T-cell Death-associated Gene 8 Research Articles

Targeting astrocytic TDAG8 with delayed CO2 postconditioning improves functional outcomes after controlled cortical impact injury in mice

T-cell death-associated gene 8 (TDAG8), a G-protein-coupled receptor sensing physiological or weak acids, regulates inflammatory responses. However, its role in traumatic brain injury (TBI) remains unknown. Our recent study showed that delayed CO2 postconditioning (DCPC) has neuroreparative effects after TBI. We hypothesized that activating astrocytic TDAG8 is a key mechanism for DCPC. WT and TDAG8−/− mice received DCPC daily by transiently inhaling 10% CO2 after controlled cortical impact (CCI). HBAAV2/9-GFAP-m-TDAG8-3xflag-EGFP was used to overexpress TDAG8 in astrocytes. The beam walking test, mNSS, immunofluorescence and Golgi-Cox staining were used to evaluate motor function, glial activation and dendritic plasticity. DCPC significantly improved motor function; increased total dendritic length, neuronal complexity and spine density; inhibited overactivation of astrocytes and microglia; and promoted the expression of astrocytic brain-derived neurotrophic factor in WT but not TDAG8−/− mice. Overexpressing TDAG8 in astrocytes surrounding the lesion in TDAG8−/− mice restored the beneficial effects of DCPC. Although the effects of DCPC on Days 14–28 were much weaker than those of DCPC on Days 3–28 in WT mice, these effects were further enhanced by overexpressing astrocytic TDAG8. Astrocytic TDAG8 is a key target of DCPC for TBI rehabilitation. Its overexpression is a strategy that broadens the therapeutic window and enhances the effects of DCPC.

A molecular characterization and clinical relevance of microglia-like cells derived from patients with panic disorder

Few studies report the microglia involvement in the pathogenesis of panic disorder (PD), although the crucial role of microglia in other neuropsychiatric diseases is being emphasized. In addition, there is no report to characterize the phenotypic and functional levels of PD patient-derived microglia to find their clinical relevance. Herein, we used a model to induce patient-derived microglia-like cells (iMGs) to clarify the molecular characteristics and function of PD-iMGs. We established iMGs from 17 PD patients and 16 healthy controls (non-psychiatric controls, HC). PD-iMGs showed increased T-cell death-associated gene-8 expression per the proposal of a previous in vivo study. In addition, we found that patient-derived iMGs showed reduced phagocytosis and increased TREM2 expression. We analyzed the phenotype of the PD-iMGs by RNA sequencing. The PD-iMGs clustered together distinct from HC-iMGs. Gene set enrichment analysis revealed the involvement of cholesterol biosynthesis and steroid metabolism in PD-iMGs. Regarding the cholesterol synthesis pathway, we discovered ACAT2 and DHCR7 as the most impacted genes related to a character of PD-iMGs compared to HC-iMGs. The ACAT2, a major cholesterol esterifier, was increased in PD-iMGs. Nevertheless, PD-iMGs did not show lipid droplet accumulation. Interestingly, ACAT2 expression was inversely correlated with the severity of depression and anxiety sensitivity to publicly observable anxiety reactions. We propose that microglia of PD patients have unique characteristics with dysregulation of cholesterol biosynthesis pathway and impaired phagocytosis, reflecting clinical phenotype.

MicroRNAs combined with the TLR4/TDAG8 mRNAs and proinflammatory cytokines are biomarkers for the rapid diagnosis of sepsis.

The early diagnosis and treatment of sepsis are of particular importance to patient survival. To obtain novel biomarkers that serve as prompt indicators of sepsis, the current study screened the differentially expressed microRNAs (DEMs) that were associated with sepsis susceptibility. The correlation between the elucidated DEMs and the inflammatory response was also examined. The present study included 40 patients with sepsis and 40 healthy controls. RNA-sequencing technology and bioinformatics analysis were applied to screen the DEMs between the two cohorts. The expression of these DEMs was subsequently verified by performing reverse transcription-quantitative PCR (RT-qPCR). In addition, IL-6, IL-21, C-X-C motif chemokine ligand-8 (CXCL8) and monocyte chemoattractant protein-1 (MCP-1) levels, along with T-cell death-associated gene 8 (TDAG8) and toll-like receptor 4 (TLR4) mRNA expression levels were assessed. The association between microRNA (miRNA/miR)-3663-3p and the secretion of various proinflammatory cytokines or TDAG8 and TLR4 mRNA expressions were subsequently evaluated by linear correlation analysis. The results revealed 305 DEMs (P<0.05; fold change >2) between patients with sepsis and healthy controls. Among these, the top 18 up- and downregulated miRNAs were selected for RT-qPCR verification. In addition, the serum content of IL-6, IL-21, CXCL8 and MCP-1, and the expression of TDAG8 and TLR4 mRNAs were significantly increased in patients with sepsis compared with healthy controls. Moreover, in patients with sepsis, a positive correlation was identified between miR-3663-3p and the secretion of inflammatory cytokines or TDAG8 and TLR4 mRNA expression. A positive correlation was also elucidated between TDAG8 and TLR4 mRNA expression and proinflammatory cytokine/chemokine secretion. Receiver operating characteristic curve analysis of miR-3663-3p expression, IL-6, IL-21, CXCL8 and MCP-1 secretion and TDAG8 and TLR4 mRNA expression demonstrated that miRNA analysis may be invaluable for the diagnosis of sepsis. Collectively, the results determined that miR-3663-3p may be a potentially powerful diagnostic and predictive biomarker of sepsis and that the combined and simultaneous detection of several biomarkers, including proteins, miRNAs and mRNA may be a reliable approach for the fast diagnosis and early identification of sepsis.

Ethylenediaminetetraacetic acid (EDTA) enhances cAMP production in human TDAG8-expressing cells

Ethylenediaminetetraacetic acid (EDTA) is a chelating agent that binds tightly to metal ions. We found that cAMP response element (CRE)-driven promoter activity by protons was enhanced by EDTA in human T-cell death-associated gene 8 (TDAG8)-overexpressed HEK293T cells. The enhancing action by EDTA was also detected by proton-induced cAMP production that is located upstream from the CRE-driven promoter activity even at physiological proton concentration pH7.4. The proton-induced CRE-driven promoter activity was not enhanced by other chelating agents, ethylene glycol tetraacetic acid (EGTA) and sodium citrate. The enhanced CRE-driven promoter activity by EDTA was not attenuated by increasing the extracellular calcium ion concentration. These results indicate that the EDTA-enhancing action may not be due to its chelating action but might rather be another EDTA-specific effect. Enhanced cAMP production by EDTA was also detected in a human leukemia cell line HL-60, in which TDAG8 and OGR1 (ovarian cancer G-protein-coupled receptor 1) were endogenously expressed, suggesting that the medical use of EDTA would influence the physiological and pathophysiological functions of hematopoietic cells.

Gut Mucosal Microbiome Is Perturbed in Rheumatoid Arthritis Mice and Partly Restored after TDAG8 Deficiency or Suppression by Salicylanilide Derivative.

Rheumatoid arthritis (RA), an autoimmune disease, is characterized by chronic joint inflammation and pain. We previously found that the deletion of T-cell death-associated gene 8 (TDAG8) significantly reduces disease severity and pain in RA mice. Whether it is by modulating gut microbiota remains unclear. In this study, 64 intestinal samples of feces, cecal content, and cecal mucus from the complete Freund’s adjuvant-induced arthritis mouse models were compared. The α- and β-diversity indices of the microbiome were significantly lower in RA mice. Cecal mucus showed a higher ratio of Firmicutes to Bacteroidetes in RA than healthy mice, suggesting the ratio could serve as an RA indicator. Four core genera, Eubacterium_Ventriosum, Alloprevotella, Rikenella, and Treponema, were reduced in content in both feces and mucus RA samples, and could serve microbial markers representing RA progression. TDAG8 deficiency decreased the abundance of proinflammation-related Eubacterium_Xylanophilum, Clostridia, Ruminococcus, Paraprevotella, and Rikenellaceae, which reduced local mucosal inflammation to relieve RA disease severity and pain. The pharmacological block of the TDAG8 function by a salicylanilide derivative partly restored the RA microbiome to a healthy composition. These findings provide a further understanding of specific bacteria interactions with host gut mucus in the RA model. The modulation by TDAG8 on particular bacteria can facilitate microbiota-based therapy.

Persistent Acidic Environment Induces Impaired Phagocytosis via ERK in Microglia.

Acidic environment evoked by stroke, traumatic brain injury, and Alzheimer's disease may change the functional properties of microglia. Nevertheless, the underlying mechanisms of functional changes in microglia remain unclear. In this study, we found that acidic stimuli (pH 6.8) increased rapidly interleukin (IL)-1β and IL-6 mRNA levels and subsequently reduced IL-10, transforming growth factor (TGF)-β1, Cx3cr1, and P2ry12 as the exposure time to acidic environment increase in BV2 cells. In addition, persistent acidic environment (pH 6.8 for 6h) induced impaired phagocytic function in BV2 cells. Short-term acidic exposure (pH 6.8 for 30min) increased cyclic AMP (cAMP) and phospho-protein kinase A (PKA) but inhibited phospho-extracellular signal-regulated kinase (p-ERK). However, under persistent acidic environment (pH 6.8 for 6h), cyclic AMP and PKA were normalized and p-ERK was increased with TDAG8 (T cell death associated gene 8; GPR65) reduction. FR 180,204, an ERK inhibitor, rescued the persistent acidic environment-induced functional changes in BV2 cells and its effect was recapitulated in primary neonatal microglia. Thus, we propose that ERK targeting may be an alternative strategy to restore microglial dysfunction in thecentral nervous system (CNS) acidic environment in various neurological disorders.

Extracellular acidity in tumor tissue upregulates programmed cell death protein1 expression on tumor cells via proton-sensing G protein-coupled receptors.

Acidity in the tumor microenvironment has been reported to promote cancer growth and metastasis. In our study, we examined a potential relation between extracellular acidity and expression level of the immune checkpoint molecule programmed cell death protein1 (PD-L1) in murine squamous cell carcinoma (SCC) and melanoma cell lines. PD-L1 expression in the tumor cells was upregulated by culturing in a low pH culture medium. Tumor-bearing mice were allowed to ingest sodium bicarbonate, resulting in neutralization of acidity in the tumor tissue, a decrease in PD-L1 expression in tumor cells and suppression of tumor growth in vivo. Proton-sensing G protein-coupled receptors, T-cell death-associated gene 8 (TDAG8) and ovarian cancer G-protein-coupled receptor 1 (OGR1), were upregulated by low pH, and essentially involved in the acidity-induced elevation of PD-L1 expression in the tumor cells. Human head and neck SCC RNAseq data from the Cancer Genome Atlas also suggested a statistically significant correlation between expression levels of the proton sensors and PD-L1 mRNA expression. These findings strongly suggest that neutralization of acidity in tumor tissue may result in reduction of PD-L1 expression, potentially leading to inhibition of an immune checkpoint and augmentation of antitumor immunity.

The protective role of proton-sensing TDAG8 in the brain injury in a mouse ischemia reperfusion model

Extracellular acidification in the brain has been observed in ischemia; however, the physiological and pathophysiological implications of the pH reduction remain largely unknown. Here, we analyzed the roles of proton-sensing G protein-coupled receptors, including T-cell death-associated gene 8 (TDAG8), ovarian cancer G protein-coupled receptor 1 (OGR1), and G protein-coupled receptor 4 (GPR4) in a mouse ischemia reperfusion model. Cerebral infarction and dysfunctional behavior with transient middle cerebral artery occlusion (tMCAO) and subsequent reperfusion were exacerbated by the deficiency of TDAG8, whereas no significant effect was observed with the deficiency of OGR1 or GPR4. We confirmed that the pH of the predicted infarction region was 6.5. TDAG8 mRNA was observed in Iba1-positive microglia in the mouse brain. The tMCAO increased the mRNA expression of tumor necrosis factor-α in the ipsilateral cerebral hemisphere and evoked morphological changes in microglia in an evolving cerebral injury. These tMCAO-induced actions were significantly enhanced by the TDAG8 deficiency. Administration of minocycline, which is known to inhibit microglial activation, improved the cerebral infarction and dysfunctional behavior induced by tMCAO in the TDAG8-deficient mouse. Thus, acidic pH/TDAG8 protects against cerebral infarction caused by tMCAO, at least due to the mechanism involving the inhibition of microglial functions.

Expression profiles of proton-sensing G-protein coupled receptors in common skin tumors

The proton-sensing GPCRs (pH-GPCRs) GPR4 (GPR19), TDAG8 (GPR65, T-cell death associated gene 8), OGR1 (GPR68, ovarian cancer GPCR1), and G2A (GPR132, G2 accumulation protein) are involved in sensing and transducing changes in extracellular pH (pHe). Extracellular acidification is a central hallmark of solid cancer. pH-GPCR function has been associated with cancer cell proliferation, adhesion, migration and metastasis, as well as with modulation of the immune system. Little is known about the expression levels and role of pH-GPCRs in skin cancer. To better understand the functions of pH-GPCRs in skin cancer in vivo, we examined the expression-profiles of GPR4, TDAG8, OGR1 and G2A in four common skin tumors, i.e. squamous cell carcinoma (SCC), malignant melanoma (MM), compound nevus cell nevi (NCN), basal cell carcinoma (BCC). We performed immunohistochemistry and immunofluorescence staining on paraffin-embedded tissue samples acquired from patients suffering from SCC, MM, NCN or BCC. We show the expression of pH-GPCRs in four common skin cancers. Different expression patterns in the investigated skin cancer types indicate that the different pH-GPCRs may have distinct functions in tumor progression and serve as novel therapeutic targets.

TDAG8 deficiency reduces satellite glial number and pro-inflammatory macrophage number to relieve rheumatoid arthritis disease severity and chronic pain

BackgroundThe autoimmune disease rheumatoid arthritis (RA) affects approximately 1% of the global population. RA is characterized with chronic joint inflammation and often associated with chronic pain. The imbalance of pro-inflammatory and anti-inflammatory macrophages is a feature of RA progression. Glial cells affecting neuronal sensitivity at both peripheral and central levels may also be important for RA progression and associated pain. Genetic variants in the T cell death-associated gene 8 (TDAG8) locus are found to associate with spondyloarthritis. TDAG8 was also found involved in RA disease progression and associated hyperalgesia in the RA mouse model. However, its modulation in RA remains unclear.MethodsTo address this question, we intra-articularly injected complete Freund’s adjuvant (CFA) into TDAG8+/+, TDAG8−/− or wild-type mice, followed by pain behavioral tests. Joints and dorsal root ganglia were taken, sectioned, and stained with antibodies to observe the number of immune cells, macrophages, and satellite glial cells (SGCs). For compound treatments, compounds were intraperitoneally or orally administered weekly for 9 consecutive weeks after CFA injection.ResultsWe demonstrated that TDAG8 deletion slightly reduced RA pain in the early phase but dramatically attenuated RA progression and pain in the chronic phase (> 7 weeks). TDAG8 deletion inhibited an increase in SGC number and inhibition of SGC function attenuated chronic phase of RA pain, so TDAG8 could regulate SGC number to control chronic pain. TDAG8 deletion also reduced M1 pro-inflammatory macrophage number at 12 weeks, contributing to the attenuation of chronic RA pain. Such results were further confirmed by using salicylanilide derivatives, CCL-2d or LCC-09, to suppress TDAG8 expression and function.ConclusionsThis study demonstrates that TDAG8 deletion reduced SGC and M1 macrophage number to relieve RA disease severity and associated chronic pain. M1 macrophages are critical for the development and maintenance of RA disease and pain, but glial activation is also required for the chronic phase of RA pain.

Temporal expression patterns of distinct cytokines and M1/M2 macrophage polarization regulate rheumatoid arthritis progression.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial joints and often associated with chronic pain. Chronic joint inflammation is attributed to severe proliferation of synoviocytes and resident macrophages and infiltration of immune cells. These cells secrete pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and IL-17 to overcome actions of anti-inflammatory cytokines, thereby maintaining chronic inflammation and pain. The imbalance between pro-inflammatory cytokines (produced by M1 macrophages) and anti-inflammatory cytokines (produced by M2 macrophages) is a feature of RA progression, but the switch time of M1/M2 polarization and which receptor regulates the switch remain unsolved. Here we used an established RA mouse model to demonstrate that TNF-α expression was responsible for the initial acute stage of inflammation and pain (1-4weeks), IL-17 expression the transition stage (4-12weeks), and IL-6 expression the later maintenance stage (> 12weeks). The switch time of M1/M2 polarization occurred at 4-8weeks. We also identified a potential compound, anthra[2,1-c][1,2,5] thiadiazole-6,11-dione (NSC745885), that specifically inhibited T-cell death-associated gene 8 (TDAG8) function and expression. NSC745885 decreased joint inflammation and destruction and attenuated pain by reducing cytokine production and regulating the M1/M2 polarization switch. TDAG8 may participate in regulating the M1/M2 polarization and temporal expression of distinct cytokines to control RA progression.

Immunomodulatory T cell death associated gene-8 (TDAG8) receptor in depression-associated behaviors

Converging evidence supports neuroimmune factors in depression psychopathology. We previously reported reduced depression-like behavior in immunomodulatory G-protein-coupled receptor, T cell death-associated gene-8 (TDAG8) deficient mice. Here, we expand on those findings by investigating depression- and anxiety-associated behaviors, and cytokine profiles in TDAG8-deficient mice. TDAG8-deficiency reduced depression- and anxiety-associated behaviors in the forced swim test (FST), open-field test and elevated zero maze. Interestingly, cytokine expression, particularly IL-6, was attenuated within hippocampus and spleen in TDAG8-deficient mice following the FST. There were no differences in immune-cell frequencies. Collectively, these data suggest a contributory role of TDAG8 in neuroimmune regulation and depression-associated physiology.

Expression of proton-sensing G-protein-coupled receptors in selected skin tumors.

In humans, there are four known proton-sensing G-Protein-coupled receptors (pH-GPCRs): GPR4 (GPR19), TDAG8 (GPR65, T-cell death-associated gene 8), OGR1 (GPR68, ovarian cancer GPCR1) and G2A (GPR132, G2 accumulation protein). They are known to be involved in sensing changes of extracellular proton concentrations in the acidic microenvironment of tumors, which leads to altered cell proliferation, migration, metastasis, immune cell function and inflammation. However, little is known about the expression of pH-GPCRs in the skin and especially skin cancers. We studied the expression of pH-GPCRs in selected skin cancers, that is Merkel cell carcinoma (MCC), dermatofibrosarcoma protuberans (DFSP), atypical fibroxanthoma (AFX) and pleomorphic dermal sarcoma (PDS). We did immunohistochemistry and immunofluorescence to analyse the expression of GPR4, TDAG8, OGR1 and G2A using paraffin-embedded tissue samples (n=4, exceptions: PDS GPR4/GPR65 n=5, AFX GPR132 n=3) from patients suffering from MCC, DFSP, AFX and PDS. (a) GPR4 was expressed on all AFX and PDS specimens. All AFX and MCC showed a positive expression of G2A. All PDS exhibited a strong positive expression of G2A. (b) MCCs neither expressed GPR4 nor TDAG8. All DFSP showed no expression of TDAG8. (c) For any other combination of GPCR and skin disease, we found positive/negative mixed results. These are the first results on pH-GPCRs in selected skin cancers. We provide evidence that these GPCRs are differentially expressed on the various types of skin cancers and that they can potentially be addressed as a therapeutic target in extensive disease.

Contextual tumor suppressor function of T cell death-associated gene 8 (TDAG8) in hematological malignancies

BackgroundExtracellular acidosis is a condition found within the tumor microenvironment due to inadequate blood perfusion, hypoxia, and altered tumor cell metabolism. Acidosis has pleiotropic effects on malignant progression; therefore it is essential to understand how acidosis exerts its diverse effects. TDAG8 is a proton-sensing G-protein-coupled receptor that can be activated by extracellular acidosis.MethodsTDAG8 gene expression was analyzed by bioinformatic analyses and quantitative RT-PCR in human hematological malignancies. Retroviral transduction was used to restore TDAG8 expression in U937, Ramos and other blood cancer cells. Multiple in vitro and in vivo tumorigenesis and metastasis assays were employed to evaluate the effects of TDAG8 expression on blood cancer progression. Western blotting, immunohistochemistry and biochemical approaches were applied to elucidate the underlying mechanisms associated with the TDAG8 receptor pathway.ResultsTDAG8 expression is significantly reduced in human blood cancers in comparison to normal blood cells. Severe acidosis, pH 6.4, inhibited U937 cancer cell proliferation while mild acidosis, pH 6.9, stimulated its proliferation. However, restoring TDAG8 gene expression modulated the U937 cell response to mild extracellular acidosis and physiological pH by reducing cell proliferation. Tumor xenograft experiments further revealed that restoring TDAG8 expression in U937 and Ramos cancer cells reduced tumor growth. It was also shown U937 cells with restored TDAG8 expression attached less to Matrigel, migrated slower toward a chemoattractant, and metastasized less in severe combined immunodeficient mice. These effects correlated with a reduction in c-myc oncogene expression. The mechanistic investigation indicated that Gα13/Rho signaling arbitrated the TDAG8-mediated c-myc oncogene repression in response to acidosis.ConclusionsThis study provides data to support the concept that TDAG8 functions as a contextual tumor suppressor down-regulated in hematological malignancies and potentiation of the TDAG8 receptor pathway may be explored as a potential anti-tumorigenic approach in blood cancers.

TDAG8, TRPV1, and ASIC3 involved in establishing hyperalgesic priming in experimental rheumatoid arthritis

Rheumatoid arthritis (RA), characterized by chronic inflammation of synovial joints, is often associated with ongoing pain and increased pain sensitivity. High hydrogen ion concentration (acidosis) found in synovial fluid in RA patients is associated with disease severity. Acidosis signaling acting on proton-sensing receptors may contribute to inflammation and pain. Previous studies focused on the early phase of arthritis (<5 weeks) and used different arthritis models, so elucidating the roles of different proton-sensing receptors in the chronic phase of arthritis is difficult. We intra-articularly injected complete Freund’s adjuvant into mice once a week for 4 weeks to establish chronic RA pain. Mice with knockout of acid-sensing ion channel 3 (ASIC3) or transient receptor potential/vanilloid receptor subtype 1 (TRPV1) showed attenuated chronic phase (>6 weeks) of RA pain. Mice with T-cell death-associated gene 8 (TDAG8) knockout showed attenuated acute and chronic phases of RA pain. TDAG8 likely participates in the initiation of RA pain, but all three genes, TDAG8, TRPV1, and ASIC3, are essential to establish hyperalgesic priming to regulate the chronic phase of RA pain.

The proton-sensing G protein-coupled receptor T-cell death-associated gene 8 (TDAG8) shows cardioprotective effects against myocardial infarction

Myocardial infarction (MI) is an ischaemic heart condition caused by the occlusion of coronary arteries. Following MI, lactic acid from anaerobic glycolysis increases and infiltrating immune cells produce severe inflammation, which leads to acidosis in the ischaemic heart. However, the physiological implication of this pH reduction remains largely unknown. T-cell death-associated gene 8 (TDAG8) is a proton-sensing G protein-coupled receptor found on cardiac macrophages that recognise increases in extracellular protons. We demonstrated that TDAG8 negatively regulates the transcription of the chemokine Ccl20. The infarcted hearts of TDAG8 KO mice showed an increase in CCL20 expression and the number of infiltrating IL-17A-producing γδT cells that express CCR6, a receptor for CCL20. Accordingly, excessive IL-17A production, which is linked to the functional deterioration after MI, was observed in MI-operated TDAG8 KO mice. The survival rate and cardiac function significantly decreased in TDAG8 KO mice compared with those in wild-type mice after MI. Thus, our results suggest that TDAG8 is a key regulator of MI and a potential therapeutic target.

Acid-sensing T cell death associated gene-8 receptor expression in panic disorder

BackgroundWhile disruption of acid-base homeostasis has been pathoetiologically implicated in panic disorder (PD), the mechanism by which pH imbalance is translated to panic pathophysiology is poorly understood. Recently, in a translational rodent model of PD, we reported a role of microglial acid sensing G-protein coupled receptor, T cell death associated gene-8 (TDAG8) in panic-associated behavior and physiology. However, the clinical validity of the TDAG8 receptor has not been investigated. ObjectiveTo assess TDAG8 in PD, we evaluated TDAG8 receptor expression in adolescents and young adults with PD and healthy comparison subjects. MethodsRelative expression of TDAG8 mRNA was determined in peripheral blood mononuclear cells from patients with PD, and compared to expression in healthy subjects. Linear models were utilized to evaluate the relationship between TDAG8 expression and panic disorder symptom severity scale (PDSS) score as well as other potential explanatory variables (e.g., CRP, body mass index, sex, age). Models were refined based on the estimated parameter significance, evidence of omitted variable bias and Bayesian/Akaike information criteria. ResultsRelative to healthy comparison subjects (n=17), expression of TDAG8 mRNA was significantly increased in patients with PD (n=15) (1.60±0.65 vs. 1.01±0.50, p=0.008). TDAG8 mRNA expression predicted PD symptom severity in a fixed effect model incorporating age and sex (p=0.003). ConclusionsCollectively, our results suggest greater TDAG8 expression in patients with PD compared to healthy subjects, and directly link TDAG8 expression and the severity of the PD symptoms. Further investigation of the TDAG8 receptor in panic pathophysiology is warranted.

TDAG8 involved in initiating inflammatory hyperalgesia and establishing hyperalgesic priming in mice

Chronic pain, resulting from injury, arthritis, and cancer, is often accompanied by inflammation. High concentrations of protons found in inflamed tissues results in tissue acidosis, a major cause of pain and hyperalgesia. Acidosis signals may mediate a transition from acute to chronic hyperalgesia (hyperalgesic priming) via proton-sensing G-protein-coupled receptors (GPCRs). The expression of T-cell death-associated gene 8 (TDAG8), a proton-sensing GPCR, is increased during inflammatory hyperalgesia. Attenuating TDAG8 expression in the spinal cord inhibits bone cancer pain, but whether TDAG8 is involved in inflammatory hyperalgesia or hyperalgesic priming remains unclear. In this study, we used TDAG8-knockout or -knockdown to explore the role of TDAG8 in pain. Suppressed TDAG8 expression delayed the onset of inflammatory hyperalgesia and shortened hyperalgesic time in mice. In a dual acid-injection model (acid [pH 5.0] injected twice, 5 days apart), shRNA inhibition of TDAG8 shortened the duration of the second hyperalgesia. Similar results were found in TDAG8-deficient mice. The dual administration of TDAG8 agonist also confirmed that TDAG8 is involved in hyperalgsic priming. Accordingly, TDAG8 may mediate acidosis signals to initiate inflammatory hyperalgesia and establish hyperalgesic priming.

Relationship between TDAG8 and endogenous neuron-protective mechanism against oxygen-glucose deprivation and restoration-induced apoptosis in rat neurons

Objective To investigate the relationship between T-cell death-associated gene 8 (TDAG8) and endogenous neuron-protective mechanism against oxygen-glucose deprivation and restoration (OGD/R)-induced apoptosis in rat neurons. Methods The primary cortical neurons obtained from fetal rats were seeded in 6-well plates at a density of 1×105 cells/ml and divided into 5 groups using a random number table: control group (group C, n=24), group OGD/R (n=48), TDAG8 agonist BTB09089 group (group BTB, n=24), TDAG8-siRNA group (group siRNA, n=24), and blank vehicle group (group V, n=24). The medium was replaced with glucose- and serum-free Locke′s buffer, and the neurons were exposed to 95% N2-5% CO2 in an air-tight incubator at 37 ℃ for 60 min followed by routine culture to establish the model of OGD/R.In BTB, siRNA and V groups, 20 μmol/L TDAG8 agonist BTB09089, 200 pmol/L TDAG8-siRNA, and 6 μl/200 μl transfection reagent were added, respectively, at 24 h before oxygen-glucose restoration.At 6 h of oxygen-glucose restoration, the neuronal viability and amount of lactic dehydrogenase (LDH) released were measured, and the expression of TDAG8 and caspase-3 mRNA in neurons was detected by fluorescent quantitative real-time polymerase chain reaction.In group OGD/R, the expression of TDAG8 and caspase-3 was measured by Western blot at 0, 3, 6, 12 and 24 h of oxygen-glucose restoration.In C, OGD/R, BTB, siRNA and V groups, the expression of TDAG8, caspase-3 and p-Akt was detected at 6 h of oxygen-glucose restoration. Results In group OGD/R, the expression of TDAG8 was gradually up-regulated after oxygen-glucose restoration, and the expression of caspase-3 peaked at 6 h of oxygen-glucose restoration.Compared with group C, the neuronal viability was significantly decreased, the amount of LDH released was significantly increased, and the expression of TDAG8 and caspase-3 protein and mRNA and p-Akt was significantly up-regulated in OGD/R, V and siRNA groups (P 0.05). Conclusion TDAG8 is partially involved in the endogenous neuron-protective mechanism against OGD/R-induced apoptosis in rat neurons, which may be related to activation of Akt signaling pathway. Key words: Receptors, G-protein-coupled; Apoptosis; Neurons; Reperfusion injury

Microglial Acid Sensing Regulates Carbon Dioxide-Evoked Fear

BackgroundCarbon dioxide (CO2) inhalation, a biological challenge and pathologic marker in panic disorder, evokes intense fear and panic attacks in susceptible individuals. The molecular identity and anatomic location of CO2-sensing systems that translate CO2-evoked fear remain unclear. We investigated contributions of microglial acid sensor T cell death–associated gene-8 (TDAG8) and microglial proinflammatory responses in CO2-evoked behavioral and physiological responses. MethodsCO2-evoked freezing, autonomic, and respiratory responses were assessed in TDAG8-deficient (–/–) and wild-type (+/+) mice. Involvement of TDAG8-dependent microglial activation and proinflammatory cytokine interleukin (IL)-1β with CO2-evoked responses was investigated using microglial blocker, minocycline, and IL-1β antagonist IL-1RA. CO2-chemosensitive firing responses using single-cell patch clamping were measured in TDAG8–/– and TDAG8+/+ mice to gain functional insights. ResultsTDAG8 expression was localized in microglia enriched within the sensory circumventricular organs. TDAG8–/– mice displayed attenuated CO2-evoked freezing and sympathetic responses. TDAG8 deficiency was associated with reduced microglial activation and proinflammatory cytokine IL-1β within the subfornical organ. Central infusion of microglial activation blocker minocycline and IL-1β antagonist IL-1RA attenuated CO2-evoked freezing. Finally, CO2-evoked neuronal firing in patch-clamped subfornical organ neurons was dependent on acid sensor TDAG8 and IL-1β. ConclusionsOur data identify TDAG8-dependent microglial acid sensing as a unique chemosensor for detecting and translating hypercapnia to fear-associated behavioral and physiological responses, providing a novel mechanism for homeostatic threat detection of relevance to psychiatric conditions such as panic disorder.