Increased activity of IRE1 improves the clinical presentation of EAE.

Activation of the endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme-1α (IRE1α) contributes to neuronal development and is known to induce neuronal remodeling in vitro and in vivo. On the contrary, excessive IRE1 activity is often detrimental and may contribute to neurodegeneration. To determine the consequences of increased activation of IRE1α, we used a mouse model expressing a C148S variant of IRE1α with increased and sustained activation. Surprisingly, the mutation did not affect the differentiation of highly secretory antibody-producing cells but exhibited a beneficial effect in a mouse model of experimental autoimmune encephalomyelitis (EAE). Although mechanical allodynia was unaffected, significant improvement in motor function was found in IRE1C148S mice with EAE relative to wild type (WT) mice. Coincident with this improvement, there was reduced microgliosis in the spinal cord of IRE1C148S mice, with reduced expression of proinflammatory cytokine genes. This was accompanied by reduced axonal degeneration and enhanced 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) levels, suggesting improved myelin integrity. Interestingly, while the IRE1C148S mutation is expressed in all cells, the reduction in proinflammatory cytokines and in the microglial activation marker ionized calcium-binding adapter molecule (IBA1), along with preservation of phagocytic gene expression, all point to microglia as the cell type contributing to the clinical improvement in IRE1C148S animals. Our data suggest that sustained increase in IRE1α activity can be beneficial in vivo, and that this protection is cell type and context dependent. Considering the overwhelming but conflicting evidence for the role of ER stress in neurological diseases, a better understanding of the function of ER stress sensors in physiological contexts is clearly needed.

The Impact of Aging on Multiple Sclerosis.

PURPOSE OF REVIEW: Multiple sclerosis (MS) is a chronic, immune-mediated demyelinating disorder of the central nervous system. Age is one of the most important factors in determining MS phenotype. This review provides an overview of how age influences MS clinical characteristics, pathology, and treatment. RECENT FINDINGS: New methods for measuring aging have improved our understanding of the aging process in MS. New studies have characterized the molecular and cellular composition of chronic active or smoldering plaques in MS. These lesions are important contributors to disability progression in MS. These studies highlight the important role of immunosenescence and the innate immune system in sustaining chronic inflammation. Given these changes in immune function, several studies have assessed optimal treatment strategies in aging individuals with MS. MS phenotype is intimately linked with chronologic age and immunosenescence. While there are many unanswered questions, there has been much progress in understanding this relationship which may lead to more effective treatments for progressive disease.

Extracellular vesicles from hiPSC-NSCs can prevent peripheral inflammation-induced cognitive dysfunction with inflammasome inhibition and improved neurogenesis in the hippocampus.

Extracellular vesicles (EVs) released by human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSCs) are enriched with miRNAs and proteins capable of mediating robust antiinflammatory activity. The lack of tumorigenic and immunogenic properties and ability to permeate the entire brain to incorporate into microglia following intranasal (IN) administrations makes them an attractive biologic for curtailing chronic neuroinflammation in neurodegenerative disorders. We tested the hypothesis that IN administrations of hiPSC-NSC-EVs can alleviate chronic neuroinflammation and cognitive impairments induced by the peripheral lipopolysaccharide (LPS) challenge. Adult male, C57BL/6J mice received intraperitoneal injections of LPS (0.75 mg/kg) for seven consecutive days. Then, the mice received either vehicle (VEH) or hiPSC-NSC-EVs (~ 10 × 109 EVs/administration, thrice over 6 days). A month later, mice in all groups were investigated for cognitive function with behavioral tests and euthanized for histological and biochemical studies. Mice receiving VEH after LPS displayed deficits in associative recognition memory, temporal pattern processing, and pattern separation. Such impairments were associated with an increased incidence of activated microglia presenting NOD-, LRR-, and pyrin domain containing 3 (NLRP3) inflammasomes, elevated levels of NLRP3 inflammasome mediators and end products, and decreased neurogenesis in the hippocampus. In contrast, the various cognitive measures in mice receiving hiPSC-NSC-EVs after LPS were closer to naive mice. Significantly, these mice displayed diminished microglial activation, NLRP3 inflammasomes, proinflammatory cytokines, and a level of neurogenesis matching age-matched naïve controls. Thus, IN administrations of hiPSC-NSC-EVs are an efficacious approach to reducing chronic neuroinflammation-induced cognitive impairments.

SFRP1 modulates astrocyte-to-microglia crosstalk in acute and chronic neuroinflammation.

Neuroinflammation is a common feature of many neurodegenerative diseases. It fosters a dysfunctional neuron-microglia-astrocyte crosstalk that, in turn, maintains microglial cells in a perniciously reactive state that often enhances neuronal damage. The molecular components that mediate this critical communication are not fully explored. Here, we show that secreted frizzled-related protein 1 (SFRP1), a multifunctional regulator of cell-to-cell communication, is part of the cellular crosstalk underlying neuroinflammation. In mouse models of acute and chronic neuroinflammation, SFRP1, largely astrocyte-derived, promotes and sustains microglial activation, and thus a chronic inflammatory state. SFRP1 promotes the upregulation of components of the hypoxia-induced factor-dependent inflammatory pathway and, to a lower extent, of those downstream of the nuclear factor-kappa B. We thus propose that SFRP1 acts as an astrocyte-to-microglia amplifier of neuroinflammation, representing a potential valuable therapeutic target for counteracting the harmful effect of chronic inflammation in several neurodegenerative diseases.

Antagonization of OX1 Receptor Potentiates CB2 Receptor Function in Microglia from APPSw/Ind Mice Model.

Microdialysis assays demonstrated a possible role of orexin in the regulation of amyloid beta peptide (Aß) levels in the hippocampal interstitial fluid in the APP transgenic model. CB2R is overexpressed in activated microglia, showing a neuroprotective effect. These two receptors may interact, forming CB2-OX1-Hets and becoming a new target to combat Alzheimer's disease. Aims: Demonstrate the potential role of CB2-OX1-Hets expression and function in microglia from animal models of Alzheimer's disease. Receptor heteromer expression was detected by immunocytochemistry, bioluminescence resonance energy transfer (BRET) and proximity ligation assay (PLA) in transfected HEK-293T cells and microglia primary cultures. Quantitation of signal transduction events in a heterologous system and in microglia cells was performed using the AlphaScreen® SureFire® kit, western blot, the GCaMP6 calcium sensor and the Lance Ultra cAMP kit (PerkinElmer). The formation of CB2-OX1 receptor complexes in transfected HEK-293T cells has been demonstrated. The tetrameric complex is constituted by one CB2R homodimer, one OX1R homodimer and two G proteins, a Gi and a Gq. The use of TAT interfering peptides showed that the CB2-OX1 receptor complex interface is TM4-TM5. At the functional level it has been observed that the OX1R antagonist, SB334867, potentiates the action induced by CB2R agonist JWH133. This effect is observed in transfected HEK-293T cells and microglia, and it is stronger in the Alzheimer's disease (AD) animal model APPSw/Ind where the expression of the complex assessed by the proximity ligation assay indicates an increase in the number of complexes compared to resting microglia. The CB2-OX1 receptor complex is overexpressed in microglia from AD animal models where OX1R antagonists potentiate the neuroprotective actions of CB2R activation. Taken together, these results point to OX1R antagonists as drugs with therapeutic potential to combat AD. Data access statement: Raw data will be provided by the corresponding author upon reasonable requirement.

Glia and extracellular matrix molecules: What are their importance for the electrographic and MRI changes in the epileptogenic zone?

Glial cells and extracellular matrix (ECM) molecules are crucial for the maintenance of brain homeostasis. Especially because of their actions regarding neurotransmitter and ionic control, and synaptic function, these cells can potentially contribute to the hyperexcitability seen in the epileptogenic, while ECM changes are linked to synaptic reorganization. The present review will explore glial and ECM homeostatic roles and their potential contribution to tissue plasticity. Finally, we will address how glial, and ECM changes in the epileptogenic zone can be seen in magnetic resonance imaging (MRI), pointing out their importance as markers for the extension of the epileptogenic area. This article is part of the Special Issue "NEWroscience 2018".

HDAC2 hyperexpression alters hippocampal neuronal transcription and microglial activity in neuroinflammation-induced cognitive dysfunction.

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.

Prominent microglial activation in cortical white matter is selectively associated with cortical atrophy in primary progressive aphasia.

AIMS: Primary progressive aphasia (PPA) is a clinical syndrome characterized by selective language impairments associated with focal cortical atrophy favouring the language dominant hemisphere. PPA is associated with Alzheimer's disease (AD), frontotemporal lobar degeneration (FTLD) and significant accumulation of activated microglia. Activated microglia can initiate an inflammatory cascade that may contribute to neurodegeneration, but their quantitative distribution in cortical white matter and their relationship with cortical atrophy remain unknown. We investigated white matter activated microglia and their association with grey matter atrophy in 10 PPA cases with either AD or FTLD-TDP pathology. METHODS: Activated microglia were quantified with optical density measures of HLA-DR immunoreactivity in two regions with peak cortical atrophy, and one nonatrophied region within the language dominant hemisphere of each PPA case. Nonatrophied contralateral homologues of the language dominant regions were examined for hemispheric asymmetry. RESULTS: Qualitatively, greater densities of activated microglia were observed in cortical white matter when compared to grey matter. Quantitative analyses revealed significantly greater densities of activated microglia in the white matter of atrophied regions compared to nonatrophied regions in the language dominant hemisphere (P < 0.05). Atrophied regions of the language dominant hemisphere also showed significantly more activated microglia compared to contralateral homologues (P < 0.05). CONCLUSIONS: White matter activated microglia accumulate more in atrophied regions in the language dominant hemisphere of PPA. While microglial activation may constitute a response to neurodegenerative processes in white matter, the resultant inflammatory processes may also exacerbate disease progression and contribute to cortical atrophy.

Basal astrocyte and microglia activation in the central nervous system of Familial Hemiplegic Migraine Type I mice.

BACKGROUND: Gain-of-function missense mutations in the α1A subunit of neuronal CaV2.1 channels, which define Familial Hemiplegic Migraine Type 1 (FHM1), result in enhanced cortical glutamatergic transmission and a higher susceptibility to cortical spreading depolarization. It is now well established that neurons signal to surrounding glial cells, namely astrocytes and microglia, in the central nervous system, which in turn become activated and in pathological conditions can sustain neuroinflammation. We and others previously demonstrated an increased activation of pro-algogenic pathways, paralleled by augmented macrophage infiltration, in both isolated trigeminal ganglia and mixed trigeminal ganglion neuron-satellite glial cell cultures of FHM1 mutant mice. Hence, we hypothesize that astrocyte and microglia activation may occur in parallel in the central nervous system. METHODS: We have evaluated signs of reactive glia in brains from naïve FHM1 mutant mice in comparison with wild type animals by immunohistochemistry and Western blotting. RESULTS: Here we show for the first time signs of reactive astrogliosis and microglia activation in the naïve FHM1 mutant mouse brain. CONCLUSIONS: Our data reinforce the involvement of glial cells in migraine, and suggest that modulating such activation may represent an innovative approach to reduce pathology.

DNA binding protein HMGB1 secreted by activated microglia promotes the apoptosis of hippocampal neurons in diabetes complicated with OSA.

Type 2 diabetes mellitus (T2DM) complicated with obstructive sleep apnea (OSA) may cause neuronal apoptosis and cognitive deficits, but the underlying mechanisms remain unclear. We aimed to determine the relationship between the activation of microglia and the apoptosis of hippocampal neurons, specifically in terms of high mobility group box-1 (HMGB1), after high glucose (HG) and intermittent hypoxia (IH) exposure. Diabetic KK-Ay mice and non-diabetic C57BL/6J mice (C57 mice) underwent IH or normoxia (control) exposure for 4 weeks. Cognitive function, microglial activation and hippocampal neuronal apoptosis were assessed after IH or normoxia exposure. Compared with C57 control mice, KK-Ay control mice exhibited increased cognitive dysfunction, microglial activation and hippocampal neuronal apoptosis. There were no differences between untreated KK-Ay control mice and C57 mice that had been exposed to IH. The abovementioned responses were aggravated in IH-exposed KK-Ay mice compared with control KK-Ay mice. In vitro, a cellular co-culture experiment showed that HG combined with IH could activate BV2 microglia, leading to the release of neuroinflammatory factors (ROS, TNF-α, IL-1ß) and mediating the apoptosis of HT22 cells via the PI3K/Akt/GSK-3ß signaling pathway. Meanwhile, HMGB1 was actively secreted into the extracellular environment from activated BV2 microglia. As a proinflammatory factor, it was able to sustain microglial activation by directly acting on those cells. The activation promoted positive feedback and aggravated neuronal damage further. In a cellular monoculture or co-culture system, HMGB1 siRNA was able to alleviate the activation of BV2 cells and the apoptosis of HT22 cells induced by HG combined with IH. Our object is to show that inhibition of HMGB1 may break the vicious cycle to prevent or treat neuroinflammation and hippocampal neuronal apoptosis caused by T2DM complicated with OSA.

[18 F]GE180 positron emission tomographic imaging indicates a potential double-hit insult in the intrahippocampal kainate mouse model of temporal lobe epilepsy.

OBJECTIVE: Accumulating evidence suggests that brain inflammation, elicited by epileptogenic insults, is involved in epilepsy development. Noninvasive nuclear imaging of brain inflammation in animal models of epileptogenesis represents a diagnostic in vivo approach with potential for direct translation into the clinic. Here, we investigated up-regulation of the translocator protein (TSPO) indicative of microglial activation by serial [18 F]GE180 positron emission tomographic (PET) imaging in a mouse model of temporal lobe epilepsy. METHODS: As epileptogenic insult, a status epilepticus (SE) was induced in mice by intrahippocampal injection of kainate. Post-SE mice injected with kainate and sham-injected mice were subjected to [18 F]GE180 PET scans before SE and at 2 days, 5-7 days, 2 weeks, 3 weeks, 7 weeks, and 14 weeks postinsult. For data evaluation, brain regions ipsilateral and contralateral to the injection site were outlined by coregistration with a standard mouse brain atlas, and percentage of injected dose per cubic centimeter was calculated. In addition, a statistical parametric mapping analysis, comparing post-SE mice to baseline, sham mice to baseline, and post-SE to sham mice was performed. RESULTS: Following SE, elevations in [18 F]GE180 uptake were most prominent in the ipsilateral hippocampus, occurring between 2 days and at least 7 weeks after SE, with a peak at 5-7 days after SE. In the contralateral hippocampus and other epilepsy-associated brain regions, increased tracer uptake was observed with a similar time profile but to a lesser extent. Moderate enhancement of tracer uptake was also evident in mice after sham surgery. SIGNIFICANCE: TSPO in vivo imaging reliably detects brain inflammation during epileptogenesis. These inflammatory processes most prominently affect the hippocampus ipsilateral to the injection site. Inflammation induced by the traumatic insult associated with surgery synergistically contributes to total brain inflammation and may also contribute to epileptogenesis. The revealed time course of neuroinflammation will help to identify appropriate time points for anti-inflammatory, potentially antiepileptogenic treatment.

Radioligands for positron emission tomography imaging of cannabinoid type 2 receptor.

The cannabinoid type 2 (CB2) receptor is an immunomodulatory receptor mainly expressed in peripheral cells and organs of the immune system. The expression level of CB2 in the central nervous system under physiological conditions is negligible, however under neuroinflammatory conditions an upregulation of CB2 protein or mRNA mainly colocalized with activated microglial cells has been reported. Consequently, CB2 agonists have been confirmed to play a role in neuroprotective and anti-inflammatory processes. A suitable positron emission tomography radioligand for imaging CB2 would provide an invaluable research tool to explore the role of CB2 receptor expression in inflammatory disorders. In this review, we provide a summary of so far published CB2 radioligands as well as their in vitro and in vivo binding characteristics.

Sex-Dependent Effects of Bmal1-Deficiency on Mouse Cerebral Cortex Infarction in Response to Photothrombotic Stroke.

Stroke is a leading cause of disability and death worldwide. There is increasing evidence that occurrence of ischemic stroke is affected by circadian system and sex. However, little is known about the effect of these factors on structural recovery after ischemic stroke. Therefore, we studied infarction in cerebral neocortex of male and female mice with deletion of the clock gene Bmal1 (Bmal1-/-) after focal ischemia induced by photothrombosis (PT). The infarct core size was significantly smaller 14 days (d) as compared to seven days after PT, consistent with structural recovery during the sub-acute phase. However, when sexes were analyzed separately 14 days after PT, infarct core was significantly larger in wild-type (Bmal1+/+) female as compared to male Bmal1+/+ mice, and in female Bmal1+/+, as compared to female Bmal1-/- mice. Volumes of reactive astrogliosis and densely packed microglia closely mirrored the size of infarct core in respective groups. Estradiol levels were significantly higher in female Bmal1-/- as compared to Bmal1+/+ mice. Our data suggests a sex-dependent effect and an interaction between sex and genotype on infarct size, the recruitment of astrocytes and microglia, and a relationship of these cells with structural recovery probably due to positive effects of estradiol during the subacute phase.

Preferential and Increased Uptake of Hydroxyl-Terminated PAMAM Dendrimers by Activated Microglia in Rabbit Brain Mixed Glial Culture.

Polyamidoamine (PAMAM) dendrimers are multifunctional nanoparticles with tunable physicochemical features, making them promising candidates for targeted drug delivery in the central nervous system (CNS). Systemically administered dendrimers have been shown to localize in activated glial cells, which mediate neuroinflammation in the CNS. These dendrimers delivered drugs specifically to activated microglia, producing significant neurological improvements in multiple brain injury models, including in a neonatal rabbit model of cerebral palsy. To gain further insight into the mechanism of dendrimer cell uptake, we utilized an in vitro model of primary glial cells isolated from newborn rabbits to assess the differences in hydroxyl-terminated generation 4 PAMAM dendrimer (D4-OH) uptake by activated and non-activated glial cells. We used fluorescently-labelled D4-OH (D-Cy5) as a tool for investigating the mechanism of dendrimer uptake. D4-OH PAMAM dendrimer uptake was determined by fluorescence quantification using confocal microscopy and flow cytometry. Our results indicate that although microglial cells in the mixed cell population demonstrate early uptake of dendrimers in this in vitro system, activated microglia take up more dendrimer compared to resting microglia. Astrocytes showed delayed and limited uptake. We also illustrated the differences in mechanism of uptake between resting and activated microglia using different pathway inhibitors. Both resting and activated microglia primarily employed endocytotic pathways, which are enhanced in activated microglial cells. Additionally, we demonstrated that hydroxyl terminated dendrimers are taken up by primary microglia using other mechanisms including pinocytosis, caveolae, and aquaporin channels for dendrimer uptake.

Propane-2-sulfonic acid octadec-9-enyl-amide, a novel PPARα/γ dual agonist, protects against ischemia-induced brain damage in mice by inhibiting inflammatory responses.

Propane-2-sulfonic acid octadec-9-enyl-amide (N15), an analogue of oleoylethanolamide (OEA), is a novel PPARα/γ dual agonist. Our previous studies verified the positive effects of OEA on the acute and delayed stages of cerebral ischemia. However, it is not clear whether N15 is effective against ischemic cerebral injury. In the present study, male Kunming mice were subjected to middle cerebral artery occlusion (MCAO). To evaluate its preventive effects, N15 (50, 100 or 200mg/kg, ip) was administered for 3days before ischemia. To evaluate its therapeutic effects, N15 (200mg/kg, ip) was administered 1h before reperfusion or 0, 1, 2 or 4h after reperfusion. Neurological deficit scores, infarct volume and the degree of brain oedema were determined at 24h after reperfusion. Blood brain barrier (BBB) disruption was evaluated by Evans blue (EB) and FITC-dextran leakages at 6h after reperfusion. The activation/inflammatory responses of microglia/macrophages were detected using immunohistochemistry and western blot. N15 pretreatment improved neurological dysfunction, reduced infarct volume and alleviated brain oedema in a dose-dependent manner; the most effective dose was 200mg/kg. The therapeutic time window was within 2h after reperfusion. N15 treatment preserved the BBB integrity and suppressed the activation of microglia/macrophages. N15 inhibited inflammatory cytokine expression not only in MCAO mice but also in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Additionally, N15 markedly decreased the phosphorylation levels of NF-κBp65, STAT3, and ERK1/2 both in vivo and in vitro. Furthermore, the PPARα antagonist MK886 or PPARγ antagonist T0070907 respectively partly abolished the anti-inflammatory effects of N15 in vitro. Our findings demonstrated that N15 can exert neuroprotective effects against cerebral ischemic insult. Moreover, the neuroprotective effects of N15 on cerebral ischemia may be attributed to its anti-inflammatory properties, at least in part, by enhancing PPARα/γ dual signaling and inhibiting the activation of the NF-κB, STAT3, and ERK1/2 signaling pathways. These findings suggest that N15 may be a potential therapeutic choice for the prevention and treatment of ischemic stroke.

Chronic peripheral inflammation, hippocampal neurogenesis, and behavior.

Adult hippocampal neurogenesis is involved in memory and learning, and disrupted neurogenesis is implicated in cognitive impairment and mood disorders, including anxiety and depression. Some long-term peripheral illnesses and metabolic disorders, as well as normal aging, create a state of chronic peripheral inflammation. These conditions are associated with behavioral disturbances linked to disrupted adult hippocampal neurogenesis, such as cognitive impairment, deficits in learning and memory, and depression and anxiety. Pro-inflammatory cytokines released in the periphery are involved in peripheral immune system-to-brain communication by activating resident microglia in the brain. Activated microglia reduce neurogenesis by suppressing neuronal stem cell proliferation, increasing apoptosis of neuronal progenitor cells, and decreasing survival of newly developing neurons and their integration into existing neuronal circuits. In this review, we summarize evolving evidence that the state of chronic peripheral inflammation reduces adult hippocampal neurogenesis, which, in turn, produces the behavioral disturbances observed in chronic inflammatory disorders. As there are no data available on neurogenesis in humans with chronic peripheral inflammatory disease, we focus on animal models and, in parallel, consider the evidence of cognitive disturbance and mood disorders in human patients.

Ganglioside accumulation in activated glia in the developing brain: comparison between WT and GalNAcT KO mice.

Our previous studies have shown accumulation of GM2 ganglioside during ethanol-induced neurodegeneration in the developing brain, and GM2 elevation has also been reported in other brain injuries and neurodegenerative diseases. Using GM2/GD2 synthase KO mice lacking GM2/GD2 and downstream gangliosides, the current study explored the significance of GM2 elevation in WT mice. Immunohistochemical studies indicated that ethanol-induced acute neurodegeneration in postnatal day 7 (P7) WT mice was associated with GM2 accumulation in the late endosomes/lysosomes of both phagocytic microglia and increased glial fibrillary acidic protein (GFAP)-positive astrocytes. However, in KO mice, although ethanol induced robust neurodegeneration and accumulation of GD3 and GM3 in the late endosomes/lysosomes of phagocytic microglia, it did not increase the number of GFAP-positive astrocytes, and the accumulation of GD3/GM3 in astrocytes was minimal. Not only ethanol, but also DMSO, induced GM2 elevation in activated microglia and astrocytes along with neurodegeneration in P7 WT mice, while lipopolysaccharide, which did not induce significant neurodegeneration, caused GM2 accumulation mainly in lysosomes of activated astrocytes. Thus, GM2 elevation is associated with activation of microglia and astrocytes in the injured developing brain, and GM2, GD2, or other downstream gangliosides may regulate astroglial responses in ethanol-induced neurodegeneration.

E. globulus leaf EO exhibits anti-inflammatory effects by regulating GSDMD-mediated pyroptosis, thereby alleviating neurological impairment and neuroinflammation in experimental stroke mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Aromatic and medicinal plants continue to be a major component of alternative and traditional medicine in the developing countries. Eucalyptus globulus (Labill.) is being employed to cultivation and production in China. However, few studies have reported the chemical composition and anti-inflammatory activity of Eucalyptus globulus (Labill.) leaf essential oil (E. globulus leaf EO) extracted from Eucalyptus globulus. AIM OF THE STUDY: This study aimed to assess the composition of E. globulus leaf EO and identify its bacteriostatic action as well as anti-inflammatory activity. Importantly, we evaluated the effect of E. globulus leaf EO on neurological impairment and neuroinflammation in experimental stroke mice. MATERIALS AND METHODS: Gas Chromatography-Mass Spectrometer (GC-MS) analyses was employed to evaluate the chemical components of E. globulus leaf EO, and the relative content of each component was determined by area normalization method. The antimicrobial activity of E. globulus leaf EO was determined by Oxford cup method and microbroth dilution assay. Cytotoxic activity of E. globulus leaf EO on THP-1 cells or BV2 cells in vitro was determined by CCK8 assay. In addition, the lipopolysaccharide (LPS)/ATP-induced inflammation model in THP-1 cells or BV2 cells were established, and the relative expression of TNF-α, IL-1ß, MCP-1and IL-6 were confirmed by RT-PCR. Furthermore, the expression of protein GSDMD, IL-lß, NLRP3 and NFκB signaling pathway were assessed by immunoblotting. In vivo,the experimental stroke model constructed by middle cerebral artery occlusion/reperfusion (MCAO/R) in mice was employed and subsequently treated with E. globulus leaf EO (50,100 mg/kg, subcutaneous injection) for 3 days to assess neurological impairment and neuroinflammation. Behavioral and neuronal damage were assessed using grip strength test, rod trarod test, and Nissl staining. Pro-inflammatory factors in serum or ischemic brain tissue was detected by ELISA kits. RESULTS: GC-MS analyses revealed that the major compound in E. globulus leaf EO was eudesmol (71.967%). E. globulus leaf EO has antimicrobial activity against Staphylococcus aureus (gram positive bacteria, MIC = 0.0625 mg/mL), Escherichia coli (gram negative bacteria, MIC = 1 mg/mL), and Candida albicans (MIC = 4 mg/mL). E. globulus leaf EO (0.5312, 1.0625, and 2.15 mg/mL) significantly decreased the expression of inflammation-related genes, including IL-1ß, TNF-α, MCP-1, and IL-6. Furthermore, reduced levels of TLR4, Myd88, phosphorylated NF-κB P65, and IκBα were found in the E. globulus leaf EO group for BV2 cells (1.025, and 2.125 mg/mL). In addition, the expression levels of GSDMD, NLRP3, IL-1ß and AIM2 were significantly decreased in the E. globulus leaf EO group when compared with the LPS -stimulated group, regulating GSDMD-mediated pyroptosis. In vivo, E. globulus leaf EO improved neurological functional deficits, inhibited excessive activation of microglia, and reduced the secretion of pro-inflammatory factors IL-1ß, TNF-α in the ischemic tissue and serum after MCAO/R. CONCLUSION: E. globulus leaf EO has strong antibacterial and anti-inflammatory activity, which has been implicated in blocking GSDMD-mediated pyroptosis. Moreover, E. globulus leaf EO could exert neuroprotective effect on cerebral ischemia-reperfusion injury.

Exploring the Role of Guanylate-Binding Protein-2 in Activated Microglia-Mediated Neuroinflammation and Neuronal Damage.

Neuron damage by microglia, which act as macrophage cells in the brain, can result in various brain diseases. However, the function of pro-inflammatory or anti-inflammatory microglia in the neurons remains controversial. Guanylate-binding protein-2 (GBP2) is expressed and activated in the microglia in the early phase of the inflammatory response and plays an important role in controlling immune responses. In this study, we evaluated whether GBP2 initially reduces the immune response induced by microglia, and whether microglia induce pro-inflammatory functions in neurons via GBP2 expression. In lipopolysaccharide (LPS)-stimulated microglia, we assessed the expression of GBP2 and how it affects neurons via activated microglia. The biological functions of microglia due to the downregulation of the GBP2 gene were examined using short hairpin RNA (shRNA)-RNA-GBP2. Downregulated GBP2 affected the function of mitochondria in the microglia and showed reduced neuronal damage when compared to the control group in the co-culture system. Furthermore, this protein was observed to be highly expressed in the brains of dementia mice. Our results are the first to report that the downregulation of GBP2 in activated microglia has an anti-inflammatory function. This study suggests that the GBP2 gene can be used as a therapeutic target biomarker for inflammation-related neurodegenerative diseases.

Targeting microglial GLP1R in epilepsy: A novel approach to modulate neuroinflammation and neuronal apoptosis.

BACKGROUND: Epilepsy is a prevalent disorder of the central nervous system. Approximately, one-third of patients show resistance to pharmacological interventions. The pathogenesis of epilepsy is complex, and neuronal apoptosis plays a critical role. Aberrantly reactive astrocytes, induced by cytokine release from activated microglia, may lead to neuronal apoptosis. This study investigated the role of glucagon-like peptide 1 receptor (GLP1R) in microglial activation in epilepsy and its impact on astrocyte-mediated neurotoxicity. METHODS: We used human hippocampal tissue from patients with temporal lobe epilepsy and a pilocarpine-induced epileptic mouse model to assess neurobiological changes in epilepsy. BV2 microglial cells and primary astrocytes were used to evaluate cytokine release and astrocyte activation in vitro. The involvement of GLP1R was explored using the GLP1R agonist, Exendin-4 (Ex-4). RESULTS: Our findings indicated that reduced GLP1R expression in hippocampal microglia in both epileptic mouse models and human patients, correlated with increased cytokine release and astrocyte activation. Ex-4 treatment restored microglial homeostasis, decreased cytokine secretion, and reduced astrocyte activation, particularly of the A1 phenotype. These changes were associated with a reduction in neuronal apoptosis. In addition, Ex-4 treatment significantly decreased the frequency and duration of seizures in epileptic mice. CONCLUSIONS: This study highlights the crucial role of microglial GLP1R in epilepsy pathophysiology. GLP1R downregulation contributes to microglial- and astrocyte-mediated neurotoxicity, exacerbating neuronal death and seizures. Activation of GLP1R with Ex-4 has emerged as a promising therapeutic strategy to reduce neuroinflammation, protect neuronal cells, and control seizures in epilepsy. This study provides a foundation for developing novel antiepileptic therapies targeting microglial GLP1R, with the potential to improve outcomes in patients with epilepsy.