CHIT1-positive microglia drive motor neuron ageing in the primate spinal cord.

Ageing is a critical factor in spinal-cord-associated disorders1, yet the ageing-specific mechanisms underlying this relationship remain poorly understood. Here, to address this knowledge gap, we combined single-nucleus RNA-sequencing analysis with behavioural and neurophysiological analysis in non-human primates (NHPs). We identified motor neuron senescence and neuroinflammation with microglial hyperactivation as intertwined hallmarks of spinal cord ageing. As an underlying mechanism, we identified a neurotoxic microglial state demarcated by elevated expression of CHIT1 (a secreted mammalian chitinase) specific to the aged spinal cords in NHP and human biopsies. In the aged spinal cord, CHIT1-positive microglia preferentially localize around motor neurons, and they have the ability to trigger senescence, partly by activating SMAD signalling. We further validated the driving role of secreted CHIT1 on MN senescence using multimodal experiments both in vivo, using the NHP spinal cord as a model, and in vitro, using a sophisticated system modelling the human motor-neuron-microenvironment interplay. Moreover, we demonstrated that ascorbic acid, a geroprotective compound, counteracted the pro-senescent effect of CHIT1 and mitigated motor neuron senescence in aged monkeys. Our findings provide the single-cell resolution cellular and molecular landscape of the aged primate spinal cord and identify a new biomarker and intervention target for spinal cord degeneration.

Prenatal disruption of blood-brain barrier formation via cyclooxygenase activation leads to lifelong brain inflammation.

Gestational maternal immune activation (MIA) in mice induces persistent brain microglial activation and a range of neuropathologies in the adult offspring. Although long-term phenotypes are well documented, how MIA in utero leads to persistent brain inflammation is not well understood. Here, we found that offspring of mothers treated with polyriboinosinic­polyribocytidylic acid [poly(I:C)] to induce MIA at gestational day 13 exhibit blood­brain barrier (BBB) dysfunction throughout life. Live MRI in utero revealed fetal BBB hyperpermeability 2 d after MIA. Decreased pericyte­endothelium coupling in cerebral blood vessels and increased microglial activation were found in fetal and 1- and 6-mo-old offspring brains. The long-lasting disruptions result from abnormal prenatal BBB formation, driven by increased proliferation of cyclooxygenase-2 (COX2; Ptgs2)-expressing microglia in fetal brain parenchyma and perivascular spaces. Targeted deletion of the Ptgs2 gene in fetal myeloid cells or treatment with the inhibitor celecoxib 24 h after immune activation prevented microglial proliferation and disruption of BBB formation and function, showing that prenatal COX2 activation is a causal pathway of MIA effects. Thus, gestational MIA disrupts fetal BBB formation, inducing persistent BBB dysfunction, which promotes microglial overactivation and behavioral alterations across the offspring life span. Taken together, the data suggest that gestational MIA disruption of BBB formation could be an etiological contributor to neuropsychiatric disorders.

Possible role of arginase 1 positive microglia on depressive/anxiety-like behaviors in atopic dermatitis mouse model.

Atopic dermatitis (AD) and mood disorder comorbidities are typical, but the exact mechanism underlying their interplay has not been clarified. In this study, we aimed to identify the possible mechanisms of anxiety/depressive-like behaviors observed in AD, focusing on microglia. AD was induced by Dermatophagoides farinae body extract (Dfb) in NC/Nga mice and anxiety/depressive-like behaviors were analyzed by behavioral assessments such as open field test (OFT), tail suspension test (TST), sucrose preference test (SPT), and social interaction. As clinical symptoms of AD induced, anxiety/depressive-like behaviors were increased in the OFT and TST and serum glucocorticoid was elevated. AD mice showed an increased mRNA expression of interleukin-4 (IL-4) in lymph nodes but decreased arginase 1 (Arg1) mRNA expression without a change of IL-4 in the hippocampus. In addition, AD mice showed microglia with a shortened branch of de-ramified form and astrocytes with longer processes and decreased branching in the hippocampus, especially in the dentate gyrus (DG). The immunofluorescence study of the DG confirmed that Arg1 reduction was associated with microglia, but not astrocytes. Furthermore, glucocorticoid receptor reduction, increased 5-HT1AR, reduced phosphorylated cAMP response element-binding protein (pCREB), and brain-derived neurotrophic factor (BDNF) expression were identified in the hippocampus of AD mice. Notably, an immunofluorescence study confirmed that pCREB was decreased in the DG of AD mice. Collectively, our data suggest that the reduced Arg1 positive microglia might contribute to anxiety/depressive-like behaviors via pCREB/BDNF reduction in AD.

Evolutionary Origin of Insulin-Degrading Enzyme and Its Subcellular Localization and Secretion Mechanism: A Study in Microglial Cells.

The insulin-degrading enzyme (IDE) is a zinc-dependent metalloendopeptidase that belongs to the M16A metalloprotease family. IDE is markedly expressed in the brain, where it is particularly relevant due to its in vitro amyloid beta (Aß)-degrading activity. The subcellular localization of IDE, a paramount aspect to understand how this enzyme can perform its proteolytic functions in vivo, remains highly controversial. In this work, we addressed IDE subcellular localization from an evolutionary perspective. Phylogenetic analyses based on protein sequence and gene and protein structure were performed. An in silico analysis of IDE signal peptide suggests an evolutionary shift in IDE exportation at the prokaryote/eukaryote divide. Subcellular localization experiments in microglia revealed that IDE is mostly cytosolic. Furthermore, IDE associates to membranes by their cytoplasmatic side and further partitions between raft and non-raft domains. When stimulated, microglia change into a secretory active state, produces numerous multivesicular bodies and IDE associates with their membranes. The subsequent inward budding of such membranes internalizes IDE in intraluminal vesicles, which later allows IDE to be exported outside the cells in small extracellular vesicles. We further demonstrate that such an IDE exportation mechanism is regulated by stimuli relevant for microglia in physiological conditions and upon aging and neurodegeneration.

Proto-oncogene tyrosine-protein kinase SRC (Src) inhibition in microglia relieves neuroinflammation in neuropathic pain mouse models.

Chronic neuroinflammation is an important factor in the development of neuropathic pain (NP). Excess microglia activation releases a mass of pro-inflammatory cytokines during neuroinflammation process, leading to a constant painful irritation of the sensory nerve. Src belongs to a non-receptor tyrosine kinase associated with sarcoma, whereas the role of Src in neuropathic pain is controversial. We designed to testify the inflammation-regulatory role of Src in the lipopolysaccharide (LPS)-induced BV2 microglia line and the mouse model of neuropathic pain by partial sciatic nerve ligation (PNL). In BV2 microglia, Src expression was inhibited using a Src family kinase inhibitor PP2 after LPS induced inflammatory response. In vivo, the neuropathic pain in mice was induced by PNL surgery and then treated with PP2. The neuroinflammation level in vitro was detected by enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), trans-well and Western blotting (WB) assays, in vivo was examined in PNL mice using immunohistochemistry (IHC) and IF. Finally, mechanical allodynia and thermal hyperalgesia assays were used to access the functional evaluation. Inhibition of Src was decreased microglial inflammation and migration after LPS stimuli. Mechanistically, the expression of nuclear factor kappa B (NF-κB) pathway decreased after Src inhibition. The data in vivo showed that the decrease expression of Src reduced neuroinflammation and the amount of microglia in spinal dorsal horn (SDH), the mechanical allodynia of mice thereby attenuated after Src inhibition. These results indicated that the inhibition of Src took a protective effect in neuropathic pain mouse models via reducing microglia-induced neuroinflammation.

MiR-191-5p alleviates microglial cell injury by targeting Map3k12 (mitogen-activated protein kinase kinase kinase 12) to inhibit the MAPK (mitogen-activated protein kinase) signaling pathway in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Multiple reports have elucidated that microRNAs are promising biomarkers for AD diagnosis and treatment. Herein, the effect of miR-191-5p on microglial cell injury and the underlying mechanism were explored. APP/PS1 transgenic mice were utilized to establish mouse model of AD. Amyloid-ß protein 1-42 (Aß1-42)-treated microglia were applied to establish in vitro cell model of AD. MiR-191-5p expression in hippocampus and microglia was measured by reverse transcription quantitative polymerase chain reaction. The viability and apoptosis of microglia were evaluated by Cell Counting Kit-8 assays and flow cytometry analyses, respectively. The binding relationship between miR-191-5p and its downstream target mitogen-activated protein kinase kinase kinase 12 (Map3k12) was determined by luciferase reporter assays. Pathological degeneration of hippocampus was tested using hematoxylin-eosin staining and Nissl staining. Aß expression in hippocampus was examined via immunohistochemistry. In this study, miR-191-5p was downregulated in Aß1-42-stimulated microglia and hippocampal tissues of APP/PS1 mice. MiR-191-5p overexpression facilitated cell viability and inhibited apoptosis rate of Aß1-42-treated microglia. Mechanically, miR-191-5p targeted Map3k12 3'-untranslated region to downregulate Map3k12 expression. MiR-191-5p inhibited Aß1-42-induced microglial cell injury and inactivated the MAPK signaling by downregulating Map3k12. Overall, miR-191-5p alleviated Aß1-42-induced microglia cell injury by targeting Map3k12 to inhibit the MAPK signaling pathway in microglia.

Prostaglandin E2 increases the expression of cyclooxygenase-2 in cultured rat microglia.

Prostaglandin E2 (PGE2) plays pivotal roles in controlling microglial activation with the EP2 receptor, a PGE2 receptor subtype. Activated microglia are often reported to increase cyclooxygenase (COX)-2 expression, followed by PGE2 production, but it is unclear whether extracellular PGE2 is involved in microglial PGE2 synthesis. In the present study, we report that PGE2 increases COX-2 protein in microglia. In a culture system, PGE2 at 10-6 M for 3 h increased COX-2 and microsomal PGE synthase (mPGES)-1 mRNA levels, and reduced mPGES-2, but did not affect COX-1 or cytosolic PGE synthase (cPGES) in microglia. PGE2 at 10-6 M for 3 h also increased the COX-2 protein level, but did not affect COX-1, mPGES-1, mPGES-2, or cPGES. An EP2 agonist, ONO-AE1-259-01, also increased COX-2 and mPGES-1 mRNA levels, and reduced mPGES-2, but did not affect COX-1 or cPGES, whereas an EP1 agonist, ONO-DI-004, an EP3 agonist, ONO-AE-248, and an EP4 agonist, ONO-AE1-329, had no effect. Similar to PGE2, ONO-AE1-259-01 increased the COX-2 protein level, but did not affect COX-1, mPGES-1, mPGES-2, or cPGES. In addition, the effects of PGE2 were inhibited by an EP2 antagonist, PF-04418948, but not by an EP1 antagonist, ONO-8713, an EP3 antagonist, ONO-AE3-240, or an EP4 antagonist, ONO-AE3-208, at 10-6 M. On the other hand, lipopolysaccharide (LPS) increased PGE2 production, but the LPS-induced PGE2 production was not affected by ONO-8713, PF-04418948, ONO-AE3-240, or ONO-AE3-208. These results indicate that PGE2 increases COX-2 protein in microglia through the EP2 receptor supporting the idea that extracellular PGE2 has a triggering aspect for microglial activation.

Inhibition of neutral sphingomyelinase 2 reduces extracellular vesicle release from neurons, oligodendrocytes, and activated microglial cells following acute brain injury.

Extracellular Vesicles (EVs) are implicated in the spread of pathogenic proteinsin a growing number of neurological diseases. Given this, there is rising interest in developing inhibitors of Neutral Sphingomyelinase 2 (nSMase2), an enzyme critical in EV biogenesis. Our group recently discovered phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate (PDDC), the first potent, selective, orally-available, and brain-penetrable nSMase2 inhibitor, capable of dose-dependently reducing EVs release in vitro and in vivo. Herein, using multiplexed Surface Plasmon Resonance imaging (SPRi), we evaluated which brain cell-derived EVs were affected by PDDC following acute brain injury. Mice were fed PDDC-containing chow at doses which gave steady PDDC brain exposures exceeding its nSMase2 IC50. Mice were then administered an intra-striatal IL-1ß injection and two hours later plasma and brain were collected. IL-1ß injection significantly increased striatal nSMase2 activity which was completely normalized by PDDC. Using SPRi, we found that IL-1ß-induced injury selectively increased plasma levels of CD171 + and PLP1 + EVs; this EV increase was normalized by PDDC. In contrast, GLAST1 + EVs were unchanged by IL-1ß or PDDC. IL-1ß injection selectively increased EVs released from activated versus non-activated microglia, indicated by the CD11b+/IB4 + ratio. The increase in EVs from CD11b + microglia was dramatically attenuated with PDDC. Taken together, our data demonstrate that following acute injury, brain nSMase2 activity is elevated. EVs released from neurons, oligodendrocytes, and activated microglial are increased in plasma and inhibition of nSMase2 with PDDC reduced these IL-1ß-induced changes implicating nSMase2 inhibition as a therapeutic target for acute brain injury.

Inhibition of microglial ß-glucocerebrosidase hampers the microglia-mediated antioxidant and protective response in neurons.

BACKGROUND: Homozygotic mutations in the GBA gene cause Gaucher's disease; moreover, both patients and heterozygotic carriers have been associated with 20- to 30-fold increased risk of developing Parkinson's disease. In homozygosis, these mutations impair the activity of ß-glucocerebrosidase, the enzyme encoded by GBA, and generate a lysosomal disorder in macrophages, which changes morphology towards an engorged phenotype, considered the hallmark of Gaucher's disease. Notwithstanding the key role of macrophages in this disease, most of the effects in the brain have been attributed to the ß-glucocerebrosidase deficit in neurons, while a microglial phenotype for these mutations has never been reported. METHODS: We applied the bioluminescence imaging technology, immunohistochemistry and gene expression analysis to investigate the consequences of microglial ß-glucocerebrosidase inhibition in the brain of reporter mice, in primary neuron/microglia cocultures and in cell lines. The use of primary cells from reporter mice allowed for the first time, to discriminate in cocultures neuronal from microglial responses consequent to the ß-glucocerebrosidase inhibition; results were finally confirmed by pharmacological depletion of microglia from the brain of mice. RESULTS: Our data demonstrate the existence of a novel neuroprotective mechanism mediated by a direct microglia-to-neuron contact supported by functional actin structures. This cellular contact stimulates the nuclear factor erythroid 2-related factor 2 activity in neurons, a key signal involved in drug detoxification, redox balance, metabolism, autophagy, lysosomal biogenesis, mitochondrial dysfunctions, and neuroinflammation. The central role played by microglia in this neuronal response in vivo was proven by depletion of the lineage in the brain of reporter mice. Pharmacological inhibition of microglial ß-glucocerebrosidase was proven to induce morphological changes, to turn on an anti-inflammatory/repairing pathway, and to hinder the microglia ability to activate the nuclear factor erythroid 2-related factor 2 response, thus increasing the neuronal susceptibility to neurotoxins. CONCLUSION: This mechanism provides a possible explanation for the increased risk of neurodegeneration observed in carriers of GBA mutations and suggest novel therapeutic strategies designed to revert the microglial phenotype associated with ß-glucocerebrosidase inhibition, aimed at resetting the protective microglia-to-neuron communication.

Bavachin exerted anti-neuroinflammatory effects by regulation of A20 ubiquitin-editing complex.

Neuroinflammation is a major pathophysiological contributor to the progression of the central nervous system disorders. Bavachin is a natural product belonging to the flavonoid class. The anti-neuroinflammatory effect and the molecular mechanisms are not well understood. In this study, we found bavachin can exert anti-neuroinflammatory effect via inhibition of nuclear factor-kappa B (NF-κB) signaling. We found that bavachin can obviously upregulate the expression of A20 (TNFAIP3) in microglial cells. Further studies suggested siRNA-A20 knockdown treatment can attenuate the inhibitory effects of bavachin on neuroinflammation. We further found bavachin can increase the interaction of ubiquitin-editing enzyme A20 complex including A20, Tax1-binding protein 1 (TAX1BP1) and Itch, the subsequently downregulated the K63-ubiquitination of TNF receptor associated factor 6 (TRAF6) and NF-κB signaling pathway. Altogether, our results indicated that bavachin exerted anti-neuroinflammatory effects through inhibition of NF-κB signaling mediated by regulation of ubiquitin-editing enzyme A20 complex. Our finding has important clinical significance for the potential application of bavachin in the treatment of neurological disorders.

GCH1-regulated miRNAs are potential targets for microglial activation in neuropathic pain.

Neuropathic pain (NP) is a chronic pain directly caused by injury or disease of the somatosensory nervous system. Previous studies suggest that GTP cyclohydrolase I (GCH1) may play a pivotal role in microglial activation, which has been shown to be essential for NP. However, its underlying mechanisms in microglial activation remain unclear. A wide range of microRNAs (miRNAs) have been found to be involved in microglial activation-induced NP. To identify the miRNAs regulated by GCH1 and predict their functions in the progression of microglial activation, we analyzed the miRNA expression profiles of GCH1-knockdown (KD) BV2 microglial cells. Small RNA-sequencing analysis revealed 13 differentially expressed (DE) miRNAs in GCH1-KD cells. The target genes of DE miRNAs mainly participate in PI3K-Akt signaling pathway, peroxisome and ferroptosis. The miRNA-mRNA regulatory network analysis showed that GCH1, MAP4K5 and YWHAB acted as hub genes. qRT-PCR results further verified the expression levels of mmu-miR-1a-3p, mmu-miR-133a-3p, mmu-miR-7a-5p and mmu-miR-10a-5p in GCH1-KD cells, which were consistent with the sequencing data. In addition, our data indicated that overexpression of mmu-miR-133a-3p alleviated the pro-inflammatory cytokines IL-1ß and IL-6 production induced by lipopolysaccharide (LPS), indicating that mmu-miR-133a-3p has a negative effect on microglial activation. Taken together, our findings suggest that many miRNAs regulated by GCH1 may be involved in microglial activation, which may provide new potential targets for GCH1 in the pathogenesis of NP.

The PINK1-Mediated Crosstalk between Neural Cells and the Underlying Link to Parkinson's Disease.

Mitochondrial dysfunction has a fundamental role in the development of idiopathic and familiar forms of Parkinson's disease (PD). The nuclear-encoded mitochondrial kinase PINK1, linked to familial PD, is responsible for diverse mechanisms of mitochondrial quality control, ATP production, mitochondrial-mediated apoptosis and neuroinflammation. The main pathological hallmark of PD is the loss of dopaminergic neurons. However, novel discoveries have brought forward the concept that a disruption in overall brain homeostasis may be the underlying cause of this neurodegeneration disease. To sustain this, astrocytes and microglia cells lacking PINK1 have revealed increased neuroinflammation and deficits in physiological roles, such as decreased wound healing capacity and ATP production, which clearly indicate involvement of these cells in the physiopathology of PD. PINK1 executes vital functions within mitochondrial regulation that have a detrimental impact on the development and progression of PD. Hence, in this review, we aim to broaden the horizon of PINK1-mediated phenotypes occurring in neurons, astrocytes and microglia and, ultimately, highlight the importance of the crosstalk between these neural cells that is crucial for brain homeostasis.

Myeloid Arginase 1 Insufficiency Exacerbates Amyloid-ß Associated Neurodegenerative Pathways and Glial Signatures in a Mouse Model of Alzheimer's Disease: A Targeted Transcriptome Analysis.

Brain myeloid cells, include infiltrating macrophages and resident microglia, play an essential role in responding to and inducing neurodegenerative diseases, such as Alzheimer's disease (AD). Genome-wide association studies (GWAS) implicate many AD casual and risk genes enriched in brain myeloid cells. Coordinated arginine metabolism through arginase 1 (Arg1) is critical for brain myeloid cells to perform biological functions, whereas dysregulated arginine metabolism disrupts them. Altered arginine metabolism is proposed as a new biomarker pathway for AD. We previously reported Arg1 deficiency in myeloid biased cells using lysozyme M (LysM) promoter-driven deletion worsened amyloidosis-related neuropathology and behavioral impairment. However, it remains unclear how Arg1 deficiency in these cells impacts the whole brain to promote amyloidosis. Herein, we aim to determine how Arg1 deficiency driven by LysM restriction during amyloidosis affects fundamental neurodegenerative pathways at the transcriptome level. By applying several bioinformatic tools and analyses, we found that amyloid-ß (Aß) stimulated transcriptomic signatures in autophagy-related pathways and myeloid cells' inflammatory response. At the same time, myeloid Arg1 deficiency during amyloidosis promoted gene signatures of lipid metabolism, myelination, and migration of myeloid cells. Focusing on Aß associated glial transcriptomic signatures, we found myeloid Arg1 deficiency up-regulated glial gene transcripts that positively correlated with Aß plaque burden. We also observed that Aß preferentially activated disease-associated microglial signatures to increase phagocytic response, whereas myeloid Arg1 deficiency selectively promoted homeostatic microglial signature that is non-phagocytic. These transcriptomic findings suggest a critical role for proper Arg1 function during normal and pathological challenges associated with amyloidosis. Furthermore, understanding pathways that govern Arg1 metabolism may provide new therapeutic opportunities to rebalance immune function and improve microglia/macrophage fitness.

Ablation of lysozyme M-positive cells prevents aircraft noise-induced vascular damage without improving cerebral side effects.

Aircraft noise induces vascular and cerebral inflammation and oxidative stress causing hypertension and cardiovascular/cerebral dysfunction. With the present studies, we sought to determine the role of myeloid cells in the vascular vs. cerebral consequences of exposure to aircraft noise. Toxin-mediated ablation of lysozyme M+ (LysM+) myeloid cells was performed in LysMCreiDTR mice carrying a cre-inducible diphtheria toxin receptor. In the last 4d of toxin treatment, the animals were exposed to noise at maximum and mean sound pressure levels of 85 and 72 dB(A), respectively. Flow cytometry analysis revealed accumulation of CD45+, CD11b+, F4/80+, and Ly6G-Ly6C+ cells in the aortas of noise-exposed mice, which was prevented by LysM+ cell ablation in the periphery, whereas brain infiltrates were even exacerbated upon ablation. Aircraft noise-induced increases in blood pressure and endothelial dysfunction of the aorta and retinal/mesenteric arterioles were almost completely normalized by ablation. Correspondingly, reactive oxygen species in the aorta, heart, and retinal/mesenteric vessels were attenuated in ablated noise-exposed mice, while microglial activation and abundance in the brain was greatly increased. Expression of phagocytic NADPH oxidase (NOX-2) and vascular cell adhesion molecule-1 (VCAM-1) mRNA in the aorta was reduced, while NFκB signaling appeared to be activated in the brain upon ablation. In sum, we show dissociation of cerebral and peripheral inflammatory reactions in response to aircraft noise after LysM+ cell ablation, wherein peripheral myeloid inflammatory cells represent a dominant part of the pathomechanism for noise stress-induced cardiovascular effects and their central nervous counterparts, microglia, as key mediators in stress responses.

A RIPK1-regulated inflammatory microglial state in amyotrophic lateral sclerosis.

Microglial-derived inflammation has been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including amyotrophic lateral sclerosis (ALS). Using single-cell RNA sequencing, a class of Disease-Associated Microglia (DAMs) have been characterized in neurodegeneration. However, the DAM phenotype alone is insufficient to explain the functional complexity of microglia, particularly with regard to regulating inflammation that is a hallmark of many neurodegenerative diseases. Here, we identify a subclass of microglia in mouse models of ALS which we term RIPK1-Regulated Inflammatory Microglia (RRIMs). RRIMs show significant up-regulation of classical proinflammatory pathways, including increased levels of Tnf and Il1b RNA and protein. We find that RRIMs are highly regulated by TNFα signaling and that the prevalence of these microglia can be suppressed by inhibiting receptor-interacting protein kinase 1 (RIPK1) activity downstream of the TNF receptor 1. These findings help to elucidate a mechanism by which RIPK1 kinase inhibition has been shown to provide therapeutic benefit in mouse models of ALS and may provide an additional biomarker for analysis in ongoing phase 2 clinical trials of RIPK1 inhibitors in ALS.

Maternal antibodies facilitate Amyloid-ß clearance by activating Fc-receptor-Syk-mediated phagocytosis.

Maternal antibodies (MAbs) protect against infections in immunologically-immature neonates. Maternally transferred immunity may also be harnessed to target diseases associated with endogenous protein misfolding and aggregation, such as Alzheimer's disease (AD) and AD-pathology in Down syndrome (DS). While familial early-onset AD (fEOAD) is associated with autosomal dominant mutations in the APP, PSEN1,2 genes, promoting cerebral Amyloid-ß (Aß) deposition, DS features a life-long overexpression of the APP and DYRK1A genes, leading to a cognitive decline mediated by Aß overproduction and tau hyperphosphorylation. Although no prenatal screening for fEOAD-related mutations is in clinical practice, DS can be diagnosed in utero. We hypothesized that anti-Aß MAbs might promote the removal of early Aß accumulation in the central nervous system of human APP-expressing mice. To this end, a DNA-vaccine expressing Aß1-11 was delivered to wild-type female mice, followed by mating with 5xFAD males, which exhibit early Aß plaque formation. MAbs reduce the offspring's cortical Aß levels 4 months after antibodies were undetectable, along with alleviating short-term memory deficits. MAbs elicit a long-term shift in microglial phenotype in a mechanism involving activation of the FcγR1/Syk/Cofilin pathway. These data suggest that maternal immunization can alleviate cognitive decline mediated by early Aß deposition, as occurs in EOAD and DS.

Dexmedetomidine Alleviates Hypoxia-Induced Synaptic Loss and Cognitive Impairment via Inhibition of Microglial NOX2 Activation in the Hippocampus of Neonatal Rats.

BACKGROUND: Perinatal hypoxia is a universal cause of death and neurological deficits in neonates worldwide. Activation of microglial NADPH oxidase 2 (NOX2) leads to oxidative stress and neuroinflammation, which may contribute to hypoxic damage in the developing brain. Dexmedetomidine has been reported to exert potent neuroprotection in several neurological diseases, but the mechanism remains unclear. We investigated whether dexmedetomidine acts through microglial NOX2 to reduce neonatal hypoxic brain damage. METHODS: The potential role of microglial NOX2 in dexmedetomidine-mediated alleviation of hypoxic damage was evaluated in cultured BV2 microglia and neonatal rats subjected to hypoxia. In vivo, neonatal rats received dexmedetomidine (25 µg/kg, i.p.) 30 min before or immediately after hypoxia (5% O2, 2 h). Apocynin-mediated NOX inhibition and lentivirus-mediated NOX2 overexpression were applied to further assess the involvement of microglial NOX2 activation. RESULTS: Pre- or posttreatment with dexmedetomidine alleviated hypoxia-induced cognitive impairment, restored damaged synapses, and increased postsynaptic density-95 and synaptophysin protein expression following neonatal hypoxia. Importantly, dexmedetomidine treatment suppressed hypoxia-induced microglial NOX2 activation and subsequent oxidative stress and the neuroinflammatory response, as reflected by reduced 4-hydroxynonenal and ROS accumulation, and decreased nuclear NF-κB p65 and proinflammatory cytokine levels in cultured BV2 microglia and the developing hippocampus. In addition, treating primary hippocampal neurons with conditioned medium (CM) from hypoxia-activated BV2 microglia resulted in neuronal damage, which was alleviated by CM from dexmedetomidine-treated microglia. Moreover, the neuroprotective effect of dexmedetomidine was reversed in NOX2-overexpressing BV2 microglia and diminished in apocynin-pretreated neonatal rats. CONCLUSION: Dexmedetomidine targets microglial NOX2 to reduce oxidative stress and neuroinflammation and subsequently protects against hippocampal synaptic loss following neonatal hypoxia.

Retinal Ganglion Cell Loss and Microglial Activation in a SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) affects the spinal cord, brain stem, and cerebral cortex. In this pathology, both neurons and glial cells are affected. However, few studies have analyzed retinal microglia in ALS models. In this study, we quantified the signs of microglial activation and the number of retinal ganglion cells (RGCs) in an SOD1G93A transgenic mouse model at 120 days (advanced stage of the disease) in retinal whole-mounts. For SOD1G93A animals (compared to the wild-type), we found, in microglial cells, (i) a significant increase in the area occupied by each microglial cell in the total area of the retina; (ii) a significant increase in the arbor area in the outer plexiform layer (OPL) inferior sector; (iii) the presence of cells with retracted processes; (iv) areas of cell groupings in some sectors; (v) no significant increase in the number of microglial cells; (vi) the expression of IFN-γ and IL-1ß; and (vii) the non-expression of IL-10 and arginase-I. For the RGCs, we found a decrease in their number. In conclusion, in the SOD1G93A model (at 120 days), retinal microglial activation occurred, taking a pro-inflammatory phenotype M1, which affected the OPL and inner retinal layers and could be related to RGC loss.

CHRFAM7A Overexpression Attenuates Cerebral Ischemia-Reperfusion Injury via Inhibiting Microglia Pyroptosis Mediated by the NLRP3/Caspase-1 pathway.

Cerebral ischemia-reperfusion (I/R) injury is an inflammation-related disease. CHRFAM7A can regulate inflammatory responses. Therefore, the present study investigated the mechanism of CHRFAM7A in cerebral I/R injury. CHRFAM7A expression and inflammatory cytokine levels in patients with cerebral I/R injury and oxygen-glucose deprivation/reperfusion (OGD/R)-treated microglia were detected. The proliferation, inflammatory cytokine expressions, nod-like receptor protein 3 (NLRP3) level, cell pyroptosis, and viability and lactate dehydrogenase (LDH) activity in OGD/R-treated microglia were detected after CHRFAM7A overexpression. The NLRP3/Caspase-1 pathway was activated to assess the effect of CHRFAM7A on microglia. Expressions of microglial M1 phenotype marker iNOS and M2 marker Arg1 were detected. Downregulated CHRFAM7A and elevated inflammatory cytokine levels were observed in patients with cerebral I/R injury and OGD/R-treated microglia. In OGD/R-treated microglia, CHRFAM7A overexpression promoted cell proliferation and viability, reduced inflammation and LDH activity, and inhibited NLRP3 inflammasome activation and cell pyroptosis. Mechanically, CHRFAM7A inhibited microglia pyroptosis via inhibiting the NLRP3/Caspase-1 pathway and reduced cell inflammatory injury via promoting microglia polarization from M1 to M2. Overall, CHRFAM7A overexpression attenuated cerebral I/R injury by inhibiting microglia pyroptosis in a NLRP3/Caspase-1 pathway-dependent manner and promoting microglia polarization to M2 phenotype.

Moringa oleifera modulates cholinergic and purinergic enzymes activity in BV-2 microglial cells.

Microglia are immune cells that are resident in central nervous system. Activation of microglial cells are detrimental to the survival of neurons. Thus, prevention of microglia activation and/or protection against microglia activation could be potential therapeutic strategy towards the management of inflammation-mediated neurodegenerative diseases. Moringa oleifera is widely consumed as food and used in folklore medicine for treating several diseases. This study was convened to investigate the effect of aqueous extract of Moringa oleifera on cell viability, cholinergic and purinergic enzymes in BV-2 microglial cultured cell. Aqueous extract of Moringa oleifera was prepared, lyophilized and reconstituted in 0.5% dimethylsulphoxide (DMSO). Cells were treated with Moringa oleifera extracts (0.1-100 µg/mL) and assessed for cell viability and nitric oxide production. Furthermore, the effect of Moringa oleifera on enzymes of cholinergic (acetylcholinesterase) and purinergic (nucleoside triphosphate diphosphohydrolase; NTPDase, 5' nucleotidase and adenosine deaminase; ADA) systems in BV-2 microglial cells were determined. Incubation of BV-2 microglia cell with M. oleifera extract maintained cell viability, modulated cholinergic and purinergic enzymes activity. The phenolic compounds found in M. oleifera extracts, include chlorogenic acid, rutin; quercetin pentoside, kaempferol derivative and quercetin derivative. Thus, this study suggest that the potential therapeutic effect of the phenolic compounds found in M. oleifera may have been responsible for the maintenance of cell viability in BV-2 microglia cells and modulation of cholinergic as well as purinergic enzymes activity.