研究者総覧

村松 慎一 (ムラマツ シンイチ)

  • オープンイノベーションセンター 神経遺伝子治療部門 教授
Last Updated :2021/12/04

研究者情報

学位

  • 医学博士(自治医科大学)

ホームページURL

ORCID ID

J-Global ID

プロフィール

  • 神経疾患の遺伝子治療を開発しています。

研究キーワード

  • 包括脳ネットワーク   脳プロ   Gene therapy   Adeno-associated virus   ES細胞   レビー小体   アルツハイマー病   大脳基底核   PET   カニクイサル   アデノ随伴ウイルス   ドパミン   パーキンソン病   遺伝子治療   

研究分野

  • ライフサイエンス / 神経科学一般
  • ライフサイエンス / 神経科学一般
  • ライフサイエンス / 神経内科学

経歴

  • 2019年04月 - 現在  自治医科大学オープンイノベーションセンター 神経遺伝子治療部門特命教授
  • 2019年04月 - 現在  東京大学医科学研究所遺伝子・細胞治療センター客員教授
  • 2008年11月 - 現在  自治医科大学神経内科学特命教授
  • 2014年11月 - 2019年03月  東京大学医科学研究所遺伝子・細胞治療センター特任教授
  • 1995年05月 - 1997年03月  米国NIH NHLBIvisiting associate

研究活動情報

論文

  • Paul Wuh-Liang Hwu, Karl Kiening, Irina Anselm, David R Compton, Takeshi Nakajima, Thomas Opladen, Phillip L Pearl, Agathe Roubertie, Thomas Roujeau, Shin-Ichi Muramatsu
    EMBO molecular medicine 13 9 e14712  2021年09月 
    This commentary provides an overview of the putamen as an established target site for gene therapy in treating aromatic l-amino acid decarboxylase (AADC) deficiency and Parkinson's disease, two debilitating neurological disorders that involve motor dysfunction caused by dopamine deficiencies. The neuroanatomy and the function of the putamen in motor control provide good rationales for targeting this brain structure. Additionally, the efficacy and safety of intraputaminal gene therapy demonstrate that restoration of dopamine synthesis in the putamen by using low doses of adeno-associated viral vector serotype 2 to deliver the hAADC gene is well tolerated. This restoration leads to sustained improvements in motor and nonmotor symptoms of AADC deficiency and improved uptake and conversion of exogenous l-DOPA into dopamine in Parkinson's patients.
  • Hajime Miyanishi, Shin-Ichi Muramatsu, Atsumi Nitta
    Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 46 9 1594 - 1605 2021年08月 
    The global number of patients with depression increases in correlation to exposure to social stress. Chronic stress does not trigger depression in all individuals, as some remain resilient. The underlying molecular mechanisms that contribute to stress sensitivity have been poorly understood, although revealing the regulation of stress sensitivity could help develop treatments for depression. We previously found that striatal Shati/Nat8l, an N-acetyltransferase, was increased in a depression mouse model. We investigated the roles of Shati/Nat8l in stress sensitivity in mice and found that Shati/Nat8l and brain-derived neurotrophic factor (BDNF) levels in the dorsal striatum were increased in stress-susceptible mice but not in resilient mice exposed to repeated social defeat stress (RSDS). Knockdown of Shati/Nat8l in the dorsal striatum induced resilience to RSDS. In addition, blockade of BDNF signaling in the dorsal striatum by ANA-12, a BDNF-specific receptor tropomyosin-receptor-kinase B (TrkB) inhibitor, also induced resilience to stress. Shati/Nat8l is correlated with BDNF expression after RSDS, and BDNF is downstream of Shati/Nat8l pathways in the dorsal striatum; Shati/Nat8l is epigenetically regulated by BDNF via histone acetylation. Our results demonstrate that striatal Shati/Nat8l-BDNF pathways determine stress sensitivity through epigenetic regulation. The striatal Shati/Nat8l-BDNF pathway could be a novel target for treatments of depression and could establish a novel therapeutic strategy for depression patients.
  • Atsumi Nitta, Naotaka Izuo, Kohei Hamatani, Ryo Inagaki, Yuka Kusui, Kequan Fu, Takashi Asano, Youta Torii, Chikako Habuchi, Hirotaka Sekiguchi, Shuji Iritani, Shin-Ichi Muramatsu, Norio Ozaki, Yoshiaki Miyamoto
    Journal of personalized medicine 11 7 2021年06月 
    Piccolo, a presynaptic cytomatrix protein, plays a role in synaptic vesicle trafficking in the presynaptic active zone. Certain single-nucleotide polymorphisms of the Piccolo-encoding gene PCLO are reported to be associated with mental disorders. However, a few studies have evaluated the relationship between Piccolo dysfunction and psychotic symptoms. Therefore, we investigated the neurophysiological and behavioral phenotypes in mice with Piccolo suppression in the medial prefrontal cortex (mPFC). Downregulation of Piccolo in the mPFC reduced regional synaptic proteins, accompanied with electrophysiological impairments. The Piccolo-suppressed mice showed an enhanced locomotor activity, impaired auditory prepulse inhibition, and cognitive dysfunction. These abnormal behaviors were partially ameliorated by the antipsychotic drug risperidone. Piccolo-suppressed mice received mild social defeat stress showed additional behavioral despair. Furthermore, the responses of these mice to extracellular glutamate and dopamine levels induced by the optical activation of mPFC projection in the dorsal striatum (dSTR) were inhibited. Similarly, the Piccolo-suppressed mice showed decreased depolarization-evoked glutamate and -aminobutyric acid elevations and increased depolarization-evoked dopamine elevation in the dSTR. These suggest that Piccolo regulates neurotransmission at the synaptic terminal of the projection site. Reduced neuronal connectivity in the mPFC-dSTR pathway via suppression of Piccolo in the mPFC may induce behavioral impairments observed in schizophrenia.
  • Sachie Nakamura, Hitoshi Osaka, Shin-Ichi Muramatsu, Naomi Takino, Mika Ito, Eriko F Jimbo, Chika Watanabe, Shuji Hishikawa, Takeshi Nakajima, Takanori Yamagata
    Gene therapy 28 6 329 - 338 2021年06月 
    Glucose transporter 1 deficiency syndrome (GLUT1DS) is caused by haplo-insufficiency of SLC2A1, which encodes GLUT1, resulting in impaired hexose transport into the brain. Previously, we generated a tyrosine-mutant AAV9/3 vector in which SLC2A1 was expressed under the control of the endogenous GLUT1 promoter (AAV-GLUT1), and confirmed the improved motor function and cerebrospinal fluid glucose levels of Glut1-deficient mice after cerebroventricular injection of AAV-GLUT1. In preparation for clinical application, we examined the expression of transgenes after intra-cisterna magna injection of AAV-GFP (tyrosine-mutant AAV9/3-GFP with the CMV promoter) and AAV-GLUT1. We injected AAV-GFP or AAV-GLUT1 (1.63 × 1012 vector genomes/kg) into the cisterna magna of pigs to compare differential promoter activity. After AAV-GFP injection, exogenous GFP was expressed in broad areas of the brain and peripheral organs. After AAV-GLUT1 injection, exogenous GLUT1 was expressed predominantly in the brain. At the cellular level, exogenous GLUT1 was mainly expressed in the endothelium, followed by glia and neurons, which was contrasted with the neuronal-predominant expression of GFP by the CMV promotor. We consider intra-cisterna magna injection of AAV-GLUT1 to be a feasible approach for gene therapy of GLUT1DS.
  • Yoshie Kurokawa, Hitoshi Osaka, Takeshi Kouga, Eriko Jimbo, Kazuhiro Muramatsu, Sachie Nakamura, Yuki Takayanagi, Tatsushi Onaka, Shin-Ichi Muramatsu, Takanori Yamagata
    Human gene therapy 32 11-12 589 - 598 2021年06月 
    Niemann-Pick disease type C1 (NPC1) is a fatal congenital neurodegenerative disorder caused by mutations in the NPC1 gene, which is involved in cholesterol transport in lysosomes. Broad clinical manifestations of NPC1 include liver failure, pulmonary disorder, neurological deficits, and psychiatric symptoms. The main cause of death in NPC1 patients involves central nervous system (CNS) dysfunction; there is no essential treatment. We generated a tyrosine-mutant adeno-associated virus (AAV) 9/3 vector that expresses human NPC1 under a cytomegalovirus (CMV) promoter (AAV-CMV-hNPC1) and injected it into the left lateral ventricle (5 μL) and cisterna magna (10 μL) of Npc1 homo-knockout (Npc1-/-) mice. Each mouse received total 1.35 × 1011 vector genome on days 4 or 5 of life. AAV-treated Npc1-/- mice (n = 11) had an average survival of >28 weeks, while all saline-treated Npc1-/- mice (n = 11) and untreated Npc1-/- mice (n = 6) died within 16 weeks. Saline-treated and untreated Npc1-/- mice lost body weight from 7 weeks until death. However, the average body weight of AAV-treated Npc1-/- mice increased until 15 weeks. AAV-treated Npc1-/- mice also showed a significant improvement in the rotarod test performance. A pathological analysis at 11 weeks showed that cerebellar Purkinje cells were preserved in AAV-treated Npc1-/- mice. In contrast, untreated Npc1-/- mice showed an almost total loss of cerebellar Purkinje cells. Combined injection into both the lateral ventricle and cisterna magna achieved broader delivery of the vector to the CNS, leading to better outcomes than noted in previous reports, with injection into the lateral ventricles or veins alone. In AAV-treated Npc1-/- mice, vector genome DNA was detected widely in the CNS and liver. Human NPC1 RNA was detected in the brain, liver, lung, and heart. Accumulated unesterified cholesterol in the liver was reduced in the AAV-treated Npc1-/- mice. Our results suggest the feasibility of gene therapy for patients with NPC1.
  • Mika Ito, Naomi Takino, Takamasa Nomura, Akihiko Kan, Shin-Ichi Muramatsu
    Scientific reports 11 1 9322 - 9322 2021年04月 
    The natural serotypes of adeno-associated virus (AAV) or their variants, such as AAV8 and AAV5, are commonly used as vectors in the clinical programs for liver-targeted gene therapy. While AAV8 vectors are not highly efficient at targeting primary human hepatocytes, AAV3 vectors have recently demonstrated remarkable efficiency at targeting both human and non-human primate hepatocytes. However, the presence of high levels of neutralizing antibodies (NAbs) impedes transduction into hepatocytes, representing a major obstacle to the clinical application of AAV3 vectors. Herein, we engineered the viral capsid to reduce its reactivity with pre-existing NAbs, thereby enhancing the transduction efficiency. By introducing three substitutions (S472A, S587A, and N706A) on the surface loop of AAV3B capsid protein, we generated a triple mutant AAV3 (AAV.GT5) vector with less reactivity to anti-AAV capsid NAbs. While the transduction efficiency of AAV.GT5 into human hepatocellular cell lines was similar to those of parental AAV3B, it was 50-fold higher for hepatocytes derived from humanized mice compared to AAV8 vectors. Moreover, the AAV.GT5 vector yield was similar to those of the AAV2 and AAV3B vectors. Thus, high resistance to pre-existing NAbs makes AAV.GT5 a promising candidate for future liver-targeted gene therapy clinical trials.
  • Meriem Haddar, Katsunori Azuma, Naotaka Izuo, Uno Kyosuke, Takashi Asano, Shin-Ichi Muramatsu, Atsumi Nitta
    Behavioural brain research 397 112938 - 112938 2021年01月 
    A novel N-acetyltransferase, Shati/Nat8l, was identified in the brains of mice exposed to methamphetamine. Shati/Nat8l overexpression in the medial prefrontal cortex (mPFC) was found to attenuate methamphetamine-induced dependence. The mPFC is a brain region that plays an important role in cognitive function. However, the effect of Shati/Nat8l on cognition and memory has not yet been clarified. To understand the role of Shati/Nat8l in memory, we generated C57BL/6J mice with overexpressed Shati/Nat8l in the mPFC and performed memory-related experiments, including novel object-location and object-in-context tests. Furthermore, we used quantitative immunohistochemistry to assess the presynaptic and postsynaptic proteins, synaptophysin and postsynaptic density protein (PSD)-95, respectively. Shati/Nat8l overexpression in the mPFC impaired both novel object-location and object-in-context memory. Moreover, Shati/Nat8l overexpression in the mPFC reduced PSD-95 levels, but not synaptophysin levels in the mPFC. These results demonstrated that Shati/Nat8l overexpression in the mPFC is involved in location and contextual memory, and can affect the excitatory postsynaptic protein, PSD-95.
  • Yoshiyuki Onuki, Sayaka Ono, Takeshi Nakajima, Karin Kojima, Naoyuki Taga, Takahiro Ikeda, Mari Kuwajima, Yoshie Kurokawa, Mitsuhiro Kato, Kensuke Kawai, Hitoshi Osaka, Toshihiko Sato, Shin-Ichi Muramatsu, Takanori Yamagata
    Brain communications 3 3 fcab078  2021年 
    Aromatic l-amino acid decarboxylase (AADC) is an essential dopamine-synthesizing enzyme. In children with AADC deficiency, the gene delivery of AADC into the putamen, which functionally interacts with cortical regions, was found to improve motor function and ameliorate dystonia. However, how the restoration of dopamine in the putamen in association with cortico-putaminal networks leads to therapeutic effects remains unclear. Here, we examined neuroimaging data of eight patients with AADC deficiency (five males and three females, age range 4-19 years) who received the AADC gene therapy of the bilateral putamen in an open-label phase 1/2 study. Using high-resolution positron emission tomography with a specific AADC tracer, 6-[18F]fluoro-l-m-tyrosine (FMT), we showed that FMT uptake increased in the broad area of the putamen over the years. Then, with the structural connectivity-based parcellation of the putaminal area, we found that motor improvement is associated with dopaminergic restoration of the putaminal area that belongs to the prefrontal cortico-putaminal network. The prefrontal area dominantly belongs to the frontoparietal control network, which contributes to cognitive-motor control function, including motor initiation and planning. The results suggest that putaminal dopamine promotes the development of an immature motor control system, particularly in the human prefrontal cortex that is primarily affected by AADC deficiency.
  • Shunya Nakane, Kouichi Mizoguchi, Koji Abe, Naoki Atsuta, Yasuyuki Iguchi, Yoshio Ikeda, Ryuji Kaji, Satoshi Kamei, Kazuo Kitagawa, Kazumi Kimura, Masahiko Suzuki, Hiroshi Takashima, Yasuo Terayama, Kazutoshi Nishiyama, Hirokazu Furuya, Etsuro Matsubara, Shin-Ichi Muramatsu, Osamu Yamamura, Atsushi Takeda, Hidefumi Ito
    Rinsho shinkeigaku = Clinical neurology 60 10 643 - 652 2020年10月 
    Disaster countermeasures have been implemented by the Japanese Society of Neurology based on the experience of support to the areas affected by the Great East Japan Earthquake on March 11, 2011. The countermeasures activity began at the end of 2011. We, the Committee for Measures Against Disaster, officially started work in 2014. We developed a support network to urgently deal with patients with intractable neurological disease at the time of disaster and strengthen disaster measures, including effective disaster countermeasure training. During the 2016 Kumamoto earthquake, we realized the need to prepare for natural disasters, leading to a state of emergency, at normal times. A list of vulnerable people should be prepared and the individual support plan for disaster should be confirmed during normal times. Furthermore, during disaster, livelihood support is required for patients with intractable neurological disease living in evacuation centers in affected areas. Therefore, we compiled and published the book, titled "The manual of disaster countermeasures," in 2017. The Committee for Measures Against Disaster in the Japanese Society of Neurology has appointed a liaison officer for patients with intractable neurological disease in each prefecture. The liaison's role of is gathering and disseminating information on the disaster-hit areas, arranging medical support, and coordinating health activities, when natural disasters occur. It is hoped that the liaison officer will play an active role both at normal times and during disaster, even unforeseen ones. Although we hope for the best, we aim to be prepared for the worst.
  • 小田口 浩, 石毛 達也, 伊藤 雄一, 若杉 安希乃, 関根 麻理子, 花輪 壽彦, 並木 隆雄, 村松 慎一, 新井 信, 三潴 忠道, 嶋田 豊, 柴原 直利, 折笠 秀樹
    日本東洋医学雑誌 71 3 284 - 295 (一社)日本東洋医学会 2020年07月 
    我々は漢方診断ロジックの形式知化を図ることを目的とした臨床データ集積研究を計画しているが、計画段階において、研究対象となる漢方方剤の数の多さ、漢方方剤の内容についての施設間差、所見の採取方法や有効性の評価の施設間・個人間差が課題となった。今回、これらの課題を研究メンバーである全参加施設で議論し、合意形成による解決を試みた。方法は、解決すべき各課題について合意形成会議を積み重ねて全員一致で結論を得る方式とした。その結果、以下の結論が得られた。まず形式知化の対象として33個の漢方方剤が選択された。また、各漢方方剤の構成生薬などの範囲が定められた。さらに漢方医学的所見の項目とその判断基準が定められた。そして、有効性・安全性の判断基準が決定された。漢方臨床研究に当たって解決すべき諸課題について、合意による解決案を提示した。今回の結果が今後漢方研究手法を議論する土台となることを期待する。(著者抄録)
  • Meriem Haddar, Kyosuke Uno, Katsunori Azuma, Shin-Ichi Muramatsu, Atsumi Nitta
    Addiction biology 25 3 e12749  2020年05月 [査読有り][通常論文]
     
    Shati/Nat8l is a novel N-acetyltransferase identified in the brain of mice treated with methamphetamine (METH). Shati/Nat8l mRNA is expressed in various brain areas, including the prefrontal cortex (PFC), where the expression level is higher than that in other brain regions. Shati/Nat8l overexpression in the nucleus accumbens (NAc) attenuates the pharmacological response to METH via mGluR3. Meanwhile, dopamine (DA) and glutamate dysregulations have been reported in the medial prefrontal cortex (mPFC) and NAc after METH self-administration and during reinstatement. However, the mechanism, the reward system, and function of Shati/Nat8l in the mPFC is unclear. Here, we injected an adeno-associated virus (AAV) vector containing Shati/Nat8l into the mPFC of mice, to overexpress Shati/Nat8l in the mPFC (mPFC-Shati/Nat8l). Interestingly, the METH-induced conditioned place preference (CPP) was attenuated in the mPFC-Shati/Nat8l mice, but locomotor activity was not. Additionally, immunohistochemical results from mice that were injected with AAV-GFP showed fluorescence in the mPFC and other brain regions, mainly the NAc, indicating an mPFC-NAc top-down connection. Finally, in vivo microdialysis experiments revealed that Shati/Nat8l overexpression in the mPFC reduced extracellular DA levels and suppressed the METH-induced DA increase in the NAc. Moreover, decreased extracellular glutamate levels were observed in the NAc. These results indicate that Shati/Nat8l overexpression in the mPFC attenuates METH-induced CPP by decreasing extracellular DA in the NAc. In contrast, Shati/Nat8l-mPFC overexpression did not alter METH-induced hyperlocomotion. This study demonstrates that Shati/Nat8l in the mPFC attenuates METH reward-seeking behaviour but not the psychomotor activity of METH.
  • Hikari Tanaka, Hidenori Homma, Kyota Fujita, Kanoh Kondo, Shingo Yamada, Xiaocen Jin, Masaaki Waragai, Gaku Ohtomo, Atsushi Iwata, Kazuhiko Tagawa, Naoki Atsuta, Masahisa Katsuno, Naoki Tomita, Katsutoshi Furukawa, Yuko Saito, Takashi Saito, Ayaka Ichise, Shinsuke Shibata, Hiroyuki Arai, Takaomi Saido, Marius Sudol, Shin-Ichi Muramatsu, Hideyuki Okano, Elliott J Mufson, Gen Sobue, Shigeo Murayama, Hitoshi Okazawa
    Nature communications 11 1 507 - 507 2020年01月 [査読有り][通常論文]
     
    The timing and characteristics of neuronal death in Alzheimer's disease (AD) remain largely unknown. Here we examine AD mouse models with an original marker, myristoylated alanine-rich C-kinase substrate phosphorylated at serine 46 (pSer46-MARCKS), and reveal an increase of neuronal necrosis during pre-symptomatic phase and a subsequent decrease during symptomatic phase. Postmortem brains of mild cognitive impairment (MCI) rather than symptomatic AD patients reveal a remarkable increase of necrosis. In vivo imaging reveals instability of endoplasmic reticulum (ER) in mouse AD models and genome-edited human AD iPS cell-derived neurons. The level of nuclear Yes-associated protein (YAP) is remarkably decreased in such neurons under AD pathology due to the sequestration into cytoplasmic amyloid beta (Aβ) aggregates, supporting the feature of YAP-dependent necrosis. Suppression of early-stage neuronal death by AAV-YAPdeltaC reduces the later-stage extracellular Aβ burden and cognitive impairment, suggesting that preclinical/prodromal YAP-dependent neuronal necrosis represents a target for AD therapeutics.
  • Hirotaka Asai, Noriaki Ohkawa, Yoshito Saitoh, Khaled Ghandour, Emi Murayama, Hirofumi Nishizono, Mina Matsuo, Teruyoshi Hirayama, Ryosuke Kaneko, Shin-Ichi Muramatsu, Takeshi Yagi, Kaoru Inokuchi
    Molecular brain 13 1 7 - 7 2020年01月 [査読有り][通常論文]
     
    Clustered protocadherins (Pcdhs), a large group of adhesion molecules, are important for axonal projections and dendritic spread, but little is known about how they influence neuronal activity. The Pcdhβ cluster is strongly expressed in the hippocampus, and in vivo Ca2+ imaging in Pcdhβ-deficient mice revealed altered activity of neuronal ensembles but not of individual cells in this region in freely moving animals. Specifically, Pcdhβ deficiency increased the number of large-size neuronal ensembles and the proportion of cells shared between ensembles. Furthermore, Pcdhβ-deficient mice exhibited reduced repetitive neuronal population activity during exploration of a novel context and were less able to discriminate contexts in a contextual fear conditioning paradigm. These results suggest that one function of Pcdhβs is to modulate neural ensemble activity in the hippocampus to promote context discrimination.
  • Hiroyuki Kabayama, Makoto Takeuchi, Naoko Tokushige, Shin-Ichi Muramatsu, Miyuki Kabayama, Mitsunori Fukuda, Yoshiyuki Yamada, Katsuhiko Mikoshiba
    Nature communications 11 1 336 - 336 2020年01月 
    Targeting cytoplasmic protein-protein interactions with antibodies remains technically challenging, since antibodies expressed in the cytosol frequently form insoluble aggregates. Existing engineering methods are based on the notion that the estimated net charge at pH 7.4 affects stability; as such, they are unable to overcome this problem. Herein, we report a versatile method for engineering an ultra-stable cytoplasmic antibody (STAND), with a strong estimated net negative charge at pH 6.6, by fusing peptide tags with a highly negative charge and a low isoelectric point. Without the need for complicated amino acid substitutions, we convert aggregation-prone antibodies to STANDs that are useful for inhibiting in vivo transmitter release, modulating animal behaviour, and inhibiting in vivo cancer proliferation driven by mutated Kras-long recognised as an "undruggable" oncogenic protein. The STAND method shows promise for targeting endogenous cytoplasmic proteins in basic biology and for developing future disease treatments.
  • Kyosuke Uno, Hajime Miyanishi, Kengo Sodeyama, Toshiyuki Fujiwara, Toh Miyazaki, Shin-Ichi Muramatsu, Atsumi Nitta
    Behavioural brain research 376 112227 - 112227 2019年12月 
    The number of patients with depressive disorders is increasing. However, the mechanism of depression onsets has not been completely revealed. We previously identified Shati/Nat8l, an N-acetyltransferase, in the brain using an animal model of psychosis. In this study, we revealed the involvement of Shati/Nat8l in the vulnerability to major depression. Shati/Nat8l mRNA was increased only in the striatum of mice, which were exposed to chronic social defeat stress. Shati/Nat8l-overexpressed mice showed impairment in social interaction and sucrose preference after the subthreshold social defeat (microdefeat) stress. These depression-like behaviors were restored by fluvoxamine and LY341495 injection prior to these tests. Furthermore, the intracerebral administration of only fluvoxamine, but not of LY341495, to the dorsal striatum and direct infusion of LY341495 to the dorsal raphe also rescued. Taken together, Shati/Nat8l in the striatum has an important role in the vulnerability to depression onsets by regulating the origin of serotonergic neuronal system via GABAergic projection neuron in the dorsal raphe from the dorsal striatum.
  • 発達障害原因遺伝子PQBP1はアルツハイマー病態を制御する
    田中 ひかり, 近藤 和, 陳 西貴, 田川 一彦, Kerever Aurelian, 青木 茂樹, 斉藤 貴志, 西道 隆臣, 村松 慎一, 藤田 慶大, 岡澤 均
    臨床神経学 59 Suppl. S461 - S461 (一社)日本神経学会 2019年11月 [査読有り][通常論文]
  • Shati/Nat8l線条体局所的ノックダウンマウスにおけるBdnf遺伝子のアセチル化制御を介したうつ病発症に対する抵抗性の形成
    宮西 肇, 宇野 恭介, 松村 慎一, 新田 淳美
    日本臨床精神神経薬理学会・日本神経精神薬理学会合同年会プログラム・抄録集 29回・49回 261 - 261 日本臨床精神神経薬理学会・日本神経精神薬理学会 2019年10月 [査読有り][通常論文]
  • マウス前頭前皮質におけるTeneurin-4の発現減少によるうつ行動への関与
    所 一輝, 宇野 恭介, 村松 慎一, 新田 淳美
    日本臨床精神神経薬理学会・日本神経精神薬理学会合同年会プログラム・抄録集 29回・49回 261 - 261 日本臨床精神神経薬理学会・日本神経精神薬理学会 2019年10月 [査読有り][通常論文]
  • アルツハイマー病患者の脳内で発現が減少するアミノ酸N-acetyl-aspartate(NAA)がマウス由来アストロサイトにおいてニコチン性アセチルコリン受容体α7サブユニットの発現に与える影響
    楠井 優香, 宇野 恭介, 村松 慎一, 新田 淳美
    日本臨床精神神経薬理学会・日本神経精神薬理学会合同年会プログラム・抄録集 29回・49回 263 - 263 日本臨床精神神経薬理学会・日本神経精神薬理学会 2019年10月 [査読有り][通常論文]
  • マウス前頭前皮質におけるShati/Nat8l発現量変化の空間認識機能への関与
    東 克憲, Meriem Haddar, 宇野 恭介, 村松 慎一, 新田 淳美
    日本臨床精神神経薬理学会・日本神経精神薬理学会合同年会プログラム・抄録集 29回・49回 264 - 264 日本臨床精神神経薬理学会・日本神経精神薬理学会 2019年10月 [査読有り][通常論文]
  • Meriem Haddar, Kyosuke Uno, Kohei Hamatani, Shin-Ichi Muramatsu, Atsumi Nitta
    Neuropsychopharmacology reports 39 3 209 - 216 2019年09月 [査読有り][通常論文]
     
    AIM: We previously reported that methamphetamine (METH)-induced conditioned place preference was attenuated by Shati/Nat8l overexpression in the medial prefrontal cortex (mPFC). Shati/Nat8l overexpression in the mPFC expressed lower levels of both glutamate and dopamine (DA) in the nucleus accumbens (NAc) and attenuated METH-induced DA elevation. We suggested a mechanism in which a decline of glutamate levels in the NAc decreases extracellular DA levels. However, the hypothesis has not confirmed. METHODS: We conducted a recovery experiments by pre-microinjection of an mGluR group II antagonist, LY341495, into the NAc shell of mPFC-Shati/Nat8l-overexpressed mice followed by METH injection and DA levels measurement by in vivo microdialysis. RESULTS: Pretreatment with LY341495 was able to restore METH-induced DA increase. Furthermore, mice injected with an adeno-associated virus vector containing GFP (AAV-GFP vector) in the mPFC expressed a colocalization of GFP with DARPP-32 a medium spiny neuron (MSN) marker. Next, co-immunostaining of DARPP-32 and neuronal nitric oxide synthase (nNOS: expressed in a subtype of gamma-Aminobutyric acid (GABA interneurons) in ventral tegmental area (VTA) showed a colocalization of nNOS and DARPP-32. CONCLUSION: These results provided a proof that Shati/Nat8l attenuation of METH-induced DA increase is mediated by mGluR group II in the NAc. Moreover, immunohistochemical study showed a direct connection of mPFC projection neurons with NAc MSN and a connection of MSN projection neurons with a subtype of GABA interneurons in VTA.
  • Nobutake Hosoi, Koji Shibasaki, Mayu Hosono, Ayumu Konno, Yo Shinoda, Hiroshi Kiyonari, Kenichi Inoue, Shin-Ichi Muramatsu, Yasuki Ishizaki, Hirokazu Hirai, Teiichi Furuichi, Tetsushi Sadakata
    The Journal of neuroscience : the official journal of the Society for Neuroscience 39 32 6339 - 6353 2019年08月 [査読有り][通常論文]
     
    ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4+/-/ARF5-/- mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4+/-/ARF5-/- mice of both sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4+/-/ARF5-/- mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of pore-forming voltage-dependent Na+ channel subunit Nav1.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein localized in the AIS and the consequent tremors in ARF4+/-/ARF5-/- mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class II ARF proteins in ARF4+/-/ARF5-/- mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Nav1.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Nav1.6, to the AIS.SIGNIFICANCE STATEMENT We found that decreasing the expression of class II ARF proteins, through the generation of ARF4+/-/ARF5-/- mice, impairs Nav1.6 distribution to the axon initial segment (AIS) of cerebellar Purkinje cells (PCs), thereby resulting in the impairment of action potential firing of PCs. The ARF4+/-/ARF5-/- mutant mice exhibited movement-associated essential tremor (ET)-like behavior with pharmacological profiles similar to those in ET patients. The exogenous expression of ARF5 reduced the tremor phenotype and restored the localization of Nav1.6 immunoreactivity to the AIS in ARF4+/-/ARF5-/- mice. Thus, our results suggest that class II ARFs are involved in the localization of Nav1.6 to the AISs in cerebellar PCs and that the reduction of class II ARF activity leads to ET-like movement disorder.
  • Kunihiko Araki, Amane Araki, Daiyu Honda, Takako Izumoto, Atsushi Hashizume, Yasuhiro Hijikata, Shinichiro Yamada, Yohei Iguchi, Akitoshi Hara, Kazuhiro Ikumi, Kaori Kawai, Shinsuke Ishigaki, Yoko Nakamichi, Shin Tsunekawa, Yusuke Seino, Akiko Yamamoto, Yasunori Takayama, Shihomi Hidaka, Makoto Tominaga, Mica Ohara-Imaizumi, Atsushi Suzuki, Hiroshi Ishiguro, Atsushi Enomoto, Mari Yoshida, Hiroshi Arima, Shin-Ichi Muramatsu, Gen Sobue, Masahisa Katsuno
    The Journal of clinical investigation 129 9 3578 - 3593 2019年07月 [査読有り][通常論文]
     
    TAR DNA-binding protein 43 kDa (TDP-43), encoded by TARDBP, is an RNA-binding protein, the nuclear depletion of which is the histopathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder affecting both upper and lower motor neurons. Besides motor symptoms, patients with ALS often develop nonneuronal signs including glucose intolerance, but the underlying pathomechanism is still controversial, i.e., whether it is impaired insulin secretion and/or insulin resistance. Here, we showed that ALS subjects reduced early-phase insulin secretion and that the nuclear localization of TDP-43 was lost in the islets of autopsied ALS pancreas. Loss of TDP-43 inhibited exocytosis by downregulating CaV1.2 calcium channels, thereby reducing early-phase insulin secretion in a cultured β cell line (MIN6) and β cell-specific Tardbp knockout mice. Overexpression of CaV1.2 restored early-phase insulin secretion in Tardbp knocked-down MIN6 cells. Our findings suggest that TDP-43 regulates cellular exocytosis mediated by L-type voltage-dependent calcium channels and thus plays an important role in the early phase of insulin secretion by pancreatic islets. Thus, nuclear loss of TDP-43 is implicated in not only the selective loss of motor neurons but also in glucose intolerance due to impaired insulin secretion at an early stage of ALS.
  • Hashimoto S, Matsuba Y, Kamano N, Mihira N, Sahara N, Takano J, Muramatsu SI, Saido TC, Saito T
    Nature communications 10 1 2964 - 2964 2019年07月 [査読有り][通常論文]
     
    An amendment to this paper has been published and can be accessed via a link at the top of the paper.
  • Shoko Hashimoto, Yukio Matsuba, Naoko Kamano, Naomi Mihira, Naruhiko Sahara, Jiro Takano, Shin-Ichi Muramatsu, Takaomi C Saido, Takashi Saito
    Nature communications 10 1 2394 - 2394 2019年06月 [査読有り][通常論文]
     
    To understand the molecular processes that link Aβ amyloidosis, tauopathy and neurodegeneration, we screened for tau-interacting proteins by immunoprecipitation/LC-MS. We identified the carboxy-terminal PDZ ligand of nNOS (CAPON) as a novel tau-binding protein. CAPON is an adaptor protein of neuronal nitric oxide synthase (nNOS), and activated by the N-methyl-D-aspartate receptor. We observed accumulation of CAPON in the hippocampal pyramidal cell layer in the AppNL-G-F -knock-in (KI) brain. To investigate the effect of CAPON accumulation on Alzheimer's disease (AD) pathogenesis, CAPON was overexpressed in the brain of AppNL-G-F mice crossbred with MAPT (human tau)-KI mice. This produced significant hippocampal atrophy and caspase3-dependent neuronal cell death in the CAPON-expressing hippocampus, suggesting that CAPON accumulation increases neurodegeneration. CAPON expression also induced significantly higher levels of phosphorylated, oligomerized and insoluble tau. In contrast, CAPON deficiency ameliorated the AD-related pathological phenotypes in tauopathy model. These findings suggest that CAPON could be a druggable AD target.
  • Hiroki Kurosaki, Kentaro Yamaguchi, Kohei Man-Yoshi, Shin-Ichi Muramatsu, Satoshi Hara, Hiroshi Ichinose
    Neurochemistry international 125 16 - 24 2019年05月 [査読有り][通常論文]
     
    Parkinson's disease (PD) is the second common neurodegenerative disorder. Deficit of the nigro-striatal dopaminergic neurons causes the motor symptoms of PD. While the oxidative stress is thought to be deeply involved in the etiology of PD, molecular targets for the oxidative insults has not been fully elucidated. 6R-5,6,7,8-Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase (TH), the rate-limiting enzyme for production of dopamine, and easily oxidized to its dihydro-form. In this study, we examined the alteration in the metabolism of BH4 caused by a parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP reduced the dopamine content and the in vivo activity of TH in the striatum prior to degeneration of the dopaminergic neurons. We found that administration of BH4 could restore the dopamine content and in vivo TH activity in the striatum of MPTP-treated mice. Unexpectedly, when BH4 was administered with MPTP, BH4 contents in the brain were far higher than those injected without MPTP even at 23 h after the last injection. Because MPTP has been shown to increase ROS production in the dopaminergic neurons, we assumed that the increased ROS oxidizes BH4 into its dihydro-form, excreted from the dopaminergic neurons, taken-up by the neighboring cells, reduced back to BH4, and then accumulated in the brain. We also investigated the action of MPTP in mice lacking quinonoid-dihydropteridine reductase (Qdpr), an enzyme catalyzing regeneration of BH4 from quinonoid dihydrobiopterin. The dopamine depletion induced by MPTP was severer in Qdpr-deficient mice than in wild-type mice. The present data suggest that perturbation of the BH4 metabolism would be the cause of early and persistent dopamine depletion in the striatum.
  • Komura H, Kakio S, Sasahara T, Arai Y, Takino N, Sato M, Satomura K, Ohnishi T, Nabeshima YI, Muramatsu SI, Kii I, Hoshi M
    iScience 13 452 - 477 2019年03月 [査読有り][通常論文]
     
    We identified ∼30-mer amyloid-β protein (Aβ) assemblies, termed amylospheroids, from brains of patients with Alzheimer disease (AD) as toxic entities responsible for neurodegeneration and showed that Na+,K+-ATPase α3 (NAKα3) is the sole target of amylospheroid-mediated neurodegeneration. However, it remains unclear where in neurons amylospheroids form and how they reach their targets to induce neurodegeneration. Here, we present an in vitro culture system designed to chronologically follow amylospheroid formation in mature neurons expressing amyloid precursor protein bearing early-onset AD mutations. Amylospheroids were found to accumulate mainly in the trans-Golgi network of excitatory neurons and were initially transported in axons. Proteasome inhibition dramatically increased amylospheroid amounts in trans-Golgi by increasing Aβ levels and induced dendritic transport. Amylospheroids were secreted and caused the degeneration of adjacent NAKα3-expressing neurons. Interestingly, the ASPD-producing neurons later died non-apoptotically. Our findings demonstrate a link between ASPD levels and proteasome function, which may have important implications for AD pathophysiology.
  • マウス線条体のShati/Nat81はうつ様症状発症に脆弱性を示す
    宇野 恭介, 宮西 肇, 宮崎 杜夫, 袖山 健吾, 藤原 俊幸, 村松 慎一, 宮本 嘉明, 新田 淳美
    日本薬学会年会要旨集 139年会 3 92 - 92 (公社)日本薬学会 2019年03月 [査読有り][通常論文]
  • Karin Kojima, Takeshi Nakajima, Naoyuki Taga, Akihiko Miyauchi, Mitsuhiro Kato, Ayumi Matsumoto, Takahiro Ikeda, Kazuyuki Nakamura, Tetsuo Kubota, Hiroaki Mizukami, Sayaka Ono, Yoshiyuki Onuki, Toshihiko Sato, Hitoshi Osaka, Shin-Ichi Muramatsu, Takanori Yamagata
    Brain : a journal of neurology 142 2 322 - 333 2019年02月 [査読有り][通常論文]
     
    In patients with aromatic l-amino acid decarboxylase (AADC) deficiency, a decrease in catecholamines and serotonin levels in the brain leads to developmental delay and movement disorders. The beneficial effects of gene therapy in patients from 1 to 8 years of age with homogeneous severity of disease have been reported from Taiwan. We conducted an open-label phase 1/2 study of population including adolescent patients with different degrees of severity. Six patients were enrolled: four males (ages 4, 10, 15 and 19 years) and one female (age 12 years) with a severe phenotype who were not capable of voluntary movement or speech, and one female (age 5 years) with a moderate phenotype who could walk with support. The patients received a total of 2 × 1011 vector genomes of adeno-associated virus vector harbouring DDC via bilateral intraputaminal infusions. At up to 2 years after gene therapy, the motor function was remarkably improved in all patients. Three patients with the severe phenotype were able to stand with support, and one patient could walk with a walker, while the patient with the moderate phenotype could run and ride a bicycle. This moderate-phenotype patient also showed improvement in her mental function, being able to converse fluently and perform simple arithmetic. Dystonia disappeared and oculogyric crisis was markedly decreased in all patients. The patients exhibited transient choreic dyskinesia for a couple of months, but no adverse events caused by vector were observed. PET with 6-[18F]fluoro-l-m-tyrosine, a specific tracer for AADC, showed a persistently increased uptake in the broad areas of the putamen. In our study, older patients (>8 years of age) also showed improvement, although treatment was more effective in younger patients. The genetic background of our patients was heterogeneous, and some patients suspected of having remnant enzyme activity showed better improvement than the Taiwanese patients. In addition to the alleviation of motor symptoms, the cognitive and verbal functions were improved in a patient with the moderate phenotype. The restoration of dopamine synthesis in the putamen via gene transfer provides transformative medical benefit across all patient ages, genotypes, and disease severities included in this study, with the most pronounced improvements noted in moderate patients.10.1093/brain/awy331_video1awy331media15991361892001.
  • Naoya Oishi, Masanori Nomoto, Noriaki Ohkawa, Yoshito Saitoh, Yoshitake Sano, Shuhei Tsujimura, Hirofumi Nishizono, Mina Matsuo, Shin-Ichi Muramatsu, Kaoru Inokuchi
    Molecular brain 12 1 2 - 2 2019年01月 [査読有り][通常論文]
     
    Previous gain-of-function studies using an optogenetic technique showed that manipulation of the hippocampal dentate gyrus or CA1 cell ensembles is important for memory reactivation and to generate synthetic or false memory. However, gain-of-function study manipulating CA3 cell ensembles has not been reported. The CA3 area of the hippocampus comprises a recurrent excitatory circuit, which is thought to be important for the generation of associations among the stored information within one brain region. We investigated whether the coincident firing of cell ensembles in one brain region, hippocampal CA3, associates distinct events. CA3 cell ensembles responding to context exploration and during contextual fear conditioning were labeled with channelrhodopsin-2 (ChR2)-mCherry. The synchronous activation of these ensembles induced freezing behavior in mice in a neutral context, in which a foot shock had never been delivered. The recall of this artificial associative fear memory was context specific. In vivo electrophysiological recordings showed that 20-Hz optical stimulation of ChR2-mCherry-expressing CA3 neurons, which is the same stimulation protocol used in behavioral experiment, induced long-term potentiation at CA3-CA3 synapses. Altogether, these results demonstrate that the synchronous activation of ensembles in one brain region, CA3 of the hippocampus, is sufficient for the association of distinct events. The results of our electrophysiology potentially suggest that this artificial association of memory events might be induced by the strengthening of synaptic efficacy between CA3 ensembles via recurrent circuit.
  • Yosuke Ohtake, Armin Sami, Xinpei Jiang, Makoto Horiuchi, Kieran Slattery, Lena Ma, George M Smith, Michael E Selzer, Shin-Ichi Muramatsu, Shuxin Li
    Molecular therapy : the journal of the American Society of Gene Therapy 27 1 102 - 117 2019年01月 [査読有り][通常論文]
     
    Liver kinase B1 (LKB1), a downstream effector of cyclic AMP (cAMP)/PKA and phosphatidylinositol 3-kinase (PI3K) pathways, is a determinant for migration and differentiation of many cells, but its role in CNS axon regeneration is unknown. Therefore, LKB1 was overexpressed in sensorimotor cortex of adult mice five days after mid-thoracic spinal cord injury, using an AAV2 vector. Regeneration of corticospinal axons was dramatically enhanced. Next, systemic injection of a mutant-AAV9 vector was used to upregulate LKB1 specifically in neurons. This promoted long-distance regeneration of injured corticospinal fibers into caudal spinal cord in adult mice and regrowth of descending serotonergic and tyrosine hydroxylase immunoreactive axons. Either intracortical or systemic viral delivery of LKB1 significantly improved recovery of locomotor functions in adult mice with spinal cord injury. Moreover, we demonstrated that LKB1 used AMPKα, NUAK1, and ERK as the downstream effectors in the cortex of adult mice. Thus, LKB1 may be a critical factor for enhancing the growth capacity of mature neurons and may be an important molecular target in the treatment of CNS injuries.
  • Shih-Yin Ho, Yin-Hsiu Chien, Li-Kai Tsai, Shin-Ichi Muramatsu, Wuh-Liang Hwu, Horng-Huei Liou, Ni-Chung Lee
    Frontiers in cellular neuroscience 13 9 - 9 2019年 [査読有り][通常論文]
     
    Aromatic L-acid decarboxylase (AADC) deficiency causes severe motor disturbances in affected children. A putamen-targeted gene therapy improves the motor function of patients. The present study investigated the electrical properties of dopaminergic (DA) neurons in the substantia nigra compacta (SNc) of mice with an AADC deficiency (DdcKI). The basal firing of DA neurons, which determines DA release in the putamen, was abnormal in the DdcKI mice, including a low frequency and irregular firing pattern, because of a decrease in the after-hyperpolarization (AHP) amplitude of action potentials (APs). The frequency of spontaneous excitatory postsynaptic currents (sEPSCs) increased and that of spontaneous inhibitory PSCs (sIPSCs) decreased in the SNc DA neurons from the DdcKI mice, suggesting an elevation in glutamatergic excitatory stimuli and a reduction in GABAergic inhibitory stimuli, respectively. Altered expression patterns of genes encoding receptors and channels were also observed in the DdcKI mice. Administration of a widespread neuron-specific gene therapy to the brains of the DdcKI mice partially corrected these electric abnormalities. The overexcitability of SNc DA neurons in the presence of generalized dopamine deficiency likely underlies the occurrence of motor disturbances.
  • Hiroyuki Igarashi, Keiko Ikeda, Hiroshi Onimaru, Ryosuke Kaneko, Kyo Koizumi, Kaoru Beppu, Kayo Nishizawa, Yukari Takahashi, Fusao Kato, Ko Matsui, Kazuto Kobayashi, Yuchio Yanagawa, Shin-Ichi Muramatsu, Toru Ishizuka, Hiromu Yawo
    Scientific Reports 8 1 5435  2018年12月 [査読有り][通常論文]
     
    Rats are excellent animal models for experimental neuroscience. However, the application of optogenetics in rats has been hindered because of the limited number of established transgenic rat strains. To accomplish cell-type specific targeting of an optimized optogenetic molecular tool, we generated ROSA26/CAG-floxed STOP-ChRFR(C167A)-Venus BAC rats that conditionally express the step-function mutant channelrhodopsin ChRFR(C167A) under the control of extrinsic Cre recombinase. In primary cultured cortical neurons derived from this reporter rat, only Cre-positive cells expressing ChRFR(C167A) became bi-stable, that is, their excitability was enhanced by blue light and returned to the baseline by yellow~red light. In bigenic pups carrying the Phox2B-Cre driver, ChRFR(C167A) was specifically expressed in the rostral parafacial respiratory group (pFRG) in the medulla, where endogenous Phox2b immunoreactivity was detected. These neurons were sensitive to blue light with an increase in the firing frequency. Thus, this transgenic rat actuator/reporter system should facilitate optogenetic studies involving the effective in vivo manipulation of the activities of specific cell fractions using light of minimal intensity.
  • Hikari Tanaka, Kanoh Kondo, Xigui Chen, Hidenori Homma, Kazuhiko Tagawa, Aurelian Kerever, Shigeki Aoki, Takashi Saito, Takaomi Saido, Shin-Ichi Muramatsu, Kyota Fujita, Hitoshi Okazawa
    Molecular psychiatry 23 10 2090 - 2110 2018年10月 [査読有り][通常論文]
     
    Early-phase pathologies of Alzheimer's disease (AD) are attracting much attention after clinical trials of drugs designed to remove beta-amyloid (Aβ) aggregates failed to recover memory and cognitive function in symptomatic AD patients. Here, we show that phosphorylation of serine/arginine repetitive matrix 2 (SRRM2) at Ser1068, which is observed in the brains of early phase AD mouse models and postmortem end-stage AD patients, prevents its nuclear translocation by inhibiting interaction with T-complex protein subunit α. SRRM2 deficiency in neurons destabilized polyglutamine binding protein 1 (PQBP1), a causative gene for intellectual disability (ID), greatly affecting the splicing patterns of synapse-related genes, as demonstrated in a newly generated PQBP1-conditional knockout model. PQBP1 and SRRM2 were downregulated in cortical neurons of human AD patients and mouse AD models, and the AAV-PQBP1 vector recovered RNA splicing, the synapse phenotype, and the cognitive decline in the two mouse models. Finally, the kinases responsible for the phosphorylation of SRRM2 at Ser1068 were identified as ERK1/2 (MAPK3/1). These results collectively reveal a new aspect of AD pathology in which a phosphorylation signal affecting RNA splicing and synapse integrity precedes the formation of extracellular Aβ aggregates and may progress in parallel with tau phosphorylation.
  • Hirai M, Sakurada T, Muramatsu SI
    Journal of clinical and experimental neuropsychology 41 2 1 - 9 2018年09月 [査読有り][通常論文]
  • Ni-Chung Lee, Wuh-Liang Hwu, Shin-Ichi Muramatsu, Darin J. Falk, Barry J. Byrne, Chia-Hao Cheng, Nien-Chu Shih, Kai-Ling Chang, Li-Kai Tsai, Yin-Hsiu Chien
    Molecular Neurobiology 55 6 5299 - 5309 2018年06月 [査読有り][通常論文]
     
    In Pompe disease, deficient lysosomal acid α-glucosidase (GAA) activity causes glycogen accumulation in the muscles, which leads to weakness, cardiomyopathy, and respiratory failure. Although glycogen accumulation also occurs in the nervous system, the burden of neurological deficits in Pompe disease remains obscure. In this study, a neuron-specific gene therapy was administered to Pompe mice through intracerebroventricular injection of a viral vector carrying a neuron-specific promoter. The results revealed that gene therapy increased GAA activity and decreased glycogen content in the brain and spinal cord but not in the muscles of Pompe mice. Gene therapy only slightly increased the muscle strength of Pompe mice but substantially improved their performance on the rotarod, a test measuring motor coordination. Gene therapy also decreased astrogliosis and increased myelination in the brain and spinal cord of Pompe mice. Therefore, a neuron-specific treatment improved the motor coordination of Pompe mice by lowering glycogen accumulation, decreasing astrogliosis, and increasing myelination. These findings indicate that neurological deficits are responsible for a significant burden in Pompe disease.
  • Abdou K, Shehata M, Choko K, Nishizono H, Matsuo M, Muramatsu SI, Inokuchi K
    Science (New York, N.Y.) 360 6394 1227 - 1231 2018年06月 [査読有り][通常論文]
     
    Memories are integrated into interconnected networks; nevertheless, each memory has its own identity. How the brain defines specific memory identity out of intermingled memories stored in a shared cell ensemble has remained elusive. We found that after complete retrograde amnesia of auditory fear conditioning in mice, optogenetic stimulation of the auditory inputs to the lateral amygdala failed to induce memory recall, implying that the memory engram no longer existed in that circuit. Complete amnesia of a given fear memory did not affect another linked fear memory encoded in the shared ensemble. Optogenetic potentiation or depotentiation of the plasticity at synapses specific to one memory affected the recall of only that memory. Thus, the sharing of engram cells underlies the linkage between memories, whereas synapse-specific plasticity guarantees the identity and storage of individual memories.
  • Inoue R, Abdou K, Hayashi-Tanaka A, Muramatsu SI, Mino K, Inokuchi K, Mori H
    eLife 7 2018年06月 [査読有り][通常論文]
  • Nakamura Sachie, Osaka Hitoshi, Muramatsu Shin-ichi, Takino Naomi, Ito Mika, Jimbo Eriko F, Shimazaki Kuniko, Onaka Tatsushi, Ohtsuki Sumio, Terasaki Tetsuya, Yamagata Takanori
    MOLECULAR THERAPY 26 5 130 - 131 2018年05月 [査読有り][通常論文]
  • Sachie Nakamura, Shin-Ichi Muramatsu, Naomi Takino, Mika Ito, Eriko F Jimbo, Kuniko Shimazaki, Tatsushi Onaka, Sumio Ohtsuki, Tetsuya Terasaki, Takanori Yamagata, Hitoshi Osaka
    The journal of gene medicine 20 4 e3013  2018年04月 [査読有り][通常論文]
     
    BACKGROUND: We generated an adeno-associated virus (AAV) vector in which the human SLC2A1 gene, encoding glucose transporter type 1 (GLUT1), was expressed under the human endogenous GLUT1 promoter (AAV-GLUT1). We examined whether AAV-GLUT1 administration could lead to functional improvement in GLUT1-deficient mice. METHODS: We extrapolated human endogenous GLUT1 promoter sequences from rat minimal Glut1 promoter sequences. We generated a tyrosine-mutant AAV9/3 vector in which human SLC2A1-myc-DDK was expressed under the human GLUT1 promoter (AAV-GLUT1). AAV-GLUT1 was administered to GLUT1-deficient mice (GLUT1+/- mice) via intracerebroventricular injection (1.85 × 1010 vg/mouse or 6.5 × 1010 vg/mouse). We analyzed exogenous GLUT1 mRNA and protein expression in the brain and other major organs. We also examined improvements of cerebral microvasculature, motor function using rota-rod and footprint tests, as well as blood and cerebrospinal fluid (CSF) glucose levels. Additionally, we confirmed exogenous GLUT1 protein distribution in the brain and other organs after intracardiac injection (7.8 × 1011 vg/mouse). RESULTS: Exogenous GLUT1 protein was strongly expressed in the cerebral cortex, hippocampus and thalamus. It was mainly expressed in endothelial cells, and partially expressed in neural cells and oligodendrocytes. Motor function and CSF glucose levels were significantly improved following intracerebroventricular injection. Exogenous GLUT1 expression was not detected in other organs after intracerebroventricular injection of AAV-GLUT1, whereas it was detected in the liver and muscle tissue after intracardiac injection. CONCLUSIONS: Exogenous GLUT1 expression after AAV-GLUT1 injection approximated that of physiological human GLUT1 expression. Local central nervous system administration of AAV-GLUT1 improved CSF glucose levels and motor function of GLUT1-deficient mice and minimized off-target effects.
  • Takeshi Sakurada, Guenther Knoblich, Natalie Sebanz, Shin-ichi Muramatsu, Masahiro Hirai
    Neuropsychologia 111 201 - 208 2018年03月 [査読有り][通常論文]
     
    Information on how the subcortical brain encodes information required to execute actions or to evaluate others’ actions remains scanty. To clarify this link, Fitts'-law tasks for perception and execution were tested in patients with Parkinson's disease (PD). For the perception task, participants were shown apparent motion displays of a person moving their arm between two identical targets and reported whether they judged that the person could realistically move at the perceived speed without missing the targets. For the motor task, participants were required to touch the two targets as quickly and accurately as possible, similarly to the person observed in the perception task. In both tasks, the PD group exhibited, or imputed to others, significantly slower performances than those of the control group. However, in both groups, the relationships of perception and execution with task difficulty were exactly those predicted by Fitts’ law. This suggests that despite dysfunction of the subcortical region, motor simulation abilities reflected mechanisms of compensation in the PD group. Moreover, we found that patients with PD had difficulty in switching their strategy for estimating others’ actions when asked to do so.
  • Atsumi Nitta, Hiroshi Noike, Kazuyuki Sumi, Hajime Miyanishi, Takuya Tanaka, Kazuya Takaoka, Miyuki Nagakura, Noriyuki Iegaki, Jin-ichiro Kaji, Yoshiaki Miyamoto, Shin-Ichi Muramatsu, Kyosuke Uno
    89 - 111 2018年 [査読有り][通常論文]
     
    Shati/Nat8l was originally isolated as a methamphetamine-related-molecule from the nucleus accumbens of mice. Since then, Shati/Nat8l has been characterized as an N-acetyltransferase-8-like protein (Nat8l) that catalyzes N-acetylaspartate (NAA) synthesis from aspartate and acetyl-coenzyme A. It has been shown that elevated NAA levels detected by proton magnetic resonance spectroscopy (1H-MRS) brain imaging indicates increased neuronal activity. Our group produced Shati/Nat8l knock out mice (Shati/Nat8l KO mice), which exhibit hyper locomotion, anxiety behaviors, and social dysfunction. These mice have a high sensitivity to methamphetamine, as evidenced by their results in assessments of locomotor activity and conditioned place preference, as well as their elevated dopamine levels. We used an adeno-associated virus (AAV) vector containing Shati/Nat8l (AAV-Shati/Nat8l) to overexpress the protein in different brain regions such as the striatum and the nucleus accumbens, in order to investigate their involvement in methamphetamine-induced behavioral and pharmacological changes. We showed that overexpression of accumbal Shati/Nat8l attenuates methamphetamine-induced behaviors. Recent clinical studies have revealed further novel roles of Shati/Nat8l in psychiatric and neuronal diseases. We are just beginning to appreciate the various actions of this intriguing, recently discovered molecule in the central nervous system.
  • Yin-Hsiu Chien, Ni-Chung Lee, Sheng-Hong Tseng, Chun-Hwei Tai, Shin-ichi Muramatsu, Barry J Byrne, Wuh-Liang Hwu
    The Lancet Child and Adolescent Health 1 4 265 - 273 2017年12月 [査読有り][通常論文]
     
    Background Aromatic L-amino acid decarboxylase (AADC) deficiency is an inherited disease that causes depletion of neurotransmitters and severe motor dysfunction in infants and children. We previously reported compassionate use of an adeno-associated virus (AAV) vector containing the human AADC gene (AAV2-hAADC) in four children with AADC deficiency (aged 4–6 years). In this study, we aimed to establish the efficacy and safety of this treatment. Methods We did an open-label, phase 1/2 trial at the National Taiwan University Hospital (Taipei, Taiwan). We included patients who had a definitive diagnosis and clinical symptoms of AADC deficiency (hypotonia, dystonia, and oculogyric crisis), who were older than 24 months or had skull bones suitable for stereotactic surgery, and who had an anti-AAV2 antibody titre lower than 1·0 optical density. All patients received bilateral intraputaminal injections of AAV2-hAADC (1·81 × 1011 vg in total) through stereotactic brain surgery. Primary efficacy outcomes were an increase in the Peabody Developmental Motor Scales (second edition PDMS-2) score of greater than 10 points and an increase in homovanillic acid (HVA) or 5-hydroxyindoleacetic acid (5-HIAA) concentrations in the cerebrospinal fluid 12 months after gene therapy. We assessed patients at baseline and at 3, 6, 9, and 12 months after gene therapy, and every 6 months thereafter for one further year all patients who received the treatment were included in the analysis. We assessed for surgical complications (cerebrospinal fluid leakage and intracerebral haemorrhage) at days 3–7 after AAV2 gene therapy, and we assessed adverse events during the follow-up evaluations for 12 months. This study is registered with ClinicalTrials.gov, number NCT01395641. Findings Ten patients (median age 2·71 years, IQR 2·46–6·35) were enrolled from Oct 1, 2014, to Dec 2, 2015. All patients tolerated the surgeries and vector injections. One patient died from influenza B encephalitis during an endemic outbreak 10 months after treatment therefore, 9 months of data were included in the analyses for this patient. All patients met the primary efficacy endpoint: 12 months after gene therapy, PDMS-2 scores were increased by a median of 62 points (IQR 39–93 p=0·005) and HVA concentrations by a median of 25 nmol/L (IQR 11–48 p=0·012) however, there was no significant change in 5-HIAA concentrations (median difference 0, IQR 0–5 p=0·20). In total, 101 adverse events were reported, with the most common being pyrexia (16 [16%] of 101 events) and orofacial dyskinesia (ten [10%]). 12 serious adverse events occurred in six patients, including one death (treatment-unrelated encephalitis due to influenza B infection), one life-threatening pyrexia, and ten events that led to hospital admission. Transient post-gene therapy dyskinesia occurred in all patients but was resolved with risperidone. Of 31 treatment-related adverse events, only one (patient 1) was severe in intensity, and none led to hospital admission or death. Interpretation Our findings suggest that intraputaminal injection of AAV2-hAADC is well tolerated and might improve motor development in children with AADC deficiency. Funding AADC Research Fund at National Taiwan University Hospital and the National Research Programme for Biopharmaceuticals.
  • Yoshiaki Miyamoto, Noriyuki Iegaki, Kequan Fu, Yudai Ishikawa, Kazuyuki Sumi, Sota Azuma, Kyosuke Uno, Shin-Ichi Muramatsu, Atsumi Nitta
    International Journal of Neuropsychopharmacology 20 12 1027 - 1035 2017年12月 [査読有り][通常論文]
     
    Background: Several clinical studies have suggested that N-acetylaspartate and N-acetylaspartylglutamate levels in the human brain are associated with various psychiatric disorders, including major depressive disorder. We have previously identified Shati/Nat8l, an N-acetyltransferase, in the brain using an animal model of psychosis. Shati/Nat8l synthesizes N-acetylaspartate from L-aspartate and acetyl-coenzyme A. Further, N-acetylaspartate is converted into N-acetylaspartylglutamate, a neurotransmitter for metabotropic glutamate receptor 3. Methods: Because Shati/Nat8l mRNA levels were increased in the dorsal striatum of mice following the exposure to forced swimming stress, Shati/Nat8l was overexpressed in mice by the microinjection of adeno-associated virus vectors containing Shati/Nat8l gene into the dorsal striatum (dS-Shati/Nat8l mice). The dS-Shati/Nat8l mice were further assessed using behavioral and neurochemical tests. Results: The dS-Shati/Nat8l mice exhibited behavioral despair in the forced swimming and tail suspension tests and social withdrawal in the 3-chamber social interaction test. These depression-like behaviors were attenuated by the administration of a metabotropic glutamate receptor 2/3 antagonist and a selective serotonin reuptake inhibitor. Furthermore, the metabolism of N-acetylaspartate to N-acetylaspartylglutamate was decreased in the dorsal striatum of the dS-Shati/Nat8l mice. This finding corresponded with the increased expression of glutamate carboxypeptidase II, an enzyme that metabolizes Nacetylaspartylglutamate present in the extracellular space. Extracellular serotonin levels were lower in the dorsal striatum of the dS-Shati/Nat8l and normal mice that were repeatedly administered a selective glutamate carboxypeptidase II inhibitor. Conclusions: Our findings indicate that the striatal expression of N-acetylaspartate synthetase Shati/Nat8l plays a role in major depressive disorder via the metabotropic glutamate receptor 3-mediated functional control of the serotonergic neuronal system.
  • Kequan Fu, Yoshiaki Miyamoto, Kazuyuki Sumi, Eriko Saika, Shin-ichi Muramatsu, Kyosuke Uno, Atsumi Nitta
    PLOS ONE 12 12 e0189006  2017年12月 [査読有り][通常論文]
     
    Transmembrane protein 168 (TMEM168) comprises 697 amino acid residues, including some putative transmembrane domains. It is reported that TMEM168 controls methamphetamine (METH) dependence in the nucleus accumbens (NAc) of mice. Moreover, a strong link between METH dependence-induced adaptive changes in the brain and mood disorders has been evaluated. In the present study, we investigated the effects of accumbal TMEM168 in a battery of behavioral paradigms. The adeno-associated virus (AAV) Tmem168 vector was injected into the NAc of C57BL/6J mice (NAc-TMEM mice). Subsequently, the accumbal TMEM168 mRNA was increased approximately by seven-fold when compared with the NAc-Mock mice (controls). The NAc-TMEM mice reported no change in the locomotor activity, cognitive ability, social interaction, and depression-like behaviors; however, TMEM168 overexpression enhanced anxiety in the elevated-plus maze and light/dark box test. The increased anxiety was reversed by pretreatment with the antianxiety drug diazepam (0.3 mg/kg i.p.). Moreover, the NAc-TMEM mice exhibited decreased prepulse inhibition (PPI) in the startle response test, and the induced schizophrenia-like behavior was reversed by pretreatment with the antipsychotic drug risperidone (0.01 mg/kg i.p.). Furthermore, accumbal TMEM168 overexpression decreased the basal levels of extracellular GABA in the NAc and the high K+ (100 mM)-stimulated GABA elevation; however, the total contents of GABA in the NAc remained unaffected. These results suggest that the TMEM168-regulated GABAergic neuronal system in the NAc might become a novel target while studying the etiology of anxiety and sensorimotor gating deficits.
  • Kequan Fu, Yoshiaki Miyamoto, Kazuya Otake, Kazuyuki Sumi, Eriko Saika, Shohei Matsumura, Naoki Sato, Yuka Ueno, Seunghee Seo, Kyosuke Uno, Shin-ichi Muramatsu, Atsumi Nitta
    SCIENTIFIC REPORTS 7 1 13084  2017年10月 [査読有り][通常論文]
     
    Chronic exposure to methamphetamine causes adaptive changes in brain, which underlie dependence symptoms. We have found that the transmembrane protein 168 (TMEM168) is overexpressed in the nucleus accumbens of mice upon repeated methamphetamine administration. Here, we firstly demonstrate the inhibitory effect of TMEM168 on methamphetamine-induced behavioral changes in mice, and attempt to elucidate the mechanism of this inhibition. We overexpressed TMEM168 in the nucleus accumbens of mice by using an adeno-associated virus vector (NAc-TMEM mice). Methamphetamine-induced hyperlocomotion and conditioned place preference were attenuated in NAc-TMEM mice. Additionally, methamphetamine-induced extracellular dopamine elevation was suppressed in the nucleus accumbens of NAc-TMEM mice. Next, we identified extracellular matrix protein osteopontin as an interacting partner of TMEM168, by conducting immunoprecipitation in cultured COS-7 cells. TMEM168 overexpression in COS-7 cells induced the enhancement of extracellular and intracellular osteopontin. Similarly, osteopontin enhancement was also observed in the nucleus accumbens of NAc-TMEM mice, in in vivo studies. Furthermore, the infusion of osteopontin proteins into the nucleus accumbens of mice was found to inhibit methamphetamine-induced hyperlocomotion and conditioned place preference. Our studies suggest that the TMEM168-regulated osteopontin system is a novel target pathway for the therapy of methamphetamine dependence, via regulating the dopaminergic function in the nucleus accumbens.
  • Tsukasa Ohmori, Yasumitsu Nagao, Hiroaki Mizukami, Asuka Sakata, Shin-ichi Muramatsu, Keiya Ozawa, Shin-ichi Tominaga, Yutaka Hanazono, Satoshi Nishimura, Osamu Nureki, Yoichi Sakata
    SCIENTIFIC REPORTS 7 1 4159  2017年06月 [査読有り][通常論文]
     
    Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B. Mutation-specific gene editing by simultaneous induction of homology-directed repair (HDR) sufficiently increased FIX levels to correct the disease phenotype. Insertion of F9 cDNA into the intron more efficiently restored haemostasis via both processes of non-homologous end-joining (NHEJ) and HDR following DSB. Notably, these therapies also cured neonate mice with haemophilia, which cannot be achieved with conventional gene therapy with AAV vector. Ongoing haemophilia therapy targeting the antithrombin gene with antisense oligonucleotide could be replaced by SaCas9/sgRNA-expressing AAV8 vector. Our results suggest that CRISPR/Cas9-mediated genome editing using an AAV8 vector provides a flexible approach to induce DSB at target genes in hepatocytes and could be a good strategy for haemophilia gene therapy.
  • Yoshihide Sehara, Ken-ichi Fujimoto, Kunihiko Ikeguchi, Yuko Katakai, Fumiko Ono, Naomi Takino, Mika Ito, Keiya Ozawa, Shin-ichi Muramatsu
    HUMAN GENE THERAPY CLINICAL DEVELOPMENT 28 2 74 - 79 2017年06月 [査読有り][通常論文]
     
    Restoring dopamine production in the putamen through gene therapy is a straightforward strategy for ameliorating motor symptoms for Parkinson's disease (PD). In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity-based primate model of PD, we previously showed the safety and efficacy of adeno-associated viral (AAV) vector-mediated gene delivery to the putamen of three dopamine-synthesizing enzymes (tyrosine hydroxylase [TH], aromatic l-amino acid decarboxylase [AADC], and guanosine triphosphate cyclohydrolase I [GCH]) up to 10 months postprocedure. Although three of four monkeys in this study have previously undergone postmortem analysis, one monkey was kept alive for 15 years after gene therapy to evaluate long-term effects. Here, we report that this monkey showed behavioral recovery in the right-side limb that remained unchanged for 15 years, at which time euthanasia was carried out owing to onset of senility. Immunohistochemistry of the postmortem brain from this monkey revealed persistent expression of TH, AADC, and GCH genes in the lesioned putamen. Transduced neurons were broadly distributed, with the estimated transduction region occupying 91% of the left postcommissural putamen. No signs of cytotoxicity or Lewy body pathology were observed in the AAV vector-injected putamen. This study provides evidence of long-term safety and efficacy of the triple-transduction method as a gene therapy for PD.
  • Sachie Nakamura, Hitoshi Osaka, Shin-Ichi Muramatsu, Naomi Takino, Mika Ito, Shiho Aoki, Eriko F Jimbo, Kuniko Shimazaki, Tatsushi Onaka, Sumio Ohtsuki, Tetsuya Terasaki, Takanori Yamagata
    Molecular genetics and metabolism reports 10 67 - 74 2017年03月 [査読有り][通常論文]
     
    OBJECTIVE: We generated an adeno-associated virus (AAV) vector in which the human SLC2A1 gene was expressed under the synapsin I promoter (AAV-hSLC2A1) and examined if AAV-hSLC2A1 administration can lead to functional improvement in GLUT1-deficient mice. METHODS: AAV-hSLC2A1 was injected into heterozygous knock-out murine Glut1 (GLUT1+/-) mice intraperitoneally (systemic; 1.85 × 1011 vg/mouse) or intra-cerebroventricularly (local; 1.85 × 1010 vg/mouse). We analyzed GLUT1 mRNA and protein expression, motor function using rota-rod and footprint tests, and blood and cerebrospinal fluid (CSF) glucose levels. RESULTS: Vector-derived RNA was detected in the cerebrum for both injection routes. In the intra-cerebroventricular injection group, exogenous GLUT1 protein was strongly expressed in the cerebral cortex and hippocampus near the injection site. In the intraperitoneal injection group, exogenous GLUT1 protein was mildly expressed in neural cells throughout the entire central nervous system. The motor function test and CSF/blood glucose ratio were significantly improved following intra-cerebroventricular injection. CONCLUSIONS: AAV-hSLC2A1 administration produced exogenous GLUT1 in neural cells and improved CSF glucose levels and motor function of heterozygous knock-out murine Glut1 mice.
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • Haruhiro Higashida, Shigeru Yokoyama, Chiharu Tsuji, Shin-ichi Muramatsu
    JOURNAL OF PHYSIOLOGICAL SCIENCES 67 1 11 - 17 2017年01月 [査読有り][通常論文]
     
    We overview the 16-kDa proteolipid mediatophore, the transmembrane c-subunit of the V0 sector of the vacuolar proton ATPase (ATP6V0C) that was shown to mediate the secretion of acetylcholine. Acetylcholine, serotonin, and dopamine (DA) are released from cell soma and/or dendrites if ATP6V0C is expressed in cultured cells. Adeno-associated viral vector-mediated gene transfer of ATP6V0C into the caudate putamen enhanced the depolarization-induced overflow of endogenous DA in Parkinson-model mice. Motor impairment was ameliorated in hemiparkinsonian model mice when ATP6V0C was expressed with DA-synthesizing enzymes. The review discusses application in the future as a potential tool for gene therapy, cell transplantation therapy, and inducible pluripotent stem cell therapy in neurological diseases, from the view point of recent findings regarding vacuolar ATPase.
  • 【パーキンソン病-基礎・臨床の最新情報-】 パーキンソン病の遺伝子治療
    村松 慎一
    日本臨床 75 1 146  (株)日本臨床社 2017年01月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病における遺伝子治療の有効性 「Yes」の立場から
    村松 慎一
    Frontiers in Parkinson Disease 9 4 176  (株)メディカルレビュー社 2016年11月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • 村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • 村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • 村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • 村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • 村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • 村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • 村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • 村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • 村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • ALS ALSの遺伝子治療
    村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • 村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • 村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • Juliana Bosso Taniguchi, Kanoh Kondo, Kyota Fujita, Xigui Chen, Hidenori Homma, Takeaki Sudo, Ying Mao, Kei Watase, Toshihiro Tanaka, Kazuhiko Tagawa, Takuya Tamura, Shin-Ichi Muramatsu, Hitoshi Okazawa
    HUMAN MOLECULAR GENETICS 25 20 4432 - 4447 2016年10月 [査読有り][通常論文]
     
    DNA damage and repair is a critical domain of many neurodegenerative diseases. In this study, we focused on RpA1, a candidate key molecule in polyQ disease pathologies, and tested the therapeutic effect of adeno-associated virus (AAV) vector expressing RpA1 on mutant Ataxin-1 knock-in (Atxn1-KI) mice. We found significant effects on motor functions, normalized DNA damage markers (gamma H2AX and 53BP1), and improved Purkinje cell morphology; effects that lasted for 50 weeks following AAV-RpA1 infection. In addition, we confirmed that AAV-RpA1 indirectly recovered multiple cellular functions such as RNA splicing, transcription and cell cycle as well as abnormal morphology of dendrite and dendritic spine of Purkinje cells in Atxn1-KI mice. All these results suggested a possibility of gene therapy with RpA1 for SCA1.
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • ALS ALSの遺伝子治療
    村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • パーキンソン病にウイルスベクターを用いた遺伝子治療は可能か YES
    村松 慎一
    パーキンソン病・運動障害疾患コングレスプログラム・抄録集 10回 56  Movement Disorder Society of Japan (MDSJ) 2016年10月 [査読無し][通常論文]
  • 村松 慎一
    神経治療学 33 5 S90  日本神経治療学会 2016年10月 [査読無し][通常論文]
  • Taniguchi, JB, Kondo, K, Fujita, K, Chen, X, Homma, H, Sudo, T, Mao, Y, Watase, K, Tanaka, T, Tagawa, K, Tamura, T, Muramatsu, SI, Okazawa, H
    Hum. Mol. Genet. 2016年08月 [査読有り][通常論文]
     
    DNA damage and repair is a critical domain of many neurodegenerative diseases. In this study, we focused on RpA1, a candidate key molecule in polyQ disease pathologies, and tested the therapeutic effect of adeno-associated virus (AAV) vector expressing RpA1 on mutant Ataxin-1 knock-in (Atxn1-KI) mice. We found significant effects on motor functions, normalized DNA damage markers (γH2AX and 53BP1), and improved Purkinje cell morphology; effects that lasted for 50 weeks following AAV-RpA1 infection. In addition, we confirmed that AAV-RpA1 indirectly recovered multiple cellular functions such as RNA splicing, transcription and cell cycle as well as abnormal morphology of dendrite and dendritic spine of Purkinje cells in Atxn1-KI mice. All these results suggested a possibility of gene therapy with RpA1 for SCA1.
  • Taniguchi JB, Kondo K, Fujita K, Chen X, Homma H, Sudo T, Mao Y, Watase K, Tanaka T, Tagawa K, Tamura T, Muramatsu SI, Okazawa H
    Human molecular genetics 2016年08月 [査読有り][通常論文]
  • Yu Miyazaki, Xiaofei Du, Shin-ichi Muramatsu, Christopher M. Gomez
    SCIENCE TRANSLATIONAL MEDICINE 8 347 347ra94  2016年07月 [査読有り][通常論文]
     
    Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited neurodegenerative disease characterized by slowly progressive ataxia and Purkinje cell degeneration. SCA6 is caused by a polyglutamine repeat expansion within a second CACNA1A gene product, alpha 1ACT. alpha 1ACT expression is under the control of an internal ribosomal entry site (IRES) present within the CACNA1A coding region. Whereas SCA6 allele knock-in mice show indistinguishable phenotypes from wild-type littermates, expression of SCA6-associated alpha 1ACT (alpha 1ACT(SCA6)) driven by a Purkinje cell-specific promoter in mice produces slowly progressive ataxia and cerebellar atrophy. We developed an early-onset SCA6 mouse model using an adeno-associated virus (AAV)-based gene delivery system to ectopically express CACNA1A IRES-driven alpha 1ACT(SCA6) to test the potential of CACNA1A IRES-targeting therapies. Mice expressing AAV9-mediated CACNA1A IRES-driven alpha 1ACT(SCA6) exhibited early-onset ataxia, motor deficits, and Purkinje cell degeneration. We identified miR-3191-5p as a microRNA (miRNA) that targeted CACNA1A IRES and preferentially inhibited the CACNA1A IRES-driven translation of a1ACT in an Argonaute 4 (Ago4)-dependent manner. We found that eukaryotic initiation factors (eIFs), eIF4AII and eIF4GII, interacted with the CACNA1A IRES to enhance alpha 1ACT translation. Ago4-bound miR-3191-5p blocked the interaction of eIF4AII and eIF4GII with the CACNA1A IRES, attenuating IRES-driven alpha 1ACT translation. Furthermore, AAV9-mediated delivery of miR-3191-5p protected mice from the ataxia, motor deficits, and Purkinje cell degeneration caused by CACNA1A IRES-driven alpha 1ACT(SCA6). We have established proof of principle that viral delivery of an miRNA can rescue a disease phenotype through modulation of cellular IRES activity in a mouse model.
  • Hiroyuki Igarashi, Kyo Koizumi, Ryosuke Kaneko, Keiko Ikeda, Ryo Egawa, Yuchio Yanagawa, Shin-Ichi Muramatsu, Hiroshi Onimaru, Toru Ishizuka, Hiromu Yawo
    PLoS ONE 11 5 e0155687  2016年05月 [査読有り][通常論文]
     
    Despite the strength of the Cre/loxP recombination system in animal models, its application in rats trails that in mice because of the lack of relevant reporter strains. Here, we generated a floxed STOP tdTomato rat that conditionally expresses a red fluorescent protein variant (tdTomato) in the presence of exogenous Cre recombinase. The tdTomato signal vividly visualizes neurons including their projection fibers and spines without any histological enhancement. In addition, a transgenic rat line (FLAME) that ubiquitously expresses tdTomato was successfully established by injecting intracytoplasmic Cre mRNA into fertilized ova. Our rat reporter system will facilitate connectome studies as well as the visualization of the fine structures of genetically identified cells for long periods both in vivo and ex vivo. Furthermore, FLAME is an ideal model for organ transplantation research owing to improved traceability of cells/tissues.
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • 村松 慎一
    総合診療 26 3 202  (株)医学書院 2016年03月 [査読無し][通常論文]
  • Kanazawa M, Ohba H, Harada N, Kakiuchi T, Muramatsu S, Tsukada H
    Journal of nuclear medicine : official publication, Society of Nuclear Medicine 57 2 303 - 308 2016年02月 [査読有り][通常論文]
     
    We recently developed a novel PET probe, 6-C-11-methyl-m-tyrosine (C-11-6MemTyr), for quantitative imaging of presynaptic dopamine synthesis in the living brain. In the present study, C-11-6MemTyr was compared with beta-C-11-L-DOPA and 6-F-18-fluoro-L-dopa (F-18-FDOPA) in the brains of normal and Parkinson disease (PD) model monkeys (Macaca fascicularis). Methods: PD model monkeys were prepared by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, and C-11-beta-CFT was applied to assess neuronal damage as dopamine transporter (DAT) availability. C-11-6MemTyr, beta-C-11-L-DOPA, or F-18-FDOPA was injected with and without carbidopa, a specific inhibitor of peripheral aromatic L-amino acid decarboxylase. In normal and PD monkeys, the dopamine synthesis rates calculated using PET probes were analyzed by the correlation plot with DAT availability in the striatum. Results: In normal monkeys, whole-brain uptake of beta-C-11-L-DOPA and F-18-FDOPA were significantly increased by carbidopa at the clinical dose of 5 mg/kg by mouth. In contrast, C-11-6MemTyr was not affected by carbidopa at this dose, and the metabolic constant value of C-11-6MemTyr in the striatum was significantly higher than those of the other 2 PET probes. Significant reduction of the presynaptic DAT availability in the striatum was detected in MPTP monkeys, and correlation analyses demonstrated that C-11-6MemTyr could detect dopaminergic damage in the striatum with much more sensitivity than the other PET probes. Conclusion: C-11-6MemTyr is a potential PET probe for quantitative imaging of presynaptic dopamine activity in the living brain with PET.
  • Sayaka Asari Ono, Toshihiko Sato, Shin-ichi Muramatsu
    PARKINSONS DISEASE 2016年 [査読有り][通常論文]
     
    Freezing of gait (FOG) is a common disorder in Parkinson's disease (PD) and could be attributed to a reduction in brain noradrenaline. The aim of this study was to determine the relationship between aromatic L-amino acid decarboxylase (AADC) activity in the locus coeruleus (LC) and FOG in PD using high-resolution positron emission tomography with an AADC tracer, 6-[F-18]fluoro-L-m-tyrosine (FMT). We assessed 40 patients with PD and 11 age-matched healthy individuals. PD was diagnosed based on the UK Brain Bank criteria by two movement disorder experts. FOG was directly observed by the clinician and assessed using a patient questionnaire. FMT uptake in the LC, caudate, and putamen was analyzed using PMOD software on coregistered magnetic resonance images. FOG was present in 30 patients. The severity of FOG correlated with the decrease of FMT uptake in the LC regardless of disease duration and the severity of other motor impairments, indicating dysfunction of the noradrenergic network in FOG.
  • Ono SA, Sato T, Muramatsu S
    Parkinson's disease 2016 5430920  2016年 [査読有り][通常論文]
     
    Freezing of gait (FOG) is a common disorder in Parkinson's disease (PD) and could be attributed to a reduction in brain noradrenaline. The aim of this study was to determine the relationship between aromatic l-amino acid decarboxylase (AADC) activity in the locus coeruleus (LC) and FOG in PD using high-resolution positron emission tomography with an AADC tracer, 6-[18F]fluoro-l-m-tyrosine (FMT). We assessed 40 patients with PD and 11 age-matched healthy individuals. PD was diagnosed based on the UK Brain Bank criteria by two movement disorder experts. FOG was directly observed by the clinician and assessed using a patient questionnaire. FMT uptake in the LC, caudate, and putamen was analyzed using PMOD software on coregistered magnetic resonance images. FOG was present in 30 patients. The severity of FOG correlated with the decrease of FMT uptake in the LC regardless of disease duration and the severity of other motor impairments, indicating dysfunction of the noradrenergic network in FOG.
  • Sachie Nakamura, Hitoshi Osaka, Shinichi Muramatsu, Shiho Aoki, Eriko F. Jimbo, Takanori Yamagata
    Molecular Genetics and Metabolism 116 3 157 - 162 2015年11月 [査読有り][通常論文]
     
    Objective: We investigated a correlation between a mutation in the SLC2A1 gene and functional disorders in Glucose transporter I deficiency syndrome (GLUT1DS). Methods: We performed direct sequence analysis of SLC2A1 in a severe GLUT1DS patient and identified a novel frame shift mutation, c.906_907insG, p.V303fs. We created a plasmid vector carrying the c.906_907insG mutation, as well as A405D or R333W in the SLC2A1, which are found in patients with mild and moderate GLUT1DS severity, respectively. We transiently expressed these mutants and wild type SLC2A1 plasmids in a human embryonic kidney cell line (HEK293), and performed immunoblotting, immunofluorescence, and enzymatic photometric 2-deoxyglucose (2DG) uptake assays. Results: GLUT1 was not detected after transient expression of the SLC2A1 plasmid carrying c.906_907insG by either immunoblotting or immunofluorescence. The degree of glucose transport reduction as determined by enzymatic photometric 2DG assay uptake correlated with disease severity. Conclusions: Enzymatic photometric 2DG uptake study appears to be a suitable functional assay to predict the effect of SLC2A1 mutations on GLUT1 transport.
  • Ni-Chung Lee, Shin-ichi Muramatsu, Yin-Hsiu Chien, Wen-Shin Liu, Wei-Hua Wang, Chia-Hao Cheng, Meng-Kai Hu, Pin-Wen Chen, Kai-Yuan Tzen, Barry J. Byrne, Wuh-Liang Hwu
    MOLECULAR THERAPY 23 10 1572 - 1581 2015年10月 [査読有り][通常論文]
     
    Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disease that impairs synthesis of dopamine and serotonin. Children with AADC deficiency exhibit severe motor, behavioral, and autonomic dysfunctions. We previously generated an IVS6+4A>T knock-in mouse model of AADC deficiency (Ddc(KI) mice) and showed that gene therapy at the neonatal stage can rescue this phenotype. In the present study, we extended this treatment to systemic therapy on young mice. After intraperitoneal injection of AADC viral vectors into 7-day-old DdcKI mice, the treated mice exhibited improvements in weight gain, survival, motor function, autonomic function, and behavior. The yfAAV9/3-Syn-I-mAADC-treated mice showed greater neuronal transduction and higher brain dopamine levels than AAV9-CMV-hAADC-treated mice, whereas AAV9-CMV-hAADC-treated mice exhibited hyperactivity. Therefore, neurotransmitter-deficient animals can be rescued at a young age using systemic gene therapy, although a vector for preferential neuronal expression may be necessary to avoid hyperactivity caused by this treatment.
  • Ni-Chung Lee, Shin-ichi Muramatsu, Yin-Hsiu Chien, Wen-Shin Liu, Wei-Hua Wang, Chia-Hao Cheng, Meng-Kai Hu, Pin-Wen Chen, Kai-Yuan Tzen, Barry J. Byrne, Wuh-Liang Hwu
    MOLECULAR THERAPY 23 10 1572 - 1581 2015年10月 [査読有り][通常論文]
     
    Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disease that impairs synthesis of dopamine and serotonin. Children with AADC deficiency exhibit severe motor, behavioral, and autonomic dysfunctions. We previously generated an IVS6+4A>T knock-in mouse model of AADC deficiency (Ddc(KI) mice) and showed that gene therapy at the neonatal stage can rescue this phenotype. In the present study, we extended this treatment to systemic therapy on young mice. After intraperitoneal injection of AADC viral vectors into 7-day-old DdcKI mice, the treated mice exhibited improvements in weight gain, survival, motor function, autonomic function, and behavior. The yfAAV9/3-Syn-I-mAADC-treated mice showed greater neuronal transduction and higher brain dopamine levels than AAV9-CMV-hAADC-treated mice, whereas AAV9-CMV-hAADC-treated mice exhibited hyperactivity. Therefore, neurotransmitter-deficient animals can be rescued at a young age using systemic gene therapy, although a vector for preferential neuronal expression may be necessary to avoid hyperactivity caused by this treatment.
  • Kazuyuki Sumi, Kyosuke Uno, Shohei Matsumura, Yoshiaki Miyamoto, Yoko Furukawa-Hibi, Shin-Ichi Muramatsu, Toshitaka Nabeshima, Atsumi Nitta
    Neuroreport 26 13 740 - 6 2015年09月 [査読有り][通常論文]
     
    A novel N-acetyltransferase, Shati/Nat8l, was identified in the nucleus accumbens of mice repeatedly treated with methamphetamine (METH). Shati/Nat8l has been reported to inhibit the pharmacological action induced by METH. Shati/Nat8l produces N-acetylaspartate from aspartate and acetyl-CoA. Previously, we reported that overexpression of Shati/Nat8l in nucleus accumbens attenuates the response to METH by N-acetylaspartylglutamate (which is derived from N-acetylaspartate)-mGluR3 signaling in the mice brain. In the present study, to clarify the type of cells that produce Shati/Nat8l, we carried out in-situ hybridization for the detection of Shati/Nat8l mRNA along with immunohistochemical studies using serial sections of mice brain. Shati/Nat8l mRNA was detected in neuronal cells, but not in astrocytes or microglia cells. Next, we investigated the function of Shati/Nat8l in the neuronal cells in mice brain; then, we used an adeno-associated virus vector containing Shati/Nat8l for transfection and overexpression of Shati/Nat8l protein into the primary cultured neurons to investigate the contribution toward the neuronal activity of Shati/Nat8l. Overexpression of Shati/Nat8l in the mice primary cultured neurons induced axonal growth, but not dendrite elongation at day 1.5 (DIV). This finding indicated that Shati/Nat8l contributes toward neuronal development. LY341495, a selective group II mGluRs antagonist, did not abolish this axonal growth, and N-acetylaspartylglutamate itself did not abolish axon outgrowth in the same cultured system. The cultured neurons overexpressing Shati/Nat8l contained high ATP, suggesting that axon outgrowth is dependent on energy metabolism. This study shows that Shati/Nat8l in the neuron may induce axon outgrowth by ATP synthesis and not through mGluR3 signaling.
  • Ohnishi T, Yanazawa M, Sasahara T, Kitamura Y, Hiroaki H, Fukazawa Y, Kii I, Nishiyama T, Kakita A, Takeda H, Takeuchi A, Arai Y, Ito A, Komura H, Hirao H, Satomura K, Inoue M, Muramatsu S, Matsui K, Tada M, Sato M, Saijo E, Shigemitsu Y, Sakai S, Umetsu Y, Goda N, Takino N, Takahashi H, Hagiwara M, Sawasaki T, Iwasaki G, Nakamura Y, Nabeshima Y, Teplow DB, Hoshi M
    Proceedings of the National Academy of Sciences of the United States of America 112 32 E4465 - E4474 2015年08月 [査読有り][通常論文]
     
    Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid beta-protein (A beta) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical A beta oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuronspecific Na+/K+-ATPase alpha 3 subunit (NAK alpha 3). ASPD-binding to NAK alpha 3 impaired NAK alpha 3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-A beta-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAK alpha 3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAK alpha 3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAK alpha 3 interaction.
  • H. Ito, H. Shiwaku, C. Yoshida, H. Homma, H. Luo, X. Chen, K. Fujita, L. Musante, U. Fischer, S. G.M. Frints, C. Romano, Y. Ikeuchi, T. Shimamura, S. Imoto, S. Miyano, S. I. Muramatsu, T. Kawauchi, M. Hoshino, M. Sudol, A. Arumughan, E. E. Wanker, T. Rich, C. Schwartz, F. Matsuzaki, A. Bonni, V. M. Kalscheuer, H. Okazawa
    Molecular Psychiatry 20 4 459 - 471 2015年04月 [査読有り][通常論文]
     
    Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.
  • Hikaru Ito, Kyota Fujita, Kazuhiko Tagawa, Xigui Chen, Hidenori Homma, Toshikazu Sasabe, Jun Shimizu, Shigeomi Shimizu, Takuya Tamura, Shin-Ichi Muramatsu, Hitoshi Okazawa
    EMBO Molecular Medicine 7 1 78 - 101 2015年01月 [査読有り][通常論文]
     
    Mutant ataxin-1 (Atxn1), which causes spinocerebellar ataxia type 1 (SCA1), binds to and impairs the function of high-mobility group box 1 (HMGB1), a crucial nuclear protein that regulates DNA architectural changes essential for DNA damage repair and transcription. In this study, we established that transgenic or virus vector-mediated complementation with HMGB1 ameliorates motor dysfunction and prolongs lifespan in mutant Atxn1 knock-in (Atxn1-KI) mice. We identified mitochondrial DNA damage repair by HMGB1 as a novel molecular basis for this effect, in addition to the mechanisms already associated with HMGB1 function, such as nuclear DNA damage repair and nuclear transcription. The dysfunction and the improvement of mitochondrial DNA damage repair functions are tightly associated with the exacerbation and rescue, respectively, of symptoms, supporting the involvement of mitochondrial DNA quality control by HMGB1 in SCA1 pathology. Moreover, we show that the rescue of Purkinje cell dendrites and dendritic spines by HMGB1 could be downstream effects. Although extracellular HMGB1 triggers inflammation mediated by Toll-like receptor and receptor for advanced glycation end products, upregulation of intracellular HMGB1 does not induce such side effects. Thus, viral delivery of HMGB1 is a candidate approach by which to modify the disease progression of SCA1 even after the onset. Synopsis: Spinocerebellar ataxia type 1 (SCA1) is an intractable neurodegenerative disease. A gene therapy approach targeting HMGB1 against the SCA1 pathology in a mutant Atxn1 knock-in mouse model prolonged lifespan and correct DNA damage defects in the mitochondrial genome. Mitochondrial genome DNA damage is repaired by HMGB1. The abnormal gene expression profile of Purkinje cells is partially corrected by HMGB1. The mean and maximum lifespan of Atxn1-KI mice are substantially prolonged (by 60-70%) by means of the gene therapy of HMGB1. Spinocerebellar ataxia type 1 (SCA1) is an intractable neurodegenerative disease. A gene therapy approach targeting HMGB1 against the SCA1 pathology in a mutant Atxn1 knock-in mouse model prolonged lifespan and correct DNA damage defects in the mitochondrial genome.
  • Wataru Kakegawa, Nikolaos Mitakidis, Eriko Miura, Manabu Abe, Keiko Matsuda, Yukari H. Takeo, Kazuhisa Kohda, Junko Motohashi, Akiyo Takahashi, Soichi Nagao, Shin-ichi Muramatsu, Masahiko Watanabe, Kenji Sakimura, A. Radu Aricescu, Michisuke Yuzaki
    NEURON 85 2 316 - 329 2015年01月 [査読有り][通常論文]
     
    Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as a crucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesion G-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions.
  • Miyamoto Y, Iida A, Sato K, Muramatsu S, Nitta A
    The international journal of neuropsychopharmacology 18 4 2014年10月 [査読有り][通常論文]
     
    BACKGROUND: Addictive drugs lead to reinforcing properties by increasing dopamine in the nucleus accumbens, which is composed of a core and shell regions. Neurons in the nucleus accumbens are divided into 2 subtypes based on the differential gene expression of the dopamine D₁ receptors and D₂ receptors. METHODS: In the present study, we investigated the role of D₂ receptors in the nucleus accumbens core in behaviors and signal transduction induced by psychostimulant methamphetamine in mice that were microinjected with adeno-associated virus vectors containing a microRNA (miRNA) sequence for D₂ receptor (adeno-associated virus-miD2r vectors) in the nucleus accumbens core. The adeno-associated virus vectors containing a miRNA sequence for D₂ receptor-treated mice (miD₂r mice) were assessed at a reduction in D₂ receptor, but at no change in dopamine D₁ receptor, in the nucleus accumbens core compared with the adeno-associated virus-Mock vectors-treated mice (Mock mice). RESULTS: miD₂r mice exhibited a reduction in hyperlocomotion that was induced by a single treatment with methamphetamine. The development of locomotor sensitization induced by repeated treatment with methamphetamine exhibited less extension in miD₂r mice. In a place conditioning paradigm, the preferred effects of methamphetamine were significantly weaker in miD₂r mice than in Mock mice. Furthermore, the single treatment with methamphetamine-induced phosphorylation of extracellular signal regulated kinase and cyclic adenosine monophosphate response element-binding protein in the nucleus accumbens core of miD₂r mice was decreased compared with that in Mock mice. Repeated treatment with methamphetamine-induced delta FBJ murine osteosarcoma viral oncogene homolog B accumulation in the nucleus accumbens core of miD₂r mice was also attenuated. CONCLUSIONS: These findings suggest that a D₂ receptor-mediated neuronal pathway from the nucleus accumbens core plays an inhibitory role in the development of reinforcing properties.
  • Yoshiaki Miyamoto, Yudai Ishikawa, Noriyuki Iegaki, Kazuyuki Sumi, Kequan Fu, Keiji Sato, Yoko Furukawa-Hibi, Shin-ichi Muramatsu, Toshitaka Nabeshima, Kyosuke Uno, Atsumi Nitta
    INTERNATIONAL JOURNAL OF NEUROPSYCHOPHARMACOLOGY 17 8 1283 - 1294 2014年08月 [査読有り][通常論文]
     
    A novel N-acetyltransferase, Shati/Nat8l, was identified in the nucleus accumbens (NAc) of mice with methamphetamine (METH) treatment. Previously we reported that suppression of Shati/Nat8l enhanced METH-induced behavioral alterations via dopaminergic neuronal regulation. However, the physiological mechanisms of Shati/Nat8l on the dopaminergic system in the brain are unclear. In this study, we injected adeno-associated virus (AAV) vector containing Shati/Nat8l into the NAc or dorsal striatum (dS) of mice, to increase Shati/Nat8l expression. Overexpression of Shati/Nat8l in the NAc, but not in the dS, attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference in mice. Moreover, the Shati/Nat8l overexpression in the NAc attenuated the elevation of extracellular dopamine levels induced by METH in in vivo microdialysis experiments. These behavioral and neurochemical alterations due to Shati/Nat8l overexpression in the NAc were inhibited by treatment with selective group II metabotropic glutamate receptor type 2 and 3 (mGluR2/3) antagonist LY341495. In the AAV vector-injected NAc, the tissue contents of both N-acetylaspartate and N-acetylaspartylglutamate (NAAG), endogenous mGluR3 agonist, were elevated. The injection of peptidase inhibitor of NAAG or the perfusion of NAAG itself reduced the basal levels of extracellular dopamine in the NAc of naive mice. These results indicate that Shati/Nat8l in the NAc, but not in the dS, plays an important suppressive role in the behavioral responses to METH by controlling the dopaminergic system via activation of group II mGluRs.
  • Wuh-Liang Hwu, Ni-Chung Lee, Yih-Dar Shieh, Kai-Yuan Tzen, Pin-Wen Chen, Shin-ichi Muramatsu, Hiroshi Ichinose, Yin-Hsiu Chien
    Catecholamine Research in the 21st Century: Abstracts and Graphical Abstracts, 10th International Catecholamine Symposium, 2012 3 - 4 2013年12月 [査読有り][通常論文]
  • Takenari Yamashita, Hui Lin Chai, Sayaka Teramoto, Shoji Tsuji, Kuniko Shimazaki, Shin-ichi Muramatsu, Shin Kwak
    EMBO MOLECULAR MEDICINE 5 11 1710 - 1719 2013年11月 [査読有り][通常論文]
     
    Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease, and the lack of effective therapy results in inevitable death within a few years of onset. Failure of GluA2 RNA editing resulting from downregulation of the RNA-editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) occurs in the majority of ALS cases and causes the death of motor neurons via a Ca2+-permeable AMPA receptor-mediated mechanism. Here, we explored the possibility of gene therapy for ALS by upregulating ADAR2 in mouse motor neurons using an adeno-associated virus serotype 9 (AAV9) vector that provides gene delivery to a wide array of central neurons after peripheral administration. A single intravenous injection of AAV9-ADAR2 in conditional ADAR2 knockout mice (AR2), which comprise a mechanistic mouse model of sporadic ALS, caused expression of exogenous ADAR2 in the central neurons and effectively prevented progressive motor dysfunction. Notably, AAV9-ADAR2 rescued the motor neurons of AR2 mice from death by normalizing TDP-43 expression. This AAV9-mediated ADAR2 gene delivery may therefore enable the development of a gene therapy for ALS.
  • Yijin Yan, Yoshiaki Miyamoto, Atsumi Nitta, Shin-ichi Muramatsu, Keiya Ozawa, Kiyofumi Yamada, Toshitaka Nabeshima
    INTERNATIONAL JOURNAL OF NEUROPSYCHOPHARMACOLOGY 16 7 1559 - 1567 2013年08月 [査読有り][通常論文]
     
    Relapse of drug abuse after abstinence is a major challenge to the treatment of addicts. In our well-established mouse models of methamphetamine (Meth) self-administration and reinstatement, bilateral microinjection of adeno-associated virus vectors expressing GDNF (AAV-Gdnf) into the striatum significantly reduced Meth self-administration, without affecting locomotor activity. Moreover, the intrastriatal AAV-Gdnf attenuated cue-induced reinstatement of Meth-seeking behaviour in a sustainable manner. In addition, this manipulation showed that Meth-primed reinstatement of Meth-seeking behaviour was reduced. These findings suggest that the AAV vector-mediated Gdnf gene transfer into the striatum is an effective and sustainable approach to attenuate Meth self-administration and Meth-associated cue-induced relapsing behaviour and that the AAV-mediated Gdnf gene transfer in the brain may be a valuable gene therapy against drug dependence and protracted relapse in clinical settings.
  • Yoshiaki Miyamoto, Eriko Saika, Etsuro Hori, Noriyuki Iegaki, Yudai Ishikawa, Kazuyuki Sumi, Toshitaka Nabeshima, Shin-Ichi Muramatsu, Hisao Nishijo, Kyosuke Uno, Atsumi Nitta
    Japanese Journal of Neuropsychopharmacology 33 4 167 - 173 2013年08月 [査読有り][通常論文]
     
    Various molecules are involved in drug addiction induced by drugs of abuse. Therefore, the mechanism of drug addiction is still not clear, and it has been a difficulty in the development of preventive and curative drugs for drug dependence. We tried to identify the molecules associated with drug dependence, and found three molecules including shati/nat8l. Recently, it has been demonstrated that the substrate for shati/nat8l is aspaltate and shati/nat8l biosynthesizes N-acetylaspartate, which exists abundantly in the mammalian brain. In this study, we investigated the physiological function of shati/nat8l and the role of shati/nat8l in drug dependence. The overexpression of shati/nat8l in the dorsal striatum of mice led to social abnormality and depression-like behavior, and worsened a part of the motor dysfunction induced by Ca2+ channel agonist BAYK 8644. The overexpression of shati/nat8l in the nucleus accumbens of mice inhibited methamphetamine-induced behavioral and biochemical abnormalities. These findings suggest that the shati/nat8l-associated system could play a role in the regulation of mental activity and motor action, and be a new target in the development of therapeutic drugs for drug dependence.
  • Ni-Chung Lee, Yih-Dar Shieh, Yin-Hsiu Chien, Kai-Yuan Tzen, I-Shing Yu, Pin-Wen Chen, Min-Hsiu Hu, Meng-kai Hu, Shin-ichi Muramatsu, Hiroshi Ichinose, Wuh-Liang Hwu
    NEUROBIOLOGY OF DISEASE 52 177 - 190 2013年04月 [査読有り][通常論文]
     
    Aromatic L-amino acid decarboxylase (AADC) is responsible for the syntheses of dopamine and serotonin. Children with AADC deficiency exhibit compromised development, particularly with regard to their motor functions. Currently, no animal model of AADC deficiency exists. We inserted an AADC gene mutation (IVS6+4A>T) and a neomycin-resistance gene into intron 6 of the mouse AADC (Ddc) gene. In the brains of homozygous knock-in (KO mice (Ddc(IVS6/IVS6)), AADC mRNA lacked exon 6, and AADC activity was <03% of that in wild-type mice. Half of the KI mice were born alive but grew poorly and exhibited severe dysldnesia and hindlimb clasping after birth. Two-thirds of the live-born KI mice survived the weaning period, with subsequent improvements in their growth and motor functions; however, these mice still displayed cardiovascular dysfunction and behavioral problems due to serotonin deficiencies. The brain dopamine levels in the KI mice increased from 9.39% of the levels in wild-type mice at 2 weeks of age to 37.86% of the levels in wild-type mice at 8 weeks of age. Adult KI mice also exhibited an exaggerated response to apomorphine and an elevation of striatal c-Fos expression, suggesting post-synaptic adaptations. Therefore, we generated an AADC deficient mouse model, in which compensatory regulation allowed the mice to survive to adulthood. This mouse model will be useful both for developing gene therapies for AADC deficiency and for designing treatments for diseases associated with neurotransmitter deficiency. (C) 2013 Elsevier Inc. All rights reserved.
  • Nobuhisa Iwata, Misaki Sekiguchi, Yoshino Hattori, Akane Takahashi, Masashi Asai, Bin Ji, Makoto Higuchi, Matthias Staufenbiel, Shin-ichi Muramatsu, Takaomi C. Saido
    SCIENTIFIC REPORTS 3 1472  2013年03月 [査読有り][通常論文]
     
    Accumulation of amyloid-beta peptide (A beta) in the brain is closely associated with cognitive decline in Alzheimer's disease (AD). Stereotaxic infusion of neprilysin-encoding viral vectors into the hippocampus has been shown to decreaseA beta in AD-model mice, but more efficient and global delivery is necessary to treat the broadly distributed burden in AD. Here we developed an adeno-associated virus (AAV) vector capable of providing neuronal gene expression throughout the brains after peripheral administration. A single intracardiac administration of the vector carrying neprilysin gene in AD-model mice elevated neprilysin activity broadly in the brain, and reduced A beta oligomers, with concurrent alleviation of abnormal learning and memory function and improvement of amyloid burden. The exogenous neprilysin was localized mainly in endosomes, thereby effectively excluding A beta oligomers from the brain. AAV vector-mediated gene transfer may provide a therapeutic strategy for neurodegenerative diseases, where global transduction of a therapeutic gene into the brain is necessary.
  • Yasushi Kondo, Tsuyoshi Okuno, Sayaka Asari, Shin-Ichi Muramatsu
    Human Fetal Tissue Transplantation 9781447141716 193 - 203 2013年03月 [査読有り][通常論文]
     
    As treatment for advanced Parkinson's disease, neural transplantation has been investigated for more than two decades with the aim of replacing degenerated dopaminergic neurons and restoring dopaminergic neurotransmission in the striatum. Although initial open-label studies on fetal midbrain cell transplant achieved excellent outcomes, double-blind clinical trials have shown controversial success, and autopsy results have revealed that some of the grafted fetal neurons displayed pathological changes typical of Parkinson's disease. Nevertheless, advances in the field of stem cell research have raised hope for novel cell replacement therapies. Embryonic stem (ES) cells may offer a substitute for fetal midbrain cells because they can proliferate extensively in an undifferentiated state and may provide an unlimited source of dopaminergic neurons. Neurons have been efficiently derived from ES cells, and beneficial effects after transplantation have been demonstrated in animal models of Parkinson's disease. However, some obstacles remain to be overcome before stem cell therapy can be routinely and safely used in humans. Since grafts are ectopically transplanted into the striatum instead of the substantia nigra in most current protocols, surviving dopaminergic neurons is not necessarily of the same subtype as the nigral cells. Future targets for cell therapy should include some types of Parkinsonism with degeneration of striatal neurons.
  • Koji Abe, Shinichiro Uchiyama, Etsuro Matsubara, Shin-Ichi Muramatsu, Naoki Atsuta, Kazuo Kitagawa, Yasuyuki Iguchi, Hiroaki Ooboshi, Ryuji Kaji, Yasuo Terao
    Rinsho shinkeigaku = Clinical neurology 53 11 1155 - 8 2013年 [査読有り][通常論文]
     
    On March 11, 2011, big earthquake and subsequent gigantic tsunami killed more than 20,000 peoples in Tohoku area of Japan. Neurological patients were one such victim because they are usually very vulnerable to such a huge tragedy due to their physical disability including artificial ventilator-support. On occasion of the last tsunami, most cases showed "all or nothing" to lose life or to survive, and there were only a little cases who needed emergency surgical treatment. In the very early period, some neurological patients required electric power to keep their lifesupportive ventilator at evacuation house or even at home. In a week to a couple of months, many neurological patients needed continuous supply of their daily drugs which are essential to keep themselves in steady physical conditions and even for keeping their life.Japanese Neurological Society (JNS) began to establish an emergent assistant network system from January 2012 in an attempt of supplying materials, drugs and energy power to neurological patients who require both under a very early period after any natural or political disaster and a later period. For example, JNS is going to apply IT system to connect distant but safer hospitals which accept emergent patients from the center of disastrous place. JNS may also send emergency medical team to the disastrous place to save neurological patients by passing necessary medicine and materials or moving patients to safer hospitals. JNS will make such a tentative program public on our website to collect many other constructive opinions from general member of the society and neurological patients. After getting those opinions, JNS made up the exact team for this purpose after general meeting of JNS on this May 2012.Based on this team, disaster-mimic trial will be performed in Tokyo, Shizuoka, and Kochi where the next big disaster is going to hit the cities.
  • Shin-Ichi Muramatsu
    Clinical Neurology 53 11 934 - 937 2013年 [査読有り][通常論文]
     
    Kampo is a traditional form of medicine in Japan. The individual formulas of the Kampo medicines consist mainly of plant-derived crude drugs. Recently, extract products that maintain specific levels of quality have been commonly used for dosage formulation instead of decoction. Although severe side effects, including pseudoaldosteronism, interstitial pneumonitis, liver damage and mesenteric phlebosclerosis may occasionally arise in some patients, herbal formulations are generally safe compared with potent western medicines. Since the complicated interaction of a Kampo formulation is difficult to analyze pharmacologically, the use of each Kampo formula has been based on the empirical rules described in classical writings by clinicians. Currently, formula selection is not always based on the pathological recognition from the Oriental medicine perspective, because these ancient theories are not necessarily amenable to current clinical practice. Nevertheless, formula selection would be more appropriate, and the therapeutic efficacy would increase if the physicians understood the basic concepts of ki, ketsu, sui, yin-yang, hypofunction and hyperfunction, heat and cold, superficies and interior, the five parenchymatous viscera and the six stages of disease. Regardless of the primary diseases, many Kampo formulas are considered to be indicated for patients complaining of headache, dizziness and numbness, which are common symptoms in everyday practice in neurology.
  • Asako Iida, Naomi Takino, Hitomi Miyauchi, Kuniko Shimazaki, Shin-ichi Muramatsu
    BIOMED RESEARCH INTERNATIONAL 2013 974819  2013年 [査読有り][通常論文]
     
    Recombinant adeno-associated virus (AAV) vectors are powerful tools for both basic neuroscience experiments and clinical gene therapies for neurological diseases. Intravascularly administered self-complementary AAV9 vectors can cross the blood-brain barrier. However, AAV9 vectors are of limited usefulness because they mainly transduce astrocytes in adult animal brains and have restrictions on foreign DNA package sizes. In this study, we show that intracardiac injections of tyrosine-mutant pseudotype AAV9/3 vectors resulted in extensive and widespread transgene expression in the brains and spinal cords of adult mice. Furthermore, the usage of neuron-specific promoters achieved selective transduction of neurons. These results suggest that tyrosine-mutant AAV9/3 vectors may be effective vehicles for delivery of therapeutic genes, including miRNAs, into the brain and for treating diseases that affect broad areas of the central nervous system.
  • Wuh-Liang Hwu, Ni-Chung Lee, Yin-Hsiu Chien, Shin-ichi Muramatsu, Hiroshi Ichinose
    Advances in Pharmacology 68 273 - 284 2013年 [査読有り][通常論文]
     
    Aromatic l-amino acid decarboxylase (AADC) is a homodimeric pyridoxal phosphate-dependent enzyme responsible for the syntheses of dopamine and serotonin. Defects in the AADC gene result in neurotransmitter deficiencies. Patients with AADC deficiency have severe motor and autonomic dysfunctions. A mouse model of AADC deficiency was recently established. These mice grow poorly and move awkwardly during infancy. They also show high anxiety when they grow up. Because drug therapy provides little or no benefit for many patients with AADC deficiency, a gene therapy has been attempted. The gene therapy employed an adeno-associated virus viral vector that can express the human AADC protein. The vector was injected to the brain of several children with AADC deficiency. The therapy was well tolerated, and all treated patients showed improvement. In the future, the mouse model will also help the development of treatments for AADC deficiency. © 2013 Elsevier Inc.
  • 一瀬 宏, 岩下 由佳, 宗実 悠佳, 徳岡 宏文, 村松 慎一
    ビタミン 87 5 289 - 290 公益社団法人 日本ビタミン学会 2013年
  • Makoto Arai, Shuichi Katai, Shin-ichi Muramatsu, Takao Namiki, Toshihiko Hanawa, Shun-ichiro Izumi
    BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 12 207  2012年11月 [査読有り][通常論文]
     
    Background: There have been a few but not precise surveys of the current status of traditional Japanese Kampo education at medical schools in Japan. Our aim was to identify problems and suggest solutions for a standardized Kampo educational model for all medical schools throughout Japan. Methods: We surveyed all 80 medical schools in Japan regarding eight items related to teaching or studying Kampo medicine: (1) the number of class meetings, target school year(s), and type of classes; (2) presence or absence of full-time instructors; (3) curricula contents; (4) textbooks in use; (5) desire for standardized textbooks; (6) faculty development programmes; (7) course contents; and (8) problems to be solved to promote Kampo education. We conducted descriptive analyses without statistics. Results: Eighty questionnaires were collected (100%). (1) There were 0 to 25 Kampo class meetings during the 6 years of medical school. At least one Kampo class was conducted at 98% of the schools, >= 4 at 84%, >= 8 at 44%, and >= 16 at 5%. Distribution of classes was 19% and 57% for third-and fourth-year students, respectively. (2) Only 29% of schools employed full-time Kampo medicine instructors. (3) Medicine was taught on the basis of traditional Japanese Kampo medicine by 81% of the schools, Chinese medicine by 19%, and Western medicine by 20%. (4) Textbooks were used by 24%. (5) Seventy-four percent considered using standardized textbooks. (6) Thirty-three percent provided faculty development programmes. (7) Regarding course contents, "characteristics" was selected by 94%, "basic concepts" by 84%, and evidence-based medicine by 64%. (8) Among the problems to be solved promptly, curriculum standardization was selected by 63%, preparation of simple textbooks by 51%, and fostering instructors responsible for Kampo education by 65%. Conclusions: Japanese medical schools only offer students a short time to study Kampo medicine, and the impetus to include Kampo medicine in their curricula varies among schools. Future Kampo education at medical schools requires solving several problems, including curriculum standardization.
  • H. Akiko Popiel, Toshihide Takeuchi, Hiromi Fujita, Kazuhiro Yamamoto, Chiyomi Ito, Hiroshi Yamane, Shin-ichi Muramatsu, Tatsushi Toda, Keiji Wada, Yoshitaka Nagai
    PLOS ONE 7 11 e51069  2012年11月 [査読有り][通常論文]
     
    The polyglutamine (polyQ) diseases such as Huntington's disease (HD), are neurodegenerative diseases caused by proteins with an expanded polyQ stretch, which misfold and aggregate, and eventually accumulate as inclusion bodies within neurons. Molecules that inhibit polyQ protein misfolding/aggregation, such as Polyglutamine Binding Peptide 1 (QBP1) and molecular chaperones, have been shown to exert therapeutic effects in vivo by crossing of transgenic animals. Towards developing a therapy using these aggregation inhibitors, we here investigated the effect of viral vector-mediated gene therapy using QBP1 and molecular chaperones on polyQ disease model mice. We found that injection of adeno-associated virus type 5 (AAV5) expressing QBP1 or Hsp40 into the striatum both dramatically suppresses inclusion body formation in the HD mouse R6/2. AAV5-Hsp40 injection also ameliorated the motor impairment and extended the lifespan of R6/2 mice. Unexpectedly, we found even in virus non-infected cells that AAV5-Hsp40 appreciably suppresses inclusion body formation, suggesting a non-cell autonomous therapeutic effect. We further show that Hsp40 inhibits secretion of the polyQ protein from cultured cells, implying that it inhibits the recently suggested cell-cell transmission of the polyQ protein. Our results demonstrate for the first time the therapeutic effect of Hsp40 gene therapy on the neurological phenotypes of polyQ disease mice.
  • Duo Jin, Shin-ichi Muramatsu, Nobuaki Shimizu, Shigeru Yokoyama, Hirokazu Hirai, Kiyofumi Yamada, Hong-Xiang Liu, Chiharu Higashida, Minako Hashii, Akihiko Higashida, Masahide Asano, Shoji Ohkuma, Haruhiro Higashida
    NEUROCHEMISTRY INTERNATIONAL 61 6 907 - 912 2012年11月 [査読有り][通常論文]
     
    A 16-kDa proteolipid, mediatophore, in Torpedo electric organs mediates Ca2+-dependent acetylcholine release. Mediatophore is identical to the pore-forming stalk c-subunit of the V0 sector of vacuolar proton ATPase (ATP6V0C). The function of ATP6V0C in the mammalian central nervous system is not clear. Here, we report transfection of adeno-associated viral vectors harboring rat ATP6V0C into the mouse substantia nigra, in which high potassium stimulation increased overflow of endogenous dopamine (DA) measured in the striatum by in vivo microdialysis. Next, in the striatum of 6-hydroxydopamine-lesioned mice, a model of Parkinson's disease (PD), human tyrosine hydroxylase, aromatic L-amino-acid decarboxylase and guanosine triphosphate cyclohydrolase 1, together with or without ATP6V0C, were expressed in the caudoputamen for rescue. Motor performance on the accelerating rotarod test and amphetamine-induced ipsilateral rotation were improved in the rescued mice coexpressing ATP6V0C. [H-3]DA, taken up into cultured N18 neuronal tumor cells transformed to express ATP6V0C, was released by potassium stimulation. These results indicated that ATP6V0C mediates DA release from nerve terminals in the striatum of DA neurons of normal mice and from gene-transferred striatal cells of parkinsonian mice. The results suggested that ATP6V0C may be useful as a rescue molecule in addition to DA-synthetic enzymes in the gene therapy of PD. (C) 2012 Elsevier Ltd. All rights reserved.
  • Ken Nakamura, Makiko Ota, Akihiro Kawata, Eiji Isozaki, Shin-ichi Muramatsu, Shigeki Matsubara
    ARQUIVOS DE NEURO-PSIQUIATRIA 70 8 646 - 646 2012年08月 [査読有り][通常論文]
  • Yu Miyazaki, Hiroaki Adachi, Masahisa Katsuno, Makoto Minamiyama, Yue-Mei Jiang, Zhe Huang, Hideki Doi, Shinjiro Matsumoto, Naohide Kondo, Madoka Iida, Genki Tohnai, Fumiaki Tanaka, Shin-ichi Muramatsu, Gen Sobue
    NATURE MEDICINE 18 7 1136 - + 2012年07月 [査読有り][通常論文]
     
    Spinal and bulbar muscular atrophy (SBMA) is an inherited neurodegenerative disorder caused by the expansion of the polyglutamine (polyQ) tract of the androgen receptor (AR-polyQ)(1,2). Characteristics of SBMA include proximal muscular atrophy, weakness, contraction fasciculation and bulbar involvement(3). MicroRNAs (miRNAs) are a diverse class of highly conserved small RNA molecules that function as crucial regulators of gene expression in animals and plants(4). Recent functional studies have shown the potent activity of specific miRNAs as disease modifiers both in vitro and in vivo(5-8). Thus, potential therapeutic approaches that target the miRNA processing pathway have recently attracted attention(9,10). Here we describe a novel therapeutic approach using the adeno-associated virus (AAV) vector-mediated delivery of a specific miRNA for SBMA. We found that miR-196a enhanced the decay of the AR mRNA by silencing CUGBP, Elav-like family member 2 (CELF2). CELF2 directly acted on AR mRNA and enhanced the stability of AR mRNA. Furthermore, we found that the early intervention of miR-196a delivered by an AAV vector ameliorated the SBMA phenotypes in a mouse model. Our results establish the proof of principle that disease-specific miRNA delivery could be useful in neurodegenerative diseases.
  • Wuh-Liang Hwu, Shin-ichi Muramatsu, Sheng-Hong Tseng, Kai-Yuan Tzen, Ni-Chung Lee, Yin-Hsiu Chien, Richard O. Snyder, Barry J. Byrne, Chun-Hwei Tai, Ruey-Meei Wu
    SCIENCE TRANSLATIONAL MEDICINE 4 134 134ra61  2012年05月 [査読有り][通常論文]
     
    Aromatic L-amino acid decarboxylase (AADC) is required for the synthesis of the neurotransmitters dopamine and serotonin. Children with defects in the AADC gene show compromised development, particularly in motor function. Drug therapy has only marginal effects on some of the symptoms and does not change early childhood mortality. Here, we performed adeno-associated viral vector-mediated gene transfer of the human AADC gene bilaterally into the putamen of four patients 4 to 6 years of age. All of the patients showed improvements in motor performance: One patient was able to stand 16 months after gene transfer, and the other three patients achieved supported sitting 6 to 15 months after gene transfer. Choreic dyskinesia was observed in all patients, but this resolved after several months. Positron emission tomography revealed increased uptake by the putamen of 6-[F-18] fluorodopa, a tracer for AADC. Cerebrospinal fluid analysis showed increased dopamine and serotonin levels after gene transfer. Thus, gene therapy targeting primary AADC deficiency is well tolerated and leads to improved motor function.
  • Shin-Ichi Muramatsu
    Clinical Neurology 52 11 896 - 898 2012年 [査読有り][通常論文]
     
    The current clinical trials of gene therapy for Parkinson's disease (PD) are based on three strategies. 1. To restore the local production of dopamine by introducing genes associated with dopamine-synthesizing enzymes into the putamen. 2. To protect nigrostriatal projection by delivering the neurturin gene, a trophic factor for dopaminergic neurons, both in the putamen and the substantia nigra. 3. To modulate the neural activity by transducing the subthalamic nucleus with vectors expressing glutamic acid decarboxylase. A phase I clinical study was initiated in 2007 to determine the safety of intra-putaminal infusion of a recombinant adeno-associated virus (AAV) vector encoding aromaticL-amino acid decarboxylase (AADC). All six patients enrolled in the trial showed improvements from baseline in the Unified Parkinson's Disease Rating Scale motor scores in the OFF medication state at 36 months after treatment. Although this trial was a small, open-label study and the use of a non-blinded, uncontrolled analysis limits interpretation, the efficacy outcomes are encouraging and indicate that the AAV vector-mediated gene transfer of AADC may benefit advanced PD patients. A similar approach, delivering AAV vector carrying AADC gene into the putamen ameliorated the symptoms in children with AADC deficiency.
  • Masayuki Miyamoto, Tomoyuki Miyamoto, Masaoki Iwanami, Shin-ichi Muramatsu, Sayaka Asari, Imaharu Nakano, Koichi Hirata
    SLEEP MEDICINE 13 1 102 - 106 2012年01月 [査読有り][通常論文]
     
    Objectives: Transcranial sonography (TCS) has been shown to reveal hyperechogenicity of the substantia nigra (SN) in people with Parkinson's disease and in approximately 10% of healthy subjects. It is hypothesized that SN hyperechogenicity in healthy subjects and patients with idiopathic rapid eye movement (REM) sleep behaviour disorder (iRBD) patients is a marker of vulnerability for Parkinson's disease. Methods: TCS and positron emission tomography (PET) with 6-[F-18] fluoro-meta-tyrosine (FMT), which can assess the level of the presynaptic dopaminergic nerve, were performed in 19 male patients with iRBD, mean age 66.4 (standard deviation [SD] 4.9) years, to assess nigrostriatal function, Results: Nine patients had pathological SN hyperechogenicity (mean age 66.8 [SD 3.9] years; 0.31 [SD 0.12] cm(2)) and 10 patients did not have SN hyperechogenicity (mean age 66.0 [SD 5.8] years; 0.11 [SD 0.06] cm(2)). FMT uptake at the putamen and caudate was significantly lower in iRBD patients with pathological SN hyperechogenicity compared with those without SN hyperechogenicity. However, no correlation was found between SN echogenicity and FMT uptake. This is in conflict with previous findings which showed that subjects with hyperechogenicity had lower FMT uptake in the striatum. Conclusion: Pathological hyperechogenic alterations in the SN in patients with iRBD may suggest the existence of preclinical SN dysfunction as determined by FMT-PET. (C) 2011 Elsevier B.V. All rights reserved.
  • Nakamura K, Ota M, Kawata A, Isozaki E, Muramatsu S, Matsubara S
    Arquivos de Neuro-Psiquiatria 70 8 645 - 649 2012年 [査読有り][通常論文]
  • Hirofumi Tokuoka, Shin-ichi Muramatsu, Chiho Sumi-Ichinose, Hiroaki Sakane, Masayo Kojima, Yoshinori Aso, Takahide Nomura, Daniel Metzger, Hiroshi Ichinose
    JOURNAL OF BIOLOGICAL CHEMISTRY 286 50 43549 - 43558 2011年12月 [査読有り][通常論文]
     
    The tyrosine hydroxylase (TH; EC 1.14.16.2) is a rate-limiting enzyme in the dopamine synthesis and important for the central dopaminergic system, which controls voluntary movements and reward-dependent behaviors. Here, to further explore the regulatory mechanism of dopamine levels by TH in adult mouse brains, we employed a genetic method to inactivate the Th gene in the nigrostriatal projection using the Cre-loxP system. Stereotaxic injection of adeno-associated virus expressing Cre recombinase (AAV-Cre) into the substantia nigra pars compacta (SNc), where dopaminergic cell bodies locate, specifically inactivated the Th gene. Whereas the number of TH-expressing cells decreased to less than 40% in the SNc 2 weeks after the AAV-Cre injection, the striatal TH protein level decreased to 75%, 50%, and 39% at 2, 4, and 8 weeks, respectively, after the injection. Thus, unexpectedly, the reduction of TH protein in the striatum, where SNc dopaminergic axons innervate densely, was slower than in the SNc. Moreover, despite the essential requirement of TH for dopamine synthesis, the striatal dopamine contents were only moderately decreased, to 70% even 8 weeks after AAV-Cre injection. Concurrently, in vivo synthesis activity of L-dihydroxyphenylalanine, the dopamine precursor, per TH protein level was augmented, suggesting up-regulation of dopamine synthesis activity in the intact nigrostriatal axons. Collectively, our conditional Th gene targeting method demonstrates two regulatory mechanisms of TH in axon terminals for dopamine homeostasis in vivo: local regulation of TH protein amount independent of soma and trans-axonal regulation of apparent L-dihydroxyphenylalanine synthesis activity per TH protein.
  • Shigeki Matsubara, Akifumi Fujita, Shin-ichi Muramatsu, Rie Usui, Kenro Chikazawa, Mitsuaki Suzuki
    JOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH 37 8 1137 - 1140 2011年08月 [査読有り][通常論文]
     
    Posterior reversible encephalopathy syndrome, if it occurs in late pregnancy, requires pregnancy termination. Here, we report a woman without a discernable underlying condition who developed neurological deficits at 14 weeks of pregnancy. Magnetic resonance imaging demonstrated an occipitoparietal brain lesion suggestive of posterior reversible encephalopathy syndrome. Neurological symptoms ameliorated spontaneously and she continued her pregnancy to term. A decision to terminate pregnancy based on only neurological and magnetic resonance imaging findings should be avoided.
  • Shigeki Matsubara, Shin-ichi Muramatsu
    JOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH 37 6 672 - 672 2011年06月 [査読有り][通常論文]
  • 構造生物学的アプローチによるアルツハイマー病の病態解明と分子標的治療の開発
    星 美奈子, 鍋島 陽一, 菊地 和也, 村松 慎一, 廣明 秀一
    新しい医療機器研究 16 28 - 28 (財)医療機器センター情報サービス部 2011年03月
  • Asari S, Fujimoto K, Miyauchi A, Sato T, Nakano I, Muramatsu S
    BMC neurology 11 35  2011年03月 [査読有り][通常論文]
     
    Background: In idiopathic Parkinson's disease (PD) the clinical features are heterogeneous and include different predominant symptoms. The aim of the present study was to determine the relationship between subregional aromatic l-amino acid decarboxylase (AADC) activity in the striatum and the cardinal motor symptoms of PD using high-resolution positron emission tomography (PET) with an AADC tracer, 6-[F-18] fluoro-L-m-tyrosine (FMT). Methods: We assessed 101 patients with PD and 19 healthy volunteers. PD was diagnosed based on the UK Brain Bank criteria by two experts on movement disorders. Motor symptoms were measured with the Unified Parkinson's Disease Rating Scale (UPDRS). FMT uptake in the subregions of the striatum was analyzed using semi-automated software for region-of-interest demarcation on co-registered magnetic resonance images. Results: In all PD patients, FMT uptake was decreased in the posterior putamen regardless of predominant motor symptoms and disease duration. Smaller uptake values were found in the putamen contralateral to the side with more affected limbs. The severity of bradykinesia, rigidity, and axial symptoms was correlated with the decrease of FMT uptake in the putamen, particularly in the anterior part. No significant correlation was observed between tremors and FMT uptake. Conclusions: Decrease of FMT uptake in the posterior putamen appears to be most sensitive in mild PD and uptake in the anterior putamen may reflect the severity of main motor symptoms, except for tremor.
  • Shin-Ichi Muramatsu, Sayaka Asari, Ken-Ichi Fujimoto, Keiya Ozawa, Imaharu Nakano
    Gene Therapy and Regulation 5 1 57 - 65 2010年10月 [査読有り][通常論文]
     
    The cardinal motor symptoms of Parkinson's disease (PD) are associated with the profound depletion of dopamine in the striatum. The replacement of dopamine is the most straightforward strategy to improve motor performance in PD. Researchers have been developing gene therapy aimed at local production of dopamine via the introduction of dopamine-synthesizing enzyme genes into the putamen. Two phase I clinical studies have used recombinant adeno-associated virus (AAV) vectors to transfer the aromatic L-amino acid decarboxylase (AADC) gene into the putamen to restore efficient conversion of orally administered L-3,4-dihydroxyphenylalanine (L-dopa). The initial results of these studies have not only confirmed the safety of AAV vectors, but have also demonstrated the alleviation of motor symptoms associated with PD. Interestingly motor performance in the "off" medication state was improved after gene therapy, suggesting long-term modulation of dopaminergic signals in the striatal neurons was induced by gene transfer. Gene delivery of tyrosine hydroxylase (TH) and guanosine triphosphate cyclohydrolase I (GCH) in addition to AADC may help to avoid motor fluctuations associated with intermittent intake of L-dopa by continuously supplying dopamine in the putamen. A clinical study of such triple gene transfer is presently underway using equine infectious anemia virus (EIAV) vector. © 2010 World Scientific Publishing Company.
  • Shin-ichi Muramatsu, Ken-ichi Fujimoto, Seiya Kato, Hiroaki Mizukami, Sayaka Asari, Kunihiko Ikeguchi, Tadataka Kawakami, Masashi Urabe, Akihiro Kume, Toshihiko Sato, Eiju Watanabe, Keiya Ozawa, Imaharu Nakano
    MOLECULAR THERAPY 18 9 1731 - 1735 2010年09月 [査読有り][通常論文]
     
    Gene transfer of dopamine-synthesizing enzymes into the striatal neurons has led to behavioral recovery in animal models of Parkinson's disease (PD). We evaluated the safety, tolerability, and potential efficacy of adenoassociated virus (AAV) vector-mediated gene delivery of aromatic l-amino acid decarboxylase (AADC) into the putamen of PD patients. Six PD patients were evaluated at baseline and at 6 months, using multiple measures, including the Unified Parkinson's Disease Rating Scale (UPDRS), motor state diaries, and positron emission tomography (PET) with 6-[(18)F] fluoro-l-m-tyrosine (FMT), a tracer for AADC. The short-duration response to levodopa was measured in three patients. The procedure was well tolerated. Six months after surgery, motor functions in the OFF-medication state improved an average of 46% based on the UPDRS scores, without apparent changes in the short-duration response to levodopa. PET revealed a 56% increase in FMT activity, which persisted up to 96 weeks. Our findings provide class IV evidence regarding the safety and efficacy of AADC gene therapy and warrant further evaluation in a randomized, controlled, phase 2 setting.
  • Agnieszka Krzyzosiak, Monika Szyszka-Niagolov, Marta Wietrzych, Serge Gobaille, Shin-ichi Muramatsu, Wojciech Krezel
    NEURON 66 6 908 - 920 2010年06月 [査読有り][通常論文]
     
    Abnormal signaling by retinoids or n-3 polyunsaturated fatty acids has been implicated in clinical depression. The converging point in activities of these two classes of molecules is transcriptional activation of retinoid X receptors (Rxr). We show here that ablation of Rxr gamma in mice leads to depressive-like behaviors including increased despair and anhedonia, which were accompanied by reduced expression of dopamine D2 receptor in the shell of nucleus accumbens (NAc) and altered serotonin signaling. While abnormal serotonin signaling is not sufficient to generate the depressive behaviors, increasing D2r expression by chronic fluoxetine (Prozac) treatment or adenoassociated virus type2 (AAV2) mediated expression of Rxr gamma or D2r in the NAc of Rxr gamma(-/-) mice normalizes depressive-like behaviors in Rxr gamma(-/-) animals. Conversely, NAc infusion of raclopride, a D2r antagonist prevents AAV2-Rxr gamma-mediated rescue of despair behaviors in Rxr gamma(-/-) mice. Combined, our data argue that control of NAc D2r expression is critical for Rxr gamma-mediated modulation of affective behaviors.
  • Xiaogang Li, Dongsheng Fan, Weizhong Xiao, Yang Shen, Shin-ichi Muramatsu, Keiya Ozawa, Imaharu Nakano
    Neural Regeneration Research 5 8 591 - 596 2010年05月 [査読有り][通常論文]
     
    BACKGROUND: Gene therapy for Parkinson's disease is being explored as an effective strategy to restore and protect the function of neuronal cells in the substantia nigra. Regulation of gene expression is necessary for gene therapy to avoid adverse effects due to excessive synthesis of transgene products. OBJECTIVE: Here we developed recombinant adeno-associated virus (AAV) as a viral vector-mediated gene regulation system based on Cre recombinase fused to the mutated ligand-binding domain of the estrogen receptor (CreERT2) + inducing agent tamoxifen. Inducible Cre recombinase was used to reduce tyrosine hydroxylase gene expression and to prevent the excessive increase in dopamine. DESIGN, TIME AND SETTING: A genetic engineering in vitro comparative study and randomized controlled animal experiment. This study was conducted at the Gene Therapy Center, Jichi Medical School, Japan from June 2002 to June 2004. METHODS: To construct a recombinant AAV vector carrying a dopamine synthase gene. The tyrosine hydroxylase gene was inserted using a loxP fragment that could be regulated by Cre recombinase. The recombinant AAV vector carrying the CreERT2 gene was co-transduced with HEK293 cells and the corpus striatum in a rat model of Parkinson's disease, with inducing agent tamoxifen to regulate gene expression. MAIN OUTCOME MEASURES: The levels of dopamine and aromatic L-amino acid decarboxylase (AADC) activity were detected in HEK293 cell medium and in the corpus striatum in a rat model of Parkinson's disease using high-performance liquid chromatography. Immunofluorescence double staining was used to observe tyrosine hydroxylase and Cre or AADC co-expression in HEK293 cell medium. Immunohistochemical staining was employed to observe tyrosine hydroxylase and AADC expression and behavioral changes were measured in Parkinson's rats. RESULTS: Transfected AAV-CreERT2 and AAV expressing dopamine synthesis enzymes could increase the synthesis of dopamine in HEK293 medium and Parkinson's rat striatum (P < 0.01) and improve the rotational behavior of Parkinson's rats. While tamoxifen markedly reduced overproduction of dopamine caused by cotransfection of viral vectors (P < 0.01), but did not affect the expression and activity of AADC. CONCLUSION: The application of AAV vector-encoded tyrosine hydroxylase gene under the gene regulation system of Cre-ERT2, after tamoxifen treatment, can effectively control the generation of genetically modified products to reduce the production of excessive dopamine in vivo and in vitro. Therefore, this method can increase the safety of gene therapy.
  • Per Nilsson, Nobuhisa Iwata, Shin-ichi Muramatsu, Lars O. Tjernberg, Bengt Winblad, Takaomi C. Saido
    JOURNAL OF CELLULAR AND MOLECULAR MEDICINE 14 4 741 - 757 2010年04月 [査読有り][通常論文]
     
    Introduction Targets and ongoing research NGF Neurotrophic function of NGF Levels of NGF in AD Role of NGF in AD NGF as a therapeutic agent Development of NGF gene therapy In vivo gene delivery of NGF BDNF Neurotrophic function of BDNF BDNF levels in AD BDNF function in AD Towards BDNF gene therapy Neprilysin Role of neprilysin in AD Neprilysin levels in AD Gene delivery of neprilysin in AD animal models Potential gene therapy target candidates APOE ECE Cathepsin B Other A beta degrading enzymes Down-regulation of AD-associated proteins by siRNA BACE1 APP Concluding remarks Alzheimer's disease (AD) is the major cause of dementia in the elderly, leading to memory loss and cognitive decline. The mechanism underlying onset of the disease has not been fully elucidated. However, characteristic pathological manifestations include extracellular accumulation and aggregation of the amyloid beta-peptide (A beta) into plaques and intracellular accumulation and aggregation of hyperphosphorylated tau, forming neurofibrillary tangles. Despite extensive research worldwide, no disease modifying treatment is yet available. In this review, we focus on gene therapy as a potential treatment for AD, and summarize recent work in the field, ranging from proof-of-concept studies in animal models to clinical trials. The multifactorial causes of AD offer a variety of possible targets for gene therapy, including two neurotrophic growth factors, nerve growth factor and brain-derived neurotrophic factor, A beta-degrading enzymes, such as neprilysin, endothelin-converting enzyme and cathepsin B, and AD associated apolipoprotein E. This review also discusses advantages and drawbacks of various rapidly developing virus-mediated gene delivery techniques for gene therapy. Finally, approaches aiming at down-regulating amyloid precursor protein (APP) and beta-site APP cleaving enzyme 1 levels by means of siRNA-mediated knockdown are briefly summarized. Overall, the prospects appear hopeful that gene therapy has the potential to be a disease modifying treatment for AD.
  • Banafsheh Kadkhodaei, Takehito Ito, Eliza Joodmardi, Bengt Mattsson, Claude Rouillard, Manolo Carta, Shin-Ichi Muramatsu, Chiho Sumi-Ichinose, Takahide Nomura, Daniel Metzger, Pierre Chambon, Eva Lindqvist, Nils-Goeran Larsson, Lars Olson, Anders Bjorklund, Hiroshi Ichinose, Thomas Perlmann
    JOURNAL OF NEUROSCIENCE 29 50 15923 - 15932 2009年12月 [査読有り][通常論文]
     
    Transcription factors involved in the specification and differentiation of neurons often continue to be expressed in the adult brain, but remarkably little is known about their late functions. Nurr1, one such transcription factor, is essential for early differentiation of midbrain dopamine (mDA) neurons but continues to be expressed into adulthood. In Parkinson's disease, Nurr1 expression is diminished and mutations in the Nurr1 gene have been identified in rare cases of disease; however, the significance of these observations remains unclear. Here, a mouse strain for conditional targeting of the Nurr1 gene was generated, and Nurr1 was ablated either at late stages of mDA neuron development by crossing with mice carrying Cre under control of the dopamine transporter locus or in the adult brain by transduction of adeno-associated virus Cre-encoding vectors. Nurr1 deficiency in maturing mDA neurons resulted in rapid loss of striatal DA, loss of mDA neuron markers, and neuron degeneration. In contrast, a more slowly progressing loss of striatal DA and mDA neuron markers was observed after ablation in the adult brain. As in Parkinson's disease, neurons of the substantia nigra compacta were more vulnerable than cells in the ventral tegmental area when Nurr1 was ablated at late embryogenesis. The results show that developmental pathways play key roles for the maintenance of terminally differentiated neurons and suggest that disrupted function of Nurr1 and other developmental transcription factors may contribute to neurodegenerative disease.
  • Tetsuo Ito, Shigekazu Yamamoto, Tsukasa Hayashi, Mika Kodera, Hiroaki Mizukami, Keiya Ozawa, Shin-ichi Muramatsu
    ANNALS OF CLINICAL BIOCHEMISTRY 46 6 508 - 510 2009年11月 [査読有り][通常論文]
     
    Background: Recombinant adeno-associated virus vectors based on serotype 2 (AAV-2) have become leading vehicles for gene therapy. Most humans in the general population have anti-AAV-2 antibodies as a result of naturally acquired infections. Pre-existing immunity to AAV-2 might affect the functional and safety consequences of AAV-2 vector-mediated gene transfer in clinical applications. Methods: An enzyme-linked immunosorbent assay (ELISA) method was developed using microwell plates coated with intact particles of recombinant AAV-2 vectors, and horseradish peroxidase-conjugated anti-human immunoglobulin G (HRP-IgG). Neutralizing antibody titres were analysed by assessing the ability of serum antibody to inhibit transduction into HEK293 cells of AAV vectors that express beta-galactosidase. Results: Anti-AAV-2 antibodies were detected by ELISA in two of 20 healthy subjects. The positivity criterion (optical density >0.5) in ELISA corresponded to the cut-off value (320-fold dilution of serum) in the AAV-2 neutralization assay. Influences of interfering substances were not observed. Conclusion: This ELISA method may be useful for rapid screening of anti-AAV-2 neutralizing antibodies in candidates for gene therapy.
  • Akihiko Noguchi, Satoko Matsumura, Mari Dezawa, Mari Tada, Masako Yanazawa, Akane Ito, Manami Akioka, Satoru Kikuchi, Michio Sato, Shouji Ideno, Munehiro Noda, Atsushi Fukunari, Shin-ichi Muramatsu, Yutaka Itokazu, Kazuki Sato, Hitoshi Takahashi, David B. Teplow, Yo-ichi Nabeshima, Akiyoshi Kakita, Kazutomo Imahori, Minako Hoshi
    JOURNAL OF BIOLOGICAL CHEMISTRY 284 47 32895 - 32905 2009年11月 [査読有り][通常論文]
     
    Amyloid beta-protein (A beta) assemblies are thought to play primary roles in Alzheimer disease (AD). They are considered to acquire surface tertiary structures, not present in physiologic monomers, that are responsible for exerting toxicity, probably through abnormal interactions with their target(s). Therefore, A beta assemblies having distinct surface tertiary structures should cause neurotoxicity through distinct mechanisms. Aiming to clarify the molecular basis of neuronal loss, which is a central phenotype in neurodegenerative diseases such as AD, we report here the selective immunoisolation of neurotoxic 10-15-nm spherical A beta assemblies termed native amylospheroids (native ASPDs) from AD and dementia with Lewy bodies brains, using ASPD tertiary structure-dependent antibodies. In AD patients, the amount of native ASPDs was correlated with the pathologic severity of disease. Native ASPDs are anti-pan oligomer A11 antibody-negative, high mass (> 100 kDa) assemblies that induce degeneration particularly of mature neurons, including those of human origin, in vitro. Importantly, their immuno-specificity strongly suggests that native ASPDs have a distinct surface tertiary structure from other reported assemblies such as dimers, A beta-derived diffusible ligands, and A11-positive assemblies. Only ASPD tertiary structure-dependent antibodies could block ASPD-induced neurodegeneration. ASPDs bind presynaptic target(s) on mature neurons and have a mode of toxicity different from those of other assemblies, which have been reported to exert their toxicity through binding postsynaptic targets and probably perturbing glutamatergic synaptic transmission. Thus, our findings indicate that native ASPDs with a distinct toxic surface induce neuronal loss through a different mechanism from other A beta assemblies.
  • Shinobu Kuratomi, Yoko Ohmori, Masayuki Ito, Kuniko Shimazaki, Shin-ichi Muramatsu, Hiroaki Mizukami, Hideki Uosaki, Jun K. Yamashita, Yuji Arai, Koichiro Kuwahara, Makoto Takano
    CARDIOVASCULAR RESEARCH 83 4 682 - 687 2009年09月 [査読有り][通常論文]
     
    Hcn4, which encodes the hyperpolarization-activated, cyclic nucleotide-sensitive channel (I(h)), is a well-established marker of the cardiac sino-atrial node. We aimed to identify cis-elements in the genomic locus of the Hcn4 gene that regulate the transcription of Hcn4. We screened evolutionarily conserved non-coding sequences (CNSs) that are often involved in the regulation of gene expression. The VISTA Enhancer Browser identified 16 regions, termed CNS 1-16, within the Hcn4 locus. Using the luciferase reporter assay in primary neonatal rat cardiomyocytes, we found that CNS13 conferred a prominent enhancer activity (more than 30-fold) on the Hcn4 promoter. Subsequent mutation analysis revealed that the Hcn4 enhancer function was dependent on myocyte enhancer factor-2 (MEF2) and activator protein-1 (AP1) binding sequences located in CNS13. Electrophoretic mobility shift assay and chromatin immunoprecipitation confirmed that MEF2 and AP1 proteins bound CNS13. Furthermore, overexpression of a dominant negative MEF2 mutant inhibited the enhancer activity of CNS13, decreased Hcn4 mRNA expression and also decreased the amplitude of I(h) current in myocytes isolated from the inflow tract of embryonic heart. These results suggest that the novel enhancer CNS13 and MEF2 may play a critical role in the transcription of Hcn4 in the heart.
  • Shin-Ichi Muramatsu, Tsuyoshi Okuno, Yutaka Suzuki, Takashi Nakayama, Takeharu Kakiuchi, Naomi Takino, Asako Iida, Fumiko Ono, Keiji Terao, Nobuo Inoue, Imaharu Nakano, Yasushi Kondo, Hideo Tsukada
    SYNAPSE 63 7 541 - 548 2009年07月 [査読有り][通常論文]
     
    The ability of primate embryonic stem (ES) cells to differentiate into dopamine (DA)-synthesizing neurons has raised hopes of creating novel cell therapies for Parkinson's disease (PD). As the primary purpose of cell transplantation in PD is restoration of dopaminergic neurotransmission in the striatum, in vivo assessment of DA function after grafting is necessary to achieve better therapeutic effects. A chronic model of PD was produced in two cynomolgus monkeys (M-1 and M-2) by systemic administration of neurotoxin. Neural stem cells (NSCs) derived from cynomolgus ES cells were implanted unilaterally in the putamen. To evaluate DA-specific functions, we used multiple [(11)C]-labeled positron emission tomography (PET) tracers, including [beta-(11)C]L-3,4-dihydroxyphenylalanine (L-[beta-(11)C]DOPA, DA precursor ligand), [(11)C]-2 beta-carbomethoxy3 beta-(4-fluorophenyl)tropane ([(11)C]beta-CFT, DA transporter ligand) and [(11)C]raclopride (D(2) receptor ligand). At 12 weeks after grafting NSCs, PET demonstrated significantly increased uptake of L-[beta-(11)C]DOPA (M-1:41%, M-2:61%) and [(11)C]beta-CFT (M-1:31%, M-2:36%) uptake in the grafted putamen. In addition, methamphetamine challenge in M-2 induced reduced [(11)C]raclopride binding (16%) in the transplanted putamen, suggesting release of DA. These results show that transplantation of NSCs derived from cynomolgus monkey ES cells can restore DA function in the putamen of a primate model of PD. PET with multitracers is useful for functional studies in developing cell-based therapies against PD. Synapse 63:541-548, 2009. (C) 2009 Wiley-Liss, Inc.
  • Tsuyoshi Okuno, Takashi Nakayama, Nae Konishi, Hideo Michibata, Koji Wakimoto, Yutaka Suzuki, Shinji Nito, Toshio Inaba, Imaharu Nakano, Shin-ichi Muramatsu, Makoto Takano, Yasushi Kondo, Nobuo Inoue
    PLOS ONE 4 7 e6318  2009年07月 [査読有り][通常論文]
     
    Background: Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES) cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM). However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins.Methodology/Principal Findings: We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES) cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to PACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM) could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation.Conclusion/Significance: By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.
  • Fumiaki Fukushima, Kazuhito Nakao, Toru Shinoe, Masahiro Fukaya, Shin-ichi Muramatsu, Kenji Sakimura, Hirotaka Kataoka, Hisashi Mori, Masahiko Watanabe, Toshiya Manabe, Masayoshi Mishina
    PLOS ONE 4 1 e3993  2009年01月 [査読有り][通常論文]
     
    Synchronized discharges in the hippocampal CA3 recurrent network are supposed to underlie network oscillations, memory formation and seizure generation. In the hippocampal CA3 network, NMDA receptors are abundant at the recurrent synapses but scarce at the mossy fiber synapses. We generated mutant mice in which NMDA receptors were abolished in hippocampal CA3 pyramidal neurons by postnatal day 14. The histological and cytological organizations of the hippocampal CA3 region were indistinguishable between control and mutant mice. We found that mutant mice lacking NMDA receptors selectively in CA3 pyramidal neurons became more susceptible to kainate-induced seizures. Consistently, mutant mice showed characteristic large EEG spikes associated with multiple unit activities (MUA), suggesting enhanced synchronous firing of CA3 neurons. The electrophysiological balance between fast excitatory and inhibitory synaptic transmission was comparable between control and mutant pyramidal neurons in the hippocampal CA3 region, while the NMDA receptor-slow AHP coupling was diminished in the mutant neurons. In the adult brain, inducible ablation of NMDA receptors in the hippocampal CA3 region by the viral expression vector for Cre recombinase also induced similar large EEG spikes. Furthermore, pharmacological blockade of CA3 NMDA receptors enhanced the susceptibility to kainate-induced seizures. These results raise an intriguing possibility that hippocampal CA3 NMDA receptors may suppress the excitability of the recurrent network as a whole in vivo by restricting synchronous firing of CA3 neurons.
  • Yujiro Tanaka, Tamako Ikeda, Yukiko Kishi, Shigeo Masuda, Hiroaki Shibata, Kengo Takeuchi, Makoto Komura, Tadashi Iwanaka, Shin-ichi Muramatsu, Yasushi Kondo, Kazutoshi Takahashi, Shinya Yamanaka, Yutaka Hanazono
    CELL TRANSPLANTATION 18 4 381 - 389 2009年 [査読有り][通常論文]
     
    The ERas gene promotes the proliferation of and formation of teratomas by Mouse embryonic stem (ES) cells. However, its human orthologue is not expressed in human ES cells. This implies that the behavior of transplanted mouse ES cells would not accurately reflect the behavior of transplanted human ES cells and that the use of nonhuman primate models might be more appropriate to demonstrate the safety of human ES cell-based therapies. However, the expression of the ERas gene has not been examined in nonhuman primate ES cells. In this study, we cloned the cynomolgus homologue and showed that the ERas gene is expressed in cynomolgus ES cells. Notably, it is also expressed in cynomolgus ES cell-derived differentiated progeny as well as cynomolgus adult tissues. The ERas protein is detectable in various cynomolgus tissues its assessed by immunohistochemisty. Cynomolgus ES cell-derived teratoma cells, which also expressed the ERas gene at higher levels than the undifferentiated cynomolgus ES cells, did not develop tumors in NOD/Shi-scid, IL-2R gamma(null) (NOG) mice. Even when the ERas gene was overexpressed in cynomolgus stromal cells, only the plating efficiency was improved and the proliferation was not promoted. Thus, it is unlikely that ERas contributes to the tumorigenicity of cynomolgus cells. Therefore, cynomolgus ES cells are more similar to human than mouse ES cells despite that ERas is expressed in cynomolgus and mouse ES cells but not in human ES cells.
  • Shin-Ichi Muramatsu
    Clinical Neurology 49 11 890 - 892 2009年 [査読有り][通常論文]
     
    Advances in the field of stem cell research have raised hopes of creating novel cell replacement therapies for Parkinson disease (PD), although double-blinded clinical trials have met with controversial success in patients implanted with fetal midbrain tissue and autopsy results have shown that some of the grafted fetal neurons displayed pathological changes typical of PD. Dopaminergic neurons have been efficiently derived from stem cells using various methods, and beneficial effects after transplantation have been demonstrated in animal models of PD. Some obstacles remain to be overcome before stem cell therapy can be routinely and safely used to treat PD in humans. A widely used prodrug/suicide gene therapy would be applied to stem cells to reduce risk of tumor formation. Since grafts were transplanted ectopically into the striatum instead of the substantia nigra in most current protocols, surviving dopaminergic neurons would not have to be the same subtype as the nigral cells. If the main mechanism underlying any functional recovery achieved by cell therapies is restoration of dopaminergic neurotransmission, then viral vector-mediated gene delivery of dopamine-synthesizing enzymes represents a more straightforward approach. Future targets for cell therapy should include some types of Parkinsonism with degeneration of striatal neurons.
  • Kishi Y, Tanaka Y, Shibata H, Nakamura S, Takeuchi K, Masuda S, Ikeda T, Muramatsu SI, Hanazono Y
    Cell transplantation 17 9 1095 - 1102 2008年09月 [査読有り][通常論文]
  • Masaki Wakamatsu, Aiko Ishii, Shingo Iwata, Junko Sakagami, Yuriko Ukai, Mieko Ono, Daiji Kanbe, Shin-ichi Muramatsu, Kazuto Kobayashi, Takeshi Iwatsubo, Makoto Yoshimotoa
    NEUROBIOLOGY OF AGING 29 4 574 - 585 2008年04月 [査読有り][通常論文]
     
    Parkinson's disease is characterized by loss of nigral dopaminergic neurons and presence of Lewy bodies, whose major component is a-synuclein. In the present study, we generated transgenic mice termed Syn130m that express truncated, human a-synuclein (amino acid residue number: 1-130) in dopaminergic neurons. Notably, dopaminergic neurons were selectively diminished in the substantia nigra pars compacta of Syn130m, while transgenic mice that expressed comparable amount of full-length human a-synuclein did not develop such pathology. Therefore, the truncation of human (x-synuclein seems to be primarily responsible for the loss of nigral dopaminergic neurons. The nigral pathology resulted in impairment of axon terminals in the striatum and concomitant decrease in striatal dopamine content. Behaviorally, spontaneous locomotor activities of Syn130m were reduced, but the abnormality was ameliorated by treatment with L-DOPA. The loss of nigral dopaminergic neurons was not progressive and seemed to occur during embryogenesis along with the onset of expression of the transgene. Our results indicate that truncated human (x-synuclein is deleterious to the development and/or survival of nigral dopaminergic neurons. (C) 2006 Elsevier Inc. All rights reserved.
  • Muramatsu Shin-Ichi, Ono Fumiko, Takino Naomi, Nishida Hiroko, Asari Sayaka, Ikeguchi Kunihiko, Fujimoto Ken-Ichi, Tsukada Hideo, Terao Keiji, Ozawa Keiya, Nakano Imaharu
    JOURNAL OF GENE MEDICINE 10 4 433 - 433 2008年04月 [査読有り][通常論文]
  • Mizukami Hiroaki, Muramatsu Shin-Ichi, Ono Fumiko, Mimuro Jun, Sakata Yoichi, Urabe Masashi, Kume Akihiro, Terao Keiji, Nakano Imaharu, Ozawa Keiya
    JOURNAL OF GENE MEDICINE 10 4 454  2008年04月 [査読有り][通常論文]
  • Yuhe Liu, Takashi Okada, Kuniko Shimazaki, Kianoush Sheykholeslami, Tatsuya Nomoto, Shin-Ichi Muramatsu, Hiroaki Mizukami, Akihiro Kume, Shuifang Xiao, Keiichi Ichimura, Keiya Ozawa
    MOLECULAR THERAPY 16 3 474 - 480 2008年03月 [査読有り][通常論文]
     
    Since standard aminoglycoside treatment progressively causes hearing disturbance with hair cell degeneration, systemic use of the drugs is limited. Adeno-associated virus (AAV)-based vectors have been of great interest because they mediate stable transgene expression in a variety of postmitotic cells with minimal toxicity. In this study, we investigated the effects of regulated AAV1-mediated glial cell line-derived neurotrophic factor (GDNF) expression in the cochlea on aminoglycoside-induced damage. AAV1-based vectors encoding GDNF or vectors encoding GDNF with an rtTA2s- S2 Tet- on regulation system were directly microinjected into the rat cochleae through the round window at 5 x 10(10) genome copies/body. Seven days after the virus injection, a dose of 333 mg/kg of kanamycin was subcutaneously given twice daily for 12 consecutive days. GDNF expression in the cochlea was confirmed and successfully modulated by the Tet-on system. Monitoring of the auditory brain stem response revealed an improvement of cochlear function after GDNF transduction over the frequencies tested. Damaged spiral ganglion cells and hair cells were significantly reduced by GDNF expression. Our results suggest that AAV1-mediated expression of GDNF using a regulatedexpression system in the cochlea is a promising strategy to protect the cochlea from aminoglycoside-induced damage.
  • Yukiko Kishi, Yujiro Tanaka, Hiroaki Shibata, Shinichiro Nakamura, Koichi Takeuchi, Shigeo Masuda, Tamako Ikeda, Shin-ichi Muramatsu, Yutaka Hanazono
    CELL TRANSPLANTATION 17 9 1095 - 1102 2008年 [査読有り][通常論文]
     
    Embryonic stem (ES) cells have the ability to generate teratomas when transplanted into immunodeficient mice, but conditions affecting the generation remain to be elucidated. Nonhuman primate cynomolgus ES cells were transplanted into immunodeficient mice under different conditions; the number of transplanted cells, physical state (clumps or single dissociated cells), transplant site, differentiation state, and immunological state of recipient mice were all varied. The tumorigenicity was then evaluated. When cynomolgus ES cells were transplanted as clumps into the lower limb muscle in either nonobese diabetic/severe combined immunodeficiency (NOD/SCID) or NOD/SCID/gamma c(null) (NOG) mice, teratomas developed in all the animals transplanted with 1 x 10(5) or more cells, but were not observed in any mouse transplanted with 1 x 10(3) cells. However, when the cells were transplanted as dissociated cells, the number of cells necessary for teratornas to form in all mice increased to 5 x 10(5). When the clump cells were injected. subcutaneously (instead of intramuscularly), the number also increased to 5 x 10(5). When cynomolgus ES cell-derived progenitor cells (1 x 10(6)), which included residual pluripotent cells, were transplanted into the lower limb muscle of NOG or NOD/SCID mice, the incidence of teratomas differed between the strains; teratomas developed in five of five NOG mice but in only two of five NOD/SCID mice. The incidence of teratomas varied substantially depending on the transplanted cells and recipient mice. Thus, considerable care must be taken as to tumorigenicity.
  • Hirohide Sawada, Ryohei Hishida, Yoko Hirata, Kenji Ono, Hirorni Suzuki, Shin-ichi Muramatsu, Imaharu Nakano, Toshiharu Nagatsu, Makoto Sawada
    JOURNAL OF NEUROSCIENCE RESEARCH 85 8 1752 - 1761 2007年06月 [査読有り][通常論文]
     
    Microglia play an important role in the inflammatory process that occurs in Parkinson's disease (PD). Activated microglia produce cytokines and neurotrophins and may have neurotoxic or neurotrophic effects. Because microglia are most proliferative and easily activated during the neonatal period, we examined the effects of neonatal microglia activated with lipopolysaccharide (LPS) on the nigro-striatal dopamine neurons in mice treated with 1-methyl-4-phenyl-1 2,3,6-tetrahyd ropyridine (MPTP), in comparison with activated microglia from the aged mice. By MPTP administration to neonatal mice, the number of dopamine neurons in the substantia nigra (SN) was decreased significantly, whereas that in the mice treated with LPS and MPTP was recovered to normal, along with significant microglial activation. Tyrosine hydroxylase (TH) activity, the levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), and the levels of pro-inflammatory cytokines IL-1 beta and IL-6 in the midbrain were elevated in the neonates treated with LPS and MPTR On the contrary, although the number of dopamine neurons in the 60-week-old mice treated with MPTP was also decreased significantly, the microglial activation by LPS treatment caused a further decrease in their number. These results suggest that the activated microglia in neonatal mice are different from those in aged mice, with the former having neurotrophic potential toward the dopamine neurons in the SN, in contrast to the neurotoxic effect of the latter. (c) 2007 Wiley-Liss, Inc.
  • Muramatsu S
    Brain and nerve = Shinkei kenkyu no shinpo 59 425 - 430 4 2007年04月 [査読有り][通常論文]
  • Muramatsu Shin-Ichi, Ono Fumiko, Takino Naomi, Nishida Hiroko, Asari Sayaka, Ikeguchi Kunihiko, Fujimoto Ken-Ichi, Tsukada Hideo, Terao Keiji, Ozawa Keiya, Nakano Imaharu
    NEUROSCIENCE RESEARCH 58 S58 - S58 2007年 [査読有り][通常論文]
  • Hiroaki Shibata, Naohide Ageyama, Yujiro Tanaka, Yukiko Kishi, Kyoko Sasaki, Shinichiro Nakamura, Shin-ichi Muramatsu, Satoshi Hayashi, Yoshihiro Kitano, Keiji Terao, Yutaka Hanazono
    Stem cells (Dayton, Ohio) 24 6 1450 - 7 2006年06月 [査読有り][通常論文]
     
    Cynomolgus monkey embryonic stem cell (cyESC)-derived in vivo hematopoiesis was examined in an allogeneic transplantation model. cyESCs were induced to differentiate into the putative hematopoietic precursors in vitro, and the cells were transplanted into the fetal cynomolgus liver at approximately the end of the first trimester (n = 3). Although cyESC-derived hematopoietic colony-forming cells were detected in the newborns (4.1%-4.7%), a teratoma developed in all newborns. The risk of tumor formation was high in this allogeneic transplantation model, given that tumors were hardly observed in immunodeficient mice or fetal sheep that had been xeno-transplanted with the same cyESC derivatives. It turned out that the cyESC-derived donor cells included a residual undifferentiated fraction positive for stage-specific embryonic antigen (SSEA)-4 (38.2% +/- 10.3%) despite the rigorous differentiation culture. When an SSEA-4-negative fraction was transplanted (n = 6), the teratoma was no longer observed, whereas the cyESC-derived hematopoietic engraftment was unperturbed (2.3%-5.0%). SSEA-4 is therefore a clinically relevant pluripotency marker of primate embryonic stem cells (ESCs). Purging pluripotent cells with this surface marker would be a promising method of producing clinical progenitor cell preparations using human ESCs.
  • XG Li, T Okada, M Kodera, Y Nara, N Takino, C Muramatsu, K Ikeguchi, F Urano, H Ichinose, D Metzger, P Chambon, Nakano, I, K Ozawa, S Muramatsu
    MOLECULAR THERAPY 13 1 160 - 166 2006年01月 [査読有り][通常論文]
     
    Regulation of gene expression is necessary to avoid possible adverse effects of gene therapy due to excess synthesis of transgene products. To reduce transgene expression, we developed a viral vector-mediated somatic regulation system using inducible Cre recombinase. A recombinant adeno-associated virus (AAV) vector expressing Cre recombinase fused to a mutated ligand-binding domain of the estrogen receptor alpha (CreER(T2)) was delivered along with AAV vectors expressing dopamine-synthesizing enzymes to rats of a Parkinson disease model. Treatment with 4-hydroxytarnoxifen, a synthetic estrogen receptor modulator, activated Cre recombinase within the transduced neurons and induced selective excision of the tyrosine hydroxylase (TH) coding sequence flanked by IoxP sites, leading to a reduction in transgene-mediated dopamine synthesis. Using this strategy, aromatic L-amino acid decarboxylase (AADC) activity was retained so that (L)-3, 4-dihydroxyphenylalanine (L-dopa), a substrate for AADC, could be converted to dopamine in the striatum and the therapeutic effects of (L)-dopa preserved, even after reduction of TH expression in the case of dopamine overproduction. Our data demonstrate that viral vector-mediated inducible Cre recombinase can serve as an in vivo molecular switch, allowing spatial and temporal control of transgene expression, thereby potentially increasing the safety of gene therapy.
  • Muramatsu S
    Nippon rinsho. Japanese journal of clinical medicine 63 Suppl 12 622 - 625 2005年12月 [査読有り][通常論文]
  • Shin-Ichi Muramatsu
    Clinical Neurology 45 11 902 - 904 2005年11月 [査読有り][通常論文]
     
    Increasing enthusiasm in the field of stem cell research is raising the hope of novel cell replacement therapies for Parkinson's disease (PD), but it also raises both scientific and ethical concerns. In most cases, dopaminergic cells are transplanted ectopically into the striatum instead of the substantia nigra. If the main mechanism underlying any observed functional recovery with these cell replacement therapies is restoration of dopaminergic neurotransmission, then viral vector-mediated gene delivery of dopamine-synthesizing enzymes is a more straight forward approach. The development of a recombinant adeno-associated viral (AAV) vector is making gene therapy for PD a feasible therapeutic option in the clinical arena. Efficient and long-term expression of genes for dopamine-synthesizing enzymes in the striatum restored local dopamine production and allowed behavioral recovery in animal models of PD. A clinical trial to evaluate the safety and efficacy of AAV vector-mediated gene transfer of aromatic L-amino acid decarboxylase, an enzyme that converts L-dopa to dopamine, is underway. With this strategy patients would still need to take L-dopa to control their PD symptoms, however, dopamine production could be regulated by altering the dose of L-dopa. Another AAV vector-based clinical trial is also ongoing in which the subthalamic nucleus is transduced to produce inhibitory transmitters.
  • YH Liu, T Okada, K Sheykholeslami, K Shimazaki, T Nomoto, SI Muramatsu, T Kanazawa, K Takeuchi, R Ajalli, H Mizukami, A Kume, K Ichimura, K Ozawa
    MOLECULAR THERAPY 12 4 725 - 733 2005年10月 [査読有り][通常論文]
     
    Recombinant adeno-associated virus (AAV) vectors are of interest for cochlear gene therapy because of their ability to mediate the efficient transfer and long-term stable expression of therapeutic genes in a wide variety of postmitotic tissues with minimal vector-related cytotoxicity. In the present study, seven AAV serotypes (AAV1-5, 7, 8) were used to construct vectors. The expression of EGFP by the chicken P-actin promoter associated with the cytomegalovirus immediate-early enhancer in cochlear cells showed that each of these serotypes successfully targets distinct cochlear cell types. In contrast to the other serotypes, the AAV3 vector specifically transduced cochlear inner hair cells with high efficiency in vivo, while the AAV1, 2, 5, 7, and 8 vectors also transduced these and other cell types, including spiral ganglion and spiral ligament cells. There was no loss of cochlear function with respect to evoked auditory brain-stem responses over the range of frequencies tested after the injection of AAV vectors. These findings are of value for further molecular studies of cochlear inner hair cells and for gene replacement strategies to correct recessive genetic hearing loss due to monogenic mutations in these cells.
  • M Maruyama, M Higuchi, Y Takaki, Y Matsuba, H Tanji, M Nemoto, N Tomita, T Matsui, N Iwata, H Mizukami, SI Muramatsu, K Ozawa, TC Saido, H Arai, H Sasaki
    ANNALS OF NEUROLOGY 57 6 832 - 842 2005年06月 [査読有り][通常論文]
     
    Amyloid β peptide (Aβ) has been implicated in Alzheimer's disease (AD) as an initiator of the pathological cascades. Several lines of compelling evidence have supported major roles of Aβ-degrading enzyme neprilysin in the pathogenesis of sporadic AD. Here, we have shown a substantial reduction of cerebrospinal fluid (CSF) neprilysin activity (CSF-NEP) in patients with AD-converted mild cognitive impairment and early AD as compared with age-matched control subjects. The altered CSF-NEP likely reflects changes in neuronal neprilysin, since transfer of neprilysin from brain tissue into CSF was demonstrated by injecting neprilysin-carrying viral vector into the brains of neprilysin-deficient mice. Interestingly, CSF-NEP showed an elevation with the progression of AD. Along with a close association of CSF-NEP with CSF tau proteins, this finding suggests that presynaptically located neprilysin can be released into CSF as a consequence of synaptic disruption. The impact of neuronal damages on CSF-NEP was further demonstrated by a prominent increase of CSF-NEP in rats exhibiting kainate-induced neurodegeneration. Our results unequivocally indicate significance of CSF-NEP as a biochemical indicator to pursue a pathological process that involves decreased neprilysin activity and Aβ-induced synaptic toxicity, and the support the potential benefits of neprilysin up-regulation in ameliorating neuropathology in prodromal and early AD.
  • S Muramatsu, H Tsukada, Nakano, I, K Ozawa
    EXPERT OPINION ON BIOLOGICAL THERAPY 5 5 663 - 671 2005年05月 [査読有り][通常論文]
     
    Existing strategies for gene therapy in the treatment of Parkinson's disease include the delivery of genes encoding dopamine (DA)-synthesising enzymes, leading to localised production of DA in the striatum; genes encoding factors that protect nigral neurons against ongoing degeneration, such as glial cell line-derived neurotrophic factor; and genes encoding proteins that produce the inhibitory transmitter γ-aminobutylic acid (GABA) in the subthalamic nucleus (STN), thus suppressing the hyperactive STN. Recombinant adeno-associated viral (rAAV) vectors, which are derived from non-pathogenic viruses, have been shown to be suitable for clinical trials. These rAAVs have been found to transduce substantial numbers of neurons efficiently and to express transgenes in mammalian brains for long periods of time, with minimum inflammatory and immunological responses. In vivo imaging using positron emission tomography is useful for monitoring transgene expression and for assessing the functional effects of gene delivery. Vector systems that regulate transgene expression are necessary to increase safety in clinical applications, and the development of such systems is in progress.
  • K. Sasaki, M. Inoue, H. Shibata, Y. Ueda, S. I. Muramatsu, T. Okada, M. Hasegawa, K. Ozawa, Y. Hanazono
    Gene Therapy 12 3 203 - 210 2005年02月 [査読有り][通常論文]
     
    Efficient gene transfer and regulated transgene expression in primate embryonic stem (ES) cells are highly desirable for future applications of the cells. In the present study, we have examined using the nonintegrating Sendai virus (SeV) vector to introduce the green fluorescent protein (GFP) gene into non-human primate cynomolgus ES cells. The GFP gene was vigorously and stably expressed in the cynomolgus ES cells for a year. The cells were able to form fluorescent teratomas when transplanted into immunodeficient mice. They were also able to differentiate into fluorescent embryoid bodies, neurons, and mature blood cells. In addition, the GFP expression levels were reduced dose-dependently by the addition of an anti-RNA virus drug, ribavirin, to the culture. Thus, SeV vector will be a useful tool for efficient gene transfer into primate ES cells and the method of using antiviral drugs should allow further investigation for regulated SeV-mediated gene expression. © 2005 Nature Publishing Group All rights reserved.
  • H Nakai, S Fuess, TA Storm, S Muramatsu, Y Nara, MA Kay
    JOURNAL OF VIROLOGY 79 1 214 - 224 2005年01月 [査読有り][通常論文]
     
    Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with P-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 X 10(12) vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, hut great caution is required for vector spillover and tight control of tissue-specific gene expression.
  • Nara Y, Muramatsu S, Nakano I
    Nippon rinsho. Japanese journal of clinical medicine 62 1643 - 1647 9 2004年09月 [査読有り][通常論文]
  • N Iwata, H Mizukami, K Shirotani, Y Takaki, S Muramatsu, B Lu, NP Gerard, C Gerard, K Ozawa, TC Saido
    JOURNAL OF NEUROSCIENCE 24 4 991 - 998 2004年01月 [査読有り][通常論文]
     
    A local increase in amyloid-beta peptide (Abeta) is closely associated with synaptic dysfunction in the brain in Alzheimer's disease. Here, we report on the catabolic mechanism of Abeta at the presynaptic sites. Neprilysin, an Abeta-degrading enzyme, expressed by recombinant adeno-associated viral vector-mediated gene transfer, was axonally transported to presynaptic sites through afferent projections of neuronal circuits. This gene transfer abolished the increase in Abeta levels in the hippocampal formations of neprilysin-deficient mice and also reduced the increase in young mutant amyloid precursor protein transgenic mice. In the latter case, Abeta levels in the hippocampal formation contralateral to the vector-injected side were also significantly reduced as a result of transport of neprilysin from the ipsilateral side, and in both sides soluble Abeta was degraded more efficiently than insoluble Abeta. Furthermore, amyloid deposition in aged mutant amyloid precursor protein transgenic mice was remarkably decelerated. Thus, presynaptic neprilysin has been demonstrated to degrade Abeta efficiently and to retard development of amyloid pathology.
  • M Nagata, MA Takahashi, S Muramatsu, Y Ueda, Y Hanazono, K Takeuchi, K Okada, Y Suzuki, Y Kondo, M Suemori, U Ikeda, Nakano, I, E Kobayashi, M Hasegawa, K Ozawa, N Nakatsuji, K Shimada
    JOURNAL OF GENE MEDICINE 5 11 921 - 928 2003年11月 [査読有り][通常論文]
     
    Background Embryonic stem (ES) cells continually proliferate and can generate large numbers of differentiated cells. Genetic manipulation of transplantable cells derived from primate ES cells offers considerable potential for development research and regenerative cell therapy. However, protocols for efficient gene transfer into primate ES-cell-derived cells have not yet been established. Methods Spontaneously contracting areas were derived from cynomolgus monkey ES cells. Features of cardiomyocytes in the area were analyzed according to gene expression (RT-PCR), morphology (immunostaining and electron microscopy), and function (intracellular calcium transience). Beating cells were transduced using a simian immunodeficiency virus (STV) vector expressing enhanced green fluorescence protein (EGFP), then transplanted into ischemic rat myocardium. Results Beating cells derived from monkey ES cells displayed gene expression, ultrastructural and functional properties of early-stage cardiomyocytes. Highly efficient (97% cardiac phenotype) and stable transduction of these ES-cell-derived cardiomyocytes was achieved using SIV vector without altering contractile function. In addition, transduced cardiomyocytes survived in the myocardium of a rat myocardial infarction model. Conclusions A lentiviral vector system based on SIV represents a useful vehicle for genetic modification of cardiomyocytes derived from primate ES cells, and can extend the application of primate ES cells to gene therapy. Copyright (C) 2003 John Wiley Sons, Ltd.
  • YY Lu, LJ Wang, S Muramatsu, K Ikeguchi, K Fujimoto, T Okada, H Mizukami, T Matsushita, Y Hanazono, A Kume, T Nagatsu, K Ozawa, Nakano, I
    NEUROSCIENCE RESEARCH 45 1 33 - 40 2003年01月 [査読有り][通常論文]
     
    Adeno-associated virus (AAV) vector has been developed as an attractive gene delivery system with proven safety. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for amyotrophic lateral sclerosis (ALS) and other motor neuron diseases. The purpose of this report was to investigate transgenic GDNF expression at different time points post AAV mediated GDNF intramuscular delivery. An AAV vector was constructed to encode a recombinant fusion of GDNF tagged with a FLAG sequence at the C-terminal (AAV-GDNF) to distinguish it from its endogenous counterpart. A single intramuscular injection of AAV-GDNF led to substantial expression of transgenic GDNF which remained for at least 10 months in transduced gastrocnemius muscle. This transgenic GDNF was distributed in a large number of myofibers, mainly in the vicinity of the sarcolemma and predominantly concentrated at the sites of neuromuscular junctions (NMJs). Furthermore, transgenic GDNF, but not P-galactosidase expressed as a control, was detected in the motoneurons that projected axons to the injected muscles, thus, indicating retrograde axonal transportation of the transgenic GDNF. This study provides a basis for a strategy of intramuscular AAV-GDNF delivery to protect motoneurons as a possible means of ALS treatment. (C) 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • S Muramatsu, LJ Wang, K Ikeguchi, K Fujimoto, Nakano, I, T Okada, H Mizukami, Y Hanazono, A Kume, Nakano, I, K Ozawa
    VIRAL VECTORS FOR TREATING DISEASES OF THE NERVOUS SYSTEM 55 205 - 222 2003年 [査読有り][通常論文]
  • T Okada, T Nomoto, K Shimazaki, LJ Wang, YY Lu, T Matsushita, H Mizukami, M Urabe, Y Hanazono, A Kume, S Muramatsu, Nakano, I, K Ozawa
    METHODS 28 2 237 - 247 2002年10月 [査読有り][通常論文]
     
    Gene therapy is a novel method under investigation for the treatment of neurological disorders. Considerable interest has focused on the possibility of using viral vectors to deliver genes to the central nervous system. Adeno-associated virus (AAV) is a potentially useful gene transfer vehicle for neurologic gene therapies. The advantages of AAV vector include the lack of any associated disease with a wild-type virus. the ability to transduce nondividing cells, the possible integration of the gene into the host genome, and the long-term expression of transgenes. The development of novel therapeutic strategies for neurological disorder by using AAV vector has an increasing impact on gene therapy research. This article describes methods that can be used to generate rodent and non-human primate models for testing treatment strategies linked to pathophysiological events in the ischemic brain and neurode-generative disorders such as Parkinson's disease. (C) 2002 Elsevier Science (USA). All rights reserved.
  • Muramatsu S, Wang L, Ikeguchi K, Fujimoto K, Nakano I, Ozawa K
    Journal of neurology 249 Suppl 2 II36 - 40 2002年09月 [査読有り][通常論文]
  • T Asano, Y Hanazono, Y Ueda, S Muramatsu, A Kume, H Suemori, Y Suzuki, Y Kondo, K Harii, M Hasegawa, N Nakatsuji, K Ozawa
    MOLECULAR THERAPY 6 2 162 - 168 2002年08月 [査読有り][通常論文]
     
    The ability to stably introduce genetic material into primate embryonic stem (ES) cells could allow their broader application. We previously derived ES cell lines from cynomolgus monkey blastocysts. In this study, we examined lentiviral gene transfer into cynomolgus ES cells. When cynomolgus ES cells were transduced once with a simian immunodeficiency virus (SIV)-based lentivirus vector encoding the green fluorescent protein (GFP) gene, most cells (around 90%) fluoresced, and high levels of GFP expression persisted for 5 months without selection procedures. In addition, high levels of GFP expression were observed during embryoid body formation. On the other hand, transduction of mouse ES cells with the SIV-based vector resulted in lower gene transfer rates, implying that SIV vectors can transduce primate ES cells more efficiently than mouse ES cells. The use of GFP as a reporter gene allows direct and simple detection of successfully transduced ES cells and facilitates monitoring of ES cell proliferation and differentiation both in vitro and potentially in vivo. Furthermore, this highly efficient gene transfer method allows faithful gene delivery to primate ES cells with potential for both research and therapeutic application.
  • LJ Wang, YY Lu, S Muramatsu, K Ikeguchi, K Fujimoto, T Okada, H Mizukami, T Matsushita, Y Hanazono, A Kume, T Nagatsu, K Ozawa, Nakano, I
    JOURNAL OF NEUROSCIENCE 22 16 6920 - 6928 2002年08月 [査読有り][通常論文]
     
    Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive lethal disease that involves selective annihilation of motoneurons. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for ALS and other motor neuron diseases. Because adeno-associated virus (AAV) has been developed as an attractive gene delivery system with proven safety, we explored the therapeutic efficacy of intramuscular delivery of the GDNF gene mediated by an AAV vector (AAV-GDNF) in the G93A mouse model of ALS. We show here that AAV-GDNF leads to substantial and long-lasting expression of transgenic GDNF in a large number of myofibers with its accumulation at the sites of neuromuscular junctions. Detection of GDNF labeled with FLAG in the anterior horn neurons, but not beta-galactosidase expressed as a control, indicates that most of the transgenic GDNF observed there is retrogradely transported GDNF protein from the transduced muscles. This transgenic GDNF prevents motoneurons from their degeneration, preserves their axons innervating the muscle, and inhibits the treated-muscle atrophy. Furthermore, four-limb injection of AAV-GDNF postpones the disease onset, delays the progression of the motor dysfunction, and prolongs the life span in the treated ALS mice. Our finding thus indicates that AAV-mediated GDNF delivery to the muscle is a promising means of gene therapy for ALS.
  • Namekawa M, Muramatsu S, Hashimoto R, Kawakami T, Fujimoto K, Nakano I
    Rinsho shinkeigaku = Clinical neurology 42 635 - 638 7 2002年07月 [査読有り][通常論文]
  • L Wang, S Muramatsu, Y Lu, K Ikeguchi, K Fujimoto, T Okada, H Mizukami, Y Hanazono, A Kume, F Urano, H Ichinose, T Nagatsu, Nakano, I, K Ozawa
    GENE THERAPY 9 6 381 - 389 2002年03月 [査読有り][通常論文]
     
    Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AA V) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AA V vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or beta-galactosidase (AAV-LacZ), into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AA V-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4-20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AA V vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.
  • SI Muramatsu, KI Fujimoto, K Ikeguchi, N Shizuma, K Kawasaki, F Ono, Y Shen, LJ Wang, H Mizukami, A Kume, M Matsumura, Nagatsu, I, F Urano, H Ichinose, T Nagatsu, K Terao, Nakano, I, K Ozawa
    HUMAN GENE THERAPY 13 3 345 - 354 2002年02月 [査読有り][通常論文]
     
    One potential strategy for gene therapy of Parkinson's disease (PD) is the local production of dopamine (DA) in the striatum induced by restoring DA-synthesizing enzymes. In addition to tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), GTP cyclohydrolase I (GCH) is necessary for efficient DA production. Using adeno-associated virus (AAV) vectors, we previously demonstrated that expression of these three enzymes in the striatum resulted in long-term behavioral recovery in rat models of PD. We here extend the preclinical exploration to primate models of PD. Mixtures of three separate AAV vectors expressing TH, AADC, and GCH, respectively, were stereotaxically injected into the unilateral putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys. Coexpression of the enzymes in the unilateral putamen resulted in remarkable improvement in manual dexterity on the contralateral to the AAV-TH/-AADC/-GCH-injected side. Behavioral recovery persisted during the observation period (four monkeys: 48 days, 65 days, 50 days, and >10 months, each). TH-immunoreactive (TH-IR), AADC-IR, and GCH-IR cells were present in a large region of the putamen. Microdialysis demonstrated that concentrations of DA in the AAV-TH/-AADC/-GCH-injected putamen were increased compared with the control side. Our results show that AAV vectors efficiently introduce DA-synthesizing enzyme genes into the striatum of primates with restoration of motor functions. This triple transduction method may offer a potential therapeutic strategy for PD.
  • M Tanaka, HJ Borgeld, J Zhang, S Muramatsu, JS Gong, M Yoneda, M Shamoto, N Fuku, M Kurata, Y Yamada, K Nishizawa, Y Akao, N Ohishi, S Miyabayashi, H Umemoto, T Muramatsu, K Furukawa, A Kikuchi, Nakano, I, K Ozawa, K Yagi
    JOURNAL OF BIOMEDICAL SCIENCE 9 6 534 - 541 2002年 [査読有り][通常論文]
     
    The restriction endonuclease Smal has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993T-->G mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F0F1-ATPase. Our ultimate goal is to apply Smal to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with the wild-type mtDNA (heteroplasmy), and because only the mutant mtDNA, but not the wild-type mtDNA, is selectively restricted by the enzyme. For this purpose, we transiently expressed the Smal gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by the Smal enzyme showed specific elimination of the mutant mtDNA. This elimination was followed with repopulation by the wild-type mtDNA, resulting in restoration of both the normal intracellular ATP level and normal mitochondrial membrane potential. Furthermore, in vivo electroporation of the plasmids expressing mitochondrion-targeted EcoRI induced a decrease in cytochrome c oxidase activity in hamster skeletal muscles while causing no degenerative changes in nuclei. Delivery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases. Copyright (C) 2002 National Science Council, ROC and S. Karger AG, Basel.
  • Shin-Ichi Muramatsu, Lijun Wang, Kunihiko Ikeguchi, Ken-Ichi Fujimoto, Imaharu Nakano, Keiya Ozawa
    Journal of Neurology, Supplement 249 2 II36 - II40 2002年 [査読有り][通常論文]
     
    The recombinant adeno-associated viral (rAAV) vector is a powerful tool for delivering therapeutic genes into mammalian brains. In rodents and non-human primates, a substantial number of striatal neurons can be transduced with high titer rAAV vectors by simple stereotaxic injection. Efficient and long-term expression of genes for dopamine (DA)-synthesizing enzymes in the striatum restored local DA production and achieved behavioral recovery in animal models of Parkinson's disease (PD). Moreover, sustained expression of a glial cell line-derived neurotrophic factor gene in the striatum rescued nigral neurons and led to functional recovery in a rat model of PD, even when treatment was delayed until after the onset of progressive degeneration. These results suggest that gene therapy using rAAV vectors may become a novel and feasible treatment for PD.
  • K Ozawa, S Muramatsu, K Fujimoto, K Ikeguchi, Y Shen, LJ Wang, T Okada, H Mizukami, Y Hanazono, A Kume, H Ichinose, T Nagatsu, K Terao, Nakano, I
    MAPPING THE PROGRESS OF ALZHEIMER'S AND PARKINSON'S DISEASE 51 459 - 462 2002年 [査読有り][通常論文]
  • 滑川道人, 村松慎一, 田中康文, 藤本健一, 中野今治, 楠 進
    臨床眼科 56 1361 - 1364 2002年 [査読有り][通常論文]
  • M Namekawa, Y Takiyama, Y Ando, K Sakoe, S Muramatsu, K Fujimoto, M Nishizawa, Nakano, I
    JOURNAL OF THE NEUROLOGICAL SCIENCES 187 1-2 103 - 106 2001年06月 [査読有り][通常論文]
     
    We describe the unusual case of a 51-year-old woman with spinocerebellar ataxia type 1 (SCAI) who showed choreiform movements in addition to cerebellar ataxia. To date, extrapyramidal signs including involuntary movements have been rarely reported in SCA1. Surface electromyogram in our patient revealed grouped discharges whose duration was longer than that of chorea observed in HD, indicating that the involuntary movements represented choreoathetosis rather than pure chorea. These choreiform movements have not been seen in non-hereditary spinocerebellar ataxia. Therefore, if 'sporadic' cases of cerebellar ataxia show such movements, the possibility of genetic origin of the ataxia is high and a surveillance of various forms of hereditary spinocerebellar ataxia including SCAI is required. (C) 2001 Elsevier Science B.V. All rights reserved.
  • J. Mimuro, S. Muramatsu, Y. Hakamada, K. Mori, J. Kikuchi, M. Urabe, S. Madoiwa, K. Ozawa, Y. Sakata
    Gene Therapy 8 22 1690 - 1697 2001年 [査読有り][通常論文]
     
    We were able to facilitate plasminogen activator inhibitor 1 (PAI-1) promoter activity approximately by 14-fold using an enhancer element. This enhanced PAI-1 promoter has a strong basal activity, comparable to CAG promoter activity, and has a response similar to the PAI-1 promoter with respect to TGFβ1 and TNFα stimulation. The characteristics of the enhanced PAI-1 promoter are thought to be suited to timely and tissue-specific expression of anticoagulant molecules in the vascular cells. Thus, we developed recombinant adeno-associated virus (rAAV) vectors using the enhanced PAI-1 promoter and were successful in transducing vascular endothelial cells to express the thrombomodulin transgene under the regulation of the enhanced PAI-1 promoter in vitro. Thromobomodulin transgene expression driven by the enhanced PAI-1 promoter in rAAV vector-transduced cultured endothelial cells was between 600- and 1000-fold higher than constitutive thrombomodulin gene expression in cultured human umbilical vein endothelial cells and was up-regulated by TGFβ1 and TNFα stimulation which may down-regulate endogenous thrombomodulin gene expression in endothelial cells. The brain vascular endothelial cells of Mongolian gerbils could also be transduced by the same rAAV vector in vivo. Transduction of endothelial cells by rAAV vectors to express enhanced PAI-1 promoter-driven transgenes may be a useful gene therapy approach for vascular diseases.
  • Y. Shen, I. Nagatsu, F. Urano, T. Suzuki, H. Ichinose, T. Nagatsu, J. Monahan, I. Nakano, K. Ozawa, S. I. Muramatsu, K. Ikeguchi, K. I. Fujimoto, D. S. Fan, M. Ogawa, H. Mizukami, M. Urabe, A. Kume
    Human Gene Therapy 11 11 1509 - 1519 2000年07月 [査読有り][通常論文]
     
    Parkinson's disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxy-dopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.
  • Nobuyuki Taniguchi, Kouichi Itoh, Yi Wang, Kiyoka Omoto, Kouichiro Shigeta, Yasutomo Fujii, Michito Namekawa, Shinichi Muramatsu, Imaharu Nakano
    Journal of Clinical Ultrasound 28 9 488 - 491 2000年 [査読有り][通常論文]
     
    Chronic inflammatory demyelinating polyradiculoneuropathy is an autoimmune disease characterized by recurrent demyelination and remyelination with resultant thickening of the peripheral nerves. We report a case in which sonography was instrumental in demonstrating diffuse peripheral nerve hypertrophy. On sonography, both brachial plexuses were found to be diffusely hypertrophic and hypoechoic. Similar findings were noted for the median, sciatic, and femoral nerves. The brachial plexus findings were confirmed by MRI. (C) 2000 John Wiley and Sons, Inc.
  • K Ozawa, DS Fan, Y Shen, S Muramatsu, K Fujimoto, K Ikeguchi, M Ogawa, M Urabe, A Kume, Nakano, I
    JOURNAL OF NEURAL TRANSMISSION-SUPPLEMENT 58 181 - 191 2000年 [査読有り][通常論文]
     
    Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of dopamine-synthesizing enzymes [tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC)]. The most appropriate gene-delivery vehicles for neurons are adeno-associated virus (AAV) vectors, which are derived from non-pathogenic virus. Therefore, TH and AADC genes were introduced into the striatum in the lesioned side using separate AAV vectors in parkinsonian rats, and the coexpression of TH and AADC resulted in better behavioral recovery compared with TH alone. Another strategy for gene therapy of PD is the protection of dopaminergic neurons in the substantia nigra using an AAV vector containing a glial cell line-derived; neurotrophic factor (GDNF) gene. Combination of dopamine-supplement gene therapy and GDNF gene therapy would be a logical approach to the treatment of PD.
  • K Ozawa, DS Fan, Y Shen, S Muramatsu, K Fujimoto, K Ikeguchi, M Ogawa, M Urabe, A Kume, Nakano, I
    ADVANCES IN RESEARCH ON NEURODEGENERATION, VOL 7 7 181 - 191 2000年 [査読有り][通常論文]
     
    Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of dopamine-synthesizing enzymes [tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC)]. The most appropriate gene-delivery vehicles for neurons are adeno-associated virus (AAV) vectors, which are derived from non-pathogenic virus. Therefore, TH and AADC genes were introduced into the striatum in the lesioned side using separate AAV vectors in parkinsonian rats, and the coexpression of TH and AADC resulted in better behavioral recovery compared with TH alone. Another strategy for gene therapy of PD is the protection of dopaminergic neurons in the substantia nigra using an AAV vector containing a glial cell line-derived neurotrophic factor (GDNF) gene. Combination of dopamine-supplement gene therapy and GDNF gene therapy would be a logical approach to the treatment of PD.
  • Yoshihisa Takiyama, Haruo Shimazaki, Mitsuya Morita, Michiyo Soutome, Kumi Sakoe, Eisaku Esumi, Shin-ichi Muramatsu, Mitsuo Yoshida, Shuichi Igarashi, Hajime Tanaka, Shoji Tsuji, Hidenao Sasaki, Akemi Wakisaka, Imaharu Nakano, Masatoyo Nishizawa
    Journal of the Neurological Sciences 155 2 141 - 145 1998年03月 [査読有り][通常論文]
     
    We studied the relationship between the number of CAG repeat units in the MJD1 gene and clinical features of Machado-Joseph disease (MJD) in eight patients from two generations of a Japanese MID family. Because of lack of characteristic clinical signs of MJD such as dystonia, bulging eyes or facial myokymia, clinical diagnosis of MJD in this family was difficult to make prior to molecular testing for the CAG repeat expansion in the MJD1 gene. All the patients exhibited maternal transmission of MJD, and the intergenerational change in the number of CAG repeat units in the MJD1 gene was very small (+0.5 ± 0.3, mean ± S.E.M., n=4) in spite of marked genetic anticipation (-17.0 years/generation). In the present family, the degree of anticipation per repeat unit in maternal transmissions was much larger than that in maternal transmissions in the other six MJD families. This indicates that some maternal factors other than the increase of the number of CAG repeat units, which is known to be the basis of anticipation, may play a role in genetic anticipation in this MJD family.

MISC

産業財産権

受賞

  • 2019年03月 Healthy Society Award
     
    受賞者: Shin-ichi Muramatsu
  • 2019年03月 ヘルシー・ソサエティー賞
     
    受賞者: 村松慎一
  • 2011年07月 Japan Society of Gene Therapy タカラバイオ賞
     
    受賞者: 村松慎一
  • 2009年06月 米国遺伝子治療学会 Top Abstract
     
    受賞者: 村松慎一
  • 2000年06月 Japan Society of Gene Therapy 日本遺伝子治療学会賞
     
    受賞者: 村松慎一

委員歴

  • - 現在   日本遺伝子細胞治療学会   理事
  • - 2019年05月   日本東洋医学会   理事


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