Noriaki Sagata

Associations between microRNA expression and mesenchymal marker gene expression in glioblastoma

The subclassification of glioblastoma (GBM) into clinically relevant subtypes using microRNA (miRNA)- and messenger RNA (mRNA)-based integrated analysis has been attempted. Because miRNAs regulate multiple gene-signaling pathways, understanding miRNA-mRNA interactions is a prerequisite for understanding glioma biology. However, such associations have not been thoroughly examined using high-throughput integrated analysis. To identify significant miRNA-mRNA correlations, we selected and quantified signature miRNAs and mRNAs in 82 gliomas (grade II: 14, III: 16, IV: 52) using real-time reverse-transcriptase polymerase chain reaction. Quantitative expression data were integrated into a single analysis platform that evaluated the expression relationship between miRNAs and mRNAs. The 21 miRNAs include miR-15b, -21, -34a, -105, -124a, -128a, -135b, -184, -196a-b, -200a-c, -203, -302a-d, -363, -367, and -504. In addition, we examined 23 genes, including proneural markers (DLL3, BCAN, and OLIG2), mesenchymal markers (YKL-40, CD44, and Vimentin), cancer stem cell-related markers, and receptor tyrosine kinase genes. Primary GBM was characterized exclusively by upregulation of mesenchymal markers, whereas secondary GBM was characterized by significant downregulation of mesenchymal markers, miR-21, and -34a, and by upregulation of proneural markers and miR-504. Statistical analysis showed that expression of miR-128a, -504, -124a, and -184 each negatively correlated with the expression of mesenchymal markers in GBM. Our functional analysis of miR-128a and -504 as inhibitors demonstrated that suppression of miR-128a and -504 increased the expression of mesenchymal markers in glioblastoma cell lines. Mesenchymal signaling in GBM may be negatively regulated by miR-128a and -504.

Direct induction of ramified microglia-like cells from human monocytes: Dynamic microglial dysfunction in Nasu-Hakola disease

Microglia have been implicated in various neurological and psychiatric disorders in rodent and human postmortem studies. However, the dynamic actions of microglia in the living human brain have not been clarified due to a lack of studies dealing with in situ microglia. Herein, we present a novel technique for developing induced microglia-like (iMG) cells from human peripheral blood cells. An optimized cocktail of cytokines, GM-CSF and IL-34, converted human monocytes into iMG cells within 14 days. The iMG cells have microglial characterizations; expressing markers, forming a ramified morphology, and phagocytic activity with various cytokine releases. To confirm clinical utilities, we developed iMG cells from a patient of Nasu-Hakola disease (NHD), which is suggested to be directly caused by microglial dysfunction, and observed that these cells from NHD express delayed but stronger inflammatory responses compared with those from the healthy control. Altogether, the iMG-technique promises to elucidate unresolved aspects of human microglia in various brain disorders.

Brain-derived Neurotrophic Factor (BDNF) Induces Sustained Intracellular Ca2+ Elevation through the Up-regulation of Surface Transient Receptor Potential 3 (TRPC3) Channels in Rodent Microglia

Background: BDNF and Ca2+ mobilization is important for microglial function. Results: We showed BDNF elevates intracellular Ca2+ through TRPC3 channels. Conclusion: TRPC3 is important for BDNF suppression of microglial activation. Significance: TRPC3 might be important for the treatment of psychiatric disorders. Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca2+ concentration ([Ca2+]i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca2+]i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca2+ elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca2+ elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders.

Plasma Metabolites Predict Severity of Depression and Suicidal Ideation in Psychiatric Patients-A Multicenter Pilot Analysis.

Evaluating the severity of depression (SOD), especially suicidal ideation (SI), is crucial in the treatment of not only patients with mood disorders but also psychiatric patients in general. SOD has been assessed on interviews such as the Hamilton Rating Scale for Depression (HAMD)-17, and/or self-administered questionnaires such as the Patient Health Questionnaire (PHQ)-9. However, these evaluation systems have relied on a person’s subjective information, which sometimes lead to difficulties in clinical settings. To resolve this limitation, a more objective SOD evaluation system is needed. Herein, we collected clinical data including HAMD-17/PHQ-9 and blood plasma of psychiatric patients from three independent clinical centers. We performed metabolome analysis of blood plasma using liquid chromatography mass spectrometry (LC-MS), and 123 metabolites were detected. Interestingly, five plasma metabolites (3-hydroxybutyrate (3HB), betaine, citrate, creatinine, and gamma-aminobutyric acid (GABA)) are commonly associated with SOD in all three independent cohort sets regardless of the presence or absence of medication and diagnostic difference. In addition, we have shown several metabolites are independently associated with sub-symptoms of depression including SI. We successfully created a classification model to discriminate depressive patients with or without SI by machine learning technique. Finally, we produced a pilot algorithm to predict a grade of SI with citrate and kynurenine. The above metabolites may have strongly been associated with the underlying novel biological pathophysiology of SOD. We should explore the biological impact of these metabolites on depressive symptoms by utilizing a cross species study model with human and rodents. The present multicenter pilot study offers a potential utility for measuring blood metabolites as a novel objective tool for not only assessing SOD but also evaluating therapeutic efficacy in clinical practice. In addition, modification of these metabolites by diet and/or medications may be a novel therapeutic target for depression. To clarify these aspects, clinical trials measuring metabolites before/after interventions should be conducted. Larger cohort studies including non-clinical subjects are also warranted to clarify our pilot findings.

TNF-α from hippocampal microglia induces working memory deficits by acute stress in mice.

The role of microglia in stress responses has recently been highlighted, yet the underlying mechanisms of action remain unresolved. The present study examined disruption in working memory due to acute stress using the water-immersion resistant stress (WIRS) test in mice. Mice were subjected to acute WIRS, and biochemical, immunohistochemical, and behavioral assessments were conducted. Spontaneous alternations (working memory) significantly decreased after exposure to acute WIRS for 2h. We employed a 3D morphological analysis and site- and microglia-specific gene analysis techniques to detect microglial activity. Morphological changes in hippocampal microglia were not observed after acute stress, even when assessing ramification ratios and cell somata volumes. Interestingly, hippocampal tumor necrosis factor (TNF)-α levels were significantly elevated after acute stress, and acute stress-induced TNF-α was produced by hippocampal-ramified microglia. Conversely, plasma concentrations of TNF-α were not elevated after acute stress. Etanercept (TNF-α inhibitor) recovered working memory deficits in accordance with hippocampal TNF-α reductions. Overall, results suggest that TNF-α from hippocampal microglia is a key contributor to early-stage stress-to-mental responses.

Association study of polymorphisms in the neutral amino acid transporter genes SLC1A4 , SLC1A5 and the glycine transporter genes SLC6A5 , SLC6A9 with schizophrenia

BackgroundBased on the glutamatergic dysfunction hypothesis for schizophrenia pathogenesis, we have been performing systematic association studies of schizophrenia with the genes involved in glutametergic transmission. We report here association studies of schizophrenia with SLC1A4, SLC1A5 encoding neutral amino acid transporters ASCT1, ASCT2, and SLC6A5, SLC6A9 encoding glycine transporters GLYT2, GLYT1, respectively.MethodsWe initially tested the association of 21 single nucleotide polymorphisms (SNPs) distributed in the four gene regions with schizophrenia using 100 Japanese cases-control pairs and examined allele, genotype and haplotype association with schizophrenia. The observed nominal significance were examined in the full-size samples (400 cases and 420 controls).ResultsWe observed nominally significant single-marker associations with schizophrenia in SNP2 (P = 0.021) and SNP3 (P = 0.029) of SLC1A4, SNP1 (P = 0.009) and SNP2 (P = 0.022) of SLC6A5. We also observed nominally significant haplotype associations with schizophrenia in the combinations of SNP2-SNP7 (P = 0.037) of SLC1A4 and SNP1-SNP4 (P = 0.043) of SLC6A5. We examined all of the nominal significance in the Full-size Sample Set, except one haplotype with insufficient LD. The significant association of SNP1 of SLC6A5 with schizophrenia was confirmed in the Full-size Sample Set (P = 0.018).ConclusionWe concluded that at least one susceptibility locus for schizophrenia may be located within or nearby SLC6A5, whereas SLC1A4, SLC1A5 and SLC6A9 are unlikely to be major susceptibility genes for schizophrenia in the Japanese population.

Aripiprazole inhibits polyI:C-induced microglial activation possibly via TRPM7.

Viral infections during fetal and adolescent periods, as well as during the course of schizophrenia itself have been linked to the onset and/or relapse of a psychosis. We previously reported that the unique antipsychotic aripiprazole, a partial D2 agonist, inhibits the release of tumor necrosis factor (TNF)-α from interferon-γ-activated rodent microglial cells. Polyinosinic-polycytidylic acid (polyI:C) has recently been used as a standard model of viral infections, and recent in vitro studies have shown that microglia are activated by polyI:C. Aripiprazole has been reported to ameliorate behavioral abnormalities in polyI:C-induced mice. To clarify the anti-inflammatory properties of aripiprazole, we investigated the effects of aripiprazole on polyI:C-induced microglial activation in a cellular model of murine microglial cells and possible surrogate cells for human microglia. PolyI:C treatment of murine microglial cells activated the production of TNF-α and enhanced the p38 mitogen-activated protein kinase (MAPK) pathway, whereas aripiprazole inhibited these responses. In addition, polyI:C treatment of possible surrogate cells for human microglia markedly increased TNF-α mRNA expression in cells from three healthy volunteers. Aripiprazole inhibited this increase in cells from two individuals. PolyI:C consistently increased intracellular Ca2+ concentration ([Ca2+]i) in murine microglial cells by influx of extracellular Ca2+. We demonstrated that transient receptor potential in melastatin 7 (TRPM7) channels contributed to this polyI:C-induced increase in [Ca2+]i. Taken together, these data suggest that aripiprazole may be therapeutic for schizophrenia by reducing microglial inflammatory reactions, and TRPM7 may be a novel therapeutic target for schizophrenia. Further studies are needed to validate these findings.

Microglial CD206 Gene Has Potential as a State Marker of Bipolar Disorder

The pathophysiology of bipolar disorder, especially the underlying mechanisms of the bipolarity between manic and depressive states, has yet to be clarified. Microglia, immune cells in the brain, play important roles in the process of brain inflammation, and recent positron emission tomography studies have indicated microglial overactivation in the brain of patients with bipolar disorder. We have recently developed a technique to induced microglia-like (iMG) cells from peripheral blood (monocytes). We introduce a novel translational approach focusing on bipolar disorder using this iMG technique. We hypothesize that immunological conditional changes in microglia may contribute to the shift between manic and depressive states, and thus we herein analyzed gene profiling patterns of iMG cells from three patients with rapid cycling bipolar disorder during both manic and depressive states, respectively. We revealed that the gene profiling patterns are different between manic and depressive states. The profiling pattern of case 1 showed that M1 microglia is dominant in the manic state compared to the depressive state. However, the patterns of cases 2 and 3 were not consistent with the pattern of case 1. CD206, a mannose receptor known as a typical M2 marker, was significantly downregulated in the manic state among all three patients. This is the first report to indicate the importance of shifting microglial M1/M2 characteristics, especially the CD206 gene expression pattern between depressive and manic states. Further translational studies are needed to dig up the microglial roles in the underlying biological mechanisms of bipolar disorder.

Blood biomarkers of Hikikomori, a severe social withdrawal syndrome

Hikikomori, a severe form of social withdrawal syndrome, is a growing social issue in Japan and internationally. The pathophysiology of hikikomori has not yet been elucidated and an effective treatment remains to be established. Recently, we revealed that avoidant personality disorder is the most common comorbidity of hikikomori. Thus, we have postulated that avoidant personality is the personality underpinning hikikomori. First, we herein show relationships between avoidant personality traits, blood biomarkers, hikikomori-related psychological features, and behavioural characteristics assessed by a trust game in non-hikikomori volunteers. Avoidant personality traits were negatively associated with high-density lipoprotein cholesterol (HDL-C) and uric acid (UA) in men, and positively associated with fibrin degeneration products (FDP) and high sensitivity C-reactive protein (hsCRP) in women. Next, we recruited actual individuals with hikikomori, and compared avoidant personality traits, blood biomarkers, and psychological features between individuals with hikikomori and age-matched healthy controls. Individuals with hikikomori had higher avoidant personality scores in both sexes, and showed lower serum UA levels in men and lower HDL-C levels in women compared with healthy controls. This is the first report showing possible blood biomarkers for hikikomori, and opens the door to clarify the underlying biological pathophysiology of hikikomori.

Tryptophan-kynurenine and lipid related metabolites as blood biomarkers for first-episode drug-naïve patients with major depressive disorder: An exploratory pilot case-control study

BACKGROUND Early intervention in depression has been critical to prevent its negative impact including suicide. Recent blood biomarker studies for major depressive disorder (MDD) have suggested that tryptophan-kynurenine and lipid related metabolites are involved in the pathophysiology of MDD. However, there have been limited studies investigating these blood biomarkers in first-episode drug-naïve MDD, which are particularly important for early intervention in depression. METHODS As an exploratory pilot case-control study, we examined the above blood biomarkers, and analyzed how these biomarkers are associated with clinical variables in first-episode drug-naïve MDD patients, based on metabolome/lipidome analysis. RESULTS Plasma tryptophan and kynurenine levels were significantly lower in MDD group (N = 15) compared to healthy controls (HC) group (N = 19), and plasma tryptophan was the significant biomarker to identify MDD group (area under the curve = 0.740). Lower serum high density lipoprotein-cholesterol (HDL-C) was the predictive biomarker for severity of depression in MDD group (R2 = 0.444). Interestingly, depressive symptoms were variously correlated with plasma tryptophan-kynurenine and lipid related metabolites. Moreover, plasma tryptophan-kynurenine metabolites and cholesteryl esters (CEs) were significantly correlated in MDD group, but not in HC group. LIMITATIONS This study had small sample size, and we did not use the multiple test correction. CONCLUSIONS This is the first study to suggest that not only tryptophan-kynurenine metabolites but also HDL-C and CEs are important blood biomarkers for first-episode drug-naïve MDD patients. The present study sheds new light on early intervention in clinical practice in depression, and further clinical studies especially large-scale prospective studies are warranted.