Heterogeneity and mitochondrial vulnerability configurate the divergent immunoreactivity of human induced microglia-like cells.

Microglia play versatile roles in progression of and protection against neuroinflammatory diseases. Little is known, however, about the mechanisms underlying the diverse reactivity of microglia to inflammatory conditions. We investigated how human induced microglia-like (iMG) cells respond to innate immune ligands. Quantitative PCR showed that poly-I:C and lipopolysaccharide (LPS) activated the expression of IL1B and TNF. Immunoreactivity of iMG did not differ between controls (n = 11) and patients with neuroinflammatory diseases (n = 24). Flow cytometry revealed that CD14high cells expressed interleukin (IL) -1ß after LPS treatment. Immunoblotting showed that poly-I:C and LPS differentially activated inflammatory pathways but commonly induced mitochondrial instability and the expression of pyruvate kinase isoform M2 (PKM2). Furthermore, a potent stimulator of PKM2 (DASA-58) alleviated IL-1ß production after LPS treatment. These data indicate that heterogeneous cell populations and mitochondrial stability underlie the divergent immunoreactivity of human iMG in environments.

Systemic Inflammation Leads to Changes in the Intracellular Localization of KLK6 in Oligodendrocytes in Spinal Cord White Matter.

Axonal injury and demyelination occur in demyelinating diseases, such as multiple sclerosis, and the detachment of myelin from axons precedes its degradation. Paranodes are the areas at which each layer of the myelin sheath adheres tightly to axons. The destruction of nodal and paranodal structures during inflammation is an important pathophysiology of various neurological disorders. However, the underlying pathological changes in these structures remain unclear. Kallikrein 6 (KLK6), a serine protease produced by oligodendrocytes, is involved in demyelinating diseases. In the present study, we intraperitoneally injected mice with LPS for several days and examined changes in the localization of KLK6. Transient changes in the intracellular localization of KLK6 to paranodes in the spinal cord were observed during LPS-induced systemic inflammation. However, these changes were not detected in the upper part of brain white matter. LPS-induced changes were suppressed by minocycline, suggesting the involvement of microglia. Moreover, nodal lengths were elongated in LPS-treated wild-type mice, but not in LPS-treated KLK6-KO mice. These results demonstrate the potential involvement of KLK6 in the process of demyelination.

ProBDNF induces sustained elevation of intracellular Ca2+ possibly mediated by TRPM7 channels in rodent microglial cells.

Microglia are intrinsic immune cells that release factors including pro- and anti-inflammatory cytokines, nitric oxide (NO) and neurotrophins following activation in the brain. Elevation of intracellular Ca2+ concentration ([Ca2+ ]i) is important for microglial functions, such as the release of cytokines or NO from activated microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia. Interestingly, proBDNF, the precursor form of mature BDNF, and mature BDNF elicit opposing neuronal responses in the brain. Mature BDNF induces sustained intracellular Ca2+ elevation through the upregulation of the surface expression of TRPC3 channels in rodent microglial cells. In addition, TRPC3 channels are important for the BDNF-induced suppression of NO production in activated microglia. In this study, we observed that proBDNF and mature BDNF have opposite effects on the relative expression of surface p75 neurotrophin receptor (p75NTR ) in rodent microglial cells. ProBDNF induces a sustained elevation of [Ca2+ ]i through binding to the p75NTR , which is possibly mediated by Rac 1 activation and TRPM7 channels in rodent microglial cells. Flow cytometry showed that proBDNF increased the relative surface expression of TRPM7. Although proBDNF did not affect either mRNA expression of pro- and anti-inflammatory cytokines or the phagocytic activity, proBDNF potentiates the generation of NO induced by IFN-γ and TRPM7 channels could be involved in the proBDNF-induced potentiation of IFN-γ-mediated production of NO. We show direct evidence that rodent microglial cells are able to respond to proBDNF, which might be important for the regulation of inflammatory responses in the brain.

GNAO1 organizes the cytoskeletal remodeling and firing of developing neurons.

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.

Donepezil suppresses intracellular Ca2+ mobilization through the PI3K pathway in rodent microglia.

BACKGROUND: Microglia are resident innate immune cells which release many factors including proinflammatory cytokines or nitric oxide (NO) when they are activated in response to immunological stimuli. Pathophysiology of Alzheimer's disease (AD) is related to the inflammatory responses mediated by microglia. Intracellular Ca2+ signaling is important for microglial functions such as release of NO and cytokines. In addition, alteration of intracellular Ca2+ signaling underlies the pathophysiology of AD, while it remains unclear how donepezil, an acetylcholinesterase inhibitor, affects intracellular Ca2+ mobilization in microglial cells. METHODS: We examined whether pretreatment with donepezil affects the intracellular Ca2+ mobilization using fura-2 imaging and tested the effects of donepezil on phagocytic activity by phagocytosis assay in rodent microglial cells. RESULTS: In this study, we observed that pretreatment with donepezil suppressed the TNFα-induced sustained intracellular Ca2+ elevation in both rat HAPI and mouse primary microglial cells. On the other hand, pretreatment with donepezil did not suppress the mRNA expression of both TNFR1 and TNFR2 in rodent microglia we used. Pretreatment with acetylcholine but not donepezil suppressed the TNFα-induced intracellular Ca2+ elevation through the nicotinic α7 receptors. In addition, sigma 1 receptors were not involved in the donepezil-induced suppression of the TNFα-mediated intracellular Ca2+ elevation. Pretreatment with donepezil suppressed the TNFα-induced intracellular Ca2+ elevation through the PI3K pathway in rodent microglial cells. Using DAF-2 imaging, we also found that pretreatment with donepezil suppressed the production of NO induced by TNFα treatment and the PI3K pathway could be important for the donepezil-induced suppression of NO production in rodent microglial cells. Finally, phagocytosis assay showed that pretreatment with donepezil promoted phagocytic activity of rodent microglial cells through the PI3K but not MAPK/ERK pathway. CONCLUSIONS: These suggest that donepezil could directly modulate the microglial function through the PI3K pathway in the rodent brain, which might be important to understand the effect of donepezil in the brain.

Microglia-derived neuregulin expression in psychiatric disorders.

Several studies have revealed that neuregulins (NRGs) are involved in brain function and psychiatric disorders. While NRGs have been regarded as neuron- or astrocyte-derived molecules, our research has revealed that microglia also express NRGs, levels of which are markedly increased in activated microglia. Previous studies have indicated that microglia are activated in the brains of individuals with autism spectrum disorder (ASD). Therefore, we investigated microglial NRG mRNA expression in multiple lines of mice considered models of ASD. Intriguingly, microglial NRG expression significantly increased in BTBR and socially-isolated mice, while maternal immune activation (MIA) mice exhibited identical NRG expression to controls. Furthermore, we observed a positive correlation between NRG expression in microglia and peripheral blood mononuclear cells (PBMCs) in mice, suggesting that NRG expression in human PBMCs may mirror microglia-derived NRG expression in the human brain. To translate these findings for application in clinical psychiatry, we measured levels of NRG1 splice-variant expression in clinically available PBMCs of patients with ASD. Levels of NRG1 type III expression in PBMCs were positively correlated with impairments in social interaction in children with ASD (as assessed using the Autistic Diagnostic Interview-Revised test: ADI-R). These findings suggest that immune cell-derived NRGs may be implicated in the pathobiology of psychiatric disorders such as ASD.

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.

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.

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 Ca(2+) concentration ([Ca(2+)]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 [Ca(2+)]i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca(2+) 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 Ca(2+) 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.

Ameliorative effects of Fingolimod (FTY720) on microglial activation and psychosis-related behavior in short term cuprizone exposed mice.

Schizophrenia is a psychiatric disorder that affects around 1% of the population in widespread populations, with severe cases leading to long-term hospitalization and necessitation of lifelong treatment. Recent studies on schizophrenia have highlighted the involvement of inflammatory and immunoregulatory mechanisms with the onset of symptoms, and the usage of anti-inflammatory treatments are being tested against periods of rapid psychosis. In the central nervous system, microglia are the innate immune population which are activated in response to a wide range of physical and psychological stress factors and produce proinflammatory mediators such as cytokines. Microglial activation and neuroinflammation has been associated to numerous psychiatric disorders including schizophrenia, especially during psychotic episodes. Thus, novel treatments which dampen microglial activation may be of great relevance in the treatment of psychiatric disorders. Fingolimod (FTY720) is a drug used as an immunosuppressive treatment to multiple sclerosis. Recent clinical trials have focused on FTY720 as a treatment for the behavioral symptoms in schizophrenia. However, the mechanisms of Fingolimod in treating the symptoms of schizophrenia are not clear. In this study we use a recently developed neuroinflammatory psychosis model in mice: cuprizone short-term exposure, to investigate the effects of FTY720 administration. FTY720 administration was able to completely alleviate methamphetamine hypersensitivity caused by cuprizone exposure. Moreover, administration of FTY720 improved multiple measures of neuroinflammation (microglial activation, cytokine production, and leucocyte infiltration). In conclusion, our results highlight the future use of FTY720 as a direct anti-inflammatory treatment against microglial activation and psychosis.

Preliminary analysis of hippocampus synaptic apoptosis and microglial phagocytosis induced by severe restraint stress.

AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Angiogenic and inflammatory responses in human induced microglia-like (iMG) cells from patients with Moyamoya disease.

Angiogenic factors associated with Moyamoya disease (MMD) are overexpressed in M2 polarized microglia in ischemic stroke, suggesting that microglia may be involved in the pathophysiology of MMD; however, existing approaches are not applicable to explore this hypothesis. Herein we applied blood induced microglial-like (iMG) cells. We recruited 25 adult patients with MMD and 24 healthy volunteers. Patients with MMD were subdivided into progressive (N = 7) or stable (N = 18) group whether novel symptoms or radiographic advancement of Suzuki stage within 1 year was observed or not. We produced 3 types of iMG cells; resting, M1-, and M2-induced cells from monocytes, then RNA sequencing followed by GO and KEGG pathway enrichment analysis and qPCR assay were performed. RNA sequencing of M2-induced iMG cells revealed that 600 genes were significantly upregulated (338) or downregulated (262) in patients with MMD. Inflammation and immune-related factors and angiogenesis-related factors were specifically associated with MMD in GO analysis. qPCR for MMP9, VEGFA, and TGFB1 expression validated these findings. This study is the first to demonstrate that M2 microglia may be involved in the angiogenic process of MMD. The iMG technique provides a promising approach to explore the bioactivity of microglia in cerebrovascular diseases.

Anticancer effects of residual powder from Barley-Shochu distillation remnants against the orthotopic xenograft mouse models of hepatocellular carcinoma in vivo.

Barely-Shochu is a traditional Japanese liquor distilled from fermented barley with Saccharomyces cerevisiae. Barely-Shochu distillation remnants (SDR) are by-products in the manufacturing process of barley-Shochu. We have already reported on valuable powder from Shochu distillation remnants (PSDR) including antioxidative compounds such as polyphenols. In this study, we investigated the therapeutic effects of barely-PSDR against orthotopic xenograft mouse models of hepatocellular carcinoma (HCC) in vivo. We constructed a mouse model of HCC by orthotopical inoculation of HepG2 cells into the liver of SCID mice. Barely-PSDR (2250 mg/kg) was orally treated once each day for 21 d after the inoculation of HepG2 cells. The livers were removed from anaesthetized mice after the treatment with barely-PSDR and fixed in formalin. The liver sections were analyzed by hematoxylin and eosin (HE) staining and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) methods. Remarkably high reduction of tumorigenesis was obtained in the mouse models of HCC after the oral administration of barely-PSDR in vivo. Induction of apoptosis in the liver section on the mouse models treated with barely-PSDR was observed. Furthermore, prolonged survival was obtained. Thus, therapeutic effects of barely-PSDR without side effects on the orthotopic xenograft mouse models were revealed for the first time.

Modulation of inflammatory responses by fractalkine signaling in microglia.

Reactive microglia are suggested to be involved in neurological disorders, and the mechanisms underlying microglial activity may provide insights into therapeutic strategies for neurological diseases. Microglia produce immunological responses to various stimuli, which include fractalkine (FKN or CX3CL1). CX3CR1, a FKN receptor, is present in microglial cells, and when FKN is applied before lipopolysaccharide (LPS) administration, LPS-induced inflammatory responses are inhibited, suggesting that the activation of the FKN signal is beneficial. Considering the practical administration for treatment, we investigated the influence of FKN on immunoreactive microglia using murine primary microglia and BV-2, a microglial cell line. The administration of LPS leads to nitric oxide (NO) production. NO was reduced when FKN was administered 4 h after LPS administration without a change in inducible nitric oxide synthase expression. In contrast, morphological changes, migratory activity, and proliferation were not altered by delayed FKN treatment. LPS decreases the CX3CR1 mRNA concentration, and the overexpression of CX3CR1 restores the FKN-mediated decrease in NO. CX3CR1 overexpression decreased the NO production that is mediated by LPS even without the application of FKN. ATP and ethanol also reduced CX3CR1 mRNA concentrations. In conclusion, the delayed FKN administration modified the LPS-induced microglial activation. The FKN signals were attenuated by a reduction in CX3CR1 by some inflammatory stimuli, and this modulated the inflammatory response of microglial cells, at least partially.

Effect of memantine, an anti-Alzheimer's drug, on rodent microglial cells in vitro.

The pathophysiology of Alzheimer's disease (AD) is related to neuroinflammatory responses mediated by microglia. Memantine, an antagonist of N-methyl-D-aspartate (NMDA) receptors used as an anti-Alzheimer's drug, protects from neuronal death accompanied by suppression of proliferation and activation of microglial cells in animal models of AD. However, it remains to be tested whether memantine can directly affect microglial cell function. In this study, we examined whether pretreatment with memantine affects intracellular NO and Ca2+ mobilization using DAF-2 and Fura-2 imaging, respectively, and tested the effects of memantine on phagocytic activity by human ß-Amyloid (1-42) phagocytosis assay in rodent microglial cells. Pretreatment with memantine did not affect production of NO or intracellular Ca2+ elevation induced by TNF in rodent microglial cells. Pretreatment with memantine also did not affect the mRNA expression of pro-inflammatory (TNF, IL-1ß, IL-6 and CD45) or anti-inflammatory (IL-10, TGF-ß and arginase) phenotypes in rodent microglial cells. In addition, pretreatment with memantine did not affect the amount of human ß-Amyloid (1-42) phagocytosed by rodent microglial cells. Moreover, we observed that pretreatment with memantine did not affect 11 major proteins, which mainly function in the phagocytosis and degradation of ß-Amyloid (1-42), including TREM2, DAP12 and neprilysin in rodent microglial cells. To the best of our knowledge, this is the first report to suggest that memantine does not directly modulate intracellular NO and Ca2+ mobilization or phagocytic activity in rodent microglial cells. Considering the neuroinflammation hypothesis of AD, the results might be important to understand the effect of memantine in the brain.

Plasma acetylcholine and nicotinic acid are correlated with focused preference for photographed females in depressed males: an economic game study.

Interpersonal difficulties are often observed in major depressive disorder (MDD), while the underlying psychological and biological mechanisms have not yet been elucidated. In the present case-control study, a PC-based trust game was conducted for 38 drug-free MDD patients and 38 healthy controls (HC). In the trust game, participants invested money in a partner (trusting behaviors), and also rated each partner's attractiveness (preference for others). In addition, blood biomarkers including metabolites were measured. Both MDD and HC males exhibited more trusting behaviors compared to females. MDD males' preference for ordinary-attractive partners (lay-person photographs) was lower than HC males, whereas their preference for high-attractive females (fashion-model photographs) was similar levels to HC males. This tendency in MDD males could reflect a "focused (narrowed) preference for females". As for blood biomarker analysis, the levels of 37 metabolites including acetylcholine, AMP, GMP, nicotinic acid and tryptophan were significantly different between two groups. Interestingly, among male participants, acetylcholine and nicotinic acid were negatively correlated with the level of focused preference for photographed females. In sum, we have revealed some behavioral, psychological and biological traits of trusting behaviors and preference for others especially in MDD males. Larger studies should be conducted to validate our preliminary findings.

Personality classification enhances blood metabolome analysis and biotyping for major depressive disorders: two-species investigation.

BACKGROUND: The relationship between depression and personality has long been suggested, however, biomarker investigations for depression have mostly overlooked this connection. METHODS: We collected personality traits from 100 drug-free patients with major depressive disorders (MDD) and 100 healthy controls based on the Five-Factor Model (FFM) such as Neuroticism (N) and Extraversion (E), and also obtained 63 plasma metabolites profiles by LCMS-based metabolome analysis. RESULTS: Partitional clustering analysis using the NEO-FFI data classified all subjects into three major clusters. Eighty-six subjects belonging to Cluster 1 (C1: less personality-biased group) constituted half of MDD patients and half of healthy controls. C2 constituted 50 subjects mainly MDD patients (N high + E low), and C3 constituted 64 subjects mainly healthy subjects (N low + E high). Using metabolome information, the machine learning model was optimized to discriminate MDD patients from healthy controls among all subjects and C1, respectively. The performance of the model for all subjects was moderate (AUC = 0. 715), while the performance was extremely improved when limited to C1 (AUC = 0. 907). Tryptophan-pathway plasma metabolites including tryptophan, serotonin and kynurenine were significantly lower in MDD patients especially among C1. We also validated metabolomic findings using a social-defeat mice model of stress-induced depression. LIMITATIONS: A case-control study design and sample size is not large. CONCLUSIONS: Our results suggest that personality classification enhances blood biomarker analysis for MDD patients and further translational investigations should be conducted to clarify the biological relationship between personality traits, stress and depression.

CD206 Expression in Induced Microglia-Like Cells From Peripheral Blood as a Surrogate Biomarker for the Specific Immune Microenvironment of Neurosurgical Diseases Including Glioma.

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.

Forskolin rapidly enhances neuron-like morphological change of directly induced-neuronal cells from neurofibromatosis type 1 patients.

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.

Antidepressant effect of the translocator protein antagonist ONO-2952 on mouse behaviors under chronic social defeat stress.

In preclinical models, it has been reported that social defeat stress activates microglial cells in the CNS. Translocator protein 18 kDa (TSPO) is a mitochondrial protein expressed on microglia in the CNS that has been proposed to be a useful biomarker for brain injury and inflammation. We hypothesized that a TSPO antagonist, ONO-2952, would inhibit the neuroinflammation induced by microglial hyperactivation and associated depressive-like behaviors. An in vitro analysis showed that ONO-2952 suppressed the release of pro-inflammatory cytokines and mitochondrial reactive oxygen species in cultured microglia stimulated by lipopolysaccharide. In mice submitted to chronic social defeat stress, microglia predominantly expressed TSPO in limbic areas implicated in depressive-like behaviors, including the amygdala, ventral hippocampus and nucleus accumbens, in which an increase in the production of pro-inflammatory cytokines in vivo were associated. Treating animals with ONO-2952 during chronic social defeat stress ameliorated impairments in social avoidance and anxiety-like behaviors and suppressed pro-inflammatory cytokine production, suggesting that ONO-2952 exerted an anti-stress effect in this animal model of depression. Thus, targeting TSPO as a candidate for the development of antidepressants that reduce susceptibility to chronic stress could pave the way toward therapeutic interventions for relapse prophylaxis in depression.