Updated on 2021/03/30

写真a

 
FUJITA Kosuke
 
Organization
Graduate School of Medicine Program in Integrated Medicine Head and Neck and Sensory Organ Medicine Assistant Professor
Graduate School
Graduate School of Medicine
Undergraduate School
School of Medicine Department of Medicine
Title
Assistant Professor

Degree 1

  1. 博士(農学) ( 2006.9   岩手大学 ) 

Research Interests 3

  1. 遺伝子治療

  2. アデノ随伴ウイルス

  3. 網膜

Research Areas 2

  1. Life Science / Ophthalmology

  2. Life Science / Molecular biology

Research History 7

  1. Nagoya University   Assistant Professor

    2020.10

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    Country:Japan

  2. Tohoku University   Assistant Professor

    2018.4 - 2020.9

  3. 東北大学医学系研究科   助手

    2013.4 - 2018.3

  4. 大阪大学理学研究科   特任研究員

    2009.4 - 2013.3

  5. National Institute of Agrobiological Sciences

    2006.10 - 2009.3

  6. 博士号取得(岩手大学 連合農学研究科)

    2006.10

  7. 日本学術振興会   特別研究員(DC2)

    2004.10 - 2006.3

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Education 3

  1. Iwate University

    2001.4 - 2006.9

  2. Iwate University

    1999.4 - 2001.3

  3. Iwate University

    1995.4 - 1999.3

Professional Memberships 3

  1. 日本分子生物学会

  2. 日本神経科学学会

  3. 日本応用動物昆虫学会

 

Papers 40

  1. A hypomorphic variant in EYS detected by genome-wide association study contributes toward retinitis pigmentosa

    Nishiguchi Koji M., Miya Fuyuki, Mori Yuka, Fujita Kosuke, Akiyama Masato, Kamatani Takashi, Koyanagi Yoshito, Sato Kota, Takigawa Toru, Ueno Shinji, Tsugita Misato, Kunikata Hiroshi, Cisarova Katarina, Nishino Jo, Murakami Akira, Abe Toshiaki, Momozawa Yukihide, Terasaki Hiroko, Wada Yuko, Sonoda Koh-Hei, Rivolta Carlo, Tsunoda Tatsuhiko, Tsujikawa Motokazu, Ikeda Yasuhiro, Nakazawa Toru

    COMMUNICATIONS BIOLOGY   Vol. 4 ( 1 ) page: 140   2021.1

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    Publisher:Communications Biology  

    DOI: 10.1038/s42003-021-01662-9

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    Scopus

    PubMed

  2. Companion diagnosis for retinal neuroprotective treatment by real-time imaging of calpain activation using a novel fluorescent probe. Reviewed International journal

    Toshifumi Asano, Yuri Nagayo, Satoru Tsuda, Azusa Ito, Wataru Kobayashi, Kosuke Fujita, Kota Sato, Koji M Nishiguchi, Hiroshi Kunikata, Hiroyoshi Fujioka, Mako Kamiya, Yasuteru Urano, Toru Nakazawa

    Bioconjugate chemistry   Vol. 31 ( 9 ) page: 2241 - 2251   2020.9

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    Language:English   Publishing type:Research paper (scientific journal)  

    Calpain activation induces retinal ganglion cell (RGC) death, while calpain inhibition suppresses RGC death, in animal studies. However, the role of calpain in human retinal disease is unclear. This study investigated a new strategy to study the role of calpain based on real-time imaging. We synthesized a novel fluorescent probe for calpain, acetyl-L-leucyl-L-methionine-hydroxymethyl rhodamine green (Ac-LM-HMRG) and used it for real-time imaging of calpain activation. The toxicity of Ac-LM-HMRG was evaluated with a lactate dehydrogenase (LDH) cytotoxicity assay, retinal sections, and electroretinograms (ERG). Here, we performed real-time imaging of calpain activation in a rat model. First, we administered N-methyl-D-aspartate (NMDA) to induce retinal injury. Twenty minutes later, we administered an intravitreal injection of Ac-LM-HMRG. Real-time imaging was then completed with a non-invasive confocal scanning laser ophthalmoscope. The inhibitory effect of SNJ-1945 against calpain activation was also examined with the same real-time imaging method. Ac-LM-HMRG had no toxic effects. The number of Ac-LM-HMRG-positive cells in real-time imaging significantly increased after NMDA injury, and SNJ-1945 significantly lowered the number of Ac-LM-HMRG-positive cells. Real-time imaging with Ac-LM-HMRG was able to quickly quantify the NMDA-induced activation of calpain and the inhibitory effect of SNJ-1945. This technique, used as a companion diagnostic system, may aid research into the development of new neuroprotective therapies.

    DOI: 10.1021/acs.bioconjchem.0c00435

    PubMed

  3. Serum anti-recoverin antibodies is found in elderly patients with retinitis pigmentosa and cancer. Reviewed International journal

    Taimu Sato, Koji M Nishiguchi, Kosuke Fujita, Fuyuki Miya, Takashi Inoue, Erika Sasaki, Toshifumi Asano, Satoru Tsuda, Yukihiro Shiga, Hiroshi Kunikata, Mitsuru Nakazawa, Toru Nakazawa

    Acta ophthalmologica   Vol. 98 ( 6 ) page: e722 - e729   2020.9

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    Language:English   Publishing type:Research paper (scientific journal)  

    PURPOSE: To screen for anti-recoverin antibodies in elderly patients with retinitis pigmentosa (RP) with or without cancer and cross-sectionally characterize the seropositive patients clinically. METHODS: Serum from 75 RP patients who had been tested for mutations in a panel of 83 RP genes and 73 normal controls, all aged 50-80 years, were screened for anti-recoverin antibodies by Western blot using recombinant recoverin, retinal lysate from a marmoset and commercial anti-recoverin antibodies as a control. RESULTS: Three RP patients with typical pigmentary degeneration of the 75 (4.0%) were seropositive for anti-recoverin antibody. Pathogenic mutations were identified in two seropositive RP patients. All three patients had visual impairment since childhood and were diagnosed as RP by the age of 30. The severity of the retinopathy varied greatly among these three patients, ranging in visual acuity from light perception OU to 20/30 OU. Retinitis pigmentosa (RP) patients with a history of cancer were more likely to have anti-recoverin antibodies (3/14; 21.4%) than those without (0/61; 0%; p = 0.005, Fischer exact test). All 73 healthy controls with no history of cancer were also seronegative. CONCLUSION: Our results show that serum anti-recoverin antibodies can be detected in typical RP patients with identified pathogenic mutations and that a history of cancer may increase the risk of developing anti-recoverin antibodies.

    DOI: 10.1111/aos.14373

    PubMed

  4. Association of CRX genotypes and retinal phenotypes confounded by variable expressivity and electronegative electroretinogram. Reviewed International journal

    Koji M Nishiguchi, Hiroshi Kunikata, Kosuke Fujita, Kazuki Hashimoto, Yoshito Koyanagi, Masato Akiyama, Yasuhiro Ikeda, Yukihide Momozawa, Koh-Hei Sonoda, Akira Murakami, Yuko Wada, Toru Nakazawa

    Clinical & experimental ophthalmology   Vol. 48 ( 5 ) page: 644 - 657   2020.7

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    Language:English   Publishing type:Research paper (scientific journal)  

    IMPORTANCE: A framework for understanding the phenotypic features of CRX retinopathy was established. BACKGROUND: To perform a phenotype-genotype correlation analysis in two groups of patients with heterozygous mutations in distinct locations of the CRX gene, encoding the cone-rod homeobox. DESIGN: Multicentre retrospective study. PARTICIPANTS: Twenty-one Japanese patients from 14 families with a heterozygous CRX mutation. METHODS: Retrospective data analysis. MAIN OUTCOME MEASURES: Clinical records on CRX mutation, symptoms, best-corrected visual acuity (BCVA), visual field, fundus photography, fundus auto-fluorescence, optical coherence tomography and electroretinograms (ERGs). RESULTS: Six different CRX heterozygous mutations were identified in the subjects. Twelve patients from 9 families shared the p.R41W mutation and 1 patient had the p.R43C mutation, both of which affect the homeobox domain of CRX. These patients often displayed adult-onset retinal dystrophy with macular degeneration. In contrast, five patients with downstream mutations (p.S204fs, p.S213fs, p.G243X and p.L299F) displayed retinal degeneration or macular degeneration with bone-spicule pigmentation. Three asymptomatic carriers with different mutations (p.R41W, p.S213fs and p.G243X) were present in both groups. Nearly all patients and carriers had an electronegative ERG in response to a bright flash under dark adaptation. There was no cross-sectional association between patients' age and BCVA, despite progressive decline in BCVA. CONCLUSIONS AND RELEVANCE: Heterozygous mutations within or downstream of the homeobox domain in CRX relate to the difference associated retinal phenotypes, which was confounded by variable expressivity and electronegative ERGs. CRX mutations should be considered in patients with an electronegative ERG with minimal or no macular changes.

    DOI: 10.1111/ceo.13743

    PubMed

  5. A founder Alu insertion in RP1 gene in Japanese patients with retinitis pigmentosa. Reviewed

    Koji Miura Nishiguchi, Kosuke Fujita, Yasuhiro Ikeda, Hiroshi Kunikata, Yoshito Koyanagi, Masato Akiyama, Toshiaki Abe, Yuko Wada, Koh-Hei Sonoda, Toru Nakazawa

    Japanese journal of ophthalmology   Vol. 64 ( 4 ) page: 346 - 350   2020.7

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    Language:English   Publishing type:Research paper (scientific journal)  

    PURPOSE: To screen for the 328 bp Alu insertion (c.4052_4053ins328, p.Tyr1352Alafs) in RP1 in a group of retinitis pigmentosa (RP) patients who had been previously identified with a heterozygous deleterious mutation in the gene. STUDY DESIGN: Prospective, clinical and experimental study. METHODS: The Alu insertion in RP1 was screened with an optimized PCR-based method in 26 RP patients with a heterozygous deleterious mutation (nonsense or frameshift) in RP1 that had been identified in a preceding genetic study. The genetic location of the previously identified mutation and its inheritance pattern were assessed. RESULTS: Out of 26 RP patients with a heterozygous deleterious mutation in RP1, 5 (19.2%) were found to carry an additional heterozygous Alu insertion, presumably resulting in a compound heterozygous state. This included 3 patients who had been previously diagnosed as autosomal dominant RP based on genetic findings. They were re-diagnosed as having an autosomal recessive disease following our new findings. In all patients identified with the Alu insertion, the other mutations found in the preceding study were outside the defined region in exon 4 (encoding amino acids 677 to 917) in which truncation mutations have been suggested to exert a dominant negative effect. CONCLUSION: The founder Alu insertion in RP1 is an important cause of autosomal recessive RP in Japanese patients and can be missed in standard targeted resequencing. Screening optimized for this mutation is warranted, particularly in patients with a heterozygous deleterious mutation outside the defined region in exon 4 of RP1.

    DOI: 10.1007/s10384-020-00732-5

    PubMed

  6. Single AAV-mediated mutation replacement genome editing in limited number of photoreceptors restores vision in mice. Reviewed International journal

    Koji M Nishiguchi, Kosuke Fujita, Fuyuki Miya, Shota Katayama, Toru Nakazawa

    Nature communications   Vol. 11 ( 1 ) page: 482 - 482   2020.1

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    Language:English   Publishing type:Research paper (scientific journal)  

    Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end-joining (MMEJ). In blind mice, the mutation replacement rescued approximately 10% of photoreceptors, resulting in an improvement in light sensitivity and an increase in visual acuity. These effects were comparable to restoration mediated by gene supplementation, which targets a greater number of photoreceptors. This strategy may be applied for the treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible.

    DOI: 10.1038/s41467-019-14181-3

    PubMed

  7. In vivo imaging of the light response in mouse retinal ganglion cells based on a neuronal activity-dependent promoter. Reviewed International journal

    Fujita K, Nishiguchi KM, Sato K, Nakagawa Y, Nakazawa T

    Biochemical and biophysical research communications   Vol. 521 ( 2 ) page: 471 - 477   2020.1

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1016/j.bbrc.2019.10.155

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  8. Phenotypic Features of Oguchi Disease and Retinitis Pigmentosa in Patients with S-Antigen Mutations: A Long-Term Follow-up Study. Reviewed International journal

    Koji M Nishiguchi, Yasuhiro Ikeda, Kosuke Fujita, Hiroshi Kunikata, Makoto Akiho, Kazuki Hashimoto, Katsuhiro Hosono, Kentaro Kurata, Yoshito Koyanagi, Masato Akiyama, Takefumi Suzuki, Ryo Kawasaki, Yuko Wada, Yoshihiro Hotta, Koh-Hei Sonoda, Akira Murakami, Mitsuru Nakazawa, Toru Nakazawa, Toshiaki Abe

    Ophthalmology   Vol. 126 ( 11 ) page: 1557 - 1566   2019.11

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    Language:English   Publishing type:Research paper (scientific journal)  

    PURPOSE: To present phenotypic features of 22 patients with S-antigen (SAG) mutations. DESIGN: Retrospective cohort study. PARTICIPANTS: Twenty-one Japanese patients from 16 families with a homozygous c.924delA mutation and 1 patient with a homozygous c.636delT mutation in the SAG gene. METHODS: Clinical records on symptoms; best-corrected visual acuity; and Goldmann perimetry, fundus photography, fundus autofluorescence (FAF), OCT, and electroretinography results were reviewed. MAIN OUTCOME MEASURES: Best-corrected visual acuity, Goldmann perimetry results, imaging findings, and electroretinography results. RESULTS: Ten patients had Oguchi disease and 12 had retinitis pigmentosa (RP) with mean follow-up periods of 13.8 and 10.2 years, respectively. Retinitis pigmentosa patients were older (mean age, 56.0 years) than those with Oguchi disease (mean age, 22.1 years; P < 0.001) at the initial visit. Night blindness noted in childhood was the most common initial symptom for both Oguchi disease (80.0%) and RP (91.7%) patients. Best-corrected visual acuity in the logarithm of the minimum angle of resolution (logMAR) was well preserved in Oguchi disease patients (mean, 0.02 logMAR in both eyes) but reduced in most RP patients (mean, 1.32 logMAR [right eye] and 1.35 logMAR [left eye]). Similarly, the visual field in the retinal area was preserved in Oguchi disease patients (mean, 677 mm2 right eye and 667 mm2 left eye) and reduced in RP patients (mean, 369 mm2 right eye and 294 mm2 left eye). Fundus images revealed a characteristic golden sheen with no retinal degeneration in Oguchi disease patients, excluding 2 with macular degeneration detected by FAF, OCT, or both and 1 with mild retinal degeneration confirmed by OCT and fluorescein angiography. Pigmentary retinal degeneration most evident posteriorly was observed in RP patients, accompanied by a characteristic golden sheen in 12 of 14 patients undergoing ultra-widefield fundus imaging. OCT showed disrupted macular structure, and FAF revealed variable hypofluorescence. Electroretinography identified absent rod responses in both diseases, along with relative preservation of cone responses in Oguchi disease patients. Three patients showed progressive loss of the golden sheen based on fundus images, including 1 who demonstrated RP 26 years after the initial diagnosis of Oguchi disease. CONCLUSIONS: Retinitis pigmentosa with SAG mutations often shows a characteristic golden sheen surrounding posterior pigmentary retinal degeneration. Oguchi disease can show progressive degeneration in adulthood, rarely resulting in RP.

    DOI: 10.1016/j.ophtha.2019.05.027

    PubMed

  9. STEFTR: A Hybrid Versatile Method for State Estimation and Feature Extraction From the Trajectory of Animal Behavior. Reviewed International journal

    Shuhei J Yamazaki, Kazuya Ohara, Kentaro Ito, Nobuo Kokubun, Takuma Kitanishi, Daisuke Takaichi, Yasufumi Yamada, Yosuke Ikejiri, Fumie Hiramatsu, Kosuke Fujita, Yuki Tanimoto, Akiko Yamazoe-Umemoto, Koichi Hashimoto, Katsufumi Sato, Ken Yoda, Akinori Takahashi, Yuki Ishikawa, Azusa Kamikouchi, Shizuko Hiryu, Takuya Maekawa, Koutarou D Kimura

    Frontiers in neuroscience   Vol. 13 ( JUN ) page: 626 - 626   2019.6

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    Animal behavior is the final and integrated output of brain activity. Thus, recording and analyzing behavior is critical to understand the underlying brain function. While recording animal behavior has become easier than ever with the development of compact and inexpensive devices, detailed behavioral data analysis requires sufficient prior knowledge and/or high content data such as video images of animal postures, which makes it difficult for most of the animal behavioral data to be efficiently analyzed. Here, we report a versatile method using a hybrid supervised/unsupervised machine learning approach for behavioral state estimation and feature extraction (STEFTR) only from low-content animal trajectory data. To demonstrate the effectiveness of the proposed method, we analyzed trajectory data of worms, fruit flies, rats, and bats in the laboratories, and penguins and flying seabirds in the wild, which were recorded with various methods and span a wide range of spatiotemporal scales-from mm to 1,000 km in space and from sub-seconds to days in time. We successfully estimated several states during behavior and comprehensively extracted characteristic features from a behavioral state and/or a specific experimental condition. Physiological and genetic experiments in worms revealed that the extracted behavioral features reflected specific neural or gene activities. Thus, our method provides a versatile and unbiased way to extract behavioral features from simple trajectory data to understand brain function.

    DOI: 10.3389/fnins.2019.00626

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  10. Alternative methods to detect anti-TRPM1 antibodies. Reviewed International journal

    Nishiguchi KM, Fujita K, Inoue T, Nakazawa T

    Clinical & experimental ophthalmology   Vol. 47 ( 1 ) page: 148 - 149   2019.1

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1111/ceo.13365

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  11. Anti-TRPM1 antibodies in patients with retinal degeneration. Reviewed International journal

    Nishiguchi KM, Fujita K, Inoue T, Nakazawa T

    Clinical & experimental ophthalmology   Vol. 46 ( 9 ) page: 1087 - 1089   2018.12

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    DOI: 10.1111/ceo.13341

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  12. Reliable detection of low visual acuity in mice with pattern visually evoked potentials. Reviewed International journal

    Tokashiki N, Nishiguchi KM, Fujita K, Sato K, Nakagawa Y, Nakazawa T

    Scientific reports   Vol. 8 ( 1 ) page: 15948   2018.10

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1038/s41598-018-34413-8

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  13. Retained Plasticity and Substantial Recovery of Rod-Mediated Visual Acuity at the Visual Cortex in Blind Adult Mice with Retinal Dystrophy. Reviewed International journal

    Nishiguchi KM, Fujita K, Tokashiki N, Komamura H, Takemoto-Kimura S, Okuno H, Bito H, Nakazawa T

    Molecular therapy : the journal of the American Society of Gene Therapy   Vol. 26 ( 10 ) page: 2397 - 2406   2018.10

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1016/j.ymthe.2018.07.012

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  14. Ecel1 Knockdown With an AAV2-Mediated CRISPR/Cas9 System Promotes Optic Nerve Damage-Induced RGC Death in the Mouse Retina. Reviewed International journal

    Sato K, Shiga Y, Nakagawa Y, Fujita K, Nishiguchi KM, Tawarayama H, Murayama N, Maekawa S, Yabana T, Omodaka K, Katayama S, Feng Q, Tsuda S, Nakazawa T

    Investigative ophthalmology & visual science   Vol. 59 ( 10 ) page: 3943 - 3951   2018.8

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1167/iovs.18-23784

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  15. Prevalence of anti-retinal antibodies in epiretinal membranes and macular holes. Reviewed International journal

    Hiroshi Kunikata, Kosuke Fujita, Koji M Nishiguchi, Toru Nakazawa

    Clinical & experimental ophthalmology   Vol. 46 ( 5 ) page: 556 - 558   2018.7

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1111/ceo.13099

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  16. Levels of Anti-Retinal Antibodies in Retinal Detachment and Proliferative Vitreoretinopathy. Reviewed International journal

    Reo Ichinohasama, Koji M Nishiguchi, Kosuke Fujita, Naoko Aizawa, Takashi Inoue, Erika Sasaki, Hiroshi Kunikata, Toru Nakazawa

    Current eye research   Vol. 43 ( 6 ) page: 804 - 809   2018.6

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    Language:English   Publishing type:Research paper (scientific journal)  

    PURPOSE: The purpose of the study is to investigate the correlation between intraocular anti-retinal antibodies and clinical measurements in patients with rhegmatogenous retinal detachment (RRD) and proliferative vitreoretinopathy (PVR). MATERIAL AND METHODS: Aqueous humor and vitreous samples were collected from patients with RRD, PVR, and from control subjects with macular hole. The levels of total protein (TP), IgG, and anti-retinal antibodies were determined with a bicinchoninic acid assay, enzyme-linked immunosorbent assay, and dot blot, respectively. Correlations between these measurements were assessed using Pearson's correlation test. Analysis of variance followed by a post-hoc test or the Student t-test was used to compare differences between groups. RESULTS: The levels of anti-retinal antibodies and IgG were correlated with each other (P < 0.010). The IgG concentration was higher in patients with PVR than in controls in both the aqueous humor (P < 0.001) and the vitreous (P < 0.001), but not in patients with RRD. Conversely, TP levels and anti-retinal antibodies in both ocular fluids from RRD and PVR patients did not significantly differ from the controls. In a subgroup analysis, vitreal anti-retinal antibody levels were correlated with average macular thickness in the re-attached macula following surgery for macula-off RRD/PVR (P = 0.012). Furthermore, patients with post-operative cystoid macular edema had a higher level of vitreal anti-retinal antibodies than those without (P = 0.009). CONCLUSIONS: Intravitreal anti-retinal antibodies were increased in the eyes with maculopathy after surgical intervention for RRD/PVR.

    DOI: 10.1080/02713683.2018.1451544

    PubMed

  17. The neuroprotective effect of hesperidin in NMDA-induced retinal injury acts by suppressing oxidative stress and excessive calpain activation Reviewed International journal

    Shigeto Maekawa, Kota Sato, Kosuke Fujita, Reiko Daigaku, Hiroshi Tawarayama, Namie Murayama, Satoru Moritoh, Takeshi Yabana, Yukihiro Shiga, Kazuko Omodaka, Kazuichi Maruyama, Koji M. Nishiguchi, Toru Nakazawa

    Scientific Reports   Vol. 7 ( 1 ) page: 6885   2017.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Nature Publishing Group  

    We found that hesperidin, a plant-derived bioflavonoid, may be a candidate agent for neuroprotective treatment in the retina, after screening 41 materials for anti-oxidative properties in a primary retinal cell culture under oxidative stress. We found that the intravitreal injection of hesperidin in mice prevented reductions in markers of the retinal ganglion cells (RGCs) and RGC death after N-methyl-D-aspartate (NMDA)-induced excitotoxicity. Hesperidin treatment also reduced calpain activation, reactive oxygen species generation and TNF-α gene expression. Finally, hesperidin treatment improved electrophysiological function, measured with visual evoked potential, and visual function, measured with optomotry. Thus, we found that hesperidin suppressed a number of cytotoxic factors associated with NMDA-induced cell death signaling, such as oxidative stress, over-activation of calpain, and inflammation, thereby protecting the RGCs in mice. Therefore, hesperidin may have potential as a therapeutic supplement for protecting the retina against the damage associated with excitotoxic injury, such as occurs in glaucoma and diabetic retinopathy.

    DOI: 10.1038/s41598-017-06969-4

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  18. Spatially and Temporally Regulated NRF2 Gene Therapy Using Mcp-1 Promoter in Retinal Ganglion Cell Injury Reviewed International journal

    Kosuke Fujita, Koji M. Nishiguchi, Yukihiro Shiga, Toru Nakazawa

    MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT   Vol. 5   page: 130 - 141   2017.6

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

    Retinal ganglion cell degeneration triggered by axonal injury is believed to underlie many ocular diseases, including glaucoma and optic neuritis. In these diseases, retinal ganglion cells are affected unevenly, both spatially and temporally, such that healthy and unhealthy cells coexist in different patterns at different time points. Herein, we describe a temporally and spatially regulated adeno-associated virus gene therapy aiming to reduce undesired off-target effects on healthy retinal neurons. The Mcp-1 promoter previously shown to be activated in stressed retinal ganglion cells following murine optic nerve injury was combined with the neuroprotective intracellular transcription factor Nrf2. In this model, Mcp-1 promoter driven NRF2 expression targeting only stressed retinal ganglion cells showed efficacy equivalent to non-selective cytomegalovirus promoter-driven therapy for preventing cell death. However, cytomegalovirus promoter-mediated NRF2 transcription induced cellular stress responses and death of Brn3A-positive uninjured retinal ganglion cells. Such undesired effects were reduced substantially by adopting the Mcp-1 promoter. Combining a stress-responsive promoter and intracellular therapeutic gene is a versatile approach for specifically targeting cells at risk of degeneration. This strategy may be applicable to numerous chronic ocular and non-ocular conditions.

    DOI: 10.1016/j.omtm.2017.04.003

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  19. Calcium dynamics regulating the timing of decision-making in C. elegans Reviewed International journal

    Yuki Tanimoto, Akiko Yamazoe-Umemoto, Kosuke Fujita, Yuya Kawazoe, Yosuke Miyanishi, Shuhei J. Yamazaki, Xianfeng Fei, Karl Emanuel Busch, Keiko Gengyo-Ando, Junichi Nakai, Yuichi Lino, Yuishi Iwasaki, Koichi Hashimoto, Koutarou D. Kimura

    ELIFE   Vol. 6   2017.5

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELIFE SCIENCES PUBLICATIONS LTD  

    Brains regulate behavioral responses with distinct timings. Here we investigate the cellular and molecular mechanisms underlying the timing of decision-making during olfactory navigation in Caenorhabditis elegans. We find that, based on subtle changes in odor concentrations, the animals appear to choose the appropriate migratory direction from multiple trials as a form of behavioral decision-making. Through optophysiological, mathematical and genetic analyses of neural activity under virtual odor gradients, we further find that odor concentration information is temporally integrated for a decision by a gradual increase in intracellular calcium concentration ([Ca2+](i)), which occurs via L-type voltage-gated calcium channels in a pair of olfactory neurons. In contrast, for a reflex-like behavioral response, [Ca2+](i) rapidly increases via multiple types of calcium channels in a pair of nociceptive neurons. Thus, the timing of neuronal responses is determined by cell type-dependent involvement of calcium channels, which may serve as a cellular basis for decision-making.

    DOI: 10.7554/eLife.21629

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  20. The neuroprotective effect of latanoprost acts via klotho-mediated suppression of calpain activation after optic nerve transection Reviewed International journal

    Kotaro Yamamoto, Kota Sato, Masayoshi Yukita, Masayuki Yasuda, Kazuko Omodaka, Morin Ryu, Kosuke Fujita, Koji M. Nishiguchi, Shigeki Machida, Toru Nakazawa

    JOURNAL OF NEUROCHEMISTRY   Vol. 140 ( 3 ) page: 495 - 508   2017.2

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY  

    Latanoprost was first developed for use in glaucoma therapy as an ocular hypotensive agent targeting the prostaglandin F2 alpha (FP) receptor. Subsequently, latanoprost showed a neuroprotective effect, an additional pharmacological action. However, although it is well-known that latanoprost exerts an ocular hypotensive effect via the FP receptor, it is not known whether this is also true of its neuroprotective effect. Klotho was firstly identified as the gene linked to the suppression of aging phenotype: the defect of klotho gene in mice results aging phenotype such as hypokinesis, arteriosclerosis, and short lifespan. After that, the function of klotho was also reported to maintain calcium homeostasis and to exert a neuroprotective effect in various models of neurodegenerative disease. However, the function of klotho in eyes including retina is still poorly understood. Here, we show that klotho is a key factor underlying the neuroprotective effect of latanoprost during post-axotomy retinal ganglion cell (RGC) degeneration. Importantly, a quantitative RT-PCR gene expression analysis of klotho in sorted rat retinal cells revealed that the highest expression level of klotho in the retina was in the RGCs. Latanoprost acid, the biologically active form of latanoprost, inhibits post-traumatic calpain activation and concomitantly facilitates the expression and shedding of klotho in axotomized RGCs. This expression profile is a good match with the localization, not of the FP receptor, but of organic anion transporting polypeptide 2B1, known as a prostaglandin transporter, in the ocular tissue. Furthermore, an organic anion transporting polypeptide 2B1 inhibitor suppressed latanoprost acid-mediated klotho shedding exvivo, whereas an FP receptor antagonist did not. The klotho fragments shed from the RGCs reduced the intracellular level of reactive oxygen species, and a specific klotho inhibitor accelerated and increased RGC death after axotomy. We conclude that the shed klotho fragments might contribute to the attenuation of axonal injury-induced calpain activation and oxidative stress, thereby protecting RGCs from post-traumatic neuronal degeneration.

    DOI: 10.1111/jnc.13902

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  21. Genetic analysis of Japanese primary open-angle glaucoma patients and clinical characterization of risk alleles near CDKN2B-AS1, SIX6 and GAS7 Reviewed International journal

    Yukihiro Shiga, Japan Glaucoma Society Omics Group (JGS-OG), Koji M. Nishiguchi, Yosuke Kawai, Kaname Kojima, Kota Sato, Kosuke Fujita, Mai Takahashi, Kazuko Omodaka, Makoto Araie, Kenji Kashiwagi, Makoto Aihara, Takeshi Iwata, Fumihiko Mabuchi, Mitsuko Takamoto, Mineo Ozaki, Kazuhide Kawase, Nobuo Fuse, Masayuki Yamamoto, Jun Yasuda, Masao Nagasaki, Toru Nakazawa

    PLoS ONE   Vol. 12 ( 12 ) page: e0186678   2017

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    Purpose To test the genetic association between Japanese patients with primary open-angle glaucoma (POAG) and the previously reported POAG susceptibility loci and to perform genotype–phenotype analysis. Methods Genetic associations for 27 SNPs from 16 loci previously linked to POAG were assessed using genome-wide SNP data of the primary cohort (565 Japanese POAG patients and 1,104 controls). Reproducibility of the assessment was tested in 607 POAG cases and 455 controls (second cohort) with a targeted genotyping approach. For POAG-associated variants, a genotype–phenotype correlation study (additive, dominant, recessive model) was performed using the objective clinical data derived from 598 eyes of 598 POAG patients. Results Among 27 SNPs from 16 loci previously linked to POAG, genotypes for total of 20 SNPs in 13 loci were available for targeted association study. Among 8 SNPs in 3 loci that showed at least nominal association (P &lt
    5.00E-02) in the primary cohort, a representative SNP for each loci (rs2157719 for CDKN2B-AS1, rs33912345 for SIX6, and rs9913911 for GAS7) were selected. For these SNPs the association was found significant in both the second cohort analysis and meta-analysis. The genotype–phenotype analysis revealed significant correlations between CDKN2B-AS1 (rs2157719) and decreased intraocular pressure (? = -6.89 mmHg, P = 1.70E-04
    dominant model) after multiple corrections. In addition, nominal correlation was observed between CDKN2B-AS1 (rs2157719) and optic nerve head blood flow (? = -0.54 and -0.67 arbitrary units (AU), P = 2.00E-02 and 1.39E-02), between SIX6 (rs33912345) and decreased total peripapillary retinal nerve fiber layer thickness (? = -2.16 and -2.82 ?m, P = 4.68E-02 and 2.40E-02, additive and recessive model, respectively) and increased optic nerve head blood flow (? = 0.44 AU, P = 2.20E-02
    additive model) and between GAS7 (rs9913911) and increased cup volume (? = 0.03 mm3, P = 4.60E-02) and mean cup depth (? = 0.03 mm3, P = 4.11E-02
    additive model) and decreased pattern standard deviation (? = -0.87 dB, P = 2.44E-02
    dominant model). Conclusion The association between SNPs near GAS7 and POAG was found in Japanese patients for the first time. Clinical characterization of the risk variants is an important step toward understanding the pathology of the disease and optimizing treatment of patients with POAG.

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  22. Topical ocular dexamethasone decreases intraocular pressure and body weight in rats Reviewed

    Kota Sato, Koji M. Nishiguchi, Kazuichi Maruyama, Satoru Moritoh, Kosuke Fujita, Yoshikazu Ikuta, Hitoshi Kasai, Toru Nakazawa

    Journal of Negative Results in BioMedicine   Vol. 15 ( 1 ) page: 5   2016.3

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    Background: Recently, topical dexamethasone-induced ocular hypertension and a consequent loss of retinal ganglion cells (RGCs) have been described in mice. This has been proposed as a model of steroid-induced glaucoma. In this study, we set up and evaluated a similar model in rats. Results: Ten-week old Sprague Dawley (SD) rats (N = 12) were used to evaluate the effect of topical 0.1 % dexamethasone (50 μl) administered 3 times daily for 4 weeks. Sodium chloride (0.9 %) was used in another group of rats (N = 12) that served as the controls. After 1 week, we observed a progressive decrease in body weight in the dexamethasone-treated rats compared both to the pre-treatment baseline and the vehicle-treated rats. In contrast to earlier work that showed elevated Intraocular pressure (IOP) following dexamethasone instillation in mice, IOP in the rats unexpectedly fell to 11.3 ± 1.3 mmHg in the treated eyes, compared to 14.8 ± 2.4 mmHg in the untreated eyes, after 3 weeks of topical dexamethasone (P = 0.032). Blood tests performed after 4 weeks of treatment showed a 3.3-fold increase in both plasma cholesterol (P &lt
    0.001) and alanine transaminase (P = 0.019) in the dexamethasone-treated rats compared to the control rats. Meanwhile, topical steroid did not induce changes in either plasma blood glucose or glycated hemoglobin (HbA1c). We also did not detect changes in the expression of RGC markers (with real-time PCR) following the treatment. Conclusions: In contrast to mice, which previously showed increased IOP following the topical administration of dexamethasone, the rats displayed a paradoxical reduction in IOP following a similar treatment. This was accompanied by a loss of body weight without affecting the level of blood glucose.

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  23. Analysis of Macular Drusen and Blood Test Results in 945 Macaca fascicularis Reviewed

    Koji M. Nishiguchi, Yu Yokoyama, Yusuke Fujii, Kosuke Fujita, Yusuke Tomiyama, Ryo Kawasaki, Toshinori Furukawa, Fumiko Ono, Nobuhiro Shimozawa, Mutsumi Togo, Michihiro Suzuki, Toru Nakazawa

    PLoS One   Vol. 11 ( 10 ) page: e0164899   2016

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    Age-dependent formation of macular drusen caused by the focal accumulation of extracellular deposits beneath the retinal pigment epithelium precede the development of age related macular degeneration (AMD), one of the leading causes of blindness worldwide. It is established that inflammation contributes to the pathogenesis of drusen and AMD. However, development of a preemptive therapeutic strategy targeting macular drusen and AMD has been impeded by the lack of relevant animal models because most laboratory animals lack macula, an anatomic feature present only in humans and a subset of monkeys. Reportedly, macular drusen and macular degeneration develop in monkeys in an age-dependent manner. In this study, we analyzed blood test results from 945 Macaca fascicularis, 317 with and 628 without drusen. First, a trend test for drusen frequency (the Cochran-Armitage test) was applied to the quartile data for each parameter. We selected variables with an increasing or decreasing trend with higher quartiles at P &lt; 0.05, to which multivariate logistic regression analysis was applied. This revealed a positive association of age (odds ratio [OR]: 1.10 per year, 95% confidence interval [CI]: 1.07-1.12) and white blood cell count (OR: 1.01 per 1 x 10(3)/mu l, 95% CI: 1.00-1.01) with drusen. When the monkeys were divided by age, the association between drusen and white blood cell count was only evident in younger monkeys (OR: 1.01 per 1 x 10(3)/mu l, 95% CI: 1.00-1.02). In conclusion, age and white blood cell count may be associated with drusen development in M. fascicularis. Systemic inflammation may contribute to drusen formation in monkeys.

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  24. Measurement of Electroretinograms and Visually Evoked Potentials in Awake Moving Mice Reviewed

    Yusuke Tomiyama, Kosuke Fujita, Koji M. Nishiguchi, Naoyuki Tokashiki, Reiko Daigaku, Kitako Tabata, Eriko Sugano, Hiroshi Tomita, Toru Nakazawa

    PLOS ONE   Vol. 11 ( 6 ) page: e0156927   2016

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    The development of new treatments for intractable retinal diseases requires reliable functional assessment tools for animal models. In vivo measurements of neural activity within visual pathways, including electroretinogram (ERG) and visually evoked potential (VEP) recordings, are commonly used for such purposes. In mice, the ERG and VEPs are usually recorded under general anesthesia, a state that may alter sensory transduction and neurotransmission, but seldom in awake freely moving mice. Therefore, it remains unknown whether the electrophysiological assessment of anesthetized mice accurately reflects the physiological function of the visual pathway. Herein, we describe a novel method to record the ERG and VEPs simultaneously in freely moving mice by immobilizing the head using a custom-built restraining device and placing a rotatable cylinder underneath to allow free running or walking during recording. Injection of the commonly used anesthetic mixture xylazine plus ketamine increased and delayed ERG oscillatory potentials by an average of 67.5% and 36.3%, respectively, compared to unanesthetized mice, while having minimal effects on the a-wave and b-wave. Similarly, components of the VEP were enhanced and delayed by up to 300.2% and 39.3%, respectively, in anesthetized mice. Our method for electrophysiological recording in conscious mice is a sensitive and robust means to assess visual function. It uses a conventional electrophysiological recording system and a simple platform that can be built in any laboratory at low cost. Measurements using this method provide objective indices of mouse visual function with high precision and stability, unaffected by anesthetics.

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  25. Axial Spondylometaphyseal Dysplasia Is Caused by C21orf2 Mutations Reviewed

    Zheng Wang, Aritoshi Iida, Noriko Miyake, Koji M. Nishiguchi, Kosuke Fujita, Toru Nakazawa, Abdulrahman Alswaid, Mohammed A. Albalwi, Ok-Hwa Kim, Tae-Joon Cho, Gye-Yeon Lim, Bertrand Isidor, Albert David, Cecilie F. Rustad, Else Merckoll, Jostein Westvik, Eva-Lena Stattin, Giedre Grigelioniene, Ikuyo Kou, Masahiro Nakajima, Hirohumi Ohashi, Sarah Smithson, Naomichi Matsumoto, Gen Nishimura, Shiro Ikegawa

    PLOS ONE   Vol. 11 ( 3 ) page: e0150555   2016

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    Axial spondylometaphyseal dysplasia (axial SMD) is an autosomal recessive disease characterized by dysplasia of axial skeleton and retinal dystrophy. We conducted whole exome sequencing and identified C21orf2 (chromosome 21 open reading frame 2) as a disease gene for axial SMD. C21orf2 mutations have been recently found to cause isolated retinal degeneration and Jeune syndrome. We found a total of five biallelic C21orf2 mutations in six families out of nine: three missense and two splicing mutations in patients with various ethnic backgrounds. The pathogenic effects of the splicing (splice-site and branch-point) mutations were confirmed on RNA level, which showed complex patterns of abnormal splicing. C21orf2 mutations presented with a wide range of skeletal phenotypes, including cupped and flared anterior ends of ribs, lacy ilia and metaphyseal dysplasia of proximal femora. Analysis of patients without C21orf2 mutation indicated genetic heterogeneity of axial SMD. Functional data in chondrocyte suggest C21orf2 is implicated in cartilage differentiation. C21orf2 protein was localized to the connecting cilium of the cone and rod photoreceptors, confirming its significance in retinal function. Our study indicates that axial SMD is a member of a unique group of ciliopathy affecting skeleton and retina.

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  26. In vivo cellular imaging of various stress/response pathways using AAV following axonal injury in mice Reviewed

    Kosuke Fujita, Koji M. Nishiguchi, Yu Yokoyama, Yusuke Tomiyama, Satoru Tsuda, Masayuki Yasuda, Shigeto Maekawa, Toru Nakazawa

    SCIENTIFIC REPORTS   Vol. 5   page: 18141   2015.12

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    Glaucoma, a leading cause of blindness worldwide, is instigated by various factors, including axonal injury, which eventually leads to a progressive loss of retinal ganglion cells (RGCs). To study various pathways reportedly involved in the pathogenesis of RGC death caused by axonal injury, seven pathways were investigated. Pathway-specific fluorescent protein-coded reporters were each packaged into an adeno-associated virus (AAV). After producing axonal injury in the eye, injected with AAV to induce RGC death, the temporal activity of each stress-related pathway was monitored in vivo through the detection of fluorescent RGCs using confocal ophthalmoscopy. We identified the activation of ATF6 and MCP-1 pathways involved in endoplasmic reticulum stress and macrophage recruitment, respectively, as early markers of RGC stress that precede neuronal death. Conversely, inflammatory responses probed by NF-kappa B and cell-death-related pathway p53 were most prominent in the later phases, when RGC death was already ongoing. AAV-mediated delivery of stress/response reporters followed by in vivo cellular imaging is a powerful strategy to characterize the temporal aspects of complex molecular pathways involved in retinal diseases. The identification of promoter elements that are activated before the death of RGCs enables the development of pre-emptive gene therapy, exclusively targeting the early phases of diseased cells.

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  27. Modulation of different behavioral components by neuropeptide and dopamine signalings in non-associative odor learning of Caenorhabditis elegans Reviewed

    Akiko Yamazoe-Umemoto, Kosuke Fujita, Yuichi Lino, Yuishi Iwasaki, Koutarou D. Kimura

    NEUROSCIENCE RESEARCH   Vol. 99   page: 22 - 33   2015.10

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    An animal's behavior is modulated by learning; however, the behavioral component modulated by learning and the mechanisms of this modulation have not been fully understood. We show here that two types of neural signalings are required for the modulation of different behavioral components in non-associative odor learning in the nematode Caenorhabditis elegans. We have previously found that C. elegans avoid the repulsive odor 2-nonanone, and preexposure to the odor for 1 h enhances the avoidance behavior as a type of non-associative learning. Systematic quantitative analyses of behavioral components revealed that the odor preexposure caused increases in average duration of straight migration ("runs") only when the animals were migrating away from the odor source within a certain range of bearing, which likely corresponds to odor decrement. Further, genetic analyses revealed that the genes for neuropeptide or dopamine signalings are both required for the enhanced odor avoidance. Neuropeptide signaling genes were required for the preexposure-dependent increase in run duration. In contrast, dopamine signaling genes were required not for the increase in run duration but likely for maintenance of run direction. Our results suggests that multiple behavioral components are regulated by different neuromodulators even in non-associative learning in C. elegans. (C) 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

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  28. BmEts upregulates promoter activity of lebocin in Bombyx mori Reviewed

    Hiromitsu Tanaka, Aki Sagisaka, Kosuke Fujita, Seiichi Furukawa, Jun Ishibashi, Minoru Yamakawa

    INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY   Vol. 42 ( 7 ) page: 474 - 81   2012.7

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    The Ets family protein BmEts is assumed to be implicated in determination of diapause in the embryogenesis of Bombyx mori. In this study, we found that expression of BmEts was increased in the fat body and other tissues of the 5th instar larvae in response to Escherichia coli injection. Cotransfection experiments using a silkworm cell line revealed that overexpression of BmEts significantly elevated the activity of lebocin promoter but not of cecropin B1, cecropin D. attacin, and moricin promoters. Activation of the lebocin promoter by BmEts was dependent on at least two kappa B elements and the most proximal GGAA/T motif located on the 5'-upstream region. BmEts further synergistically enhanced E. coli or BmRelish1-d2 (active form)-stimulated lebocin promoter activation. Two kappa B elements were also found to be involved in promoter activation by BmRelish1-d2 and in synergistic promoter activation by BmEts and BmRelish1-d2 in the silkworm cells. Specific binding of recombinant BmEts to the proximal kappa B element and the most proximal GGAA/T motif and interaction between BmEts and BmRelish1 were also observed. To our knowledge, this is the first report of an Ets family protein directly regulating immune-related genes in invertebrates. (C) 2012 Elsevier Ltd. All rights reserved.

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  29. A Novel Method to Convert a DNA Fragment Inserted into a Plasmid to an Inverted Repeat Structure Reviewed

    Kazuya Tomimoto, Kosuke Fujita, Jun Ishibashi, Shigeo Imanishi, Minoru Yamakawa, Hiromitsu Tanaka

    MOLECULAR BIOTECHNOLOGY   Vol. 50 ( 1 ) page: 18 - 27   2012.1

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    Transfection of an expression plasmid possessing inverted repeat (IR) DNA into cultured cells leads to the overexpression of hairpin RNA and efficient suppression of target gene expression. Such DNA vector-based RNA interference (RNAi) is widely used for characterizing genes of interest in cultured cell lines. In this study, we developed a new method to convert an inserted DNA fragment (IDF) in specially designed plasmid vectors into an IR structure by using nicking endonucleases and BcaBEST DNA polymerase. This method consists of the following steps: (1) linearization of the plasmid with a nick by using a restriction enzyme and a nicking endonuclease, (2) formation of the hairpin-loop DNA at the end near the IDF of the linearized plasmid, (3) insertion of a nick at the other end of the IDF by a nicking endonuclease, (4) execution of the strand displacement reaction from the nick to synthesize IR DNA, and (5) self-ligation of the linear double-stranded DNA. The IR DNA containing expression plasmids constructed by this method effectively induced target-specific RNAi in a silkworm cell line. We further established a method to purify expression plasmids containing IR DNA. Our new methods provide techniques for the construction of long hairpin RNA (lhRNA) expression plasmids for silencing specific genes in silkworms and other organisms, and offer a fundamental methodology for constructing an lhRNA expression library from a cDNA plasmid library.

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  30. Enhancement of Odor Avoidance Regulated by Dopamine Signaling in Caenorhabditis elegans Reviewed

    Koutarou D. Kimura, Kosuke Fujita, Isao Katsura

    JOURNAL OF NEUROSCIENCE   Vol. 30 ( 48 ) page: 16365 - 75   2010.12

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    The enhancement of sensory responses after prior exposure to a stimulus is a fundamental mechanism of neural function in animals. Its molecular basis, however, has not been studied in as much depth as the reduction of sensory responses, such as adaptation or habituation. We report here that the avoidance behavior of the nematode Caenorhabditis elegans in response to repellent odors (2-nonanone or 1-octanol) is enhanced rather than reduced after preexposure to the odors. This enhancement effect of preexposure was maintained for at least 1 h after the conditioning. The enhancement of 2-nonanone avoidance was not dependent on the presence or absence of food during conditioning, which generally functions as a strong positive or negative unconditioned stimulus in the animals. These results suggest that the enhancement is acquired as a type of nonassociative learning. In addition, genetic and pharmacological analyses revealed that the enhancement of 2-nonanone avoidance requires dopamine signaling via D2-like dopamine receptor DOP-3, which functions in a pair of RIC interneurons to regulate the enhancement. Because dopamine signaling has been tightly linked with food-related information to modulate various behaviors of C. elegans, it may play different role in the regulation of the enhancement of 2-nonanone avoidance. Thus, our data suggest a new genetic and pharmacological paradigm for nonassociative enhancement of neural responses that is regulated by dopamine signaling.

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  31. EXPRESSION PROFILING OF NOVEL BACTERIA-INDUCED GENES FROM THE SILKWORM, Bombyx mori Reviewed

    Hiromitsu Tanaka, Noriko Suzuki, Yoshiro Nakajima, Masaaki Sato, Aki Sagisaka, Kosuke Fujita, Jun Ishibashi, Shigeo Imanishi, Kazuei Mita, Minoru Yamakawa

    ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY   Vol. 73 ( 3 ) page: 148 - 62   2010.3

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    In this study, we have newly identified three bacteria-induced genes from the silkworm Bombyx mori by quantitative reverse transcriptase-polymerase chain reaction. One of these, eukaryotic initiation factor 4E-1 (eIF4E-1), is assumed to encode an eIF4E family, which plays a role in the initiation of translation as a mRNA cap-binding protein. The second gene is BmFOXG1, belonging to a family of forkhead transcription factors, FOXG1. The third gene is MBF2-related (MBF2-R) whose product has high homology to a co-activator protein MBF2 from B. mori. Although BmFOXG1 was up-regulated in the fat body in response to three kinds of bacteria, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, eIF4E-1 and MBF2-R were up-regulated by E. coli and B. subtilis, but not S. aureus, suggesting that bacteria possessing meso-diaminopimelic acid-containing peptidoglycan but not lysine-containing peptidoglycan activate eIF4E-1 and MBF2-R, probably through a conserved immune deficiency pathway. We further profiled the expression of three genes in different tissues and a silkworm cell line, NIAS-Bm-aff3, in response to bacteria, and at different times after bacterial challenge in the fat body. (C) 2010 Wiley Periodicals, Inc.

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  32. Genorne-wide analysis of host gene expression in the silkworm cells infected with Bombyx mori nucleopolyhedrovirus Reviewed

    Aki Sagisaka, Kosuke Fujita, Yuki Nakamura, Jun Ishibashi, Hiroaki Noda, Shigeo Imanishi, Kazuei Mita, Minoru Yamakawa, Hiromitsu Tanaka

    VIRUS RESEARCH   Vol. 147 ( 2 ) page: 166 - 75   2010.2

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    The global transcriptional profile of host genes in the silkworm cell line during the early phase of Bombyx mori nucleopolyhedrovirus (BmNPV) infection was analyzed by oligonucleotide microarray. Our analysis showed 35 genes were significantly up-regulated and 17 genes were significantly down-regulated. This is the first report of changes in the expression of these genes in response to NPV infection. We further quantified the levels of mRNA expression by quantitative reverse transcriptase-polymerase chain reaction and confirmed that the expression of 13 (such as BmEts and BmToll10-3) genes significantly increased and 7 genes (such as Hsp20-1) significantly decreased after BmNPV infection. However, the expression levels of most genes were not dramatically changed except BmEts expression increased approximately 8.0-fold 12 h after BmNPV infection. (C) 2009 Elsevier B.V. All rights reserved.

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  33. DNA Vector-Based RNA Interference in Cell Lines Derived from Bombyx mori Reviewed

    Kosuke Fujita, Aki Sagisaka, Kazuya Tomimoto, Jun Ishibashi, Shigeo Imanishi, Minoru Yamakawa, Hiromitsu Tanaka

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   Vol. 73 ( 9 ) page: 2026 - 31   2009.9

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    Gene-knockdown technology using RNA interference (RNAi) is widely used to characterize gene functions in many organisms. In this study, we analyzed the conditions for employing DNA vector-based RNAi in silkworm cell lines using long-hairpin RNA-expressing plasmid DNA. We found that NIAS-Bm-oyanagi2 was the most effective cell line for RNAi. Expression of long-hairpin RNA containing an approximately 500 base-pair stem region suppressed expression of a reporter target gene by more than 99% in this cell line. Furthermore, the loop sequence of hairpin RNA was not as important to RNAi efficiency as previously observed in Drosophila melanogaster. DNA vector-based RNAi also induced significant suppression of endogenous clathrin in NIAS-Bm-oyanagi2. Luciferase activity from recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) containing luciferase in the clathrin-knockdown cells was significantly less than in the control cells, suggesting that clathrin is indispensable for the entry of BmNPV into silkworm cell lines.

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  34. Correlation of Differential Expression of Silkworm Antimicrobial Peptide Genes with Different Amounts of Rel Family Proteins and Their Gene Transcriptional Activity Reviewed

    Hiromitsu Tanaka, Aki Sagisaka, Yoshiro Nakajima, Kosuke Fujita, Shigeo Imanishi, Minoru Yamakawa

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   Vol. 73 ( 3 ) page: 599 - 606   2009.3

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    In the silkworm, Bombyx mori, antimicrobial peptide (AMP) genes are upregulated in the larval fat body by injection of bacteria and peptidoglycans (PGNs). The DAP-type PGN from Escherichia coli and Bacillus subtilis exhibited stronger elicitor activity for expression of AMP genes in B. mori than Lys-type PGN from Staphylococcus aureus, suggesting that differences in bacterial influence on the induction levels of these genes depend on the differences in types of PGN. BmRelish1 mRNA was more abundant than BmRel mRNAs in the larval fat bodies. Moreover, the ability of the BmRelish1 active form to enhance the promoter activity of AMP genes was higher than that of BmRels. The difference was related to the binding affinity of Rel family proteins to kappa B sites. Our results suggest that different amounts and different transcriptional activities of Rel family proteins result in differential activation of AMP genes by PGN type and bacterium species.

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  35. shRNA Expression Plasmids Generated by a Novel Method Efficiently Induce Gene-Specific Knockdown in a Silkworm Cell Line Reviewed

    Hiromitsu Tanaka, Kosuke Fujita, Aki Sagisaka, Kazuya Tomimoto, Shigeo Imanishi, Minoru Yamakawa

    MOLECULAR BIOTECHNOLOGY   Vol. 41 ( 2 ) page: 173 - 9   2009.2

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    RNAi knockdown by using shRNA expression plasmids is widely used to determine the function of individual genes in mammals. Here we developed a simple method to create an IR DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded IR DNA with short hairpin structure and Bst DNA polymerase. Furthermore, we demonstrated that the shRNA expression plasmids constructed by our method effectively induced target-specific RNAi in the silkworm cell line. We also found that sequence preference in the silkworm cell line was much lower than in mammalian cells and shRNA-induced RNAi was influenced by the length of the stem region.

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  36. Lipopolysaccharide elicits expression of immune-related genes in the silkworm, Bombyx mori Reviewed

    H. Tanaka, A. Sagisaka, K. Fujita, Y. Kaneko, S. Imanishi, M. Yamakawa

    INSECT MOLECULAR BIOLOGY   Vol. 18 ( 1 ) page: 71 - 75   2009.2

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    Lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria, was found to be unable to activate immune-related genes in Drosophila melanogaster. In contrast, highly purified LPS elicited immune-related gene expression in the fat body of Bombyx mori. However, the level of activation by highly purified LPS was lower than crude LPS and peptidoglycan. Furthermore, synthetic lipid A also activated these genes, suggesting that B. mori possesses unknown signal pathways to activate immune-related genes by LPS. Up-regulation of antimicrobial peptide genes by highly purified LPS was not confirmed in the immune-responsive cell line, NIAS-Bm-aff3, suggesting that some factors necessary for signal transduction activated by LPS are deficient in this cell line.

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  37. A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori Reviewed

    Hiromitsu Tanaka, Jun Ishibashi, Kosuke Fujita, Yoshiro Nakajima, Aki Sagisaka, Kazuya Tomimoto, Noriko Suzuki, Mikio Yoshiyama, Yoichi Kaneko, Takashi Iwasaki, Tomoya Sunagawa, Kayoko Yamaji, Ai Asaoka, Kazuei Mita, Minoru Yamakawa

    INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY   Vol. 38 ( 12 ) page: 1087 - 110   2008.12

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    A genome-wide analysis of innate immunity-related genes and gene families was conducted using the silkworm, Bombyx mod. We identified orthologs for a large number of genes involved in insect immunity that have been reported from Drosophila melanogaster (Diptera), Anopheles gambiae (Diptera), Apis mellifera (Hymenoptera) and Tribolium castaneum (Coleoptera). B. mod has a unique recognition gene and antimicrobial peptide genes that are not present in the Drosophila, Anopheles, Apis and Tribolium genomes, suggesting a lineage-specific gene evolution for lepidopteran insects. The comparative analysis of the insect immune repertoires indicated a dynamic and flexible gene expansion in recognition, modulation and effector mechanisms due to different selection pressures. Differential gene regulation by different bacterial species was found in PGRP and Serpin genes, suggesting that Bombyx has a highly selective gene regulation system depending on bacterial species. (C) 2008 Elsevier Ltd. All rights reserved.

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  38. A chitinase structurally related to the glycoside hydrolase family 48 is indispensable for the hormonally induced diapause termination in a beetle Reviewed

    K Fujita, K Shimomura, K Yamamoto, T Yamashita, K Suzuki

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   Vol. 345 ( 1 ) page: 502 - 7   2006.6

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    Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Two proteins (APAP I and II) of the glycoside hydrolase family 48 (Family GH48) were isolated from the active adults of the leaf beetle Gastrophysa atrocyanea. Full-length and cDNAs were sequenced. APAP I expression and function were examined in detail. The protein has a chitinase but not a glucanase and cellobiohydrolase activity. It is expressed in the feeding stages, including beetles whose diapause was terminated with a juvenile hormone agonist. Suppression of the APAP I expression by means of RNA interference prevented the hormonal termination of diapause. (c) 2006 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2006.04.126

    Web of Science

    PubMed

  39. A Double Chaperone Function of the sHsp Genes against Heat-Based Environmental Adversity in the Soil-Dwelling Leaf Beetles Reviewed

    Atungulu Elizabeth, Tanaka Hiromasa, Fujita Kosuke, Yamamoto Kei-ichiro, Sakata Mutsumi, Sato Erika, Hara Michihiro, Yamashita Tetsuro, Suzuki Koichi

    Journal of Insect Biotechnology and Sericology   Vol. 75 ( 1 ) page: 15 - 22   2006

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    Language:English   Publisher:The Japanese Society of Sericultural Science  

    Small heat shock proteins (sHsps), which are constitutively expressed in the absence of stress during development in many organisms, have been correlated with not only enhanced thermoresistance but also the developmental, tissue-, and cell-specific induction and expression. This study demonstrates the constitutive sHsps 21 and 23 of an entomoresouce, the leaf beetle, <i>Gastrophysa atrocyanea</i>, display a double chaperone function <i>in vitro</i>. The RNAi suppression of two sHsp genes in adults resulted in decreased thermoresistance. A strong correlation was observed between the <i>in vitro</i> chaperone function and <i>in vivo</i> thermotolerance analysis, and the results support a possible double chaperone function critical for the survival of the leaf beetles against higher temperatures.<br>

    DOI: 10.11416/jibs.75.15

  40. Continuous rearing of an entomoresource, the leaf beetle, Gastrophysa atrocyanea Motschulsky (Coleoptera : Chrysomelidae) on artificial diets Reviewed

    OJIMA Noriyuki, IKEDA Shinichi, KOIKE Osamu, FUJITA Kosuke, SUZUKI Koichi

    Applied entomology and zoology   Vol. 40 ( 1 ) page: 119 - 124   2005.2

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    Language:English   Publisher:Japanese Society of Applied Entomology and Zoology  

    The leaf beetle, Gastrophysa atrocyanea Motschulsky (Coleoptera : Chrysomelidae) has been regarded as a bioagent suitable for the extermination of certain weeds, especially Rumex obtusifolius. We examined conditions for artificial mass rearing of this insect. An artificial diet based on the host-plant leaf powder and supplemented with feeding stimulants, was developed for laboratory rearing of the leaf beetles. In particular, the hatchability of eggs laid by the fourth generation and female longevity were equivalent to those for fresh host leaves. Consequently, the fifth generation was achieved by rearing on artificial diet.

    DOI: 10.1303/aez.2005.119

    CiNii Books

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Books 1

  1. 耐性の昆虫学 第23章 休眠による微生物環境への耐性機構

    藤田幸輔, 鈴木幸一

    東海大学出版会  2008.6 

MISC 8

  1. 新規開発蛍光プローブを用いたカルパイン活性の生体内イメージング International journal

    浅野俊文, 津田聡, 伊藤梓, 佐藤孝太, 藤田幸輔, 國方彦志, 神谷真子, 浦野泰照, 中澤徹

    日本緑内障学会抄録集   Vol. 29th   page: 127   2018

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    Language:Japanese  

    J-GLOBAL

  2. 新規開発蛍光プローブを用いたカルパイン活性の評価 International journal

    津田聡, 津田聡, 浅野俊文, 伊藤梓, 佐藤孝太, 藤田幸輔, 國方彦志, 神谷真子, 浦野泰照, 中澤徹

    日本緑内障学会抄録集   Vol. 29th   page: 128   2018

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    Language:Japanese  

    J-GLOBAL

  3. パスウェイ特異的プロモーターを用いた網膜神経節細胞ストレス応答のin vivoイメージング

    藤田 幸輔, 西口 康二, 横山 悠, 富山 優介, 津田 聡, 安田 正幸, 中澤 徹

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   Vol. 88回・38回   page: [4T18p - 11(3P1293)]   2015.12

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    Language:Japanese   Publisher:(公社)日本生化学会  

  4. パスウェイ特異的レポーターによる網膜神経節細胞ストレス応答のin vivoイメージング

    西口 康二, 藤田 幸輔, 藤井 裕介, 山本 耕太郎, 津田 聡, 安田 正幸, 中澤 徹

    日本眼科学会雑誌   Vol. 119 ( 臨増 ) page: 244 - 244   2015.3

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    Language:Japanese   Publisher:(公財)日本眼科学会  

  5. Enhancement of odor avoidance regulated by dopamine signaling in the nematode C-elegans

    Kotaro Kimura, Kosuke Fujita, Yosuke Miyanishi, Akiko Yamazoe

    JOURNAL OF PHYSIOLOGICAL SCIENCES   Vol. 60   page: S73 - S73   2010

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:SPRINGER TOKYO  

    Web of Science

  6. Identifying the site of dopamine action for regulating experience-dependent odor avoidance in the nematode C. elegans

    Kosuke Fujita, Kotaro Kimura

    NEUROSCIENCE RESEARCH   Vol. 68   page: E402 - E402   2010

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:ELSEVIER IRELAND LTD  

    DOI: 10.1016/j.neures.2010.07.1785

    Web of Science

  7. カイコ免疫関与遺伝子の比較ゲノム解析

    田中博光, 石橋純, 藤田幸輔, 勾坂晶, 中島由郎, 芳山三喜雄, 冨本和也, 金子陽一, 岩崎崇, 砂川智哉, 朝岡愛, 三田和英, 山川稔

    生化学     page: 1P-0696   2008

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    Language:Japanese  

    J-GLOBAL

  8. 昆虫休眠研究とバイオテクノロジー

    鈴木 幸一, 楊 平, 田中 弘正, 藤田 幸輔

    蚕糸・昆虫バイオテック   Vol. 75 ( 1 ) page: 3 - 9   2006.4

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    Language:Japanese   Publisher:日本蚕糸学会  

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Research Project for Joint Research, Competitive Funding, etc. 1

  1. 網膜変性に対するPrime editingを用いた革新的ゲノム編集遺伝子治療の開発

    2021.1 - 2022.3

    堀科学芸術振興財団 研究費助成事業 

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    Grant amount:\1000000

KAKENHI (Grants-in-Aid for Scientific Research) 6

  1. エピゲノム編集による抹消血を用いた網膜色素変性病因遺伝子の発現解析法の開発

    Grant number:20K09765  2020.4 - 2023.3

    基盤研究(C) 

    西口 康二

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    Authorship:Coinvestigator(s)  Grant type:Competitive

    Grant amount:\100000 ( Direct Cost: \100000 )

    網膜色素変性(RP)患者由来の不死化リンパ球を対象に、ゲノム編集技術を用いて特定のRP病因遺伝子のプロモーターを非メチル化することにより同遺伝子の強制発現細胞株の樹立を可能にする技術を開発する。この細胞株を用いて、本来リンパ球では発現しないRP病因遺伝子のmRNA発現解析を行い、同患者のゲノム情報と照合することにより、逆算的に病因変異が同定可能か検証する。このアプローチの有用性が明らかになった場合、比較的簡便かつ安価な遺伝子診断補助検査法の開発が可能になり、遺伝性疾患の診断率の向上に寄与すると期待される。

  2. 網膜疾患関連遺伝子の機能解析のための新規CRISPR-Cas9ベクターの開発

    Grant number:18K09395  2018.4 - 2021.3

    基盤研究(C) 

    藤田 幸輔

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\4420000 ( Direct Cost: \3400000 、 Indirect Cost:\1020000 )

    近年、遺伝子解析技術の発展により眼疾患の遺伝的背景が急速に解明されつつある。しかし、同定された多くの疾患関連遺伝子の機能解析が十分になされていない。一方、近年、効率的な遺伝子機能解析手法としてCRISPR-Cas9システムによるゲノム編集が注目されている。これを用いて短時間かつin vivoで網膜神経細胞における遺伝子機能を評価する場合、アデノ随伴ウイルス(AAV)を用いてターゲット細胞にシステムを導入することになるが、AAVは搭載可能なインサートサイズが小さいという難点がある。本研究は、CRISPR-Cas9システムによるゲノム編集に必要な各パーツを小型化することにより1つのAAV上に搭載したAll-in-one AAVベクターを作製し、網膜細胞を対象に効率的な遺伝子機能解析を可能にする技術開発を目的とする。
    初年度は、視細胞特異的な小型プロモーターを開発し、それを組み込んだAll-in-oneゲノム編集用AAVを作製した。ゲノム編集を効果的に判定するために、視細胞変性モデルマウスを用いて、ゲノム編集による病因変異の正常化とそれによる治療効果を検討した。
    当該年度は、引き続きゲノム編集による治療効果を詳細に解析・検討した。その結果、開発したAll-in-one ゲノム編集AAVベクターにより、マウス視細胞の病因変異がおよそ10%正常に修復されたことを明らかにした。その効果について組織学的、電気生理学的、行動学的に視機能を評価したところ、従来の遺伝子治療と同程度にまで視力が回復したことが示された。加えて、オフターゲット効果が生じていないことも確認し、有用な遺伝子治療ツールとなりうることを示した。
    本研究は効率的な遺伝子機能解析の大幅な効率化とともに、遺伝子治療への応用も期待できるものである。
    当該年度はAll-in-one AAVベクターの作製を行い、年度後半からマウス網膜でのゲノム編集効果の評価に取り組む計画であった。現在までのところ、開発したAll-in-one AAVベクターを用いて視細胞のゲノム編集による遺伝子治療を行い、視機能回復に成功した。以上のように、既に遺伝子治療まで可能なAll-in-one AAVベクターが完成している。これは遺伝子機能解析にも有用である。現段階において、研究を進める上で大きな問題はなく、おおむね順調に進展していると評価した。
    今後はゲノム編集効率の向上に取り組み、より効果的なゲノム編集ツールの開発を行っていく。ゲノム編集効率の向上が見込まれる薬剤の検討や、AAVベクターの改変を行い、培養細胞を用いた系により評価する。選定した改良型AAVベクターをマウス網膜に導入し、編集効果を解析していく。
    また、網膜の他の細胞種を標的にしたゲノム編集AAVベクターの開発も同時に行っていく。

  3. Comparison of plasticity between rod and cone visual pathways

    Grant number:16K11315  2016.4 - 2019.3

    Nishiguchi Koji

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    Authorship:Coinvestigator(s)  Grant type:Competitive

    Grant amount:\300000 ( Direct Cost: \300000 )

    Mice defective of Pde6c and Gnat1 resulting in congenital lack of cone and rod function are virtually blind from birth. In this study, we treated mice of different ages by supplementing GNAT1 gene followed by assessment of visual recovery.
    As a result, the levels of functional restoration measured at the retina and visual cortex were not significantly different between mice treated at 1M, 3M, and 9M following GNAT1 gene supplementation therapy. Moreover, visual restoration at the level of visual cortex were similar to mice with only Pde6c defect.
    The results indicate that rod visual pathway in adult mice retain substantial plasticity.

  4. In vivo imaging of retinal ganglion cell function using adeno-associated virus

    Grant number:16K15730  2016.4 - 2018.3

    Nakazawa Toru

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    Authorship:Coinvestigator(s)  Grant type:Competitive

    Grant amount:\150000 ( Direct Cost: \150000 )

    Glaucoma, a leading cause of blindness worldwide, is characterized by progressive loss of the retinal ganglion cells (RGCs). To study neuronal activity in pathogenesis of RGC death, we developed in vivo imaging for quantitating activities of neuronal activity. E-SARE drives neuronal activity dependent gene expression at high levels.
    The E-SARE-driven fluorescent protein reporters was packaged into AAV. The temporal activity was monitored using confocal ophthalmoscopy. Dark-adapted animals were kept in dark for 48 hours before use. Light-adapted animals were kept under a constant light for 6 hours after dark-adaptation for 48 hours before use. E-SARE reporter expression was induced by light stimulation, and localized in RGC. Increased reporter activity occurred by 2 hours after light stimuli, and decreased at 2hours after dark-adaptation showing 44% decreases. This E-SARE reporter in vivo cellular imaging is a usuful tool to assess the RGCs function in retinal diseases.

  5. Spatially and temporally regulated gene therapy using stress response promoter in retinal injury

    Grant number:16K20301  2016.4 - 2018.3

    Fujita Kosuke

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\3900000 ( Direct Cost: \3000000 、 Indirect Cost:\900000 )

    Retinal ganglion cell (RGC) degeneration is believed to underlie many ocular diseases including glaucoma. In these diseases, RGCs are affected unevenly, both spatially and temporally, such that healthy and unhealthy RGCs coexist in different patterns at different time points. We describe a temporally and spatially regulated AAV gene therapy aiming to reduce undesired off-target effects on healthy RGCs. The Mcp-1 promoter shown to be transcribed in stressed RGCs following murine optic nerve injury was combined with the neuroprotective transcription factor NRF2. In this model, Mcp-1-driven NRF2 expression targeting only stressed RGCs showed efficacy equivalent to non-selective CMV promoter-driven therapy for preventing RGC death. However, CMV promoter-mediated NRF2 transcription induced cellular stress responses and death of uninjured RGCs. Combining a stress-responsive promoter and therapeutic gene is a versatile strategy for specifically targeting cells at risk of degeneration.

  6. Analysis of molecular responses in retinal ganglion cell death using RNAi

    Grant number:25893015  2013.8 - 2015.3

    FUJITA Kosuke

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    Authorship:Principal investigator 

    Grant amount:\2730000 ( Direct Cost: \2100000 、 Indirect Cost:\630000 )

    Glaucoma, a leading cause of blindness, is characterized by the progressive loss of retinal ganglion cells (RGCs); however, the molecular component modulated by cell death and the mechanisms of this modulation have not been fully understood.
    Recently, our laboratory performed a comprehensive gene expression analysis of the murine retina in the optic nerve crush injury (Yasuda et al., 2014). To identify molecules involved in the RGS cell death signaling characterize their modulation, we conducted RNAi and overexpression experiments using adeno-associated virus. Using mice devoid of Chop and AAV2/2 carrying Chop transgene, we found that RGC death was indeed mediated specifically through Chop signaling, suggesting that ER stress seems to play an important role in the pathogenesis of RGC death following axonal injury, which is in agreement with recent comprehensive gene expression studies.

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Industrial property rights 2

  1. GENE THERAPY EMPLOYING GENOME EDITING WITH SINGLE AAV VECTOR

    Koji Nishiguchi, Toru Nakazawa, Kosuke Fujita

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    Application no:特願2018-210670  Date applied:2018.11

  2. 外来DNA断片由来の逆方向反復配列を含む組換えベクター及びその作製方法

    田中 博光, 冨本 和也, 藤田 幸輔, 山川 稔

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    Applicant:独立行政法人農業生物資源研究所

    Application no:特願2008-072167  Date applied:2008.3

    Announcement no:特開2009-225682  Date announced:2009.10

    Patent/Registration no:特許第5422804号  Date issued:2013.12

    J-GLOBAL