Aprepitant Inhibits JNK and p38/MAPK to Attenuate Inflammation and Suppresses Inflammatory Pain

Substance P contributes to the pathogenesis of pain by acting on NK-1R, specialized sensory neurons that detect noxious stimuli. Aprepitant, an antagonist of NK-1R, is widely used to treat chemotherapy-induced nausea and vomiting. In this study, we used LPS-stimulated BV-2 microglia cell line and animal models of inflammatory pain to explore the analgesic effect of aprepitant on inflammatory pain and its underlying mechanism. The excitability of DRG neurons were measured using whole-cell patch-clamp recordings. The behavioral tests were measured and the morphological changes on inflamed paw sections were determined by HE staining. Changes in the expressions of cytokine were measured by using real-time quantitative PCR analysis and ELISA method. Immunofluorescence and western blotting were used to detect the microglia activation and MAPK. Aprepitant treatment significantly inhibited the excitability of DRG neurons. The pain behavior and the paw tissues inflammatory damage were significantly relived after the administration of aprepitant compared to formalin group. Aprepitant significantly suppressed the activation of microglia, phosphorylation of JNK and p38 MAPK, as well as the mRNA and protein expressions of MCP-1, TNF-α, IL-6, and IL-1β, in vivo and in vitro. The LPS-induced over-translocation into nucleus of NF-κBp65 was down-regulated following aprepitant treatment in BV-2 cells. The present study suggests that aprepitant attenuates inflammatory pain in mice via suppressing the phosphorylation of JNK and p38, and inhibiting the NF-κB signaling pathway.

Neurotoxic Potential of Deoxynivalenol in Murine Brain Cell Lines and Primary Hippocampal Cultures

Chronic exposure to the mycotoxin deoxynivalenol (DON) from grain-based food and feed affects human and animal health. Known consequences include entereopathogenic and immunotoxic defects; however, the neurotoxic potential of DON has only come into focus more recently due to the observation of behavioural disorders in exposed farm animals. DON can cross the blood-brain barrier and interfere with the homeostasis/functioning of the nervous system, but the underlying mechanisms of action remain elusive. Here, we have investigated the impact of DON on mouse astrocyte and microglia cell lines, as well as on primary hippocampal cultures by analysing different toxicological endpoints. We found that DON has an impact on the viability of both glial cell types, as shown by a significant decrease of metabolic activity, and a notable cytotoxic effect, which was stronger in the microglia. In astrocytes, DON caused a G1 phase arrest in the cell cycle and a decrease of cyclic-adenosine monophosphate (cAMP) levels. The pro-inflammatory cytokine tumour necrosis factor (TNF)-α was secreted in the microglia in response to DON exposure. Furthermore, the intermediate filaments of the astrocytic cytoskeleton were disturbed in primary hippocampal cultures, and the dendrite lengths of neurons were shortened. The combined results indicated DON’s considerable potential to interfere with the brain cell physiology, which helps explain the observed in vivo neurotoxicological effects.

Opening KATP Channels Induces Inflammatory Tolerance and Prevents Chronic Pain

Background: Current treatments for chronic pain are not satisfactory, prompting a frantic search for new therapeutics and new therapeutic targets. Our previous study indicates KATP channel opener has analgesic effect, but the mechanism has not been elucidated. We speculated that KATP channel opener may increase suppressor of cytokine signaling (SOCS)-3 expression to induce inflammatory tolerance and attenuate chronic pain. Methods: The plantar incision (PI) surgery-induced postoperative pain was performed to establish chronic pain model. Growth arrest–specific 6 (Gas6)-/- and Axl-/- mice were used for signaling research. The microglia cell line BV-2 was cultured for in vitro experiments.Results: KATP channel opener significantly attenuated incision-induced mechanical allodynia in mice, associated with the up-regulated expression of SOCS3. Opening KATP channels induced the expression of SOCS3 dependent on Gas6/Axl signaling pathway in microglia. Opening KATP channels inhibits incision-induced mechanical allodynia by activating Gas6/Axl-SOCS3 signaling pathway. Opening KATP channels induces inflammatory tolerance to relieve neuroinflammation and postoperative pain.Conclusions: We demonstrated that KATP channel opening activated Gas6/Axl/SOCS3 signaling to induce inflammatory tolerance and relief chronic pain. We explored a new target for anti-inflammatory and analgesia by regulating the innate immune system, and provide a theoretical basis for clinical preemptive analgesia.

Fluoxetine Potentiates Phagocytosis and Autophagy in Microglia

Fluoxetine is a classic antidepressant drug, and its immunomodulatory effects have recently been reported in many disease models. In addition, it has strong antineuroinflammatory effects in stroke and neurodegenerative animal models. However, the effect of fluoxetine on microglia phagocytosis and its molecular mechanisms have not yet been studied. In this study, we investigated whether fluoxetine has a regulatory effect on microglial function. Microglia cell lines and primary mouse microglia were treated with fluoxetine, and the production of inflammatory cytokines and neurotrophic factors and the phagocytosis of amyloid β were measured. Fluoxetine significantly attenuated the production of lipopolysaccharide-induced proinflammatory cytokines and oxidative stress in microglia. Fluoxetine also significantly potentiated microglia phagocytosis and autophagy. In addition, autophagy flux inhibitors attenuated fluoxetine-induced phagocytosis. In conclusion, fluoxetine induces autophagy and potentiates phagocytosis in microglia, which can be a novel molecular mechanism of the neuroinflammatory and neuroprotective effects of fluoxetine.

A Core Transcription Regulatory Circuitry Defining Microglia Cell Identity Inferred from the Reanalysis of Multiple Human Microglia Differentiation Protocols

Microglia, the immune cells in the brain involved in both homeostasis and injury/infection control, play a predominant role in neurodegenerative diseases. In vivo studies on microglia are limited due to the requirement of surgical intervention, which can lead to the destruction of the tissues. Over the last few years, multiple protocols—presenting a variety of strategies—have described microglia differentiation issued from human pluripotent stem cells. Herein, we have reanalyzed the transcriptomes released on six different microglia differentiation protocols and revealed a consensus core of master transcription regulatory circuitry defining microglia identity. Furthermore, we have discussed the major divergencies among the studied protocols and have provided suggestions to further enhance microglia differentiation assays.

Validation and Selection of New Reference Genes for RT-qPCR Analysis in Pediatric Glioma of Different Grades

Gliomas are heterogeneous, solid, and intracranial tumors that originate from glial cells. Malignant cells from the tumor undergo metabolic alterations to obtain the energy required for proliferation and the invasion of the cerebral parenchyma. The alterations in the expression of the genes related to the metabolic pathways can be detected in biopsies of gliomas of different CNS WHO grades. In this study, we evaluated the expression of 16 candidate reference genes in the HMC3 microglia cell line. Then, statistical algorithms such as BestKeeper, the comparative ΔCT method, geNorm, NormFinder, and RefFinder were applied to obtain the genes most suitable to be considered as references for measuring the levels of expression in glioma samples. The results show that PKM and TPI1 are two novel genes suitable for genic expression studies on gliomas. Finally, we analyzed the expression of genes involved in metabolic pathways in clinical samples of brain gliomas of different CNS WHO grades. RT-qPCR analysis showed that in CNS WHO grade 3 and 4 gliomas, the expression levels of HK1, PFKM, GAPDH, G6PD, PGD1, IDH1, FASN, ACACA, and ELOVL2 were higher than those of CNS WHO grade 1 and 2 glioma biopsies. Hence, our results suggest that reference genes from metabolic pathways have different expression profiles depending on the stratification of gliomas and constitute a potential model for studying the development of this type of tumor and the search for molecular targets to treat gliomas.

p38-TFEB Pathways Promote Microglia Activation Through Inhibiting CMA-mediated NLRP3 Degradation in Parkinson's Disease

BackgroundParkinson’s disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), accompanied by chronic neuroinflammation, autophagy dysfunction and α-synuclein accumulation in the form of Lewy bodies. Previous studies showed that misfolded α-synuclein upregulates the inflammatory and autophagy dysfunction in microglial cell. The NLRP3 inflammasome signaling pathway plays a crucial role in the neuroinflammatory process in the central nervous system. However, the inter-relationship between autophagy deficiency and neuroinflammation induced by α-synuclein accumulation is not well understood.MethodsWe investigated the impact of p38-TFEB-NLRP3 pathways on neuroinflammation in the α-synucleinA53T PD models, using a combination of immunoblotting, immunofluorescence, immunocytochemistry, flow cytometry, ELISA, and a series of behavioral texts.ResultsIn the present study, we showed NLRP3 was degraded through chaperone-mediated autophagy (CMA) in microglia cell. Furthermore, p38-TFEB pathways inhibited CMA-mediated NLRP3 degradation in Parkinson's disease. Overexpress mice and BV2 cells with α-synuclein A53T mutant active P38, which inhibit the master transcriptional activator of autophagy, TEEB in BV2 cells. Notably, inhibition p38 had a protective effect on Parkinson's disease model, which depend on suppressing the activation of NLRP3 inflmmasome pathway. Importantly, both p38 inhibitor SB203580 and NLRP3 inhibitor MCC950 not only prevent neurodegeneration in vitro, but also alleviates movement impairment in α-synucleinA53T-tg mice model of Parkinson’s disease. ConclusionOur research reveals an endogenous regulatory mechanism of NLRP3 turnover and microglia-dopaminergic neuron interaction, which may be a potential therapeutic strategy for Parkinson’s disease.

Purinoreceptors and ectonucleotidases control ATP-induced calcium waveforms and calcium-dependent responses in microglia

Adenosine triphosphate (ATP) drives microglia motility and cytokine production by activating P2X- and P2Y- class purinergic receptors with extracellular ATP and its metabolites. Purinergic receptor activation gives rise to diverse intracellular Ca2+ signals, or waveforms, that differ in amplitude, duration, and frequency. Whether and how these diverse waveforms influence microglia function is not well established. We developed a computational model trained with published primary murine microglia studies. We simulate how purinoreceptors influence Ca2+ signaling and migration and how purinoreceptor expression modifies these processes. Our simulation confirmed that P2 receptors encode the amplitude and duration of the ATP-induced calcium waveforms. Our simulations also implicate CD39, an ectonucleotidase that rapidly degrades ATP, as a regulator of purinergic receptor-induced Ca2+ responses. We, therefore, next evaluated how purinoreceptors and ectonucleotidase work in tandem. Our modeling results indicate that small transients are sufficient to promote motility, while large and sustained transients are needed for cytokine responses. Lastly, we predict how these phenotypical responses vary in a BV2 microglia cell line using published P2 receptor mRNA data to illustrate how our computer model can be extrapolated to diverse microglia subtypes. These findings provide important insights into how differences in purinergic receptor expression influence the microglial responses to ATP.

miR-335 Targets LRRK2 and Mitigates Inflammation in Parkinson’s Disease

Parkinson’s disease (PD) is mainly driven by dopaminergic neuronal degeneration in the substantia nigra pars compacta accompanied by chronic neuroinflammation. Despite being mainly sporadic, approximately 10% of all cases are defined as heritable forms of PD, with mutations in the leucine-rich repeat kinase (LRRK2) gene being the most frequent known cause of familial PD. MicroRNAs (miRNAs or miRs), including miR-335, are frequently deregulated in neurodegenerative diseases, such as PD. Here, we aimed to dissect the protective role of miR-335 during inflammation and/or neurodegenerative events in experimental models of PD. Our results showed that miR-335 is significantly downregulated in different PD-mimicking conditions, including BV2 microglia cells stimulated with lipopolysaccharide (LPS) and/or overexpressing wild-type LRRK2. Importantly, these results were confirmed in serum of mice injected with 1-methyl-1-4-phenyl-1,2,3,6-tetrahydripyridine hydrochloride (MPTP), and further validated in patients with idiopathic PD (iPD) and those harboring mutations in LRRK2 (LRRK2-PD), thus corroborating potential clinical relevance. Mechanistically, miR-335 directly targeted LRRK2 mRNA. In the BV2 and N9 microglia cell lines, miR-335 strongly counteracted LPS-induced proinflammatory gene expression, and downregulated receptor interacting protein 1 (RIP1) and RIP3, two important players of necroptotic and inflammatory signaling pathways. Further, miR-335 inhibited LPS-mediated ERK1/2 activation. LRRK2-Wt-induced proinflammatory gene expression was also significantly reduced by miR-335 overexpression. Finally, in SH-SY5Y neuroblastoma cells, miR-335 decreased the expression of pro-inflammatory genes triggered by α-synuclein. In conclusion, we revealed novel roles for miR-335 in both microglia and neuronal cells that strongly halt the effects of classical inflammatory stimuli or LRRK2-Wt overexpression, thus attenuating chronic neuroinflammation.