P2X7 receptor deficiency attenuates cisplatin-induced kidney injury via inhibiting NLRP3 inflammasome activation.

Nephrotoxicity is a major constraint of cisplatin application in many solid tumors. Since the lack of preventive strategies, the necessity exists to identify critical molecular targets involved in cisplatin nephrotoxicity. The Purinergic ligand-gcotedion channel 7 receptor (P2X7R) is a ligand-gated ion channel that is predominantly implicated in inflammation and cell death. Our aim is to investigate the role P2X7R in cisplatin-induced acute and chronic kidney injury, as well as the underlying mechanism. In this study, we found that cisplatin can cause an increase in the expression of P2X7R in mouse kidney tissue, and P2X7R knockout can alleviate acute renal function damage caused by cisplatin, as well as the expression of kidney injury molecule 1 (KIM-1) and interleukin-18 (IL-18). Cisplatin can cause an increase in the expression of nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome in mouse kidney tissue. Compared with wild-type mice, P2X7R -/- mice showed decreased expression of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cleaved Caspase-1, and cleaved IL-1ß in kidney tissue after cisplatin administration, and the apoptosis of renal tubular epithelial cells were also decreased. In addition, we also found that NLRP3 knockout can improve cisplatin induced degeneration, detachment, and necrosis of renal tubular epithelial cells. Furthermore, P2X7R -/- mice also showed reduced renal fibrosis and better long-term renal prognosis. In conclusion, our study identified that P2X7R knockout can improve cisplatin induced acute renal injury and chronic renal fibrosis by inhibiting the activation of NLRP3 inflammasome.

P2X7-deficiency improves plasticity and cognitive abilities in a mouse model of Tauopathy.

Alzheimer's disease is the most common form of dementia characterized by intracellular aggregates of hyperphosphorylated Tau protein and extracellular accumulation of amyloid ß (Aß) peptides. We previously demonstrated that the purinergic receptor P2X7 (P2X7) plays a major role in Aß-mediated neurodegeneration but the relationship between P2X7 and Tau remained overlooked. Such a link was supported by cortical upregulation of P2X7 in patients with various type of frontotemporal lobar degeneration, including mutation in the Tau-coding gene, MAPT, as well as in the brain of a Tauopathy mouse model (THY-Tau22). Subsequent phenotype analysis of P2X7-deficient Tau mice revealed the instrumental impact of this purinergic receptor. Indeed, while P2X7-deficiency had a moderate effect on Tau pathology itself, we observed a significant reduction of microglia activation and of Tau-related inflammatory mediators, particularly CCL4. Importantly, P2X7 deletion ultimately rescued synaptic plasticity and memory impairments of Tau mice. Altogether, the present data support a contributory role of P2X7 dysregulation on processes governing Tau-induced brain anomalies. Due to the convergent role of P2X7 blockade in both Aß and Tau background, P2X7 inhibitors might prove to be ideal candidate drugs to curb the devastating cognitive decline in Alzheimer's disease and Tauopathies.

P2X7 Receptors Amplify CNS Damage in Neurodegenerative Diseases.

ATP is a (co)transmitter and signaling molecule in the CNS. It acts at a multitude of ligand-gated cationic channels termed P2X to induce rapid depolarization of the cell membrane. Within this receptor-channel family, the P2X7 receptor (R) allows the transmembrane fluxes of Na+, Ca2+, and K+, but also allows the slow permeation of larger organic molecules. This is supposed to cause necrosis by excessive Ca2+ influx, as well as depletion of intracellular ions and metabolites. Cell death may also occur by apoptosis due to the activation of the caspase enzymatic cascade. Because P2X7Rs are localized in the CNS preferentially on microglia, but also at a lower density on neuroglia (astrocytes, oligodendrocytes) the stimulation of this receptor leads to the release of neurodegeneration-inducing bioactive molecules such as pro-inflammatory cytokines, chemokines, proteases, reactive oxygen and nitrogen molecules, and the excitotoxic glutamate/ATP. Various neurodegenerative reactions of the brain/spinal cord following acute harmful events (mechanical CNS damage, ischemia, status epilepticus) or chronic neurodegenerative diseases (neuropathic pain, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis) lead to a massive release of ATP via the leaky plasma membrane of neural tissue. This causes cellular damage superimposed on the original consequences of neurodegeneration. Hence, blood-brain-barrier permeable pharmacological antagonists of P2X7Rs with excellent bioavailability are possible therapeutic agents for these diseases. The aim of this review article is to summarize our present state of knowledge on the involvement of P2X7R-mediated events in neurodegenerative illnesses endangering especially the life quality and duration of the aged human population.

P2X7 Receptor Activity Limits Accumulation of T Cells within Tumors.

Extracellular ATP (eATP) is a signaling molecule that variably affects all cells of the immune system either directly or after hydrolysis to adenosine. Although eATP is virtually absent in the interstitium of normal tissues, it can be present in the hundreds of micromolar range in tumors, a concentration compatible with activation of the ATP-gated ionotropic P2X7 receptor. Here, we show that P2X7 activity in tumor-infiltrating lymphocytes (TIL) induces cellular senescence and limits tumor suppression. P2X7 stimulation affected cell cycling of effector T cells and resulted in generation of mitochondrial reactive oxygen species and p38 MAPK-dependent upregulation of cyclin-dependent kinase inhibitor 1A (Cdkn1a, encoding for p21Waf1/Cip1). Lack of P2X7 promoted a transcriptional signature that correlated with enhanced cytotoxic T-cell response in human solid tumors. In mice, transfer of tumor-specific T cells with deletion of P2rx7 significantly reduced tumor growth and extended survival. Collectively, these findings uncover a purinergic checkpoint that can be targeted to improve the efficacy of cancer immunotherapy strategies. SIGNIFICANCE: These findings suggest that the purinergic checkpoint P2X7 may be targeted to enhance T-cell-mediated cancer immunotherapy and improve T effector cell accumulation in the tumor microenvironment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/18/3906/F1.large.jpg.

Long-lived T follicular helper cells retain plasticity and help sustain humoral immunity.

CD4+ memory T cells play an important role in protective immunity and are a key target in vaccine development. Many studies have focused on T central memory (Tcm) cells, whereas the existence and functional significance of long-lived T follicular helper (Tfh) cells are controversial. Here, we show that Tfh cells are highly susceptible to NAD-induced cell death (NICD) during isolation from tissues, leading to their underrepresentation in prior studies. NICD blockade reveals the persistence of abundant Tfh cells with high expression of hallmark Tfh markers to at least 400 days after infection, by which time Tcm cells are no longer found. Using single-cell RNA-seq, we demonstrate that long-lived Tfh cells are transcriptionally distinct from Tcm cells, maintain stemness and self-renewal gene expression, and, in contrast to Tcm cells, are multipotent after recall. At the protein level, we show that folate receptor 4 (FR4) robustly discriminates long-lived Tfh cells from Tcm cells. Unexpectedly, long-lived Tfh cells concurrently express a distinct glycolytic signature similar to trained immune cells, including elevated expression of mTOR-, HIF-1-, and cAMP-regulated genes. Late disruption of glycolysis/ICOS signaling leads to Tfh cell depletion concomitant with decreased splenic plasma cells and circulating antibody titers, demonstrating both unique homeostatic regulation of Tfh and their sustained function during the memory phase of the immune response. These results highlight the metabolic heterogeneity underlying distinct long-lived T cell subsets and establish Tfh cells as an attractive target for the induction of durable adaptive immunity.

Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7R-Dependent STING Activation by Tumor-Derived cGAMP.

Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas-/- mice. Abolishing cGAMP production in Cgas-/- tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.

Enrichment of intestinal Lactobacillus by enhanced secretory IgA coating alters glucose homeostasis in P2rx7-/- mice.

The secretory immunoglobulin A (SIgA) in mammalian gut protects the organism from infections and contributes to host physiology by shaping microbiota composition. The mechanisms regulating the adaptive SIgA response towards gut microbes are poorly defined. Deletion of P2rx7, encoding for the ATP-gated ionotropic P2X7 receptor, leads to T follicular helper (Tfh) cells expansion in the Peyer's patches (PPs) of the small intestine, enhanced germinal centre (GC) reaction and IgA secretion; the resulting alterations of the gut microbiota in turn affects host metabolism. Here, we define gut microbiota modifications that correlate with deregulated SIgA secretion and metabolic alterations in P2rx7-/- mice. In particular, Lactobacillus shows enhanced SIgA coating in P2rx7-/- with respect to wild-type (WT) mice. The abundance of SIgA-coated lactobacilli positively correlates with Tfh cells number and body weight, suggesting Lactobacillus-specific SIgA response conditions host metabolism. Accordingly, oral administration of intestinal Lactobacillus isolates from P2rx7-/- mice to WT animals results in altered glucose homeostasis and fat deposition. Thus, enhanced SIgA production by P2X7 insufficiency promotes Lactobacillus colonization that interferes with systemic metabolic homeostasis. These data indicate that P2X7 receptor-mediated regulation of commensals coating by SIgA is important in tuning the selection of bacterial taxa, which condition host metabolism.

P2X7 Receptor Indirectly Regulates the JAM-A Protein Content via Modulation of GSK-3ß.

The alveolar epithelial cells represent an important part of the alveolar barrier, which is maintained by tight junction proteins, particularly JAM-A, occludin, and claudin-18, which regulate paracellular permeability. In this study, we report on a strong increase in epithelial JAM-A expression in P2X7 receptor knockout mice when compared to the wildtype. Precision-cut lung slices of wildtype and knockout lungs and immortal epithelial lung E10 cells were treated with bleomycin, the P2X7 receptor inhibitor oxATP, and the agonist BzATP, respectively, to evaluate early changes in JAM-A expression. Biochemical and immunohistochemical data showed evidence for P2X7 receptor-dependent JAM-A expression in vitro. Inhibition of the P2X7 receptor using oxATP increased JAM-A, whereas activation of the receptor decreased the JAM-A protein level. In order to examine the role of GSK-3ß in the expression of JAM-A in alveolar epithelial cells, we used lithium chloride for GSK-3ß inhibiting experiments, which showed a modulating effect on bleomycin-induced alterations in JAM-A levels. Our data suggest that an increased constitutive JAM-A protein level in P2X7 receptor knockout mice may have a protective effect against bleomycin-induced lung injury. Bleomycin-treated precision-cut lung slices from P2X7 receptor knockout mice responded with a lower increase in mRNA expression of JAM-A than bleomycin-treated precision-cut lung slices from wildtype mice.

P2RX7 Deletion in T Cells Promotes Autoimmune Arthritis by Unleashing the Tfh Cell Response.

Rheumatoid arthritis (RA) is an autoimmune disease that affects ~1% of the world's population. B cells and autoantibodies play an important role in the pathogenesis of RA. The P2RX7 receptor is an ATP-gated cation channel and its activation results in the release of pro-inflammatory molecules. Thus, antagonists of P2RX7 have been considered to have potential as novel anti-inflammatory therapies. Although originally identified for its role in innate immunity, P2RX7 has recently been found to negatively control Peyer's patches (PP) T follicular helper cells (Tfh), which specialize in helping B cells, under homeostatic conditions. We have previously demonstrated that PP Tfh cells are required for the augmentation of autoimmune arthritis mediated by gut commensal segmented filamentous bacteria (SFB). Thus, we hypothesized that P2RX7 is required to control autoimmune disease by keeping the Tfh cell response in check. To test our hypothesis, we analyzed the impact of P2RX7 deficiency in vivo using both the original K/BxN autoimmune arthritis model and T cell transfers in the K/BxN system. We also examined the impact of P2RX7 ablation on autoimmune development in the presence of the gut microbiota SFB. Our data illustrate that contrary to exerting an anti-inflammatory effect, P2RX7 deficiency actually enhances autoimmune arthritis. Interestingly, SFB colonization can negate the difference in disease severity between WT and P2RX7-deficient mice. We further demonstrated that P2RX7 ablation in the absence of SFB caused reduced apoptotic Tfh cells and enhanced the Tfh response, leading to an increase in autoantibody production. It has been shown that activation of TIGIT, a well-known T cell exhaustion marker, up-regulates anti-apoptotic molecules and promotes T cell survival. We demonstrated that the reduced apoptotic phenotype of P2rx7-/- Tfh cells is associated with their increased expression of TIGIT. This suggested that while P2RX7 was regulating the Tfh population by promoting cell death, TIGIT may have been opposing P2RX7 by inhibiting cell death. Together, these results demonstrated that systemic administration of general P2RX7 antagonists may have detrimental effects in autoimmune therapies, especially in Tfh cell-dependent autoimmune diseases, and cell-specific targeting of P2RX7 should be considered in order to achieve efficacy for P2RX7-related therapy.

P2X7 receptor deletion suppresses γ-radiation-induced hyposalivation.

Head and neck cancer treatments typically involve a combination of surgery and radiotherapy, often leading to collateral damage to nearby tissues causing unwanted side effects. Radiation damage to salivary glands frequently leads to irreversible dysfunction by poorly understood mechanisms. The P2X7 receptor (P2X7R) is a ligand-gated ion channel activated by extracellular ATP released from damaged cells as "danger signals." P2X7R activation initiates apoptosis and is involved in numerous inflammatory disorders. In this study, we utilized P2X7R knockout (P2X7R-/-) mice to determine the role of the receptor in radiation-induced salivary gland damage. Results indicate a dose-dependent increase in γ-radiation-induced ATP release from primary parotid gland cells of wild-type but not P2X7R-/- mice. Despite these differences, apoptosis levels are similar in parotid glands of wild-type and P2X7R-/- mice 24-72 h after radiation. However, γ-radiation caused elevated prostaglandin E2 (PGE2) release from primary parotid cells of wild-type but not P2X7R-/- mice. To attempt to uncover the mechanism underlying differential PGE2 release, we evaluated the expression and activities of cyclooxygenase and PGE synthase isoforms. There were no consistent trends in these mediators following radiation that could explain the reduction in PGE2 release in P2X7R-/- mice. Irradiated P2X7R-/- mice have stimulated salivary flow rates similar to unirradiated controls, whereas irradiated wild-type mice have significantly decreased salivary flow rates compared with unirradiated controls. Notably, treatment with the P2X7R antagonist A438079 preserves stimulated salivary flow rates in wild-type mice following γ-radiation. These data suggest that P2X7R antagonism is a promising approach for preventing γ-radiation-induced hyposalivation.

Lack of P2X7 Receptors Protects against Renal Fibrosis after Pyelonephritis with α-Hemolysin-Producing Escherichia coli.

Severe urinary tract infections are commonly caused by sub-strains of Escherichia coli secreting the pore-forming virulence factor α-hemolysin (HlyA). Repeated or severe cases of pyelonephritis can cause renal scarring that subsequently can lead to progressive failure. We have previously demonstrated that HlyA releases cellular ATP directly through its membrane pore and that acute HlyA-induced cell damage is completely prevented by blocking ATP signaling. Local ATP signaling and P2X7 receptor activation play a key role in the development of tissue fibrosis. This study investigated the effect of P2X7 receptors on infection-induced renal scarring in a murine model of pyelonephritis. Pyelonephritis was induced by injecting 100 million HlyA-producing, uropathogenic E. coli into the urinary bladder of BALB/cJ mice. A similar degree of pyelonephritis and mortality was confirmed at day 5 after infection in P2X7+/+ and P2X7-/- mice. Fibrosis was first observed 2 weeks after infection, and the data clearly demonstrated that P2X7-/- mice and mice exposed to the P2X7 antagonist, brillian blue G, show markedly less renal fibrosis 14 days after infection compared with controls (P < 0.001). Immunohistochemistry revealed comparable early neutrophil infiltration in the renal cortex from P2X7+/+ and P2X7-/- mice. Interestingly, lack of P2X7 receptors resulted in diminished macrophage infiltration and reduced neutrophil clearance in the cortex of P2X7-/- mice. Hence, this study suggests the P2X7 receptor to be an appealing antifibrotic target after renal infections.

CD8+, but not CD4+ effector/memory T cells, express the CD44highCD45RBhigh phenotype with aging, which displays reduced expression levels of P2X7 receptor and ATP-induced cellular responses.

Previously we reported that the sensitivity of CD4+ T cells to ATP does not depend on P2X7 receptor (P2X7R) expression levels but on their activation and differentiation stages. Therefore, here we have investigated a potential relationship between the sensitivity of CD8+ T cells to ATP and their stages of differentiation. Thus, the CD8+ subpopulation exhibits a drastically reduced sensitivity to ATP with aging, which parallels the strong increase of an effector/memory CD8+ subset expressing high levels of CD44 cell adhesion molecule and CD45RB transmembrane phosphatase (CD44hiCD45RBhi). Using l-selectin/CD62L, CC-chemokine receptor 7, and CD127/IL-7 receptor-α markers, we showed that effector/memory CD8+ T cells belong to a central or effector memory subset. In contrast, the CD44hiCD45RBhi effector/memory subset is absent or poorly expressed in the CD4+ T subpopulation regardless of age. While ATP treatment can trigger channel and pore formation, CD62L shedding, phosphatidylserine exposure, and cell death in the CD44loCD45RBhi-naive CD8+ subset, it is unable to induce these cellular activities in the CD44hiCD45RBhi effector/memory CD8+ subset. Importantly, both CD44loCD45RBhi-naive and CD44hiCD45RBhi effector/memory subsets express similar low levels of P2X7R, demonstrating that the sensitivity of CD8+ T cells to ATP depends on the stage of differentiation instead of P2X7R expression levels.-Mellouk, A., Bobé, P. CD8+, but not CD4+ effector/memory T cells, express the CD44highCD45RBhigh phenotype with aging, which displays reduced expression levels of P2X7 receptor and ATP-induced cellular responses.

The TWIK2 Potassium Efflux Channel in Macrophages Mediates NLRP3 Inflammasome-Induced Inflammation.

Potassium (K+) efflux across the plasma membrane is thought to be an essential mechanism for ATP-induced NLRP3 inflammasome activation, yet the identity of the efflux channel has remained elusive. Here we identified the two-pore domain K+ channel (K2P) TWIK2 as the K+ efflux channel triggering NLRP3 inflammasome activation. Deletion of Kcnk6 (encoding TWIK2) prevented NLRP3 activation in macrophages and suppressed sepsis-induced lung inflammation. Adoptive transfer of Kcnk6-/- macrophages into mouse airways after macrophage depletion also prevented inflammatory lung injury. The K+ efflux channel TWIK2 in macrophages has a fundamental role in activating the NLRP3 inflammasome and consequently mediates inflammation, pointing to TWIK2 as a potential target for anti-inflammatory therapies.

The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells.

Extracellular ATP (eATP) is an ancient 'danger signal' used by eukaryotes to detect cellular damage1. In mice and humans, the release of eATP during inflammation or injury stimulates both innate immune activation and chronic pain through the purinergic receptor P2RX72-4. It is unclear, however, whether this pathway influences the generation of immunological memory, a hallmark of the adaptive immune system that constitutes the basis of vaccines and protective immunity against re-infection5,6. Here we show that P2RX7 is required for the establishment, maintenance and functionality of long-lived central and tissue-resident memory CD8+ T cell populations in mice. By contrast, P2RX7 is not required for the generation of short-lived effector CD8+ T cells. Mechanistically, P2RX7 promotes mitochondrial homeostasis and metabolic function in differentiating memory CD8+ T cells, at least in part by inducing AMP-activated protein kinase. Pharmacological inhibitors of P2RX7 provoked dysregulated metabolism and differentiation of activated mouse and human CD8+ T cells in vitro, and transient P2RX7 blockade in vivo ameliorated neuropathic pain but also compromised production of CD8+ memory T cells. These findings show that activation of P2RX7 by eATP provides a common currency that both alerts the nervous and immune system to tissue damage, and promotes the metabolic fitness and survival of the most durable and functionally relevant memory CD8+ T cell populations.

Small molecule inducers of ABCA1 and apoE that act through indirect activation of the LXR pathway.

apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer's disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.

P2X7R: independent modulation of aquaporin 5 expression in CdCl2-injured alveolar epithelial cells.

The expression of aquaporin 5 in alveolar epithelial type I cells under conditions of cadmium-induced injury has not yet been discovered. We investigated the effect of the P2X7R agonist BzATP under this condition, since P2X7R is involved in altered regulation of aquaporin 5 in pulmonary fibrosis. CdCl2/TGF-ß1 treatment of lung epithelial MLE-12 cells was leading to increasing P2X7R, and aquaporin 5 protein levels. The aquaporin 5 expression was P2X7R-independent in MLE-12 cells under cadmium, as was shown in blocking experiments with oxATP. Further, the expression of both proteins increased after 24 h CdCl2/TGF-ß1 treatment of precision-cut lung slices, but decreased after 72 h. Using immunohistochemistry, the activation of the P2X7R with the agonist BzATP modulated the aquaporin 5 immunoreactivity in the alveolar epithelium of precision-cut lung slices from wild-type but not from P2X7R knockout mice. Similarly, aquaporin 5 protein was reduced in BzATP-treated immortal lung epithelial E10 cells. Surprisingly, untreated alveolar epithelial type II cells of P2X7R knockouts exhibited a pronounced apical immunoreactivity in addition to the remaining alveolar epithelial type I cells. BzATP exposure did not alter this distribution pattern, but increased the number of apoptotic alveolar epithelial type II cells in wild-type lung slices.

Papel dos receptores purinérgicos em modelo animal de doença de Parkinson / Role of purinergic receptors in an animal model of Parkinsons Disease

A Doença de Parkinson é uma doença altamente incapacitante e de grande prevalência. Pouco se sabe sobre sua etiologia e os tratamentos atuais consistem na diminuição dos sintomas, uma vez que ainda não foi encontrada uma maneira de reverter o déficit de neurônios dopaminérgicos observados nos pacientes acometidos. Sabe-se que os receptores purinérgicos são encontrados por todo o sistema nervoso central, não só no indivíduo adulto como também em diferentes estágios do desenvolvimento embrionário e estão envolvidos com proliferação e diferenciação celular. Este trabalho estudou a participação dos receptores purinérgicos em modelo animal de doença de Parkinson por lesão dos neurônios dopaminérgicos da via nigroestriatal com 6-OH dopamina (6-OHDA). Realizamos a análise do perfil de expressão gênica dos diferentes receptores após a lesão e subsequente modulação. Observamos expressão gênica alterada dos receptores P2X7 e P2Y6 até 5 semanas após a lesão. O uso do antagonista do receptor P2X7 Brilliant Blue G (BBG) induziu a regeneração da via nigroestriatal e o uso do antagonista do receptor P2Y6 MRS2578 preveniu a morte dos neurônios. Como esses efeitos foram acompanhados pela inativação de células microgliais, supõe-se que o controle do microambiente neuroinflamatório causado pela injeção de 6-OHDA seja a principal causa do efeito antiparkinsoniano observado pelo tratamento com BBG e MRS2578. Além disso, o transplante celular com células precursoras neuraisnão foi capaz de reverter o comportamento hemiparkinsoniano dos animais lesionados. Apesar do uso concomitante com BBG reduzir o comportamento, parece que esse efeito deve-se ao BBG per se, uma vez que o tratamento somente com o antagonista de P2X7 foi mais eficaz. De maneira geral, a modulação da atividade dos receptores purinérgicos se mostrou uma ferramenta promissora na pesquisa de cura e compreensão das bases moleculares da Doença de Parkinson

Parkinson's disease is a highly disabling and prevalent disease. Little is known about its etiology and the current treatments consist in the reduction of the symptoms, since there is no known method to reverse the dopaminergic neurons deficit observed in patients. Purinergic receptors are found throughout the central nervous system, not only in the adult individual but also at different stages of embryonic development, and are involved in proliferation and differentiation. This work investigated the role of purinergic receptors in the animal model of Parkinson's disease induced by 6-OH dopamine (6-OHDA) injection and consequent death of dopaminergic neurons of the nigrostriatal pathway. Patterns of purinergic receptors gene expression after the lesion and subsequent modulation were analyzed. We observed altered gene expression of P2X7 and P2Y6 receptors within 5 weeks of injury. The use of the P2X7 receptor antagonist Brilliant Blue G (BBG) induced the regeneration of the nigrostriatal pathway and treatment with P2Y6 receptor antagonist MRS2578 prevented the death of the neurons. Since these effects were accompanied by the inactivation of microglial cells, it is assumed that the control of neuroinflammatory milieu caused by the 6-OHDA injection is the main cause of the antiparkinsonian effect observed by the treatment with BBG and MRS2578. In addition, transplantation with neural precursor cells was not able to reverse the hemiparkinsonian behavior of injured animals. Although concomitant use with BBG improved cell engraftment, it appears that this effect is due to BBG per se, since treatment with only this P2X7receptor antagonist was more effective. In general, modulation of purinergic receptor activity showed to be a promising tool in the research of cure and understanding of the molecular bases of Parkinson's Disease

Deficiency of the Purinergic Receptor 2X7 Attenuates Nonalcoholic Steatohepatitis Induced by High-Fat Diet: Possible Role of the NLRP3 Inflammasome.

Molecular mechanisms driving transition from simple steatosis to nonalcoholic steatohepatitis (NASH), a critical step in the progression of nonalcoholic fatty liver disease (NAFLD) to cirrhosis, are poorly defined. This study aimed at investigating the role of the purinergic receptor 2X7 (PR2X7), through the NLRP3 inflammasome, in the development of NASH. To this end, mice knockout for the Pr2x7 gene (Pr2x7-/-) and coeval wild-type (WT) mice were fed a high-fat diet (HFD) or normal-fat diet for 16 weeks. NAFLD grade and stage were lower in Pr2x7-/- than WT mice, and only 1/7 Pr2x7-/- animals showed evidence of NASH, as compared with 4/7 WT mice. Molecular markers of inflammation, oxidative stress, and fibrosis were markedly increased in WT-HFD mice, whereas no or significantly reduced increments were detected in Pr2x7-/- animals, which showed also decreased modulation of genes of lipid metabolism. Deletion of Pr2x7 gene was associated with blunted or abolished activation of NLRP3 inflammasome and expression of its components, which were induced in liver sinusoidal endothelial cells challenged with appropriate stimuli. These data show that Pr2x7 gene deletion protects mice from HFD-induced NASH, possibly through blunted activation of NLRP3 inflammasome, suggesting that PR2X7 and NLRP3 may represent novel therapeutic targets.

The loss of P2X7 receptor expression leads to increase intestinal glucose transit and hepatic steatosis.

In intestinal epithelial cells (IEC), it was reported that the activation of the P2X7 receptor leads to the internalization of the glucose transporter GLUT2, which is accompanied by a reduction of IEC capacity to transport glucose. In this study, we used P2rx7 -/- mice to decipher P2X7 functions in intestinal glucose transport and to evaluate the impacts on metabolism. Immunohistochemistry analyses revealed the presence of GLUT2 at the apical domain of P2rx7 -/- jejunum enterocytes. Positron emission tomography and biodistribution studies demonstrated that glucose was more efficiently delivered to the circulation of knockout animals. These findings correlated with increase blood glucose, insulin, triglycerides and cholesterol levels. In fact, P2rx7 -/- mice had increased serum triglyceride and cholesterol levels and displayed glucose intolerance and resistance to insulin. Finally, P2rx7 -/- mice developed a hepatic steatosis characterized by a reduction of Acaca, Acacb, Fasn and Acox1 mRNA expression, as well as for ACC and FAS protein expression. Our study suggests that P2X7 could play a central role in metabolic diseases.