The Lysosome Rupture-activated TAK1-JNK Pathway Regulates NLRP3 Inflammasome Activation

Lysosome rupture triggers NLRP3 inflammasome activation in macrophages. However, the underlying mechanism is not fully understood. Here we showed that the TAK1-JNK pathway, a MAPK signaling pathway, is activated through lysosome rupture and that this activation is necessary for the complete activation of the NLRP3 inflammasome through the oligomerization of an adapter protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). We also revealed that the activation of the TAK1-JNK pathway is sustained through Ca²⁺ ions and that calcium/calmodulin-dependent protein kinase type II functions upstream of the TAK1-JNK pathway and specifically regulates lysosome rupture-induced NLRP3 inflammasome activation. These data suggest a novel role for the TAK1-JNK pathway as a critical regulator of NLRP3 inflammasome activation.     

Encephalomyocarditis Virus Viroporin 2B Activates NLRP3 Inflammasome

Nod-like receptors (NLRs) comprise a large family of intracellular pattern- recognition receptors. Members of the NLR family assemble into large multiprotein complexes, termed the inflammasomes. The NLR family, pyrin domain-containing 3 (NLRP3) is triggered by a diverse set of molecules and signals, and forms the NLRP3 inflammasome. Recent studies have indicated that both DNA and RNA viruses stimulate the NLRP3 inflammasome, leading to the secretion of interleukin 1 beta (IL-1β) and IL-18 following the activation of caspase-1. We previously demonstrated that the proton-selective ion channel M2 protein of influenza virus activates the NLRP3 inflammasome. However, the precise mechanism by which NLRP3 recognizes viral infections remains to be defined. Here, we demonstrate that encephalomyocarditis virus (EMCV), a positive strand RNA virus of the family Picornaviridae, activates the NLRP3 inflammasome in mouse dendritic cells and macrophages. Although transfection with RNA from EMCV virions or EMCV-infected cells induced robust expression of type I interferons in macrophages, it failed to stimulate secretion of IL-1β. Instead, the EMCV viroporin 2B was sufficient to cause inflammasome activation in lipopolysaccharide-primed macrophages. While cells untransfected or transfected with the gene encoding the EMCV non-structural protein 2A or 2C expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells transfected with the gene encoding the EMCV 2B or influenza virus M2 protein. 2B proteins of other picornaviruses, poliovirus and enterovirus 71, also caused the NLRP3 redistribution. Elevation of the intracellular Ca²⁺ level, but not mitochondrial reactive oxygen species and lysosomal cathepsin B, was important in EMCV-induced NLRP3 inflammasome activation. Chelation of extracellular Ca²⁺ did not reduce virus-induced IL-1β secretion. These results indicate that EMCV activates the NLRP3 inflammasome by stimulating Ca²⁺ flux from intracellular storages to the cytosol, and highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.     

Mechanisms of NLRP3 Inflammasome Activation: Its Role in the Treatment of Alzheimer’s Disease

Alzheimer’s disease (AD) is a common neurodegenerative disease of progressive dementia which is characterized pathologically by extracellular neuritic plaques containing aggregated amyloid beta (Aβ) and intracellular hyperphosphorylated tau protein tangles in cerebrum. It has been confirmed that microglia-specific nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome-mediated chronic neuroinflammation plays a crucial role in the pathogenesis of AD. Stimulated by Aβ deposition, NLRP3 assembles and activates within microglia in the AD brain, leading to caspase-1 activation along with downstream interleukin (IL)-1β secretion, and subsequent inflammatory events. Activation of the NLRP3 inflammasome mediates microglia to exhibit inflammatory M1 phenotype, with high expression of caspase-1 and IL-1β. This leads to Aβ deposition and neuronal loss in the amyloid precursor protein (APP)/human presenilin-1 (PS1) mouse model of AD. However, NLRP3 or caspase-1 deletion in APP/PS1 mice promotes microglia to transform to an anti-inflammatory M2 phenotype, with decreased secretion of caspase-1 and IL-1β. It also results in improved cognition, enhanced Aβ clearance, and a lower cerebral inflammatory response. This result suggests that the NLRP3 inflammasome may be an appropriate target for reducing neuroinflammation and alleviating pathological processes in AD. In the present review, we summarize the generally accepted regulatory mechanisms of NLRP3 inflammasome activation, and explore its role in neuroinflammation. Furthermore, we speculate on the possible roles of microglia-specific NLRP3 activation in AD pathogenesis and consider potential therapeutic interventions targeting the NLRP3 inflammasome in AD.

     

Cathepsin B links oxidative stress to the activation of NLRP3 inflammasome

Oxidative stress-mediated activation of NLRP3 inflammasome in microglia is critical in the development of neurodegerative diseases such as Alzheimer's disease (AD), Parkinson disease (PD). However, the mechanism underlying oxidative stress activates NLRP3 inflammasome remains exclusive. Here we demonstrated cathepsin B (CTSB) as a regulator of the activation of NLRP3 inflammasome by H₂O₂·H₂O₂ induced IL-1β secretion in NLRP3 inflammasome-dependent manner·H₂O₂ treatment increased CTSB activity, which in turn activated NLRP3 inflammasome, and subsequently processed pro-caspase-1 cleavage into caspase-1, resulting in IL-1 β secretion. Genetic inhibition or pharmacological inhibition of CTSB blocked the cleavage of pro-caspase-1 into caspase-1 and subsequent IL-1 β secretion induced by H₂O₂. Importantly, CTSB activity, IL-1β levels and malondialdehyde (MDA) were remarkably elevated in plasma of AD patients compared to healthy controls, while glutathione was significantly lower than healthy controls. Correlation analyses showed that CTSB activity was positively correlated with IL-1β and MDA levels, but negatively correlated with GSH levels in plasma of AD patients. Taken together, our results indicate that oxidative stress activates NLRP3 through upregulating CTSB activity. Our results identify an important biological function of CTSB in neuroinflammation, suggesting that CTSB is a potential target in AD therapy.     

Loss of Allograft Inflammatory Factor-1 Ameliorates Experimental Autoimmune Encephalomyelitis by Limiting Encephalitogenic CD4 T-Cell Expansion

Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), is mediated by myelin-specific autoreactive T cells that cause inflammation and demyelination in the central nervous system (CNS), with significant contributions from activated microglia and macrophages. The molecular bases for expansion and activation of these cells, plus trafficking to the CNS for peripheral cells, are not fully understood. Allograft inflammatory factor-1 (Aif-1) (also known as ionized Ca²⁺ binding adapter-1 [Iba-1]) is induced in leukocytes in MS and EAE; here we provide the first assessment of Aif-1 function in this setting. After myelin oligodendrocyte glycoprotein peptide (MOG_35–55) immunization, Aif-1–deficient mice were less likely than controls to develop EAE and had less CNS leukocyte infiltration and demyelination; their spinal cords contained fewer CD4 T cells and microglia and more CD8 T cells. These mice also showed significantly less splenic CD4 T-cell expansion and activation, plus decreased proinflammatory cytokine expression. These findings identify Aif-1 as a potent molecule that promotes expansion and activation of CD4 T cells, plus elaboration of a proinflammatory cytokine milieu, in MOG_35–55-induced EAE and as a potential therapeutic target in MS.     

Distinct properties of Ca2+–calmodulin binding to N- and C-terminal regulatory regions of the TRPV1 channel

Transient receptor potential (TRP) vanilloid 1 (TRPV1) is a molecular pain receptor belonging to the TRP superfamily of nonselective cation channels. As a polymodal receptor, TRPV1 responds to heat and a wide range of chemical stimuli. The influx of calcium after channel activation serves as a negative feedback mechanism leading to TRPV1 desensitization. The cellular calcium sensor calmodulin (CaM) likely participates in the desensitization of TRPV1. Two CaM-binding sites are identified in TRPV1: the N-terminal ankyrin repeat domain (ARD) and a short distal C-terminal (CT) segment. Here, we present the crystal structure of calcium-bound CaM (Ca²⁺–CaM) in complex with the TRPV1-CT segment, determined to 1.95-Å resolution. The two lobes of Ca²⁺–CaM wrap around a helical TRPV1-CT segment in an antiparallel orientation, and two hydrophobic anchors, W787 and L796, contact the C-lobe and N-lobe of Ca²⁺–CaM, respectively. This structure is similar to canonical Ca²⁺–CaM-peptide complexes, although TRPV1 contains no classical CaM recognition sequence motif. Using structural and mutational studies, we established the TRPV1 C terminus as a high affinity Ca²⁺–CaM-binding site in both the isolated TRPV1 C terminus and in full-length TRPV1. Although a ternary complex of CaM, TRPV1-ARD, and TRPV1-CT had previously been postulated, we found no biochemical evidence of such a complex. In electrophysiology studies, mutation of the Ca²⁺–CaM-binding site on TRPV1-ARD abolished desensitization in response to repeated application of capsaicin, whereas mutation of the Ca²⁺–CaM-binding site in TRPV1-CT led to a more subtle phenotype of slowed and reduced TRPV1 desensitization. In summary, our results show that the TRPV1-ARD is an important mediator of TRPV1 desensitization, whereas TRPV1-CT has higher affinity for CaM and is likely involved in separate regulatory mechanisms.     

Morphine and Fentanyl Repeated Administration Induces Different Levels of NLRP3-Dependent Pyroptosis in the Dorsal Raphe Nucleus of Male Rats via Cell-Specific Activation of TLR4 and Opioid Receptors

Morphine promotes neuroinflammation after NOD-like receptor protein 3 (NLRP3) oligomerization in glial cells, but the capacity of other opioids to induce neuroinflammation and its relationship to the development of analgesic tolerance is unknown. We studied the effects of morphine and fentanyl on NLRP3 inflammasome activation in glial and neuronal cells in the dorsal raphe nucleus (DRN), a region involved in pain regulation. Male Wistar rats received i.p. injections of morphine (10 mg/kg) or fentanyl (0.1 mg/kg) 3?×?daily for 7 days and were tested for nociception. Two hours after the last (19th) administration, we analyzed NLRP3 oligomerization, caspase-1 activation and gasdermin D-N (GSDMD-N) expression in microglia (CD11b positive cells), astrocytes (GFAP-positive cells) and neurons (NeuN-positive cells). Tolerance developed to both opioids, but only fentanyl produced hyperalgesia. Morphine and fentanyl activated NLRP3 inflammasome in astrocytes and serotonergic (TPH-2-positive) neurons, but fentanyl effects were more pronounced. Both opioids increased GFAP and CD11b immunoreactivity, caspase-1 and GSDMD activation, indicating pyroptotic cell death. The opioid receptor antagonist (?)-naloxone, but not the TLR4 receptor antagonist (+)-naloxone, prevented microglia activation and NLRP3 oligomerization. Only (+)-naloxone prevented astrocytes’ activation. The anti-inflammatory agent minocycline and the NLRP3 inhibitor MCC950 delayed tolerance to morphine and fentanyl antinociception and prevented fentanyl-induced hyperalgesia. MCC950 also prevented opioid-induced NLRP3 oligomerization. In conclusion, morphine and fentanyl differentially induce cell-specific activation of NLRP3 inflammasome and pyroptosis in the DRN through TLR4 receptors in astrocytes and through opioid receptors in neurons, indicating that neuroinflammation is involved in opioid-induced analgesia and fentanyl-induced hyperalgesia after repeated administrations.

     

Activated human CD4+CD45RO+ memory T-cells indirectly inhibit NLRP3 inflammasome activation through downregulation of P2X7R signalling

Inflammasomes are multi-protein complexes that control the production of pro-inflammatory cytokines such as IL-1β. Inflammasomes play an important role in the control of immunity to tumors and infections, and also in autoimmune diseases, but the mechanisms controlling the activation of human inflammasomes are largely unknown. We found that human activated CD4+CD45RO+ memory T-cells specifically suppress P2X7R-mediated NLRP3 inflammasome activation, without affecting P2X7R-independent NLRP3 or NLRP1 inflammasome activation. The concomitant increase in pro-IL-1β production induced by activated memory T-cells concealed this effect. Priming with IFNβ decreased pro-IL-1β production in addition to NLRP3 inflammasome inhibition and thus unmasked the inhibitory effect on NLRP3 inflammasome activation. IFNβ suppresses NLRP3 inflammasome activation through an indirect mechanism involving decreased P2X7R signaling. The inhibition of pro-IL-1β production and suppression of NLRP3 inflammasome activation by IFNβ-primed human CD4+CD45RO+ memory T-cells is partly mediated by soluble FasL and is associated with down-regulated P2X7R mRNA expression and reduced response to ATP in monocytes. CD4+CD45RO+ memory T-cells from multiple sclerosis (MS) patients showed a reduced ability to suppress NLRP3 inflammasome activation, however their suppressive ability was recovered following in vivo treatment with IFNβ. Thus, our data demonstrate that human P2X7R-mediated NLRP3 inflammasome activation is regulated by activated CD4+CD45RO+ memory T cells, and provide new information on the mechanisms mediating the therapeutic effects of IFNβ in MS.     

A Highlights from MBoC Selection: Regulation of the epithelial Ca2+ channel TRPV5 by reversible histidine phosphorylation mediated by NDPK-B and PHPT1

The kidney, together with bone and intestine, plays a crucial role in maintaining whole-body calcium (Ca²⁺) homoeostasis, which is primarily mediated by altering the reabsorption of Ca²⁺ filtered by the glomerulus. The transient receptor potential-vanilloid-5 (TRPV5) channel protein forms a six- transmembrane Ca²⁺-permeable channel that regulates urinary Ca²⁺ excretion by mediating active Ca²⁺ reabsorption in the distal convoluted tubule of the kidney. Here we show that the histidine kinase, nucleoside diphosphate kinase B (NDPK-B), activates TRPV5 channel activity and Ca²⁺ flux, and this activation requires histidine 711 in the carboxy-terminal tail of TRPV5. In addition, the histidine phosphatase, protein histidine phosphatase 1, inhibits NDPK-B–activated TRPV5 in inside/out patch experiments. This is physiologically relevant to Ca²⁺ reabsorption in vivo, as short hairpin RNA knockdown of NDPK-B leads to decreased TRPV5 channel activity, and urinary Ca²⁺ excretion is increased in NDPK-B^−/− mice fed a high-Ca²⁺ diet. Thus these findings identify a novel mechanism by which TRPV5 and Ca²⁺ reabsorption is regulated by the kidney and support the idea that histidine phosphorylation plays other, yet-uncovered roles in mammalian biology.     

The role of NLRP3-CASP1 in inflammasome-mediated neuroinflammation and autophagy dysfunction in manganese-induced,hippocampal-dependent impairment of learning and memory ability

Central nervous system (CNS) inflammation and autophagy dysfunction are known to be involved in the pathology of neurodegenerative diseases. Manganese (Mn), a neurotoxic metal, has the potential to induce microglia-mediated neuroinflammation as well as autophagy dysfunction. NLRP3 (NLR family, pyrin domain containing 3)- CASP1 (caspase 1) inflammasome-mediated neuroinflammation in microglia has specific relevance to neurological diseases. However, the mechanism driving these phenomena remains poorly understood. We demonstrate that Mn activates the NLRP3-CASP1 inflammasome pathway in the hippocampus of mice and BV2 cells by triggering autophagy-lysosomal dysfunction. The autophagy-lysosomal dysfunction is induced by lysosomal damage caused by excessive Mn accumulation, damaging the structure and normal function of these organelles. Additionally, we show that the release of lysosomal CTSB (cathepsin B) plays an important role in Mn-induced NLRP3-CASP1 inflammasome activation, and that the increased autophagosomes in the cytoplasm are not the main cause of NLRP3-CASP1 inflammasome activation. The accumulation of proinflammatory cytokines, such as IL1B (interleukin 1 β) and IL18 (interleukin 18), as well as the dysfunctional autophagy pathway may damage hippocampal neuronal cells, thus leading to hippocampal-dependent impairment in learning and memory, which is associated with the pathogenesis of Alzheimer disease (AD).     

RAGE-TXNIP axis drives inflammation in Alzheimer’s by targeting Aβ to mitochondria in microglia

Alzheimer’s disease (AD) is the most common form of dementia characterized by progressive memory loss and cognitive decline. Although neuroinflammation and oxidative stress are well-recognized features of AD, their correlations with the early molecular events characterizing the pathology are not yet well clarified. Here, we characterize the role of RAGE–TXNIP axis in neuroinflammation in relation to amyloid-beta (Aβ) burden in both in vivo and in vitro models. In the hippocampus of 5xFAD mice microglial activation, cytokine secretion, and glial fibrillary acidic protein-enhanced expression are paralleled with increased TXNIP expression. TXNIP silencing or its pharmacological inhibition prevents neuroinflammation in those mice. TXNIP is also associated with RAGE and Aβ. In particular, RAGE–TXNIP axis is required for targeting Aβ in mitochondria, leading to mitochondrial dysfunction and oxidative stress. Silencing of TXNIP or inhibition of RAGE activation reduces Aβ transport from the cellular surface to mitochondria, restores mitochondrial functionality, and mitigates Aβ toxicity. Furthermore, Aβ shuttling into mitochondria promotes Drp1 activation and exacerbates mitochondrial dysfunction, which induces NLRP3 inflammasome activation, leading to secretion of IL-1β and activation of the pyroptosis-associated protein Gasdermin D (GSDMD). Downregulation of RAGE–TXNIP axis inhibits Aβ-induced mitochondria dysfunction, inflammation, and induction of GSDMD. Herein we unveil a new pathway driven by TXNIP that links the mitochondrial transport of Aβ to the activation of Drp1 and the NLRP3 inflammasome, promoting the secretion of IL-1β and the pyroptosis pathway associated with GSDMD cleavage. Altogether these data shed new light on a novel mechanism of action of RAGE–TXNIP axis in microglia, which is intertwined with Aβ and ultimately causes mitochondria dysfunction and NLRP3 inflammasome cascade activation, suggesting TXNIP as a druggable target to be better deepened for AD.Subject terms: Cellular neuroscience, Inflammasome     

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)¹. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)². Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers³. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation⁴ and pathologies such as inflammation⁵. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages⁶ and microglia⁷. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer''s disease and brain tumors.     

Activation of the NLRP3 inflammasome in microglia: the role of ceramide

Inflammation within the CNS is a major component of many neurodegenerative diseases. A characteristic feature is the generation of microglia‐derived factors that play an essential role in the immune response. IL‐1β is a pro‐inflammatory cytokine released by activated microglia, able to exacerbate injury at elevated levels. In the presence of caspase‐1, pro‐IL‐1β is cleaved to the mature cytokine following NOD‐like receptor pyrin domain containing 3 (NLRP3) inflammasome activation. Growing evidence suggests that ceramide plays a critical role in NLRP3 inflammasome assembly, however, the relationship between ceramide and inflammasome activation in microglia remains unknown. Here, we investigated potential mechanistic links between ceramide as a modulator of NLRP3 inflammasome assembly and the resulting secretion of IL‐1β using small bioactive enzyme stimulators and inhibitors of ceramide signaling in wild‐type and apoptosis‐associated speck‐like protein containing a CARD knockout (ASC^?/?) primary microglia. To induce the expression of inflammasome components, microglia were primed prior to experiments. Treatment with sodium palmitate (PA) induced de novo ceramide synthesis via modulation of its synthesizing protein serine palmitoyl transferase resulting in increased IL‐1β secretion in microglia. Exposure of microglia to the serine palmitoyl transferase‐inhibitor l ‐cycloserine significantly prevented PA‐induced IL‐1β secretion. Application of the ceramide analogue C2 and the sphingosine‐1‐phosphate‐receptor agonist Fingolimod (FTY720) up‐regulated levels of IL‐1β and cleaved caspase‐1 in wild‐type microglia, whereas ASC^?/? microglia were unaffected. HPA‐12 inhibition of ceramide transport did not affect inflammasome activation. Taken together, our findings reveal a critical role for ceramide as a positive modulator of NLRP3 inflammasome assembly and the resulting release of IL‐1β.

     

Activation of TRPV4 channel in pancreatic INS-1E beta cells enhances glucose-stimulated insulin secretion via calcium-dependent mechanisms

Transient receptor potential channel vanilloid type 4 (TRPV4) is a Ca²⁺- and Mg²⁺-permeable cation channel that influences oxidative metabolism and insulin sensitivity. The role of TRPV4 in pancreatic beta cells is largely unknown. Here, we characterize the role of TRPV4 in controlling intracellular Ca²⁺ and insulin secretion in INS-1E beta cells. Osmotic, thermal or pharmacological activation of TRPV4 caused a rapid rise of intracellular Ca²⁺ and enhanced glucose-stimulated insulin secretion. In the presence of the TRPV channel blocker ruthenium red (RuR) or after suppression of TRPV4 protein production, TRPV4 activators failed to increase [Ca²⁺]_i and insulin secretion in INS-1E cells.     

NLRP3 Inflammasome Inhibitor Ameliorates Amyloid Pathology in a Mouse Model of Alzheimer’s Disease

The activation of the NLRP3 inflammasome signaling pathway plays an important role in the neuroinflammation in Alzheimer’s disease (AD). In this study, we investigated the effects of JC-124, a rationally designed NLRP3 inflammasome inhibitor, on AD-related deficits in CRND8 APP transgenic mice (TgCRND8). We first demonstrated increased formation and activation of NLRP3 inflammasome in TgCRND8 mice compared to non-transgenic littermate controls, which was inhibited by the treatment with JC-124. Importantly, JC-124 treatment led to decreased levels of Aβ deposition and decreased levels of soluble and insoluble Aβ_1–42 in the brain of CRND8 mice which was accompanied by reduced β-cleavage of APP, reduced activation of microglia but enhanced astrocytosis. Oxidative stress was decreased and synaptophysin was increased in the CRND8 mice after JC-124 treatment, demonstrating a neuroprotective effect. Overall, these data demonstrated beneficial effects of JC-124 as a specific NLRP3 inflammasome inhibitor in AD mouse model and supported the further development of NLRP3 inflammasome inhibitors as a viable option for AD therapeutics.     

N4-acetylcytidine is required for sustained NLRP3 inflammasome activation via HMGB1 pathway in microglia

Persistent inflammasome activation contributes to chronic, low grade inflammation. However, it is unclear how the inflammasome activation is sustained after initiation. Here we reported that N⁴-acetylcytidine (N4A), a nucleoside metabolite, activated microglia and sustained NLRP3 inflammasome activation by inducing HMGB1 signaling. Released HMGB1 through N4A activated NFκB and induced NLRP3 expression. HMGB1 silencing abolished N4A-stimulated NFκB activation, NLRP3 and persistent HMGB1 expression. In addition, inhibiting NLRP3 expression by RNAi abrogated N4A-mediated HMGB1 expression. Lack of NLRP3 inflammasome adaptor named apoptosis-associated speck-like protein containing a CARD (ASC) abrogated N4A-induced HMGB1 expression, NFκB activation, and NLRP3 expression. Taken together, our results reveal a novel role of N4A in activation of NLRP3 inflamasome via HMGB1 feedback.     

Characterization of Functional TRPV1 Channels in the Sarcoplasmic Reticulum of Mouse Skeletal Muscle

TRPV1 represents a non-selective cation channel activated by capsaicin, acidosis and high temperature. In the central nervous system where TRPV1 is highly expressed, its physiological role in nociception is clearly identified. In skeletal muscle, TRPV1 appears implicated in energy metabolism and exercise endurance. However, how as a Ca²⁺ channel, it contributes to intracellular calcium concentration ([Ca²⁺]_i) maintenance and muscle contraction remains unknown. Here, as in rats, we report that TRPV1 is functionally expressed in mouse skeletal muscle. In contrast to earlier reports, our analysis show TRPV1 presence only at the sarcoplasmic reticulum (SR) membrane (preferably at the longitudinal part) in the proximity of SERCA1 pumps. Using intracellular Ca²⁺ imaging, we directly accessed to the channel functionality in intact FDB mouse fibers. Capsaicin and resiniferatoxin, both agonists as well as high temperature (45°C) elicited an increase in [Ca²⁺]_i. TRPV1-inhibition by capsazepine resulted in a strong inhibition of TRPV1-mediated functional responses and abolished channel activation. Blocking the SR release (with ryanodine or dantrolene) led to a reduced capsaicin-induced Ca²⁺ elevation suggesting that TRPV1 may participate to a secondary SR Ca²⁺ liberation of greater amplitude. In conclusion, our experiments point out that TRPV1 is a functional SR Ca²⁺ leak channel and may crosstalk with RyR1 in adult mouse muscle fibers.     

Plasma membrane stretch activates transient receptor potential vanilloid and ankyrin channels in Merkel cells from hamster buccal mucosa

Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons. Many transient receptor potential (TRP) channels have been identified in mammals; however, the activation properties of these channels in oral mucosal MCs remain to be clarified. We investigated the biophysical and pharmacological properties of TRP vanilloid (TRPV)-1, TRPV2, TRPV4, TRP ankyrin (TRPA)-1, and TRP melastatin (TRPM)-8 channels, which are sensitive to osmotic and mechanical stimuli by measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) using fura-2. We also analyzed their localization patterns through immunofluorescence. MCs showed immunoreaction for TRPV1, TRPV2, TRPV4, TRPA1, and TRPM8 channels. In the presence of extracellular Ca²⁺, the hypotonic test solution evoked Ca²⁺ influx. The [Ca²⁺]_i increases were inhibited by TRPV1, TRPV2, TRPV4, or TRPA1 channel antagonists, but not by the TRPM8 channel antagonist. Application of TRPV1, TRPV2, TRPV4, TRPA1, or TRPM8 channel selective agonists elicited transient increases in [Ca²⁺]_i only in the presence of extracellular Ca²⁺. The results indicate that membrane stretching in MCs activates TRPV1, TRPV2, TRPV4, and TRPA1 channels, that it may be involved in synaptic transmission to sensory neurons, and that MCs could contribute to the mechanosensory transduction sequence.     

Prion Pathogenesis in the Absence of NLRP3/ASC Inflammasomes

The accumulation of the scrapie prion protein PrP^Sc, a misfolded conformer of the cellular prion protein PrP^C, is a crucial feature of prion diseases. In the central nervous system, this process is accompanied by conspicuous microglia activation. The NLRP3 inflammasome is a multi-molecular complex which can sense heterogeneous pathogen-associated molecular patterns and culminates in the activation of caspase 1 and release of IL 1β. The NLRP3 inflammasome was reported to be essential for IL 1β release after in vitro exposure to the amyloidogenic peptide PrP^106-126 and to recombinant PrP fibrils. We therefore studied the role of the NLRP3 inflammasome in a mouse model of prion infection. Upon intracerebral inoculation with scrapie prions (strain RML), mice lacking NLRP3 (Nlrp3^-/-) or the inflammasome adaptor protein ASC (Pycard^-/-) succumbed to scrapie with attack rates and incubation times similar to wild-type mice, and developed the classic histologic and biochemical features of prion diseases. Genetic ablation of NLRP3 or ASC did not significantly impact on brain levels of IL 1β at the terminal stage of disease. Our results exclude a significant role for NLRP3 and ASC in prion pathogenesis and invalidate their claimed potential as therapeutic target against prion diseases.     

Discrete Control of TRPV4 Channel Function in the Distal Nephron by Protein Kinases A and C

We have recently documented that the Ca²⁺-permeable TRPV4 channel, which is abundantly expressed in distal nephron cells, mediates cellular Ca²⁺ responses to elevated luminal flow. In this study, we combined Fura-2-based [Ca²⁺]_i imaging with immunofluorescence microscopy in isolated split-opened distal nephrons of C57BL/6 mice to probe the molecular determinants of TRPV4 activity and subcellular distribution. We found that activation of the PKC pathway with phorbol 12-myristate 13-acetate significantly increased [Ca²⁺]_i responses to flow without affecting the subcellular distribution of TRPV4. Inhibition of PKC with bisindolylmaleimide I diminished cellular responses to elevated flow. In contrast, activation of the PKA pathway with forskolin did not affect TRPV4-mediated [Ca²⁺]_i responses to flow but markedly shifted the subcellular distribution of the channel toward the apical membrane. These actions were blocked with the specific PKA inhibitor H-89. Concomitant activation of the PKA and PKC cascades additively enhanced the amplitude of flow-induced [Ca²⁺]_i responses and greatly increased basal [Ca²⁺]_i levels, indicating constitutive TRPV4 activation. This effect was precluded by the selective TRPV4 antagonist HC-067047. Therefore, the functional status of the TRPV4 channel in the distal nephron is regulated by two distinct signaling pathways. Although the PKA-dependent cascade promotes TRPV4 trafficking and translocation to the apical membrane, the PKC-dependent pathway increases the activity of the channel on the plasma membrane.