Microglial Surface Research Articles

Isolation and Whole-Cell Patch-Clamp Recording of Hippocampal Microglia from Adult Mice.

Microglia are resident immune cells in the brain that interact with neurons to maintain the homeostasis of the central nervous system (CNS). Studies show that the microglial surface expresses potassium channels that regulate microglial activation, while abnormalities in these potassium channels can lead to neural diseases. Currently, whole-cell patch-clamp recordings of microglia are mostly performed on cultured primary microglia from fetal or newborn mice due to difficulties in conducting electrophysiological evaluations on acutely isolated microglia. This study introduces an easy-to-follow protocol for isolating hippocampal microglia from adult mice and performing whole-cell patch-clamp recordings on the isolated cells. Briefly, the brain was removed from a mouse after decapitation, the hippocampus was dissected bilaterally, and microglia were isolated using an adult mouse brain dissociation kit. The microglia were then purified using a magnetic-activated cell sorting (MACS) method and seeded onto coverslips. Successful microglial isolation was confirmed by immunofluorescent staining with anti-CD11 and anti-Iba1 antibodies. A cover slip was placed in a recording chamber, and the whole-cell potassium currents of the acutely isolated microglia were recorded under voltage-clamp conditions.

An engineered cellular carrier delivers miR-138–5p to enhance mitophagy and protect hypoxic-injured neurons via the DNMT3A/Rhebl1 axis

Mitophagy influences the progression and prognosis of ischemic stroke (IS). However, whether DNA methylation in the brain is associated with altered mitophagy in hypoxia-injured neurons remains unclear. Here, miR-138–5p was found to be highly expressed in exosomes secreted by astrocytes stimulated with oxygen and glucose deprivation/re-oxygenation (OGD/R), which could influence the recovery of OGD/R-injured neurons through autophagy. Mechanistically, miR-138–5p promotes the stable expression of Ras homolog enriched in brain like 1(Rhebl1) through DNA-methyltransferase-3a (DNMT3A), thereby enhancing ubiquitin-dependent mitophagy to maintain mitochondrial homeostasis. Furthermore, we employed glycosylation engineering and bioorthogonal click reactions to load mirna onto the surface of microglia and deliver them to injured region utilising the inflammatory chemotactic properties of microglia to achieve drug-targeted delivery to the central nervous system (CNS). Our findings demonstrate miR-138–5p improves mitochondrial function in neurons through the miR-138–5p/DNMT3A/Rhebl1 axis. Additionally, our engineered cell vector-targeted delivery system could be promising for treating IS. Statement of significanceIn this study, we demonstrated that miR-138–5p in exosomes secreted by astrocytes under hypoxia plays a critical role in the treatment of hypoxia-injured neurons. And we find a new target of miR-138–5p, DNMT3A, which affects neuronal mitophagy and thus exerts a protective effect by regulating the methylation of Rbebl1. Furthermore, we have developed a carrier delivery system by combining miR-138–5p with the cell membrane of microglia and utilized the inflammatory chemotactic properties of microglia to deliver this system to the brain via intravenous injection. This groundbreaking study not only provides a novel therapeutic approach for ischemia-reperfusion treatment but also establishes a solid theoretical foundation for further research on targeted drug delivery for central nervous system diseases with promising clinical applications.

Role of microglia in diabetic neuropathic pain.

Approximately one-third of the patients with diabetes worldwide suffer from neuropathic pain, mainly categorized by spontaneous and stimulus-induced pain. Microglia are a class of immune effector cells residing in the central nervous system and play a pivotal role in diabetic neuropathic pain (DNP). Microglia specifically respond to hyperglycemia along with inflammatory cytokines and adenosine triphosphate produced during hyperglycemic damage to nerve fibers. Because of the presence of multiple receptors on the microglial surface, microglia are dynamically and highly responsive to their immediate environment. Following peripheral sensitization caused by hyperglycemia, microglia are affected by the cascade of inflammatory factors and other substances and respond accordingly, resulting in a change in their functional state for DNP pathogenesis. Inhibition of receptors such as P2X reporters, reducing cytokine expression levels in the microglial reactivity mechanisms, and inhibiting their intracellular signaling pathways can effectively alleviate DNP. A variety of drugs attenuate DNP by inhibiting the aforementioned processes induced by microglial reactivity. In this review, we summarize the pathological mechanisms by which microglia promote and maintain DNP, the drugs and therapeutic techniques available, and the latest advances in this field.

Research progress of microglial surface receptors in perioperative neurocognitive disorders

AbstractPerioperative neurocognitive disorder (PND) is a common complication in the perioperative period, which not only prolongs the hospitalization of patients, increases the cost of treatment, but even increases the postoperative mortality of patients, bringing a heavy burden to families and society. Mechanism exploration involves anesthesia and surgery that lead to microglial activation, promote the synthesis and secretion of inflammatory factors, cause an inflammatory cascade, aggravate nerve cell damage, and lead to cognitive dysfunction. It is believed that microglia‐mediated neuroinflammatory responses play a vital role in the formation of PND. Microglia surface receptors are essential mediators for microglia to receive external stimuli, regulate microglial functional status, and carry out intercellular signal transmission. Various microglial surface receptors trigger neuroinflammation, damage neurons, and participate in the development and progression of PND by activating microglia. In this study, the roles of immunoglobulin receptors, chemokine receptors, purinergic receptors, and pattern recognition receptors in microglia surface receptors in PND were reviewed, to provide a reference for the mechanism research, prevention, and treatment of PND.

The Pathogenesis in Alzheimer's Disease: TREM2 as a Potential Target.

Alzheimer's disease (AD) is ranked as the third-most expensive illness and sixth leading cause of mortality. It is associated with the deposition of extracellular amyloid-β (Aβ) in neural plaques (NPs), as well as intracellular hyperphosphorylated tau proteins that form neurofibrillary tangles (NFTs). As a new target in regulating neuroinflammation in AD, triggering receptor expressed on myeloid cells 2 (TREM2) is highly and exclusively expressed on the microglial surface. TREM2 interacts with adaptor protein DAP12 to initiate signal pathways that mainly dominant microglia phenotype and phagocytosis mobility. Furthermore, TREM2 gene mutations confer increased AD risk, and TREM2 deficiency exhibits more dendritic spine loss around neural plaques. Mechanisms for regulating TREM2 to alleviate AD has evolved as an area of AD research in recent years. Current medications targeting Aβ or tau proteins are unable to reverse AD progression. Emerging evidence implicating neuroinflammation may provide novel insights, as early microglia-related inflammation can be induced decades prior to the commencement of AD-related cognitive damage. Physical exercise can exert a neuroprotective effect over the course of AD progression. This review aims to (1) summarize the pathogenesis of AD and recent updates in the field, (2) assess the concept that AD cognitive impairment is closely correlated with microglia-related inflammation, and (3) review TREM2 functions and its role between exercise and AD, which is likely to be an ideal candidate target.

Glial cell response and microthrombosis in aneurysmal subarachnoid hemorrhage patients: An autopsy study.

Neuroinflammation and microthrombosis may be underlying mechanisms of brain injury after aneurysmal subarachnoid hemorrhage (aSAH), but they have not been studied in relation to each other. In postmortem brain tissue, we investigated neuroinflammation by studying the microglial and astrocyte response in the frontal cortex of 11 aSAH and 10 control patients. In a second study, we investigated the correlation between microthrombosis and microglia by studying the microglial surface area around vessels with and without microthrombosis in the frontal cortex and hippocampus of 8 other aSAH patients. In comparison with controls, we found increased numbers of microglia (mean ± SEM 50 ± 8 vs 20 ± 5 per 0.0026 mm³, p < 0.01), an increased surface area (%) of microglia (mean ± SEM 4.2 ± 0.6 vs 2.2 ± 0.4, p < 0.05), a higher intensity of the astrocytic intermediate filament protein glial fibrillary acidic protein (GFAP) (mean ± SEM 184 ± 28 vs 92 ± 23 arbitrary units, p < 0.05), and an increased GFAP surface area (%) (mean ± SEM 21.2 ± 2.6 vs 10.7 ± 2.1, p < 0.01) in aSAH tissue. Microglia surface area was approximately 40% larger around vessels with microthrombosis than those without microthrombosis (estimated marginal means [95% CI]; 6.1 [5.4-6.9] vs 4.3 [3.6-5.0], p < 0.001). Our results show that the microglial and astrocyte surface areas increased after aSAH and that microthrombosis and microglia are interrelated.

Chronic glial activation causes cholinergic cell loss in the basal forebrain during pathological aging

AbstractBackgroundNeuroinflammation, described by chronic glial reactivation lead to gradual neurodegeneration during pathological aging, contributing to disorders like Alzheimer’s disease (AD). Cholinergic cell loss in the basal forebrain (BF) including the medial septum (MS) is associated with AD; however the underlying mechanism is still unknown. Hence, this study investigated the effects of aging and chronic neuroinflammation on the MS cholinergic cell numbers and morphology.MethodAged, 12 and 24 months WT and GFAP‐IL6 mice (presenting IL‐6 overexpression under the GFAP promoter, resulting in chronic glial activation) were used. Immunohistochemistry combined with unbiased stereology and 3D morphology analysis were used to estimate the number and morphology of Iba1+ microglia and ChAT+ cholinergic cells in the MS. Transcriptomic analysis of the MS was performed using qPCR.ResultStereological estimation of MS microglia number displayed a significant 25% increase in GFAP‐IL6 mice compared to control WT (12M). A significant 30% increase was obtained for GFAP‐IL6 (24M) mice compared to control (24M). Furthermore, a significant 23% increase in microglia was described for GFAP‐IL6 (24M) compared to the GFAP‐IL6 (12M) mice. A significant 28% and 38% decrease in microglial 3D surface area, and volume respectively were observed between WT and GFAP‐IL (12M) mice, as well as 26% and 34% decrease respectively between WT (12M) and (24M), resembling reactive microglial morphology. Meanwhile, cholinergic cell number displayed a significant 31% decrease in GFAP‐IL6 (12M) mice compared to WT (12M). While a significant 30% decrease was displayed in GFAP‐IL6 (24M) mice compared to WT (24M) mice. There was a tendency for the cholinergic cell number to decrease with ageing among control and GFAP‐IL6 cohorts. Cholinergic cell morphology displayed significant 58% and 67% reduction in the 3D dendritic field surface area, and volume respectively between WT (12M) and GFAP‐IL (12M) mice, as well as 41% and 46% decrease respectively between WT (12M) and (24M), displaying patterns like neuronal degeneration.ConclusionThese findings demonstrate a significant impact of both aging and chronic glial activation alone on cholinergic cell number and morphology in the MS, resembling the cholinergic cell loss in AD, which can exacerbate throughout ageing leading to neurodegenerative impairments.

Potential role of the hydroxyl carboxylic acid receptor type 2 (HCAR2) in microglia pathophysiology: A possible cross-talk with C-X-C chemokine receptor 1 (CXCR1)

Following insults or injury, microglia cells are activated contributing to the cytotoxic response or by promoting an immune-mediated damage resolution. Microglia cells express HCA2R, a hydroxy carboxylic acid (HCA) receptor, which has been shown to mediate neuroprotective and anti-inflammatory effects.In this study we found that HCAR2 expression levels were increased in cultured rat microglia cells after Lipopolysaccharide (LPS) exposure. In a similar fashion, the treatment with MK 1903, a potent full agonist of HCAR2, increased the receptor protein levels. Moreover, HCAR2 stimulation prevented i) cells viability ii) morphological activation iii) pro/anti-inflammatory mediators production in LPS-treated cells. Likewise, HCAR2 stimulation reduced the proinflammatory mediators mRNA expression induced by neuronal chemokine fractalkine (FKN), a neuronal derived chemokine activating its unique receptor, chemokine receptor 1 (CX3CR1) on microglia surface. Interestingly, electrophysiological recordings in vivo revealed that MK1903 was able to prevent the increase of the nociceptive neurons (NS) firing activity mediated by the spinal FKN application in healthy rats.Collectively, our data demonstrate that HCAR2 is functionally expressed in microglia, by showing its capability to shift microglia toward an anti-inflammatory phenotype. Moreover, we indicated the contribute of HCAR2 in the FKN signaling and suggested a possible HCAR2/CX3CR1 functional interaction.This study paves the way for further investigations aimed at understanding the role HCAR2 as potential target in neuroinflammation-based CNS disorders.This article is part of the Special Issue on “The receptor-receptor interaction as a new target for therapy”.

P2Y6 receptor-dependent microglial phagocytosis of synapses mediates synaptic and memory loss in aging.

Aging causes loss of brain synapses and memory, and microglial phagocytosis of synapses may contribute to this loss. Stressed neurons can release the nucleotide UTP, which is rapidly converted into UDP, that in turn activates the P2Y6 receptor (P2Y6 R) on the surface of microglia, inducing microglial phagocytosis of neurons. However, whether the activation of P2Y6 R affects microglial phagocytosis of synapses is unknown. We show here that inactivation of P2Y6 R decreases microglial phagocytosis of isolated synapses (synaptosomes) and synaptic loss in neuronal-glial co-cultures. In vivo, wild-type mice aged from 4 to 17 months exhibited reduced synaptic density in cortical and hippocampal regions, which correlated with increased internalization of synaptic material within microglia. However, this aging-induced synaptic loss and internalization were absent in P2Y6 R knockout mice, and these mice also lacked any aging-induced memory loss. Thus, P2Y6 R appears to mediate aging-induced loss of synapses and memory by increasing microglial phagocytosis of synapses. Consequently, blocking P2Y6 R has the potential to prevent age-associated memory impairment.

Continued P2X7 activation leads to mitochondrial fission and compromising microglial phagocytosis after subarachnoid haemorrhage.

Subarachnoid haemorrhage (SAH) has a high rate of disability and mortality. Extremely damaging molecules, including adenosine triphosphate (ATP), are released from extravasated red blood cells and nerve cells, which activate microglia and induce sterile tissue injury and organ dysfunction. P2X purinoceptor 7 (P2X7) is one of the most important purine receptors on the microglial surface and is involved in the proinflammatory activation of microglia. While P2X7 can also affect microglial phagocytosis, the mechanism is not clear. Here, we demonstrated that microglial phagocytosis is progressively impaired under continued BzATP exposure and P2X7 activation. Furthermore, we found that P2X7 activation leads to increased intracellular Ca2+ levels and activates Calcineurin, which dephosphorylates dynamin-related protein 1 (DRP1) S637. The dephosphorylation of DRP1 at S637 leads to increased mitochondrial fission and decreased mitochondrial function, which may be responsible for the decreased microglial phagocytosis. Finally, we pharmacologically inhibited P2X7 activation in mice, which resulted in rescue of mitochondrial function and decreased microglial proliferation, but improved phagocytosis after SAH. Our study confirmed that P2X7 activation after SAH leads to the impairment of microglial phagocytosis through mitochondrial fission and verified that P2X7 inhibition restores microglial phagocytosis both in vitro and in vivo.

Microglia are SYK of Aβ and cell debris

During neurodegenerative disease, resident CNS macrophages termed "microglia" assume a neuroprotective role and engulf toxic protein aggregates and cell debris. In this issue of Cell, two groups independently show how spleen tyrosine kinase (SYK) acts downstream of microglial surface receptors to propagate this neuroprotective program invivo.

TREM2-induced activation of microglia contributes to synaptic integrity in cognitively intact aged individuals with Alzheimer's neuropathology.

The existence of individuals who remain cognitively intact despite presenting histopathological signs of Alzheimer's disease (AD), here referred to as "Nondemented with AD neuropathology" (NDAN), suggests that some mechanisms are triggered to resist cognitive impairment. Exposed phosphatidylserine (ePS) represents a neuronal "eat-me" signal involved in microglial-mediated phagocytosis of damaged synapses. A possible mediator of this process is TREM2, a microglial surface receptor activated by ligands including PS. Based on TREM2 role in the scavenging function of microglia, we hypothesize that an efficient microglial phagocytosis of damaged synapses underlies synaptic resilience in NDAN, thus protecting from memory deficits. Using immunofluorescence microscopy, we performed a comparative study of human post-mortem frontal cortices of aged-matched, AD and NDAN individuals. We studied the distribution of activated microglia (IBA1, IBA1+ /CD68+ cells) and phagocytic microglia-related proteins (TREM2, DAP12), demonstrating higher microglial activation and TREM2 expression in NDAN versus AD. A study of the preservation of synapses around plaques, assessed using MAP2 and βIII tubulin as dendritic and axonal markers, respectively, and PSD95 as a postsynaptic marker, revealed preserved axonal/dendritic structure around plaques in NDAN versus AD. Moreover, high levels of PSD95 around NDAN plaques and the colocalization of PSD95 with CD68 indicated a prompt removal of damaged synapses by phagocytic microglia. Furthermore, Annexin V assay on aged-matched, AD and NDAN individuals synaptosomes revealed increased levels of ePS in NDAN, confirming damaged synapses engulfment. Our results suggest a higher efficiency of TREM2-induced phagocytic microglia in removing damaged synapses, underlying synaptic resilience in NDAN individuals.

Time dependent analysis of rat microglial surface markers in traumatic brain injury reveals dynamics of distinct cell subpopulations

Traumatic brain injury (TBI) results in a cascade of cellular responses, which produce neuroinflammation, partly due to the activation of microglia. Accurate identification of microglial populations is key to understanding therapeutic approaches that modify microglial responses to TBI and improve long-term outcome measures. Notably, previous studies often utilized an outdated convention to describe microglial phenotypes. We conducted a temporal analysis of the response to controlled cortical impact (CCI) in rat microglia between ipsilateral and contralateral hemispheres across seven time points, identified microglia through expression of activation markers including CD45, CD11b/c, and p2y12 receptor and evaluated their activation state using additional markers of CD32, CD86, RT1B, CD200R, and CD163. We identified unique sub-populations of microglial cells that express individual or combination of activation markers across time points. We further portrayed how the size of these sub-populations changes through time, corresponding to stages in TBI response. We described longitudinal changes in microglial population after CCI in two different locations using activation markers, showing clear separation into cellular sub-populations that feature different temporal patterns of markers after injury. These changes may aid in understanding the symptomatic progression following TBI and help define microglial subpopulations beyond the outdated M1/M2 paradigm.

Dynamics of neuroinflammation in Alzheimer's disease

Findings from genome-wide association studies have implicated genetic variants that are highly expressed in microglia in the pathogenesis of Alzheimer's disease. Among these, a rare missense mutation in TREM2 increases the risk of Alzheimer's disease nearly three times, on par with the effect of APOɛ4, the most common Alzheimer's disease risk allele.1 TREM2 encodes a microglial surface receptor, the activation of which promotes microglial activation, survival, and phagocytosis. Cleavage of this surface receptor by ADAM10 and ADAM17 releases soluble TREM2, which is detectable in CSF and reflects microglial signalling.

Research Progress on the Role of Microglia Membrane Proteins or Receptors in Neuroinflammation and Degeneration.

Microglia are intrinsic immune cells of the central nervous system and play a dual role (pro-inflammatory and anti-inflammatory) in the homeostasis of the nervous system. Neuroinflammation mediated by microglia serves as an important stage of ischemic hypoxic brain injury, cerebral hemorrhage disease, neurodegeneration and neurotumor of the nervous system and is present through the whole course of these diseases. Microglial membrane protein or receptor is the basis of mediating microglia to play the inflammatory role and they have been found to be upregulated by recognizing associated ligands or sensing changes in the nervous system microenvironment. They can then allosterically activate the downstream signal transduction and produce a series of complex cascade reactions that can activate microglia, promote microglia chemotactic migration and stimulate the release of proinflammatory factor such as TNF-α, IL-β to effectively damage the nervous system and cause apoptosis of neurons. In this paper, several representative membrane proteins or receptors present on the surface of microglia are systematically reviewed and information about their structures, functions and specific roles in one or more neurological diseases. And on this basis, some prospects for the treatment of novel coronavirus neurological complications are presented.

TREM2-induced activation of microglia contributes to synaptic resilience in non-demented individuals with Alzheimer's neuropathology.

The existence of individuals who remain cognitively intact despite presenting histopathological signs of Alzheimer's disease (AD), here referred to as "Non demented with AD neuropathology" (NDAN), suggests that some unknown mechanisms are triggered to resist cognitive impairment. Synaptic dysfunction has been identified as one of the major AD causes and it is established that microglia, attracted to plaques, engulf damaged synapses. In this context, exposed phosphatidylserine (ePS) represents a neuronal "eat-me" signal involved in microglial-mediated phagocytosis of damaged synapses. A possible candidate mediator of this process is represented by TREM2, a microglial surface receptor activated by ligands including PS. Based on TREM2 role in the scavenging function of microglia, we hypothesize that an efficient microglial phagocytosis of damaged synapses underlies synaptic resilience in NDAN, thus protecting from memory deficits. Using immunofluorescence microscopy, a comparative study of human post-mortem frontal cortices of aged-matched individuals, AD and NDAN individuals has been performed. The distribution of activated microglia (IBA1 and IBA1/CD68 positive cells) and the expression of phagocytic microglia-related proteins (TREM2 and DAP12) were evaluated. To test the efficacy of microglia in removing debris and damaged synapses, preservation of synapses around plaques was assessed using MAP2 and tubulin βIII as dendritic and axonal markers respectively, and PSD95 as a post synaptic marker. Furthermore, taking advantage of flow cytometry techniques, a study of the ePS using Annexin V assay has been performed. Immunofluorescence analyses indicate higher microglial activation and TREM2 expression in NDAN individuals, as well as preserved axonal and dendritic structure around plaques vs. AD. High levels of PSD95 around NDAN plaques and the colocalization of PSD95 and CD68 may suggest a prompt removal of damaged synapses by hyperactive phagocytic microglia. Annexin V assay on synaptosomes isolated from aged-matched, AD and NDAN individuals indicates changes in the ePS in NDAN individuals, confirming the engulfment of damaged synapses. Our results suggest a higher efficiency of TREM2-induced phagocytic microglia in removing damaged synapses, underlying synaptic resilience in NDAN individuals.

Discovery of Active Ingredients Targeted TREM2 by SPR Biosensor-UPLC/MS Recognition System, and Investigating the Mechanism of Anti-Neuroinflammatory Activity on the Lignin-Amides from Datura metel Seeds.

As a new target protein for Alzheimer’s disease (AD), the triggering receptor expressed on myeloid Cells 2 (TREM2) was expressed on the surface of microglia, which was shown to regulate neuroinflammation, be associated with a variety of neuropathologic, and regarded as a potential indicator for monitoring AD. In this study, a novel recognition system based on surface plasmon resonance (SPR) for the TREM2 target spot was established coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-MS), in order to screen the active ingredients targeting TREM2 from Datura metel seeds. The results showed that four lignan-amides were discovered as candidate compounds by SPR biosensor-UPLC/MS recognition analysis. According to the guidance of the active ingredients discovered by the system, the lignin-amides from Datura metel seeds (LDS) were preliminarily identified as containing 27 lignan-amides, which were enriched compositions by the HP-20 of Datura metel seeds. Meanwhile, the anti-inflammatory activity of LDS was evaluated in BV2 microglia induced by LPS. Our experimental results demonstrated that LDS could reduce NO release in LPS-treated BV2 microglia cells and significantly reduce the expression of the proteins of inducible Nitric Oxide Synthase (iNOS), cyclooxygenase 2 (COX-2), microtubule-associated protein tau (Tau), and ionized calcium-binding adapter molecule 1 (IBA-1). Accordingly, LDS might increase the expression of TREM2/DNAX-activating protein of 12 kDa (DAP12) and suppress the Toll-like receptor SX4 (TLR4) pathway and Recombinant NLR Family, Pyrin Domain Containing Protein 3 (NLRP3)/cysteinyl aspartate specific proteinase-1 (Caspase-1) inflammasome expression by LDS in LPS-induced BV2 microglial cells. Then, the inhibitory release of inflammatory factors Interleukin 1 beta (IL-1β), Interleukin 6 (IL-6), and Tumor necrosis factor-alpha (TNFα) inflammatory cytokines were detected to inhibit neuroinflammatory responses. The present results propose that LDS has potential as an anti-neuroinflammatory agent against microglia-mediated neuroinflammatory disorders.

Insights from molecular docking and molecular dynamics on the potential of vitexin as an antagonist candidate against lipopolysaccharide (LPS) for microglial activation in neuroinflammation

BackgroundNeuroinflammation has been identified to be the key player in most neurodegenerative diseases. If neuroinflammation is left to be unresolved, chronic neuroinflammation will be establish. Such situation is due to the overly-activated microglia which have the tendency to secrete an abundance amount of pro-inflammatory cytokines into the neuron microenvironment. The abundance of pro-inflammatory cytokines will later cause toxic and death to neurons. Toll-like receptor 4 (TLR4)/MD-2 complex found on the cell surface of microglia is responsible for the attachment of LPS and activation of nuclear factor-κB (NF-κB) downstream signalling pathway. Albeit vitexin has been shown to possess anti-inflammatory property, however, little is known on its ability to bind at the binding site of TLR4/MD-2 complex of microglia as well as to be an antagonist for LPS.ResultsThe present study reveals that both vitexin and donepezil are able to bind at the close proximity of LPS binding site located at the TLR4/MD-2 complex with the binding energy of − 4.35 and − 9.14 kcal/mol, respectively. During molecular dynamic simulations, both vitexin and donepezil formed stable complex with TLR4/MD-2 throughout the 100 ns time length with the root mean square deviation (RMSD) values of 2.5 Å and 4.0 Å, respectively. The root mean square fluctuation (RMSF) reveals that both compounds are stable. Interestingly, the radius of gyration (rGyr) for donepezil shows notable fluctuations when compare with vitexin. The MM-GBSA results showed that vitexin has higher binding energy in comparison with donepezil.ConclusionsTaken together, the findings suggest that vitexin is able to bind at the binding site of TLR4/MD-2 complex with more stability than donepezil throughout the course of 100 ns simulation. Hence, vitexin has the potential to be an antagonist candidate for LPS.

Validation of Induced Microglia-Like Cells (iMG Cells) for Future Studies of Brain Diseases.

Microglia are the primary resident immune cells of the central nervous system that maintain physiological homeostasis in the brain and contribute to the pathogenesis of many psychiatric disorders and neurodegenerative diseases. Due to the lack of appropriate human cellular models, it is difficult to study the basic pathophysiological processes linking microglia to brain diseases. In this study, we adopted a microglia-like cellular model derived from peripheral blood monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-34 (IL-34). We characterized and validated this in vitro cellular model by morphology, immunocytochemistry, gene expression profiles, and functional study. Our results indicated that the iMG cells developed typical microglial ramified morphology, expressed microglial specific surface markers (P2RY12 and TMEM119), and possessed phagocytic activity. Principal component analyses and multidimensional scaling analyses of RNA-seq data showed that iMG cells were distinct from monocytes and induced macrophages (iMacs) but clustered closer to human microglia and hiPSC-induced microglia. Heatmap analyses also found that iMG cells, but not monocytes, were closely clustered with human primary microglia. Further pathway and relative expression analysis indicated that unique genes from iMG cells were involved in the regulation of the complement system, especially in the synapse and ion transport. Overall, our data demonstrated that the iMG model mimicked many features of the brain resident microglia, highlighting its utility in the study of microglial function in many brain diseases, such as schizophrenia and Alzheimer's disease (AD).

MEK1/2 activity modulates TREM2 cell surface recruitment

Rare sequence variants in the microglial cell surface receptor TREM2 have been shown to increase the risk for Alzheimer’s disease (AD). Disease-linked TREM2 mutations seem to confer a partial loss of function, and increasing TREM2 cell surface expression and thereby its function(s) might have therapeutic benefit in AD. However, druggable targets that could modulate microglial TREM2 surface expression are not known. To identify such targets, we conducted a screen of small molecule compounds with known pharmacology using human myeloid cells, searching for those that enhance TREM2 protein at the cell surface. Inhibitors of the kinases MEK1/2 displayed the strongest and most consistent increases in cell surface TREM2 protein, identifying a previously unreported pathway for TREM2 regulation. Unexpectedly, inhibitors of the downstream effector ERK kinases did not have the same effect, suggesting that noncanonical MEK signaling regulates TREM2 trafficking. In addition, siRNA knockdown experiments confirmed that decreased MEK1 and MEK2 were required for this recruitment. In iPSC-derived microglia, MEK inhibition increased cell surface TREM2 only modestly, so various cytokines were used to alter iPSC microglia phenotype, making cells more sensitive to MEK inhibitor-induced TREM2 recruitment. Of those tested, only IFN-gamma priming prior to MEK inhibitor treatment resulted in greater TREM2 recruitment. These data identify the first known mechanisms for increasing surface TREM2 protein and TREM2-regulated function in human myeloid cells and are the first to show a role for MEK1/MEK2 signaling in TREM2 activity.