NLRP3 Inflammasome Activation In Microglia Research Articles

Cycloastragenol reduces microglial NLRP3 inflammasome activation in Parkinson's disease models by promoting autophagy and reducing Scrib-driven ROS

BackgroundIn Parkinson's disease (PD), microglial autophagy is crucial for the maintenance of cellular redox homeostasis. Meanwhile, cycloastragenol (CAG), a triterpenoid saponin and the principal active component of Astragalus, reduces the activation of NLRP3 inflammasomes. Nevertheless, the specific molecular mechanisms underlying the CAG-mitigated microglial neuroinflammation remains obscure in PD. PurposeThis study explored the role of CAG in the activation of microglial NLRP3 inflammasome and the mechanisms underlying its therapeutic potential for PD treatment. Study designThe effect of CAG was assessed in α-Syn-induced primary microglia and PD models. MethodsAAV1/2-hsyn-SNCA (A53T) was stereo-injected into the striatum of mice to induce PD models and CAG was orally administered. The mice underwent quantitative 4D proteomics analysis and behavioral assessments. The primary microglia and neuron cultures were analyzed by western blotting, immunofluorescence, transmission electron microscopy, etc. ResultsCAG reduced phagocytosis-induced reactive oxygen species (ROS) by suppressing the microglial Scribble (Scrib) and p22phox expression. Concurrently, CAG enhanced autophagy, promoted α-Syn clearance, and reduced mitochondrial damage. These synergistic effects downregulated NLRP3 inflammasome activation, in turn reducing gasdermin D cleavage, caspase-1 activation, and the release of interleukin-1β and interleukin-18. Further investigation revealed that CAG shielded neurons from α-Syn toxicity, thus attenuating behavioral impairments observed in the mouse PD model. ConclusionCAG mitigates neuroinflammation by inhibiting ROS-induced NLRP3 inflammasome activation in microglia via promoting microglial autophagy and reducing the activity of Scrib-associated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which signifies a promising alternative approach to PD management.

Polyphyllin I alleviates neuroinflammation after cerebral ischemia–reperfusion injury via facilitating autophagy-mediated M2 microglial polarization

Microglial activation and polarization play a central role in poststroke inflammation and neuronal damage. Modulating microglial polarization from pro-inflammatory to anti-inflammatory phenotype is a promising therapeutic strategy for the treatment of cerebral ischemia. Polyphyllin I (PPI), a steroidal saponin, shows multiple bioactivities in various diseases, but the potential function of PPI in cerebral ischemia is not elucidated yet. In our study, the influence of PPI on cerebral ischemia–reperfusion injury was evaluated. Mouse middle cerebral artery occlusion (MCAO) model and oxygen–glucose deprivation and reoxygenation (OGD/R) model were constructed to mimic cerebral ischemia–reperfusion injury in vivo and in vitro. TTC staining, TUNEL staining, RT-qPCR, ELISA, flow cytometry, western blot, immunofluorescence, hanging wire test, rotarod test and foot-fault test, open-field test and Morris water maze test were performed in our study. We found that PPI alleviated cerebral ischemia–reperfusion injury and neuroinflammation, and improved functional recovery of mice after MCAO. PPI modulated microglial polarization towards anti-inflammatory M2 phenotype in MCAO mice in vivo and post OGD/R in vitro. Besides, PPI promoted autophagy via suppressing Akt/mTOR signaling in microglia, while inhibition of autophagy abrogated the effect of PPI on M2 microglial polarization after OGD/R. Furthermore, PPI facilitated autophagy-mediated ROS clearance to inhibit NLRP3 inflammasome activation in microglia, and NLRP3 inflammasome reactivation by nigericin abolished the effect of PPI on M2 microglia polarization. In conclusion, PPI alleviated post-stroke neuroinflammation and tissue damage via increasing autophagy-mediated M2 microglial polarization. Our data suggested that PPI had potential for ischemic stroke treatment.

Liver-specific adiponectin gene therapy suppresses microglial NLRP3-inflammasome activation for treating Alzheimer’s disease

Adiponectin (APN) is an adipokine which predominantly expresses in adipocytes with neuroprotective and anti-inflammatory effects. We have recently indicated that circulatory trimeric APN can enter the brain by crossing the blood–brain barrier (BBB) and modulate microglia-mediated neuroinflammation. Here, we found that the microglial NLR family pyrin domain containing 3 (NLRP3)-inflammasome activation was exacerbated in APN−/−5xFAD mice in age-dependent manner. The focus of this study was to develop a new and tractable therapeutic approach for treating Alzheimer’s disease (AD)-related pathology in 5xFAD mice using peripheral APN gene therapy. We have generated and transduced adeno-associated virus (AAV2/8) expressing the mouse mutated APN gene (APNC39S) into the liver of 5xFAD mice that generated only low-molecular-weight trimeric APN (APNTri). Single dose of AAV2/8-APNC39S in the liver increased circulatory and cerebral APN levels indicating the overexpressed APNTri was able to cross the BBB. Overexpression of APNTri decreased both the soluble and fibrillar Aβ in the brains of 5xFAD mice. AAV2/8-APNTri treatment reduced Aβ-induced IL-1β and IL-18 secretion by suppressing microglial NLRP3-inflammasome activation. The memory functions improved significantly in AAV-APNTri-treated 5xFAD mice with reduction of dystrophic neurites. These findings demonstrate that peripheral gene delivery to overexpress trimeric APN can be a potential therapy for AD.Graphical

Vagus nerve stimulation as a promising neuroprotection for ischemic stroke via α7nAchR-dependent inactivation of microglial NLRP3 inflammasome.

Ischemic stroke is a major cause of disability and death worldwide, and its management requires urgent attention. Previous studies have shown that vagus nerve stimulation (VNS) exerts neuroprotection in ischemic stroke by inhibiting neuroinflammation and apoptosis. In this study, we evaluated the timing for VNS intervention in ischemic stroke, and the underlying mechanisms ofVNS-induced neuroprotection. Mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 60 min. The left vagus nerve at cervical level was exposed and attached to an electrode connected to a low-frequency electrical stimulator. Vagus nerve stimulation (VNS) was given for 60 min before, during and after tMCAO (Pre-VNS, Dur-VNS, Post-VNS). Neurological function was assessed 24 h after reperfusion. We found that all the three VNS significantly protected against the tMCAO-induced injury evidenced by improved neurological function and reduced infarct volume. Moreover, the Pre-VNS was the most effective against the ischemic injury. We found that tMCAO activated microglia in the ischemic core and penumbra regions of the brain, followed by the NLRP3 inflammasome activation-induced neuroinflammation, which finally triggered neuronal death. VNS treatment preserved α7nAChR expression in the penumbra regions, inhibited NLRP3 inflammasome activation and ensuing neuroinflammation, rescuing cerebral neurons. The role of α7nAChR in microglial NLRP3 inflammasome activation in ischemic stroke was further validated using genetic manipulations, including Chrna7 knockout mice and microglial Chrna7 overexpression mice, as well as pharmacological interventions using the α7nAChR inhibitor methyllycaconitine and agonist PNU-282987. Collectively, this study demonstrates the potential of VNS as a safe and effective strategy to treat ischemic stroke, and presents a new approach targeting microglial NLRP3 inflammasome, which might be therapeutic for other inflammation-related diseases.

Quercetin Ameliorates Cognitive Impairment in Depression by Targeting HSP90 to Inhibit NLRP3 Inflammasome Activation.

Cognitive dysfunction was a common symptom of major depressive disorder (MDD). In previous studies, psychological stress leads to activation and proliferation of microglial cells in different brain regions. Quercetin, a bioflavonoid derived from vegetables and fruits, exerts anti-inflammatory effects in various diseases. To demonstrate the role of quercetin in thehippocampal inflammatory response in depress mice. The chronic unpredictable stress (CUS) depressive mice model built is used to explore the protective effects of quercetin on depression. Neurobehavioral test, protein expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and heat shock protein 90 (HSP90), and cytokines (IL-6, IL-1β, MCP-1, and TNF-α) were assessed. Quercetin ameliorated depressive-like behavior and cognitive impairment, and quercetin attenuates neuroinflammation and by targeting HSP90 to inhibit NLRP3 inflammasome activation. Quercetin inhibited the increase of HSP90 levels in the hippocampus and reverses inflammation-induced cognitive impairment. Besides, quercetin inhibited the increased level of cytokines (IL-6, IL-1β, MCP-1, and TNF-α) in the hippocampus of the depressive model mouse and the increased level of cytokines (IL-6, IL-1β, and MCP-1) in microglia. The current study indicated that quercetin mitigated depressive-like behavior and by targeting HSP90 to inhibit NLRP3 inflammasome activation in microglia and depressive mice model, meanwhile ameliorated cognitive impairment in depression. Quercetin has huge potential for the novel pharmacological efficacy of antidepressant therapy.

1. Advanced age and ethanol exposure potentiates markers of NLRP3 inflammasome activation in microglia

1. Advanced age and ethanol exposure potentiates markers of NLRP3 inflammasome activation in microglia

Echinacoside exerts neuroprotection via suppressing microglial α-synuclein/TLR2/NF-κB/NLRP3 axis in parkinsonian models

BackgroundEchinacoside (ECH), a natural active compound, was found to exert neuroprotection in Parkinson's disease (PD). However, the underlying molecular mechanisms remain controversial. PurposeThis study aimed to explore the roles of ECH in PD and its engaged mechanisms. ConclusionIn vivo, MPTP was adapted to construct subacute PD mouse model to explore the regulation of ECH on NLRP3 inflammasome. In vitro, α-synuclein (α-syn)/MPP+ was used to mediate the activation of NLRP3 inflammasome in BV2 cells, and the mechanism of ECH regulation of it was explored with molecular docking, immunofluorescence, Western blotting, and small molecule inhibitors. ConclusionThe activation of microglial NLRP3 inflammasome could be evoked by MPTP in vitro, but its toxic metabolite MPP+ alone cannot trigger the activation of NLRP3 inflammasome in vitro, which requires α-synuclein (α-syn) priming. Exogenous α-syn could evoke microglial TLR2/NF-κB/NLRP3 axis, playing the priming role in MPP+ -mediated NLRP3 inflammasome activation. ECH can suppress the upregulation of α-syn in MPTP-treated mice and BV2 microglia. It can also suppress the activation of the TLR2/NF-κB/NLRP3 axis induced by α-syn. ConclusionECH exerts neuroprotective effects by downregulating the TLR2/NF-κB/NLRP3 axis via reducing the expression of α-syn in the PD models.

Mitophagy-dependent mitochondrial ROS mediates 2,5-hexanedione-induced NLRP3 inflammasome activation in BV2 microglia

We recently revealed a pivotal role of NLRP3 inflammasome in the neurotoxicity induced by n-hexane, owing to its activation and release of pro-inflammatory cytokines. However, the mechanisms of how the activation of NLRP3 inflammasome was triggered by 2,5-hexanedione (HD), the toxic product of n-hexane metabolism, remain to be explored. Here, we investigated whether mitochondrial reactive oxygen species (mtROS) was involved in HD-elicited NLRP3 inflammasome activation in microglia. We demonstrated that exposure to HD at 4 and 8 mM elevated production of mtROS in BV2 microglia. Scavenging mtROS by Mito-TEMPO, an mtROS scavenger, dramatically reduced HD-induced NLRP3 expression, caspase-1 activation and interleukin-1β production, pointing a crucial role of mtROS in NLRP3 inflammasome activation. Mechanistic study revealed that HD intoxication promoted activation of mitophagy. HD induced expression of Beclin-1, LC3II, and two mitophagy-related proteins, i.e., Pink1 and Parkin and simultaneously, reduced p62 expression in both whole cell and isolated mitochondria of microglia. Furthermore, inhibition of mitophagy by 3-methyladenine (3-MA) greatly reduced production of mtROS, expression of mitochondrial fission-related proteins, dynamin-related protein 1 (Drp1) and fission protein 1 (Fis1) and activation of NLRP3 inflammasome in HD-intoxicated microglia. Blocking mitochondrial fission by Mdivi-1 also prevented HD-induced mtROS production and NLRP3 inflammasome activation in microglia. In conclusion, our data indicated that HD triggered activation of NLRP3 inflammasome through mitophagy-dependent mtROS production, offering an important insight for the immunopathogenesis of environmental toxins-induced neuroinflammation and neurotoxicity.

Cystathionine β-Synthase Suppresses NLRP3 Inflammasome Activation via Redox Regulation in Microglia.

Aims: Cystathionine β-synthase (CBS) is essential for homocysteine (Hcy) transsulfuration, yielding cysteine as a common precursor of hydrogen sulfide (H2S), glutathione (GSH), and other sulfur molecules, which produce neuroprotective effects in neurological conditions. We previously reported a disruption of microglial CBS/H2S signaling in a Parkinson's disease (PD) mouse model. Yet, it remains unclear whether CBS affects nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome activity and other pathologies in PD. Results: Microglial CBS expression decreased after lipopolysaccharide (LPS) stimulation. Elevated GSSG (the oxidized GSH) content and decreased H2S generation were found in the brains of microglial cbs conditional-knockout (cbscKO) mice, whereas serum and brain Hcy levels remained unaltered. Moreover, microglial cbscKO mice were susceptible to NLRP3 inflammasome activation and dopaminergic neuron losses caused by LPS injection into the substantia nigra, whereas cbs overexpression or activation produced opposite effects. In vitro studies showed that cbs overexpression or activation suppressed microglial NLRP3 inflammasome activation and interleukin (IL)-1β secretion by reducing mitochondrial reactive oxygen species (mitoROS) level. Conversely, ablation of cbs enhanced NLRP3 expression and mitoROS generation and augmented microglial NLRP3 inflammasome activity in response to adenosine triphosphate challenge, which was blocked by the mitoROS scavenger. Innovation and Conclusion: The study demonstrated an elevated GSSG level and reduced H2S generation, which correlated with a susceptible status of microglia in the brain of cbscKO mice. Our findings reveal a critical role of CBS in restraining the microglial NLRP3 inflammasome by controlling redox homeostasis and highlight that activation or upregulation of CBS may become a potential strategy for PD treatment.

NPLC0393 from Gynostemma pentaphyllum ameliorates Alzheimer's disease-like pathology in mice by targeting protein phosphatase magnesium-dependent 1A phosphatase.

Alzheimer's disease (AD) is a neurodegenerative disease with clinical hallmarks of progressive cognitive impairment and memory loss. Gynostemma pentaphyllum ameliorates cognitive impairment, but the mechanisms remain obscure. Here, we determine the effect of triterpene saponin NPLC0393 from G. pentaphyllum on AD-like pathology in 3×Tg-AD mice and elucidate the underlying mechanisms. NPLC0393 was administered daily in vivo by intraperitoneal injection for 3 months and its amelioration on the cognitive impairment in 3×Tg-AD mice was assessed by new object recognition (NOR), Y-maze, Morris water maze (MWM), and elevated plus-maze (EPM) tests. The mechanisms were investigated by RT-PCR, western blot, and immunohistochemistry techniques, while verified by the 3×Tg-AD mice with protein phosphatase magnesium-dependent 1A (PPM1A) knockdown (KD) through brain-specific injection of adeno-associated virus (AAV)-ePHP-KD-PPM1A. NPLC0393 ameliorated AD-like pathology targeting PPM1A. It repressed microglial NLRP3 inflammasome activation by reducing NLRP3 transcription during priming and promoting PPM1A binding to NLRP3 to disrupt NLRP3 assembly with apoptosis-associated speck-like protein containing a CARD and pro-caspase-1. Moreover, NPLC0393 suppressed tauopathy by inhibiting tau hyperphosphorylation through PPM1A/NLRP3/tau axis and promoting microglial phagocytosis of tau oligomers through PPM1A/nuclear factor-κB/CX3CR1 pathway. PPM1A mediates microglia/neurons crosstalk in AD pathology, whose activation by NPLC0393 represents a promising therapeutic strategy for AD.

Microglial NLRP3 inflammasome activation mediates diabetes-induced depression-like behavior via triggering neuroinflammation

BackgroundAbundant evidence suggests that the prevalence and risk of depression in people with diabetes is high. However, the pathogenesis of diabetes-related depression remains unclear. Since neuroinflammation is associated with the pathophysiology of diabetic complications and depression, this study aims to elucidate the neuroimmune mechanism of diabetes-related depression. MethodsMale C57BL/6 mice were injected with streptozotocin to establish a diabetes model. After screening, diabetic mice were treated with the NLRP3 inhibitor MCC950. Then, metabolic indicators and depression-like behaviors were evaluated in these mice, as well as their central and peripheral inflammation. To explore the mechanism of high glucose-induced microglial NLRP3 inflammasome activation, we performed in vitro studies focusing on its canonical upstream signal I (TLR4/MyD88/NF-κB) and signal II (ROS/PKR/P2X7R/TXNIP). ResultsDiabetic mice exhibited depression-like behaviors and activation of NLRP3 inflammasome in hippocampus. In vitro high-glucose (50 mM) environment primed microglial NLRP3 inflammasome by promoting NF-κB phosphorylation in a TLR4/MyD88-independent manner. Subsequently, high glucose activated the NLRP3 inflammasome via enhancing intracellular ROS accumulation, upregulating P2X7R, as well as promoting PKR phosphorylation and TXNIP expression, thereby facilitating the production and secretion of IL-1β. Inhibition of NLRP3 with MCC950 significantly restored hyperglycemia-induced depression-like behavior and reversed the increase in IL-1β levels in the hippocampus and serum. ConclusionThe activation of NLRP3 inflammasome, probably mainly in hippocampal microglia, mediates the development of depression-like behaviors in STZ-induced diabetic mice. Targeting the microglial inflammasome is a feasible strategy for the treatment of diabetes-related depression.

Curcumin protects against rotenone-induced Parkinson's disease in mice by inhibiting microglial NLRP3 inflammasome activation and alleviating mitochondrial dysfunction

Parkinson's disease (PD) is a common neurodegenerative disorder worldwide. Currently, treatment options can only relieve symptoms but cannot prevent, slow, or halt the neurodegenerative process of PD. Much evidence has suggested that microglia-mediated neuroinflammation is involved in the pathophysiology of PD. As an anti-inflammatory agent, curcumin may exert a neuroprotective effect on PD. However, its mechanism has yet to be demonstrated clearly. Our results indicated that curcumin alleviated rotenone-induced behavioral defects, dopamine neuron loss, and microglial activation. Besides, the NF-κB signaling pathway, the NLRP3 inflammasome, and pro-inflammatory cytokines, including IL-18 and IL-1β, contributed to the microglia-mediated neuroinflammation in PD. Furthermore, Drp1-mediated mitochondrial fission causing mitochondrial dysfunction also had an etiological role in the process. This study suggests that curcumin protects against rotenone-induced PD by inhibiting microglial NLRP3 inflammasome activation and alleviating mitochondrial dysfunction in mice. Thus, curcumin may be a neuroprotective drug with promising prospects in PD.

Parkin regulates microglial NLRP3 and represses neurodegeneration in Parkinson's disease.

Microglial hyperactivation of the NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) inflammasome contributes to the pathogenesis of Parkinson's disease (PD). Recently, neuronally expressed NLRP3 was demonstrated to be a Parkin polyubiquitination substrate and a driver of neurodegeneration in PD. However, the role of Parkin in NLRP3 inflammasome activation in microglia remains unclear. Thus, we aimed to investigate whether Parkin regulates NLRP3 in microglia. We investigated the role of Parkin in NLRP3 inflammasome activation through the overexpression of Parkin in BV2 microglial cells and knockout of Parkin in primary microglia after lipopolysaccharide (LPS) treatment. Immunoprecipitation experiments were conducted to quantify the ubiquitination levels of NLRP3 under various conditions and to assess the interaction between Parkin and NLRP3. In vivo experiments were conducted by administering intraperitoneal injections of LPS in wild-type and Parkin knockout mice. The Rotarod test, pole test, and open field test were performed to evaluate motor functions. Immunofluorescence was performed for pathological detection of key proteins. Overexpression of Parkin mediated NLRP3 degradation via K48-linked polyubiquitination in microglia. The loss of Parkin activity in LPS-induced mice resulted in excessive microglial NLRP3 inflammasome assembly, facilitating motor impairment, and dopaminergic neuron loss in the substantia nigra. Accelerating Parkin-induced NLRP3 degradation by administration of a heat shock protein (HSP90) inhibitor reduced the inflammatory response. Parkin regulates microglial NLRP3 inflammasome activation through polyubiquitination and alleviates neurodegeneration in PD. These results suggest that targeting Parkin-mediated microglial NLRP3 inflammasome activity could be a potential therapeutic strategy for PD.

Integrin Mac1 mediates paraquat and maneb-induced learning and memory impairments in mice through NADPH oxidase–NLRP3 inflammasome axis-dependent microglial activation

IntroductionThe mechanisms of cognitive impairments in Parkinson’s disease (PD) remain unknown. Accumulating evidence revealed that brain neuroinflammatory response mediated by microglial cells contributes to cognitive deficits in neuropathological conditions and macrophage antigen complex-1 (Mac1) is a key factor in controlling microglial activation.ObjectivesTo explore whether Mac1-mediated microglial activation participates in cognitive dysfunction in PD using paraquat and maneb-generated mouse PD model.MethodsCognitive performance was measured in wild type and Mac1−/− mice using Morris water maze test. The role and mechanisms of NADPH oxidase (NOX)–NLRP3 inflammasome axis in Mac1-mediated microglial dysfunction, neuronal damage, synaptic degeneration and phosphorylation (Ser129) of α-synuclein were explored by immunohistochemistry, Western blot and RT-PCR.ResultsGenetic deletion of Mac1 significantly ameliorated learning and memory impairments, neuronal damage, synaptic loss and α-synuclein phosphorylation (Ser129) caused by paraquat and maneb in mice. Subsequently, blocking Mac1 activation was found to mitigate paraquat and maneb-elicited microglial NLRP3 inflammasome activation in both in vivo and in vitro. Interestingly, stimulating activation of NOX by phorbol myristate acetate abolished the inhibitory effects of Mac1 blocking peptide RGD on paraquat and maneb-provoked NLRP3 inflammasome activation, indicating a key role of NOX in Mac1-mediated NLRP3 inflammasome activation. Furthermore, NOX1 and NOX2, two members of NOX family, and downstream PAK1 and MAPK pathways were recognized to be essential for NOX to regulate NLRP3 inflammasome activation. Finally, a NLRP3 inflammasome inhibitor glybenclamide abrogated microglial M1 activation, neurodegeneration and phosphorylation (Ser129) of α-synuclein elicited by paraquat and maneb, which were accompanied by improved cognitive capacity in mice.ConclusionsMac1 was involved in cognitive dysfunction in a mouse PD model through NOX–NLRP3 inflammasome axis-dependent microglial activation, providing a novel mechanistic basis of cognitive decline in PD.

CB2R activation ameliorates late adolescent chronic alcohol exposure-induced anxiety-like behaviors during withdrawal by preventing morphological changes and suppressing NLRP3 inflammasome activation in prefrontal cortex microglia in mice

BackgroundChronic alcohol exposure (CAE) during late adolescence increases the risk of anxiety development. Alcohol-induced prefrontal cortex (PFC) microglial activation, characterized by morphological changes and increased associations with neurons, plays a critical role in the pathogenesis of anxiety. Alcohol exposure increases NLRP3 inflammasome expression, increasing cytokine secretion by activated microglia. Cannabinoid type 2 receptor (CB2R), an essential receptor of the endocannabinoid system, regulates microglial activation and neuroinflammatory reactions. We aimed to investigate the role of CB2R activation in ameliorating late adolescent CAE-induced anxiety-like behaviors and microglial activation in C57BL/6J mice. MethodsSix-week-old C57BL/6J mice were acclimated for 7 days and then were administered alcohol by gavage (4 g/kg, 25 % w/v) for 28 days. The mice were intraperitoneally injected with the specific CB2R agonist AM1241 1 h before alcohol treatment. Anxiety-like behaviors during withdrawal were assessed by open field test and elevated plus maze test 24 h after the last alcohol administration. Microglial activation, microglia-neuron interactions, and CB2R and NLRP3 inflammasome-related molecule expression in the PFC were measured using immunofluorescence, immunohistochemical, qPCR, and Western blotting assays. Microglial morphology was evaluated by Sholl analysis and the cell body-to-total cell size index. Additionally, N9 microglia were activated by LPS in vitro, and the effects of AM1241 on NLRP3 and N9 microglial activation were investigated. ResultsAfter CAE, mice exhibited severe anxiety-like behaviors during withdrawal. CAE induced obvious microglia-neuron associations, and increased expression of microglial activation markers, CB2R, and NLRP3 inflammasome-related molecules in the PFC. Microglia also showed marked filament retraction and reduction and cell body enlargement after CAE. AM1241 treatment ameliorated anxiety-like behaviors in CAE model mice, and it prevented microglial morphological changes, reduced microglial activation marker expression, and suppressed the microglial NLRP3 inflammasome activation and proinflammatory cytokine secretion induced by CAE. AM1241 suppressed the LPS-induced increase in NLRP3 inflammasome-related molecules, IL-1β release, and M1 phenotype markers (iNOS and CD86) in N9 cell, which was reversed by CB2R antagonist treatment. ConclusionsCAE caused anxiety-like behaviors in late adolescent mice at least partly by inducing microglial activation and increasing microglia-neuron associations in the PFC. CB2R activation ameliorated these effects by preventing morphological changes and suppressing NLRP3 inflammasome activation in PFC microglia.

Icariin ameliorates behavioral deficits and neuropathology in a mouse model of multiple sclerosis

Multiple sclerosis (MS) is a systemic inflammatory illness of the central nervous system that involves demyelinating lesions in the myelin-rich white matter and pathology in the grey matter. Despite significant advancements in drug research for MS, the disease's complex pathophysiology makes it difficult to treat the progressive forms of the disease. In this study, we identified a natural flavonoid compound icariin (ICA) as a potent effective agent for MS in ameliorating the deterioration of symptoms including the neurological deficit score and the body weight in a murine experimental autoimmune encephalomyelitis (EAE) model. These improvements were associated with decreased demyelination in the corpus callosum and neuron loss in the hippocampus and cortex confirmed by immunohistochemistry analysis. Meanwhile, it was observed that the activation of microglia in cerebral cortex and hippocampus were inhibited followed by the neuroinflammatory cytokines downregulation such as IL-1β, IL-6 and TNF-α after ICA treatment, which was probably attributable to the suppression of microglial NLRP3 inflammasome activation. Additionally, molecular docking also revealed the binding force of ICA to NLRP3 inflammasome protein complexes in vitro. Taken together, our findings have demonstrated that ICA, as pleiotropic agent, prevents EAE-induced MS by improving demyelination and neuron loss, which interferes with the neuroinflammation via microglial NLRP3 inflammasome activation.

Sulforaphane attenuates microglia-mediated neuronal damage by down-regulating the ROS/autophagy/NLRP3 signal axis in fibrillar Aβ-activated microglia

The neuroinflammatory hypothesis of Alzheimer’s disease (AD) posits that amyloid-beta (Aβ) phagocytosis along with subsequent lysosomal damage and NLRP3 inflammasome activation plays important roles in Aβ-induced microglia activation and microglia-induced neurotoxicity. Sulforaphane (SFN) has neuroprotective effects for AD. However, whether SFN can inhibit its cytotoxic autophagy and NLRP3 inflammasome activation in microglia remain unknown. In this study, results showed SFN played an indirect, protective role on neurons via a series of impacts on Aβ-activated microglia, including inhibition of autophagy initiation as well as autophagic lysosomal membrane permeability and subsequent NLRP3/caspase-1 inflammasomes activation. M1 phenotype polarization was also inhibited. Our results demonstrated that SFN could inhibit the cytostatic autophagy-induced NLRP3 signaling pathway in Aβ-activated microglia by decreasing reactive oxygen species (ROS) production. These results provide novel insight into the potential role of SFN in AD therapy.

Amyloid‐β activates NLRP3 inflammasomes by affecting microglial immunometabolism through the Syk‐AMPK pathway

AbstractBackgroundNeuroinflammation is considered one of major factors in the pathogenesis of Alzheimer’s disease (AD). In particular, inflammasome activation, including NLRP3 inflammasome in microglia, is regarded as fundamental for the pro‐inflammatory response of immune cells (Hanslik & Ulland, 2020). Aβ functions to primarily activate microglia in AD and reports have been made that Aβ can activate NLRP3 inflammasome(Halle et al., 2008). However, the precise molecular mechanism through which the NLRP3 inflammasome is associated with AD pathologies remains unclear.MethodPrimary microglia were treated with LPS and Aβ, and the induced signaling pathway were examined by RT‐PCR, western blot and immunostaining. Effect of flufenamic acid (FA) were examined and various inhibitors of Syk, AMPK and NLRP3 were used for the verification of the hypothesis. AD mouse model expressing Aβ and tau pathology were intraperitoneally treated with FA. After behavior tests, their brains were obtained for western blot and immunostaining.ResultAβ activates the NLRP3 inflammasome in microglia by activating Syk and inhibiting AMPK. Deactivated AMPK induces metabolic dysregulation, mitochondrial fragmentation and reactive oxygen species formation, leading to the activation of the NLRP3 inflammasome. In addition, flufenamic acid (FA), a member of non‐steroid anti‐inflammatory drugs, was found to effectively inhibit activation of the microglial NLRP3 inflammasome by regulating Syk and AMPK. Importantly, FA has marked therapeutic effects on major AD pathologies including amyloid plaque formation, tau phosphorylation, gliosis and memory dysfunction in vivo in microglia‐dependent way.ConclusionThe molecular mechanism of microglial NLRP3 inflammasome activation by Aβ was demonstrated to be Syk‐AMPK pathway. Also, FA can be repurposed for inhibiting microglial activation of the NLRP3 inflammasome, which is a potential treatment for AD.*ReferencesHanslik, K. L., & Ulland, T. K. (2020). The Role of Microglia and the Nlrp3 Inflammasome in Alzheimer’s Disease. Frontiers in Neurology, 11.Halle, A., Hornung, V., Petzold, G. C., Stewart, C. R., Monks, B. G., Reinheckel, T., Fitzgerald, K. A., Latz, E., Moore, K. J., & Golenbock, D. T. (2008). The NALP3 inflammasome is involved in the innate immune response to amyloid‐β. Nature immunology, 9(8), 857.

Intranasally administered human MSC-derived extracellular vesicles inhibit NLRP3-p38/MAPK signaling after TBI and prevent chronic brain dysfunction

Traumatic brain injury (TBI) leads to lasting brain dysfunction with chronic neuroinflammation typified by nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) inflammasome activation in microglia. This study probed whether a single intranasal (IN) administration of human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs) naturally enriched with activated microglia-modulating miRNAs can avert chronic adverse outcomes of TBI. Small RNA sequencing confirmed the enrichment of miRNAs capable of modulating activated microglia in hMSC-EV cargo. IN administration of hMSC-EVs into adult mice ninety minutes after the induction of a unilateral controlled cortical impact injury resulted in their incorporation into neurons and microglia in both injured and contralateral hemispheres. A single higher dose hMSC-EV treatment also inhibited NLRP3 inflammasome activation after TBI, evidenced by reduced NLRP3, apoptosis-associated speck-like protein containing a CARD, activated caspase-1, interleukin-1 beta, and IL-18 levels in the injured brain. Such inhibition in the acute phase of TBI endured in the chronic phase, which could also be gleaned from diminished NLRP3 inflammasome activation in microglia of TBI mice receiving hMSC-EVs. Proteomic analysis and validation revealed that higher dose hMSC-EV treatment thwarted the chronic activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway by IL-18, which decreased the release of proinflammatory cytokines. Inhibition of the chronic activation of NLRP3-p38/MAPK signaling after TBI also prevented long-term cognitive and mood impairments. Notably, the animals receiving higher doses of hMSC-EVs after TBI displayed better cognitive and mood function in all behavioral tests than animals receiving the vehicle after TBI. A lower dose of hMSC-EV treatment also partially improved cognitive and mood function. Thus, an optimal IN dose of hMSC-EVs naturally enriched with activated microglia-modulating miRNAs can inhibit the chronic activation of NLRP3-p38/MAPK signaling after TBI and prevent lasting brain dysfunction.

Heat-killed Lactobacillus murinus confers neuroprotection against dopamine neuronal loss by targeting NLRP3 inflammasome.

The intestinal flora has become very active in studies related to Parkinson's disease (PD) in recent years. The microbe-gut-brain axis is closely related to the maintenance of brain homeostasis as well as PD pathogenesis. Alterations in gut bacteria can contribute to neuroinflammation and dopamine (DA) neurodegeneration. Lactobacillus murinus, a gram-positive bacterium, is a commensal gut bacteria present in the mammalian gut and considered as a potential probiotic due to its beneficial effects, including anti-inflammatory and antibacterial actions. In this study, the effects of live L. murinus and heat-killed L. murinus on DA neuronal damage in rats and the underlying mechanisms were investigated. Data showed that heat-killed L. murinus ameliorated 6-hydroxydopamine-induced motor dysfunctions and loss of substantia nigra DA neurons, while no protection was shown in live L. murinus treatment. At the same time, heat-killed L. murinus reduced the activation of NLRP3 inflammasome in microglia and the secretion of pro-inflammatory factors, thus inhibiting the development of neuroinflammation. Furthermore, heat-killed L. murinus failed to display its original neuroprotective properties in NLRP3 inflammasome knockout mice. Together, heat-killed L. murinus conferred neuroprotection against DA neuronal loss via the inhibition of microglial NLRP3 inflammasome activation. These findings provide a promising potential for future applications of L. murinus, and also beneficial strategy for PD treatment.