Takaomi C Saido

RIKEN, Вако, Saitama, Japan

Are you Takaomi C Saido?

Claim your profile

Publications (374)1935.35 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ. Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed ScaleS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. ScaleS permitted optical reconstructions of aged and diseased brain in Alzheimer's disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimer's disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by ScaleS. These findings suggest that ScaleS is a simple and reproducible method for accurate visualization of biological tissue.
    Nature Neuroscience 09/2015; DOI:10.1038/nn.4107 · 16.10 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Calpain mediates the collagen synthesis and cell proliferation and plays an important role in pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). In the present study, we investigate whether and how calpain is activated by PAH mediators in pulmonary artery smooth muscle cells (PASMCs). These data showed that smooth muscle-specific knockout of calpain attenuated and knockout of calpastatin potentiated pulmonary vascular remodeling and pulmonary hypertension. Treatment of PASMCs with the PAH mediators platelet-derived growth factor (PDGF), serotonin, H2O2, endothelin-1 and interleukin-6 caused significant increases in calpain activity, cell proliferation, and collagen-I protein level without changes in protein levels of calpain-1 and -2. The calcium chelator BAPTA/AM did not affect calpain activation but the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 and knocking down of calpain-2 prevented calpain activation in PAH mediator-treated PASMCs. Mass spectrometry data showed that the phosphorylation of calpain-2 at serine 50 (Ser50) was increased and the phosphorylation of calpain-2 at serine 369 (Ser369) was decreased in PDGF-treated PASMCs. The PDGF-induced increase in Ser50 phosphorylation of calpain-2 was prevented by the ERK1/2 inhibitor PD98059, while dephosphorylation of calpain-2 at Ser369 was blocked by the protein phosphatase 2A (PP2A) inhibitor fostriecin. Furthermore, smooth muscle of pulmonary arteries in PAH animal model and PAH patients showed higher levels of P-Ser50-calpain-2 and lower levels of P-Ser369-calpain-2. These data support that calpain modulates pulmonary vascular remodeling in PAH. PAH mediator-induced activation of calpain is caused by ERK1/2-dependent phosphorylation of calpain-2 at Ser50 and PP2A-dependent dephosphorylation of calpain-2 at Ser369 in pulmonary vascular remodeling of PAH.
    American Journal of Respiratory Cell and Molecular Biology 08/2015; DOI:10.1165/rcmb.2015-0151OC · 3.99 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Alzheimer's disease (AD) is a neurodegenerative disease displaying extracellular plaques formed by the neurotoxic amyloid β-peptide (Aβ), and intracellular neurofibrillary tangles consisting of protein tau. However, how these pathologies relates to the massive neuronal death that occurs in AD brains remain elusive. Neprilysin is the major Aβ degrading enzyme and a lack thereof increases Aβ levels in the brain twofold. To identify altered protein expression levels induced by increased Aβ levels, we performed a proteomic analysis of the brain of the AD mouse model APPsw and compared it to that of APPsw mice lacking neprilysin. To this end we established an LC-MS/MS method to analyze brain homogenate, using an (18) O-labeled internal standard to accurately quantify the protein levels. To distinguish between alterations in protein levels caused by increased Aβ levels and those induced by neprilysin deficiency independently of Aβ, the brain proteome of neprilysin deficient APPsw mice was also compared to that of neprilysin deficient mice. By this approach we identified approximately 600 proteins and the levels of 300 of these were quantified. Pathway analysis showed that many of the proteins with altered expression were involved in neurological disorders, and that tau, presenilin and APP were key regulators in the identified networks. The data have been deposited to the ProteomeXchange Consortium with identifiers PXD000968 and PXD001786. Interestingly, the levels of several proteins, including some not previously reported to be linked to AD, were associated with increased Aβ levels. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Proteomics 07/2015; DOI:10.1002/pmic.201400211 · 3.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Calpains are ubiquitous proteases involved in cell proliferation, adhesion and motility. In the brain, calpains have been associated with neuronal damage in both acute and neurodegenerative disorders, but their physiological function in the nervous system remains elusive. During brain ischemia, there is a large increase in the levels of intracellular calcium, leading to the activation of calpains. Inhibition of these proteases has been shown to reduce neuronal death in a variety of stroke models. On the other hand, after stroke, neural stem cells (NSC) increase their proliferation and newly formed neuroblasts migrate towards the site of injury. However, the process of forming new neurons after injury is not efficient and finding ways to improve it may help with recovery after lesion. Understanding the role of calpains in the process of neurogenesis may therefore open a new window for the treatment of stroke. We investigated the involvement of calpains in NSC proliferation and neuroblast migration in two highly neurogenic regions in the mouse brain, the dentate gyrus (DG) and the subventricular zone (SVZ). We used mice that lack calpastatin, the endogenous calpain inhibitor, and calpains were also modulated directly, using calpeptin, a pharmacological calpain inhibitor. Calpastatin deletion impaired both NSC proliferation and neuroblast migration. Calpain inhibition increased NSC proliferation, migration speed and migration distance in cells from the SVZ. Overall, our work suggests that calpains are important for neurogenesis and encourages further research on their neurogenic role. Prospective therapies targeting calpain activity may improve the formation of new neurons following stroke, in addition to affording neuroprotection.
    Frontiers in Cellular Neuroscience 02/2015; 9. DOI:10.3389/fncel.2015.00022 · 4.29 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The β-site amyloid precursor protein cleaving enzyme-1 (BACE1), an essential protease for the generation of amyloid-β (Aβ) peptide, is a major drug target for Alzheimer's disease (AD). However, there is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N-acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT-III (Mgat3), revealed that cleavage of Aβ-precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in Aβ plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin-2, are normally cleaved in GnT-III-deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT-III-deficient mice remain healthy, GnT-III may be a novel and promising drug target for AD therapeutics. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.
    EMBO Molecular Medicine 02/2015; 7(2). DOI:10.15252/emmm.201404438 · 8.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Alzheimer's disease (AD) is characterized by β-amyloid plaques and intraneuronal τ aggregation usually associated with cerebral amyloid angiopathy (CAA). Both β-amyloid plaques and CAA deposits contain fibrillar aggregates of the amyloid β-peptide (Aβ). Aβ plaques and CAA develop first in neocortical areas of preclinical AD patients and, then, expand in a characteristic sequence into further brain regions with end-stage pathology in symptomatic AD patients. Aβ aggregates are not restricted to amyloid plaques and CAA. Soluble and several types of insoluble non-plaque- and non-CAA-associated Aβ aggregates have been described. Amyloid fibrils are products of a complex self-assembly process that involves different types of transient intermediates. Amongst these intermediate species are protofibrils and oligomers. Different variants of Aβ peptides may result from alternative processing or from mutations that lead to rare forms of familial AD. These variants can exhibit different self-assembly and aggregation properties. In addition, several post-translational modifications of Aβ have been described that result, for example, in the production of N-terminal truncated Aβ with pyroglutamate modification at position 3 (AβN3pE) or of Aβ phosphorylated at serine 8 (pSer8Aβ). Both AβN3pE and pSer8Aβ show enhanced aggregation into oligomers and fibrils. However, the earliest detectable soluble and insoluble Aβ aggregates in the human brain exhibit non-modified Aβ, whereas AβN3pE and pSer8Aβ are detected in later stages. This finding indicates the existence of different biochemical stages of Aβ aggregate maturation with pSer8Aβ being related mainly to cases with symptomatic AD. The conversion from preclinical to symptomatic AD could thereby be related to combined effects of increased Aβ concentration, maturation of aggregates and spread of deposits into additional brain regions. Thus, the inhibition of Aβ aggregation and maturation before entering the symptomatic stage of the disease as indicated by the accumulation of pSer8Aβ may represent an attractive treatment strategy for preventing disease progression.
    Acta Neuropathologica 12/2014; 129(2). DOI:10.1007/s00401-014-1375-y · 10.76 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Alzheimer disease (AD) is biochemically characterized by increased levels of amyloid β (Aβ) peptide, which aggregates into extracellular Aβ plaques in AD brains. Before plaque formation, Aβ accumulates intracellularly in both AD brains and in the brains of AD model mice, which may contribute to disease progression. Autophagy, which is impaired in AD, clears cellular protein aggregates and participates in Aβ metabolism. In addition to a degradative role of autophagy in Aβ metabolism we recently showed that Aβ secretion is inhibited in mice lacking autophagy-related gene 7 (Atg7) in excitatory neurons in the mouse forebrain. This inhibition of Aβ secretion leads to intracellular accumulation of Aβ. Here, we used fluorescence and immunoelectron microscopy to elucidate the subcellular localization of the intracellular Aβ accumulation which accumulates in Aβ precursor protein mice lacking Atg7. Autophagy deficiency causes accumulation of p62(+) aggregates, but these aggregates do not contain Aβ. However, knockdown of Atg7 induced Aβ accumulation in the Golgi and a concomitant reduction of Aβ in multivesicular bodies. This indicates that Atg7 influences the transport of Aβ possibly derived from Golgi to multivesicular bodies. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
    American Journal Of Pathology 11/2014; 185(2). DOI:10.1016/j.ajpath.2014.10.011 · 4.59 Impact Factor
  • Source
  • Source
    Kaori Tsukakoshi · Takashi Saito · Takaomi Saido
  • Source
    Takashi Saito · Yukio Matsuba · Naomi Mihira · Takaomi Saido
    Alzheimer's and Dementia 07/2014; 10(4):P477. DOI:10.1016/j.jalz.2014.05.693 · 12.41 Impact Factor
  • Source
    Alzheimer's and Dementia 07/2014; 10(4):P193. DOI:10.1016/j.jalz.2014.04.227 · 12.41 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Amyloid-β (Aβ) peptides represent key players in the pathogenesis of Alzheimer’s disease (AD), and mounting evidence indicates that soluble Aβ oligomers mediate the toxicity. Prefoldin (PFD) is a molecular chaperone that prevents aggregation of misfolded proteins. Here we investigated the role of PFD in Aβ aggregation. First, we demonstrated that PFD is expressed in mouse brain by Western blotting and immunohistochemistry and found that PFD is upregulated in AD model APP23 transgenic mice. Then we investigated the effect of recombinant human PFD (hPFD) on Aβ(1–42) aggregation in vitro and found that hPFD inhibited Aβ fibrillation and induced formation of soluble Aβ oligomers. Interestingly, cell viability measurements using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that Aβ oligomers formed by hPFD were 30–40% less toxic to cultured rat pheochromocytoma (PC12) cells or primary cortical neurons from embryonic C57BL/6CrSlc mice than previously reported Aβ oligomers (formed by archaeal PFD) and Aβ fibrils (p < 0.001). Thioflavin T measurements and immunoblotting indicated different structural properties for the different Aβ oligomers. Our findings show a relation between cytotoxicity of Aβ oligomers and structure and suggest a possible protective role of PFD in AD.
    Biochemistry 06/2014; 53(21):3520. DOI:10.1021/bi5005415 · 3.02 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Enzymatic proteolysis by calpains, Ca2+-dependent intracellular cysteine proteases, has been implicated in pathological processes such as cellular degeneration or death. Here, we investigated the role of calpain activation in the hearts subjected to myocardial infarction. We produced myocardial infarction in Cast−/− mice deficient for calpastatin, the specific endogenous inhibitory protein for calpains, and Cast+/+ mice. The activity of cardiac calpains in Cast+/+ mice was not elevated within 1 day but showed a gradual elevation after 7 days following myocardial infarction, which was further pronounced in Cast−/− mice. Although the prevalence of cardiomyocyte death was indistinguishable between Cast−/− and Cast+/+ mice, Cast−/− mice exhibited profound contractile dysfunction and chamber dilatation and showed a significant reduction in survival rate after myocardial infarction as compared with Cast+/+ mice. Notably, immunofluorescence revealed that at 28 days after myocardial infarction, calpains were activated in cardiomyocytes exclusively at the border zone and that Cast−/− mice showed higher intensity and a broader extent of calpain activation at the border zone than Cast+/+ mice. In the border zone of Cast−/− mice, pronounced activation of calpains was associated with a decrease in N-cadherin expression and up-regulation of molecular markers for cardiac hypertrophy and fibrosis. In cultured rat neonatal cardiomyocytes, calpain activation by treatment with ionomycin induced cleavage of N-cadherin and decreased expression levels of β-catenin and connexin 43, which was attenuated by calpain inhibitor. These results thus demonstrate that activation of calpains disassembles cell-cell adhesion at intercalated discs by degrading N-cadherin and thereby promotes left ventricular remodeling after myocardial infarction.
    Journal of Biological Chemistry 06/2014; 289(28). DOI:10.1074/jbc.M114.567206 · 4.57 Impact Factor
  • Source
    Per Nilsson · Takaomi C. Saido
    [Show abstract] [Hide abstract]
    ABSTRACT: Alzheimer's disease (AD) is a neurodegenerative disease exhibiting amyloid beta (Aβ) peptide accumulation as a key characteristic. Autophagy, which is dysregulated in AD, participates in the metabolism of Aβ. Unexpectedly, we recently found that autophagy, in addition to its degradative function, also mediates the secretion of Aβ. This finding adds Aβ to an increasing number of biomolecules, the secretion of which is mediated by autophagy. We also showed that inhibition of Aβ secretion through genetic deletion of autophagy leads to intracellular Aβ accumulation, which enhanced neurodegeneration induced by autophagy deficiency. Hence, autophagy may play a central role in two pathological hallmarks of AD: Aβ amyloidosis and neurodegeneration. Herein, we summarize the role of autophagy in AD with focus on Aβ metabolism in light of the recently established role of autophagy in protein secretion. We discuss potential routes for autophagy-mediated Aβ secretion and suggest experimental approaches to further elucidate its mechanisms.
    BioEssays 06/2014; 36(6). DOI:10.1002/bies.201400002 · 4.73 Impact Factor
  • Per Nilsson · Takashi Saito · Takaomi C Saido
    [Show abstract] [Hide abstract]
    ABSTRACT: Amyloid β-peptide (Aβ) accumulation is a key characteristic of Alzheimer's disease (AD); therefore, mouse models of AD exhibiting Aβ pathology are valuable tools for unraveling disease mechanisms. However, the overexpression of Aβ precursor protein (APP) used in previous mouse models may cause Aβ-independent artifacts that influence data interpretation. To circumvent these problems, we used an APP knock-in (KI) strategy to introduce mutations to the mouse APP gene to develop a new generation of AD mouse models. These new models, termed APP(NL-F) and APP(NL-G-F), have endogenous APP levels and develop robust Aβ amyloidosis, which induce synaptic degeneration and memory impairments. Thus, we suggest that these novel APP KI mice will serve as important tools to elucidate molecular mechanisms of AD.
    ACS Chemical Neuroscience 05/2014; 5(7). DOI:10.1021/cn500105p · 4.36 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Experimental studies of Alzheimer's disease have largely depended on transgenic mice overexpressing amyloid precursor protein (APP). These mice, however, suffer from artificial phenotypes because, in addition to amyloid β peptide (Aβ), they overproduce other APP fragments. We generated knock-in mice that harbor Swedish and Beyreuther/Iberian mutations with and without the Arctic mutation in the APP gene. The mice showed typical Aβ pathology, neuroinflammation and memory impairment in an age-dependent manner.
    Nature Neuroscience 04/2014; 17(5). DOI:10.1038/nn.3697 · 16.10 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic mice (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated-aggregation of αSyn indicating a therapeutic potential of calpain inhibition in PD.
    Human Molecular Genetics 03/2014; 23(15). DOI:10.1093/hmg/ddu112 · 6.39 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The hallmark of Alzheimer's disease (AD) pathology is an accumulation of amyloid b (Ab) and phosphorylated tau, which are encoded by the amyloid precursor protein (APP) and microtubule-associated protein tau (MAPT) genes, respectively. Less than 5% of all AD cases are familial in nature, i.e. caused by mutations in APP, PSEN1 or PSEN2. Almost all mutations found in them are related to an overproduction of Ab 1 – 42 , which is prone to aggre-gation. While these genes are mutation free, their function, or those of related genes, could be compromised in sporadic AD as well. In this study, pyrosequencing analysis of post-mortem brains revealed aberrant CpG methylation in APP, MAPT and GSK3B genes of the AD brain. These changes were further evaluated by a newly developed in vitro-specific DNA methylation system, which in turn highlighted an enhanced expression of APP and MAPT. Cell nucleus sorting of post-mortem brains revealed that the methylation changes of APP and MAPT occurred in both neuronal and non-neuronal cells, whereas GSK3B was abnormally methylated in non-neuronal cells. Further analysis revealed an association between abnormal APP CpG methylation and apo-lipoprotein E 14 allele (APOE 14)-negative cases. The presence of a small number of highly methylated neurons among normal neurons contribute to the methylation difference in APP and MAPT CpGs, thus abnormally methy-lated cells could compromise the neural circuit and/or serve as 'seed cells' for abnormal protein propagation. Our results provide a link between familial AD genes and sporadic neuropathology, thus emphasizing an epigen-etic pathomechanism for sporadic AD. INTRODUCTION
    Human Molecular Genetics 02/2014; 23(3):648-656. · 6.39 Impact Factor
  • Source
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Understanding the substrate recognition mechanism of γ-secretase is a key step for establishing substrate-specific inhibition of amyloid β-protein (Aβ) production. However, it is widely believed that γ-secretase is a promiscuous protease and that its substrate-specific inhibition is elusive. Here we show that γ-secretase distinguishes the ectodomain length of substrates and preferentially captures and cleaves substrates containing a short ectodomain. We also show that a subset of peptides containing the CDCYCxxxxCxCxSC motif binds to the amino terminus of C99 and inhibits Aβ production in a substrate-specific manner. Interestingly, these peptides suppress β-secretase-dependent cleavage of APP, but not that of sialyltransferase 1. Most importantly, intraperitoneal administration of peptides into mice results in a significant reduction in cerebral Aβ levels. This report provides direct evidence of the substrate preference of γ-secretase and its mechanism. Our results demonstrate that the ectodomain of C99 is a potent target for substrate-specific anti-Aβ therapeutics to combat Alzheimer's disease.
    Nature Communications 10/2013; 4:2529. DOI:10.1038/ncomms3529 · 11.47 Impact Factor

Publication Stats

18k Citations
1,935.35 Total Impact Points


  • 1998–2014
    • RIKEN
      • • Laboratory for Proteolytic Neuroscience
      • • Brain Science Institute (BSI)
      Вако, Saitama, Japan
    • Tokyo Metropolitan Institute
      Edo, Tōkyō, Japan
    • Toyota Physical and Chemical Institute
      Seto, Aichi, Japan
  • 2005–2012
    • University of Kentucky
      • Spinal Cord and Brain Injury Research Center
      Lexington, Kentucky, United States
  • 2011
    • Tokyo Metropolitan University
      • Department of Biological Sciences
      Edo, Tōkyō, Japan
  • 1990–2007
    • Tokyo Metropolitan Institute of Medical Science
      Edo, Tōkyō, Japan
  • 2001–2004
    • Case Western Reserve University
      • Institute of Pathology
      Cleveland, Ohio, United States
  • 2000
    • William Penn University
      Filadelfia, Pennsylvania, United States
    • Gunma University
      • Department of Neurology
      Maebashi, Gunma Prefecture, Japan
  • 1997–1998
    • The Graduate University for Advanced Studies
      Миура, Kanagawa, Japan
    • Harvard Medical School
      • Department of Psychiatry
      Boston, Massachusetts, United States
  • 1995
    • Hyogo College of Medicine
      • Department of Neurosurgery
      Nishinomiya, Hyōgo, Japan
    • The Scripps Research Institute
      • Department of Molecular and Experimental Medicine
      لا هویا, California, United States
  • 1992–1994
    • The University of Tokyo
      • • Institute of Molecular and Cellular Biosciences
      • • Institute of Medical Science
      Edo, Tōkyō, Japan