Establishing a whole blood CD4+ T cell immunity measurement to predict response to anti-PD-1.

BACKGROUND: Biomarkers that can accurately predict the efficacy of immune checkpoint inhibitors (ICIs) against programmed death 1 (PD-1) ligand in cancer immunotherapy are urgently needed. We have previously reported a novel formula that predicts the response to treatment with second-line nivolumab with high sensitivity and specificity in patients with non-small cell lung cancer (NSCLC) previously treated with chemotherapy. The formula was based on the percentages of CD62LlowCD4+ T cells (effector T cells; %Teff) and CD4+CD25+FOXP3+ T cells (regulatory T cells; %Treg) in the peripheral blood before treatment estimated using the peripheral blood mononuclear cell (PBMC) method. Here, we investigated the applicability of the formula (K-index) to predict the response to treatment with another ICI to expand its clinical applicability. Furthermore, we developed a simpler assay method based on whole blood (WB) samples to overcome the limitations of the PBMC method, such as technical difficulties, in obtaining the K-index. METHODS: The K-index was evaluated using the PBMC method in 59 patients with NSCLC who received first-line pembrolizumab treatment. We also assessed the K-index using the WB method and estimated the correlation between the measurements obtained using both methods in 76 patients with lung cancer. RESULTS: This formula consistently predicted the response to first-line pembrolizumab therapy in patients with NSCLC. The WB method correlated well with the PBMC method to obtain %Teff, %Treg, and the formula value. The WB method showed high repeatability (coefficient of variation, < 10%). The data obtained using WB samples collected in tubes containing either heparin or EDTA-2K and stored at room temperature (18-24 °C) for one day after blood sampling did not differ. Additionally, the performance of the WB method was consistent in different flow cytometry instruments. CONCLUSIONS: The K-index successfully predicted the response to first-line therapy with pembrolizumab, as reported earlier for the second-line therapy with nivolumab in patients with NSCLC. The WB method established in this study can replace the cumbersome PBMC method in obtaining the K-index. Overall, this study suggests that the K-index can predict the response to anti-PD-1 therapy in various cancers, including NSCLC.

Th1 cytokine endotype discriminates and predicts severe complications in COVID-19

Treatment of severe and critical cases of coronavirus disease 2019 (COVID-19) is still a top priority in public health. Previously, we reported distinct Th1 cytokines related to the pathophysiology of severe COVID-19 condition. In the present study, we investigated the association of Th1 and Th2 cytokine/chemokine endotypes with cell-mediated immunity via multiplex immunophenotyping, single-cell RNA-Seq analysis of peripheral blood mononuclear cells, and analysis of the clinical features of COVID-19 patients. Based on serum cytokine and systemic inflammatory markers, COVID-19 cases were classified into four clusters of increasing (I-IV) severity. Two prominent clusters were of interest and could be used as prognostic reference for a targeted treatment of severe COVID-19 cases. Cluster III reflected severe/critical pathology and was characterized by decreased in CCL17 levels and increase in IL-6, C-reactive protein CXCL9, IL-18, and IL-10 levels. The second cluster (Cluster II) showed mild to moderate pathology and was characterized by predominated CXCL9 and IL-18 levels, levels of IL-6 and CRP were relatively low. Cluster II patients received anti-inflammatory treatment in early-stage, which may have led prevent disease prognosis which is accompanied to IL-6 and CRP induction. In Cluster III, a decrease in the proportion of effector T cells with signs of T cell exhaustion was observed. This study highlights the mechanisms of endotype clustering based on specific inflammatory markers in related the clinical outcome of COVID-19.

Salvianolic acid B improves the survival rate, acute kidney dysfunction, inflammation and NETosis-mediated antibacterial action in a crush syndrome rat model.

Crush syndrome (CS) is a potentially lethal condition characterized by muscle cell damage resulting from decompression following compression. Patients with CS can develop cardiac failure, kidney dysfunction, shock, systemic inflammation and sepsis. Salvianolic acid B (SalB) has cardiac and kidney protective effects and anti-oxidative, anti-inflammatory, anti-apoptotic and anti-bacterial properties. The present study aimed to demonstrate the survival benefit of SalB in the CS rat model, which comprised anesthetized rats with bilateral hindlimb compression by a rubber tourniquet for 5 h. The rats examined were randomly divided into four groups: i) Sham; ii) sham treated with SalB; iii) CS rat model without treatment; and iv) CS rat model treated with SalB. Under continuous monitoring and recording of arterial blood pressures, blood and tissue samples were collected for biochemical analyses at designated timepoints before and after reperfusion. SalB administration improved the survival rate, kidney function (by treating shock and metabolic acidosis) and inflammation (by reducing mitochondrial dysfunction and endothelial damage). Reduced incidence of cardiac failure due to hyperkalemia was associated with reduced muscle injury via the prevention of mitochondrial dysfunction. Additionally, indirect antibacterial action by the neutrophil extracellular trap system (NETs) was observed. SalB administration to the CS rat model led to a substantial improvement in survival following CS by decreasing kidney and cardiac dysfunctions, inflammation, and endothelial dysfunction by improving the mitochondrial function and through antibacterial effects via NETs.

Schizophrenia-Like Behavioral Impairments in Mice with Suppressed Expression of Piccolo in the Medial Prefrontal Cortex.

Piccolo, a presynaptic cytomatrix protein, plays a role in synaptic vesicle trafficking in the presynaptic active zone. Certain single-nucleotide polymorphisms of the Piccolo-encoding gene PCLO are reported to be associated with mental disorders. However, a few studies have evaluated the relationship between Piccolo dysfunction and psychotic symptoms. Therefore, we investigated the neurophysiological and behavioral phenotypes in mice with Piccolo suppression in the medial prefrontal cortex (mPFC). Downregulation of Piccolo in the mPFC reduced regional synaptic proteins, accompanied with electrophysiological impairments. The Piccolo-suppressed mice showed an enhanced locomotor activity, impaired auditory prepulse inhibition, and cognitive dysfunction. These abnormal behaviors were partially ameliorated by the antipsychotic drug risperidone. Piccolo-suppressed mice received mild social defeat stress showed additional behavioral despair. Furthermore, the responses of these mice to extracellular glutamate and dopamine levels induced by the optical activation of mPFC projection in the dorsal striatum (dSTR) were inhibited. Similarly, the Piccolo-suppressed mice showed decreased depolarization-evoked glutamate and -aminobutyric acid elevations and increased depolarization-evoked dopamine elevation in the dSTR. These suggest that Piccolo regulates neurotransmission at the synaptic terminal of the projection site. Reduced neuronal connectivity in the mPFC-dSTR pathway via suppression of Piccolo in the mPFC may induce behavioral impairments observed in schizophrenia.

Functional maintenance of calcium store by ShcB adaptor protein in cerebellar Purkinje cells.

Intracellular Ca2+ levels are changed by influx from extracellular medium and release from intracellular stores. In the central nervous systems, Ca2+ release is involved in various physiological events, such as neuronal excitability and transmitter release. Although stable Ca2+ release in response to stimulus is critical for proper functions of the nervous systems, regulatory mechanisms relating to Ca2+ release are not fully understood in central neurons. Here, we demonstrate that ShcB, an adaptor protein expressed in central neurons, has an essential role in functional maintenance of Ca2+ store in cerebellar Purkinje cells (PCs). ShcB-knockout (KO) mice showed defects in cerebellar-dependent motor function and long-term depression (LTD) at cerebellar synapse. The reduced LTD was accompanied with an impairment of intracellular Ca2+ release. Although the expression of Ca2+ release channels and morphology of Ca2+ store looked intact, content of intracellular Ca2+ store and activity of sarco/endoplasmic reticular Ca2+-ATPase (SERCA) were largely decreased in the ShcB-deficient cerebellum. Furthermore, when ShcB was ectopically expressed in the ShcB-KO PCs, the Ca2+ release and its SERCA-dependent component were restored. These data indicate that ShcB plays a key role in the functional maintenance of ER Ca2+ store in central neurons through regulation of SERCA activity.

Overexpression of transmembrane protein 168 in the mouse nucleus accumbens induces anxiety and sensorimotor gating deficit.

Transmembrane protein 168 (TMEM168) comprises 697 amino acid residues, including some putative transmembrane domains. It is reported that TMEM168 controls methamphetamine (METH) dependence in the nucleus accumbens (NAc) of mice. Moreover, a strong link between METH dependence-induced adaptive changes in the brain and mood disorders has been evaluated. In the present study, we investigated the effects of accumbal TMEM168 in a battery of behavioral paradigms. The adeno-associated virus (AAV) Tmem168 vector was injected into the NAc of C57BL/6J mice (NAc-TMEM mice). Subsequently, the accumbal TMEM168 mRNA was increased approximately by seven-fold when compared with the NAc-Mock mice (controls). The NAc-TMEM mice reported no change in the locomotor activity, cognitive ability, social interaction, and depression-like behaviors; however, TMEM168 overexpression enhanced anxiety in the elevated-plus maze and light/dark box test. The increased anxiety was reversed by pretreatment with the antianxiety drug diazepam (0.3 mg/kg i.p.). Moreover, the NAc-TMEM mice exhibited decreased prepulse inhibition (PPI) in the startle response test, and the induced schizophrenia-like behavior was reversed by pretreatment with the antipsychotic drug risperidone (0.01 mg/kg i.p.). Furthermore, accumbal TMEM168 overexpression decreased the basal levels of extracellular GABA in the NAc and the high K+ (100 mM)-stimulated GABA elevation; however, the total contents of GABA in the NAc remained unaffected. These results suggest that the TMEM168-regulated GABAergic neuronal system in the NAc might become a novel target while studying the etiology of anxiety and sensorimotor gating deficits.

Behavioral impairment in SHATI/NAT8L knockout mice via dysfunction of myelination development.

We have identified SHATI/NAT8L in the brain of mice treated with methamphetamine. Recently, it has been reported that SHATI is N-acetyltransferase 8-like protein (NAT8L) that produces N-acetylaspatate (NAA) from aspartate and acetyl-CoA. We have generated SHATI/NAT8L knockout (Shati -/-) mouse which demonstrates behavioral deficits that are not rescued by single NAA supplementation, although the reason for which is still not clarified. It is possible that the developmental impairment results from deletion of SHATI/NAT8L in the mouse brain, because NAA is involved in myelination through lipid synthesis in oligodendrocytes. However, it remains unclear whether SHATI/NAT8L is involved in brain development. In this study, we found that the expression of Shati/Nat8l mRNA was increased with brain development in mice, while there was a reduction in the myelin basic protein (MBP) level in the prefrontal cortex of juvenile, but not adult, Shati -/- mice. Next, we found that deletion of SHATI/NAT8L induces several behavioral deficits in mice, and that glyceryltriacetate (GTA) treatment ameliorates the behavioral impairments and normalizes the reduced protein level of MBP in juvenile Shati -/- mice. These findings suggest that SHATI/NAT8L is involved in myelination in the juvenile mouse brain via supplementation of acetate derived from NAA. Thus, reduction of SHATI/NAT8L induces developmental neuronal dysfunction.

Striatal N-Acetylaspartate Synthetase Shati/Nat8l Regulates Depression-Like Behaviors via mGluR3-Mediated Serotonergic Suppression in Mice.

Background: Several clinical studies have suggested that N-acetylaspartate and N-acetylaspartylglutamate levels in the human brain are associated with various psychiatric disorders, including major depressive disorder. We have previously identified Shati/Nat8l, an N-acetyltransferase, in the brain using an animal model of psychosis. Shati/Nat8l synthesizes N-acetylaspartate from L-aspartate and acetyl-coenzyme A. Further, N-acetylaspartate is converted into N-acetylaspartylglutamate, a neurotransmitter for metabotropic glutamate receptor 3. Methods: Because Shati/Nat8l mRNA levels were increased in the dorsal striatum of mice following the exposure to forced swimming stress, Shati/Nat8l was overexpressed in mice by the microinjection of adeno-associated virus vectors containing Shati/Nat8l gene into the dorsal striatum (dS-Shati/Nat8l mice). The dS-Shati/Nat8l mice were further assessed using behavioral and neurochemical tests. Results: The dS-Shati/Nat8l mice exhibited behavioral despair in the forced swimming and tail suspension tests and social withdrawal in the 3-chamber social interaction test. These depression-like behaviors were attenuated by the administration of a metabotropic glutamate receptor 2/3 antagonist and a selective serotonin reuptake inhibitor. Furthermore, the metabolism of N-acetylaspartate to N-acetylaspartylglutamate was decreased in the dorsal striatum of the dS-Shati/Nat8l mice. This finding corresponded with the increased expression of glutamate carboxypeptidase II, an enzyme that metabolizes N-acetylaspartylglutamate present in the extracellular space. Extracellular serotonin levels were lower in the dorsal striatum of the dS-Shati/Nat8l and normal mice that were repeatedly administered a selective glutamate carboxypeptidase II inhibitor. Conclusions: Our findings indicate that the striatal expression of N-acetylaspartate synthetase Shati/Nat8l plays a role in major depressive disorder via the metabotropic glutamate receptor 3-mediated functional control of the serotonergic neuronal system.

Involvement of the accumbal osteopontin-interacting transmembrane protein 168 in methamphetamine-induced place preference and hyperlocomotion in mice.

Chronic exposure to methamphetamine causes adaptive changes in brain, which underlie dependence symptoms. We have found that the transmembrane protein 168 (TMEM168) is overexpressed in the nucleus accumbens of mice upon repeated methamphetamine administration. Here, we firstly demonstrate the inhibitory effect of TMEM168 on methamphetamine-induced behavioral changes in mice, and attempt to elucidate the mechanism of this inhibition. We overexpressed TMEM168 in the nucleus accumbens of mice by using an adeno-associated virus vector (NAc-TMEM mice). Methamphetamine-induced hyperlocomotion and conditioned place preference were attenuated in NAc-TMEM mice. Additionally, methamphetamine-induced extracellular dopamine elevation was suppressed in the nucleus accumbens of NAc-TMEM mice. Next, we identified extracellular matrix protein osteopontin as an interacting partner of TMEM168, by conducting immunoprecipitation in cultured COS-7 cells. TMEM168 overexpression in COS-7 cells induced the enhancement of extracellular and intracellular osteopontin. Similarly, osteopontin enhancement was also observed in the nucleus accumbens of NAc-TMEM mice, in in vivo studies. Furthermore, the infusion of osteopontin proteins into the nucleus accumbens of mice was found to inhibit methamphetamine-induced hyperlocomotion and conditioned place preference. Our studies suggest that the TMEM168-regulated osteopontin system is a novel target pathway for the therapy of methamphetamine dependence, via regulating the dopaminergic function in the nucleus accumbens.

Methamphetamine induces Shati/Nat8L expression in the mouse nucleus accumbens via CREB- and dopamine D1 receptor-dependent mechanism.

Shati/Nat8L significantly increased in the nucleus accumbens (NAc) of mice after repeated methamphetamine (METH) treatment. We reported that Shati/Nat8L overexpression in mouse NAc attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference. We recently found that Shati/Nat8L overexpression in NAc regulates the dopaminergic neuronal system via the activation of group II mGluRs by elevated N-acetylaspartylglutamate following N-acetylaspartate increase due to the overexpression. These findings suggest that Shati/Nat8L suppresses METH-induced responses. However, the mechanism by which METH increases the Shati/Nat8L mRNA expression in NAc is unclear. To investigate the regulatory mechanism of Shati/Nat8L mRNA expression, we performed a mouse Shati/Nat8L luciferase assay using PC12 cells. Next, we investigated the response of METH to Shati/Nat8L expression and CREB activity using mouse brain slices of NAc, METH administration to mice, and western blotting for CREB activity of specific dopamine receptor signals in vivo and ex vivo. We found that METH activates CREB binding to the Shati/Nat8L promoter to induce the Shati/Nat8L mRNA expression. Furthermore, the dopamine D1 receptor antagonist SCH23390, but not the dopamine D2 receptor antagonist sulpiride, inhibited the upregulation of Shati/Nat8L and CREB activities in the mouse NAc slices. Thus, the administration of the dopamine D1 receptor agonist SKF38393 increased the Shati/Nat8L mRNA expression in mouse NAc. These results showed that the Shati/Nat8L mRNA was increased by METH-induced CREB pathway via dopamine D1 receptor signaling in mouse NAc. These findings may contribute to development of a clinical tool for METH addiction.

Decreased DNA Methylation in the Shati/Nat8l Promoter in Both Patients with Schizophrenia and a Methamphetamine-Induced Murine Model of Schizophrenia-Like Phenotype.

The number of patients with schizophrenia has increased over the past decade. Previously, many studies have been performed to establish its diagnostic criteria, prophylactic methods, and effective therapies. In this study, we analyzed whether the ratios of DNA methylation in CpG islands of the Shati/Nat8l is decreased in model mice of schizophrenia-like phenotype using genomic DNA collected from brain regions and peripheral blood, since the mouse model of schizophrenia-like phenotype, mice treated repeatedly with methamphetamine showed increase of Shati/Nat8l mRNA expression in our previous experiment. The ratios of Shati/Nat8l CpG island methylation were significantly decreased in both the nucleus accumbens and the peripheral blood of model mice compared with those of control mice. We also investigated Shati/Nat8l methylation in the blood of patients with schizophrenia. We found that Shati/Nat8l CpG island methylation ratios were lower in the patients with schizophrenia than in the healthy controls, which is consistent with our findings in the mice model. To our knowledge, this is the first study to show similar alterations in methylation status of a particular genomic DNA site in both the brain and peripheral blood of mice. Furthermore, the same phenomenon was observed in corresponding human genomic sequences of the DNA extracted from the peripheral blood of patients with schizophrenia. Based on our findings, DNA methylation profiles of the CpG island of Shati/Nat8l might be a diagnostic biomarker of schizophrenia.

Pseudoginsenoside-F11 inhibits methamphetamine-induced behaviors by regulating dopaminergic and GABAergic neurons in the nucleus accumbens.

RATIONALE: Although dependence to methamphetamine (METH) is associated with serious psychiatric symptoms and is a global health and social problem, no effective therapeutic approaches have been identified. Pseudoginsenoside-F11 (PF11) is an ocotillol-type saponin that is isolated from Panax quinquefolius (American ginseng) and was shown to have neuroprotective effects to promote learning and memory and to antagonize the pharmacological effects of morphine. Furthermore, PF11 also shows protective effects against METH-induced neurotoxicity in mice. However, the effects of PF11 on METH-induced preference and dopamine (DA) release have not been defined. OBJECTIVES: We investigated the effects of PF11 administration on METH-induced hyperlocomotion and conditioned place preference (CPP) in mice. Subsequently, extracellular DA and gamma-aminobutyric acid (GABA) levels were determined in the nucleus accumbens (NAc) of mice after co-administration of PF11 and METH using in vivo microdialysis analyses. Moreover, the effects of PF11 administration on the µ-opioid neuronal responses, DAMGO (µ-opioid receptor agonist; [D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin)-induced hyperlocomotion and accumbal extracellular DA increase were investigated to elucidate how PF11 inhibits METH-induced dependence by dopaminergic neuronal hyperfunction. RESULTS: Co-administration of PF11 and METH for 6 days attenuated METH-induced locomotor sensitization compared with treatment with METH alone. In the CPP test, PF11 administration also inhibited METH-induced place preference. In vivo microdialysis analyses indicated that co-administration of PF11 and METH for 7 days prevented METH-induced extracellular DA increase in the NAc and repeated PF11 administration with or without METH for 7 days increased extracellular GABA levels in the NAc, whereas single administration of PF11 did not. Furthermore, DAMGO-induced hyperlocomotion and accumbal extracellular DA increase were significantly inhibited by acute PF11 administration. CONCLUSIONS: The present data suggest that PF11 inhibits METH-induced hyperlocomotion, preference, and accumbal extracellular DA increase by regulating GABAergic neurons and µ-opioid receptors.

Induction of neuronal axon outgrowth by Shati/Nat8l by energy metabolism in mice cultured neurons.

A novel N-acetyltransferase, Shati/Nat8l, was identified in the nucleus accumbens of mice repeatedly treated with methamphetamine (METH). Shati/Nat8l has been reported to inhibit the pharmacological action induced by METH. Shati/Nat8l produces N-acetylaspartate from aspartate and acetyl-CoA. Previously, we reported that overexpression of Shati/Nat8l in nucleus accumbens attenuates the response to METH by N-acetylaspartylglutamate (which is derived from N-acetylaspartate)-mGluR3 signaling in the mice brain. In the present study, to clarify the type of cells that produce Shati/Nat8l, we carried out in-situ hybridization for the detection of Shati/Nat8l mRNA along with immunohistochemical studies using serial sections of mice brain. Shati/Nat8l mRNA was detected in neuronal cells, but not in astrocytes or microglia cells. Next, we investigated the function of Shati/Nat8l in the neuronal cells in mice brain; then, we used an adeno-associated virus vector containing Shati/Nat8l for transfection and overexpression of Shati/Nat8l protein into the primary cultured neurons to investigate the contribution toward the neuronal activity of Shati/Nat8l. Overexpression of Shati/Nat8l in the mice primary cultured neurons induced axonal growth, but not dendrite elongation at day 1.5 (DIV). This finding indicated that Shati/Nat8l contributes toward neuronal development. LY341495, a selective group II mGluRs antagonist, did not abolish this axonal growth, and N-acetylaspartylglutamate itself did not abolish axon outgrowth in the same cultured system. The cultured neurons overexpressing Shati/Nat8l contained high ATP, suggesting that axon outgrowth is dependent on energy metabolism. This study shows that Shati/Nat8l in the neuron may induce axon outgrowth by ATP synthesis and not through mGluR3 signaling.

Knockdown of dopamine D2 receptors in the nucleus accumbens core suppresses methamphetamine-induced behaviors and signal transduction in mice.

BACKGROUND: Addictive drugs lead to reinforcing properties by increasing dopamine in the nucleus accumbens, which is composed of a core and shell regions. Neurons in the nucleus accumbens are divided into 2 subtypes based on the differential gene expression of the dopamine D1 receptors and D2 receptors. METHODS: In the present study, we investigated the role of D2 receptors in the nucleus accumbens core in behaviors and signal transduction induced by psychostimulant methamphetamine in mice that were microinjected with adeno-associated virus vectors containing a microRNA (miRNA) sequence for D2 receptor (adeno-associated virus-miD2r vectors) in the nucleus accumbens core. The adeno-associated virus vectors containing a miRNA sequence for D2 receptor-treated mice (miD2r mice) were assessed at a reduction in D2 receptor, but at no change in dopamine D1 receptor, in the nucleus accumbens core compared with the adeno-associated virus-Mock vectors-treated mice (Mock mice). RESULTS: miD2r mice exhibited a reduction in hyperlocomotion that was induced by a single treatment with methamphetamine. The development of locomotor sensitization induced by repeated treatment with methamphetamine exhibited less extension in miD2r mice. In a place conditioning paradigm, the preferred effects of methamphetamine were significantly weaker in miD2r mice than in Mock mice. Furthermore, the single treatment with methamphetamine-induced phosphorylation of extracellular signal regulated kinase and cyclic adenosine monophosphate response element-binding protein in the nucleus accumbens core of miD2r mice was decreased compared with that in Mock mice. Repeated treatment with methamphetamine-induced delta FBJ murine osteosarcoma viral oncogene homolog B accumulation in the nucleus accumbens core of miD2r mice was also attenuated. CONCLUSIONS: These findings suggest that a D2 receptor-mediated neuronal pathway from the nucleus accumbens core plays an inhibitory role in the development of reinforcing properties.

Behavioral phenotypes for negative symptoms in animal models of schizophrenia.

A devastating psychiatric disorder, schizophrenia is characterized by three major symptoms, positive and negative symptoms and cognitive deficit. Almost all current therapeutic drugs for schizophrenia have efficacy for positive symptoms, and weak efficacy for negative and cognitive deficit. In particular, social withdrawal, diminished motivation, and anhedonia as the depressive aspects of negative symptoms are resistant to the treatment of antipsychotic drugs. Therefore, there is a need for development of new therapeutic drugs for negative symptoms of schizophrenia, and it is necessary to have comprehensive animal models to understand the neurobiological foundations of their symptoms. In this review, we represent the behavioral phenotypes in available animal models of schizophrenia for drug discovery, focusing on the depressive aspects of negative symptoms. We mention here animal models based on the pathology and epidemiology of schizophrenia, e.g., the pharmacological, neurodevelopmental, genetic, and gene-environment combination models. The animal models of schizophrenia are developed by various approaches and are assessed, but there are few models demonstrating negative symptoms with sensitivities to available therapeutic drugs. The development of comprehensive animal model reflecting negative symptoms and of novel compounds that can remedy them provide certain insight into the neurobiological process of schizophrenia and also point the way to a new therapeutic strategy.

Overexpression of Shati/Nat8l, an N-acetyltransferase, in the nucleus accumbens attenuates the response to methamphetamine via activation of group II mGluRs in mice.

A novel N-acetyltransferase, Shati/Nat8l, was identified in the nucleus accumbens (NAc) of mice with methamphetamine (METH) treatment. Previously we reported that suppression of Shati/Nat8l enhanced METH-induced behavioral alterations via dopaminergic neuronal regulation. However, the physiological mechanisms of Shati/Nat8l on the dopaminergic system in the brain are unclear. In this study, we injected adeno-associated virus (AAV) vector containing Shati/Nat8l into the NAc or dorsal striatum (dS) of mice, to increase Shati/Nat8l expression. Overexpression of Shati/Nat8l in the NAc, but not in the dS, attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference in mice. Moreover, the Shati/Nat8l overexpression in the NAc attenuated the elevation of extracellular dopamine levels induced by METH in in vivo microdialysis experiments. These behavioral and neurochemical alterations due to Shati/Nat8l overexpression in the NAc were inhibited by treatment with selective group II metabotropic glutamate receptor type 2 and 3 (mGluR2/3) antagonist LY341495. In the AAV vector-injected NAc, the tissue contents of both N-acetylaspartate and N-acetylaspartylglutamate (NAAG), endogenous mGluR3 agonist, were elevated. The injection of peptidase inhibitor of NAAG or the perfusion of NAAG itself reduced the basal levels of extracellular dopamine in the NAc of naive mice. These results indicate that Shati/Nat8l in the NAc, but not in the dS, plays an important suppressive role in the behavioral responses to METH by controlling the dopaminergic system via activation of group II mGluRs.

Functional analysis of deep intronic SNP rs13438494 in intron 24 of PCLO gene.

The single nucleotide polymorphism (SNP) rs13438494 in intron 24 of PCLO was significantly associated with bipolar disorder in a meta-analysis of genome-wide association studies. In this study, we performed functional minigene analysis and bioinformatics prediction of splicing regulatory sequences to characterize the deep intronic SNP rs13438494. We constructed minigenes with A and C alleles containing exon 24, intron 24, and exon 25 of PCLO to assess the genetic effect of rs13438494 on splicing. We found that the C allele of rs13438494 reduces the splicing efficiency of the PCLO minigene. In addition, prediction analysis of enhancer/silencer motifs using the Human Splice Finder web tool indicated that rs13438494 induces the abrogation or creation of such binding sites. Our results indicate that rs13438494 alters splicing efficiency by creating or disrupting a splicing motif, which functions by binding of splicing regulatory proteins, and may ultimately result in bipolar disorder in affected people.

CD45RA-Foxp3(high) activated/effector regulatory T cells in the CCR7 + CD45RA-CD27 + CD28+central memory subset are decreased in peripheral blood from patients with rheumatoid arthritis.

Human CD4+ T cells can be classified as either naïve, central memory (TCM), or effector memory (TEM) cells. To identify the CD4+ T cell subsets most important in the pathogenesis of rheumatoid arthritis (RA), we phenotypically defined human CD4+ T cells as functionally distinct subsets, and analyzed the distribution and characteristics of each subset in the peripheral blood. We classified CD4+ T cells into six novel subsets based on the expression of CD45RA, CCR7, CD27, and CD28. The CCR7 + CD45RA-CD27 + CD28+ TCM subset comprised a significantly smaller proportion of CD4+ T cells in RA patients compared to healthy controls. The frequency of TNF-α-producing cells in the CCR7-CD45RA-CD27 + CD28+ TEM subset was significantly increased in RA. Furthermore, within the CCR7 + CD45RA-CD27 + CD28+ TCM subset, which was decreased in periperal blood from RA, the proportions of total Foxp3+ Treg cells and CD45RA-Foxp3(high) activated/effector Treg cells were significantly lower in RA patients. Our findings suggest that the increased proportion of TNF-α-producing cells and the decreased proportion of CD45RA-Foxp3(high) activated/effector Treg cells in particular subsets may have critical roles in the pathogenesis of RA.

One-pot stereoselective synthesis of 2-acylaziridines and 2-acylpyrrolidines from N-(propargylic)hydroxylamines.

The stereoselective direct transformation of N-(propargylic)hydroxylamines into cis-2-acylaziridines was achieved by the combined use of AgBF4 and CuCl. Copper salts were found to promote the transformation of the intermediary 4-isoxazolines into 2-acylaziridines and both 3-aryl- and 3-alkyl-substituted 2-acylaziridines could be prepared by using this method. Furthermore, subsequent 1,3-dipolar cycloaddition of azomethine ylides that were generated in situ from the intermediary 2-acylaziridines with maleimides was achieved in a stereoselective one-pot procedure to afford the corresponding 2-acylpyrrolidines, which consisted of an octahydropyrrolo[3,4-c]pyrrole skeleton.

Intrastriatal gene delivery of GDNF persistently attenuates methamphetamine self-administration and relapse in mice.

Relapse of drug abuse after abstinence is a major challenge to the treatment of addicts. In our well-established mouse models of methamphetamine (Meth) self-administration and reinstatement, bilateral microinjection of adeno-associated virus vectors expressing GDNF (AAV-Gdnf) into the striatum significantly reduced Meth self-administration, without affecting locomotor activity. Moreover, the intrastriatal AAV-Gdnf attenuated cue-induced reinstatement of Meth-seeking behaviour in a sustainable manner. In addition, this manipulation showed that Meth-primed reinstatement of Meth-seeking behaviour was reduced. These findings suggest that the AAV vector-mediated Gdnf gene transfer into the striatum is an effective and sustainable approach to attenuate Meth self-administration and Meth-associated cue-induced relapsing behaviour and that the AAV-mediated Gdnf gene transfer in the brain may be a valuable gene therapy against drug dependence and protracted relapse in clinical settings.