Microglial Neuroinflammation Research Articles

Microglial circ-UBE2K exacerbates depression by regulating parental gene UBE2K via targeting HNRNPU.

Background: Knowledge about the pathogenesis of depression and treatments for this disease are lacking. Epigenetics-related circRNAs are likely involved in the mechanism of depression and have great potential as treatment targets, but their mechanism of action is still unclear. Methods: Circular RNA UBE2K (circ-UBE2K) was screened from peripheral blood of patients with major depressive disorder (MDD) and brain of depression model mice through high-throughput sequencing. Microinjection of circ-UBE2K overexpression lentivirus and adeno-associated virus for interfering with microglial circ-UBE2K into the mouse hippocampus was used to observe the role of circ-UBE2K in MDD. Sucrose preference, forced swim, tail suspension and open filed tests were performed to evaluate the depressive-like behaviors of mice. Immunofluorescence and Western blotting analysis of the effects of circ-UBE2K on microglial activation and immune inflammation. Pull-down-mass spectrometry assay, RNA immunoprecipitation (RIP) test and fluorescence in situ hybridization (FISH) were used to identify downstream targets of circ-UBE2K/ HNRNPU (heterogeneous nuclear ribonucleoprotein U) axis. Results: In this study, through high-throughput sequencing and large-scale screening, we found that circ-UBE2K levels were significantly elevated both in the peripheral blood of patients with MDD and in the brains of depression model mice. Functionally, circ-UBE2K-overexpressing mice exhibited worsened depression-like symptoms, elevated brain inflammatory factor levels, and abnormal microglial activation. Knocking down circ-UBE2K mitigated these changes. Mechanistically, we found that circ-UBE2K binds to heterogeneous nuclear ribonucleoprotein U (HNRNPU) to form a complex that upregulates the expression of the parental gene ubiquitin conjugating enzyme E2 K (UBE2K), leading to abnormal microglial activation and neuroinflammation and promoting the occurrence and development of depression. Conclusions: The findings of the present study revealed that the expression of circUBE2K, which combines with HNRNPU to form the circUBE2K/HNRNPU complex, is increased in microglia after external stress, thus regulating the expression of the parental gene UBE2K and mediating the abnormal activation of microglia to induce neuroinflammation, promoting the development of MDD. These results indicate that circ-UBE2K plays a newly discovered role in the pathogenesis of depression.

N-Glycan profile of the cell membrane as a probe for lipopolysaccharide-induced microglial neuroinflammation uncovers the effects of common fatty acid supplementation.

Altered N-glycosylation of proteins on the cell membrane is associated with several neurodegenerative diseases. Microglia are an ideal model for studying glycosylation and neuroinflammation, but whether aberrant N-glycosylation in microglia can be restored by diet remains unknown. Herein, we profiled the N-glycome, proteome, and glycoproteome of the human microglia following lipopolysaccharide (LPS) induction to probe the impact of dietary and gut microbe-derived fatty acids-oleic acid, lauric acid, palmitic acid, valeric acid, butyric acid, isobutyric acid, and propionic acid-on neuroinflammation using liquid chromatography-tandem mass spectrometry. LPS changed N-glycosylation in the microglial glycocalyx altering high mannose and sialofucosylated N-glycans, suggesting the dysregulation of mannosidases, fucosyltransferases, and sialyltransferases. The results were consistent as we observed the restoration effect of the fatty acids, especially oleic acid, on the LPS-treated microglia, specifically on the high mannose and sialofucosylated glycoforms of translocon-associated proteins, SSRA and SSRB along with the cell surface proteins, CD63 and CD166. In addition, proteomic analysis and in silico modeling substantiated the potential of fatty acids in reverting the effects of LPS on microglial N-glycosylation. Our results showed that N-glycosylation is likely affected by diet by restoring alterations following LPS challenge, which may then influence the disease state.

ROLE OF MICROGLIA IN SEPSIS-ASSOCIATED ENCEPHALOPATHY PATHOGENESIS: AN UPDATE.

Sepsis-associated encephalopathy (SAE) is a serious complication of sepsis, which is characterized by cognitive dysfunction, a poor prognosis, and high incidences of morbidity and mortality. Substantial levels of systemic inflammatory factors induce neuroinflammatory responses during sepsis, ultimately disrupting the central nervous system's (CNS) homeostasis. This disruption results in brain dysfunction through various underlying mechanisms, contributing further to SAE's development. Microglia, the most important macrophage in the CNS, can induce neuroinflammatory responses, brain tissue injury, and neuronal dysregulation, resulting in brain dysfunction. They serve an important regulatory role in CNS homeostasis and can be activated through multiple pathways. Consequently, activated microglia are involved in several pathogenic mechanisms related to SAE and play a crucial role in its development. This article discusses the role of microglia in neuroinflammation, dysfunction of neurotransmitters, disruption of the blood-brain barrier, abnormal control of cerebral blood flow, mitochondrial dysfunction, and reduction in the number of good bacteria in the gut as main pathogenic mechanisms of SAE and focuses on studies targeting microglia to ameliorate SAE to provide a theoretical basis for targeted microglial therapy for SAE.

Central activation of the fatty acid sensor GPR120 suppresses microglia reactivity and alleviates sickness- and anxiety-like behaviors

G protein-coupled receptor 120 (GPR120, Ffar4) is a sensor for long-chain fatty acids including omega-3 polyunsaturated fatty acids (n-3 PUFAs) known for beneficial effects on inflammation, metabolism, and mood. GPR120 mediates the anti-inflammatory and insulin-sensitizing effects of n-3 PUFAs in peripheral tissues. The aim of this study was to determine the impact of GPR120 stimulation on microglial reactivity, neuroinflammation and sickness- and anxiety-like behaviors by acute proinflammatory insults. We found GPR120 mRNA to be enriched in both murine and human microglia, and in situ hybridization revealed GPR120 expression in microglia of the nucleus accumbens (NAc) in mice. In a manner similar to or exceeding n-3 PUFAs, GPR120 agonism (Compound A, CpdA) strongly attenuated lipopolysaccharide (LPS)-induced proinflammatory marker expression in primary mouse microglia, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and inhibited nuclear factor-ĸB translocation to the nucleus. Central administration of CpdA to adult mice blunted LPS-induced hypolocomotion and anxiety-like behavior and reduced TNF-α, IL-1β and IBA-1 (microglia marker) mRNA in the NAc, a brain region modulating anxiety and motivation and implicated in neuroinflammation-induced mood deficits. GPR120 agonist pre-treatment attenuated NAc microglia reactivity and alleviated sickness-like behaviors elicited by central injection TNF-α and IL-1β. These findings suggest that microglial GPR120 contributes to neuroimmune regulation and behavioral changes in response to acute infection and elevated brain cytokines. GPR120 may participate in the protective action of n-3 PUFAs at the neural and behavioral level and offers potential as treatment target for neuroinflammatory conditions.

Sestrin2 attenuates depressive-like behaviors and neuroinflammation in CUMS mice through inhibiting ferroptosis.

Sestrin2 (SESN2) is a stress-inducible protein and acts as a neuroprotective regulator. The present study aimed to explore the antidepressant activity of SESN2 and its relevant mechanism. Depression mouse model was established by chronic unpredictable mild stress (CUMS) for a successive 5 weeks. Behaviors tests were conducted to examine depressive-like behaviors including sugar preference test, tail suspension test and open field test. The expression of SESN2 and ferroptosis-related proteins was examined by western blot. The production of cytokines was measured by ELISA. Iron deposition was assessed using Prussian blue staining and Fe 2+ content was measured using commercial kits. Lipid peroxidation was evaluated by thiobarbituric acid reactive substances assay. BV-2 cells were treated with LPS to induce microglial activation, which was evaluated by the iba-1 level adopting immunofluorescence assay. The ferroptosis inducer Erastin was adopted for the pretreatment in BV-2 cells to conduct a rescue experiment. SESN2 was downregulated in CUMS-induced mice, and SESN2 overexpression dramatically ameliorated CUMS-induced depression-like behaviors. Meanwhile, SESN2 reduced the production of pro-inflammatory cytokines and iba-1 level in hippocampus of CUMS mice, as well as reducing iron deposition and lipid peroxidation, demonstrating that SESN2 reduced microglial activation, neuroinflammation and ferroptosis in CUMS mice. Similarly, SESN2 also restricted iba-1 level, pro-inflammatory cytokines production, and ferroptosis in LPS-induced BV-2 cells, which was partly reversed by additional treatment of Erastin. These findings suggest that SESN2 possesses potent antidepressant property through inhibiting ferroptosis and neuroinflammation.

Mesenchymal stromal cells suppress microglial activation and tumor necrosis factor production

Background aimsWhite matter diseases are commonly associated with microglial activation and neuroinflammation. Mesenchymal stromal cells (MSCs) have immunomodulatory properties and thus have the potential to be developed as cell therapy for white matter disease. MSCs interact with resident macrophages to alter the trajectory of inflammation; however, the impact MSCs have on central nervous system macrophages and the effect this has on the progression of white matter disease are unclear. MethodsIn this study, we utilized numerous assays of varying complexity to model different aspects of white matter disease. These assays ranged from an in vivo spinal cord acute demyelination model to a simple microglial cell line activation assay. Our goal was to investigate the influence of human umbilical cord tissue MSCs on the activation of microglia. ResultsMSCs reduced the production of tumor necrosis factor (TNF) by microglia and decreased demyelinated lesions in the spinal cord after acute focal injury. To determine if MSCs could directly suppress the activation of microglia and to develop an efficient potency assay, we utilized isolated primary microglia from mouse brains and the Immortalized MicroGlial Cell Line (IMG). MSCs suppressed the activation of microglia and the release of TNF after stimulation with lipopolysaccharide, a toll-like receptor agonist. ConclusionsIn this study, we demonstrated that MSCs altered the immune response after acute injury in the spinal cord. In numerous assays, MSCs suppressed activation of microglia and release of the pro-inflammatory cytokine TNF. Of these assays, IMG could be standardized and used as an effective potency assay to determine the efficacy of MSCs for treating white matter disease or other neuroinflammatory conditions associated with microglial activation.

Low-intensity pulsed ultrasound treatment mitigates ventricular arrhythmias via inhibiting microglia-mediated neuroinflammation in heart failure rat model

BackgroundSympathetic overactivation plays an important role in heart failure (HF)-induced ventricular arrhythmias (VAs). Microglia-mediated neuroinflammation could contribute to sympathetic overactivation. A previous study demonstrated that low-intensity pulsed ultrasound (LIPUS) could inhibit neuroinflammation. However, whether LIPUS could attenuate HF-induced VAs via inhibiting microglia-mediated neuroinflammation remains largely unknown. MethodsForth Sprague-Dawley male rats were averagely randomized into four groups: CTL (control) group, CTL + LIPUS group, HF group and HF + LIPUS. Surgical ligation of the coronary artery was used for induction of HF. In vivo electrophysiological study was performed to check VAs susceptibility. Left stellate ganglion (LSG) neural activity and heart rate variability (HRV) were used to test sympathetic nerve activity. ResultsCompared to the HF group, LIPUS treatment significantly ameliorated HF-induced cardiac hypertrophy, fibrosis, and dysfunction. In addition, LIPUS treatment markedly inhibited HF-induced VAs susceptibility and reversed gap junction remodeling. LIPUS treatment obviously inhibited microglial activation and neuroinflammation in PVN, sympathetic hyperactivity in the LSG and proinflammatory cytokines releases in the ventricle. P2X7/NLRP3 signaling pathway may be involved in the anti-arrhythmic effect of LIPUS treatment following HF. ConclusionsOur data demonstrated that LIPUS treatment protected against HF-induced VAs via alleviating microglia-mediated neuroinflammation, sympathetic overactivation and proinflammatory cytokines releases through inhibiting P2X7/NLRP3 signaling. This study provides novel insight into the therapeutic potential of LIPUS.

Neurotropin Alleviates Bortezomib-Induced Neurotoxicity Via Microglial Neuroinflammation

Objective: To investigate the effect of neurotropin (NTP) on the prevention and amelioration of neurotoxicity induced by bortezomib (BTZ)-based chemotherapy regimens and the possible mechanism of action, and to provide new ideas for the prevention mechanism and treatment of bortezomib-induced peripheral neuropathy (BIPN). Methods: The BIPN model was constructed by injecting bortezomib （ BTZ group ）or bortezomib+saline ( BTZ+NS group)into the tail vein of C57BL/6 mice. After successful modeling, NTP was added to take intraperitoneal injections for 4 weeks, which were defined to be NTP group. In order to assess the severity of BIPN, systemic toxicity, pain behavioral and sensorimotor kinetic changes in mice of different group were observed while transmission electron microscopy examination was used to observe the damage of peripheral nerve ultrastructure and myelin thickness. Immunofluorescence staining method was used to detected the activation of spinal cord microglial and their expression level of inflammatory factors such as IL-1β, IL-6 and TNF-α. ELISA was used to detected the expression of IL-1β, IL-6 and TNF-α in the dorsal horn homogenate of the spinal cord; Western blot to detect the protein expression of pathway-associated proteins JAK/STAT. Results: The Compared with the Control group, the BTZ and BTZ+NS groups showed significant decreases in mechanical, thermal pain thresholds and motor coordination compared with the Control group (P<0.05), and ultrastructural damage to the peripheral nerves, with demyelinating lesions and a significant reduction in the overall thickness of myelin sheaths in myelinated fibers (0.62±0.08 Vs 0.56±0.12, P＜0.01). NTP administration significantly improved pain behavioral changes, reduced ultrastructural damage, reversed demyelinating lesions and significantly increased myelin thickness (0.56±0.06 Vs 0.62±0.08, P＜0.01) in model mice compared with BTZ group. Immunofluorescence staining suggested that microglia in the dorsal horn of the spinal cord of mice in the BTZ group and the BTZ+NS group were significantly activated, which were shown the fluorescence ratio of IBA-1 + was significantly increased (2.55±0.61 Vs 0.55±0.04，2.42±0.89 Vs 0.55±0.04，P＜0.0001), while NTP significantly improved the activation of microglia in the model mice (0.71±0.13 Vs 2.55±0.61, 0.71±0.13 Vs 2.42±0.89, P<0.0001). The co-staining results suggest that the increase in inflammatory factors may originate from microglia. ELISA results that the expression level of IL-6 and TNF-α in NTP administration group significantly decreased compared with the group of BTZ group (IL-6: 37.87±23.2 Vs 111.8±6.0, P＜0.05; TNF-α: 547.9±17.11 Vs 998.8±333.4, P＜0.05) and the BTZ+NS group (IL-6: 37.87±23.2 Vs 88.3±15.58, P<0.05; TNF-α: 547.9±17.11 Vs 768.3±37.45, P<0.05) in the dorsal horn homogenate of the spinal cord. The results of spinal cord proteomics showed that neurotropin could induce the changes of differential proteins, such as PI3K-AKT, MAPK, JAK/STAT, which were related to inflammation. Moreover, these differential proteins can be significantly enriched in the JAK/STAT KEGG Pathway. The KEGG pathway of differential protein enrichment in the spinal cord of differential BIPN mice was detected by proteomics chip after NTP treatment, and the JAK/STAT pathway was significantly down-regulated after NTP treatment. Western blot showed that the protein expression of JAK1/STAT3 decreased after NTP administration. Conclusion: Neurotropin alleviates bortezomib induced neurotoxicity through microglial neuroinflammation, and may reduce the release of glial inflammation by regulating JAK1/STAT3 signaling pathway, thus playing a therapeutic role in BIPN. Repeated experiments are needed for further research. Keywords: Neurotropin, Bortezomib, Peripheral neuropathy, Neuroinflammation

Triggering Receptor Expressed on Myeloid Cells 2 Deficiency Exacerbates Methamphetamine-Induced Activation of Microglia and Neuroinflammation.

Methamphetamine (METH) is a highly addictive psychostimulant and one of the most widely abused drugs worldwide. The continuous use of METH eventually leads to neurotoxicity and drug addiction. Studies have shown that neurotoxicity is strongly associated with METH-induced neuroinflammation, and microglia are the key drivers of neuroinflammation. Triggering receptor expressed on myeloid cells 2 (TREM2) is reported to play a key role in activation of microglia and neuroinflammation. Yet, the molecular mechanisms by which METH causes neuroinflammation and neurotoxicity remain elusive. In the current study, we investigated the role of TREM2 in neuroinflammation induced by METH in BV2 cells and the wild-type (WT) C57BL/6J mice, CX3CR1GFP/+ transgenic mice, and TREM2 knockout (KO) mice. Postmortem samples from the frontal cortex of humans with a history of METH use were also analyzed to determine the levels of TREM2, TLR4, IBA1, and IL-1β. The expression levels of TREM2, TLR4, IBA1, IL-1β, iNOS, and Arg-1 were then assessed in the BV2 cells and frontal cortex of mice and human METH users. Results revealed that the expression levels of TREM2, TLR4, IBA1, and IL-1β were significantly elevated in METH-using individuals and BV2 cells. Microglia were clearly activated in the frontal cortex of WT C57BL/6 mice and CX3CR1GFP/+ transgenic mice, and the protein levels of IBA1, TREM2, TLR4, and IL-1β were elevated in the METH-induced mouse models. Moreover, TREM2-KO mice showed further increased microglial activation, neuroinflammation, and excitotoxicity induced by METH. Thus, these findings suggest that TREM2 may be a target for regulating METH-induced neuroinflammation.

Systemic treatment with a selective TNFR2 agonist alters the central and peripheral immune responses and transiently improves functional outcome after experimental ischemic stroke

Ischemic stroke often leaves survivors with permanent disabilities and therapies aimed at limiting detrimental inflammation and improving functional outcome are still needed. Tumor necrosis factor (TNF) levels increase rapidly after ischemic stroke, and while signaling through TNF receptor 1 (TNFR1) is primarily detrimental, TNFR2 signaling mainly has protective functions. We therefore investigated how systemic stimulation of TNFR2 with the TNFR2 agonist NewSTAR2 affects ischemic stroke in mice. We found that NewSTAR2 treatment induced changes in peripheral immune cell numbers and transiently affected microglial numbers and neuroinflammation. However, this was not sufficient to improve long-term functional outcome after stroke in mice.

Anti-epileptic and Neuroprotective Effects of Ultra-low Dose NADPH Oxidase Inhibitor Dextromethorphan on Kainic Acid-induced Chronic Temporal Lobe Epilepsy in Rats.

Neuroinflammation mediated by microglia and oxidative stress play pivotal roles in the development of chronic temporal lobe epilepsy (TLE). We postulated that kainic acid (KA)-Induced status epilepticus triggers microglia-dependent inflammation, leading to neuronal damage, a lowered seizure threshold, and the emergence of spontaneous recurrent seizures (SRS). Extensive evidence from our laboratory suggests that dextromethorphan (DM), even in ultra-low doses, has anti-inflammatory and neuroprotective effects in many animal models of neurodegenerative disease. Our results showed that administration of DM (10 ng/kg per day; subcutaneously via osmotic minipump for 4 weeks) significantly mitigated the residual effects of KA, including the frequency of SRS and seizure susceptibility. In addition, DM-treated rats showed improved cognitive function and reduced hippocampal neuronal loss. We found suppressed microglial activation-mediated neuroinflammation and decreased expression of hippocampal gp91phox and p47phox proteins in KA-induced chronic TLE rats. Notably, even after discontinuation of DM treatment, ultra-low doses of DM continued to confer long-term anti-seizure and neuroprotective effects, which were attributed to the inhibition of microglial NADPH oxidase 2 as revealed by mechanistic studies.

Direct inhibition of microglial activation by a μ receptor selective agonist alleviates inflammatory-induced pain hypersensitivity

Opioids are widely used in the treatment of moderate and severe pain. Nociceptive stimulation has been reported to potentially promote microglial activation and neuroinflammation, which also causes chronic pain sensitization. The aim of this study was to demonstrate whether the novel μ receptor agonist MEL-0614 could inhibit activated microglia directly and the associated signaling pathway. Mice were administered lipopolysaccharide and formalin to induce allodynia. Von Frey test was used to detect the anti-allodynia effect of MEL-0614 before and after LPS and formalin injection. In the spinal cord, the levels of proinflammatory cytokines and microglial activation were determined after MEL-0614 administration. BV2 and primary microglia were cultured to further explore the effect of MEL-0614 on LPS-induced microglial activation and key signaling pathways involved. MEL-0614 partially prevented and reversed allodynia induced by LPS and formalin in vivo, which was not inhibited by the μ receptor antagonist CTAP. Minocycline was effective in reversing the established allodynia. MEL-0614 also downregulated the activation of microglia and related proinflammatory cytokines in the spinal cord. Additionally, in BV2 and primary microglia, MEL-0614 inhibited the LPS-induced upregulation of proinflammatory factors, which was unaffected by CTAP. The NLR family pyrin domain containing 3 (NLRP3) related signaling pathway may be involved in the interaction between MEL-0614 and microglia. The opioid agonist MEL-0614 inhibited the activation of microglia and the subsequent upregulation of proinflammatory factors both in vivo and in vitro. Notably, this effect is partially mediated by the μ receptor.

CKLF induces microglial activation via triggering defective mitophagy and mitochondrial dysfunction

ABSTRACT Although microglial activation is induced by an increase in chemokines, the role of mitophagy in this process remains unclear. This study aimed to elucidate the role of microglial mitophagy in CKLF/CKLF1 (chemokine-like factor 1)-induced microglial activation and neuroinflammation, as well as the underlying molecular mechanisms following CKLF treatment. This study determined that CKLF, an inducible chemokine in the brain, leads to an increase in mitophagy markers, such as DNM1L, PINK1 (PTEN induced putative kinase 1), PRKN, and OPTN, along with a simultaneous increase in autophagosome formation, as evidenced by elevated levels of BECN1 and MAP1LC3B (microtubule-associated protein 1 light chain 3 beta)-II. However, SQSTM1, a substrate of autophagy, was also accumulated by CKLF treatment, suggesting that mitophagy flux was reduced and mitophagosomes accumulated. These findings were confirmed by transmission electron microscopy and confocal microscopy. The defective mitophagy observed in our study was caused by impaired lysosomal function, including mitophagosome-lysosome fusion, lysosome generation, and acidification, resulting in the accumulation of damaged mitochondria in microglial cells. Further analysis revealed that pharmacological blocking or gene-silencing of mitophagy inhibited CKLF-mediated microglial activation, as evidenced by the expression of the microglial marker AIF1 (allograft inflammatory factor 1) and the mRNA of proinflammatory cytokines (Tnf and Il6). Ultimately, defective mitophagy induced by CKLF results in microglial activation, as observed in the brains of adult mice. In summary, CKLF induces defective mitophagy, microglial activation, and inflammation, providing a potential approach for treating neuroinflammatory diseases. Abbreviation: 3-MA: 3-methyladenine; AIF1: allograft inflammatory factor 1; ANOVA: analysis of variance; BAF: bafilomycin A1; BSA: bovine serum albumin; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; CKLF/CKLF1: chemokine-like factor 1; CNS: central nervous system; DMEM: Dulbecco’s Modified Eagle Medium; DNM1L: dynamin 1 like; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescence protein; IRF3: interferon regulatory factor 3; IgG: immunoglobulin G; LAMP1: lysosomal-associated membrane protein 1; LAPTM4A: lysosomal-associated protein transmembrane 4A; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; Mdivi-1: mitochondrial division inhibitor 1; mRFP: monomeric red fluorescent protein; mtDNA: mitochondrial DNA; MTORC1: mechanistic target of rapamycin kinase complex 1; OPTN: optineurin; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PINK1: PTEN induced putative kinase 1; PLL: poly-L-lysine; PRKN: parkin RBR E3 ubiquitin protein ligase; qPCR: quantitative polymerase chain reaction; ROS: reactive oxygen species; SQSTM1: sequestosome 1; TBK1: TANK-binding kinase 1; TFEB: transcription factor EB; VDAC: voltage-dependent anion channel

To re-examine the intersection of microglial activation and neuroinflammation in neurodegenerative diseases from the perspective of pyroptosis.

Neurodegenerative diseases (NDs), such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and motor neuron disease, are diseases characterized by neuronal damage and dysfunction. NDs are considered to be a multifactorial disease with diverse etiologies (immune, inflammatory, aging, genetic, etc.) and complex pathophysiological processes. Previous studies have found that neuroinflammation and typical microglial activation are important mechanisms of NDs, leading to neurological dysfunction and disease progression. Pyroptosis is a new mode involved in this process. As a form of programmed cell death, pyroptosis is characterized by the expansion of cells until the cell membrane bursts, resulting in the release of cell contents that activates a strong inflammatory response that promotes NDs by accelerating neuronal dysfunction and abnormal microglial activation. In this case, abnormally activated microglia release various pro-inflammatory factors, leading to the occurrence of neuroinflammation and exacerbating both microglial and neuronal pyroptosis, thus forming a vicious cycle. The recognition of the association between pyroptosis and microglia activation, as well as neuroinflammation, is of significant importance in understanding the pathogenesis of NDs and providing new targets and strategies for their prevention and treatment.

Interleukin-4 Modulates Neuroinflammation by Inducing Phenotypic Transformation of Microglia Following Subarachnoid Hemorrhage

Neuroinflammation, a key pathological feature following subarachnoid hemorrhage (SAH), can be therapeutically targeted by inhibiting microglia M1 polarization and promoting phenotypic transformation to M2 microglia. Interleukin-4 (IL-4) is a pleiotropic cytokine known to its regulation of physiological functions of the central nervous system (CNS) and mediate neuroinflammatory processes. However, its specific role in neuroinflammation and microglia responses following SAH remains unexplored. In this investigation, we established both in vivo and in vitro SAH models and employed a comprehensive array of assessments, including ELISA, neurofunctional profiling, immunofluorescence staining, qRT-PCR, determination of phagocytic capacity, and RNA-Seq analyses. The findings demonstrate an elevated expression of IL-4 within cerebrospinal fluid (CSF) subsequent to SAH. Furthermore, exogenous administration of IL-4 ameliorates post-SAH neurofunctional deficits, attenuates cellular apoptosis, fosters M2 microglia phenotype conversion, and mitigates neuroinflammatory responses. The RNA-Seq analysis signifies that IL-4 governs the modulation of neuroinflammation in microglia within an in vitro SAH model through intricate cascades of signaling pathways, encompassing interactions between cytokines and cytokine receptors. These discoveries not only augment comprehension of the neuropathogenesis associated with post-SAH neuroinflammation but also present novel therapeutic targets for the management thereof.Graphical

Long-Term Neuropsychiatric Developmental Defects after Neonatal Organophosphate Exposure: Mitigation by Synthetic Neurosteroids.

Children are much more susceptible to the neurotoxic effects of organophosphate (OP) pesticides and nerve agents than adults. OP poisoning in children leads to acute seizures and neuropsychiatric sequela, including the development of long-term disabilities and cognitive impairments. Despite these risks, there are few chronic rodent models that use pediatric OP exposure for studying neurodevelopmental consequences and interventions. Here, we investigated the protective effect of the neurosteroid ganaxolone (GX) on the long-term developmental impact of neonatal exposure to the OP compound, diisopropyl-fluorophosphate (DFP). Pediatric postnatal day-28 rats were acutely exposed to DFP, and at 3 and 10 months after exposure, they were evaluated using a series of cognitive and behavioral tests with or without the postexposure treatment of GX. Analysis of the neuropathology was performed after 10 months. DFP-exposed animals displayed significant long-term deficits in mood, anxiety, depression, and aggressive traits. In spatial and nonspatial cognitive tests, they displayed striking impairments in learning and memory. Analysis of brain sections showed significant loss of neuronal nuclei antigen(+) principal neurons, parvalbumin(+) inhibitory interneurons, and neurogenesis, along with increased astrogliosis, microglial neuroinflammation, and mossy fiber sprouting. These detrimental neuropathological changes are consistent with behavioral dysfunctions. In the neurosteroid GX-treated cohort, behavioral and cognitive deficits were significantly reduced and were associated with strong protection against long-term neuroinflammation and neurodegeneration. In conclusion, this pediatric model replicates the salient features of children exposed to OPs, and the protective outcomes from neurosteroid intervention support the viability of developing this strategy for mitigating the long-term effects of acute OP exposure in children. SIGNIFICANCE STATEMENT: An estimated 3 million organophosphate exposures occur annually worldwide, with children comprising over 30% of all victims. Our understanding of the neurodevelopmental consequences in children exposed to organophosphates is limited. Here, we investigated the long-term impact of neonatal exposure to diisopropyl-fluorophosphate in pediatric rats. Neurosteroid treatment protected against major deficits in behavior and memory and was well correlated with neuropathological changes. Overall, this pediatric model is helpful to screen novel therapies to mitigate long-term developmental deficits of organophosphate exposure.

Long Non-Coding RNA MALAT1 Protects Against Spinal Cord Injury via Suppressing microRNA-125b-5p Mediated Microglial M1 Polarization, Neuroinflammation, and Neural Apoptosis.

Our previous studies have discovered that long non-coding RNA (lncRNA) MALAT1 and its target microRNA-125b-5p (miR-125b-5p) are implicated in neurological diseases via regulating neuroinflammation and neuronal injury. This study aimed to further explore the relationship between lncRNA MALAT1 and miR-125b-5p, as well as their effect on microglial activation, neuroinflammation, and neural apoptosis in spinal cord injury (SCI).Primary microglia from Sprague Dawley rats were stimulated with lipopolysaccharide (LPS). Then, microglia were transfected with lncRNA MALAT1 overexpression or knock-down adenovirus-associated virus with or without miR-125b-5p mimic. The culture medium of microglia was incubated with primary neurons. SCI rats were established for in vivo validation.LncRNA MALAT1 expression was reduced by LPS treatment in a dose-dependent manner. LncRNA MALAT1 overexpression suppressed the microglial M1 polarization (decreased iNOS but increased ARG1), neuroinflammation (declined PTGS2, TNF-α, IL-1β, and IL-6), and microglia-induced neural apoptosis (lower TUNEL positive cells and C-caspase3 but higher BCL2) under LPS treatment; its knock-down displayed the opposite trend. Moreover, lncRNA MALAT1 directly bound to and negatively regulated miR-125b-5p. MiR-125b-5p mimic promoted microglial M1 polarization, neuroinflammation, and microglia-induced neural apoptosis following LPS treatment; also, it could attenuate the effect of lncRNA MALAT1. Further in vivo study displayed that lncRNA MALAT1 overexpression elevated the Basso-Beattie-Bresnahan motor function score and improved neural injury. Also, in vivo validation indicated a similar effect of lncRNA MALAT1 on microglial polarization and neuroinflammation as in vitro.LncRNA MALAT1 improves SCI recovery via miR-125b-5p mediated microglial M1 polarization, neuroinflammation, and neural apoptosis.

The Combination of Citicoline and Nicotinamide Mononucleotide Induces Neurite Outgrowth and Mitigates Vascular Cognitive Impairment via SIRT1/CREB Pathway.

Vascular dementia (VD) is characterized with vascular cognitive impairment (VCI), which currently has few effective therapies in clinic. Neuronal damage and white matter injury are involved in the pathogenesis of VCI. Citicoline has been demonstrated to exhibit neuroprotection and neurorepair to improve cognition in cerebrovascular diseases. Nicotinamide adenine dinucleotide (NAD+)-dependent sirtuin (SIRT) signaling pathway constitutes a strong intrinsic defense system against various stresses including neuroinflammation in VCI. Our hypothesis is that the combined use of citicoline and the precursor of NAD+, nicotinamide mononucleotide (NMN), could enhance action on cognitive function in VCI. We investigated the synergistic effect of these two drugs in the rat model of VCI by bilateral common carotid artery occlusion (BCCAO). Citicoline significantly enhanced neurite outgrowth in Neuro-2a cells, and the combination of citicoline and NMN remarkably induced neurite outgrowth in Neuro-2a cells and primary cortical neuronal cells with an optimal proportion of 4:1. In the rat model of BCCAO, when two drugs in combination of 160mg/kg citicoline and 40mg/kg NMN, this combination administrated at 7days post-BCCAO significantly improved the cognitive impairment in BCCAO rats compared with vehicle group by the analysis of the Morris water maze and the novel object recognition test. This combination also decreased microglial activation and neuroinflammation, and protected white matter integrity indicated by the increased myelin basic protein (MBP) expression through activation of SIRT1/TORC1/CREB signaling pathway. Our results suggest that the combination of citicoline and NMN has a synergistic effect for the treatment of VD associated with VCI.

RU.521 mitigates subarachnoid hemorrhage-induced brain injury via regulating microglial polarization and neuroinflammation mediated by the cGAS/STING/NF-κB pathway

BackgroundThe poor prognosis of subarachnoid hemorrhage (SAH) is often attributed to neuroinflammation. The cGAS-STING axis, a cytoplasmic pathway responsible for detecting dsDNA, plays a significant role in mediating neuroinflammation in neurological diseases. However, the effects of inhibiting cGAS with the selective small molecule inhibitor RU.521 on brain injury and the underlying mechanisms after SAH are still unclear.MethodsThe expression and microglial localization of cGAS following SAH were investigated with western blot analysis and immunofluorescent double-staining, respectively. RU.521 was administered after SAH. 2’3’-cGAMP, a second messenger converted by activated cGAS, was used to activate cGAS-STING. The assessments were carried out by adopting various techniques including neurological function scores, brain water content, blood–brain barrier permeability, western blot analysis, TUNEL staining, Nissl staining, immunofluorescence, morphological analysis, Morris water maze test, Golgi staining, CCK8, flow cytometry in the in vivo and in vitro settings.ResultsFollowing SAH, there was an observed increase in the expression levels of cGAS in rat brain tissue, with peak levels observed at 24 h post-SAH. RU.521 resulted in a reduction of brain water content and blood–brain barrier permeability, leading to an improvement in neurological deficits after SAH. RU.521 had beneficial effects on neuronal apoptosis and microglia activation, as well as improvements in microglial morphology. Additionally, RU.521 prompted a shift in microglial phenotype from M1 to M2. We also noted a decrease in the production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, and an increase in the level of the anti-inflammatory cytokine IL-10. Finally, RU.521 treatment was associated with improvements in cognitive function and an increase in the number of dendritic spines in the hippocampus. The therapeutic effects were mediated by the cGAS/STING/NF-κB pathway and were found to be abolished by 2’3’-cGAMP. In vitro, RU.521 significantly reduced apoptosis and neuroinflammation.ConclusionThe study showed that SAH leads to neuroinflammation caused by microglial activation, which contributes to early brain injury. RU.521 improved neurological outcomes and reduced neuroinflammation by regulating microglial polarization through the cGAS/STING/NF-κB pathway in early brain injury after SAH. RU.521 may be a promising candidate for the treatment of neuroinflammatory pathology after SAH.BdJw4U5DXrR7YY_5KbyxVXVideo

The citrus flavonoid, nobiletin inhibits neuronal inflammation by preventing the activation of NF-κB

Nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone) is one of the flavonoids found in shikuwasa, a popular citrus fruit in Okinawa, Japan. It exerts various pharmacological effects, such as anti-tumor, antioxidant, and anti-inflammatory activities. We herein investigated whether nobiletin attenuated lipopolysaccharide (LPS)-induced inflammatory responses in the murine microglial cell line BV-2 and neuroinflammation in mice induced by an intracerebral injection of LPS. In BV-2 cells, nobiletin significantly inhibited the LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) by preventing the mRNA expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. Nobiletin also inhibited the LPS-induced mRNA expression of CCL2, CXCL1, IL-6, and TNFα. Nobiletin markedly attenuated the transcriptional activity of the NF-κB p65 subunit without affecting the degradation of IκBα or the nuclear localization of the NF-κB p65 subunit. Nobiletin also inhibited the LPS-induced activation of JNK, but not ERK or p38, in BV-2 cells. Furthermore, the administration of nobiletin significantly suppressed the accumulation of microglia and induction of the mRNA expression of CCL2, CXCL1, IL-6, and TNFα in the murine brain induced by injecting LPS into the striatum. Collectively, these results suggest the potential of nobiletin as a candidate anti-inflammatory drug for the prevention of neuroinflammation.