyrus of CMS mice. Such blockade abolished the promoted effects of ASD-treated microglia on NSPC proliferation, survival, and neurogenesis. The pro-neurogenic and anti-depressant effects of ASD were blocked by GW9962. These results suggested that ASD acts via the PPAR-γ pathway to induce a pro-neurogenic microglia in dentate gyrus of CMS mice that can increase BDNF expression and promote NSPC proliferation, survival, and neurogenesis.

Voltage-Gated Proton Channel Hv1 Regulates Neuroinflammation and Dopaminergic Neurodegeneration in Parkinson's Disease Models.

Although the precise mechanisms for neurodegeneration in Parkinson's disease (PD) are unknown, evidence suggests that neuroinflammation is a critical factor in the pathogenic process. Here, we sought to determine whether the voltage-gated proton channel, Hv1 (HVCN1), which is expressed in microglia and regulates NADPH oxidase, is associated with dopaminergic neurodegeneration. We utilized data mining to evaluate the mRNA expression of HVCN1 in the brains of PD patients and controls and uncovered increased expression of the gene encoding Hv1, HVCN1, in the brains of PD patients compared to controls, specifically in male PD patients. In an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 4 × 16 mg/kg) mouse model of PD, Hvcn1 gene expression was increased 2-fold in the striatum. MPTP administration to wild-type (WT) mice resulted in a ~65% loss of tyrosine hydroxylase positive neurons (TH+) in the substantia nigra (SN), while a ~39% loss was observed in Hv1 knockout (KO) mice. Comparable neuroprotective effects of Hv1 deficiency were found in a repeated-dose LPS model. Neuroprotection was associated with decreased pro-inflammatory cytokine levels and pro-oxidant factors in both neurotoxicant animal models. These in vivo results were confirmed in primary microglial cultures, with LPS treatment increasing Hvcn1 mRNA levels and Hv1 KO microglia failing to exhibit the LPS-mediated inflammatory response. Conditioned media from Hv1 KO microglia treated with LPS resulted in an attenuated loss of cultured dopamine neuron cell viability compared to WT microglia. Taken together, these data suggest that Hv1 is upregulated and mediates microglial pro-inflammatory cytokine production in parkinsonian models and therefore represents a novel target for neuroprotection.

Abstract TP206: Scrnaseq Revealed Increased Microglial Expression Of Interferon-induced Transmembrane 3 (ifitm3) In Stroke And Other Inflammatory Conditions In The Brain.

Microglia (MG) play critical roles in modulating inflammation and interferon signaling is a driver of neuroinflammation in various neurodegenerative diseases. Recently, Ifitm3 (a member of the “interferon-inducible transmembrane” family) has been revealed as a molecular mediator between neuroinflammation and neurodegeneration. However, expression of Ifitm3 in MG under inflammatory conditions, is not well described. Methods: To define the expression of Ifitm3 following stroke, permanent distal middle cerebral artery occlusion was performed with aged mice (18-22 mos). Brains were isolated at post-stroke day (PSD) 14 for single-cell RNAseq (scRNAseq), and at PSD 3, 7, and 14 for immunostaining and qRT-PCR. Primary microglial culture was established to evaluate mRNA levels of Ifitm3 and other pro-inflammatory genes to cytokine treatment. Results: Our scRNAseq revealed that Ifitm3 is among the top 10 genes upregulated in MG following stroke in aged brains. To validate these findings, we utilized a stroke model in aged mice and primary cell culture. We found that ischemic injury causes an induction of Ifitm3 in the striatum and peri-infarcted area following stroke, with peak induction PSD 7 (n=3-5, p<0.01). We also examined the induction of other Ifitm gene mRNA in brains following stroke. Interestingly, among Ifitm family members, only Ifitm3 was markedly induced in the damaged cortex of PSD 3 brains (n=4-6). We found that Ifitm3 expression is induced in primary MG following treatment with proinflammatory mediators (TNFα/IFNγ) for 6 hours (n=5-6, p<0.05) or for 24 hours (n=7-8, p<0.01). TNFα/IFNγ treatment also induced increased expression of inflammasome component gene, Il1b and Casp1 . There was a close correlation between Ifitm3 and Casp1 expression, suggesting a potential of Ifitm3 in inflammasome activation. Conclusions: We demonstrated the profound induction of Ifitm3 in activated MG within the stroke brain in aged mice and in response to inflammatory factors in primary microglia culture. These studies identify Ifitm3 as a novel player involved in microglial modulation in response to a variety of inflammatory conditions. Future studies are warranted to determine if Ifitm3 deletion or inhibition can reduce neuroinflammation in the stroke brain.

Adalimumab combined with erythropoietin improves recovery from spinal cord injury by suppressing microglial M1 polarization-mediated neural inflammation and apoptosis.

Adalimumab (ADM), a humanized antibody against tumour necrosis factor (TNF), is widely applied in treating inflammatory and autoimmune diseases, but its usage in spinal cord injury (SCI) is rarely reported. Hence, this study aimed to explore the effect of ADM with or without erythropoietin (EPO) on microglial polarization, neuroinflammation, neural apoptosis, and functional recovery in SCI. Primary microglia were stimulated with lipopolysaccharide (LPS) and then treated with ADM, EPO, or ADM combined with EPO. Then, primary neurons were incubated in the microglial culture medium. SCI rats were established and then treated with ADM, EPO or ADM combined with EPO. ADM suppressed LPS-induced microglial M1 polarization, as reflected by downregulated iNOS and CD86 expression, and neuroinflammation, as reflected by decreased TNF-α, IL-1β, and IL-6 expression, in a dose-dependent manner. Moreover, ADM inhibited microglia-induced neural apoptosis, as reflected by TUNEL assay results and the expression of apoptotic markers (C-Caspase3 and Bcl2), in a dose-dependent manner. EPO monotherapy displayed an effect similar to that of ADM monotherapy. Furthermore, ADM combined with EPO therapy exhibited greater effects than either monotherapy in terms of inhibiting microglial M1 polarization, neuroinflammation, and neural apoptosis. In vivo experiments confirmed the findings of the in vitro experiments and showed that ADM combined with EPO improved SCI functional recovery and neural injury compared with monotherapy. ADM combined with EPO improves recovery from SCI by suppressing microglial M1 polarization-mediated neural inflammation and apoptosis.

The Expression of Cellular Prion Protein, PrPC, Favors pTau Propagation and Blocks NMDAR Signaling in Primary Cortical Neurons.

The N-methyl-D-aspartate receptor (NMDAR) is a target in current treatments for Alzheimer's disease (AD). The human prion protein (PrPC) has an important role in the pathophysiology of AD. We hypothesized that PrPC modulates NMDA signaling, thus being a process associated with Alzheimer's disease. NMDAR signaling was characterized in the absence or presence of PrPC in cAMP level determination, mitogen-activated protein kinase (MAPK) pathway and label-free assays in homologous and heterologous systems. Bioluminescence resonance energy transfer was used to detect the formation of NMDAR-PrPC complexes. AXIS™ Axon Isolation Devices were used to determine axonal transport of Tau and pTau proteins in cortical primary neurons in the absence or presence of PrPC. Finally, proximity ligation assays were used to quantify NMDA-PrPC complex formation in neuronal primary cultures isolated from APPSw/Ind transgenic mice, an Alzheimer's disease model expressing the Indiana and Swedish mutated version of the human amyloid precursor protein (APP). We discovered a direct interaction between the PrPC and the NMDAR and we found a negative modulation of NMDAR-mediated signaling due to the NMDAR-PrPC interaction. In mice primary neurons, we identified NMDA-PrPC complexes where PrPC was capable of blocking NMDAR-mediated effects. In addition, we observed how the presence of PrPC results in increased neurotoxicity and neuronal death. Similarly, in microglial primary cultures, we observed that PrPC caused a blockade of the NMDA receptor link to the MAPK signaling cascade. Interestingly, a significant increase in NMDA-PrPC macromolecular complexes was observed in cortical neurons isolated from the APPSw,Ind transgenic model of AD. PrPC can interact with the NMDAR, and the interaction results in the alteration of the receptor functionality. NMDAR-PrPC complexes are overexpressed in neurons of APPSw/Ind mouse brain. In addition, PrPC exacerbates axonal transport of Tau and pTau proteins.

Single-cell microglial transcriptomics during demyelination defines a microglial state required for lytic carcass clearance

BackgroundMicroglia regulate the response to injury and disease in the brain and spinal cord. In white matter diseases microglia may cause demyelination. However, how microglia respond and regulate demyelination is not fully understood.MethodsTo understand how microglia respond during demyelination, we fed mice cuprizone—a potent demyelinating agent—and assessed the dynamics of genetically fate-mapped microglia. We then used single-cell RNA sequencing to identify and track the microglial subpopulations that arise during demyelination. To understand how microglia contribute to the clearance of dead oligodendrocytes, we ablated microglia starting at the peak of cuprizone-induced cell death and used the viability dye acridine orange to monitor apoptotic and lytic cell morphologies after microglial ablation. Lastly, we treated serum-free primary microglial cultures to model distinct aspects of cuprizone-induced demyelination and assessed the response.ResultsThe cuprizone diet generated a robust microglial response by week 4 of the diet. Single-cell RNA sequencing at this time point revealed the presence of several cuprizone-associated microglia (CAM) clusters. These clusters expressed a transcriptomic signature indicative of cytokine regulation and reactive oxygen species production with altered lysosomal and metabolic changes consistent with ongoing phagocytosis. Using acridine orange to monitor apoptotic and lytic cell death after microglial ablation, we found that microglia preferentially phagocytose lytic carcasses. In culture, microglia exposed to lytic carcasses partially recapitulated the CAM state, suggesting that phagocytosis contributes to this distinct microglial state during cuprizone demyelination.ConclusionsMicroglia serve multiple roles during demyelination, yet their transcriptomic state resembles other neurodegenerative conditions. The phagocytosis of cellular debris is likely a universal cause for a common neurodegenerative microglial state.

INPP5D inhibition attenuates amyloid pathology through the regulation of microglial functions.

AbstractBackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, robust microgliosis, neuroinflammation, and neuronal loss. Genome‐wide association studies recently highlighted a prominent role for microglia in late‐onset AD (LOAD). Specifically, inositol polyphosphate‐5‐phosphatase (INPP5D) is selectively expressed in brain microglia and one of its common intronic variants (rs35349669; OR=1.08, 95%CI=1.06‐1.11) has been reported to be associated with increased risk of LOAD. INPP5D is linked to the triggering receptor expressed on myeloid cells 2 (TREM2) signaling, but little is known about the function of INPP5D in microglia and how INPP5D regulates TREM2‐related AD pathogenesis. Therefore, we aim to understand the role of INPP5D in microglia and AD pathology.MethodWe performed differential gene expression analysis to investigate INPP5D expression in LOAD and its association with plaque density using transcriptomic data from the Accelerating Medicines Partnership for Alzheimer’s Disease (AMP‐AD) cohort. We also performed quantitative real‐time PCR, immunoblotting, and immunofluorescence assays to assess INPP5D expression and microglial markers in the 5xFAD amyloid mouse model with INPP5D deficiency. Behavioral deficits were assessed by the Y‐maze assay in all animal cohorts at 4‐ and 6‐month of age. The primary microglial culture was used the evaluate the amyloid uptake ability of microglia.Result INPP5D gene expression was upregulated in LOAD and positively correlated with amyloid plaque density. Inpp5d expression increased as the disease progressed in the 5xFAD mice, and selectively in plaque‐associated microglia. Inpp5d deficiency mitigated the plaque burdens and neuropathology in the 5xFAD mice and further protected against behavioral deficits induced by amyloid pathology. Inpp5d deficiency modulates the microglial signaling, including Trem2 expression and Syk phosphorylation, and further upregulates the expression of the post‐synaptic marker. In the primary microglial culture, Inpp5d inhibition increased the uptake of fibril amyloid beta‐peptide.ConclusionOur findings show that INPP5D expression increases throughout the AD progression and is predominantly in plaque‐associated microglia. Importantly, INPP5D deficiency reduces Abeta pathology through the regulation of microglial functions, highlighting INPP5D as a potential therapeutic target.

High levels of HDAC expression correlate with microglial aging

ABSTRACT Background Cellular damage gradually accumulates with aging, promoting a time-dependent functional decline of the brain. Microglia play an essential regulatory role in maintaining cognitive activity by phagocytosing cell debris and apoptotic cells during neurogenesis. The activities of different histone deacetylases (HDACs) regulate microglial function during development and neurodegeneration. However, no studies have described the role of HDACs in microglia during physiological aging. Research design and methods HDAC and microglial marker levels were examined in microglial cells after inducing senescence in vitro and in mouse and human hippocampal biopsies in vivo, using quantitative real-time PCR. Publicly available datasets were used to determine HDAC expression in different brain areas during physiological aging. Results HDAC expression increased upon the induction of senescence with bleomycin or serial passage in microglial cultures. High levels of HDACs were detected in mice and aged human brain samples. Human hippocampal samples showed a positive correlation between the expression of HDAC1, 3, and 7 and microglial and senescence markers. HDAC1 and 3 levels are enriched in the purified aged microglial population. Conclusions Several HDACs, particularly HDAC1, are elevated in microglia upon senescence induction in vitro and with aging in vivo, and correlate with microglial and senescence biomarkers.