p38 Inhibition Decreases Tau Toxicity in Microglia and Improves Their Phagocytic Function

Alzheimer’s disease (AD) and other tauopathies are histopathologically characterized by tau aggregation, along with a chronic inflammatory response driven by microglia. Over the past few years, the role of microglia in AD has been studied mainly in relation to amyloid-β (Aβ) pathology. Consequently, there is a substantial knowledge gap concerning the molecular mechanisms involved in tau-mediated toxicity and neuroinflammation, thus hindering the development of therapeutic strategies. We previously demonstrated that extracellular soluble tau triggers p38 MAPK activation in microglia. Given the activation of this signaling pathway in AD and its involvement in neuroinflammation processes, here we evaluated the effect of p38 inhibition on primary microglia cultures subjected to tau treatment. Our data showed that the toxic effect driven by tau in microglia was diminished through p38 inhibition. Furthermore, p38 blockade enhanced microglia-mediated tau phagocytosis, as reflected by an increase in the number of lysosomes. In conclusion, these results contribute to our understanding of the functions of p38 in the central nervous system (CNS) beyond tau phosphorylation in neurons and provide further insights into the potential of p38 inhibition as a therapeutic strategy to halt neuroinflammation in tauopathies.

Melatonin Modulates Microglia Activation in Neuro-inflammation by Regulating the ER Stress/PPARd/SIRT1 Signaling Pathway

Background: Activated microglia-mediated neuro-inflammation plays a vital aspect in regulating the micromilieu of the central nervous system. Neuro-inflammation involves distinct alterations of microglial phenotypes, containing nocuous pro-inflammatory (M1) phenotype and neuroprotective anti-inflammatory (M2) phenotype. Currently, there is no effective treatment for modulating such alterations. Little evidence shows that melatonin prevents the detrimental cascade of activated microglia-mediated neuro-inflammation. Methods: The expression levels of M1/M2 marker of primary microglia influenced by Melatonin were detected via qPCR. Functional activities were explored by western blotting, luciferase activity, EMSA, and ChIP assay. Structure interaction was assessed by molecular docking and LIGPLOT analysis. ER stress detection was examined by ultrastructure TEM, calapin activity, and ERSE assay. The neurobehavioral evaluations and immunofluorescence staining in animals were used for investigation of Melatonin on the neuroinflammation in vivo. Results: Melatonin had targeted on Peroxisome Proliferator Activated Receptor Delta (PPARd) activity, boosted LPS-stimulated alterations in polarization from the M1 to the M2 phenotype, and thereby inhibited NFkB–IKKb activation in primary microglia. The PPARd agonist L-165041 or over-expression of PPARd plasmid (ov-PPARd) showed similar results. Molecular docking screening, dynamic simulation approaches, and biological studies of melatonin showed that the activated site was located at PPARd (phospho-Thr256-PPARd). Furthermore, we found that activated microglia had lowered PPARd activity as well as the downstream SIRT1 formation via enhancing ER stress. Melatonin, PPARd agonist and ov-PPARd all effectively reversed the above-mentioned effects. Melatonin blocked ER stress by regulating calapin activity and expression in LPS-activated microglia. Additionally, melatonin or L-165041 ameliorated the neurobehavioral deficits in LPS-aggravated neuroinflammatory mice through blocking microglia activities, and also promoted phenotype changes to M2-predominant microglia. Conclusions Melatonin suppressed neuro-inflammation in vitro and in vivo by tuning microglial activation through the ER stress-dependent PPARd/SIRT1 signaling cascade. We proposed that this treatment strategy is an encouraging pharmacological approach for the remedy of neuro-inflammation associated disorders.

Autism-Like Behavior in the Offspring of CYP11A1-Overexpressing Pregnant Rats

Autism spectrum disorders (ASD) represent a complex group of neurodevelopmental disorders that are characterized by impaired social behavior and communication as well as repetitive behavior and restricted interests. Prenatal exposure to high levels of testosterone and preeclampsia are thought to be risk factors of ASD. We had previously reported that overexpression of the mitochondrial cholesterol side-chain cleavage enzyme (CYP11A1) could lead to both preeclampsia-like symptoms and increased testosterone levels in pregnant rats. In this study, we investigated the association between high CYP11A1 levels in pregnant rats and autism-like behavior in their offspring. Timed-pregnant Sprague-Dawley (SD) rats were injected with CYP11A1 gene-carrying adenoviruses on gestational day 8.5, and their offspring were then compared with those from timed-pregnant control SD rats. Compared with their control counterparts, the offspring of the CYP11A1-ovexpressing dams displayed more symptoms of anxiety and spent less time in social interactions and more time in self-grooming and rearing, all indicators of autism-like behavior. Sequencing of the transcriptome in primary microglia from the offspring of CYP11A1-overexpressing dams revealed that immune pathways were highly activated, and the gamma-aminobutyric acid type A (GABAA) receptor genes were among the top differentially expressed genes. Using primary microglia cultures generated from neonatal rats, tumor necrosis factor-alpha expression was found to be elevated in the cells transfected with CYP11A1-carrying adenoviruses. Additionally, the offspring of CYP11A1-overexpressing dams displayed dysregulated GABAA receptor expression. Taken together, these results suggest that abnormal CYP11A1 gene expression in pregnant rats could lead to microglial immune activation and dysregulated GABAA receptor expression in their offspring and thereby anxiety and autism-related behavior. Our study suggests that the pathways regulated by CYP11A1 could be promising preventative and therapeutic targets for ASD.

Characterization of the Leucocyte Immunoglobulin-like Receptor B4 (Lilrb4) Expression in Microglia

As resident innate immune cells of the CNS, microglia play important essential roles during physiological and pathological situations. Recent reports have described the expression of Lilrb4 in disease-associated and aged microglia. Here, we characterized the expression of Lilrb4 in microglia in vitro and in vivo in comparison with bone marrow-derived monocytes and peritoneal macrophages in mice. Using BV2 cells, primary microglia cultures as well as ex vivo isolated microglia and myeloid cells in combination with qPCR and flow cytometry, we were able to provide a comprehensive characterization of Lilrb4 expression in distinct mouse myeloid cells. Whereas microglia in vivo display low expression of Lilrb4, primary microglia cultures present high levels of surface LILRB4. Among the analyzed peripheral myeloid cells, peritoneal macrophages showed the highest expression levels of Lilrb4. Moreover, LPS treatment and inhibition of microglial TGFβ signaling resulted in significant increases of LILRB4 cell surface levels. Taken together, our data indicate that LILRB4 is a reliable surface marker for activated microglia and further demonstrate that microglial TGFβ signaling is involved in the regulation of Lilrb4 expression during LPS-induced microglia activation.

Diethyl Succinate Modulates Microglial Polarization and Activation by Reducing Mitochondrial Fission and Cellular ROS

Succinate is a metabolite in the tricarboxylic acid cycle (TCA) which plays a central role in mitochondrial activity. Excess succinate is known to be transported out of the cytosol, where it activates a succinate receptor (SUCNR1) to enhance inflammation through macrophages in various contexts. In addition, the intracellular role of succinate beyond an intermediate metabolite and prior to its extracellular release is also important to the polarization of macrophages. However, the role of succinate in microglial cells has not been characterized. Lipopolysaccharide (LPS) stimulates the elevation of intracellular succinate levels. To reveal the function of intracellular succinate associated with LPS-stimulated inflammatory response in microglial cells, we assessed the levels of ROS, cytokine production and mitochondrial fission in the primary microglia pretreated with cell-permeable diethyl succinate mimicking increased intracellular succinate. Our results suggest that elevated intracellular succinate exerts a protective role in the primary microglia by preventing their conversion into the pro-inflammatory M1 phenotype induced by LPS. This protective effect is SUCNR1-independent and mediated by reduced mitochondrial fission and cellular ROS production.

Direct Inhibition of Microglia Activation by Pretreatment With Botulinum Neurotoxin A for the Prevention of Neuropathic Pain

Peripheral injection of botulinum neurotoxin A (BoNT/A) has been demonstrated to have a long-term analgesic effect in treating neuropathic pain. Around peripheral nerves, BoNT/A is taken up by primary afferent neurons and inhibits neuropeptide release. Moreover, BoNT/A could also be retrogradely transported to the spinal cord. Recent studies have suggested that BoNT/A could attenuates neuropathic pain by inhibiting the activation of spinal glial cells. However, it remains unclear whether BoNT/A directly interacts with these glial cells or via their interaction with neurons. Our aim here is to determine the direct effect of BoNT/A on primary microglia and astrocytes. We show that BoNT/A pretreatment significantly inhibits lipopolysaccharide (LPS) -induced activation and pro-inflammatory cytokine release in primary microglia (1 U/mL BoNT/A in medium), while it has no effect on the activation of astrocytes (2 U/mL BoNT/A in medium). Moreover, a single intrathecal pre-administration of a low dose of BoNT/A (1 U/kg) significantly prohibited the partial sciatic nerve ligation (PSNL)- induced upregulation of pro-inflammatory cytokines in both the spinal cord dorsal horn and dorsal root ganglions (DRGs), which in turn prevented the PSNL-induced mechanical allodynia and thermal hyperalgesia. In conclusion, our results indicate that BoNT/A pretreatment prevents PSNL-induced neuropathic pain by direct inhibition of spinal microglia activation.

Maraviroc, An Inhibitor of Chemokine Receptor Type 5, Alleviates Neuroinflammatory Response after Cerebral Ischemia/Reperfusion Injury via Regulating JNK Signaling

Neuroinflammation is a key factor that contributes to the secondary injury after cerebral ischemia/reperfusion (CI/R) injury. Chemokine receptor type 5(CCR5) has shown its pro-inflammatory effects during central nervous system (CNS) diseases. However, the role of CCR5 in CI/R injury is still unclear. In this study, we administered maraviroc (MVC,APEXBIO,UK-427857), a CCR5 antagonist, to the middle cerebral artery occlusion(MCAO) mice. In vivo studies showed that MVC was successively intraperitoneally (i.p.) with different doses (5, 20, or 50 mg/kg body weight) for 3 days after mice MCAO. MVC showed its neuroprotective effects in alleviating neurological deficits and infarct volumes after MCAO. The level of apoptosis and inflammation were remarkably decreased by MVC treatment after CI/R injury. Subsequently, primary microglia were stimulated with different doses of MVC (0.2, 2, 20 or 200nM) for 12h after oxygen-glucose deprivation/reoxygenation model (OGD/R) in vitro. MVC significantly increased the viability of primary microglia after (OGD/R). The expression of pro-inflammatory cytokines (IL-1β and IL-6) in microglia were down-regulated by MVC treatment. Mechanistically, MVC also inhibited the secretion of IL-1β and IL-6 by microglia after OGD stimulation. Furthermore, the key components of NF-κB pathway were measured in vivo and in vitro after MCAO and OGD. MVC significantly inhibited the activity of NF-κB pathway in the above pathological environments. Finally, our data indicated that MVC treatment decreased the activation of JNK signaling pathway after CI/R injury in vivo and in vitro. The JNK activator anisomycin (AN，Beyotime，SC0132-5mg) reversed the neuroprotective effects of MVC, indicating that the JNK pathway is involved in the anti-inflammatory and anti-apoptotic mechanisms of MVC in CI/R injury. Our data demonstrated that CCR5 inhibition exhibits neuroprotective effects after CI/R injury. MVC, which is widely used for HIV treatment by its anti-virus effect, is a potential drug for the treatment of ischemic stroke in the future clinical trials.

Diosgenin Prevents Microglial Activation and Protects Dopaminergic Neurons from Lipopolysaccharide-Induced Neural Damage In Vitro and In Vivo

Background: The prevention of age-related neurodegenerative disorders is an important issue in an aging society. Microglia-mediated neuroinflammation resulting in dopaminergic neuron loss may lead to the pathogenesis of Parkinson’s disease (PD). Lipopolysaccharide (LPS), an endotoxin, induces neuroinflammatory microglial activation, contributing to dopaminergic neuron damage. Diosgenin is a phytosteroid sapogenin with a wide spectrum of pharmacological activities, e.g., anti-inflammatory activity. However, the preventive effect of diosgenin on neuroinflammation is not clear. Thus, in this study, we further investigated the neuroprotective effect of diosgenin on LPS-induced neural damage in vitro and in vivo. Methods: For in vitro experiments, primary mesencephalic neuron-glia cultures and primary microglia cultures isolated from Sprague–Dawley rats were used. Cells were pretreated with diosgenin and then stimulated with LPS. The expression of proinflammatory cytokines or tyrosine hydroxylase (TH) in the cells was analyzed. In vivo, rats were fed a diet containing 0.1% (w/w) diosgenin for 4 weeks before being administered a unilateral substantia nigra (SN) injection of LPS. Four weeks after the LPS injection, the rats were assessed for lesion severity using the amphetamine-induced rotation test and TH immunohistochemistry. Results: Diosgenin pretreatment prevented LPS-induced neurite shortening in TH-positive neurons in mesencephalic neuron-glia cultures. In addition, pretreatment of primary microglia with diosgenin significantly reduced tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression. Moreover, diosgenin pretreatment significantly suppressed LPS-induced extracellular signal-regulated kinase (ERK) activation. In vivo, the intranigral injection of LPS in rats fed a diosgenin-containing diet significantly improved motor dysfunction and reduced TH expression in SN. Conclusion: These results support the effectiveness of diosgenin in protecting dopaminergic neurons from LPS-induced neuroinflammation.

Endothelial Progenitor Cell Transplantation Attenuated Synaptic Loss by Enhancing CR3 Dependent Microglial Phagocytosis in Mice

Background: Endothelial progenitor cell (EPC) transplantation has been shown to have therapeutic effects in cerebral ischemia. However, whether the therapeutic effect of EPCs is a result of the modulation of microglia activity remain elusive. Methods: Adult male mice (n=184) underwent 90 minute-middle cerebral artery occlusion and EPCs were transplanted into the peri-infarct region immediately after the surgery. Microglia migration and phagocytosis were evaluated in the ischemic brain in vivo and underwent oxygen-glucose-deprivation culture condition in vitro. Complement receptor 3 was examined in ischemic brain and cultured primary microglia. Complement receptor 3 agonist leukadherin-1 was intraperitoneally injected to mice immediately after ischemia to imitate the EPC effect. Expression of synapse remodeling related synaptophysin and PSD-95 proteins was detected in the EPC and leukadherin-1 treated mice, separately. Results: EPC transplantation increased the number of microglia in the peri-infarct region of the brain at 3 days after focal ischemia (p<0.05). The ability of phagocytizing apoptotic cells of microglia was higher in EPCs transplanted group at 3 days after ischemia compared to the controls (p<0.05). In vitro study showed that cultured microglia displayed a higher migration (p<0.05) and phagocytosis ability (p<0.05) under the stimulation of EPC conditioned medium or cultured EPCs compared to the controls. Complement receptor 3 expression in the ischemic mouse brain with EPC transplantation (p<0.05), and primary microglia treated by EPC conditioned medium or cultured EPCs was up-regulated (p<0.05). Leukadherin-1 reduced brain atrophy volume at 14 days (p<0.05) and ameliorated neurological deficiency during 14 days after cerebral ischemia (p<0.05). Both EPC transplantation and leukadherin-1 injection increased synaptophysin (p<0.05) and PSD-95 expressions (p<0.05) at 14 days after focal ischemia. Conclusion: We concluded that EPC transplantation promoted regulating complement receptor 3 mediated microglial phagocytosis at acute phase, and subsequently benefited for attenuating synaptic loss at the recovery phase of ischemic stroke, which provided a novel therapeutic mechanism of EPC for cerebral ischemia.