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Cortical excitatory neurons (Cx neurons) are the most dominant neuronal cell type in the cerebral cortex, which play a central role in cognition, perception, intellectual behavior and emotional processing. Robustin vitroinduction of Cx neurons may facilitate as a tool for the elucidation of brain development and pathomechanism of the intractable neurodevelopmental and neurodegenerative disorders including Alzheimer’s disease, and thus potentially contribute to drug development. Here, we report a defined method for efficient induction of Cx neurons from the feeder-free-conditioned human embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). By using this method, human ES/iPS cells could be differentiated into ~99% MAP2-positive neurons by three weeks, and these induced neurons, within five weeks, presented various characteristics of mature excitatory neurons such as strong expression of glutamatergic neuron-specific markers (subunits of AMPA and NDMA receptors and CAMKIIα), highly synchronized spontaneous firing and excitatory postsynaptic current (EPSC). Moreover, the Cx neurons showed susceptibility to the toxicity of Aβ42oligomers and excitotoxicity of excessive glutamates, which is another advantage in terms of toxicity test and searching for the therapeutic agents. Taken together, this study provides a novel research platform for the study of neural development and degeneration based on the feeder-free human ES/iPS cell system.

BackgroundSeveral genetic factors are associated with the pathogenesis of sporadic amyotrophic lateral sclerosis (ALS) and its phenotypes, such as disease progression. Here, in this study, we aimed to identify the genes that affect the survival of patients with sporadic ALS.MethodsWe enrolled 1076 Japanese patients with sporadic ALS with imputed genotype data of 7 908 526 variants. We used Cox proportional hazards regression analysis with an additive model adjusted for sex, age at onset and the first two principal components calculated from genotyped data to conduct a genome-wide association study. We further analysed messenger RNA (mRNA) and phenotype expression in motor neurons derived from induced pluripotent stem cells (iPSC-MNs) of patients with ALS.ResultsThree novel loci were significantly associated with the survival of patients with sporadic ALS—FGF1at 5q31.3 (rs11738209, HR=2.36 (95% CI, 1.77 to 3.15), p=4.85×10−9),THSD7Aat 7p21.3 (rs2354952, 1.38 (95% CI, 1.24 to 1.55), p=1.61×10−8) andLRP1at 12q13.3 (rs60565245, 2.18 (95% CI, 1.66 to 2.86), p=2.35×10−8).FGF1andTHSD7Avariants were associated with decreased mRNA expression of each gene in iPSC-MNs and reduced in vitro survival of iPSC-MNs obtained from patients with ALS. The iPSC-MN in vitro survival was reduced when the expression ofFGF1andTHSD7Awas partially disrupted. The rs60565245 was not associated withLRP1mRNA expression.ConclusionsWe identified three loci associated with the survival of patients with sporadic ALS, decreased mRNA expression ofFGF1andTHSD7Aand the viability of iPSC-MNs from patients. The iPSC-MN model reflects the association between patient prognosis and genotype and can contribute to target screening and validation for therapeutic intervention.

We investigated the prognostic effects of hyperuricemia and high or low body mass index (BMI) in peripheral artery disease (PAD) after endovascular therapy (EVT). Between July 2015–2016, 357 consecutive patients with PAD who underwent EVT were enrolled. Patients were divided into 2 groups: BMI < 25 kg/m2 (low BMI) and ≥ 25 kg/m2 (high BMI); they were also divided into 2 more groups based on the presence/absence of hyperuricemia. The primary and secondary endpoints were major adverse cardiovascular and limb events (MACLE), and all-cause death at 3 years post-EVT. Patients with hyperuricemia had significantly lower freedom from MACLE than patients without hyperuricemia at 3 years (57.0 vs 71.9%, p = .0068). The overall survival of patients with hyperuricemia was significantly lower than that of patients without hyperuricemia (63.9 vs 81.7%, p = .0012). Patients with hyperuricemia who had low BMI experienced significantly lower freedom from MACLE than those without hyperuricemia who had low BMI (48.2 vs 69.9%, p = .002). The overall survival of patients with hyperuricemia who had low BMI was significantly lower than that of patients without hyperuricemia who had low BMI (55.2 vs 77.1%, p = .003). Patients with hyperuricemia had significantly more MACLE and a lower survival at 3 years than patients without hyperuricemia, even if they had a low BMI.

BackgroundWe previously used an induced pluripotent stem cell-based drug repurposing approach to demonstrate that ropinirole hydrochloride (ropinirole) attenuated amyotrophic lateral sclerosis (ALS)-specific pathological phenotypes. Here, we assessed the safety and feasibility of ropinirole in ALS patients to verify its efficacy.MethodsWe conducted a randomized feasibility trial of ALS. Twenty participants with ALSFRS-R scores greater than 2 points were randomly assigned using dynamic allocation to receive ropinirole or placebo for 24 weeks in the double-blind period. Upon completion, participants could choose to participate in the following 24-week open-label active extension period. The primary outcomes were safety and tolerability. The secondary outcomes for the feasibility trial objective were the change in the ALS Functional Rating Scale-Revised (ALSFRS-R) score, composite functional endpoint, combined assessment of function and survival, event-free survival, and time to ≤50% forced vital capacity (blinded outcome assessment). This study is registered with the UMIN Clinical Trials Registry, UMIN000034954.FindingsTwenty-one participants were randomized into two groups (ropinirole group; n=14) and received ropinirole (n=13) or placebo (n=7) and the data of all participants were analysed using mixed-effects models for repeated measures together. Overall, the incidences of adverse events, most of which had been reported previously, were similar within both groups. Notably, the incidence of gastrointestinal disorders (mainly, temporary mild nausea and diarrhoea) was high at 76·9% in the ropinirole group (14·3% in the placebo group). Regarding the feasibility of verifying efficacy, there were no significant differences in the ALSFRS-R score and combined assessment of function and survival scores during the double-blind period for 6 months, while the participants in the ropinirole group had lived an additional 27·9 weeks without disease progression events compared with the placebo group (log-rank test, 95% confidence interval, 4·3–37·4) at 12 months (secondary outcome).InterpretationRopinirole is safe and tolerable for patients with ALS and this trial indicates feasibility for a subsequent large-scale trial.FundingThis study was funded by The Japan Agency for Medical Research and Development and K Pharma Inc.

Background The characteristic structure of motor neurons (MNs), particularly of the long axons, becomes damaged in the early stages of amyotrophic lateral sclerosis (ALS). However, the molecular pathophysiology of axonal degeneration remains to be fully elucidated. Method Two sets of isogenic human-induced pluripotent stem cell (hiPSCs)-derived MNs possessing the single amino acid difference (p.H517D) in the fused in sarcoma ( FUS ) were constructed. By combining MN reporter lentivirus, MN specific phenotype was analyzed. Moreover, RNA profiling of isolated axons were conducted by applying the microfluidic devices that enable axon bundles to be produced for omics analysis. The relationship between the target gene, which was identified as a pathological candidate in ALS with RNA-sequencing, and the MN phenotype was confirmed by intervention with si-RNA or overexpression to hiPSCs-derived MNs and even in vivo . The commonality was further confirmed with other ALS-causative mutant hiPSCs-derived MNs and human pathology. Findings We identified aberrant increasing of axon branchings in FUS -mutant hiPSCs-derived MN axons compared with isogenic controls as a novel phenotype. We identified increased level of Fos-B mRNA, the binding target of FUS, in FUS -mutant MNs. While Fos-B reduction using si-RNA or an inhibitor ameliorated the observed aberrant axon branching, Fos-B overexpression resulted in aberrant axon branching even in vivo . The commonality of those phenotypes was further confirmed with other ALS causative mutation than FUS . Interpretation Analyzing the axonal fraction of hiPSC-derived MNs using microfluidic devices revealed that Fos-B is a key regulator of FUS -mutant axon branching. Fund Japan Agency for Medical Research and development; Japanese Ministry of Education, Culture, Sports, Science and Technology Clinical Research, Innovation and Education Center, Tohoku University Hospital; Japan Intractable Diseases (Nanbyo) Research Foundation; the Kanae Foundation for the Promotion of Medical Science; and “Inochi-no-Iro” ALS research grant.

Amyotrophic lateral sclerosis (ALS) is a heterogeneous motor neuron disease for which no effective treatment is available, despite decades of research into SOD1-mutant familial ALS (FALS). The majority of ALS patients have no familial history, making the modeling of sporadic ALS (SALS) essential to the development of ALS therapeutics. However, as mutations underlying ALS pathogenesis have not yet been identified, it remains difficult to establish useful models of SALS. Using induced pluripotent stem cell (iPSC) technology to generate stem and differentiated cells retaining the patients’ full genetic information, we have established a large number of in vitro cellular models of SALS. These models showed phenotypic differences in their pattern of neuronal degeneration, types of abnormal protein aggregates, cell death mechanisms, and onset and progression of these phenotypes in vitro among cases. We therefore developed a system for case clustering capable of subdividing these heterogeneous SALS models by their in vitro characteristics. We further evaluated multiple-phenotype rescue of these subclassified SALS models using agents selected from non-SOD1 FALS models, and identified ropinirole as a potential therapeutic candidate. Integration of the datasets acquired in this study permitted the visualization of molecular pathologies shared across a wide range of SALS models. iPSC-derived motor neurons from over 30 heterogeneous sporadic ALS cases exhibit pathologies correlated with clinical disease progression, are more similar to FUS/TDP-43 familial ALS than SOD1-ALS and are corrected by repurposing of ropinirole.

Summary Human pluripotent stem cells (hPSCs) represent a potentially valuable cell source for applications in cell replacement therapy, drug development, and disease modeling. For all these uses, it is necessary to develop reproducible and robust protocols for differentiation into desired cell types. However, differentiation protocols remain unstable and inefficient, which makes minimizing the differentiation variance among hPSC lines and obtaining purified terminally differentiated cells extremely time consuming. Here, we report a simple treatment with three small molecules—SB431542, dorsomorphine, and CHIR99021—that enhanced hPSC differentiation into three germ layers with a chemically transitional embryoid-body-like state (CTraS). Induction of CTraS reduced the innate differentiation propensities of hPSCs (even unfavorably differentiated hPSCs) and shifted their differentiation into terminally differentiated cells, particularly neurons. In addition, CTraS induction accelerated in vitro pathological expression concurrently with neural maturation. Thus, CTraS can promote the latent potential of hPSCs for differentiation and potentially expand the utility and applicability of hPSCs., Graphical Abstract, Highlights • CTraS induction enhances hPSC differentiation into three germ layers without bias • CTraS induction is applicable to a wide range of hPSCs even without colony selection • Developing a robust neural induction protocol via CTraS for hPSC disease modeling • CTraS induction promotes in vitro pathological expression with maturation and aging, Simple treatment with three small molecules enhanced hPSC differentiation into three germ layers, namely CTraS. CTraS reduced the innate differentiation propensities of hPSCs and shifted them into terminal differentiations. CTraS induction accelerated in vitro pathological expression with maturation and aging. Thus, CTraS can bring out the latent potential of hPSCs.

Visual Abstract, Bipolar disorder (BP) and schizophrenia (SCZ) are major psychiatric disorders, but the molecular mechanisms underlying the complicated pathologies of these disorders remain unclear. It is difficult to establish adequate in vitro models for pathological analysis because of the heterogeneity of these disorders. In the present study, to recapitulate the pathologies of these disorders in vitro, we established in vitro models by differentiating mature neurons from human induced pluripotent stem cells (hiPSCs) derived from BP and SCZ patient with contributive copy number variations, as follows: two BP patients with PCDH15 deletion and one SCZ patient with RELN deletion. Glutamatergic neurons and GABAergic neurons were induced from hiPSCs under optimized conditions. Both types of induced neurons from both hiPSCs exhibited similar phenotypes of MAP2 (microtubule-associated protein 2)-positive dendrite shortening and decreasing synapse numbers. Additionally, we analyzed isogenic PCDH15- or RELN-deleted cells. The dendrite and synapse phenotypes of isogenic neurons were partially similar to those of patient-derived neurons. These results suggest that the observed phenotypes are general phenotypes of psychiatric disorders, and our in vitro models using hiPSC-based technology may be suitable for analysis of the pathologies of psychiatric disorders.

Introduction In patients with peripheral artery disease (PAD) it is known that CVD is one of prognostic factors. But, it is unclear whether left ventricular ejection fraction (LVEF) affects prognosis of PAD patients. So we investigated that LVEF affects prognosis of PAD patients. Methods From July 2015 to July 2016, 371 consecutive PAD patients who performed endovascular treatment (EVT) were enrolled in I-PAD registry. We could conduct follow up survey about 337 (age 73.8±9.6, men 72.4%) patients and divided two groups according to their LVEF (group with LVEF≤40%, n=18, group without LVEF≤40%, n=319). The primary end point was major adverse limb events (MALE: TLR, TVR, major amputations) and secondary end point was all-cause death. Results The median follow-up period was 13.6±5.7 months. The 18 months MALE and all-cause death rate were significantly higher in the group with low LVEF than group without low LVEF (61.1% vs 21.6% p Conclusion LVEF was significantly associated with MALE and all-cause death in patients with PAD.

Introduction In patients with critical limb ischemia (CLI) it is known that malnutrition, low BMI, inflammation and so on are prognostic factors. But, it is unclear whether left ventricular ejection fraction (LVEF) affects prognosis of CLI patients. So we investigated that LVEF affects prognosis of CLI patients. Methods From July 2015 to July 2016, 371 consecutive peripheral artery disease patients who performed endovascular treatment (EVT) were enrolled in I-PAD registry. 179 of them were patients with CLI. We could conduct follow up survey about 126 (age 75.5±11.1, men 63.5%) and divided two groups according to their LVEF (group with LVEF≤40%, n=13, group without LVEF≤40%, n=113). The primary end point was major adverse limb events (MALE: TLR, TVR, major amputations) and secondary end point was all-cause death. Results The median follow-up period was 11.5±6.7 months. The 18 months MALE rate was significant higher in the group with low LVEF than group without low LVEF (76.9% vs 37.2% p Conclusion LVEF was associated with MALE in patients with CLI.

Background Diabetes mellitus (DM) is known to be one of the risks of arteriosclerosis. However, it is still unknown whether DM is a risk factor also in secondary prevention of frail patients after endovascular treatments (EVT) Purpose To investigate impact of diabetes on patients with frail after EVT. Methods From July 2015 to July 2016, 371 consecutive PAD patients who performed EVT were enrolled in I-PAD registry. We could conduct follow up survey 361 patients (446 lesions) and divided into 2 groups; with diabetes (185 patients, 226 lesions) or without diabetes (176 patients, 220 lesions) and analyzed. And among them,we selected 96 patients with frail and divided into 2 groups; with diabetes (49 patients, 70 lesions) or without diabetes (46 patients, 58 lesions) and analyzed. We defined frail patients as the patients with Clinical Frailty Scale 5 (mild frail) or higher. The primary end point was all-cause-death and major adverse limb events (MALE: TLR, TVR, major amputations) at 1 year. Result At 1 years in the patients group with diabetes, overall survival and freedom from MALE were significantly lower (81.7% vs 95.8% P Conclusion The prognosis of patients with diabetes after EVT was worse than the patient without. On the other hand, the prognosis of frail patients with diabetes after EVT was no difference with the frail patient without diabetes in this study.

Induced pluripotent stem cells (iPSCs) are increasingly used in the study of disease mechanisms and the development of effective disease-modifying therapies for neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Recently, three candidate anti-ALS drugs – ropinirole (ROPI), retigabine, and bosutinib – have been identified in iPSC-based drug screens and are now being evaluated in clinical trials for safety and effectiveness. We review the preclinical data, clinical research design, and rationale for ROPI as an anti-ALS drug candidate compared with those of the other two drugs. We also discuss the use of iPSCs for understanding and monitoring treatment response as well as for new insights into the development of new drugs and therapeutic interventions for major neurodegenerative diseases.

The neuropathological hallmarks of Parkinson's disease (PD) include the appearance of α-synuclein (α-SYN)-positive Lewy bodies (LBs) and the loss of catecholaminergic neurons. Thus, a potential mechanism promoting the uptake of extracellular α-SYN may exist in susceptible neurons. Of the various differentially expressed proteins, we are interested in flotillin (FLOT)-1 because this protein is highly expressed in the brainstem catecholaminergic neurons and is strikingly up-regulated in PD brains. In this study, we found that extracellular monomeric and fibrillar α-SYN can potentiate FLOT1-dopamine transporter (DAT) binding and pre-endocytic clustering of DAT on the cell surface, thereby facilitating DAT endocytosis and down-regulating its transporter activity. Moreover, we demonstrated that α-SYN itself exploited the DAT endocytic process to enter dopaminergic neuron-like cells, and both FLOT1 and DAT were found to be the components of LBs. Altogether, these findings revealed a novel role of extracellular α-SYN on cellular trafficking of DAT and may provide a rationale for the cell type-specific, functional, and pathologic alterations in PD.-Kobayashi, J., Hasegawa, T., Sugeno, N., Yoshida, S., Akiyama, T., Fujimori, K., Hatakeyama, H., Miki, Y., Tomiyama, A., Kawata, Y., Fukuda, M., Kawahata, I., Yamakuni, T., Ezura, M., Kikuchi, A., Baba, T., Takeda, A., Kanzaki, M., Wakabayashi, K., Okano, H., Aoki, M. Extracellular α-synuclein enters dopaminergic cells by modulating flotillin-1-assisted dopamine transporter endocytosis.

Summary Modeling of neurological diseases using induced pluripotent stem cells (iPSCs) derived from the somatic cells of patients has provided a means of elucidating pathogenic mechanisms and performing drug screening. T cells are an ideal source of patient-specific iPSCs because they can be easily obtained from samples. Recent studies indicated that iPSCs retain an epigenetic memory relating to their cell of origin that restricts their differentiation potential. The classical method of differentiation via embryoid body formation was not suitable for T cell-derived iPSCs (TiPSCs). We developed a neurosphere-based robust differentiation protocol, which enabled TiPSCs to differentiate into functional neurons, despite differences in global gene expression between TiPSCs and adult human dermal fibroblast-derived iPSCs. Furthermore, neurons derived from TiPSCs generated from a juvenile patient with Parkinson's disease exhibited several Parkinson's disease phenotypes. Therefore, we conclude that TiPSCs are a useful tool for modeling neurological diseases., Highlights • TiPSCs are a suitable source for neural disease modeling • Developing a robust neurosphere-based induction protocol • An optimized differentiation protocol can overcome clonal variations • Modeling Parkinson's disease using TiPSC-derived neurons, In this article, Okano, Akamatsu, and colleagues show that an established neural induction protocol was able to induce neural cells from TiPSCs as well as fibroblast-derived iPSCs. We also observed that neurons derived from PARK2-TiPSCs can exhibit several Parkinson's disease-specific phenotypes observed in fibroblast-derived PARK2-iPSCs. Thus, T cells, which are easily accessible in patients, are suitable source for neural disease modeling.

Summary The CNS contains many diverse neuronal subtypes, and most neurological diseases target specific subtypes. However, the mechanism of neuronal subtype specificity of disease phenotypes remains elusive. Although in vitro disease models employing human pluripotent stem cells (PSCs) have great potential to clarify the association of neuronal subtypes with disease, it is currently difficult to compare various PSC-derived subtypes. This is due to the limited number of subtypes whose induction is established, and different cultivation protocols for each subtype. Here, we report a culture system to control the regional identity of PSC-derived neurons along the anteroposterior (A-P) and dorsoventral (D-V) axes. This system was successfully used to obtain various neuronal subtypes based on the same protocol. Furthermore, we reproduced subtype-specific phenotypes of amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD) by comparing the obtained subtypes. Therefore, our culture system provides new opportunities for modeling neurological diseases with PSCs., Graphical Abstract, Highlights • The regional identity of PSC-derived neurons can be controlled precisely • Phenotypes between different neuronal subtypes were compared successfully • Neuronal subtype-specific phenotypes of ALS and AD were reproduced in vitro • A novel tool is offered to study subtype specificity of disease phenotypes, Okano, Akamatsu, and colleagues established a culture system to control the regional identity of PSC-derived neurons. This system was used to obtain a variety of neuronal subtypes based on the same protocol. By comparing these subtypes, neurological disease phenotypes were reproduced in vitro in a subtype-specific manner. Therefore, this system is useful for the study of subtype specificity of disease phenotypes.

The characteristic structure of motor neurons (MNs), particularly of the long axons, becomes damaged in the early stages of amyotrophic lateral sclerosis (ALS). However, the molecular pathophysiology of axonal degeneration remains to be fully elucidated. In the present study, two sets of isogenic human-induced pluripotent stem cell (hiPSCs)-derived MNs were compared, and we found an abnormal increase in axon branching in the ALS-causative FUS-mutant lines. Moreover, we applied microfluidic devices that enable axon bundles to be produced for omics analysis and conducted RNA profiling of isolated axons. Thus, we identified increased level of Fos-B mRNA, the binding target of FUS, in FUS-mutant MNs. While Fos-B reduction using si-RNA or an inhibitor ameliorated the observed aberrant axon branching, Fos-B overexpression resulted in aberrant axon branching even in vivo. Analyzing the axonal fraction of hiPSC-derived MNs using microfluidic devices revealed that Fos-B is a key regulator of FUS-mutant axon branching. Funding Statement: This work was supported by Research and Study Project of Tokai University Educational System General Research Organization [to H.M]. Declaration of Interests: H.Ok. is a paid Scientific Advisor of SanBio Co.Ltd and K Pharma Inc. Y.O. is a scientific advisor for Kohjin Bio Co., Ltd. Other authors declare no competing financial interests. Ethics Approval Statement: All protocols were approved by the ethics committees of Tohoku University School of Medicine (No. 2010–306) and the Keio University School of Medicine (N0. 20080016).

Diabetes is known to be one of the risks of arteriosclerosis. However, the therapeutic goal of HbA1c in diabetic elderly is still unclear, in particular, in secondary prevention after percutaneous coronary intervention (PCI). Between August 2012 and July 2013, a total of 1665 consecutive patients

The extracellular L-glutamate (L-Glu) concentration is elevated in neuroinflammation, thereby causing excitotoxicity. One of the mechanisms is down-regulation of astrocyte L-Glu transporters. Some antidepressants have anti-inflammatory effects. We therefore investigated effects of various antidepressants on the down-regulation of astrocyte L-Glu transporters in the in vitro neuroinflammation model. Among these antidepressants, only paroxetine was effective. We previously demonstrated that the down-regulation of astrocyte L-Glu transporters was caused by L-Glu released from activated microglia. We here clarified that only paroxetine inhibited L-Glu release from microglia. This is the novel action of paroxetine, which may bring advantages on the therapy of neuroinflammation.

The cerebral cortex is subdivided into distinct areas that have particular functions. The rostrocaudal (R-C) gradient of fibroblast growth factor 8 (FGF8) signaling defines this areal identity during neural development. In this study, we recapitulated cortical R-C patterning in human pluripotent stem cell (PSC) cultures. Modulation of FGF8 signaling appropriately regulated the R-C markers, and the patterns of global gene expression resembled those of the corresponding areas of human fetal brains. Furthermore, we demonstrated the utility of this culture system in modeling the area-specific forebrain phenotypes [presumptive upper motor neuron (UMN) phenotypes] of amyotrophic lateral sclerosis (ALS). We anticipate that our culture system will contribute to studies of human neurodevelopment and neurological disease modeling.

The current understanding is that hyperuricemia(HUA) is associated with an increased risk of cardiovascular disease. However, it remains unclear whether HUA carries a risk of cardiovascular disease in patients with diabetes mellitus(DM). Three hundred five patients underwent percutaneous coronary

Background In the central nervous system, astrocytic L-glutamate (L-Glu) transporters maintain extracellular L-Glu below neurotoxic levels, but their function is impaired with neuroinflammation. Microglia become activated with inflammation; however, the correlation between activated microglia and the impairment of L-Glu transporters is unknown. Methods We used a mixed culture composed of astrocytes, microglia, and neurons. To quantify L-Glu transporter function, we measured the extracellular L-Glu that remained 30 min after an application of L-Glu to the medium (the starting concentration was 100 μM). We determined the optimal conditions of lipopolysaccharide (LPS) treatment to establish an inflammation model without cell death. We examined the predominant subtypes of L-Glu transporters and the changes in the expression levels of these transporters in this inflammation model. We then investigated the role of activated microglia in the changes in L-Glu transporter expression and the underlying mechanisms in this inflammation model. Results Because LPS (10 ng/mL, 72 h) caused a significant increase in the levels of L-Glu remaining but did not affect cell viability, we adopted this condition for our inflammation model without cell death. GLAST was the predominant L-Glu transporter subtype, and its expression decreased in this inflammation model. As a result of their release of L-Glu, activated microglia were shown to be essential for the significant decrease in L-Glu uptake. The serial application of L-Glu caused a significant decrease in L-Glu uptake and GLAST expression in the astrocyte culture. The hemichannel inhibitor carbenoxolone (CBX) inhibited L-Glu release from activated microglia and ameliorated the decrease in GLAST expression in the inflammation model. In addition, the elevation of the astrocytic intracellular L-Glu itself caused the downregulation of GLAST. Conclusions Our findings suggest that activated microglia trigger the elevation of extracellular L-Glu through their own release of L-Glu, and astrocyte L-Glu transporters are downregulated as a result of the elevation of astrocytic intracellular L-Glu levels, causing a further increase of extracellular L-Glu. Our data suggest the new hypothesis that activated microglia collude with astrocytes to cause the elevation of extracellular L-Glu in the early stages of neuroinflammation.

Summary Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disorder. Although its neuropathology is well understood, the cellular and molecular mechanisms are yet to be elucidated due to limitations in the currently available human genetic data. In this study, we generated induced pluripotent stem cells (iPSC) from two familial ALS (FALS) patients with a missense mutation in the fused-in sarcoma (FUS) gene carrying the heterozygous FUS H517D mutation, and isogenic iPSCs with the homozygous FUS H517D mutation by genome editing technology. These cell-derived motor neurons mimicked several neurodegenerative phenotypes including mis-localization of FUS into cytosolic and stress granules under stress conditions, and cellular vulnerability. Moreover, exon array analysis using motor neuron precursor cells (MPCs) combined with CLIP-seq datasets revealed aberrant gene expression and/or splicing pattern in FALS MPCs. These results suggest that iPSC-derived motor neurons are a useful tool for analyzing the pathogenesis of human motor neuron disorders., Graphical Abstract, Highlights • Modeling familial ALS (FALS) using iPSC-derived motor neurons • Aberrant gene expression and/or splicing in FALS motor neuron precursor cells • Mis-localization of mutant FUS protein in FALS motor neurons • Increased apoptosis of FALS motor neurons, Okano, Yano, and colleagues established iPSCs from two familial ALS patients with the missense mutation in the FUS gene and differentiated them into motor neurons as a novel in vitro model for ALS. Moreover, this model enables the pursuit of correlations and new discovery of molecular pathophysiology in various cell biological phenomena by endogenous mutation in patient-derived motor neurons.

Patient-specific induced pluripotent stem cells (iPSCs) facilitate understanding of the etiology of diseases, discovery of new drugs and development of novel therapeutic interventions. A frequently used starting source of cells for generating iPSCs has been dermal fibroblasts (DFs) isolated from skin biopsies. However, there are also numerous repositories containing lymphoblastoid B-cell lines (LCLs) generated from a variety of patients. To date, this rich bioresource of LCLs has been underused for generating iPSCs, and its use would greatly expand the range of targeted diseases that could be studied by using patient-specific iPSCs. However, it remains unclear whether patient’s LCL-derived iPSCs (LiPSCs) can function as a disease model. Therefore, we generated Parkinson’s disease patient-specific LiPSCs and evaluated their utility as tools for modeling neurological diseases. We established iPSCs from two LCL clones, which were derived from a healthy donor and a patient carrying PARK2 mutations, by using existing non-integrating episomal protocols. Whole genome sequencing (WGS) and comparative genomic hybridization (CGH) analyses showed that the appearance of somatic variations in the genomes of the iPSCs did not vary substantially according to the original cell types (LCLs, T-cells and fibroblasts). Furthermore, LiPSCs could be differentiated into functional neurons by using the direct neurosphere conversion method (dNS method), and they showed several Parkinson’s disease phenotypes that were similar to those of DF-iPSCs. These data indicate that the global LCL repositories can be used as a resource for generating iPSCs and disease models. Thus, LCLs are the powerful tools for generating iPSCs and modeling neurological diseases. Electronic supplementary material The online version of this article (doi:10.1186/s13041-016-0267-6) contains supplementary material, which is available to authorized users.