Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells.

Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.

Oxidized thioredoxin 1 places a leash on NLRP1 inflammasome activity.

The biology of the NACHT domain and leucine-rich repeat (NLR) and pyrin domain-containing 1 (NLRP1) inflammasome has perplexed researchers since this inflammasome was first described about two decades ago. The identification of oxidized thioredoxin 1 (TRX1) as a suppressor of NLRP1 recently linked cellular redox homeostasis to NLRP1 inflammasome signaling. Now, Zhang et al. present a molecular structure of TRX1-bound NLRP1 with unprecedented detail. This structure gives key insight into regulatory mechanisms governing NLRP1 activation and offers enormous potential for structure-based anti-inflammatory drug design.

Autophagy dysfunction contributes to NLRP1 inflammasome-linked depressive-like behaviors in mice.

BACKGROUND: Major depressive disorder (MDD) is a common but severe psychiatric illness characterized by depressive mood and diminished interest. Both nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 1 (NLRP1) inflammasome and autophagy have been reported to implicate in the pathological processes of depression. However, the mechanistic interplay between NLRP1 inflammasome, autophagy, and depression is still poorly known. METHODS: Animal model of depression was established by chronic social defeat stress (CSDS). Depressive-like behaviors were determined by social interaction test (SIT), sucrose preference test (SPT), open field test (OFT), forced swim test (FST), and tail-suspension test (TST). The protein expression levels of NLRP1 inflammasome complexes, pro-inflammatory cytokines, phosphorylated-phosphatidylinositol 3-kinase (p-PI3K)/PI3K, phosphorylated-AKT (p-AKT)/AKT, phosphorylated-mechanistic target of rapamycin (p-mTOR)/mTOR, brain-derived neurotrophic factor (BDNF), phosphorylated-tyrosine kinase receptor B (p-TrkB)/TrkB, Bcl-2-associated X protein (Bax)/B-cell lymphoma-2 (Bcl2) and cleaved cysteinyl aspartate-specific proteinase-3 (caspase-3) were examined by western blotting. The mRNA expression levels of pro-inflammatory cytokines were tested by quantitative real-time PCR. The interaction between proteins was detected by immunofluorescence and coimmunoprecipitation. Neuronal injury was assessed by Nissl staining. The autophagosomes were visualized by transmission electron microscopy. Nlrp1a knockdown was performed using an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA-eGFP infusion. RESULTS: CSDS exposure caused a bidirectional change in hippocampal autophagy function, which was activated in the initial period but impaired at the later stage. In addition, CSDS exposure increased the expression levels of hippocampal NLRP1 inflammasome complexes, pro-inflammatory cytokines, p-PI3K, p-AKT and p-mTOR in a time-dependent manner. Interestingly, NLRP1 is immunoprecipitated with mTOR but not PI3K/AKT and CSDS exposure facilitated the immunoprecipitation between them. Hippocampal Nlrp1a knockdown inhibited the activity of PI3K/AKT/mTOR signaling, rescued the impaired autophagy and ameliorated depressive-like behavior induced by CSDS. In addition, rapamycin, an autophagy inducer, abolished NLRP1 inflammasome-driven inflammatory reactions, alleviated depressive-like behavior and exerted a neuroprotective effect. CONCLUSIONS: Autophagy dysfunction contributes to NLRP1 inflammasome-linked depressive-like behavior in mice and the regulation of autophagy could be a valuable therapeutic strategy for the management of depression.

NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype.

BACKGROUND: Senescence is a cellular aging-related process triggered by different stresses and characterized by the secretion of various inflammatory factors referred to as senescence-associated secretory phenotype (SASP), some of which are produced by the NLRP3 inflammasome. Here, we present evidence that the NLRP1 inflammasome is a DNA damage sensor and a key mediator of senescence. METHODS: Senescence was induced in fibroblasts in vitro and in mice. Cellular senescence was assessed by Western blot analysis of several proteins, including p16, p21, p53, and SASP factors, released in the culture media or serum. Inflammasome components, including NLRP1, NLRP3 and GSDMD were knocked out or silenced using siRNAs. RESULTS: In vitro and in vivo results suggest that the NLRP1 inflammasome promotes senescence by regulating the expression of p16, p21, p53, and SASP factors in a Gasdermin D (GSDMD)-dependent manner. Mechanistically, the NLRP1 inflammasome is activated in response to genomic damage detected by the cytosolic DNA sensor cGMP-AMP (cGAMP) synthase (cGAS). CONCLUSION: Our findings show that NLRP1 is a cGAS-dependent DNA damage sensor during senescence and a mediator of SASP release through GSDMD. This study advances the knowledge on the biology of the NLRP1 inflammasome and highlights this pathway as a potential pharmcological target to modulate senescence.

Inhibition of NLRP1 inflammasome improves autophagy dysfunction and Aß disposition in APP/PS1 mice.

Increasing evidence has shown that the NOD-like receptor protein 1 (NLRP1) inflammasome is associated with Aß generation and deposition, which contributes to neuronal damage and neuronal-inflammation in Alzheimer's disease (AD). However, the specific mechanism of NLRP1 inflammasome in the pathogenesis of AD is still unclear. It has been reported that autophagy dysfunction can aggravate the pathological symptoms of AD and plays an important role in regulating Aß generation and clearance. We hypothesized that NLRP1 inflammasome activation may induce autophagy dysfunction contributing to the progression of AD. In the present study, we observed the relationship between Aß generation and NLRP1 inflammasome activation, as well as AMPK/mTOR mediated-autophagy dysfunction in WT 9-month-old (M) mice, APP/PS1 6 M and APP/PS1 9 M mice. Additionally, we further studied the effect of NLRP1 knockdown on cognitive function, Aß generation, neuroinflammation and AMPK/mTOR mediated autophagy in APP/PS1 9 M mice. Our results indicated that NLRP1 inflammasome activation and AMPK/mTOR mediated-autophagy dysfunction are closely implicated in Aß generation and deposition in APP/PS1 9 M mice, but not in APP/PS1 6 M mice. Meanwhile, we found that knockdown of NLRP1 significantly improved learning and memory impairments, decreased the expressions of NLRP1, ASC, caspase-1, p-NF-κB, IL-1ß, APP, CTF-ß, BACE1 and Aß1-42, and decreased the level of p-AMPK, Beclin 1 and LC3 II, and increased the level of p-mTOR and P62 in APP/PS1 9 M mice. Our study suggested that inhibition of NLRP1 inflammasome activation improves AMPK/mTOR mediated-autophagy dysfunction, resulting in the decrease of Aß generation, and NLRP1 and autophagy might be important targets to delay the progression of AD.

The Role and Mechanism of Hyperoside against Depression-like Behavior in Mice via the NLRP1 Inflammasome.

BACKGROUND AND OBJECTIVES: Hypericum perforatum (HP) is widely used for depressive therapy. Nevertheless, the antidepressant effect and potential mechanism of hyperoside (Hyp), the main active component of HP, have not been determined. MATERIALS AND METHODS: We performed ultra-performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry (UPLC-Q-TOF-MS/MS) technology to analyze the components in HP. Using data mining and network pharmacology methods, combined with Cytoscape v3.7.1 and other software, the active components, drug-disease targets, and key pathways of HP in the treatment of depression were evaluated. Finally, the antidepressant effects of Hyp and the mechanism involved were verified in chronic-stress-induced mice. RESULTS: We identified 12 compounds from HP. Hyp, isoquercetin, and quercetin are the main active components of HP. The Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), the Analysis Platform, DrugBank, and other databases were analyzed using data mining, and the results show that the active components of HP and depression are linked to targets such as TNF-, IL-2, TLR4, and so on. A potential signaling pathway that was most relevant to the antidepressant effects of Hyp is the C-type lectin receptor signaling pathway. Furthermore, the antidepressant effects of Hyp were examined, and it is verified for the first time that Hyp significantly alleviated depressive-like behaviors in chronic-stress-induced mice, which may be mediated by inhibiting the NLRP1 inflammasome through the CXCL1/CXCR2/BDNF signaling pathway. CONCLUSION: Hyp is one of the main active components of HP, and Hyp has antidepressant effects through the NLRP1 inflammasome, which may be connected with the CXCL1/CXCR2/BDNF signaling pathway.

NLRP1 inflammasome involves in learning and memory impairments and neuronal damages during aging process in mice.

BACKGROUND: Brain aging is an important risk factor in many human diseases, such as Alzheimer's disease (AD). The production of excess reactive oxygen species (ROS) mediated by nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) and the maturation of inflammatory cytokines caused by activation of the NOD-like receptor protein 1 (NLRP1) inflammasome play central roles in promoting brain aging. However, it is still unclear when and how the neuroinflammation appears in the brain during aging process. METHODS: In this study, we observed the alterations of learning and memory impairments, neuronal damage, NLRP1 inflammasome activation, ROS production and NOX2 expression in the young 6-month-old (6 M) mice, presenile 16 M mice, and older 20 M and 24 M mice. RESULTS: The results indicated that, compared to 6 M mice, the locomotor activity, learning and memory abilities were slightly decreased in 16 M mice, and were significantly decreased in 20 M and 24 M mice, especially in the 24 M mice. The pathological results also showed that there were no significant neuronal damages in 6 M and 16 M mice, while there were obvious neuronal damages in 20 M and 24 M mice, especially in the 24 M group. Consistent with the behavioral and histological changes in the older mice, the activity of ß-galactosidase (ß-gal), the levels of ROS and IL-1ß, and the expressions of NLRP1, ASC, caspase-1, NOX2, p47phox and p22phox were significantly increased in the cortex and hippocampus in the older 20 M and 24 M mice. CONCLUSION: Our study suggested that NLRP1 inflammasome activation may be closely involved in aging-related neuronal damage and may be an important target for preventing brain aging.

Sarsasapogenin ameliorates diabetes-associated memory impairment and neuroinflammation through down-regulation of PAR-1 receptor.

Sarsasapogenin (Sar), a natural steroidal compound, shows neuroprotection, cognition-enhancement, antiinflammation, antithrombosis effects, and so on. However, whether Sar has ameliorative effects on diabetes-associated cognitive impairment remains unknown. In this study, we found that Sar ameliorated diabetes-associated memory impairment in streptozotocin-induced diabetic rats, evidenced by increased numbers of crossing platform and percentage of time spent in the target quadrant in Morris water maze tests, and suppressed the nucleotide-binding domain and leucine-rich repeat containing protein 1 (NLRP1) inflammasome in hippocampus and cerebral cortex. Furthermore, Sar inhibited advanced glycation end-products and its receptor (AGEs/RAGE) axis and suppressed up-regulation of thrombin receptor protease-activated receptor 1 (PAR-1) in cerebral cortex. On the other hand, Sar mitigated high glucose-induced neuronal damages, NLRP1 inflammasome activation, and PAR-1 up-regulation in high glucose-cultured SH-SY5Y cells, but did not affect thrombin activity. Moreover, the effects of Sar were similar to those of a selective PAR-1 antagonist vorapaxar. Further studies indicated that activation of the NLRP1 inflammasome and NF-κB mediated the effect of PAR-1 up-regulation in high glucose condition by using PAR-1 knockdown assay. In summary, this study demonstrated that Sar prevented memory impairment caused by diabetes, which was achieved through suppressing neuroinflammation from activated NLRP1 inflammasome and NF-κB regulated by cerebral PAR-1. HIGHLIGHTS: Sarsasapogenin ameliorated memory impairment caused by diabetes in rats. Sarsasapogenin mitigated neuronal damages and neuroinflammation by down-regulating cerebral PAR-1. The NLRP1 inflammasome and NF-κB signaling mediated the pro-inflammatory effects of PAR-1. Sarsasapogenin was a pleiotropic neuroprotective agent and memory enhancer in diabetic rodents.

Spinal cord NLRP1 inflammasome contributes to dry skin induced chronic itch in mice.

BACKGROUND: Dry skin itch is one of the most common skin diseases and elderly people are believed to be particularly prone to it. The inflammasome has been suggested to play an important role in chronic inflammatory disorders including inflammatory skin diseases such as psoriasis. However, little is known about the role of NLRP1 inflammasome in dry skin-induced chronic itch. METHODS: Dry skin-induced chronic itch model was established by acetone-ether-water (AEW) treatment. Spontaneous scratching behavior was recorded by video monitoring. The expression of nucleotide oligomerization domain (NOD)-like receptor protein 1 (NLRP1) inflammasome complexes, transient receptor potential vanilloid type 1 (TRPV1), and the level of inflammatory cytokines were determined by western blot, quantitative real-time PCR, and enzyme-linked immunosorbent assay (ELISA) kits. Nlrp1a knockdown was performed by an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA-eGFP infusion. H.E. staining was used to evaluate skin lesion. RESULTS: AEW treatment triggers spontaneous scratching and significantly increases the expression of NLRP1, ASC, and caspase-1 and the levels of IL-1ß, IL-18, IL-6, and TNF-α in the spinal cord and the skin of mice. Spinal cord Nlrp1a knockdown prevents AEW-induced NLRP1 inflammasome assembly, TRPV1 channel activation, and spontaneous scratching behavior. Capsazepine, a specific antagonist of TRPV1, can also inhibit AEW-induced inflammatory response and scratching behavior. Furthermore, elderly mice and female mice exhibited more significant AEW-induced scratching behavior than young mice and male mice, respectively. Interestingly, AEW-induced increases in the expression of NLRP1 inflammasome complex and the levels of inflammatory cytokines were more remarkable in elderly mice and female mice than in young mice and male mice, respectively. CONCLUSIONS: Spinal cord NLRP1 inflammasome-mediated inflammatory response contributes to dry skin-induced chronic itch by TRPV1 channel, and it is also involved in age and sex differences of chronic itch. Inhibition of NLRP1 inflammasome may offer a new therapy for dry skin itch.

NLRP1 inflammasome contributes to chronic stress-induced depressive-like behaviors in mice.

BACKGROUND: Major depressive disorder (MDD) is a highly prevalent psychiatric disorder, and inflammation has been considered crucial components of the pathogenesis of depression. NLRP1 inflammasome-driven inflammatory response is believed to participate in many neurological disorders. However, it is unclear whether NLRP1 inflammasome is implicated in the development of depression. METHODS: Animal models of depression were established by four different chronic stress stimuli including chronic unpredictable mild stress (CUMS), chronic restrain stress (CRS), chronic social defeat stress (CSDS), and repeat social defeat stress (RSDS). Depressive-like behaviors were determined by sucrose preference test (SPT), forced swim test (FST), tail-suspension test (TST), open-field test (OFT), social interaction test (SIT), and light-dark test (LDT). The expression of NLRP1 inflammasome complexes, BDNF, and CXCL1/CXCR2 were tested by western blot and quantitative real-time PCR. The levels of inflammatory cytokines were tested by enzyme-linked immunosorbent assay (ELISA) kits. Nlrp1a knockdown was performed by an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA-eGFP infusion. RESULTS: Chronic stress stimuli activated hippocampal NLRP1 inflammasome and promoted the release of pro-inflammatory cytokines IL-1ß, IL-18, IL-6, and TNF-α in mice. Hippocampal Nlrp1a knockdown prevented NLRP1 inflammasome-driven inflammatory response and ameliorated stress-induced depressive-like behaviors. Also, chronic stress stimuli caused the increase in hippocampal CXCL1/CXCR2 expression and low BDNF levels in mice. Interestingly, Nlrp1a knockdown inhibited the up-regulation of CXCL1/CXCR2 expression and restored BDNF levels in the hippocampus. CONCLUSIONS: NLRP1 inflammasome-driven inflammatory response contributes to chronic stress induced depressive-like behaviors and the mechanism may be related to CXCL1/CXCR2/BDNF signaling pathway. Thus, NLRP1 inflammasome could become a potential antidepressant target.

Sinomenine exerts anticonvulsant profile and neuroprotective activity in pentylenetetrazole kindled rats: involvement of inhibition of NLRP1 inflammasome.

BACKGROUND: Epilepsy is a common neurological disorder and is not well controlled by available antiepileptic drugs (AEDs). Inflammation is considered to be a critical factor in the pathophysiology of epilepsy. Sinomenine (SN), a bioactive alkaloid with anti-inflammatory effect, exerts neuroprotective activity in many nervous system diseases. However, little is known about the effect of SN on epilepsy. METHODS: The chronic epilepsy model was established by pentylenetetrazole (PTZ) kindling. Morris water maze (MWM) was used to test spatial learning and memory ability. H.E. staining and Hoechst 33258 staining were used to evaluate hippocampal neuronal damage. The expression of nucleotide oligomerization domain (NOD)-like receptor protein 1 (NLRP1) inflammasome complexes and the level of inflammatory cytokines were determined by western blot, quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: SN (20, 40, and 80 mg/kg) dose-dependently disrupts the kindling acquisition process, which decreases the seizure scores and the incidence of fully kindling. SN also increases the latency of seizure and decreases the duration of seizure in fully kindled rats. In addition, different doses of SN block the hippocampal neuronal damage and minimize the impairment of spatial learning and memory in PTZ kindled rats. Finally, PTZ kindling increases the expression of NLRP1 inflammasome complexes and the levels of inflammatory cytokines IL-1ß, IL-18, IL-6, and TNF-α, which are all attenuated by SN in a dose- dependent manner. CONCLUSIONS: SN exerts anticonvulsant and neuroprotective activity in PTZ kindling model of epilepsy. Disrupting the kindling acquisition, which inhibits NLRP1 inflammasome-mediated inflammatory process, might be involved in its effects.

Chronic glucocorticoid exposure activates BK-NLRP1 signal involving in hippocampal neuron damage.

BACKGROUND: Neuroinflammation mediated by NLRP1 (nucleotide-binding oligomerization domain (NOD)-like receptor protein 1) inflammasome plays an important role in many neurological diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD). Our previous studies showed that chronic glucocorticoid (GC) exposure increased brain inflammation via NLRP1 inflammasome and induce neurodegeneration. However, little is known about the mechanism of chronic GC exposure on NLRP1 inflammasome activation in hippocampal neurons. METHODS: Hippocampal neurons damage was assessed by LDH kit and Hoechst 33258 staining. The expression of microtubule-associated protein 2 (MAP2), inflammasome complex protein (NLRP1, ASC and caspase-1), inflammatory cytokines (IL-1ß), and large-conductance Ca2+ and voltage-activated K+ channel (BK channels) protein was detected by Western blot. The inflammatory cytokines (IL-1ß and IL-18) were examined by ELISA kit. The mRNA levels of NLRP1, IL-1ß, and BK were detected by real-time PCR. BK channel currents were recorded by whole-cell patch-clamp technology. Measurement of [K+]i was performed by ion-selective electrode (ISE) technology. RESULTS: Chronic dexamethasone (DEX) treatment significantly increased LDH release and neuronal apoptosis and decreased expression of MAP2. The mechanistic studies revealed that chronic DEX exposure significantly increased the expression of NLRP1, ASC, caspase-1, IL-1ß, L-18, and BK protein and NLRP1, IL-1ß and BK mRNA levels in hippocampal neurons. Further studies showed that DEX exposure results in the increase of BK channel currents, with the subsequent K+ efflux and a low concentration of intracellular K+, which involved in activation of NLRP1 inflammasome. Moreover, these effects of chronic DEX exposure could be blocked by specific BK channel inhibitor iberiotoxin (IbTx). CONCLUSION: Our findings suggest that chronic GC exposure may increase neuroinflammation via activation of BK-NLRP1 signal pathway and promote hippocampal neurons damage, which may be involved in the development and progression of AD.

Chronic glucocorticoids exposure enhances neurodegeneration in the frontal cortex and hippocampus via NLRP-1 inflammasome activation in male mice.

Neuroinflammation plays an important role in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD) and depression. Chronic glucocorticoids (GCs) exposure has deleterious effects on the structure and function of neurons and is associated with development and progression of AD. However, little is known about the proinflammatory effects of chronic GCs exposure on neurodegeneration in brain. Therefore, the aim of this study was to evaluate the effects of chronic dexamethasone (DEX) treatment (5mg/kg, s.c. for 7, 14, 21 and 28 days) on behavior, neurodegeneration and neuroinflammatory parameters of nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 1 (NLRP-1) inflammasome in male mice. The results showed that DEX treatment for 21 and 28 days significantly reduced the spontaneous motor activity and exploratory behavior of the mice. In addition, these mice showed significant neurodegeneration and a decrease of microtubule-associated protein 2 (MAP2) in the frontal cortex and hippocampus CA3. DEX treatment for 7, 14, 21 and 28 days significantly decreased the mRNA and protein expression of glucocorticoid receptor (GR). Moreover, DEX treatment for 21 and 28 days significantly increased the proteins expression of NLRP-1, Caspase-1, Caspase-5, apoptosis associated speck-like protein (ASC), nuclear factor-κB (NF-κB), p-NF-κB, interleukin-1ß (IL-1ß), IL-18 and IL-6 in the frontal cortex and hippocampus brain tissue. DEX treatment for 28 days also significantly increased the mRNA expression levels of NLRP-1, Caspase-1, ASC and IL-1ß. These results suggest that chronic GCs exposure may increase brain inflammation via NLRP-1 inflammasome activation and induce neurodegeneration.

Celecoxib inhibits NLRP1 inflammasome pathway in MDA-MB-231 Cells.

NLRP1 is predominantly overexpressed in breast cancer tissue, and the evaluated activation of NLRP1 inflammasome is associated with tumor growth, angiogenesis, and metastasis. Therefore, targeting NLRP1 activation could be a crucial strategy in anticancer therapy. In this study, we investigated the hypothesis that NLRP1 pathway may contribute to the cytotoxic effects of celecoxib and nimesulide in MDA-MB-231 cells. First of all, IC50 values and inhibitory effects on the colony-forming ability of drugs were evaluated in cells. Then, the alterations in the expression levels of NLRP1 inflammasome components induced by drugs were investigated. Subsequently, the release of inflammatory cytokine IL-1ß and the activity of caspase-1 in drug-treated cells were measured. According to our results, celecoxib and nimesulide selectively inhibited the viability of MDA-MB-231 cells. These drugs remarkably inhibited the colony-forming ability of cells. The expression levels of NLRP1 inflammasome components decreased in celecoxib-treated cells, accompanied by decreased caspase-1 activity and IL-1ß release. In contrast, nimesulide treatment led to the upregulation of the related protein expressions with unchanged caspase-1 activity and increased IL-1ß secretion. Our results indicated that the NLRP1 inflammasome pathway might contribute to the antiproliferative effects of celecoxib in MDA-MB-231 cells but is not a crucial mechanism for nimesulide.

Li, P HY-021068 alleviates cerebral ischemia-reperfusion injury by inhibiting NLRP1 inflammasome and restoring autophagy function in mice.

Cerebral ischemia-reperfusion injury (CIRI) is a severe pathological condition that involves oxidative stress, inflammatory response, and neuronal damage. HY-021068 belongs to a new drug of chemical class 1, which is a potential thromboxane synthase inhibitor. Our preliminary experiment found that HY-021068 has significant anti-neuroinflammatory and neuroprotective effects. However, the protective effect and mechanism of HY-021068 in CIRI remain unclear. To investigate the protective effect and mechanism of HY-021068 in CIRI mice. In mice, CIRI was induced by bilateral common carotid artery occlusion and reperfusion. Mice were treated with HY-021068 or LV-NLRP1-shRNA (lentivirus-mediated shRNA transfection to knock down NLRP1 expression). The locomotor activity, neuronal damage, pathological changes, postsynaptic density protein-95 (PSD-95) expression, NLRP1 inflammasome activation, autophagy markers, and apoptotic proteins were assessed in CIRI mice. In this study, treatment with HY-021065 and LV-NLRP1-shRNA significantly improved motor dysfunction and neuronal damage after CIRI in mice. HY-021065 and NLRP1 knockdown significantly ameliorated the pathological damage and increased PSD-95 expression in the cortex and hippocampus CA1 and CA3 regions. The further studies showed that compared with the CIRI model group, HY-021065 and NLRP1 knockdown treatment inhibited the expressions of NLRP1, ASC, caspase-1, and IL-1ß, restored the expressions of p-AMPK/AMPK, Beclin1, LC3II/LC3I, p-mTOR/m-TOR and P62, and regulated the expressions of BCL-2, Caspase3, and BAX in brain tissues of CIRI mice in CIRI mice. These results suggest that HY-021068 exerts a protective role in CIRI mice by inhibiting NLRP1 inflammasome activation and regulating autophagy function and neuronal apoptosis. HY-021068 is expected to become a new therapeutic drug for CIRI.

Increased NLRP1 mRNA and Protein Expression Suggests Inflammasome Activation in the Dorsolateral Prefrontal and Medial Orbitofrontal Cortex in Schizophrenia.

Schizophrenia is a complex mental condition, with key symptoms marked for diagnosis including delusions, hallucinations, disorganized thinking, reduced emotional expression, and social dysfunction. In the context of major developmental hypotheses of schizophrenia, notably those concerning maternal immune activation and neuroinflammation, we studied NLRP1 expression and content in the postmortem brain tissue of 10 schizophrenia and 10 control subjects. In the medial orbitofrontal cortex (Brodmann's area 11/12) and dorsolateral prefrontal cortex (area 46) from both hemispheres of six schizophrenia subjects, the NLRP1 mRNA expression was significantly higher than in six control brains (p < 0.05). As the expression difference was highest for the medial orbitofrontal cortex in the right hemisphere, we assessed NLRP1-immunoreactive pyramidal neurons in layers III, V, and VI in the medial orbitofrontal cortex in the right hemisphere of seven schizophrenia and five control brains. Compared to controls, we quantified a significantly higher number of NLRP1-positive pyramidal neurons in the schizophrenia brains (p < 0.01), suggesting NLRP1 inflammasome activation in schizophrenia subjects. Layer III pyramidal neuron dysfunction aligns with working memory deficits, while impairments of pyramidal neurons in layers V and VI likely disrupt predictive processing. We propose NLRP1 inflammasome as a potential biomarker and therapeutic target in schizophrenia.

Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model." However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite's replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

Ligustroflavone exerts neuroprotective activity through suppression of NLRP1 inflammasome in ischaemic stroke mice.

Inflammation is thought to play an important role in the pathophysiology of ischaemic stroke, which is a main cause of disability and morbidity worldwide. Inhibition of the NOD-like receptor protein 1 (NLRP1) inflammasome has been reported to alleviate the inflammatory response in cell and animal models. Ligustroflavone (LIG) is a compound derived from Ligustrum lucidum, which shows anti-inflammatory activity and may play a beneficial role in a number of neurological diseases. To date, the potential for LIG to act through NLRP1 as a treatment for ischemic stroke has not been studied. The present study established an ischaemic stroke model by middle cerebral artery occlusion (MCAO). Modified neurological severity scoring, open-field and the Rotarod test were used to assess neurological deficits. Staining with Hoechst 33258 and western blotting were used to evaluate neuronal damage. Expression levels of NLRP1 inflammasome complexes and inflammatory cytokines were determined using western blotting, enzyme-linked immunosorbent assay and reverse transcription-quantitative PCR. Treatment with LIG minimized the impairment of neurological function and blocked neuronal damage in MCAO mice. In addition, treatment with LIG attenuated the upregulation of expression levels of the NLRP1 inflammasome complexes and the inflammatory cytokines TNF-α, IL-18, IL-6 and IL-1ß. Overall, LIG played an important role in anti-inflammatory and neuroprotective activity in MCAO models of ischaemic stroke.

Chronic hypoxia of endothelial cells boosts HIF-1α-NLRP1 circuit in Alzheimer's disease.

Cerebral microvasculature of patients with Alzheimer's disease (AD) exhibits reduced capillary diameter and impaired blood flow. Molecular mechanisms of ischemic vessels affecting AD progressions have not been well established yet. In the present study, we found that in vivo triple (PS1M146V, APPswe, tauP301L) transgenic AD mouse model (3x-Tg AD) brains and retinas showed hypoxic vessels expressing hypoxyprobe and hypoxia inducible factor-1α (HIF-1α). To mimic in vivo hypoxic vessels, we used in vitro oxygen-glucose deprivation (OGD)-treated endothelial cells. HIF-1α protein was increased through reactive oxygen species (ROS) producing NADPH oxidases (NOX) (i.e., Nox2, Nox4). OGD-induced HIF-1α upregulated Nox2 and Nox4, demonstrating crosstalk between HIF-1α and NOX (i.e., Nox2, Nox4). Interestingly, NLR family pyrin domain containing 1 (NLRP1) protein was promoted by OGD, and such effect was blocked by downregulation of Nox4 and HIF-1α. Knockdown of NLRP1 also diminished OGD-mediated protein levels of Nox2, Nox4, and HIF-1α in human brain microvascular endothelial cells. These results showed interplay among HIF-1α, Nox4 and NLRP1 in OGD-treated endothelial cells. Expression of NLRP3 was not detected well in hypoxic endothelial cells of 3x-Tg AD retinas or OGD-treated endothelial cells. Instead, hypoxic endothelial cells of 3x-Tg AD brains and retinas markedly expressed NLRP1, the adaptor molecule apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and interleukin-1ß (IL-1ß). Taken together, our results suggest that AD brains and retinas can trigger chronic hypoxia especially in microvascular endothelial cells, consequently leading to NLRP1 inflammasome formation and upregulation of ASC-caspase-1-IL-1ß cascades. In addition, NLRP1 can stimulate HIF-1α expression and form HIF-1α-NLRP1 circuit. These consequences might further destroy vascular system in AD.

Ginsenoside Rg1 alleviates learning and memory impairments and Aß disposition through inhibiting NLRP1 inflammasome and autophagy dysfunction in APP/PS1 mice.

Alzheimer's disease (AD) is a common neurodegenerative disorder. Amyloid ß (Aß) deposition is considered an important pathological feature of AD. Growing evidence has linked neuroinflammation and autophagy to Aß deposition in the progression of AD. However, there are few drug options for inhibiting neuroinflammation and autophagy to prevent AD. Ginsenoside Rg1 (Rg1), a steroidal saponin extracted from ginseng, has been reported to possess multiple neuroprotective effects. The present study aimed to evaluate whether Rg1 treatment could attenuate cognitive disorders and neuronal injuries by inhibiting NLRP1 inflammasome and autophagy dysfunction in an AD model of APP/PS1 mice. The results of behavioral tests indicated that Rg1 treatment for 12 weeks could significantly improve olfactory dysfunction as well as learning and memory impairments. The results of histopathological tests indicated that Rg1 treatment could reduce Aß deposition and neuronal damages in APP/PS1­9M mice. Additionally, the results of immunoblot, reverse transcription­quantitative PCR or immunohistochemistry demonstrated that Rg1 treatment significantly downregulated the expression levels of inflammation­related proteins of NLRP1, caspase1, IL­1ß and TNF­α, as well as autophagy­related proteins of p­AMPK/AMPK, Beclin1 and LC3 II/LC3 I, and increased the expression levels of p­mTOR/mTOR and P62 in APP/PS1­9M mice. In addition, the molecular docking analysis showed that there was favorable binding result between Rg1 and NLRP1. The present study suggested that Rg1 may alleviate learning and memory impairments and Aß disposition by inhibiting NLRP1 inflammasome and improving autophagy dysfunction, suggesting that Rg1 may be a potential therapeutic agent for delaying AD.