The microglial P2Y6 receptor as a therapeutic target for neurodegenerative diseases.

Neurodegenerative diseases are associated with chronic neuroinflammation in the brain, which can result in microglial phagocytosis of live synapses and neurons that may contribute to cognitive deficits and neuronal loss. The microglial P2Y6 receptor (P2Y6R) is a G-protein coupled receptor, which stimulates microglial phagocytosis when activated by extracellular uridine diphosphate, released by stressed neurons. Knockout or inhibition of P2Y6R can prevent neuronal loss in mouse models of Alzheimer's disease (AD), Parkinson's disease, epilepsy, neuroinflammation and aging, and prevent cognitive deficits in models of AD, epilepsy and aging. This review summarises the known roles of P2Y6R in the physiology and pathology of the brain, and its potential as a therapeutic target to prevent neurodegeneration and other brain pathologies.

Mechanical stimulation-induced purinome priming fosters osteogenic differentiation and osteointegration of mesenchymal stem cells from the bone marrow of post-menopausal women.

BACKGROUND: Mechanical stimulation (MS) significantly increases the release of adenine and uracil nucleotides from bone marrow-derived mesenchymal stem cells (BM-MSCs) undergoing osteogenic differentiation. Released nucleotides acting via ionotropic P2X7 and metabotropic P2Y6 purinoceptors sensitive to ATP and UDP, respectively, control the osteogenic commitment of BM-MSCs and, thus, bone growth and remodelling. Yet, this mechanism is impaired in post-menopausal (Pm)-derived BM-MSCs, mostly because NTPDase3 overexpression decreases the extracellular accumulation of nucleotides below the levels required to activate plasma membrane-bound P2 purinoceptors. This prompted us to investigate whether in vitro MS of BM-MSCs from Pm women could rehabilitate their osteogenic commitment and whether xenotransplantation of MS purinome-primed Pm cells promote repair of critical bone defects in an in vivo animal model. METHODS: BM-MSCs were harvested from the neck of femora of Pm women (70 ± 3 years old) undergoing total hip replacement. The cells grew, for 35 days, in an osteogenic-inducing medium either submitted (SS) or not (CTR) to MS (90 r.p.m. for 30 min) twice a week. Increases in alkaline phosphatase activity and in the amount of osteogenic transcription factors, osterix and osteopontin, denoted osteogenic cells differentiation, while bone nodules formation was ascertain by the alizarin red-staining assay. The luciferin-luciferase bioluminescence assay was used to quantify extracellular ATP. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC. The density of P2Y6 and P2X7 purinoceptors in the cells was assessed by immunofluorescence confocal microscopy. MS-stimulated BM-MSCs from Pm women were xenotransplanted into critical bone defects drilled in the great trochanter of femora of one-year female Wistar rats; bone repair was assessed by histological analysis 10 days after xenotransplantation. RESULTS: MS-stimulated Pm BM-MSCs in culture (i) release 1.6-fold higher ATP amounts, (ii) overexpress P2X7 and P2Y6 purinoceptors, (iii) exhibit higher alkaline phosphatase activity and overexpress the osteogenic transcription factors, osterix and osteopontin, and (iv) form larger bone nodules, than CTR cells. Selective blockage of P2X7 and P2Y6 purinoceptors with A438079 (3 µM) and MRS 2578 (0.1 µM), respectively, prevented the osteogenic commitment of cultured Pm BM-MSCs. Xenotransplanted MS purinome-primed Pm BM-MSCs accelerated the repair of critical bone defects in the in vivo rat model. CONCLUSIONS: Data suggest that in vitro MS restores the purinergic cell-to-cell communication fostering the osteogenic differentiation and osteointegration of BM-MSCs from Pm women, a strategy that may be used in bone regeneration and repair tactics.

Uracil as a biomarker for spatial pyrimidine metabolism in the development of gingivobuccal oral squamous cell carcinoma.

No biomarker has yet been identified that allows accurate diagnosis and prognosis of oral cancers. In this study, we investigated the presence of key metabolites in oral cancer using proton nuclear magnetic resonance (NMR) spectroscopy to identify metabolic biomarkers of gingivobuccal oral squamous cell carcinoma (GB-OSCC). NMR spectroscopy revealed that uracil was expressed in 83.09% of tumor tissues and pyrimidine metabolism was active in GB-OSCC; these results correlated well with immunohistochemistry (IHC) and RNA sequencing data. Based on further gene and protein analyses, we proposed a pathway for the production of uracil in GB-OSCC tissues. Uridinetriphosphate (UTP) is hydrolyzed to uridine diphosphate (UDP) by CD39 in the tumor microenvironment (TME). We hypothesized that UDP enters the cell with the help of the UDP-specific P2Y6 receptor for further processing by ENTPD4/5 to produce uracil. As the ATP reserves diminish, the weakened immune cells in the TME utilize pyrimidine metabolism as fuel for antitumor activity, and the same mechanism is hijacked by the tumor cells to promote their survival. Correspondingly, the differential expression of ENTPD4 and ENTPD5 in immune and tumor cells, respectively, indicatedtheir involvement in disease progression. Furthermore, higher uracil levels were detected in patients with lymph node metastasis, indicating that metastatic potential is increased in the presence of uracil. The presence of uracil and/or expression patterns of intermediate molecules in purine and pyrimidine pathways, such asCD39, CD73, and P2Y6 receptors together with ENTPD4 and ENTPD5, hold promise as biomarker(s) for oral cancer diagnosis and prognosis.

Astilbin inhibited neutrophil extracellular traps in gouty arthritis through suppression of purinergic P2Y6 receptor.

BACKGROUND: Gouty arthritis (GA), a common inflammatory condition triggered by monosodium urate crystal accumulation, often necessitates safer treatment alternatives due to the limitations of current therapies. Astilbin, a flavonoid from Smilax glabra Roxb, has demonstrated potential in traditional Chinese medicine for its anti-inflammatory properties. However, the anti-GA effect and its underlying mechanism have not been fully elucidated. PURPOSE: This study aimed to investigate the therapeutic potential of astilbin in GA, focusing on its effects on neutrophil extracellular traps (NETs), as well as the potential molecular target of GA both in vitro and in vivo. STUDY DESIGN: Firstly, astilbin inhibited the citrullinated histone H3 (Cit h3) protein levels and reduced the NETs formation in neutrophils stimulated by monosodium urate (MSU). Secondly, we wondered the effect of astilbin on migration of neutrophils and dimethyl-sulfoxide (DMSO)-differentiated HL-60 (dHL-60) cells under the stimulation of MSU. Then, the effect of astilbin on suppressing NETs through purinergic P2Y6 receptor (P2Y6R) and Interlukin-8 (IL-8)/ CXC chemokine receptor 2 (CXCR2) pathway was investigated. Also, the relationship between P2Y6R and IL-8/CXCR2 was explored in dHL-60 cells under stimulation of MSU. Finally, we testified the effect of astilbin on reducing NETs in GA through suppressing P2Y6R and then down-regulating IL-8/CXCR2 pathway. METHODS: MSU was used to induce NETs in neutrophils and dHL-60 cells. Real-time formation of NETs and migration of neutrophils were monitored by cell living imaging with or without MSU. Then, the effect of astilbin on NETs formation, P2Y6R and IL-8/CXCR2 pathway were detected by immunofluorescence (IF) and western blotting. P2Y6R knockdown dHL-60 cells were established by small interfering RNA to investigate the association between P2Y6R and IL-8/CXCR2 pathway. Also, plasmid of P2Y6R was used to overexpress P2Y6R in dHL-60 cells, which was employed to explore the role of P2Y6R in astilbin inhibiting NETs. Within the conditions of knockdown and overexpression of P2Y6R, migration and NETs formation were assessed by transmigration assay and IF staining, respectively. In vivo, MSU-induced GA mice model was established to assess the effect of astilbin on inflammation by haematoxylin-eosin and ELISA. Additionally, the effects of astilbin on neutrophils infiltration, NETs, P2Y6R and IL-8/CXCR2 pathway were analyzed by IF, ELISA, immunohistochemistry (IHC) and western blotting. RESULTS: Under MSU stimulation, astilbin significantly suppressed the level of Cit h3 and NETs formation including the fluorescent expressions of Cit h3, neutrophils elastase, myeloperoxidase, and intra/extracellular DNA. Also, results showed that MSU caused NETs release in neutrophils as well as a trend towards recruitment of dHL-60 cells to MSU. Astilbin could markedly decrease expressions of P2Y6R and IL-8/CXCR2 pathway which were upregulated by MSU. By silencing P2Y6R, the expression of IL-8/CXCR2 pathway and migration of dHL-60 cells were inhibited, leading to the suppression of NETs. These findings indicated the upstream role of P2Y6R in the IL-8/CXCR2 pathway. Moreover, overexpression of P2Y6R was evidently inhibited by astilbin, causing a downregulation in IL-8/CXCR2 pathway, migration of dHL-60 cells and NETs formation. These results emphasized that astilbin inhibited the IL-8/CXCR2 pathway primarily through P2Y6R. In vivo, astilbin administration led to marked reductions in ankle swelling, inflammatory infiltration as well as neutrophils infiltration. Expressions of P2Y6R and IL-8/CXCR2 pathway were evidently decreased by astilbin and P2Y6R inhibitor MRS2578 either alone or in combination. Also, astilbin and MRS2578 showed notable effect on reducing MSU-induced NETs formation and IL-8/CXCR2 pathway whether used alone or in combination, parallelly demonstrating that astilbin decreased NETs formation mainly through P2Y6R. CONCLUSION: This study revealed that astilbin suppressed NETs formation via downregulating P2Y6R and subsequently the IL-8/CXCR2 pathway, which evidently mitigated GA induced by MSU. It also highlighted the potential of astilbin as a promising natural therapeutic for GA.

P2Y6 Receptor-Dependent Microglial Phagocytosis of Synapses during Development Regulates Synapse Density and Memory.

During brain development, excess synapses are pruned (i.e., removed), in part by microglial phagocytosis, and dysregulated synaptic pruning can lead to behavioral deficits. The P2Y6 receptor (P2Y6R) is known to regulate microglial phagocytosis of neurons, and to regulate microglial phagocytosis of synapses in cell culture and in vivo during aging. However, currently it is unknown whether P2Y6R regulates synaptic pruning during development. Here, we show that P2Y6R KO mice of both sexes had strongly reduced microglial internalization of synaptic material, measured as Vglut1 within CD68-staining lysosomes of microglia at postnatal day 30 (P30), suggesting reduced microglial phagocytosis of synapses. Consistent with this, we found an increased density of synapses in the somatosensory cortex and the CA3 region and dentate gyrus of the hippocampus at P30. We also show that adult P2Y6R KO mice have impaired short- and long-term spatial memory and impaired short- and long-term recognition memory compared with WT mice, as measured by novel location recognition, novel object recognition, and Y-maze memory tests. Overall, this indicates that P2Y6R regulates microglial phagocytosis of synapses during development, and this contributes to memory capacity.SIGNIFICANCE STATEMENT The P2Y6 receptor (P2Y6R) is activated by uridine diphosphate released by neurons, inducing microglial phagocytosis of such neurons or synapses. We tested whether P2Y6R regulates developmental synaptic pruning in mice and found that P2Y6R KO mice have reduced synaptic material within microglial lysosomes, and increased synaptic density in the brains of postnatal day 30 mice, consistent with reduced synaptic pruning during development. We also found that adult P2Y6R KO mice had reduced memory, consistent with persistent deficits in brain function, resulting from impaired synaptic pruning. Overall, the results suggest that P2Y6R mediates microglial phagocytosis of synapses during development, and the absence of this results in memory deficits in the adult.

The Role of P2Y6 Receptors in the Mechanisms of the Neuroprotective Effect of Citicoline.

Spontaneous bioelectrical activity of the brain and the duration of gasping were recorded in mice during modeling of global strangulation ischemia of the brain against the background of preventive administration of citicoline. The maximum neuroprotective effect of citicoline was observed when it was administered 60 min before the simulation of ischemia and was completely prevented by preliminary administration of a selective P2Y6 receptor antagonist MRS2578. The obtained experimental data attest to the leading role of receptor mechanisms in the implementation of neuroprotective activity of citicoline.

Sepsis modulates aortic AT1 and P2Y6 receptors to produce vascular hyporeactivity in mice.

PURPOSE: Hyporeactivity to vasopressors leading to multiple organ failure is a serious clinical implication in sepsis. Though the regulatory role of purinoceptors in inflammation is reported, their involvement in sepsis-induced vasoplegia is still unknown. Thus we investigated the effect of sepsis on vascular AT1 and P2Y6 receptors. MATERIALS AND METHODS: Polymicrobial sepsis was induced by cecal ligation and puncture in mice. Vascular reactivity was assessed by organ bath study and aortic mRNA expression of AT1 and P2Y6 was quantified by qRT-PCR. RESULTS: Both angiotensin-II and UDP produced higher contractions in the absence of endothelium as well as following inhibition of nitric oxide synthase. Angiotensin-II mediated aortic contraction was antagonized by losartan (AT1 antagonist), but not by PD123319 (AT2 antagonist) whereas UDP-induced aortic contraction was significantly inhibited by MRS2578 (P2Y6 antagonist). In addition, MRS2578 significantly inhibited the contractile response of Ang-II. Compared to SO mice, angiotensin-II and UDP-induced maximum contraction were found to be significantly attenuated in sepsis. Accordingly, aortic mRNA expression of AT1a receptors was significantly down-regulated while that of P2Y6 receptors was significantly increased in sepsis. 1400 W (a selective iNOS inhibitor) significantly reversed angiotensin-II-induced vascular hyporeactivity in sepsis without affecting UDP-induced hypo-reactivity. CONCLUSION: Sepsis-induced vascular hyporeactivity to angiotensin-II is mediated by enhanced expression of iNOS. Moreover, AT1R-P2Y6 cross talk/heterodimerization could be a novel target for regulating vascular dysfunction in sepsis.

Ligand-dependent intracellular trafficking of the G protein-coupled P2Y6 receptor.

Endosomal trafficking is intricately linked to G protein-coupled receptors (GPCR) fate and signaling. Extracellular uridine diphosphate (UDP) acts as a signaling molecule by selectively activating the GPCR P2Y6. Despite the recent interest for this receptor in pathologies, such as gastrointestinal and neurological diseases, there is sparse information on the endosomal trafficking of P2Y6 receptors in response to its endogenous agonist UDP and synthetic selective agonist 5-iodo-UDP (MRS2693). Confocal microscopy and cell surface ELISA revealed delayed internalization kinetics in response to MRS2693 vs. UDP stimulation in AD293 and HCT116 cells expressing human P2Y6. Interestingly, UDP induced clathrin-dependent P2Y6 internalization, whereas receptor stimulation by MRS2693 endocytosis appeared to be associated with a caveolin-dependent mechanism. Internalized P2Y6 was associated with Rab4, 5, and 7 positive vesicles independent of the agonist. We have measured a higher frequency of receptor expression co-occurrence with Rab11-vesicles, the trans-Golgi network, and lysosomes in response to MRS2693. Interestingly, a higher agonist concentration reversed the delayed P2Y6 internalization and recycling kinetics in the presence of MRS2693 stimulation without changing its caveolin-dependent internalization. This work showed a ligand-dependent effect affecting the P2Y6 receptor internalization and endosomal trafficking. These findings could guide the development of bias ligands that could influence P2Y6 signaling.

Knockout of Purinergic P2Y6 Receptor Fails to Improve Liver Injury and Inflammation in Non-Alcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a disease that progresses from nonalcoholic fatty liver (NAFL) and which is characterized by inflammation and fibrosis. The purinergic P2Y6 receptor (P2Y6R) is a pro-inflammatory Gq/G12 family protein-coupled receptor and reportedly contributes to intestinal inflammation and cardiovascular fibrosis, but its role in liver pathogenesis is unknown. Human genomics data analysis revealed that the liver P2Y6R mRNA expression level is increased during the progression from NAFL to NASH, which positively correlates with inductions of C-C motif chemokine 2 (CCL2) and collagen type I α1 chain (Col1a1) mRNAs. Therefore, we examined the impact of P2Y6R functional deficiency in mice crossed with a NASH model using a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Feeding CDAHFD for 6 weeks markedly increased P2Y6R expression level in mouse liver, which was positively correlated with CCL2 mRNA induction. Unexpectedly, the CDAHFD treatment for 6 weeks increased liver weights with severe steatosis in both wild-type (WT) and P2Y6R knockout (KO) mice, while the disease marker levels such as serum AST and liver CCL2 mRNA in CDAHFD-treated P2Y6R KO mice were rather aggravated compared with those of CDAHFD-treated WT mice. Thus, P2Y6R may not contribute to the progression of liver injury, despite increased expression in NASH liver.

P2Y6 receptor-dependent microglial phagocytosis of synapses mediates synaptic and memory loss in aging.

Aging causes loss of brain synapses and memory, and microglial phagocytosis of synapses may contribute to this loss. Stressed neurons can release the nucleotide UTP, which is rapidly converted into UDP, that in turn activates the P2Y6 receptor (P2Y6 R) on the surface of microglia, inducing microglial phagocytosis of neurons. However, whether the activation of P2Y6 R affects microglial phagocytosis of synapses is unknown. We show here that inactivation of P2Y6 R decreases microglial phagocytosis of isolated synapses (synaptosomes) and synaptic loss in neuronal-glial co-cultures. In vivo, wild-type mice aged from 4 to 17 months exhibited reduced synaptic density in cortical and hippocampal regions, which correlated with increased internalization of synaptic material within microglia. However, this aging-induced synaptic loss and internalization were absent in P2Y6 R knockout mice, and these mice also lacked any aging-induced memory loss. Thus, P2Y6 R appears to mediate aging-induced loss of synapses and memory by increasing microglial phagocytosis of synapses. Consequently, blocking P2Y6 R has the potential to prevent age-associated memory impairment.

Mechanisms of sympathoexcitation via P2Y6 receptors.

Many drugs used in cardiovascular therapy, such as angiotensin receptor antagonists and beta-blockers, may exert at least some of their actions through effects on the sympathetic nervous system, and this also holds true for e.g., P2Y12 antagonists. A new target at the horizon of cardiovascular drugs is the P2Y6 receptor which contributes to the development of arteriosclerosis and hypertension. To learn whether P2Y6 receptors in the sympathetic nervous system might contribute to actions of respective receptor ligands, responses of sympathetic neurons to P2Y6 receptor activation were analyzed in primary cell culture. UDP in a concentration dependent manner caused membrane depolarization and enhanced numbers of action potentials fired in response to current injections. The excitatory action was antagonized by the P2Y6 receptor antagonist MRS2578, but not by the P2Y2 antagonist AR-C118925XX. UDP raised intracellular Ca2+ in the same range of concentrations as it enhanced excitability and elicited inward currents under conditions that favor Cl- conductances, and these were reduced by a blocker of Ca2+-activated Cl- channels, CaCCInh-A01. In addition, UDP inhibited currents through KV7 channels. The increase in numbers of action potentials caused by UDP was not altered by the KV7 channel blocker linopirdine, but was enhanced in low extracellular Cl- and was reduced by CaCCInh-A01 and by an inhibitor of phospholipase C. Moreover, UDP enhanced release of previously incorporated [3H] noradrenaline, and this was augmented in low extracellular Cl- and by linopirdine, but attenuated by CaCCInh-A01. Together, these results reveal sympathoexcitatory actions of P2Y6 receptor activation involving Ca2+-activated Cl- channels.

The microglial P2Y6 receptor mediates neuronal loss and memory deficits in neurodegeneration.

Microglia are implicated in neurodegeneration, potentially by phagocytosing neurons, but it is unclear how to block the detrimental effects of microglia while preserving their beneficial roles. The microglial P2Y6 receptor (P2Y6R) - activated by extracellular UDP released by stressed neurons - is required for microglial phagocytosis of neurons. We show here that injection of amyloid beta (Aß) into mouse brain induces microglial phagocytosis of neurons, followed by neuronal and memory loss, and this is all prevented by knockout of P2Y6R. In a chronic tau model of neurodegeneration (P301S TAU mice), P2Y6R knockout prevented TAU-induced neuronal and memory loss. In vitro, P2Y6R knockout blocked microglial phagocytosis of live but not dead targets and reduced tau-, Aß-, and UDP-induced neuronal loss in glial-neuronal cultures. Thus, the P2Y6 receptor appears to mediate Aß- and tau-induced neuronal and memory loss via microglial phagocytosis of neurons, suggesting that blocking this receptor may be beneficial in the treatment of neurodegenerative diseases.

Uridine Diphosphate Promotes Rheumatoid Arthritis Through P2Y6 Activation.

BACKGROUND: Uridine diphosphate (UDP) is an extracellular nucleotide signaling molecule implicated in diverse biological processes via specific activation of pyrimidinergic receptor P2Y, G Protein-Coupled, 6 (P2Y6). There is very little knowledge about the function and mechanism of UDP in rheumatoid arthritis (RA). METHODS: This study used a quasi-targeted liquid chromatography-mass spectrometry (LC-MS) approach to investigate the unique expression of metabolites in RA synovial fluids (SF) (n = 10) with samples from osteoarthritis (OA) as controls (n = 10). RA fibroblast-like synoviocytes (FLSs) were collected from synovial tissues (n = 5) and cultured with UDP or MRS2578, a P2Y6 antagonist, and FLSs from OA were used as controls (n = 5). Rats with collagen-induced arthritis (CIA) were injected with UDP, MRS2578 or both (n = 9 for each group). P2Y6 expression was examined using real-time PCR, Western blotting and immunohistochemistry. Cell proliferation, apoptosis and migration of RA FLSs were measured using CCK-8 assay, real-time cell analysis, flow cytometry, wound healing assay and Transwell assay, respectively. The UDP levels in the culture medium, synovial fluid (n = 36) and peripheral blood (n = 36) of RA and CIA rats were measured using a Transcreener UDP Assay. Levels of proinflammatory cytokines were measured using a flow assay. Interleukin-6 (IL-6) levels were measured using ELISA and flow. RESULTS: LC-MS analysis detected significantly increased UDP levels in RA SF compared with OA SF, and the level was positively correlated with anticyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF)levels in RA. The increased UDP concentration was verified in the blood and synovial fluids of RA patients compared with samples from OA patients and healthy volunteers, respectively. UDP stimulated cell proliferation, migration and IL-6 secretion in RA FLSs and inhibited their apoptosis in culture, and MRS2578 inhibited these effects of UDP. UDP injection accelerated CIA and stimulated IL-6 production rather than other proinflammatory cytokines in the rat model, but simultaneous injection of MRS2578 suppressed these effects and alleviated CIA. P2Y6 expression was increased in RA and CIA synovial tissues. CONCLUSION: These results suggest that UDP is highly expressed in RA and stimulates RA pathogenesis by promoting P2Y6 activities to increase IL-6 production.

Structure-activity relationships of pyrimidine nucleotides containing a 5'-α,ß-methylene diphosphonate at the P2Y6 receptor.

The Gq-coupled P2Y6 receptor (P2Y6R) is a component of the purinergic signaling system and functions in inflammatory, cardiovascular and metabolic processes. UDP, the native P2Y6R agonist and P2Y14R partial agonist, is subject to hydrolysis by ectonucleotidases. Therefore, we have synthesized UDP/CDP analogues containing a stabilizing α,ß-methylene bridge as P2Y6R agonists and identified compatible affinity-enhancing pyrimidine modifications. A distal binding region on the receptor was explored with 4-benzyloxyimino cytidine 5'-diphosphate analogues and their potency determined in a calcium mobilization assay. A 4-trifluoromethyl-benzyloxyimino substituent in 25 provided the highest human P2Y6R potency (MRS4554, 0.57 µM), and a 5-fluoro substitution of the cytosine ring in 28 similarly enhanced potency, with >175- and 39-fold selectivity over human P2Y14R, respectively. However, 3-alkyl (31-33, 37, 38), ß-d-arabinofuranose (39) and 6-aza (40) substitution prevented P2Y6R activation. Thus, we have identified new α,ß-methylene bridged N4-extended CDP analogues as P2Y6R agonists that are highly selective over the P2Y14R.

NTPDase1 Modulates Smooth Muscle Contraction in Mice Bladder by Regulating Nucleotide Receptor Activation Distinctly in Male and Female.

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5'-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y1, and P2Y6 receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1-/-) mice compared to wild type controls. The strongest responses were obtained with uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP), suggesting the involvement of P2Y6 receptors, which was confirmed with P2ry6-/- bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y2 and/or P2Y4, and P2Y12 in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1-/-, and P2ry6-/- female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y6 receptor activation were weaker in female bladders.

P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis.

INTRODUCTION: Clearance of damaged cells and debris is beneficial for the functional recovery after ischemic brain injury. However, the specific phagocytic receptor that mediates microglial phagocytosis after ischemic stroke is unknown. AIM: To investigate whether P2Y6 receptor-mediated microglial phagocytosis is beneficial for the debris clearance and functional recovery after ischemic stroke. RESULTS: The expression of the P2Y6 receptor in microglia increased within 3 days after transient middle cerebral artery occlusion. Inhibition of microglial phagocytosis by the selective inhibitor MRS2578 enlarged the brain atrophy and edema volume after ischemic stroke, subsequently aggravated neurological function as measured by modified neurological severity scores and Grid walking test. MRS2578 treatment had no effect on the expression of IL-1α, IL-1ß, IL-6, IL-10, TNF-α, TGF-ß, and MPO after ischemic stroke. Finally, we found that the expression of myosin light chain kinase decreased after microglial phagocytosis inhibition in the ischemic mouse brain, which suggested that myosin light chain kinase was involved in P2Y6 receptor-mediated phagocytosis. CONCLUSION: Our results indicate that P2Y6 receptor-mediated microglial phagocytosis plays a beneficial role during the acute stage of ischemic stroke, which can be a therapeutic target for ischemic stroke.

P2Y6 and P2X7 Receptor Antagonism Exerts Neuroprotective/ Neuroregenerative Effects in an Animal Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by decreased dopamine bioavailability in the substantia nigra and the striatum. Taking into account that adenosine-5'-triphosphate (ATP) and its metabolites are intensely released in the 6-hydroxydopamine (6-OHDA) animal model of PD, screening of purinergic receptor gene expression was performed. Effects of pharmacological P2Y6 or P2X7 receptor antagonism were studied in preventing or reversing hemiparkinsonian behavior and dopaminergic deficits in this animal model. P2X7 receptor antagonism with Brilliant Blue G (BBG) at a dose of 75 mg/kg re-established the dopaminergic nigrostriatal pathway in rats injured with 6-OHDA. Selective P2Y6 receptor antagonism by MRS2578 prevented dopaminergic neuron death in SH-SY5Y cells in vitro and in vivo in the substantia nigra of rats injured with 6-OHDA. Moreover, in vivo analysis showed that both treatments were accompanied by a reduction of microglial activation in the substantia nigra. Altogether, these data provide evidence that antagonism of P2X7 or P2Y6 receptors results in neuroregenerative or neuroprotective effects, respectively, possibly through modulation of neuroinflammatory responses.

Exacerbated intestinal inflammation in P2Y6 deficient mice is associated with Th17 activation.

Extracellular nucleotides are released as constitutive danger signals by various cell types and activate nucleotide (P2) receptors such as P2Y6 receptor. P2Y6 activation on monocytes induces the secretion of the chemokine CXCL8 which may propagate intestinal inflammation. Also, P2Y6 expression is increased in infiltrating T cells of Crohn's disease patients. As inflammatory bowel disease (IBD) is associated with immune cell recruitment, we hypothesised that P2Y6 would participate to the establishment of inflammation in this disease. To address this, we used P2Y6 deficient (P2ry6--/-) mice in the dextran sodium sulfate (DSS) murine model of IBD. In disagreement with our hypothesis, P2Y6 deficient mice were more susceptible to inflammation induced by DSS than WT mice. DSS treated-P2ry6-/- mice showed increased histological damage and increased neutrophil and macrophage infiltration that correlated with increased mRNA levels of the chemokines KC and MCP-1. DSS treated-P2ry6-/- mice exhibited also higher levels of Th17/Th1 lymphocytes in their colon which correlated with increased levels of IFN-γ and IL-17A in the sera as well as increased mRNA levels of IFN-γ, IL-17A, IL-6, IL-23 and IL-1ß in P2ry6-/- colons. This inflammation was also accompanied by a decreased cell proliferation and goblet cell number. Importantly, injection of anti-IL-17 intraperitoneally partially protected P2ry6-/- mice from DSS-induced colitis. Taken together, in the absence of P2Y6, an exacerbated intestinal inflammation to DSS was observed which correlated with increased recruitment of Th17/Th1 lymphocytes. These data suggest a protective role of P2Y6 expressed on leukocytes in intestinal inflammation.

A promising drug candidate for the treatment of glaucoma based on a P2Y6-receptor agonist.

Extracellular nucleotides can regulate the production/drainage of the aqueous humor via activation of P2 receptors, thus affecting the intraocular pressure (IOP). We evaluated 5-OMe-UDP(α-B), 1A, a potent P2Y6-receptor agonist, for reducing IOP and treating glaucoma. Cell viability in the presence of 1A was measured using [3-(4, 5-dimethyl-thiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide] (MTT) assay in rabbit NPE ciliary non-pigmented and corneal epithelial cells, human retinoblastoma, and liver Huh7 cells. The effect of 1A on IOP was determined in acute glaucomatous rabbit hyaluronate model and phenol-induced chronic glaucomatous rabbit model. The origin of activity of 1A was investigated by generation of a homology model of hP2Y6-R and docking studies. 1A did not exert cytotoxic effects up to 100 mM vs. trusopt and timolol in MTT assay in ocular and liver cells. In normotensive rabbits, 100 µM 1A vs. xalatan, trusopt, and pilocarpine reduced IOP by 45 vs. 20-30%, respectively. In the phenol animal model, 1A (100 µM) showed reduction of IOP by 40 and 20%, following early and late administration, respectively. Docking results suggest that the high activity and selectivity of 1A is due to intramolecular interaction between Pα-BH3 and C5-OMe which positions 1A in a most favorable site inside the receptor. P2Y6-receptor agonist 1A effectively and safely reduces IOP in normotense, acute, and chronic glaucomatous rabbits, and hence may be suggested as a novel approach for the treatment of glaucoma.

P2Y6 receptor activation is involved in the development of neuropathic pain induced by chronic constriction injury of the sciatic nerve in rats.

Purinergic signaling in spinal cord microglia plays an important role in the pathogenesis of neuropathic pain. Among all P2 receptors, P2Y6 receptor is expressed in rat dorsal spinal cord. However, it's not clear that the role of P2Y6 receptor in the chronic constriction injury (CCI) model of neuropathic pain rats. We evaluated the effect of repeated intrathecal administration of MRS2578 (selective P2Y6 receptor antagonist) on CCI-induced nociceptive behaviors in rats. After CCI, MRS2578 (10-11-10-4 M) was administration. The thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were assessed. The expression of P2Y6 receptor and Iba-1 at rat dorsal spinal cord was observed by using RT-PCR. We found that intrathecal injection of MRS2578 suppressed CCI-induced mechanical allodynia and thermal hyperalgesia with a dose-dependent manner. The CCI rats presented increased expression of P2Y6 receptor and Iba-1 at the mRNA level in the ipsilateral dorsal spinal cord than that in sham group. Treatment with either minocycline or SB203580 effectively inhibited P2Y6 receptor expression compared to CCI rats. Intrathecal injection of UDP enhanced mechanical and thermal allodynia than that in CCI group. To the further study, intrathecal injection of UDP causes mechanical allodynia and thermal hyperalgesia in naive rats. The increased expression of P2Y6 receptor and Iba-1 were observed in UDP-treated rats. Intrathecal injection of MRS2578 alleviates pain response in UDP-treated rats. These observations suggested that P2Y6 receptor in dorsal spinal cord contribute to mechanical allodynia and thermal hyperalgesia in CCI-induced neuropathic pain.