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Ageing is a critical factor in spinal-cord-associated disorders 1 , 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., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

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 polyriboinosinic–polyribocytidylic acid [poly(I:C)] to induce MIA at gestational day 13 exhibit blood–brain barrier (BBB) dysfunction throughout life. Live MRI in utero revealed fetal BBB hyperpermeability 2 d after MIA. Decreased pericyte–endothelium 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.

Allograft inflammatory factor-1 (AIF-1) is a Ca2+-binding peptide that constitutes a potential modulator of macrophage activation and function during the immune response of the brain. Peptides termed microglia response factor-1 or ionized calcium-binding adaptor molecule-1 have been reported to be identical with AIF-1. We have investigated the expression of AIF-1 in the rat C6 glioblastoma and 9L gliosarcoma tumor models and additionally assessed AIF-1 expression in a diverse range of human astrocytomas by immunohistochemistry. AIF-1 was expressed by activated microglial cells and a subset of infiltrating macrophages in areas of infiltrative tumor growth and in compact tumor areas in both rat and human gliomas. Double-labeling experiments in rats and humans characterized the nature and the functional status of AIF-1+ cells. AIF-1 expression was detected in cells expressing major histocompatibility complex class II molecules and in a subset of activated macrophages/microglial cells. All MRP-8+ cells coexpressed AIF-1. In humans, there was a strong correlation of AIF-1-expressing activated macrophages/microglial cells with tumor malignancy (P < 0.0001). These results suggest that AIF-1 defines a distinct subset of tumor-associated activated macrophages/ microglial cells. [ABSTRACT FROM AUTHOR]

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 (Aβ)-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.

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-HT 1A R, 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., (© 2022. The Pharmaceutical Society of Korea.)

Prostaglandin E 2 (PGE 2 ) plays pivotal roles in controlling microglial activation with the EP 2 receptor, a PGE 2 receptor subtype. Activated microglia are often reported to increase cyclooxygenase (COX)-2 expression, followed by PGE 2 production, but it is unclear whether extracellular PGE 2 is involved in microglial PGE 2 synthesis. In the present study, we report that PGE 2 increases COX-2 protein in microglia. In a culture system, PGE 2 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. PGE 2 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 EP 2 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 EP 1 agonist, ONO-DI-004, an EP 3 agonist, ONO-AE-248, and an EP 4 agonist, ONO-AE1-329, had no effect. Similar to PGE 2 , 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 PGE 2 were inhibited by an EP 2 antagonist, PF-04418948, but not by an EP 1 antagonist, ONO-8713, an EP 3 antagonist, ONO-AE3-240, or an EP 4 antagonist, ONO-AE3-208, at 10 -6 M. On the other hand, lipopolysaccharide (LPS) increased PGE 2 production, but the LPS-induced PGE 2 production was not affected by ONO-8713, PF-04418948, ONO-AE3-240, or ONO-AE3-208. These results indicate that PGE 2 increases COX-2 protein in microglia through the EP 2 receptor supporting the idea that extracellular PGE 2 has a triggering aspect for microglial activation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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-β protein 1-42 (Aβ1-42)-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. Aβ expression in hippocampus was examined via immunohistochemistry. In this study, miR-191-5p was downregulated in Aβ1-42-stimulated microglia and hippocampal tissues of APP/PS1 mice. MiR-191-5p overexpression facilitated cell viability and inhibited apoptosis rate of Aβ1-42-treated microglia. Mechanically, miR-191-5p targeted Map3k12 3'-untranslated region to downregulate Map3k12 expression. MiR-191-5p inhibited Aβ1-42-induced microglial cell injury and inactivated the MAPK signaling by downregulating Map3k12. Overall, miR-191-5p alleviated Aβ1-42-induced microglia cell injury by targeting Map3k12 to inhibit the MAPK signaling pathway in microglia.

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 IC 50 . Mice were then administered an intra-striatal IL-1β injection and two hours later plasma and brain were collected. IL-1β injection significantly increased striatal nSMase2 activity which was completely normalized by PDDC. Using SPRi, we found that IL-1β-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-1β or PDDC. IL-1β 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-1β-induced changes implicating nSMase2 inhibition as a therapeutic target for acute brain injury., (Copyright © 2021. Published by Elsevier Inc.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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-1β 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., (© 2021 The Author(s).)

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 β-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 β-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 β-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 β-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 β-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 β-glucocerebrosidase inhibition, aimed at resetting the protective microglia-to-neuron communication., (© 2021. The Author(s).)

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.

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.

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-β (Aβ) 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 Aβ associated glial transcriptomic signatures, we found myeloid Arg1 deficiency up-regulated glial gene transcripts that positively correlated with Aβ plaque burden. We also observed that Aβ 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ma, Hunt, Kovalenko, Liang, Selenica, Orr, Zhang, Gensel, Feola, Gordon, Morgan, Bickford and Lee.)

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 LysMCre iDTR 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.

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.

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., Competing Interests: Competing interest statement: J.Y. is a consultant for Denali Therapeutics and Sanofi. J.Y. and J.M.H. are co-authors on a 2018 Nomenclature review article.

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-β (Aβ) deposition, DS features a life-long overexpression of the APP and DYRK1A genes, leading to a cognitive decline mediated by Aβ 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-Aβ MAbs might promote the removal of early Aβ accumulation in the central nervous system of human APP-expressing mice. To this end, a DNA-vaccine expressing Aβ 1-11 was delivered to wild-type female mice, followed by mating with 5xFAD males, which exhibit early Aβ plaque formation. MAbs reduce the offspring's cortical Aβ 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 Aβ deposition, as occurs in EOAD and DS.

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  μ g/kg, i.p.) 30 min before or immediately after hypoxia (5% O 2 , 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- κ B 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., Competing Interests: The authors declared no conflict of interest., (Copyright © 2021 Xiaohui Chen et al.)

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-1β; 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.

Bupivacaine (Bup) has a certain research basis in pain-related diseases, but it has not been studied in painful diabetic neuropathy. In this study, we investigated the role of Bupivacaine in painful diabetic neuropathy. Mouse model with painful diabetic neuropathy was established, and then treated with different concentrations of Bupivacaine. The blood glucose level in the tail vein and the changes in body weight was measured. The mechanical allodynia, thermal hyperalgesia and thermal allodynia was assessed by pain behavioral tests. Microglia were treated with high glucose (HG) and different concentrations of Bupivacaine. The levels of inflammatory cytokines were detected by using Enzyme-linked immunosorbent assays. Dual luciferase reporter assay explored the relationship between miR-23a and phosphodiesterase 4 B (PDE4B). The results displayed that Bupivacaine ameliorated the mechanical allodynia, thermal hyperalgesia, and thermal allodynia in mice with painful diabetic neuropathy, and is more effective at low concentration. Moreover, low concentration of Bupivacaine inhibited inflammation and promoted miR-23a expression in mice with painful diabetic neuropathy and in microglia induced by HIGH GLUCOSE. Overexpression of miR-23a reduced the levels of inflammatory cytokines by down-regulating PDE4B expression. Knockdown of miR-23a reversed the inhibition effect of Bupivacaine on microglial inflammation. These results revealed that low concentration of Bupivacaine inhibited microglial inflammation through down-regulating PDE4B via miR-23a, thereby attenuated painful diabetic neuropathy., (Copyright © 2020. Published by Elsevier B.V.)

Microglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice had upregulated mTOR complex 1 signaling controlling translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual yet contrasting effect on microglia priming: it caused an NF-κB-dependent upregulation of priming genes at the mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation, and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating that upregulation of mTOR-dependent translation is an essential aspect of microglia priming in aging and neurodegeneration.

Microglial surveillance is a key feature of brain physiology and disease. Here, we found that G i -dependent microglial dynamics prevent neuronal network hyperexcitability. By generating Mg PTX mice to genetically inhibit G i in microglia, we show that sustained reduction of microglia brain surveillance and directed process motility induced spontaneous seizures and increased hypersynchrony after physiologically evoked neuronal activity in awake adult mice. Thus, G i -dependent microglia dynamics may prevent hyperexcitability in neurological diseases.

Subarachnoid hemorrhage (SAH) is an acute and severe disease with high disability and mortality. Inflammatory reactions have been proven to occur throughout SAH. Extracellular vesicles derived from mesenchymal stem cells (MSCs-EVs) have shown broad potential for the treatment of brain dysfunction and neuroprotective effects through neurogenesis and angiogenesis after stroke. However, the mechanisms of EVs in neuroinflammation during the acute phase of SAH are not well known. Our present study was designed to investigate the effects of MSCs-EVs on neuroinflammation and the polarization regulation of microglia to the M2 phenotype and related signaling pathways after SAH in rats. The SAH model was induced by an improved method of intravascular perforation, and MSCs-EVs were injected via the tail vein. Post-SAH assessments included neurobehavioral tests as well as brain water content, immunohistochemistry, PCR and Western blot analyses. Our results showed that MSCs-EVs alleviated the expression of inflammatory cytokines in the parietal cortex and hippocampus 24 h and 48 h after SAH and that MSCs-EVs inhibited NF-κB and activated AMPK to reduce inflammation after SAH. Furthermore, MSC-EVs regulated the polarization of microglia toward the M2 phenotype by downregulating interleukin-1β, cluster of differentiation 16, cluster of differentiation 11b, and inducible nitric oxide synthase and upregulating the expression of cluster of differentiation 206 and arginase-1. Additionally, MSCs-EVs inhibited the neuroinflammatory response and had neuroprotective effects in the brain tissues of rats after SAH. This study may support their use as a potential treatment strategy for early SAH in the future., (Copyright © 2020. Published by Elsevier Masson SAS.)

Abnormal activation of the transcriptional factor STAT3 (signal transducer and activator of transcription 3) was recently associated with Alzheimer Disease (AD). STAT3 phosphorylation is critical for cytokine secretion linked to neuroinflammation. Moreover, STAT3 may act as a transcriptional regulator of BACE1 (β-APP cleaving enzyme-1), the key enzyme in amyloid β (Aβ) production. We have previously shown that neuroinflammation and increased brain BACE1 levels triggered by LPS-induced systemic inflammation in wild-type mice are associated with an enhanced STAT3 activation. Using this LPS model, the goal of this study was to investigate if a STAT3 inhibitor administration could be protective against neuroinflammation and abnormal BACE1 regulation. Our results show that intraperitoneal injection of Stattic, a molecule that selectively inhibits the activation of STAT3, decreases LPS-induced microglial activation in the hippocampus. In addition, STAT3 inhibition reduced brain levels of cytokines IL-6, IL-1β and TNF-α triggered by LPS systemic administration. A significant reduction of BACE1 levels was observed in the hippocampus of mice treated with LPS and Stattic compared to those exposed to LPS alone. Taking together, our results show that Stattic can protect hippocampus against two pathological hallmarks of AD, and pave the way for further explorations of the therapeutic potential of STAT3 inhibition in AD., (Copyright © 2020 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Background: Neuroinflammation in the spinal cord following acute brachial plexus injury (BPI) remains a vital cause that leads to motor dysfunction and neuropathic pain. In this study, we aim to explore the role of long non-coding RNA JHDM1D antisense 1 (JHDM1D-AS1) in mediating BPI-induced neuroinflammation and neuronal injury., Methods: A total brachial plexus root avulsion (tBPRA) model in adult rats and IL-1β-treated motor neuron-like NSC-34 cells and LPS-treated microglia cell line BV2 were conducted for in vivo and in vitro experiments, respectively. The expressions of JHDM1D-AS1, miR-101-3p and DUSP1, p38, NF-κB, TNF-α, IL-1β, and IL-6 were detected by RT-PCR and western blot seven days after tBPI. Immunohistochemistry (IHC) was used to detect neuronal apoptosis. CCK8 assay, Tunel assay and LDH kit were used for the detection of neuronal injury. The targeted relationships between JHDM1D-AS1 and miR-101-3p, miR-101-3p and DUSP1 were verified by RNA immunoprecipitation (RIP) and dual-luciferase reporter gene assay., Results: We found significant downregulated expression of JHDM1D-AS1 and DUSP1 but upregulated expression of miR-101-3p in the spinal cord after tBPI. Overexpression of JHDM1D-AS1 had a prominent neuroprotective effect by suppressing neuronal apoptosis and microglial inflammation through reactivation of DUSP1. Further exploration revealed that JHDM1D-AS1 may act as a competitive endogenous RNA targeting miR-101-3p, which bound on the 3'UTR of DUSP1 mRNA. In addition, overexpression of miR-101-3p could reverse the neuroprotective effects of JHDM1D-AS1 upregulation by blocking DUSP1., Conclusions: JHDM1D-AS1 exerted neuroprotective and anti-inflammatory effects in a rat model of tBPI by regulating miR-101-3p/DUSP1 axis., (Copyright © 2020. Published by Elsevier B.V.)

Microglia are rapidly activated following ischaemic stroke and participate in the induction of neuroinflammation, which exacerbates the injury of ischaemic stroke. However, the mechanisms regulating ischaemic microglia remain unclear. In the present study, middle cerebral artery occlusion and oxygen and glucose deprivation models were established for in vivo and vitro monitoring of experimental stroke. We applied recombinant human thioredoxin-1 (rhTrx-1) and Necrostatin-1 (Nec-1, inhibitor of RIPK1) to examine the role of receptor-interacting protein kinase 1 (RIPK1) in the development of inflammation in ischaemic microglia via explored the inflammatory responses and the associated mechanisms. Molecular docking results indicated that rhTrx-1 could directly bind to RIPK1. In vivo and vitro data revealed that rhTrx-1 reduced necroptosis, mitochondrial membrane potential damage, reactive oxygen species accumulation and NLR Family, pyrin domain-containing 3 protein (NLRP3) inflammasome activation and regulated the microglial M1/M2 phenotypic changes by inhibiting RIPK1 expression in ischaemic microglia. Consistent with these findings, further in vivo experiments revealed that rhTrx-1 treatment attenuated cerebral ischaemic injury by inhibiting the inflammatory response. Our data demonstrated the role of RIPK1 in microglia-induced neuroinflammation following cerebral ischaemia. Administration of rhTrx-1 provides neuroprotection in ischaemic stroke-induced microglial neuroinflammation by inhibiting RIPK1 expression., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

N-Acylethanolamine acid amidase (NAAA) deactivates the endogenous peroxisome proliferator-activated receptor-α (PPAR-α) agonist palmitoylethanolamide (PEA). NAAA-regulated PEA signaling participates in the control of peripheral inflammation, but evidence suggests also a role in the modulation of neuroinflammatory pathologies such as multiple sclerosis (MS). Here we show that disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS is accompanied by induction of NAAA expression in spinal cord, which in presymptomatic animals is confined to motor neurons and oligodendrocytes but, as EAE progresses, extends to microglia/macrophages and other cell types. As previously reported for NAAA inhibition, genetic NAAA deletion delayed disease onset and attenuated symptom intensity in female EAE mice, suggesting that accrued NAAA expression may contribute to pathology. To further delineate the role of NAAA in EAE, we generated a mouse line that selectively overexpresses the enzyme in macrophages, microglia and other monocyte-derived cells. Non-stimulated alveolar macrophages from these Naaa CD11b+ mice contain higher-than-normal levels of inducible nitric oxide synthase and display an activated morphology. Furthermore, intranasal lipopolysaccharide injections cause greater alveolar leukocyte accumulation in Naaa CD11b+ than in control mice. Naaa CD11b+ mice also display a more aggressive clinical response to EAE induction, compared to their wild-type littermates. The results identify NAAA as a critical control step in EAE pathogenesis, and point to this enzyme as a possible target for the treatment of MS., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Neuroinflammation is critical for the pathogenesis of ischemia brain damage. Over-activated microglia-mediated inflammation plays a very important role in ischemia cerebral injuries. 6-Gingerol, obtained from edible ginger (Zingiber Officinale) exhibits protective effects against inflammation. In this study, we found that 6-Gingerol could reduce the size of infarction (P = 0.0184) and improve neurological functions (P = 0.04) at the third day after ischemic brain injury in vivo. Since 6-Gingerol has the anti-inflammatory effects, we further investigated its impacts on neuroinflammation mediated by microglia both in vivo and in vitro. We found that the levels of pro-inflammatory cytokines Interleukin-1 beta (IL-1β, P = 0.0213), Interleukin-6 (IL-6, P = 0.0316), and inducible NO synthase (iNOS, P = 0.0229) in the infarct penumbra were lower in 6-Gingerol treated groups. Furthermore, microglia induced pro-inflammatory cytokines, such as IL-6, IL-1β, incremental intercellular nitric oxide (NO), as well as iNOS were blocked by the treatment of 6-Gingerol in lipopolysaccharide (LPS) stimulated microglia. In terms of mechanism, 6-Gingerol potently suppressed phosphorylation of serine-threonine protein kinase (Akt) - mammalian target of rapamycin (mTOR) - signal transducer and activator of transcription 3 (STAT3) in LPS-treated microglia. Taken together, the present study suggested that 6-Gingerol improved cerebral ischemia injury by suppressing microglia-mediated neuroinflammation by down-regulating Akt-mTOR-STAT3 pathway., (Copyright © 2020. Published by Elsevier B.V.)

HIV-associated neurocognitive disorders (HAND) remain prevalent despite implementation of antiretroviral therapy (ART). Development of HAND is linked to mitochondrial dysfunction and oxidative stress in the brain; therefore, upregulation of antioxidant defenses is critical to curtail neuronal damage. Superoxide dismutase 2 (SOD2) is a mitochondrial antioxidant enzyme essential for maintaining cellular viability. We hypothesized that SOD2 was upregulated during retroviral infection. Using a simian immunodeficiency virus (SIV)-infected macaque model of HIV, quantitative PCR showed elevated SOD2 mRNA in cortical gray ([GM], 7.6-fold for SIV vs uninfected) and white matter ([WM], 77-fold for SIV vs uninfected) during SIV infection. Further, SOD2 immunostaining was enhanced in GM and WM from SIV-infected animals. Double immunofluorescence labeling illustrated that SOD2 primarily colocalized with astrocyte marker glial fibrillary acidic protein (GFAP) in SIV-infected animals. Interestingly, in ART-treated SIV-infected animals, brain SOD2 RNA levels were similar to uninfected animals. Additionally, using principal component analysis in a transcriptomic approach, SOD2 and GFAP expression separated SIV-infected from uninfected brain tissue. Projection of these data into a HIV dataset revealed similar expression changes, thereby validating the clinical relevance. Together, our findings suggest that novel SOD2-enhancing therapies may reduce neuroinflammation in ART-treated HIV-infected patients., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

Aberrant microglial activation drives neuroinflammation and neurodegeneration in Alzheimer's disease (AD). The present study is aimed at investigating whether the herbal formula Qi-Fu-Yin (QFY) could inhibit the inflammatory activation of cultured BV-2 microglia. A network pharmacology approach was employed to predict the active compounds of QFY, protein targets, and affected pathways. The representative pathways and molecular functions of the targets were analyzed by Gene Ontology (GO) and pathway enrichment. A total of 145 active compounds were selected from seven herbal ingredients of QFY. Targets (e.g., MAPT, APP, ACHE, iNOS, and COX-2) were predicted for the selected active compounds based on the relevance to AD and inflammation. As a validation, fractions were sequentially prepared by aqueous extraction, ethanolic precipitation, and HPLC separation, and assayed for downregulating two key proinflammatory biomarkers iNOS and COX-2 in lipopolysaccharide- (LPS-) challenged BV-2 cells by the Western blotting technique. Moreover, the compounds of QFY in 90% ethanol downregulated iNOS in BV-2 cells but showed no activity against COX-2 induction. Among the herbal ingredients of QFY, Angelicae Sinensis Radix and Ginseng Radix et Rhizoma contributed to the selective inhibition of iNOS induction. Furthermore, chemical analysis identified ginsenosides, especially Rg3, as antineuroinflammatory compounds. The herbal formula QFY may ameliorate neuroinflammation via downregulating iNOS in microglia., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Fung Yin Ngo et al.)

Ischemic stroke is a major cause of death and disability worldwide. Necroptosis is known as a form of cell death, playing an essential role in regulating ischemia-induced brain injury. Triad3A is a ubiquitin ligase of the RING-in-between-RING family, and regulates necroptotic cell death under different pathological conditions, including neurodegenerative disorders. In the present study, the effects of Triad3A on experimental stroke were explored on a mouse model with middle cerebral artery occlusion (MCAO). The results indicated that Triad3A expression was markedly induced in the ischemic brain after MCAO operation. The neurons and microglia cells were the major cellular sources for Triad3A induction. Triad3A knockdown enhanced the infarction area, cell death, microglia activity, and the expression levels of pro-inflammatory markers including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS), CD32 and CD68 in MCAO mice. Triad3A and necroptosis were triggered in mouse microglia cells treated with oxygen and glucose deprivation (OGD), and in TNFα-incubated mouse hippocampal neuronal cells treated with Z-VAD-fmk, known as a pan-caspase inhibitor. Moreover, Triad3A knockdown accelerated cell death in microglial cells and neurons under these stresses. Furthermore, pre-treatment with necroptosis inhibitor markedly inhibited the cell death promoted by Triad3A silence in brain of mice with MCAO operation, demonstrating that Triad3A could regulate necroptosis to meditate the progression of cerebral I/R injury. Collectively, these finding illustrated that Triad3A could be served as a potential target for stroke therapy., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work., (Copyright © 2020. Published by Elsevier Masson SAS.)

Neurological disorders are associated with increased oxidative stress. Reactive oxidants damage tissue and promote cell death, but it is apparent that oxidants can have more subtle effects on cell function through the modulation of redox-sensitive signalling pathways. Cells of the blood-brain barrier regulate the brain microenvironment but become dysfunctional during neurological disease. The blood-brain barrier is maintained by many cell types, and is modulated by redox-sensitive pathways, ranging from the cytoskeletal elements responsible for establishing a barrier, to growth factor and cytokine signalling pathways that influence neurovascular cells. During neurological disease, blood-brain barrier cells are exposed to exogenously generated oxidants from immune cells, as well as increasing endogenously oxidant production. These oxidants impair the function of the blood-brain barrier, leading to increased leakage and reduced blood flow. Reducing the impact of oxidants on the function of blood-brain barrier cells may provide new strategies for delaying the progression of neurological disease., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Leucine-rich repeat kinase 2 ( LRRK2 ) encodes a complex protein that includes kinase and GTPase domains. Genome-wide association studies have identified dominant LRRK2 alleles that predispose their carriers to late-onset idiotypic Parkinson's disease (PD) and also to autoimmune disorders such as Crohn's disease. Considerable evidence indicates that PD initiation and progression involve activation of innate immune functions in microglia, which are brain-resident macrophages. Here we asked whether LRRK2 modifies inflammatory signaling and how this modification might contribute to PD and Crohn's disease. We used RNA-Seq-based high-resolution transcriptomics to compare gene expression in activated primary macrophages derived from WT and Lrrk2 knockout mice. Remarkably, expression of a single gene, Rap guanine nucleotide exchange factor 3 ( Rapgef3 ), was strongly up-regulated in the absence of LRRK2 and down-regulated in its presence. We observed similar regulation of Rapgef3 expression in cells treated with a highly specific inhibitor of LRRK2 protein kinase activity. Rapgef3 encodes an exchange protein, activated by cAMP 1 (EPAC-1), a guanine nucleotide exchange factor that activates the small GTPase Rap-1. Rap-1 mediates cell adhesion, polarization, and directional motility, and our results indicate that LRRK2 modulates chemotaxis of microglia and macrophages. Dominant PD-associated LRRK2 alleles may suppress EPAC-1 activity, further restricting motility and preventing efficient migration of microglia to sites of neuronal damage. Functional analysis in vivo in a subclinical infection model also indicated that Lrrk2 subtly modifies the inflammatory response. These results indicate that LRRK2 modulates the expression of genes involved in murine immune cell chemotaxis., (© 2020 Levy et al.)

The pro-inflammatory immune response driven by microglia is a key contributor to the pathogenesis of several neurodegenerative diseases. Though the research of microglia spans over a century, the last two decades have increased our understanding exponentially. Here, we discuss the phenotypic transformation from homeostatic microglia towards reactive microglia, initiated by specific ligand binding to pattern recognition receptors including toll-like receptor-4 (TLR4) or triggering receptors expressed on myeloid cells-2 (TREM2), as well as pro-inflammatory signaling pathways triggered such as the caspase-mediated immune response. Additionally, new research disciplines such as epigenetics and immunometabolism have provided us with a more holistic view of how changes in DNA methylation, microRNAs, and the metabolome may influence the pro-inflammatory response. This review aimed to discuss our current knowledge of pro-inflammatory microglia from different angles, including recent research highlights such as the role of exosomes in spreading neuroinflammation and emerging techniques in microglia research including positron emission tomography (PET) scanning and the use of human microglia generated from induced pluripotent stem cells (iPSCs). Finally, we also discuss current thoughts on the impact of pro-inflammatory microglia in neurodegenerative diseases.

Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms., Competing Interests: The authors declare no competing interest.

Long noncoding RNAs (lncRNAs) have been involved in the development of multiple pathological processes including neuropathic pain. The aim of the present study is to investigate the role of lncRNA down-regulated in liver cancer stem cells (DILC) in the progression of neuropathic pain and its underlying mechanism. Neuropathic pain rat model was established with the bilateral chronic constriction injury (bCCI) method. The results from quantitative PCR analysis in the spinal cord showed that DILC was significantly up-regulated in rats with bCCI compared with the sham group. DILC down-regulation mediated by intrathecal administration of DILC siRNA significantly increased the mechanical shrinkage threshold (MWT) and paw withdrawal threshold latency (PWTL), decreased the positive frequency for nerve sensitivity to cold and suppressed the expression of inflammatory genes in bCCI rats. Down-regulation of DILC induced suppressor of cytokine signaling (SOCS3) expression and inhibited the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) in spinal cord tissues. Western blotting showed that down-regulation of DILC by DILC siRNA transfection induced SOCS3 expression and inhibited the expression of p-Janus kinase 2 (p-JAK2) and p-STAT3 and their downstream genes in primary microglia. Furthermore, down-regulation of DILC increased the viability of primary microglia, suppressed apoptosis, and inhibited the production of interleukin (IL)-6 and IL-1β in microglia. In contrast, overexpression of DILC showed the opposite functions to those of DILC knockdown. In conclusion, silence of lncRNA DILC attenuates neuropathic pain via SOCS3-induced suppression of the JAK2/STAT3 pathway., (© 2020 The Author(s).)

Hyperactivation of microglia in the brain is closely related to neuroinflammation and leads to neuronal dysfunction. Costunolide (CTL) is a natural sesquiterpene lactone with wide pharmacological activities including anti-inflammation and antioxidation. In this study, we found that CTL significantly inhibited the production of inflammatory mediators including nitric oxide, IL-6, TNF-α, and PGE2 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Moreover, CTL effectively attenuated IKKβ/NF-κB signaling pathway activation. To identify direct cellular target of CTL, we performed high-throughput reverse virtual screening assay using scPDB protein structure library, and found cyclin-dependent kinase 2 (CDK2) was the most specific binding protein for CTL. We further confirmed the binding ability of CTL with CDK2 using cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) assays. Surface plasmon resonance analysis also supported that CTL specifically bound to CDK2 with a dissociation constant at micromole level. Furthermore, knocking down CDK2 obviously reversed the anti-inflammation effect of CTL via AKT/IKKβ/NF-κB signaling pathway on BV-2 cells. Collectively, these results indicate that CTL inhibits microglia-mediated neuroinflammation through directly targeting CDK2, and provide insights into the role of CDK2 as a promising anti-neuroinflammation therapeutic target.

HIV-1 Associated Neurocognitive Disorder (HAND) is a common and clinically detrimental complication of HIV infection. Viral proteins, including Tat, released from infected cells, cause neuronal toxicity. Substance abuse in HIV-infected patients greatly influences the severity of neuronal damage. To repurpose small molecule inhibitors for anti-HAND therapy, we employed MOLIERE, an AI-based literature mining system that we developed. All human genes were analyzed and prioritized by MOLIERE to find previously unknown targets connected to HAND. From the identified high priority genes, we narrowed the list to those with known small molecule ligands developed for other applications and lacking systemic toxicity in animal models. To validate the AI-based process, the selective small molecule inhibitor of DDX3 helicase activity, RK-33, was chosen and tested for neuroprotective activity. The compound, previously developed for cancer treatment, was tested for the prevention of combined neurotoxicity of HIV Tat and cocaine. Rodent cortical cultures were treated with 6 or 60 ng/ml of HIV Tat and 10 or 25 μM of cocaine, which caused substantial toxicity. RK-33 at doses as low as 1 μM greatly reduced the neurotoxicity of Tat and cocaine. Transcriptome analysis showed that most Tat-activated transcripts are microglia-specific genes and that RK-33 blocks their activation. Treatment with RK-33 inhibits the Tat and cocaine-dependent increase in the number and size of microglia and the proinflammatory cytokines IL-6, TNF-α, MCP-1/CCL2, MIP-2, IL-1α and IL-1β. These findings reveal that inhibition of DDX3 may have the potential to treat not only HAND but other neurodegenerative diseases. Graphical Abstract RK-33, selective inhibitor of Dead Box RNA helicase 3 (DDX3) protects neurons from combined Tat and cocaine neurotoxicity by inhibition of microglia activation and production of proinflammatory cytokines.

In the central nervous system, activated microglia and astrocytes produce proinflammatory mediators such as inducible nitric oxide (iNOS) and cytokines. Uncontrolled release of these mediators induced by immune challenge can lead to increased vulnerability to complex brain disorders such as schizophrenia. In this study, BALB/c mice were injected intraperitoneally (i.p) with the viral mimetic polyriboinosinic-polyribocytidilic acid (poly(I:C)) or saline. At postnatal day 30 (PND0), the animals were sacrificed and the hippocampus, corpus callosum, striatum, cortex, fimbria and ventricle were immunostained for Iba-1, a microglial marker, glial fibrillary acidic protein (GFAP), an astrocyte marker, and iNOS, an activation marker for NO. Additionally, serum cytokine profiling (Interleukin-2 (IL-2), IL- 4, IL-6, interferon gamma (IFN-γ), tumour necrosis factor (TNF), IL-17A and IL-10) was determined using serum samples from poly(I:C)-treated and control mice. Our results demonstrated that poly(I:C) induced overactivation of differential proinflammatory responses in microglia and astrocytes, which could be strongly enhanced by a postnatal poly(I:C) administration before PND 30 in one part of the animals investigated. Specifically, there was significant iNOS upregulation in hippocampus, cortex and corpus callosum of poly(I:C)-affected off-springs. These inflammatory alterations were accompanied by increased circulating levels of the proinflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). This study provides insight into the role of microglia and astrocytes in an animal model of schizophrenia and an understanding of the regulation of iNOS expression in glial cells and cytokine networks. This knowledge could help identify novel targets for anti-oxidative and anti-inflammatory therapeutic schizophrenia intervention., Competing Interests: Conflict of interests We all declare to have no conflict of interests with regards to this work., (Copyright © 2020. Published by Elsevier B.V.)

Failure of remyelination underlies the progressive nature of demyelinating diseases such as multiple sclerosis. Macrophages and microglia are crucially involved in the formation and repair of demyelinated lesions. Here we show that myelin uptake temporarily skewed these phagocytes toward a disease-resolving phenotype, while sustained intracellular accumulation of myelin induced a lesion-promoting phenotype. This phenotypic shift was controlled by stearoyl-CoA desaturase-1 (SCD1), an enzyme responsible for the desaturation of saturated fatty acids. Monounsaturated fatty acids generated by SCD1 reduced the surface abundance of the cholesterol efflux transporter ABCA1, which in turn promoted lipid accumulation and induced an inflammatory phagocyte phenotype. Pharmacological inhibition or phagocyte-specific deficiency of Scd1 accelerated remyelination ex vivo and in vivo. These findings identify SCD1 as a novel therapeutic target to promote remyelination., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Bogie et al.)

Microglial dysregulation, pertaining to impairment in phagocytosis, clearance and containment of amyloid-β (Aβ), and activation of neuroinflammation, has been posited to contribute to the pathogenesis of Alzheimer's disease (AD). Detailed cellular mechanisms that are disrupted during the disease course to display such impairment in microglia, however, remain largely undetermined. We hypothesize that loss of hematopoietic cell kinase (HCK), a phagocytosis-regulating member of the Src family tyrosine kinases that mediate signals from triggering receptor expressed on myeloid cells 2 and other immunoreceptors, impairs microglial homeostasis and Aβ clearance, leading to the accelerated buildup of Aβ pathology and cognitive decline during the early stage of neuropathological development. To elucidate the pivotal role of HCK in AD, we generated a constitutive knockout of HCK in the Tg2576 mouse model of AD. We found that HCK deficiency accelerated cognitive decline along with elevated Aβ level and plaque burden, attenuated microglial Aβ phagocytosis, induced iNOS expression in microglial clusters, and reduced pre-synaptic protein at the hippocampal regions. Our findings substantiate that HCK plays a prominent role in regulating microglial neuroprotective functions and attenuating early AD neuropathology.

The excellent optical property and relatively low toxicity of CdTe/ZnS core/shell quantum dots (QDs) make them an advanced fluorescent probe in the application of biomedicines, particularly in neuroscience. Thus, it is important to evaluate the biosafety of CdTe/ZnS QDs on the central nervous system (CNS). Our previous studies have suggested that the high possibility of CdTe/ZnS QDs being transported into the brain across the blood-brain barrier resulted in microglial activation and a shift of glycometabolism, but their underlying mechanism remains unclear. In this study, when mice were injected intravenously with CdTe/ZnS QDs through tail veins, the microglial activation, polarized into both M1 phenotype and M2 phenotype, and the neuronal impairment were observed in the hippocampus. Meanwhile, the increased pro- and anti-inflammatory cytokines released from BV2 microglial cells treated with CdTe/ZnS QDs also indicated that QD exposure was capable of inducing microglial activation in vitro. We further demonstrated that the glycolytic shift from oxidative phosphorylation switching into aerobic glycolysis was required in the microglial activation into M1 phenotype induced by CdTe/ZnS QD treatment, which was mediated through the mTOR signaling pathway. The findings, taken together, provide a mechanistic insight regarding the CdTe/ZnS QDs inducing microglial activation and the role of the glycolytic shift in it., (© 2019 John Wiley & Sons, Ltd.)

Neurodegenerative diseases are characterized by neuronal degeneration as well as neuroinflammation. While CD38 is strongly expressed in brain cells including neurons, astrocytes as well as microglial cells, the role played by CD38 in neurodegeneration and neuroinflammation remains elusive. Yet, CD38 expression increases as a consequence of aging which is otherwise the primary risk associated with neurodegenerative diseases, and several experimental data demonstrated that CD38 knockout mice are protected from neurodegenerative and neuroinflammatory insults. Moreover, nicotinamide adenine dinucleotide, whose levels are tightly controlled by CD38, is a recognized and potent neuroprotective agent, and NAD supplementation was found to be beneficial against neurodegenerative diseases. The aims of this review are to summarize the physiological role played by CD38 in the brain, present the arguments indicating the involvement of CD38 in neurodegeneration and neuroinflammation, and to discuss these observations in light of CD38 complex biology., Competing Interests: S.G, L.B., D.T. are founders and shareholders of Encefa. A.-L.P. is employed by Encefa. Encefa is a biotechnology company developing an anti-CD38 antibody against neurodegenerative diseases.

Cerebral ischemia induces a robust neuroinflammatory response that is largely mediated by the activation of CNS resident microglia. Activated microglia produce pro-inflammatory molecules to cause neuronal damage. Identifying regulators of microglial activation bears great potential in discovering promising candidates for neuroprotection post cerebral ischemia. Previous studies demonstrate abnormal elevation of glutaminase 1 (GLS1) in microglia in chronic CNS disorders including Alzheimer's disease and HIV-associated neurocognitive disorders. Ectopic expression of GLS1 induced microglia polarization into pro-inflammatory phenotype and exosome release in vitro . However, whether GLS1 is involved in neuroinflammation in acute brain injury remains unknown. Here, we observed activation of microglia, elevation of GLS1 expression, and accumulation of pro-inflammatory exosomes in rat brains 72 h post focal cerebral ischemia. Treatment with CB839, a glutaminase inhibitor, reversed ischemia-induced microglial activation, inflammatory response, and exosome release. Furthermore, we found that the application of exosome secretion inhibitor, GW4869, displayed similar anti-inflammatory effects to that of CB839, suggesting GLS1-mediated exosome release may play an important role in the formation of neuroinflammatory microenvironment. Therefore, GLS1 may serve as a key mediator and promising target of neuroinflammatory response in cerebral ischemia., (Copyright © 2020 Gao, Li, Zhu, Wang, Huang, Zhao, Sheng, Song, Ji, Li, Yang, Ye, Qi, Zhang, Xia and Zheng.)

Literature studies suggest important protective effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on inflammatory pathways affecting joint and cerebral diseases. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. Therefore the aim of this study was to identify the molecular targets of PEMFs anti-neuroinflammatory action. The effects of PEMF exposure in cytokine production by lipopolysaccharide (LPS)-activated N9 microglial cells as well as the pathways involved, including adenylyl cyclase (AC), phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and delta (PKC-δ), p38, ERK1/2, JNK1/2 mitogen activated protein kinases (MAPK), Akt and caspase 1, were investigated. In addition, the ability of PEMFs to modulate ROS generation, cell invasion and phagocytosis, was addressed. PEMFs reduced the LPS-increased production of TNF-α and IL-1β in N9 cells, through a pathway involving JNK1/2. Furthermore, they decreased the LPS-induced release of IL-6, by a mechanism not dependent on AC, PLC, PKC-ε, PKC-δ, p38, ERK1/2, JNK1/2, Akt and caspase 1. Importantly, a significant effect of PEMFs in the reduction of crucial cell functions specific of microglia like ROS generation, cell invasion and phagocytosis was found. PEMFs inhibit neuroinflammation in N9 cells through a mechanism involving, at least in part, the activation of JNK MAPK signalling pathway and may be relevant to treat a variety of diseases characterized by neuroinflammation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Peripheral nerve injury elicits spinal microgliosis, contributing to neuropathic pain. The aurora kinases A (AURKA), B (AURKB), and C (AURKC) are potential therapeutic targets in proliferating cells. However, their role has not been clarified in microglia. The aim of this study was to examine the regulation of aurora kinases and their roles and druggability in spinal microgliosis and neuropathic pain. Sprague-Dawley rats received chronic constriction injury (CCI). Gene expression of aurora kinases A-C was evaluated by quantitative RT-PCR and western blot, respectively, in spinal cords at 1, 3, 7, and 14 days after CCI. AURKB gene and protein expression was up-regulated concomitantly with the development of spinal microgliosis and neuropathic pain. Using lentiviral over-expression and adeno-associated viral knockdown approaches, the function of AURKB was further investigated by western blot, immunohistochemistry, RNA sequencing, and pain behavior tests. We found that AURKB over-expression in naive rats caused spinal microgliosis and pain hypersensitivity, whereas AURKB knockdown reduced microgliosis and alleviated CCI-induced neuropathic pain. Accordingly, RNA sequencing data revealed down-regulation of genes critically involved in signaling pathways associated with spinal microgliosis and neuropathic pain after AURKB knockdown in CCI rats. To examine its therapeutic potential for treatment of neuropathic pain, animals were treated intrathecally with the pharmacological AURKB inhibitor AZD1152-HQPA resulting in the alleviation of CCI-induced pain. Taken together, our findings indicated that AURKB plays a critical role in spinal microgliosis and neuropathic pain. Targeting AURKB may be an efficient method for treatment of neuropathic pain subsequent to peripheral nerve injury., (© 2019 International Society for Neurochemistry.)

Background: Inflammation can induce cognitive dysfunction in patients who undergo surgery. Previous studies have demonstrated that both acute peripheral inflammation and anaesthetic insults, especially isoflurane (ISO), are risk factors for memory impairment. Few studies are currently investigating the role of ISO under acute peri-inflammatory conditions, and it is difficult to predict whether ISO can aggravate inflammation-induced cognitive deficits. HDACs, which are essential for learning, participate in the deacetylation of lysine residues and the regulation of gene transcription. However, the cell-specific mechanism of HDACs in inflammation-induced cognitive impairment remains unknown., Methods: Three-month-old C57BL/6 mice were treated with single versus combined exposure to LPS injected intraperitoneally (i.p.) to simulate acute abdominal inflammation and isoflurane to investigate the role of anaesthesia and acute peripheral inflammation in cognitive impairment. Behavioural tests, Western blotting, ELISA, immunofluorescence, qRT-PCR, and ChIP assays were performed to detect memory, the expressions of inflammatory cytokines, HDAC2, BDNF, c-Fos, acetyl-H3, microglial activity, Bdnf mRNA, c-fos mRNA, and Bdnf and c-fos transcription in the hippocampus., Results: LPS, but not isoflurane, induced neuroinflammation-induced memory impairment and reduced histone acetylation by upregulating histone deacetylase 2 (HDAC2) in dorsal hippocampal CaMKII + neurons. The hyperexpression of HDAC2 in neurons was mediated by the activation of microglia. The decreased level of histone acetylation suppressed the transcription of Bdnf and c-fos and the expressions of BDNF and c-Fos, which subsequently impaired memory. The adeno-associated virus ShHdac2, which suppresses Hdac2 after injection into the dorsal hippocampus, reversed microglial activation, hippocampal glutamatergic BDNF and c-Fos expressions, and memory deficits., Conclusions: Reversing HDAC2 in hippocampal CaMKII + neurons exert a neuroprotective effect against neuroinflammation-induced memory deficits.

Trained immunity and immune tolerance have been identified as long-term response patterns of the innate immune system. The causes of these opposing reactions remain elusive. Here, we report about differential inflammatory responses of microglial cells derived from neonatal mouse brain to increasing doses of the endotoxin LPS. Prolonged priming with ultra-low LPS doses provokes trained immunity, i.e., increased production of pro-inflammatory mediators in comparison to the unprimed control. In contrast, priming with high doses of LPS induces immune tolerance, implying decreased production of inflammatory mediators and pronounced release of anti-inflammatory cytokines. Investigation of the signaling processes and cell functions involved in these memory-like immune responses reveals the essential role of phosphoinositide 3-kinase γ (PI3Kγ), one of the phosphoinositide 3-kinase species highly expressed in innate immune cells. Together, our data suggest profound influence of preceding contacts with pathogens on the immune response of microglia. The impact of these interactions-trained immunity or immune tolerance-appears to be shaped by pathogen dose., (Copyright © 2019 Lajqi, Lang, Haas, Williams, Hudalla, Bauer, Groth, Wetzker and Bauer.)