The diversity and clinical implications of genetic variants influencing clopidogrel bioactivation and response in the Emirati population.

BACKGROUND: Clopidogrel is a widely prescribed prodrug that requires activation via specific pharmacogenes to exert its anti-platelet function. Genetic variations in the genes encoding its transporter, metabolizing enzymes, and target receptor lead to variability in its activation and platelet inhibition and, consequently, its efficacy. This variability increases the risk of secondary cardiovascular events, and therefore, some variations have been utilized as genetic biomarkers when prescribing clopidogrel. METHODS: Our study examined clopidogrel-related genes (CYP2C19, ABCB1, PON1, and P2Y12R) in a cohort of 298 healthy Emiratis individuals. The study used whole exome sequencing (WES) data to comprehensively analyze pertinent variations of these genes, including their minor allele frequencies, haplotype distribution, and their resulting phenotypes. RESULTS: Our data shows that approximately 37% (n = 119) of the cohort are likely to benefit from the use of alternative anti-platelet drugs due to their classification as intermediate or poor CYP2C19 metabolizers. Additionally, more than 50% of the studied cohort exhibited variants in ABCB1, PON1, and P2YR12 genes, potentially influencing clopidogrel's transport, enzymatic clearance, and receptor performance. CONCLUSIONS: Recognizing these alleles and genotype frequencies may explain the clinical differences in medication response across different ethnicities and predict adverse events. Our findings underscore the need to consider genetic variations in prescribing clopidogrel, with potential implications for implementing personalized anti-platelet therapy among Emiratis based on their genetic profiles.

Electroacupuncture Alleviates Pain by Suppressing P2Y12R-Dependent Microglial Activation in Monoarthritic Rats.

Electroacupuncture (EA) effectively improves arthritis-induced hyperalgesia and allodynia by repressing spinal microglial activation, which plays a crucial role in pain hypersensitivity following tissue inflammation. However, the mechanism by which EA suppresses spinal microglial activation in monoarthritis (MA) remains unclear. In the present study, a rat model of MA was established through unilateral ankle intra-articular injection of complete Freund's adjuvant (CFA). The relationship among P2Y12 receptor (P2Y12R) expression, spinal microglial activation, and EA analgesia was investigated using quantitative real-time PCR (qRT‒PCR), western blotting, immunofluorescence (IF), and behavioral testing. The results found that EA treatment at the ipsilateral "Huantiao" (GB30) and "Yanglingquan" (GB34) acupoints markedly attenuated pain and spinal microglia M1 polarization in MA rats. In particular, P2Y12R expression was significantly increased at the mRNA and protein levels in the spinal dorsal horn in MA rats, whereas EA treatment effectively repressed the MA-induced upregulation of P2Y12R. IF analysis further revealed that most P2Y12R was expressed in microglia in the spinal dorsal horn. Pharmacological inhibition of P2Y12R by its antagonist (AR-C69931MX) decreased MA-induced spinal microglial activation and subsequent proinflammatory cytokine production. Consequently, AR-C69931MX significantly intensified the anti-pain hypersensitive function of EA in MA rats. Taken together, these results demonstrate that EA alleviates MA-induced pain by suppressing P2Y12R-dependent microglial activation.

Role and recent progress of P2Y12 receptor in cancer development.

P2Y12 receptor (P2Y12R) is an adenosine-activated G protein-coupled receptor (GPCR) that plays a central role in platelet function, hemostasis, and thrombosis. P2Y12R activation can promote platelet aggregation and adhesion to cancer cells, promote tumor angiogenesis, and affect the tumor immune microenvironment (TIME) and tumor drug resistance, which is conducive to the progression of cancers. Meanwhile, P2Y12R inhibitors can inhibit this effect, suggesting that P2Y12R may be a potential therapeutic target for cancer. P2Y12R is involved in cancer development and metastasis, while P2Y12R inhibitors are effective in inhibiting cancer. However, a new study suggests that long-term use of P2Y12R inhibitors may increase the risk of cancer and the mechanism remains to be explored. In this paper, we reviewed the structural and functional characteristics of P2Y12R and its role in cancer. We explored the role of P2Y12R inhibitors in different tumors and the latest advances by summarizing the basic and clinical studies on the effects of P2Y12R inhibitors on tumors.

Microglial remodeling of actin network by Tau oligomers, via G protein-coupled purinergic receptor, P2Y12R-driven chemotaxis.

Alzheimer's disease (AD) is associated with age-related neurodegeneration, synaptic deformation and chronic inflammation mediated by microglia and infiltrated macrophages in the brain. Tau oligomers can be released from damaged neurons via various mechanisms such as exosomes, neurotransmitter, membrane leakage etc. Microglia sense the extracellular Tau through several cell-surface receptors and mediate chemotaxis and phagocytosis. The purinergic receptor P2Y12R recently gained interest in neurodegeneration for neuro-glial communication and microglial chemotaxis towards the site of plaque deposition. To understand the effect of extracellular Tau oligomers in microglial migration, the P2Y12R-mediated actin remodeling, reorientation of tubulin network and rate of migration were studied in the presence of ATP. The extracellular Tau species directly interacted with P2Y12R and also induced this purinoceptor expression in microglia. Microglial P2Y12R colocalized with remodeled membrane-associated actin network as a component of migration in response to Tau oligomers. As an inducer of P2Y12R, ATP facilitated the localization of P2Y12R in lamellipodia and filopodia during accelerated microglial migration. The direct interaction of extracellular Tau oligomers with microglial P2Y12R would facilitate the signal transduction in both way, directional chemotaxis and receptor-mediated phagocytosis. These unprecedented findings emphasize that microglia can modulate the membrane-associated actin structure and incorporate P2Y12R to perceive the axis and rate of chemotaxis in Tauopathy.

GSDMD knockdown attenuates phagocytic activity of microglia and exacerbates seizure susceptibility in TLE mice.

BACKGROUND: Temporal lobe epilepsy (TLE) is often characterized pathologically by severe neuronal loss in the hippocampus. Phagocytic activity of microglia is essential for clearing apoptotic neuronal debris, allowing for repair and regeneration. Our previous research has shown that gasdermin D (GSDMD)-mediated pyroptosis is involved in the pathogenesis of TLE. However, whether GSDMD-mediated pyroptosis influences the accumulation of apoptotic neurons remains unclear. Therefore, the present study was designed to investigate whether phagocytic activity of microglia is involved in GSDMD-mediated pyroptosis and the pathogenesis of TLE. METHODS: To establish a TLE model, an intra-amygdala injection of kainic acid (KA) was performed. The Racine score and local field potential (LFP) recordings were used to assess seizure severity. Neuronal death in the bilateral hippocampus was assessed by Nissl staining and TUNEL staining. Microglial morphology and phagocytic activity were detected by immunofluorescence and verified by lipopolysaccharide (LPS) and the P2Y12R agonist 2MeSADP. RESULTS: GSDMD knockdown augmented the accumulation of apoptotic neurons and seizure susceptibility in TLE mice. Microglia activated and transition to the M1 type with increased pro-inflammatory cytokines. Furthermore, GSDMD knockdown attenuated the migration and phagocytic activity of microglia. Of note, LPS-activated microglia attenuated seizure susceptibility and the accumulation of apoptotic neurons in TLE after GSDMD knockdown. A P2Y12R selective agonist, 2MeSADP, enhanced the migration and phagocytic activity of microglia. CONCLUSIONS: Our results demonstrate that GSDMD knockdown exacerbates seizure susceptibility and the accumulation of apoptotic neurons by attenuating phagocytic activity of microglia. These findings suggest that GSDMD plays a protective role against KA-induced seizure susceptibility.

Microglial P2Y12 Receptor Regulates Seizure-Induced Neurogenesis and Immature Neuronal Projections.

Seizures are common in humans with various etiologies ranging from congenital aberrations to acute injuries that alter the normal balance of brain excitation and inhibition. A notable consequence of seizures is the induction of aberrant neurogenesis and increased immature neuronal projections. However, regulatory mechanisms governing these features during epilepsy development are not fully understood. Recent studies show that microglia, the brain's resident immune cell, contribute to normal neurogenesis and regulate seizure phenotypes. However, the role of microglia in aberrant neurogenic seizure contexts has not been adequately investigated. To address this question, we coupled the intracerebroventricular kainic acid model with current pharmacogenetic approaches to eliminate microglia in male mice. We show that microglia promote seizure-induced neurogenesis and subsequent seizure-induced immature neuronal projections above and below the pyramidal neurons between the DG and the CA3 regions. Furthermore, we identify microglial P2Y12 receptors (P2Y12R) as a participant in this neurogenic process. Together, our results implicate microglial P2Y12R signaling in epileptogenesis and provide further evidence for targeting microglia in general and microglial P2Y12R in specific to ameliorate proepileptogenic processes.SIGNIFICANCE STATEMENT Epileptogenesis is a process by which the brain develops epilepsy. Several processes have been identified that confer the brain with such epileptic characteristics, including aberrant neurogenesis and increased immature neuronal projections. Understanding the mechanisms that promote such changes is critical in developing therapies to adequately restrain epileptogenesis. We investigated the role of purinergic P2Y12 receptors selectively expressed by microglia, the resident brain immune cells. We report, for the first time, that microglia in general and microglial P2Y12 receptors in specific promote both aberrant neurogenesis and increased immature neuronal projections. These results indicate that microglia enhance epileptogenesis by promoting these processes and suggest that targeting this immune axis could be a novel therapeutic strategy in the clinic.

The synergistic effects of clopidogrel with montelukast may be beneficial for asthma treatment.

Platelets modulate asthma pathogenesis by forming the platelet-eosinophil aggregation (PEA), which facilitates the activation of eosinophils. Platelets exhibit the purinergic receptor (P2Y12R), which responds to cysteinyl leukotriene E4 (LTE4 ). We have suggested that the combination of an antiplatelet drug (clopidogrel, [Clo]) and montelukast (Mon) would synergistically suppress asthma. BALB/c mice were intraperitoneally sensitized with ovalbumin (OVA) on days 0 and 14 and subsequently challenged on days 28-30 and 42-44. Mice were administered with Clo (10 mg/kg), Mon (10 mg/kg) or both drugs (Clo/Mon) orally 30 minutes before the OVA (1%) challenge on days 42-44. Mice were assayed for airway hyper-responsiveness (AHR) to methacholine and airway inflammation. Clopidogrel and montelukast attenuated the increased AHR; the combined treatment was more effective than a single treatment for total and eosinophil counts (all P < 0.05). Levels of interleukin (IL)-4, IL-5, IL-13, platelet factor 4, eosinophil peroxidase and LTE4 increased in the bronchoalveolar lavage fluid of asthmatic mice, but these levels decreased in mice treated with Clo/Mon (all P < 0.05). Goblet cell hyperplasia decreased in response to Clo/Mon. Mouse platelets and eosinophils were isolated and co-cultured for an in vitro assay with 10 µmol/L adenosine diphosphate (ADP), LTE4 (200 nmol/L), Mon (1 µmol/L), Clo (1 µmol/L) and Clo/Mon (1 µmol/L). Flow cytometry revealed that the increased formation of the PEA (%) was fully mediated by ADP and partly mediated by LTE4 . Clo/Mon reduced ADP-induced PEA formation and P-selectin expression (P < 0.05). In conclusion, Clo/Mon synergistically relieved asthma by inhibiting ADP-mediated PEA formation.

Purinergic receptors P2Y12R and P2X7R: potential targets for PET imaging of microglia phenotypes in multiple sclerosis.

BACKGROUND: Microglia are major players in the pathogenesis of multiple sclerosis (MS) and may play a dual role in disease progression. The activation status of microglia in vivo is highly dynamic and occurs as a continuum, with the pro-inflammatory and anti-inflammatory phenotypes on either end of this spectrum. Little is known about in vivo dynamics of microglia phenotypes in MS due to the lack of diagnostic tools. Positron emission tomography (PET) imaging is a powerful non-invasive technique that allows real-time imaging of microglia activation phenotypes in the central nervous system, depending on the availability of selective PET tracers. Our objective is to investigate and characterize the expression of the purinergic receptors P2Y12R and P2X7R as potential targets for PET tracer development and subsequent PET imaging in order to evaluate the dynamics of microglia status in vivo. METHODS: We used immunohistochemical analysis to explore the expression of P2Y12R and P2X7R in experimental autoimmune encephalomyelitis (EAE) post-mortem tissues and different stages of well-characterized MS lesions. We evaluated by quantitative real-time polymerase chain reaction the expression of P2Y12R and P2X7R in human polarized microglia, and we performed autoradiography binding assay with radiolabeled P2Y12R and P2X7R antagonists using MS and rat EAE tissues. RESULTS: Here, we demonstrate that P2X7R is associated with a pro-inflammatory phenotype of human microglia in vitro, and is highly expressed in microglia in MS lesions as well as during the peak of EAE. In contrast, P2Y12R was associated with an anti-inflammatory phenotype in human microglia in vitro and was expressed at lower levels in active inflammatory MS lesions compared to normal-appearing white matter (NAWM) and similarly in EAE, while its expression increased in the remission phase of EAE. Binding of radiolabeled tracers specific for P2Y12R and P2X7R on ex vivo tissues validated the value of these receptors as PET imaging targets for microglia phenotypes in vivo. CONCLUSION: Our results suggest that P2Y12R and P2X7R are excellent targets for PET imaging to discriminate distinct microglia phenotypes in MS. Ultimately, this may provide insight into the role of microglia in disease progression and monitor novel treatment strategies to alter microglia phenotype.

Trimethylamine-N-oxide aggravated the sympathetic excitation in D-galactose induced aging rats by down-regulating P2Y12 receptor in microglia.

This study aimed to determine whether trimethylamine N-oxide (TMAO) was involved in sympathetic activation in aging and the underlying mechanisms. Our hypothesis is TMAO reduces P2Y12 receptor (P2Y12R) and induces microglia-mediated inflammation in the paraventricular nucleus (PVN), then leading to sympathetic activation in aging. This study involved 18 young adults and 16 old adults. Aging rats were established by injecting D-galactose (D-gal, 200 mg/kg/d) subcutaneously for 12 weeks. TMAO (120 mg/kg/d) or 1% 3, 3-dimethyl-l-butanol (DMB) was administrated via drinking water for 12 weeks to investigate their effects on neuroinflammation and sympathetic activation in aging rats. Plasma TMAO, NE and IL-1ß levels were higher in old adults than in young adults. In addition, standard deviation of all normal to normal intervals (SDNN) and standard deviation of the average of normal to normal intervals (SDANN) were lower in old adults and negatively correlated with TMAO, indicating sympathetic activation in old adults, which is associated with an increase in TMAO levels. Treatment of rats with D-gal showed increased senescence-associated protein levels and microglia-mediated inflammation, as well as decreased P2Y12R protein levels in PVN. Plasma TMAO, NE and IL-1ß levels were increased, accompanied by enhanced renal sympathetic nerve activity (RSNA). While TMAO treatment exacerbated the above phenomenon, DMB mitigated it. These findings suggest that TMAO contributes to sympathetic hyperactivity in aging by downregulating P2Y12R in microglia and increasing inflammation in the PVN. These results may provide promising new target for the prevention and treatment of aging and aging-related diseases.

The role and pharmacological properties of P2Y12 receptor in cancer and cancer pain.

The G protein-coupled P2Y12 receptor (P2Y12R) was cloned in platelets and found to play a key role in maintaining platelet function in hemostasis and thrombosis, and these effects could be mediated by the P2Y12R. However, it has recently been found that P2Y12R-mediated the progression of tumor through interactions between platelets and tumor and stromal cells, as well as through products secreted by platelets. During tumor progression, tumor cells or other cells in the tumor microenvironment (such as immune cells) can secrete large amounts of ATP into the extracellular matrix, and extracellular ATP can be hydrolyzed into ADP. ADP is a P2Y12R activator and plays an important regulatory role in the proliferation and metastasis of tumor cells. P2Y12R is involved in platelet-cancer cell crosstalk and become a potential target for anticancer therapy. Moreover, tumor progression can induce pain, which seriously affects the quality of life of patients. P2Y12R is expressed in microglia and mediates the activities of microglial and participates in the occurrence of cancer pain. Conversely, inhibiting P2Y12R activation and down-regulating its expression has the effect of inhibiting tumor progression and pain. Therefore, P2Y12R can be a common therapeutic target for both. In this article, we explored the potential link between P2Y12R and cancer, discussed the intrinsic link of P2Y12R in cancer pain and the pharmacological properties of P2Y12R antagonists in the treatment of both.

Tau aggregates improve the Purinergic receptor P2Y12-associated podosome rearrangements in microglial cells.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is associated with protein misfolding, plaque accumulation, neuronal dysfunction, synaptic loss, and cognitive decline. The pathological cascade of AD includes the intracellular Tau hyperphosphorylation and its subsequent aggregation, extracellular Amyloid-ß plaque formation and microglia-mediated neuroinflammation. The extracellular release of aggregated Tau is sensed by surveilling microglia through the involvement of various cell surface receptors. Among all, purinergic P2Y12R signaling is involved in microglial chemotaxis towards the damaged neurons. Microglial migration is highly linked with membrane-associated actin remodeling leading to the phagocytosis of extracellular Tau species. Here, we studied the formation of various actin structures such as podosome, lamellipodia and filopodia, in response to extracellular Tau monomers and aggregates. Microglial podosomes are colocalized with actin nucleator protein WASP, Arp2 and TKS5 adaptor protein during Tau-mediated migration. Moreover, the P2Y12 receptors were associated with F-actin-rich podosome structures, which signify the potential of Tau aggregates in microglial chemotaxis through the involvement of actin remodeling.

Microglia modulate general anesthesia through P2Y12 receptor.

General anesthesia (GA) is an unconscious state produced by anesthetic drugs, which act on neurons to cause overall suppression of neuronal activity in the brain. Recent studies have revealed that GA also substantially enhances the dynamics of microglia, the primary brain immune cells, with increased process motility and territory surveillance. However, whether microglia are actively involved in GA modulation remains unknown. Here, we report a previously unrecognized role for microglia engaging in multiple GA processes. We found that microglial ablation reduced the sensitivity of mice to anesthetics and substantially shortened duration of loss of righting reflex (LORR) or unconsciousness induced by multiple anesthetics, thereby promoting earlier emergence from GA. Microglial repopulation restored the regular anesthetic recovery, and chemogenetic activation of microglia prolonged the duration of LORR. In addition, anesthesia-accompanying analgesia and hypothermia were also attenuated after microglial depletion. Single-cell RNA sequencing analyses showed that anesthesia prominently affected the transcriptional levels of chemotaxis and migration-related genes in microglia. By pharmacologically targeting different microglial motility pathways, we found that blocking P2Y12 receptor (P2Y12R) reduced the duration of LORR of mice. Moreover, genetic ablation of P2Y12R in microglia also promoted quicker recovery in mice from anesthesia, verifying the importance of microglial P2Y12R in anesthetic regulation. Our work presents the first evidence that microglia actively participate in multiple processes of GA through P2Y12R-mediated signaling and expands the non-immune roles of microglia in the brain.

Radiosynthesis and initial preclinical evaluation of [11C]AZD1283 as a potential P2Y12R PET radiotracer.

INTRO: Chronic neuroinflammation and microglial dysfunction are key features of many neurological diseases, including Alzheimer's Disease and multiple sclerosis. While there is unfortunately a dearth of highly selective molecular imaging biomarkers/probes for studying microglia in vivo, P2Y12R has emerged as an attractive candidate PET biomarker being explored for this purpose. Importantly, P2Y12R is selectively expressed on microglia in the CNS and undergoes dynamic changes in expression according to inflammatory context (e.g., toxic versus beneficial/healing states), thus having the potential to reveal functional information about microglia in living subjects. Herein, we identified a high affinity, small molecule P2Y12R antagonist (AZD1283) to radiolabel and assess as a candidate radiotracer through in vitro assays and in vivo positron emission tomography (PET) imaging of both wild-type and total knockout mice and a non-human primate. METHODS: First, we evaluated the metabolic stability and passive permeability of non-radioactive AZD1283 in vitro. Next, we radiolabeled [11C]AZD1283 with radioactive precursor [11C]NH4CN and determined stability in formulation and human plasma. Finally, we investigated the in vivo stability and kinetics of [11C]AZD1283 via dynamic PET imaging of naïve wild-type mice, P2Y12R knockout mouse, and a rhesus macaque. RESULTS: We determined the half-life of AZD1283 in mouse and human liver microsomes to be 37 and > 160 min, respectively, and predicted passive CNS uptake with a small amount of active efflux, using a Caco-2 assay. Our radiolabeling efforts afforded [11C]AZD1283 in an activity of 12.69 ± 10.64 mCi with high chemical and radiochemical purity (>99%) and molar activity of 1142.84 ± 504.73 mCi/µmol (average of n = 3). Of note, we found [11C]AZD1283 to be highly stable in vitro, with >99% intact tracer present after 90 min of incubation in formulation and 60 min of incubation in human serum. PET imaging revealed negligible brain signal in healthy wild-type mice (n = 3) and a P2Y12 knockout mouse (0.55 ± 0.37%ID/g at 5 min post injection). Strikingly, high signal was detected in the liver of all mice within the first 20 min of administration (peak uptake = 58.28 ± 18.75%ID/g at 5 min post injection) and persisted for the remaining duration of the scan. Ex vivo gamma counting of mouse tissues at 60 min post-injection mirrored in vivo data with a mean %ID/g of 0.9% ± 0.40, 0.02% ± 0.01, and 106 ± 29.70% in the blood, brain, and liver, respectively (n = 4). High performance liquid chromatography (HPLC) analysis of murine blood and liver metabolite samples revealed a single radioactive peak (relative area under peak: 100%), representing intact tracer. Finally, PET imaging of a rhesus macaque also revealed negligible CNS uptake/binding in monkey brain (peak uptake = 0.37 Standard Uptake Values (SUV)). CONCLUSION: Despite our initial encouraging liver microsome and Caco-2 monolayer data, in addition to the observed high stability of [11C]AZD1283 in formulation and human serum, in vivo brain uptake was negligible and rapid accumulation was observed in the liver of both naïve wildtype and P2Y12R knockout mice. Liver signal appeared to be independent of both metabolism and P2Y12R expression due to the confirmation of intact tracer in this tissue for both wildtype and P2Y12R knockout mice. In Rhesus Macaque, negligible uptake of [11C]AZD1283 brain indicates a lack of potential for translation or its further investigation in vivo. P2Y12R is an extremely promising potential PET biomarker, and the data presented here suggests encouraging metabolic stability for this scaffold; however, the mechanism of liver uptake in mice should be elucidated prior to further analogue development.

Microglial control of neuronal development via somatic purinergic junctions.

Microglia, the resident immune cells of the brain, play important roles during development. Although bi-directional communication between microglia and neuronal progenitors or immature neurons has been demonstrated, the main sites of interaction and the underlying mechanisms remain elusive. By using advanced methods, here we provide evidence that microglial processes form specialized contacts with the cell bodies of developing neurons throughout embryonic, early postnatal, and adult neurogenesis. These early developmental contacts are highly reminiscent of somatic purinergic junctions that are instrumental for microglia-neuron communication in the adult brain. The formation and maintenance of these junctions is regulated by functional microglial P2Y12 receptors, and deletion of P2Y12Rs disturbs proliferation of neuronal precursors and leads to aberrant cortical cytoarchitecture during development and in adulthood. We propose that early developmental formation of somatic purinergic junctions represents an important interface for microglia to monitor the status of immature neurons and control neurodevelopment.

G-protein coupled purinergic P2Y12 receptor interacts and internalizes TauRD-mediated by membrane-associated actin cytoskeleton remodeling in microglia.

In Alzheimer's disease, the microtubule-associated protein, Tau misfolds to form aggregates and filaments in the intra- and extracellular region of neuronal cells. Microglial cells are the resident brain macrophage cells involved in constant surveillance and activated by the extracellular deposits. Purinergic receptors are involved in the chemotactic migration of microglial cells towards the site of inflammation. From our recent study, we have observed that the microglial P2Y12 receptor is involved in phagocytosis of full-length Tau species such as monomers, oligomers and aggregates by actin-driven chemotaxis. This study shows the interaction of repeat-domain of Tau (TauRD) with the microglial P2Y12 receptor and the corresponding residues for interaction have been analysed by various in-silico approaches. In the cellular studies, TauRD was found to interact with microglial P2Y12R and induces its cellular expression confirmed by co-immunoprecipitation and western blot analysis. Furthermore, the P2Y12R-mediated TauRD internalization has demonstrated activation of microglia with an increase in the Iba1 level, and TauRD becomes accumulated at the peri-nuclear region for the degradation. Similarly, immunofluorescence microscopic studies emphasized that TauRD is phagocytosed by microglial P2Y12R via the membrane-associated actin remodeling as filopodia extension. Upon internalization, we have demonstrated the P2Y12R signaling-mediated degradation of accumulated TauRD by lysosomal pathway. Altogether, microglial P2Y12R interacts with TauRD and mediates directed migration and activation for its internalization and degradation.

Novel Thienopyrimidine-Based PET Tracers for P2Y12 Receptor Imaging in the Brain.

The P2Y12 receptor (P2Y12R) is uniquely expressed on microglia in the brain, and its expression level directly depends on the microglial activation state. Therefore, P2Y12R provides a promising imaging marker for distinguishing the pro- and anti-inflammatory microglial phenotypes, both of which play crucial roles in neuroinflammatory diseases. In this study, three P2Y12R antagonists were selected from the literature, radiolabeled with carbon-11 or fluorine-18, and evaluated in healthy Wistar rats. Brain imaging was performed with and without blocking of efflux transporters P-glycoprotein and breast cancer resistance protein using tariquidar. Low brain uptake in healthy rats was observed for all tracers at baseline conditions, whereas blocking of efflux transporters resulted in a strong (6-7 fold) increase in brain uptake for both of them. Binding of the most promising tracer, [18F]3, was further evaluated by in vitro autoradiography on rat brain sections, ex vivo metabolite studies, and in vivo P2Y12R blocking studies. In vitro binding of [18F]3 on rat brain sections indicated high P2Y12R targeting with approximately 70% selective and specific binding. At 60 min post-injection, over 95% of radioactivity in the brain accounted for an intact tracer. In blood plasma, still 40% intact tracer was found, and formed metabolites did not enter the brain. A moderate P2Y12R blocking effect was observed in vivo by positron emission tomography (PET) imaging with [18F]3 (p = 0.04). To conclude, three potential P2Y12R PET tracers were obtained and analyzed for P2Y12R targeting in the brain. Unfortunately, the brain uptake appeared low. Future work will focus on the design of P2Y12R inhibitors with improved physicochemical characteristics to reduce efflux transport and increase brain penetration.

Microglia in the RVLM of SHR have reduced P2Y12R and CX3CR1 expression, shorter processes, and lower cell density.

The RVLM of spontaneously hypertensive rats (SHR) contains over-active C1 neurons, which model the pathology of essential hypertension. Hypertension involves chronic low-grade neuroinflammation. Inflammation in the brain is produced and maintained primarily by microglia. We assessed microglial gene expression (P2Y12R and CX3CR1) and morphology in the RVLM of SHR compared to normotensive Wistar-Kyoto rats (WKY). The gene expression of the metabotropic purinergic receptor P2Y12 and the fractalkine receptor CX3CR1 was downregulated in the RVLM of SHR compared to WKY (by 37.3% and 30.9% respectively). P2Y12R and CX3CR1 are required for normal microglial function, and reduced P2Y12R expression is associated with changes in microglial activity. Histological analysis showed a 22.9% reduction in microglial cell density, along with 18.7% shorter microglial processes, a phenotypic indicator of activation, in the RVLM of SHR compared to WKY. These results indicate a subtle loss of function, or a mild state of inflammation, in the RVLM microglia of SHR.

Associations of P2Y12R gene polymorphisms with susceptibility to coronary heart disease and clinical efficacy of antiplatelet treatment with clopidogrel.

OBJECTIVE: To investigate the correlations of three P2Y12 receptor (P2Y12R) gene polymorphisms (rs7428575 T>G, rs2046934 C>T, and rs3732759 A>G) with susceptibility to coronary artery disease (CHD) and clinical efficacy of clopidogrel treatment for CHD. METHODS: From May 2014 to May 2015, 178 CHD patients (the case group) and 182 healthy controls (the control group) were selected from our hospital. The platelet-rich plasma (PRP) turbidimetry was used to measure the rate of adenosine diphosphate (ADP)-induced platelet aggregation before and after clopidogrel treatment. Clopidogrel-sensitive group was defined as a 10% or greater decrease in the rate of platelet aggregation after 10 days of clopidogrel treatment, while clopidogrel-resistant group was defined as a <10% decrease. Genotyping was performed by denaturing high-performance liquid chromatography (DHPLC). A haplotype analysis of P2Y12R gene polymorphisms was performed using SHEsis software. RESULTS: There were significant differences in genotype and allele frequencies of rs2046934 C>T and rs3732759 A>G between the case and control groups (all P<.05). Haplotypes GTA and TTA were negatively associated with CHD risk (both P<.05), but haplotype TCA was positively associated with CHD risk (P=.005). CHD patients in the clopidogrel-sensitive group had higher frequencies of TT genotype of rs2046934 C>T and lower frequencies of GG genotype of rs3732759 A>G than those in the clopidogrel-resistant group (both P<.05). CONCLUSIONS: P2Y12R gene rs2046934 C>T and rs3732759 A>G polymorphisms might be associated with the risk of CHD and the efficacy of clopidogrel treatment for CHD.