Sensing of ATP via the Purinergic Receptor P2RX7 Promotes CD8+ Trm Cell Generation by Enhancing Their Sensitivity to the Cytokine TGF-ß.

Tissue-resident memory (Trm) CD8+ T cells mediate protective immunity in barrier tissues, but the cues promoting Trm cell generation are poorly understood. Sensing of extracellular adenosine triphosphate (eATP) by the purinergic receptor P2RX7 is needed for recirculating CD8+ T cell memory, but its role for Trm cells is unclear. Here we showed that P2RX7 supported Trm cell generation by enhancing CD8+ T cell sensing of TGF-ß, which was necessary for tissue residency. P2RX7-deficient Trm cells progressively decayed in non-lymphoid tissues and expressed dysregulated Trm-specific markers. P2RX7 was required for efficient re-expression of the receptor TGF-ßRII through calcineurin signaling. Forced Tgfbr2 expression rescued P2RX7-deficient Trm cell generation, and TGF-ß sensitivity was dictated by P2RX7 agonists and antagonists. Forced Tgfbr2 also rescued P2RX7-deficient Trm cell mitochondrial function. Sustained P2RX7 signaling was required for long-term Trm cell maintenance, indicating that P2RX7 signaling drives induction and CD8+ T cell durability in barrier sites.

The microRNA-211-5p/P2RX7/ERK/GPX4 axis regulates epilepsy-associated neuronal ferroptosis and oxidative stress.

Ferroptosis is an iron-dependent cell death mechanism involving the accumulation of lipid peroxides. As a critical regulator, glutathione peroxidase 4 (GPX4) has been demonstrated to be downregulated in epilepsy. However, the mechanism of ferroptosis in epilepsy remains unclear. In this study, bioinformatics analysis, analysis of epilepsy patient blood samples and cell and mouse experiments revealed strong associations among epilepsy, ferroptosis, microRNA-211-5p and purinergic receptor P2X 7 (P2RX7). P2RX7 is a nonselective ligand-gated homotrimeric cation channel, and its activation mainly increases neuronal activity during epileptic seizures. In our study, the upregulation of P2RX7 in epilepsy was attributed to the downregulation of microRNA (miR)-211-5p. Furthermore, P2RX7 has been found to regulate GPX4/HO-1 by alleviating lipid peroxidation induced by suppression of the MAPK/ERK signaling pathway in murine models. The dynamic decrease in miR-211-5p expression induces hypersynchronization and both nonconvulsive and convulsive seizures, and forebrain miR-211-5p suppression exacerbates long-lasting pentylenetetrazole-induced seizures. Additionally, in this study, induction of miR-211-5p expression or genetic-silencing of P2RX7 significantly reduced the seizure score and duration in murine models through the abovementioned pathways. These results suggest that the miR-211-5p/P2RX7 axis is a novel target for suppressing both ferroptosis and epilepsy.

P2RX7 gene variants associate with altered inflammasome assembly and reduced pyroptosis in chronic nonbacterial osteomyelitis (CNO).

Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1ß and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care.

Embers of the Past: Early Childhood Traumas Interact with Variation in P2RX7 Gene Implicated in Neuroinflammation on Markers of Current Suicide Risk.

Both early childhood traumatic experiences and current stress increase the risk of suicidal behaviour, in which immune activation might play a role. Previous research suggests an association between mood disorders and P2RX7 gene encoding P2X7 receptors, which stimulate neuroinflammation. We investigated the effect of P2RX7 variation in interaction with early childhood adversities and traumas and recent stressors on lifetime suicide attempts and current suicide risk markers. Overall, 1644 participants completed questionnaires assessing childhood adversities, recent negative life events, and provided information about previous suicide attempts and current suicide risk-related markers, including thoughts of ending their life, death, and hopelessness. Subjects were genotyped for 681 SNPs in the P2RX7 gene, 335 of which passed quality control and were entered into logistic and linear regression models, followed by a clumping procedure to identify clumps of SNPs with a significant main and interaction effect. We identified two significant clumps with a main effect on current suicidal ideation with top SNPs rs641940 and rs1653613. In interaction with childhood trauma, we identified a clump with top SNP psy_rs11615992 and another clump on hopelessness containing rs78473339 as index SNP. Our results suggest that P2RX7 variation may mediate the effect of early childhood adversities and traumas on later emergence of suicide risk.

Protocol for Evaluating In Vivo the Activation of the P2RX7 Immunomodulator.

BACKGROUND: P2RX7 is a purinergic receptor with pleiotropic activities that is activated by high levels of extracellular ATP that are found in inflamed tissues. P2RX7 has immunomodulatory and anti-tumor proprieties and is therefore a therapeutic target for various diseases. Several compounds are developed to either inhibit or enhance its activation. However, studying their effect on P2RX7's activities is limited to in vitro and ex vivo studies that require the use of unphysiological media that could affect its activation. Up to now, the only way to assess the activity of P2RX7 modulators on the receptor in vivo was in an indirect manner. RESULTS: We successfully developed a protocol allowing the detection of P2RX7 activation in vivo in lungs of mice, by taking advantage of its unique macropore formation ability. The protocol is based on intranasal delivery of TO-PRO™-3, a non-permeant DNA intercalating dye, and fluorescence measurement by flow cytometry. We show that ATP enhances TO-PRO™-3 fluorescence mainly in lung immune cells of mice in a P2RX7-dependant manner. CONCLUSIONS: The described approach has allowed the successful analysis of P2RX7 activity directly in the lungs of WT and transgenic C57BL6 mice. The provided detailed guidelines and recommendations will support the use of this protocol to study the potency of pharmacologic or biologic compounds targeting P2RX7.

P2RX7 promotes osteosarcoma progression and glucose metabolism by enhancing c-Myc stabilization.

BACKGROUND: Osteosarcoma is the most common malignant tumor in bone and its prognosis has reached a plateau in the past few decades. Recently, metabolic reprogramming has attracted increasing attention in the field of cancer research. In our previous study, P2RX7 has been identified as an oncogene in osteosarcoma. However, whether and how P2RX7 promotes osteosarcoma growth and metastasis through metabolic reprogramming remains unexplored. METHODS: We used CRISPR/Cas9 genome editing technology to establish P2RX7 knockout cell lines. Transcriptomics and metabolomics were performed to explore metabolic reprogramming in osteosarcoma. RT-PCR, western blot and immunofluorescence analyses were used to determine gene expression related to glucose metabolism. Cell cycle and apoptosis were examined by flowcytometry. The capacity of glycolysis and oxidative phosphorylation were assessed by seahorse experiments. PET/CT was carried out to assess glucose uptake in vivo. RESULTS: We demonstrated that P2RX7 significantly promotes glucose metabolism in osteosarcoma via upregulating the expression of genes related to glucose metabolism. Inhibition of glucose metabolism largely abolishes the ability of P2RX7 to promote osteosarcoma progression. Mechanistically, P2RX7 enhances c-Myc stabilization by facilitating nuclear retention and reducing ubiquitination-dependent degradation. Furthermore, P2RX7 promotes osteosarcoma growth and metastasis through metabolic reprogramming in a predominantly c-Myc-dependent manner. CONCLUSIONS: P2RX7 plays a key role in metabolic reprogramming and osteosarcoma progression via increasing c-Myc stability. These findings provide new evidence that P2RX7 might be a potential diagnostic and/or therapeutic target for osteosarcoma. Novel therapeutic strategies targeting metabolic reprogramming appear to hold promise for a breakthrough in the treatment of osteosarcoma.

Soybean lectin-triggered IL-6 secretion induces autophagy to kill intracellular mycobacteria through P2RX7 dependent activation of the JAK2/STAT3/Mcl-1 pathway.

Cytokine therapy and cytokine-mediated autophagy have been used as prominent host-directed therapy (HDT) approaches to restrain M. tb growth in the host cell. In the present study, we have dissected the anti-tubercular activity of Soybean lectin (SBL) through cytokine-mediated autophagy induction in differentiated THP-1 (dTHP-1) cells. A significant increase in IL-6 expression was observed in both uninfected and mycobacteria infected dTHP-1 cells through the P2RX7 mediated pathway via PI3K/Akt/CREB-dependent signalling after SBL treatment. Inhibition of IL-6 level using IL-6 neutralizing antibody or associated signalling significantly enhanced the mycobacterial load in SBL-treated dTHP-1 cells. Further, autocrine signalling of IL-6 through its receptor-induced Mcl-1 expression activated autophagy via JAK2/STAT3 pathway, and inhibition of this pathway affected autophagy. Finally, blocking the IL-6-regulated autophagy through NSC 33994 (a JAK2 inhibitor) or S63845 (an Mcl-1 inhibitor) led to a notable increase in intracellular mycobacterial growth in SBL-treated cells. Taken together, these results indicate that SBL interacts with P2RX7 to regulate PI3K/Akt/CREB network to release IL-6 in dTHP-1 cells. The released IL-6, in turn, activates the JAK2/STAT3/Mcl-1 pathway upon interaction with IL-6Rα to modulate autophagy that ultimately controls mycobacterial growth in macrophages.

Glomerulonephritis and autoimmune vasculitis are independent of P2RX7 but may depend on alternative inflammasome pathways.

P2RX7, an ionotropic receptor for extracellular adenosine triphosphate (ATP), is expressed on immune cells, including macrophages, monocytes, and dendritic cells and is upregulated on nonimmune cells following injury. P2RX7 plays a role in many biological processes, including production of proinflammatory cytokines such as interleukin (IL)-1ß via the canonical inflammasome pathway. P2RX7 has been shown to be important in inflammation and fibrosis and may also play a role in autoimmunity. We have developed and phenotyped a novel P2RX7 knockout (KO) inbred rat strain and, taking advantage of the human-resembling unique histopathological features of rat models of glomerulonephritis, we induced three models of disease: nephrotoxic nephritis, experimental autoimmune glomerulonephritis, and experimental autoimmune vasculitis. We found that deletion of P2RX7 does not protect rats from models of experimental glomerulonephritis or the development of autoimmunity. Notably, treatment with A-438079, a P2RX7 antagonist, was equally protective in WKY WT and P2RX7 KO rats, revealing its 'off-target' properties. We identified a novel ATP/P2RX7/K+ efflux-independent and caspase-1/8-dependent pathway for the production of IL-1ß in rat dendritic cells, which was absent in macrophages. Taken together, these results comprehensively establish that inflammation and autoimmunity in glomerulonephritis is independent of P2RX7 and reveals the off-target properties of drugs previously known as selective P2RX7 antagonists. Rat mononuclear phagocytes may be able to utilise an 'alternative inflammasome' pathway to produce IL-1ß independently of P2RX7, which may account for the susceptibility of P2RX7 KO rats to inflammation and autoimmunity in glomerulonephritis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Asiaticoside hampers epithelial-mesenchymal transition by promoting PPARG expression and suppressing P2RX7-mediated TGF-ß/Smad signaling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) accounts for 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes because of its high propensity to develop metastases. Here, the anticancer effects of asiaticoside (AC) against TNBC and the possible underlying mechanism were examined. We found that AC inhibited the TGF-ß1 expression and the SMAD2/3 phosphorylation in TNBC cells, thereby impairing the TGF-ß/SMAD signaling. AC inhibited the migration, invasion, and epithelial-mesenchymal transition (EMT) of TNBC cells by suppressing the TGF-ß/SMAD signaling. Meanwhile, AC inhibited the lung metastasis of TNBC cells in vivo and the expression of p-SMAD2/3 and vimentin, and increased the expression of E-cadherin and ZO-1 in the lung. Peroxisome proliferator activated receptor gamma (PPARG) was identified as a potential target of AC. AC increased PPARG expression, while PPARG knockdown attenuated the therapeutic effect of AC. AC-mediated PPARG overexpression suppressed the transcription of P2X purinoceptor 7 (P2RX7). The restoration of P2RX7 reversed the therapeutic effect of AC. These results suggested that AC blocked P2RX7-mediated TGF-ß/SMAD signaling by increasing PPARG expression, thereby suppressing EMT in TNBC.

The Role of IL-18 in P2RX7-Mediated Antitumor Immunity.

Cancer is the leading cause of death worldwide despite the variety of treatments that are currently used. This is due to an innate or acquired resistance to therapy that encourages the discovery of novel therapeutic strategies to overcome the resistance. This review will focus on the role of the purinergic receptor P2RX7 in the control of tumor growth, through its ability to modulate antitumor immunity by releasing IL-18. In particular, we describe how the ATP-induced receptor activities (cationic exchange, large pore opening and NLRP3 inflammasome activation) modulate immune cell functions. Furthermore, we recapitulate our current knowledge of the production of IL-18 downstream of P2RX7 activation and how IL-18 controls the fate of tumor growth. Finally, the potential of targeting the P2RX7/IL-18 pathway in combination with classical immunotherapies to fight cancer is discussed.

Animal Models for the Investigation of P2X7 Receptors.

The P2X7 receptor is a trimeric ligand-gated cation channel activated by extracellular adenosine 5'-triphosphate. The study of animals has greatly advanced the investigation of P2X7 and helped to establish the numerous physiological and pathophysiological roles of this receptor in human health and disease. Following a short overview of the P2X7 distribution, roles and functional properties, this article discusses how animal models have contributed to the generation of P2X7-specific antibodies and nanobodies (including biologics), recombinant receptors and radioligands to study P2X7 as well as to the pharmacokinetic testing of P2X7 antagonists. This article then outlines how mouse and rat models have been used to study P2X7. These sections include discussions on preclinical disease models, polymorphic P2X7 variants, P2X7 knockout mice (including bone marrow chimeras and conditional knockouts), P2X7 reporter mice, humanized P2X7 mice and P2X7 knockout rats. Finally, this article reviews the limited number of studies involving guinea pigs, rabbits, monkeys (rhesus macaques), dogs, cats, zebrafish, and other fish species (seabream, ayu sweetfish, rainbow trout and Japanese flounder) to study P2X7.

P2X7 receptor activation increases expression of caveolin-1 and formation of macrophage lipid rafts, thereby boosting CD39 activity.

Macrophages are tissue-resident immune cells that are crucial for the initiation and maintenance of immune responses. Purinergic signaling modulates macrophage activity and impacts cellular plasticity. The ATP-activated purinergic receptor P2X7 (also known as P2RX7) has pro-inflammatory properties, which contribute to macrophage activation. P2X7 receptor signaling is, in turn, modulated by ectonucleotidases, such as CD39 (also known as ENTPD1), expressed in caveolae and lipid rafts. Here, we examined P2X7 receptor activity and determined impacts on ectonucleotidase localization and function in macrophages primed with lipopolysaccharide (LPS). First, we verified that ATP boosts CD39 activity and caveolin-1 protein expression in LPS-primed macrophages. Drugs that disrupt cholesterol-enriched domains - such as nystatin and methyl-ß-cyclodextrin - decreased CD39 enzymatic activity in all circumstances. We noted that CD39 colocalized with lipid raft markers (flotillin-2 and caveolin-1) in macrophages that had been primed with LPS followed by treatment with ATP. P2X7 receptor inhibition blocked these ATP-mediated increases in caveolin-1 expression and inhibited the colocalization with CD39. Further, we found that STAT3 activation is significantly attenuated caveolin-1-deficient macrophages treated with LPS or LPS+BzATP. Taken together, our data suggest that P2X7 receptor triggers the initiation of lipid raft-dependent mechanisms that upregulates CD39 activity and could contribute to limit macrophage responses restoring homeostasis.

Specific Dystrophins Selectively Associate with Inhibitory and Excitatory Synapses of the Mouse Cerebellum and their Loss Alters Expression of P2X7 Purinoceptors and Pro-Inflammatory Mediators.

Duchenne muscular dystrophy (DMD) patients, having mutations of the DMD gene, present with a range of neuropsychiatric disorders, in addition to the quintessential muscle pathology. The neurobiological basis remains poorly understood because the contributions of different DMD gene products (dystrophins) to the different neural networks underlying such symptoms are yet to be fully characterised. While full-length dystrophin clusters in inhibitory synapses, with inhibitory neurotransmitter receptors, the precise subcellular expression of truncated DMD gene products with excitatory synapses remains unresolved. Furthermore, inflammation, involving P2X purinoceptor 7 (P2RX7) accompanies DMD muscle pathology, yet any association with brain dystrophins is yet to be established. The aim of this study was to investigate the comparative expression of different dystrophins, alongside ionotropic glutamate receptors and P2RX7s, within the cerebellar circuitry known to express different dystrophin isoforms. Immunoreactivity for truncated DMD gene products was targeted to Purkinje cell (PC) distal dendrites adjacent to, or overlapping with, signal for GluA1, GluA4, GluN2A, and GluD2 receptor subunits. P2X7R immunoreactivity was located in Bergmann glia profiles adjacent to PC-dystrophin immunoreactivity. Ablation of all DMD gene products coincided with decreased mRNA expression for Gria2, Gria3, and Grin2a and increased GluD2 immunoreactivity. Finally, dystrophin-null mice showed decreased brain mRNA expression of P2rx7 and several inflammatory mediators. The data suggest that PCs target different dystrophin isoforms to molecularly and functionally distinct populations of synapses. In contrast to muscle, dystrophinopathy in brain leads to the dampening of the local immune system.

Influence of clinical factors on the protective or deleterious impact of genetic variants in orthodontically induced external root resorption: an observational study.

BACKGROUND: Prediction of susceptibility to Orthodontically Induced External Apical Root Resorption (OIEARR) has been hampered by the complex architecture of this multifactorial phenotype. The aim of this study was to analyze the impact of the interaction of multiple variables in the susceptibility to OIEARR. METHODS: The study evaluated 195 patients requiring orthodontic treatment. Nine clinical and treatment variables, single nucleotide polymorphisms (SNPs) from five genes and variables interactions were analyzed as risk factors for OIEARR using a multiple linear regression model. RESULTS: The model explained 29% of OIEARR variability (ANOVA: p < 0.01). Duration of treatment was the most important predictor and gender was the second, closely followed by premolar extraction. For genes encoding osteoprotegerin (OPG), the receptor activator of nuclear factor κ B (RANK) and the IL1 receptor antagonist (IL1RN), the effect of analyzed variants changed from protective to deleterious depending on the duration of treatment and the age of the patient. CONCLUSIONS: This work shows that in OIEARR the impact of genetic susceptibility factors is dynamic changing according to clinical variables.

Regulation of the Acute Sickness Response by the P2RX7 Receptor.

BACKGROUND: The acute sickness response to infection is a stereotyped set of illness manifestations initiated by proinflammatory signals in the periphery but mediated centrally. P2RX7 is a highly polymorphic gene encoding an ATP-gated cationic pore, widely expressed on immune cells and the brain, and regulating the NLRP3 inflammasome, as well as diverse neural functions. METHODS: Associations between P2RX7 genotype, pore activity, and illness manifestations were examined in a cohort with acute viral and bacterial infections (n = 484). Genotyping of 12 P2RX7 function-modifying single-nucleotide polymorphisms (SNPs) was used to identify haplotypes and diplotypes. Leucocyte pore activity was measured by uptake of the fluorescent dye, YO-PRO-1, and by ATP-induced interleukin-1ß (IL-1ß) release. Associations were sought with scores describing the symptom domains, or endophenotypes, derived from principal components analysis. RESULTS: Among the 12 SNPs, a 4-SNP haplotype block with 5 variants was found in 99.5% of the subjects. These haplotypes and diplotypes were closely associated with variations in pore activity and IL-1ß production. Homozygous diplotypes were associated with overall illness severity as well as fatigue, pain, and mood disturbances. CONCLUSIONS: P2RX7 signaling plays a significant role in the acute sickness response to infection, likely acting in both the immune system and the brain.

Murine iNKT cells are depleted by liver damage via activation of P2RX7.

Invariant natural killer T cells (iNKT) constitute up to 50% of liver lymphocytes and contribute to immunosurveillance as well as pathogenesis of the liver. Systemic activation of iNKT cells induces acute immune-mediated liver injury. However, how tissue damage events regulate iNKT cell function and homeostasis remains unclear. We found that specifically tissue-resident iNKT cells in liver and spleen express the tissue-damage receptor P2RX7 and the P2RX7-activating ectoenzyme ARTC2. P2RX7 expression restricted formation of iNKT cells in the liver suggesting that liver iNKT cells are actively restrained under homeostatic conditions. Deliberate activation of P2RX7 in vivo by exogenous NAD resulted in a nearly complete iNKT cell ablation in liver and spleen in a P2RX7-dependent manner. Tissue damage generated by acetaminophen-induced liver injury reduced the number of iNKT cells in the liver. The tissue-damage-induced iNKT cell depletion was driven by P2RX7 and localized to the site of injury, as iNKT cells in the spleen remained intact. The depleted liver iNKT cells reconstituted only slowly compared to other lymphocytes such as regulatory T cells. These findings suggest that tissue-damage-mediated depletion of iNKT cells acts as a feedback mechanism to limit iNKT cell-induced pathology resulting in the establishment of a tolerogenic environment.

The relationship between CD4+ follicular helper T cells and CD8+ resident memory T cells: sisters or distant cousins?

Independent studies over the last decade have characterized the properties of non-circulating CD8+ 'resident' memory T cells (TRM), which offer barrier protective immunity in non-lymphoid tissues and CD4+ follicular helper T cells (TFH), which mediate B-cell help in lymphoid sites. Despite their very different biological roles in the immune system, intriguing parallels have been noted between the trafficking properties and differentiation cues of these populations, parallels which have only sharpened with recent findings. In this review, we explore the features that underlie these similarities and discuss whether these indicate meaningful homologies in the development of CD8+ TRM and CD4+ TFH or reflect resemblances which are only 'skin-deep'.

Regulatory function of praja ring finger ubiquitin ligase 2 mediated by the P2rx3/P2rx7 axis in mouse hippocampal neuronal cells.

Praja2 (Pja2), a member of the growing family of mammalian RING E3 ubiquitin ligases, is reportedly involved in not only several types of cancer but also neurological diseases and disorders, but the genetic mechanism underlying the regulation of Pja2 in the nervous system remains unclear. To study the cellular and molecular functions of Pja2 in mouse hippocampal neuronal cells (MHNCs), we used gain- and loss-of-function manipulations of Pja2 in HT-22 cells and tested their regulatory effects on three Alzheimer's disease (AD) genes and cell proliferation. The results revealed that the expression of AD markers, including amyloid beta precursor protein (App), microtubule-associated protein tau (Mapt), and gamma-secretase activating protein (Gsap), could be inhibited by Pja2 overexpression and activated by Pja2 knockdown. In addition, HT-22 cell proliferation was enhanced by Pja2 upregulation and suppressed by its downregulation. We also evaluated and quantified the targets that responded to the enforced expression of Pja2 by RNA-Seq, and the results showed that purinergic receptor P2X, ligand-gated ion channel 3 and 7 (P2rx3 and P2rx7), which show different expression patterns in the critical calcium signaling pathway, mediated the regulatory effect of Pja2 in HT-22 cells. Functional studies indicated that Pja2 regulated HT-22 cells development and AD marker genes by inhibiting P2rx3 but promoting P2rx7, a gene downstream of P2rx3. In conclusion, our results provide new insights into the regulatory function of the Pja2 gene in MHNCs and thus underscore the potential relevance of this molecule to the pathophysiology of AD.

Ca2+ flux through splice variants of the ATP-gated ionotropic receptor P2X7 is regulated by its cytoplasmic N terminus.

Activation of ionotropic P2X receptors increases free intracellular Ca2+ ([Ca2+] i ) by initiating a transmembrane cation flux. We studied the "a" and "k" splice variants of the rat purinergic P2X7 receptor (rP2X7aR and rP2X7kR) to exhibit a significant difference in Ca2+ flux through this channel. This difference is surprising because the variants share absolute sequence identity in the area of the pore that defines ionic selectivity. Here, we used patch-clamp fluorometry and chimeric receptors to show that the fraction of the total current carried by Ca2+ is a function of the primary sequence of the cytoplasmic N terminus. Using scanning mutagenesis, we identified five sites within the N terminus that respond to mutagenesis with a decrease in fractional calcium current and an increase in permeability to the polyatomic cation, N-methyl-d-glucamine (NMDG+), relative to Na+ (PNMDG/PNa). We tested the hypothesis that these sites line the permeation pathway by measuring the ability of thiol-reactive MTSET+ to alter the current of cysteine-substituted variants, but we detected no effect. Finally, we studied the homologous sites of the rat P2X2 receptor (rP2X2R) and observed that substitutions at Glu17 significantly reduced the fractional calcium current. Taken together, our results suggest that a change in the structure of the N terminus alters the ability of an intra-pore Ca2+ selectivity filter to discriminate among permeating cations. These results are noteworthy for two reasons: they identify a previously unknown outcome of mutagenesis of the N-terminal domain, and they suggest caution when assigning structure to function for truncated P2X receptors that lack a part of the N terminus.

Muscle death participates in myofibrillar abnormalities in FHL1 knockout mice.

BACKGROUND: Mutations in the four and-a-half LIM domain protein 1 (FHL1) gene or FHL1 protein deletion have been identified as the cause of rare hereditary myopathies or cardiomyopathies. In our previous study, autophagy activation was associated with myofibrillar abnormalities in FHL1 knockout (KO) mice. P2RX7 induces cell death, such as autophagy, pyroptosis or apoptosis via cell-specific downstream signaling; however, the roles of P2RX7 in pyroptosis or apoptosis in myofibrillar abnormalities in FHL1 KO mice have not been well elucidated. METHODS: In this study, skeletal muscle and heart of 2.5 months old WT and FHL1 KO male mice histomorphology were examined by hematoxylin and eosin staining. The indicators for pyroptosis (NLRP3; ASC; cleaved-caspase1; IL-1ß), apoptosis (Apaf-1; Bcl-2; caspase9; cleaved-caspase3), and P2RX7 were detected in the triceps (Tri), tibialis anterior muscles (TA), and heart by western blot and/or immunohistochemistry in WT and FHL1 KO male mice. RESULTS: Indicators for pyroptosis (ASC; cleaved-caspase1; IL-1ß) and apoptosis (Apaf-1 and cleaved-caspase3), as well as P2RX7 were upregulated in Tri, tibialis TA, and heart in FHL1 KO mice, indicating pyroptosis and apoptosis play important roles in myofibrillar abnormalities in FHL1 KO mice. CONCLUSIONS: P2RX7 may participate in myofibrillar abnormalities by activating pyroptosis and apoptosis in FHL1 KO mice. These findings have basic implications for the understanding of myopathies induced by FHL1 deficiency and provide new avenues for the treatment of these hereditary myopathies by modulating P2RX7.