Microglial Proteins Research Articles

Temporal changes in the microglial proteome of male and female mice after a diffuse brain injury using label-free quantitative proteomics.

Traumatic brain injury (TBI) triggers neuroinflammatory cascades mediated by microglia, which promotes tissue repair in the short-term. These cascades may exacerbate TBI-induced tissue damage and symptoms in the months to years post-injury. However, the progression of the microglial function across time post-injury and whether this differs between biological sexes is not well understood. In this study, we examined the microglial proteome at 3-, 7-, or 28-days after a midline fluid percussion injury (mFPI) in male and female mice using label-free quantitative proteomics. Data are available via ProteomeXchange with identifier PXD033628. We identified a reduction in microglial proteins involved with clearance of neuronal debris via phagocytosis at 3- and 7-days post-injury. At 28 days post-injury, pro-inflammatory proteins were decreased and anti-inflammatory proteins were increased in microglia. These results indicate a reduction in microglial clearance of neuronal debris in the days post-injury with a shift to anti-inflammatory function by 28 days following TBI. The changes in the microglial proteome that occurred across time post-injury did not differ between biological sexes. However, we did identify an increase in microglial proteins related to pro-inflammation and phagocytosis as well as insulin and estrogen signaling in males compared with female mice that occurred with or without a brain injury. Although the microglial response was similar between males and females up to 28 days following TBI, biological sex differences in the microglial proteome, regardless of TBI, has implications for the efficacy of treatment strategies targeting the microglial response post-injury.

Microglial proteins Iba1 and TSPO (PBR) are differentially altered in the temporal cortex and cerebellum according to Braak stage

AbstractBackgroundMicroglia are the resident immune cells in the brain and contribute to the neuropathology of Alzheimer’s disease (AD) by mechanisms not completely known. Iba1 is a microglial marker associated with homeostatic and motility functions. Whereas TSPO (PBR) is a marker of the mitochondrial membrane in microglia, macrophages and endothelial cells and is proposed to be associated with the activated subtype of microglia.MethodTo assess both Iba1 and PBR, we retrieved human temporal cortex and cerebellum samples from 57 cases at different stages of the disease, based on the Braak staging (Group 0‐II n=19, Group III‐IV n=20, Group V‐VI n=18), using immunohistochemical analysis. Additionally, a V‐Plex Mesoscale Discovery (MSD) analysis was performed to assess the inflammatory environment.ResultThere was a significant increase in PBR percentage load (P<0.001) across the Braak stages in the temporal cortex (average load 0.93%) but not in the cerebellum (average load 0.76%). Conversely, there was a significant increase in cerebellar Iba1 load (average load 0.96%, P=0.012) but not in the temporal cortex (average load 1.12%). Furthermore, it was observed that Iba1 forms clusters, putatively around amyloid plaques, whereas this phenomenon was not seen with the PBR marker. The only significant inflammatory molecule was the cytokine IL15 in the temporal cortex (P=0.024), which increased across all Braak stages. However, when the MSD data was correlated with the Iba1 and PBR markers it showed that GM‐CSF was significantly correlated with PBR in the temporal cortex (P<0.0099) and IL‐16 was significantly correlated with Iba1 in the cerebellum (P<0.0012).ConclusionThis emphasises the outcome that, in AD, PBR may contribute to inflammation in the temporal cortex and Iba1 may be correlated to inflammation in the cerebellum. The findings indicate that TSPO relates to microglial activation in the brain which is Braak‐stage specific and consequently supports the use of TSPO ligands for positron emission tomography (PET) imaging of Alzheimer’s disease‐relevant microglial activity in vivo. Moreover, the absence of an increase in PBR load in the cerebellum could qualify this structure as a reference region for PET quantification.

Icaritin inhibits neuroinflammation in a rat cerebral ischemia model by regulating microglial polarization through the GPER–ERK–NF-κB signaling pathway

BackgroundActivated microglia play a key role in initiating the inflammatory cascade following ischemic stroke and exert proinflammatory or anti-inflammatory effects, depending on whether they are polarized toward the M1 or M2 phenotype. The present study investigated the regulatory effect of icaritin (ICT) on microglial polarization in rats after cerebral ischemia/reperfusion injury (CI/RI) and explored the possible anti-inflammatory mechanisms of ICT.MethodsA rat model of transient middle cerebral artery occlusion (tMCAO) was established. Following treatment with ICT, a G protein-coupled estrogen receptor (GPER) inhibitor or an extracellular signal-regulated kinase (ERK) inhibitor, the Garcia scale and rotarod test were used to assess neurological and locomotor function. 2,3,5-Triphenyltetrazolium chloride (TTC) and Fluoro-Jade C (FJC) staining were used to evaluate the infarct volume and neuronal death. The levels of inflammatory factors in the ischemic penumbra were evaluated using enzyme-linked immunosorbent assays (ELISAs). In addition, western blotting, immunofluorescence staining and quantitative PCR (qPCR) were performed to measure the expression levels of markers of different microglial phenotypes and proteins related to the GPER–ERK–nuclear factor kappa B (NF-κB) signaling pathway.ResultsICT treatment significantly decreased the cerebral infarct volume, brain water content and fluorescence intensity of FJC; improved the Garcia score; increased the latency to fall and rotation speed in the rotarod test; decreased the levels of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), Iba1, CD40, CD68 and p-P65-NF-κB; and increased the levels of CD206 and p-ERK. U0126 (an inhibitor of ERK) and G15 (a selective antagonist of GPER) antagonized these effects.ConclusionsThese findings indicate that ICT plays roles in inhibiting the inflammatory response and achieving neuroprotection by regulating GPER–ERK–NF-κB signaling and then inhibiting microglial activation and M1 polarization while promoting M2 polarization, which provides a new therapeutic for against cerebral ischemic stroke.Graphical

Spatial Analysis of Neural Cell Proteomic Profiles Following Ischemic Stroke in Mice Using High-Plex Digital Spatial Profiling

Stroke is ranked as the fifth leading cause of death and the leading cause of adult disability in the USA. The progression of neuronal damage after stroke is recognized to be a complex integration of glia, neurons, and the surrounding extracellular matrix, therefore potential treatments must target the detrimental effects created by these interactions. In this study, we examined the spatial cellular and neuroinflammatory mechanisms occurring early after ischemic stroke utilizing Nanostring Digital Spatial Profiling (DSP) technology. Male C57bl/6 mice were subjected to photothrombotic middle cerebral artery occlusion (MCAO) and sacrificed at 3 days post-ischemia. Spatial distinction of the ipsilateral hemisphere was studied according to the regions of interest: the ischemic core, peri-infarct tissues, and peri-infarct normal tissue (PiNT) in comparison to the contralateral hemisphere. We demonstrated that the ipsilateral hemisphere initiates distinct spatial regulatory proteomic profiles with DSP technology that can be identified consistently with the immunohistochemical markers, FJB, GFAP, and Iba-1. The core border profile demonstrated an induction of neuronal death, apoptosis, autophagy, immunoreactivity, and early degenerative proteins. Most notably, the core border resulted in a decrease of the neuronal proteins Map2 and NeuN; an increase in the autophagy proteins BAG3 and CTSD; an increase in the microglial and peripheral immune invasion proteins Iba1, CD45, CD11b, and CD39; and an increase in the neurodegenerative proteins BACE1, APP, amyloid β 1–42, ApoE, and hyperphosphorylated tau protein S-199. The peri-infarct region demonstrated increased astrocytic, immunoreactivity, apoptotic, and neurodegenerative proteomic profiles, with an increase in BAG3, GFAP, and hyperphosphorylated tau protein S-199. The PiNT region displayed minimal changes compared to the contralateral cortex with only an increase in GFAP. In this study, we showed that mechanisms known to be associated with stroke, such as apoptosis and inflammation, occur in distinct spatial domains of the injured brain following ischemia. We also demonstrated the dysregulation of specific autophagic pathways that may lead to neurodegeneration in peri-infarct brain tissues. Taken together, these data suggest that identifying post-ischemic mechanisms occurring in a spatiotemporal manner may lead to more precise targets for successful therapeutic interventions to treat stroke.

Retrovirus-derived RTL5 and RTL6 genes are novel constituents of the innate immune system in the eutherian brain.

Retrotransposon Gag-like 5 [RTL5, also known as sushi-ichi-related retrotransposon homolog 8 (SIRH8)] and RTL6 (also known as SIRH3) are eutherian-specific genes presumably derived from a retrovirus and phylogenetically related to each other. They, respectively, encode a strongly acidic and extremely basic protein, and are well conserved among the eutherians. Here, we report that RTL5 and RTL6 are microglial genes with roles in the front line of innate brain immune response. Venus and mCherry knock-in mice exhibited expression of RTL5-mCherry and RTL6-Venus fusion proteins in microglia and appeared as extracellular dots and granules in the central nervous system. These proteins display a rapid response to pathogens such as lipopolysaccharide (LPS), double-stranded (ds) RNA analog and non-methylated CpG DNA, acting both cooperatively and/or independently. Experiments using Rtl6 or Rtl5 knockout mice provided additional evidence that RTL6 and RTL5 act as factors against LPS and dsRNA, respectively, in the brain, providing the first demonstration that retrovirus-derived genes play a role in the eutherian innate immune system. Finally, we propose a model emphasizing the importance of extra-embryonic tissues as the origin site of retrovirus-derived genes. This article has an associated 'The people behind the papers' interview.

Complement C1q-dependent excitatory and inhibitory synapse elimination by astrocytes and microglia in Alzheimer’s disease mouse models

Microglia and complement can mediate neurodegeneration in Alzheimer’s disease (AD). By integrative multi-omics analysis, here we show that astrocytic and microglial proteins are increased in TauP301S synapse fractions with age and in a C1q-dependent manner. In addition to microglia, we identified that astrocytes contribute substantially to synapse elimination in TauP301S hippocampi. Notably, we found relatively more excitatory synapse marker proteins in astrocytic lysosomes, whereas microglial lysosomes contained more inhibitory synapse material. C1q deletion reduced astrocyte–synapse association and decreased astrocytic and microglial synapses engulfment in TauP301S mice and rescued synapse density. Finally, in an AD mouse model that combines β-amyloid and Tau pathologies, deletion of the AD risk gene Trem2 impaired microglial phagocytosis of synapses, whereas astrocytes engulfed more inhibitory synapses around plaques. Together, our data reveal that astrocytes contact and eliminate synapses in a C1q-dependent manner and thereby contribute to pathological synapse loss and that astrocytic phagocytosis can compensate for microglial dysfunction.

Perinatal Morphine Exposure Leads to Sex-Dependent Executive Function Deficits and Microglial Changes in Mice.

Children exposed prenatally to opioids are at an increased risk for behavioral problems and executive function deficits. The prefrontal cortex (PFC) and amygdala (AMG) regulate executive function and social behavior and are sensitive to opioids prenatally. Opioids can bind to toll-like receptor 4 (TLR4) to activate microglia, which may be developmentally important for synaptic pruning. Therefore, we tested the effects of perinatal morphine exposure on executive function and social behavior in male and female mouse offspring, along with microglial-related and synaptic-related outcomes. Dams were injected once daily subcutaneously with saline (n = 8) or morphine (MO; 10 mg/kg; n = 12) throughout pregestation, gestation, and lactation until offspring were weaned on postnatal day 21 (P21). Male MO offspring had impairments in attention and accuracy in the five-choice serial reaction time task, while female MO offspring were less affected. Targeted gene expression analysis at P21 in the PFC identified alterations in microglial-related and TLR4-related genes, while immunohistochemical analysis in adult brains indicated decreased microglial Iba1 and phagocytic CD68 proteins in the PFC and AMG in males, but females had an increase. Further, both male and female MO offspring had increased social preference. Overall, these data demonstrate male vulnerability to executive function deficits in response to perinatal opioid exposure and evidence for disruptions in neuron–microglial signaling.

The expression and phosphorylation of SMAD3 protein in microglia and astrocytes of the rat hippocampus after transient global cerebral ischemia

SMAD3 protein transduces signals from TGF-β and activins. In vitro studies have shown that SMAD3 plays an important role in regulating of micoglia and astrocytic function. However, there is little information on the association between SMAD3 signaling and the pathophysiology of the glial cells in the post-ischemic hippocampus. In this study, we examined the time-course changes in the expression and phosphorylation of SMAD3 in the rat hippocampus using a rat model of global cerebral ischemia. Most pyramidal neuronal cells in the CA1 region died within 7 days after ischemia. The number of SMAD3- or phosphorylated SMAD3 (p-SMAD3)-immunopositive microglia or astrocytes increased in the CA1 region 7 days after ischemia. Real-time PCR analysis showed an increase in the level of TGF-β1 mRNA in the hippocampus after ischemia. Intracerebroventricular injection of SB525334, a selective inhibitor of TGF-β receptor I kinase (ALK5), reduced the ischemia-induced p-SMAD3 immunoreactivity in the microglia and astrocytes. By contrast, intracerebroventricular injection of SB525334 did not affect the ischemia-induced neuronal cell death. These results suggest that ischemia-induced SMAD3 phosphorylation in the microglia and astrocytes of post-ischemic hippocampi is associated with tissue repair and not neuroprotection.

Impacts of autofluorescence on fluorescence based techniques to study microglia

BackgroundMicroglia, the resident immune cells in the central nervous system, accrue autofluorescent granules inside their cytoplasm throughout their lifespan. In this report, we studied the impacts of autofluorescence on widely used fluorescence-based techniques to study microglia, including flow cytometry, immunofluorescence staining, and live imaging.ResultsThe failed attempt of using fluorescein isothiocyanate (FITC) conjugated antibody to detect lymphocyte-activation gene 3 protein in microglia prompted us to compare the sensitivity of FITC, phycoerythrin (PE) and allophycocyanin (APC) conjugated antibodies to detect surface protein expression in microglia. We found that PE outperformed FITC and APC as the fluorophore conjugated to antibody for flow cytometry by overcoming the interference from microglia autofluorescence. To identify the location and source of microglia autofluorescence, we did confocal imaging and spectral analysis of microglia autofluorescence on fixed brain tissues, revealing that microglia autofluorescence emitted from cytoplasmic granules and displayed a multi-peak emission spectrum. We recommended removing autofluorescence by lipofuscin removing agents when staining intracellular proteins in microglia with the immunofluorescence techniques. On live brain slices, autofluorescent granules reduced the amplitudes of calcium signals in microglial somata derived from GCaMP6s fluorescence and thus needed to be excluded when selecting regions of interest (ROI).ConclusionsIn conclusion, autofluorescence is a critical factor to consider when designing experiments and interpreting results based on fluorescence-based techniques to study microglia.

Targeting Forkhead box O1-aquaporin 5 axis mitigates neuropathic pain in a CCI rat model through inhibiting astrocytic and microglial activation

ABSTRACT Forkhead box O1 (FoxO1) is a critical molecule in modulating cell growth, differentiation and metabolism, acting as a vital transcription factor. This study explored the role of FoxO1 in chronic constriction injury (CCI)-induced neuropathic pain (NP). Microglial and astrocyte activation was achieved with lipopolysaccharide (LPS, 100 ng/mL) to establish an in-vitro NP model. Morphological alterations in LPS-induced microglia and astrocytes were assayed by light microscopy. The levels of inflammatory cytokines and proteins in microglia and astrocytes were gauged by enzyme-linked immunosorbent assay (ELISA), and Western blot (WB). The CCI-induced NP rat model was constructed for investigating the FoxO1-AQP5 axis in NP. LPS markedly expanded the expression of inflammatory factors and boosted the expression of FoxO1 and AQP5 in microglia and astrocytes. Inhibition of FoxO1 or AQP5 dramatically decreased the LPS-induced inflammation in microglia and astrocytes. In vivo, CCI exacerbated the inflammatory response and NP symptoms and substantially raised the contents of FoxO1 and AQP5 in rats’ spinal cord tissues. Intrathecal administration of the Sirt1 agonist Resveratrol abated CCI-induced activation of FoxO1 and AQP5, abrogated CCI-induced mechanical hyperalgesia and thermal hyperalgesia, depressed microglial and astrocyte activation, and declined the generation of pro-inflammatory mediators in spinal cord tissues. Mechanistically, blocking the FoxO1-AQP5 pathway inactivated the ERK and p38 MAPK pathways. Suppressing the FoxO1-AQP5 axis alleviated CCI-induced NP and inflammatory responses by modulating the ERK and p38 MAPK signaling pathways.

Increasing Severity of Spinal Cord Injury Results in Microglia/Macrophages With Annular-Shaped Morphology and No Change in Expression of CD40 and Tumor Growth Factor-β During the Chronic Post-injury Stage.

Determination of the quantitative composition of phenotypically and morphologically different populations of resident microglia and infiltrating macrophages in spinal cord injury (SCI) of various degrees of severity could lead to much needed novel therapeutic interventions in neurotrauma. In this regard, we investigated the CD40 and TGF-β expressing populations of microglia/macrophages and their morphological states in a rat model of SCI of varying severity. We are the first to describe the annular-shaped microglia/macrophages, the morphology of which was formed due to the spatial orientation of the processes that form round or oval micro-territories, which include disintegrating myelin fibers. This type of cell morphology was found only in the injured spinal cord and mainly in the white matter. At the same time, an assessment of the number of annular-shaped microglia/macrophages and the diameter of micro-territories formed by their processes showed an elevation in these indicators as the severity of SCI increased. While we did not find significant quantitative changes in the populations of Iba1+/CD40+ and Iba1+/TGF-β+ microglia/macrophages with increased severity of SCI in the chronic period (60 dpi), we did determine changes in the expression of cytokines and mRNAs of genes-encoding microglial marker proteins, finding the greatest changes on days 7 and 14 after SCI between experimental groups with varying severity.

Natural bear bile powder suppresses neuroinflammation in lipopolysaccharide-treated mice via regulating TGR5/AKT/NF-κB signaling pathway

Ethnopharmacological relevanceAccording to the Tang Dynasty classics Dietetic Material Medica and the Ming Dynasty classics Compendium of Materia Medica records, bear bile powder (BBP) has been used to treat a variety of diseases, such as febrile seizures, the pathogenesis of which is associated to neuroinflammation. However, the mechanism of BBP on alleviating neuroinflammation remains unclear. Aims of the studyMicroglia can be activated by peripheral lipopolysaccharide (LPS) and play an important role in the pathogenesis of neuroinflammation. The purpose of this study is to investigate the effects and mechanism of BBP in inhibiting LPS-induced microglia inflammation in vitro and in vivo. Materials and methodsThe anti-microglia inflammatory effects and mechanism of BBP were assessed in LPS-treated BV2 microglial cells and in LPS-treated mice. The mRNA expression levels of the inflammatory factor and the protein expressions of cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), takeda G-protein coupled receptor 5 (TGR5), nuclear factor-κB (NF-κB), inhibitor of NF-κB (IκBɑ), protein kinase B (AKT) in BV2 cells, mouse hippocampus and cortex were detected. The NF-κB transcription activity and NF-κB nuclear translocation were observed. ResultsOur findings showed that BBP reduces branched process retraction and NO in LPS-treated BV2 cells, inhibits the protein expression of ionized calcium binding adaptor molecule 1 in the hippocampus of LPS-treated mice. Moreover, we observed that BBP decreases tumor necrosis factor α, interleukin (IL)-6 and IL-1β mRNA levels, deceases iNOS and COX-2 protein levels, increases TGR5 protein levels, suppresses the phosphorylation of AKT, NF-κB and IκBɑ protein in microglia both in vitro and in vivo. Further, we found that triamterene, the inhibitor of TGR5, abolishes the effects of BBP in LPS- treated BV2 cells. ConclusionBBP inhibits LPS-induced microglia activation, and the mechanism of its action is partly through TGR5/AKT/NF-κB signaling pathway.

The roles of microglia and astrocytes in phagocytosis and myelination: Insights from the cuprizone model of multiple sclerosis.

In human demyelinating diseases such as multiple sclerosis (MS), an imbalance between demyelination and remyelination can trigger progressive degenerative processes. The clearance of myelin debris (phagocytosis) from the site of demyelination by microglia is critically important to achieve adequate remyelination and to slow the progression of the disease. However, how microglia phagocytose the myelin debris, and why clearance is impaired in MS, is not fully known; likewise, the role of the microglia in remyelination remains unclear. Recent studies using cuprizone (CPZ) as an animal model of central nervous system demyelination revealed that the up‐regulation of signaling proteins in microglia facilitates effective phagocytosis of myelin debris. Moreover, during demyelination, protective mediators are released from activated microglia, resulting in the acceleration of remyelination in the CPZ model. In contrast, inadequate microglial activation or recruitment to the site of demyelination, and the production of toxic mediators, impairs remyelination resulting in progressive demyelination. In addition to the microglia‐mediated phagocytosis, astrocytes play an important role in the phagocytic process by recruiting microglia to the site of demyelination and producing regenerative mediators. The current review is an update of these emerging findings from the CPZ animal model, discussing the roles of microglia and astrocytes in phagocytosis and myelination.

Effects of enriched environment on microglia and functional white matter recovery in rats with post stroke cognitive impairment

BackgroundWhite matter damage is an important contributor to cognitive impairment after stroke. This study was designed to explore the beneficial effects of enriched environment (EE) on white matter recovery and cognitive dysfunction after stroke, and further explore the potential mechanism of EE on white matter recovery from the perspective of microglia and microglia-mediated neuroinflammation. MethodsMale SD rats underwent middle cerebral artery occlusion (MCAO) or sham surgery. During the MCAO operation, a laser Doppler blood flow meter was used to monitor the blood flow to ensure the success of the model. At 72 h after the operation, 3 rats were selected for TTC staining to identify the infarct size. One week after surgery, the rats were randomly assigned into four different groups—MCAO+standard environment (SE), MCAO+enriched environment (EE), Sham+SE and Sham+EE for 4 weeks. At four weeks after MCAO surgery, neurological function deficiency condition and cognitive function were assessed using Longa score and Morris Water Maze prior to euthanasia. The loss or regeneration of myelin was stained with LFB, the expression of myelin regeneration-related protein and microglia protein was quantified by western blot and immunofluorescence, and the level of inflammatory factors was measured by ELISA. ResultsEE treatment remarkably decreased the neurological deficit score, ameliorated the cognitive functional deficit in MCAO rats. Furthermore, EE alleviated white matter lesions and demyelination, increased myelin basic protein expression and decreased the number of activated microglia in the hippocampus of MCAO rats. In addition, ELISA analysis indicated that EE decreased the level of IL-1β, IL-6, which further suggests that EE may reduce the level of pro-inflammatory factors by affecting the expression of microglia marker, IBA1, provide a benefit physiological environment for myelin recovery, and improve post stroke cognitive impairment. ConclusionsOur results suggest that exposure to EE substantially reduced the damage to brain tissue caused by activation of microglia activation, decreased the level of pro-inflammatory cytokins, which may induced by microglia, protected and promote white matter recovery to improve cognitive function after stroke. Our findings also indicate exposure to EE is beneficial for patients with white matter impairment characterised by white matter disease-related inflammation.

Extracellular vesicles from adipose-derived stem cells promote microglia M2 polarization and neurological recovery in a mouse model of transient middle cerebral artery occlusion

BackgroundAdipose-derived stem cells (ADSCs) and their extracellular vesicles (EVs) have therapeutic potential in ischemic brain injury, but the underlying mechanism is poorly understood. The current study aimed to explore the contribution of miRNAs in ADSC-EVs to the treatment of cerebral ischemia.MethodsAfter the intravenous injection of ADSC-EVs, therapeutic efficacy was evaluated by neurobehavioral tests and brain atrophy volume. The polarization of microglia was assessed by immunostaining and qPCR. We further performed miRNA sequencing of ADSC-EVs and analyzed the relationship between the upregulated miRNAs in ADSC-EVs and microglial polarization-related proteins using Ingenuity Pathway Analysis (IPA).ResultsThe results showed that ADSC-EVs reduced brain atrophy volume, improved neuromotor and cognitive functions after mouse ischemic stroke. The loss of oligodendrocytes was attenuated after ADSC-EVs injection. The number of blood vessels, as well as newly proliferated endothelial cells in the peri-ischemia area were higher in the ADSC-EVs treated group than that in the PBS group. In addition, ADSC-EVs regulated the polarization of microglia, resulting in increased repair-promoting M2 phenotype and decreased pro-inflammatory M1 phenotype. Finally, STAT1 and PTEN were highlighted as two downstream targets of up-regulated miRNAs in ADSC-EVs among 85 microglia/macrophage polarization related proteins by IPA. The inhibition of STAT1 and PTEN by ADSC-EVs were confirmed in cultured microglia.ConclusionsIn summary, ADSC-EVs reduced ischemic brain injury, which was associated with the regulation of microglial polarization. miRNAs in ADSC-EVs partly contributed to their function in regulating microglial polarization by targeting PTEN and STAT1.

The Expression and Phosphorylation of Smad3 Proteins in Microglia and Astrocytes of the Rat Hippocampus after Transient Global Cerebral Ischemia

The Expression and Phosphorylation of Smad3 Proteins in Microglia and Astrocytes of the Rat Hippocampus after Transient Global Cerebral Ischemia

Gas6/TAM Signalling Negatively Regulates Inflammatory Induction of GM-CSF in Mouse Brain Microglia.

Microglia and astrocytes are the main CNS glial cells responsible for the neuroinflammatory response, where they release a plethora of cytokines into the CNS inflammatory milieu. The TAM (Tyro3, Axl, Mer) receptors and their main ligand Gas6 are regulators of this response, however, the underlying mechanisms remain to be determined. We investigated the ability of Gas6 to modulate the CNS glial inflammatory response to lipopolysaccharide (LPS), a strong pro-inflammatory agent, through a qPCR array that explored Toll-like receptor signalling pathway-associated genes in primary cultured mouse microglia. We identified the Csf2 gene, encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), as a major Gas6 target gene whose induction by LPS was markedly blunted by Gas6. Both the Csf2 gene induction and the suppressive effect of Gas6 on this were emulated through measurement of GM-CSF protein release by cells. We found distinct profiles of GM-CSF induction in different glial cell types, with microglia being most responsive during inflammation. Also, Gas6 markedly inhibited the LPS-stimulated nuclear translocation of NF-κB p65 protein in microglia. These results illustrate microglia as a major resident CNS cellular source of GM-CSF as part of the neuroinflammatory response, and that Gas6/TAM signalling inhibits this response through suppression of NF-κB signalling.

Structural biology of cell surface receptors implicated in Alzheimer's disease.

Alzheimer's disease is a common and devastating age-related disease with no effective disease-modifying treatments. Human genetics has implicated a wide range of cell surface receptors as playing a role in the disease, many of which are involved in the production or clearance of neurotoxins in the brain. Amyloid precursor protein, a membrane-bound signaling molecule, is at the very heart of the disease: hereditary mutations in its gene are associated with a greatly increased risk of getting the disease. A proteolytic breakdown product of amyloid precursor protein, the neurotoxic Aβ peptide, has been the target for many drug discovery efforts. Antibodies have been designed to target Aβ production with some success, although they have not proved efficacious in clinical trials with regards to cognitive benefits to date. Many of the recently identified genes associated with late-onset Alzheimer's disease risk are integral to the innate immune system. Some of these genes code for microglial proteins, such as the strongest genetic risk factor for the disease, namely APOE, and the cell surface receptors CD33 and TREM2 which are involved in clearance of the Aβ peptide from the brain. In this review, we show how structural biology has provided key insights into the normal functioning of these cell surface receptors and provided a framework for developing novel treatments to combat Alzheimer's disease.

Altered synaptic connectivity and brain function in mice lacking microglial adapter protein Iba1

Growing evidence indicates that microglia impact brain function by regulating synaptic pruning and formation as well as synaptic transmission and plasticity. Iba1 (ionized Ca+2-binding adapter protein 1), encoded by the Allograft inflammatory factor 1 (Aif1) gene, is an actin-interacting protein in microglia. Although Iba1 has long been used as a cellular marker for microglia, its functional role remains unknown. Here, we used global, Iba1-deficient (Aif1-/-) mice to characterize microglial activity, synaptic function, and behavior. Microglial imaging in acute hippocampal slices and fixed tissues from juvenile mice revealed that Aif1-/- microglia display reductions in ATP-induced motility and ramification, respectively. Biochemical assays further demonstrated that Aif1-/- brain tissues exhibit an altered expression of microglial-enriched proteins associated with synaptic pruning. Consistent with these changes, juvenile Aif1-/- mice displayed deficits in the excitatory synapse number and synaptic drive assessed by neuronal labeling and whole-cell patch-clamp recording in acute hippocampal slices. Unexpectedly, microglial synaptic engulfment capacity was diminished in juvenile Aif1-/- mice. During early postnatal development, when synapse formation is a predominant event in the hippocampus, the excitatory synapse number was still reduced in Aif1-/- mice. Together, these findings support an overall role of Iba1 in excitatory synaptic growth in juvenile mice. Lastly, postnatal synaptic deficits persisted in adulthood and correlated with significant behavioral changes in adult Aif1-/- mice, which exhibited impairments in object recognition memory and social interaction. These results suggest that Iba1 critically contributes to microglial activity underlying essential neuroglia developmental processes that may deeply influence behavior.

Differential Stimulation of Pluripotent Stem Cell-Derived Human Microglia Leads to Exosomal Proteomic Changes Affecting Neurons.

Microglial exosomes are an emerging communication pathway, implicated in fulfilling homeostatic microglial functions and transmitting neurodegenerative signals. Gene variants of triggering receptor expressed on myeloid cells-2 (TREM2) are associated with an increased risk of developing dementia. We investigated the influence of the TREM2 Alzheimer’s disease risk variant, R47Hhet, on the microglial exosomal proteome consisting of 3019 proteins secreted from human iPS-derived microglia (iPS-Mg). Exosomal protein content changed according to how the iPS-Mg were stimulated. Thus lipopolysaccharide (LPS) induced microglial exosomes to contain more inflammatory signals, whilst stimulation with the TREM2 ligand phosphatidylserine (PS+) increased metabolic signals within the microglial exosomes. We tested the effect of these exosomes on neurons and found that the exosomal protein changes were functionally relevant and influenced downstream functions in both neurons and microglia. Exosomes from R47Hhet iPS-Mg contained disease-associated microglial (DAM) signature proteins and were less able to promote the outgrowth of neuronal processes and increase mitochondrial metabolism in neurons compared with exosomes from the common TREM2 variant iPS-Mg. Taken together, these data highlight the importance of microglial exosomes in fulfilling microglial functions. Additionally, variations in the exosomal proteome influenced by the R47Hhet TREM2 variant may underlie the increased risk of Alzheimer’s disease associated with this variant.