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Several studies reported stress-induced microglial phagocytosis, but the biochemical mechanisms by which stress alters microglial synaptic phagocytosis are not fully uncovered. Local or limited apoptosis without cell death was observed at neuronal synapses in previous studies, and proposed as an upstream mechanism for microglial synapse elimination. In this micro-report, we aimed to preliminary examine local synaptic apoptosis in the mouse hippocampus following severe restraint stress, and its effect on microglial phagocytosis.Mice were exposed to 10-day water immersion restraint stress (WIRS). Brain sections including stratum lucidum in the hippocampal CA3 subfield were stained with antibodies against cleaved caspase 3, ionized calcium-binding adapter molecule1 (Iba1), lysosomal-associated membrane protein1 (LAMP1), vesicular glutamate transporter1 (VGLUT1). Co-localization among these proteins were calculated.Our image analysis revealed that synaptic apoptosis was increased while there were no significant changes in microglial phagocytic activity and synaptic phagocytosis following 10-day WIRS.Severe restraint stress enhanced pre-synaptic apoptosis in mouse CA3 stratum lucidum region, but did not promote microglial phagocytosis. The phenomenon microglia fail to phagocytose weakened and unnecessary synapses may be related to pathology of stress.

Aim Neurofibromatosis type 1 (NF1) is a multifaceted disease, and frequently comorbid with neurodevelopmental disorders such as autism spectrum disorder (ASD) and learning disorder. Dysfunction of adenylyl cyclase (AC) is one of the candidate pathways in abnormal development of neuronal cells in the brain of NF1 patients, while its dynamic abnormalities have not been observed. Direct conversion technology can generate induced‐neuronal (iN) cells directly from human fibroblasts within 2 weeks. Just recently, we have revealed that forskolin, an AC activator, rescues the gene expression pattern of iN cells derived from NF1 patients (NF1‐iN cells). In this microreport, we show the dynamic effect of forskolin on NF1‐iN cells. Methods iN cells derived from healthy control (HC‐iN cells) and NF1‐iN cells were treated with forskolin (final concentration 10 μM), respectively. Morphological changes of iN cells were captured by inverted microscope with CCD camera every 2 minutes for 90 minutes. Results Prior to forskolin treatment, neuron‐like spherical‐form cells were observed in HC‐iN cells, but most NF1‐iN cells were not spherical‐form but flatform. Only 20 minutes after forskolin treatment, the morphology of the iN cells were dramatically changed from flatform to spherical form, especially in NF1‐iN cells. Conclusion The present pilot data indicate that forskolin or AC activators may have therapeutic effects on the growth of neuronal cells in NF1 patients. Further translational research should be conducted to validate our pilot findings for future drug development of ASD., Neurofibromatosis type 1 (NF1) is highly comorbid with neurodevelopmental disorders such as autism spectrum disorder (ASD) and learning disorder, and underlying mechanisms have not been well clarified. We herein showed that forskolin, an AC activator, rapidly enhances neuron‐like morphological change of directly induced‐neuronal (iN) cells from NF1 patients. The present pilot data using the direct conversion technology indicate that forskolin or AC activators may have therapeutic effects on the growth of neuronal cells in NF1 patients.

Direct conversion techniques, which generate induced-neuronal (iN) cells from human fibroblasts in less than two weeks, are expected to discover unknown neuronal phenotypes of neuropsychiatric disorders. Here, we present unique gene expression and cell morphology profiles in iN cells derived from neurofibromatosis type 1 (NF1) patients. NF1 is a single-gene multifaceted disorder with relatively high co-occurrence of autism spectrum disorder (ASD). Adenylyl cyclase (AC) dysfunction is one of the candidate pathways in abnormal neuronal development in the brains of NF1 patients. In our study, microarray-based transcriptomic analysis of iN cells from healthy controls (males) and NF1 patients (males) revealed significantly different gene expression of 149 (110 were upregulated and 39 were downregulated). In iN cells derived from NF1 patients (NF1-iN cells), there was a change in the expression level of 90 genes with the addition of forskolin, an AC activator. Furthermore, treatment with forskolin dramatically changed the cell morphology, especially that of NF1-iN cells, from flat-form to spherical-form. Current pilot data indicate the potential therapeutic effect of forskolin or AC activators on neuronal growth in NF1 patients. Further translational research is needed to validate the pilot findings for future drug development of ASD.

Targeting the unique glioma immune microenvironment is a promising approach in developing breakthrough immunotherapy treatments. However, recent advances in immunotherapy, including the development of immune checkpoint inhibitors, have not improved the outcomes of patients with glioma. A way of monitoring biological activity of immune cells in neural tissues affected by glioma should be developed to address this lack of sensitivity to immunotherapy. Thus, in this study, we sought to examine the feasibility of non-invasive monitoring of glioma-associated microglia/macrophages (GAM) by utilizing our previously developed induced microglia-like (iMG) cells. Primary microglia (pMG) were isolated from surgically obtained brain tissues of 22 patients with neurological diseases. iMG cells were produced from monocytes extracted from the patients’ peripheral blood. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed a significant correlation of the expression levels of representative markers for M1 and M2 microglia phenotypes between pMG and the corresponding iMG cells in each patient (Spearman’s correlation coefficient = 0.5225, P <0.0001). Synchronous upregulation of CD206 expression levels was observed in most patients with glioma (6/9, 66.7%) and almost all patients with glioblastoma (4/5, 80%). Therefore, iMG cells can be used as a minimally invasive tool for monitoring the disease-related immunological state of GAM in various brain diseases, including glioma. CD206 upregulation detected in iMG cells can be used as a surrogate biomarker of glioma.

Developmental and epileptic encephalopathy (DEE) represents a group of neurodevelopmental disorders characterized by infantile-onset intractable seizures and unfavorable prognosis of psychomotor development. To date, hundreds of genes have been linked to the onset of DEE. GNAO1 is a DEE-associated gene encoding the alpha-O1 subunit of guanine nucleotide-binding protein (GαO ). Despite the increasing number of reported children with GNAO1 encephalopathy, the molecular mechanisms underlying their neurodevelopmental phenotypes remain elusive. We herein present that co-immunoprecipitation and mass spectrometry analyses identified another DEE-associated protein, SPTAN1, as an interacting partner of GαO . Silencing of endogenous Gnao1 attenuated the neurite outgrowth and calcium-dependent signaling. Inactivation of GNAO1 in human-induced pluripotent stem cells gave rise to anomalous brain organoids that only weakly expressed SPTAN1 and Ankyrin-G. Furthermore, GNAO1-deficient organoids failed to conduct synchronized firing to adjacent neurons. These data indicate that GαO and other DEE-associated proteins organize the cytoskeletal remodeling and functional polarity of neurons in the developing brain.

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-naive 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-naive 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-naive 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.

Advance in the exome-wide sequencing analysis contributes to identifying hundreds of genes that are associated with early-onset epileptic encephalopathy and neurodevelopmental disorders. On the basis of massive sequencing data, functional interactions among different genes are suggested to explain the common molecular pathway underlying the pathogenic process of these disorders. However, the relevance of such interactions with the phenotypic severity or variety in an affected individual remains elusive. In this report, we present a 45-year-old woman with neurofibromatosis type 1 (NF1), infantile-onset epileptic encephalopathy, and severe developmental delay. Whole-exome sequencing identified de novo pathogenic mutations in NF1 and the Schaaf-Yang syndrome-associated gene, MAGEL2. Literature-curated interaction data predicted that NF1 and MAGEL2 proteins were closely connected in this network via their common interacting proteins. Direct conversion of fibroblasts into neurons in vitro showed that neuronal cells from 9 patients with NF1 expressed significantly lower levels of MAGEL2 (54%, p = 0.0047) than those from healthy individuals. These data provide the first evidence that pathogenic mutations of NF1 deregulate the expression of other neurodevelopmental disease-associated genes. De novo mutations in multiple genes may lead to severe developmental phenotypes through their cumulative effects or synergistic interactions.

Direct conversion technique to produce induced-neuronal (iN) cells from human fibroblasts within 2 weeks is expected to discover unknown neuronal phenotypes of neuropsychiatric disorders. Here, we present unique gene expression profiles in iN cells from patients with neurofibromatosis type 1 (NF1), a single-gene multifaceted disorder with comparatively high co-occurrence of autism spectrum disorder (ASD). Microarray-based transcriptomic analysis on iN cells from male healthy controls and male NF1 patients (NF1-iN cells) revealed that 149 genes expressions were significantly different (110 upregulated and 39 downregulated). We validated that mRNA of MEX3D (mex-3 RNA binding family member D) was lower in NF1-iN cells by real-time PCR with 12 sex-mixed samples. In NF1-iN cells on day 14, higher expression of FOS mRNA was observed with lower expression of MEX3D mRNA. Interestingly, BCL2 mRNA was higher in NF1-iN cells on day 5 (early-period) but not on day 14. Our data suggest that aberrant molecular signals due to NF1 mutations may disturb gene expressions, a subset of which defines continuum of the neuronal phenotypes of NF1 with ASD. Further translational studies using induced pluripotent stem (iPS) cell-derived neuronal cells are needed to validate our preliminary findings especially confirming meanings of analysis using early-period iN cells.

Fibromyalgia is a refractory disease characterized by chronic intractable pain and psychological suffering, the cause of which has not yet been elucidated due to its complex pathology. Activation of immune cells in the brain called microglia has attracted attention as a potential underlying pathological mechanism in chronic pain. Until recently, however, technological and ethical considerations have limited the ability to conduct research using human microglia. To overcome this limitation, we have recently developed a technique to create human-induced microglia-like (iMG) cells from human peripheral blood monocytes. In this study, we created the iMG cells from 14 patients with fibromyalgia and 10 healthy individuals, and compared the activation of iMG cells between two groups at the cellular level. The expression of tumor necrosis factor (TNF)-α at mRNA and protein levels significantly increased in ATP-stimulated iMG cells from patients with fibromyalgia compared to cells from healthy individuals. Interestingly, there was a moderate correlation between ATP-induced upregulation of TNF-α expression and clinical parameters of subjective pain and other mental manifestations of fibromyalgia. These findings suggest that microglia in patients with fibromyalgia are hypersensitive to ATP. TNF-α from microglia may be a key factor underlying the complex pathology of fibromyalgia.

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.

INTRODUCTION: As recent advancement of multimodal treatments including immune-check point inhibitors have not led to massive outcome improvement of glioma. Targeting the peculiar immune microenvironment of glioma is a promising approach to innovate a breakthrough treatment, however, there remains to be technical and ethical burdens for monitoring the bioactivities of immune cells in neural tissues. Herein, we examined the feasibility of non-invasive monitoring of glioma-associated microglia/macrophages (GAM) properties through utilization of our originally developed induced-microglia-like (iMG) cells technique. METHODS: We isolated primary microglia (pMG) from surgically-obtained brain tissues of 15 patients with neurosurgical diseases. We induced iMG cells from monocyte extracted from their corresponding peripheral blood by MACS treatment. Expression profiles of representative markers for an M1 and M2 microglia phenotype were analyzed in both pMG and iMG cells by qPCR. RESULTS: q-PCR revealed that a significant correlation of expression level of microglial markers between pMG and the corresponding iMG in each patient. Synchronous upregulations of CD206 were exclusively detected in patients with glioma. CONCLUSION: The present study suggested that iMG cells can be a less-invasive monitor tool for disease-related bioactivity of microglia, thereby seem to be utilized as an intermedium for investigation of relationship between microglia and neuronal diseases including glioma. CD206 upregulation detected by iMG technique can surrogate the specific microenvironment of glioma surrounding tissues and might be utilized as a future biomarker of glioma.

Background Neuroinflammation is suggested to be a crucial factor in the pathophysiology of major depressive disorder (MDD). Analysis of neuron-derived exosomes (NDE) in peripheral blood has recently been highlighted to reveal the pathophysiology of brain diseases without using brain biopsy. Currently, human NDE studies require a considerable amount of peripheral blood to measure multiple substances inside exosomes. Previously, NDE-based clinical studies focusing on MDD have not been reported. Methods As an exploratory pilot case-control study between healthy controls (HC) and drug-free MDD patients (each; N = 34), we searched for NDE-related blood biomarkers with a small amount of peripheral blood using a novel sandwich immunoassay between anti-neuron antibody and antibodies against CD81 (an exosome marker) and against other proteins related to neuroinflammation and synaptic functions. Results Most neuron-related blood biomarkers had moderately to strongly positive correlation with CD81 (NDE), thus we normalized the above biomarkers by CD81 (quantity of each biomarker/CD81) to predict NDE-related blood substances. Interleukin 34 (IL34)/CD81 levels were significantly higher in MDD group compared to HC group. Synaptophysin (SYP), SYP/CD81, and tumor necrosis factor receptor 1 (TNFR1)/CD81 were positively correlated with severities of depression and/or various sub-symptoms. Limitations We did not actually extract NDE from peripheral blood. Conclusions Using a small amount of peripheral blood, we have successfully detected possible NDE-related blood biomarkers. This is the first study to suggest that not only SYP and TNFR1 but also IL34 are important blood biomarkers for patients with MDD. Further studies are warranted to evaluate the present study.

Mitochondrial dysfunction is a critical step in the pathogenesis of many neurodegenerative diseases. The p32/ C1qbp gene functions as an essential RNA and protein chaperone in mitochondrial translation, and is indispensable for embryonic development. However, little is known about the consequences of mitochondrial dysfunction of p32 deletion in the brain development. Here, we found that mice lacking p32 in the central nervous system (p32cKO mice) showed white matter degeneration accompanied by progressive oligodendrocyte loss, axon degeneration and vacuolation in the mid brain and brain stem regions. Furthermore, p32cKO mice died within 8 weeks of birth. We also found that p32-deficient oligodendrocytes and neurons showed reduced oligodendrocyte differentiation and axon degeneration in primary culture. We show that mitochondrial disruption activates an adaptive program known as the integrated stress response (ISR). Mitochondrial respiratory chain function in oligodendrocytes and neurons is, therefore, essential for myelination and axon maintenance, respectively, suggesting that mitochondrial respiratory chain dysfunction in the central nervous system contributes to leukoencephalopathy.

Summary Advance in the exome‐wide sequencing analysis contributes to identifying hundreds of genes that are associated with early‐onset epileptic encephalopathy and neurodevelopmental disorders. On the basis of massive sequencing data, functional interactions among different genes are suggested to explain the common molecular pathway underlying the pathogenic process of these disorders. However, the relevance of such interactions with the phenotypic severity or variety in an affected individual remains elusive. In this report, we present a 45‐year‐old woman with neurofibromatosis type 1 (NF1), infantile‐onset epileptic encephalopathy, and severe developmental delay. Whole‐exome sequencing identified de novo pathogenic mutations in NF1 and the Schaaf‐Yang syndrome‐associated gene, MAGEL2. Literature‐curated interaction data predicted that NF1 and MAGEL2 proteins were closely connected in this network via their common interacting proteins. Direct conversion of fibroblasts into neurons in vitro showed that neuronal cells from 9 patients with NF1 expressed significantly lower levels of MAGEL2 (54%, p = 0.0047) than those from healthy individuals. These data provide the first evidence that pathogenic mutations of NF1 deregulate the expression of other neurodevelopmental disease‐associated genes. De novo mutations in multiple genes may lead to severe developmental phenotypes through their cumulative effects or synergistic interactions.

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 (PET) studies have indicated microglial over-activation in the brain of patients with bipolar disorder. We have recently developed a technique to induce human 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 case 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.

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.

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.