研究者詳細 - 岡野　栄之

論文 - 岡野　栄之

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Clinical utility of neuronal cells directly converted from fibroblasts of patients for neuropsychiatric disorders

Kano S., Yuan M., Cardarelli R. A., Maegawa G., Higurashi N., Gaval-Cruz M., Wilson A. M., Tristan C., Kondo M. A., Chen Y., Koga M., Obie C., Ishizuka K., Seshadri S., Srivastava R., Kato T. A., Horiuchi Y., Sedlak T. W., Lee Y., Rapoport J. L., Hirose S., Okano H., Valle D., O’Donnell P., Sawa A., Kai M.

Current Molecular Medicine 15 （ 2 ） 138 - 145 2015年

ISSN 1566-5240

<p>Methodologies for generating functional neuronal cells directly from human fibroblasts [induced neuronal (iN) cells] have been recently developed, but the research so far has only focused on technical refinements or recapitulation of known pathological phenotypes. A critical question is whether this novel technology will contribute to elucidation of novel disease mechanisms or evaluation of therapeutic strategies. Here we have addressed this question by studying Tay-Sachs disease, a representative lysosomal storage disease, and Dravet syndrome, a form of severe myoclonic epilepsy in infancy, using human iN cells with feature of immature postmitotic glutamatergic neuronal cells. In Tay-Sachs disease, we have successfully characterized canonical neuronal pathology, massive accumulation of GM2 ganglioside, and demonstrated the suitability of this novel cell culture for future drug screening. In Dravet syndrome, we have identified a novel functional phenotype that was not suggested by studies of classical mouse models and human autopsied brains. Taken together, the present study demonstrates that human iN cells are useful for translational neuroscience research to explore novel disease mechanisms and evaluate therapeutic compounds. In the future, research using human iN cells with well-characterized genomic landscape can be integrated into multidisciplinary patient-oriented research on neuropsychiatric disorders to address novel disease mechanisms and evaluate therapeutic strategies.</p>

Control of the Survival and Growth of Human Glioblastoma Grafted Into the Spinal Cord of Mice by Taking Advantage of Immunorejection

岡野栄之

CELL TRANSPLANTATION 24 （ 7 ） 1299 - 1311 2015年

研究論文（学術雑誌）, ISSN 0963-6897

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atlasBREX: Automated template-derived brain extraction in animal MRI

Lohmeier J., Kaneko T., Hamm B., Makowski M., Okano H.

Scientific Reports （Scientific Reports） 9 （ 1 ） 2019年12月

© 2019, The Author(s). We proposed a generic template-derived approach for (semi-) automated brain extraction in animal MRI studies and evaluated our implementation with different animal models (macaque, marmoset, rodent) and MRI protocols (T1, T2). While conventional MR-neuroimaging studies perform brain extraction as an initial step priming subsequent image-registration from subject to template, our proposed approach propagates an anatomical template to (whole-head) individual subjects in reverse order, which is challenging due to the surrounding extracranial tissue, greater differences in contrast pattern and larger areas with field inhomogeneity. As a novel approach, the herein introduced brain extraction algorithm derives whole-brain segmentation using rigid and non-rigid deformation based on unbiased anatomical atlas building with a priori estimates from study-cohort and an initial approximate brain extraction. We evaluated our proposed method in comparison to several other technical approaches including “Marker based watershed scalper”, “Brain-Extraction-Tool”, “3dSkullStrip”, “Primatologist-Toolbox”, “Rapid Automatic Tissue Segmentation” and “Robust automatic rodent brain extraction using 3D pulse-coupled neural networks” with manual skull-stripping as reference standard. ABX demonstrated best performance with accurate (≥92%) and consistent results throughout datasets and across species, age and MRI protocols. ABX was made available to the public with documentation, templates and sample material (https://www.github.com/jlohmeier/atlasBREX).

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The adeno-associated virus rh10 vector is an effective gene transfer system for chronic spinal cord injury

Hoshino Y., Nishide K., Nagoshi N., Shibata S., Moritoki N., Kojima K., Tsuji O., Matsumoto M., Kohyama J., Nakamura M., Okano H.

Scientific Reports （Scientific Reports） 9 （ 1 ） 2019年12月

© 2019, The Author(s). Treatment options for chronic spinal cord injury (SCI) remain limited due to unfavourable changes in the microenvironment. Gene therapy can overcome these barriers through continuous delivery of therapeutic gene products to the target tissue. In particular, adeno-associated virus (AAV) vectors are potential candidates for use in chronic SCI, considering their safety and stable gene expression in vivo. Given that different AAV serotypes display different cellular tropisms, it is extremely important to select an optimal serotype for establishing a gene transfer system during the chronic phase of SCI. Therefore, we generated multiple AAV serotypes expressing ffLuc-cp156, a fusion protein of firefly luciferase and Venus, a variant of yellow fluorescent protein with fast and efficient maturation, as a reporter, and we performed intraparenchymal injection in a chronic SCI mouse model. Among the various serotypes tested, AAVrh10 displayed the highest photon count on bioluminescence imaging. Immunohistological analysis revealed that AAVrh10 showed favourable tropism for neurons, astrocytes, and oligodendrocytes. Additionally, with AAVrh10, the area expressing Venus was larger in the injury epicentre and extended to the surrounding tissue. Furthermore, the fluorescence intensity was significantly higher with AAVrh10 than with the other vectors. These results indicate that AAVrh10 may be an appropriate serotype for gene delivery to the chronically injured spinal cord. This promising tool may be applied for research and development related to the treatment of chronic SCI.

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Role of cyclooxygenase-2-mediated prostaglandin E2-prostaglandin E receptor 4 signaling in cardiac reprogramming

Muraoka N., Nara K., Tamura F., Kojima H., Yamakawa H., Sadahiro T., Miyamoto K., Isomi M., Haginiwa S., Tani H., Kurotsu S., Osakabe R., Torii S., Shimizu S., Okano H., Sugimoto Y., Fukuda K., Ieda M.

Nature Communications （Nature Communications） 10 （ 1 ） 2019年12月

© 2019, The Author(s). Direct cardiac reprogramming from fibroblasts can be a promising approach for disease modeling, drug screening, and cardiac regeneration in pediatric and adult patients. However, postnatal and adult fibroblasts are less efficient for reprogramming compared with embryonic fibroblasts, and barriers to cardiac reprogramming associated with aging remain undetermined. In this study, we screened 8400 chemical compounds and found that diclofenac sodium (diclofenac), a non-steroidal anti-inflammatory drug, greatly enhanced cardiac reprogramming in combination with Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Hand2. Intriguingly, diclofenac promoted cardiac reprogramming in mouse postnatal and adult tail-tip fibroblasts (TTFs), but not in mouse embryonic fibroblasts (MEFs). Mechanistically, diclofenac enhanced cardiac reprogramming by inhibiting cyclooxygenase-2, prostaglandin E2/prostaglandin E receptor 4, cyclic AMP/protein kinase A, and interleukin 1β signaling and by silencing inflammatory and fibroblast programs, which were activated in postnatal and adult TTFs. Thus, anti-inflammation represents a new target for cardiac reprogramming associated with aging.

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Robust and efficient knock-in in embryonic stem cells and early-stage embryos of the common marmoset using the CRISPR-Cas9 system

Yoshimatsu S., Okahara J., Sone T., Takeda Y., Nakamura M., Sasaki E., Kishi N., Shiozawa S., Okano H.

Scientific Reports （Scientific Reports） 9 （ 1 ） 2019年12月

© 2019, The Author(s). Genome editing technology greatly facilitates the genetic modification of various cells and animals. The common marmoset (Callithrix jacchus), a small non-human primate which exhibits high reproductive efficiency, is a widely used animal model in biomedical research. Developing genome editing techniques in the common marmoset will further enhance its utility. Here, we report the successful establishment of a knock-in (KI) method for marmoset embryonic stem cells (ESCs), which is based on the CRISPR-Cas9 system. The use of CRISPR-Cas9, mediated by homologous recombination (HR), enhanced the KI efficiency in marmoset ESCs. Furthermore, we succeeded in performing KI in early-stage marmoset embryos. In the course of the experiments, we found that HR in the marmoset ESCs is innately highly efficient. This suggested that the marmoset possesses a repair mechanism for DNA double-strand breaks. The current study will facilitate the generation of genetically modified marmosets and gene function analysis in the marmoset.

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Dual usage of a stage-specific fluorescent reporter system based on a helper-dependent adenoviral vector to visualize osteogenic differentiation

Sone T., Shin M., Ouchi T., Sasanuma H., Miyamoto A., Ohte S., Tsukamoto S., Nakanishi M., Okano H., Katagiri T., Mitani K.

Scientific Reports （Scientific Reports） 9 （ 1 ） 2019年12月

© 2019, The Author(s). We developed a reporter system that can be used in a dual manner in visualizing mature osteoblast formation. The system is based on a helper-dependent adenoviral vector (HDAdV), in which a fluorescent protein, Venus, is expressed under the control of the 19-kb human osteocalcin (OC) genomic locus. By infecting human and murine primary osteoblast (POB) cultures with this reporter vector, the cells forming bone-like nodules were specifically visualized by the reporter. In addition, the same vector was utilized to efficiently knock-in the reporter into the endogenous OC gene of human induced pluripotent stem cells (iPSCs), by homologous recombination. Neural crest-like cells (NCLCs) derived from the knock-in reporter iPSCs were differentiated into osteoblasts forming bone-like nodules and could be visualized by the expression of the fluorescent reporter. Living mature osteoblasts were then isolated from the murine mixed POB culture by fluorescence-activated cell sorting (FACS), and their mRNA expression profile was analyzed. Our study presents unique utility of reporter HDAdVs in stem cell biology and related applications.

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Cell therapy for spinal cord injury using induced pluripotent stem cells

Nagoshi N., Tsuji O., Nakamura M., Okano H.

Regenerative Therapy （Regenerative Therapy） 11 75 - 80 2019年12月

© 2019 The Japanese Society for Regenerative Medicine For the past few decades, spinal cord injury (SCI) has been believed to be an incurable traumatic condition, but with recent developments in stem cell biology, the field of regenerative medicine has gained hopeful momentum in the development of a treatment for this challenging pathology. Among the treatment candidates, transplantation of neural precursor cells has gained remarkable attention as a reasonable therapeutic intervention to replace the damaged central nervous system cells and promote functional recovery. Here, we highlight transplantation therapy techniques using induced pluripotent stem cells to treat SCI and review the recent research giving consideration to future clinical applications.

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Ropinirole hydrochloride remedy for amyotrophic lateral sclerosis – Protocol for a randomized, double-blind, placebo-controlled, single-center, and open-label continuation phase I/IIa clinical trial (ROPALS trial)

Morimoto S., Takahashi S., Fukushima K., Saya H., Suzuki N., Aoki M., Okano H., Nakahara J.

Regenerative Therapy （Regenerative Therapy） 11 143 - 166 2019年12月

© 2019 The Japanese Society for Regenerative Medicine Introduction: Amyotrophic lateral sclerosis (ALS) is an intractable and incurable neurological disease. It is a progressive disease characterized by muscle atrophy and weakness caused by selective vulnerability of upper and lower motor neurons. In disease research, it has been common to use mouse models carrying mutations in responsible genes for familial ALS as pathological models of ALS. However, there is no model that has reproduced the actual conditions of human spinal cord pathology. Thus, we developed a method of producing human spinal motor neurons using human induced pluripotent stem cells (iPSCs) and an innovative experimental technique for drug screening. As a result, ropinirole hydrochloride was eventually discovered after considering such results as its preferable transitivity in the brain and tolerability, including possible adverse reactions. Therefore, we explore the safety, tolerability and efficacy of ropinirole hydrochloride as an ALS treatment in this clinical trial. Methods: The ROPALS trial is a single-center double-blind randomized parallel group-controlled trial of the safety, tolerability, and efficacy of the ropinirole hydrochloride extended-release tablet (Requip CR) at 2- to 16-mg doses in patients with ALS. Twenty patients will be recruited for the active drug group (fifteen patients) and placebo group (five patients). All patients will be able to receive the standard ALS treatment of riluzole if not changed the dosage during this trial. The primary outcome will be safety and tolerability at 24 weeks, defined from the date of randomization. Secondary outcome will be the efficacy, including any change in the ALS Functional Rating Scale-Revised (ALSFRS-R), change in the Combined Assessment of Function and Survival (CAFS), and the composite endpoint as a sum of Z-transformed scores on various clinical items. Notably, we will perform an explorative search for a drug effect evaluation using the patient-derived iPSCs to prove this trial concept. Eligible patients will have El Escorial Possible, clinically possible and laboratory-supported, clinically probable, or clinically definite amyotrophic lateral sclerosis with disease duration less than 60 months (inclusive), an ALSFRS-R score ≥2 points on all items and age from 20 to 80 years. Conclusion: Patient recruitment began in December 2018 and the last patient is expected to complete the trial protocol in November 2020. Trial registration: Current controlled trials UMIN000034954 and JMA-IIA00397 Protocol version: version 1.6 (Date; 5/Apr/2019).

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Generation of a human iPS cell line (CGMH.SLC26A4919-2) from a Pendred syndrome patient carrying SLC26A4 c.919-2A>G splice-site mutation

Cheng Y., Chan Y., Hu C., Lu Y., Saeki T., Hosoya M., Saegusa C., Fujioka M., Okano H., Weng S., Hsu C., Chang K., Wu C.

Stem Cell Research （Stem Cell Research） 40 2019年10月

ISSN 18735061

© 2019 SLC26A4 is the second most frequent gene implicated in congenital hearing loss after GJB2 mutations. Here, we report the generation of induced pluripotent stem cells (iPSCs), from a patient who was carrying a homozygous c.919-2A>G variant in the SLC26A4 gene. This is the most common variant of SLC26A4 gene in the Chinese population and the second most prevalent one in other Asian countries. The established patient-derived iPSC displayed all the features of pluripotent stem cell markers and had the ability to differentiate into all of the three germ layers and possessed a normal karyotype.

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Analysis of skeletal-muscle condition after excessive loading of the lower legs by sequential magnetic resonance imaging

Hata J., Endo K., Tsuji O., Arakawa S., Sato M., Yagi K., Fujiyoshi K., Okano H., Nakamura M.

Journal of Orthopaedic Science （Journal of Orthopaedic Science） 24 （ 5 ） 873 - 880 2019年09月

ISSN 09492658

© 2019 Objectives: To determine the changes of muscle conditions of lower leg after stretch shortening cycle exercises by MRI. Subjects and methods: This study included 20 healthy male adult volunteers. After conducting control MRI, each subject performed 3 sets of exercise loading, each set consisting of 100 repetitions of rebound jumping on one foot. MRIs were performed immediately after exercise loading (0 h), 6, 30, and 168 h later. After constructing T1/T2/Fractional Anisotropy (FA)/Apparent Diffusion Coefficient (ADC) maps, the changes of three skeletal muscles of the leg (the tibialis anterior [TA], soleus [SOL], and gastrocnemius [GA]) were quantitatively evaluated in each map at each time point. Results: The T1 and T2 values were prolonged after exercise loading, and there was a delay in the recovery of T1 at 6 and 30 h after exercise loading, as compared to those of T2 values over time. The ADC values were elevated in all three muscles immediately after exercise loading, then recovered more slowly than T1 and T2, and still had not returned to baseline 168 h after exercise loading. The FA value decreased in all three muscles after exercise loading, with the greatest decrease occurring immediately after exercise loading. As with the ADC values, the FA values were slow to recover from the decrease, and had not returned to baseline levels 168 h post-loading. Conclusion: The delay of T1 value recovery suggested that the T1 value may reflect the muscle condition like fatigue and damage. Changes in the ADC and FA values over time suggested that structural changes such as minute muscular injuries can be detected by diffusion-weighted MRI. Meanwhile, the changes observed in the T1 and T2 values suggested that the measured relaxation time data reflected not only the water volume in the muscle, but also the muscle condition after exercise loading.

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Establishment of induced pluripotent stem cells from common marmoset fibroblasts by RNA-based reprogramming

Nakajima M., Yoshimatsu S., Sato T., Nakamura M., Okahara J., Sasaki E., Shiozawa S., Okano H.

Biochemical and Biophysical Research Communications （Biochemical and Biophysical Research Communications） 515 （ 4 ） 593 - 599 2019年08月

ISSN 0006291X

© 2019 Elsevier Inc. The common marmoset (marmoset; Callithrix jacchus) shows anatomical and physiological features that are in common with humans. Establishing induced pluripotent stem cells (iPSCs) from marmosets holds promise for enhancing the utility of the animal model for biomedical and preclinical studies. However, in spite of the presence of some previous reports on marmoset iPSCs, the reprogramming technology in marmosets is still under development. In particular, the efficacy of RNA-based reprogramming has not been thoroughly investigated. In this study, we attempted RNA-based reprogramming for deriving iPSCs from marmoset fibroblasts. Although we failed to derive iPSC colonies from marmoset fibroblasts by using a conventional RNA-based reprogramming method previously validated in human fibroblasts, we succeeded in deriving colony-forming cells with a modified induction medium supplemented with a novel set of small molecules. Importantly, following one-week culture of the colony-forming cells in conventional embryonic stem cell (ESC) medium, we obtained iPSCs which express endogenous pluripotent markers and show a differentiation potential into all three germ layers. Taken together, our results indicate that RNA-based reprogramming, which is valuable for deriving transgene-free iPSCs, is applicable to marmosets.

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Semi-supervised deep learning of brain tissue segmentation

Ito R., Nakae K., Hata J., Okano H., Ishii S.

Neural Networks （Neural Networks） 116 25 - 34 2019年08月

ISSN 08936080

© 2019 Elsevier Ltd Brain image segmentation is of great importance not only for clinical use but also for neuroscience research. Recent developments in deep neural networks (DNNs) have led to the application of DNNs to brain image segmentation, which required extensive human annotations of whole brain images. Annotating three-dimensional brain images requires laborious efforts by expert anatomists because of the differences among images in terms of their dimensionality, noise, contrast, or ambiguous boundaries that even prevent these experts from necessarily attaining consistency. This paper proposes a semi-supervised learning framework to train a DNN based on a relatively small number of annotated (labeled) images, named atlases, but also a relatively large number of unlabeled images by leveraging image registration to attach pseudo-labels to images that were originally unlabeled. We applied our proposed method to two different datasets: open human brain images and our original marmoset brain images. When provided with the same number of atlases for training, we found our method achieved superior and more stable segmentation results than those by existing registration-based and DNN-based methods.

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Aberrant axon branching via Fos-B dysregulation in FUS-ALS motor neurons

Akiyama T., Suzuki N., Ishikawa M., Fujimori K., Sone T., Kawada J., Funayama R., Fujishima F., Mitsuzawa S., Ikeda K., Ono H., Shijo T., Osana S., Shirota M., Nakagawa T., Kitajima Y., Nishiyama A., Izumi R., Morimoto S., Okada Y., Kamei T., Nishida M., Nogami M., Kaneda S., Ikeuchi Y., Mitsuhashi H., Nakayama K., Fujii T., Warita H., Okano H., Aoki M.

EBioMedicine （EBioMedicine） 45 362 - 378 2019年07月

© 2019 The Authors 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.

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Degradation of Extracellular Matrix by Matrix Metalloproteinase 2 Is Essential for the Establishment of the Blood-Brain Barrier in Drosophila

Kanda H., Shimamura R., Koizumi-Kitajima M., Okano H.

iScience （iScience） 16 218 - 229 2019年06月

© 2019 The Author(s) The blood-brain barrier (BBB) is an essential system that isolates the central nervous system from the internal environment. Increasing evidence has begun to reveal the molecules that are required for BBB integrity. However, how these components are regulated remains unclear. Here we report that a matrix metalloproteinase, Mmp2, is essential for the establishment of the BBB in Drosophila. In the absence of mmp2, the BBB becomes leaky, which allows the tracer to penetrate the brain. Moreover, the expression pattern of a junctional component, Neuroglian, is altered. We also find that the regulation of the amounts of particular extracellular matrix components is critical for BBB establishment. Furthermore, the process of mesenchymal-epithelial transition of BBB-forming cells is perturbed in the absence of Mmp2. These data indicate that the presence of Mmp(s), which is typically considered to be a risk factor for BBB degradation, is essential for BBB integrity in Drosophila. Biological Sciences; Molecular Biology; Cell Biology

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Involvement of p38 in Age-Related Decline in Adult Neurogenesis via Modulation of Wnt Signaling

Kase Y., Otsu K., Shimazaki T., Okano H.

Stem Cell Reports （Stem Cell Reports） 12 （ 6 ） 1313 - 1328 2019年06月

ISSN 22136711

© 2019 The Author(s) Neurogenesis in specific brain regions in adult mammals decreases with age. Progressive reduction in the proliferation of neural stem and progenitor cells (NS/PCs) is a primary cause of this age-associated decline. However, the mechanism responsible for this reduction is poorly understood. We identify p38 MAPK as a key factor in the proliferation of neural progenitor cells (NPCs) in adult neurogenic niches. p38 expression in adult NS/PCs is downregulated during aging. Deletion of p38α in NS/PCs specifically reduces the proliferation of NPCs but not stem cells. Conversely, forced expression of p38α in NS/PCs in the aged mouse subventricular zone (SVZ) restores NPC proliferation and neurogenesis, and prevents age-dependent SVZ atrophy. We also found that p38 is necessary for suppressing the expression of Wnt antagonists DKK1 and SFRP3, which inhibit the proliferation of NPCs. Age-related reduction in p38 thus leads to decreased adult neurogenesis via downregulation of Wnt signaling. Kase et al. show that p38 expression in neural stem and progenitor cells (NS/PCs) in the adult brain decreases with aging. This reduction specifically causes proliferation defect in neural progenitor cells (NPCs), leading to the age-dependent decline of adult neurogenesis. Conversely, overexpression of p38α in NS/PCs in the aged brain restores NPC proliferation without exhaustion of neural stem cells.

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Cell-specific overexpression of COMT in dopaminergic neurons of Parkinson's disease

Kuzumaki N., Suda Y., Iwasawa C., Narita M., Sone T., Watanabe M., Maekawa A., Matsumoto T., Akamatsu W., Igarashi K., Tamura H., Takeshima H., Tawfik V., Ushijima T., Hattori N., Okano H., Narita M.

Brain : a journal of neurology （Brain : a journal of neurology） 142 （ 6 ） 1675 - 1689 2019年06月

© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com. The mechanism by which dopaminergic neurons are selectively affected in Parkinson's disease is not fully understood. In this study, we found a dramatic increase in the expression of catechol-O-methyltransferase (COMT), along with a lower level of DNA methylation, in induced pluripotent stem cell-derived dopaminergic neurons from patients with parkin (PARK2) gene mutations compared to those from healthy controls. In addition, a significant increase in the expression of COMT was found in dopaminergic neurons of isogenic PARK2 induced pluripotent stem cell lines that mimicked loss of function of PARK2 by CRISPR Cas9 technology. In dopamine transporter (DAT)-Cre mice, overexpression of COMT, specifically in dopaminergic neurons of the substantia nigra, produced cataleptic behaviours associated with impaired motor coordination. These findings suggest that upregulation of COMT, likely resulting from DNA hypomethylation, in dopaminergic neurons may contribute to the initial stage of neuronal dysfunction in Parkinson's disease.

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Estimating the concentration of therapeutic range using disease-specific iPS cells: Low-dose rapamycin therapy for Pendred syndrome

Hosoya M., Saeki T., Saegusa C., Matsunaga T., Okano H., Fujioka M., Ogawa K.

Regenerative Therapy （Regenerative Therapy） 10 54 - 63 2019年06月

© 2018 The Japanese Society for Regenerative Medicine Introduction: Pendred syndrome is an autosomal-recessive disease characterized by congenital hearing loss and thyroid goiter. Previously, cell stress susceptibilities were shown to increase in patient-derived cells with intracellular aggregation using an in vitro acute cochlear cell model derived from patient-specific pluripotent stem (iPS) cells. Moreover, we showed that rapamycin can relieve cell death. However, studies regarding long-term cell survival without cell stressors that mimic the natural course of disease or the rational minimum concentration of rapamycin that prevents cell death are missing. Methods: In this report, we first investigated the rational minimum concentration of rapamycin using patient-specific iPS cells derived-cochlear cells with three different conditions of acute stress. We next confirmed the effects of rapamycin in long-term cell survival and phenotypes by using cochlear cells derived from three different patient-derived iPS cells. Results: We found that inner ear cells derived from Pendred syndrome patients are more vulnerable than those from healthy individuals during long-term culturing; however, this susceptibility was relieved via treatment with low-dose rapamycin. The slow progression of hearing loss in patients may be explained, in part, by the vulnerability observed in patient cells during long-term culturing. We successfully evaluated the rational minimum concentration of rapamycin for treatment of Pendred syndrome. Conclusion: Our results suggest that low-dose rapamycin not only decreases acute symptoms but may prevent progression of hearing loss in Pendred syndrome patients.

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Visualization of spatiotemporal dynamics of human glioma stem cell invasion

Tamura R., Miyoshi H., Sampetrean O., Shinozaki M., Morimoto Y., Iwasawa C., Fukaya R., Mine Y., Masuda H., Maruyama T., Narita M., Saya H., Yoshida K., Okano H., Toda M.

Molecular Brain （Molecular Brain） 12 （ 1 ） 2019年05月

© 2019 The Author(s). Glioblastoma exhibits phenotypic and genetic heterogeneity, aggressive invasiveness, therapeutic resistance, and tumor recurrence, which can be explained by the existence of glioma stem cells (GSCs). In this study, we visualized the spatiotemporal dynamics of invasion of human GSCs in an orthotopic xenograft mouse model using time-lapse imaging of organotypic brain slice cultures and three-dimensional imaging of optically cleared whole brains. GSCs implanted in the striatum exhibited directional migration toward axon bundles, perivascular area, and the subventricular zone around the inferior horn of the lateral ventricle. GSCs migrated in a helical pattern around axon bundles in the striatum and invaded broadly in both the rostral and caudal directions. GSCs in the corpus callosum migrated more rapidly and unidirectionally toward the contralateral side with pseudopod extension. These characteristics of GSC invasion shared histological features observed in glioblastoma patients. Spatiotemporal visualization techniques can contribute to the elucidation of the mechanisms underlying GSC invasion that may lead to the development of effective therapy for glioblastoma.

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Correlative study using structural MRI and super-resolution microscopy to detect structural alterations induced by long-term optogenetic stimulation of striatal medium spiny neurons

Abe Y., Komaki Y., Seki F., Shibata S., Okano H., Tanaka K.

Neurochemistry International （Neurochemistry International） 125 163 - 174 2019年05月

ISSN 01970186

© 2019 Elsevier Ltd Striatal medium spiny neurons (MSNs) control motor function. Hyper- or hypo-activity of MSNs coincides with basal ganglia-related movement disorders. Based on the assumption that lasting alterations in neuronal activity lead to structural changes in the brain, understanding these structural alterations may be used to infer MSN functional abnormalities. To infer MSN function from structural data, understanding how long-lasting alterations in MSN activity affect brain morphology is essential. To address this, we utilized a simplified model of functional induction by stimulating MSNs expressing channelrhodopsin 2 (ChR2). Subsequent structural alterations which induced long-term activity changes in these MSNs were investigated in the striatal pathway and its associated regions by diffusion tensor imaging (DTI) and histological assessment with super-resolution microscopy. DTI detected changes in the striatum, substantia nigra, and motor cortex. Histological assessment found a reduction in the diameter of myelinated cortical axons as well as MSN dendrites and axons. The structural changes showed a high correlation between DTI parameters and histological data. These results demonstrated that long-term neural activation in the MSNs alters the diameter of MSN and cortical neurons fibers. This study provides a tool for understanding the causal relationship between functional and structural alterations.

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