Faculty Profiles - SUZUKI, Hiromi

Signal Sequence-Dependent Orientation of Signal Peptide Fragments to Exosomes. Reviewed

Ono K, Niwa M, Suzuki H, Kobayashi NB, Yoshida T, Sawada M

International Journal of Molecular Sciences Vol. 23 ( 6 ) page： 3137 2022.3

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

Signal peptides (SPs) not only mediate targeting to the endoplasmic reticulum (ER) but also play important roles as biomarkers and substances with physiological activity in extracellular fluids including blood. SPs are thought to be degraded intracellularly, making it unclear how they are transported from the ER to the extracellular fluid. In a recent study, we showed that a C-terminal fragment of the SP of a type I membrane protein, amyloid precursor protein (APP), was secreted into the extracellular fluid via exosomes using transformed HEK293 cells expressing APP SP flanking a reporter protein. In the present study, we demonstrate that a N-terminal fragment of the SP from a type II membrane protein, human placental secreted alkaline phosphatase (SEAP), is contained in exosomes and secreted into the extracellular fluid using HEK-Blue hTLR3 cells, which express both a human toll-like receptor 3 gene and an inducible SEAP reporter gene. When HEK-Blue hTLR3 cells were stimulated with a TLR3 ligand, a N-terminal fragment of SEAP SP in exosomes was increased in parallel with SEAP secretion in a concentration-dependent manner. These results indicated that SP fragments are exosomal components. In addition, migrating SP fragments were determined by characteristics of the signal–anchor sequence of membrane proteins. Furthermore, we found that SP fragments could bind to calmodulin (CALM), which is a cytosolic protein and also a component of exosomes, suggesting its involvement in the transportation of SP fragments from the endoplasmic reticulum to exosomes.

DOI： 10.3390/ijms23063137

Other Link： https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8950404/

Secretion of signal peptides via extracellular vesicles Reviewed International journal

Ono K, Niwa M, Suzuki H, Kobayashi NB, Yoshida T, Sawada M.

Biochem Biophys Res Commun . Vol. 560 page： 21 - 26 2021.5

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

Signal peptides (SPs) consist of short peptide sequences present at the N-terminal of newly synthesizing proteins and act as a zip code for the translocation of the proteins to the endoplasmic reticulum (ER). It was thought that the SPs are intracellularly degraded after translocation to the ER; however, recent studies showed cleaved SPs have diverse roles for controlling cell functions in auto- and/or intercellular manners. In addition, it still remains obscure how SP fragments translocate away from the site where they are produced. Extracellular vesicles (EV) are important for intercellular communication and can transport functional molecules to specific cells. In this study, we show that SPs are involved in EV from T-REx AspALP cells that were transfected with a human APP SP-inducible expression vector. There was no difference in the average particle size or particle concentration of EV collected from T-REx AspALP cells and T-REx Mock cells. When the SP content in the EV was examined by mass spectrometry, the C-terminal fragment of APP SP was identified in the exosomes (SEV) of T-REx AspALP cells. In our preparation of SEV fractions, no ER-specific proteins were detected; therefore, SPs may be included in SEV but not in the debris of degraded ER. This is the first indication that SPs are secreted from cells via EV.

DOI： 10.1016/j.bbrc.2021.04.073.

Peripheral benzodiazepine receptor/18 kDa translocator protein positron emission tomography imaging in a rat model of acute brain injury Reviewed International coauthorship

Nomura M, Toyama H, Suzuki H, Yamada T, Hatano K, Alan A Wilson, Ito K, Sawada M

Ann Null Med. Vol. 35 ( 1 ) page： 8 - 16 2021.1

Distribution of Signal Peptides in Microvesicles from Activated Macrophage Cells Invited Reviewed

International Journal of Molecular Science Vol. 24 ( 15 ) 2023.7

Calmodulin as a Key Regulator of Exosomal Signal Peptides. Invited Reviewed International journal

Kenji Ono, Mikio Niwa, Hiromi Suzuki, Nahoko Bailey Kobayashi, Tetsuhiko Yoshida, Makoto Sawada

Cells Vol. 12 ( 1 ) page： 158 2022.12

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

Signal peptides (SPs) and their fragments play important roles as biomarkers and substances with physiological functions in extracellular fluid. We previously reported that SP fragments were released into extracellular fluid via exosomes and bound to calmodulin (CaM), an exosomal component, in a cell-free system. However, it currently remains unclear whether CaM intracellularly interacts with SP fragments or is involved in the trafficking of these fragments to exosomes. Therefore, the present study examined the binding of CaM to SP fragments in T-REx AspALP cells, transformed HEK293 cells expressing amyloid precursor protein (APP) SP flanking a reporter protein, and their exosomes. APP SP fragments were detected in exosomes from T-REx AspALP cells in the absence of W13, a CaM inhibitor, but were present in lower amounts in exosomes from W13-treated cells. Cargo proteins, such as Alix, CD63, and CD81, were increased in W13-treated T-REx AspALP cells but were decreased in their exosomes. Furthermore, CaM interacted with heat shock protein 70 and CD81 in T-REx AspALP cells and this increased in the presence of W13. APP SP fragments were detected in intracellular CaM complexes in the absence of W13, but not in its presence. These results indicate that CaM functions as a key regulator of the transport of SP fragments into exosomes and plays novel roles in the sorting of contents during exosomal biogenesis.

DOI： 10.3390/cells12010158

Inhibition of cellular inflammatory mediator production and amelioration of learning deficit in flies by deep sea Aspergillus-derived cyclopenin. Reviewed International coauthorship

Wang L, Li M, Lin Y, Du S, Liu Z, Ju J, Suzuki H, Sawada M, Umezawa K

The Journal of antibiotics Vol. 73 ( 9 ) page： 622 - 629 2020.9

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

In the course of screening lipopolysaccharide (LPS)-induced nitric oxide (NO) production inhibitors, two related benzodiazepine derivatives, cyclopenol and cyclopenin, were isolated from the extract of a deep marine-derived fungal strain, Aspergillus sp. SCSIOW2. Cyclopenol and cyclopenin inhibited the LPS-induced formation of NO and secretion of IL-6 in RAW264.7 cells at nontoxic concentrations. In terms of the mechanism underlying these effects, cyclopenol and cyclopenin were found to inhibit the upstream signal of NF-κB activation. These compounds also inhibited the expression of IL-1β, IL-6, and inducible nitric oxide synthase (iNOS) in mouse microglia cells, macrophages in the brain. In relation to the cause of Alzheimer’s disease, amyloid-β-peptide is known to induce inflammation in the brain. Therefore, the present study investigated the ameliorative effects of these inhibitors on an in vivo Alzheimer’s model using flies. Learning deficits were induced by the overexpression of amyloid-β42 in flies, and cyclopenin but not cyclopenol was found to rescue learning impairment. Therefore, novel anti-inflammatory activities of cyclopenin were identified, which may be useful as a candidate of anti-inflammatory agents for neurodegenerative diseases.

DOI： 10.1038/s41429-020-0302-9

Dipeptidyl peptidase-4 inhibition prevents nonalcoholic steatohepatitis-associated liver fibrosis and tumor development in mice independently of its anti-diabetic effects Reviewed International coauthorship

Mitsuhiro Kawakubo, Miyako Tanaka, Kozue Ochi, Akiko Watanabe, Marie Saka-Tanaka, Yohei Kanamori, Naoki Yoshioka, Satoko Yamashita, Moritaka Goto, Michiko Itoh, Ibuki Shirakawa, Sayaka Kanai, Hiromi Suzuki, Makoto Sawada, Ayaka Ito, Masatoshi Ishigami, Mitsuhiro Fujishiro, Hiroshi Arima, Yoshihiro Ogawa, Takayoshi Suganami

Science Report Vol. 10 ( 1 ) page： 983 2020.1

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

Nonalcoholic steatohepatitis (NASH) is a hepatic phenotype of the metabolic syndrome, and increases the risk of cirrhosis and hepatocellular carcinoma (HCC). Although increasing evidence points to the therapeutic implications of certain types of anti-diabetic agents in NASH, it remains to be elucidated whether their effects on NASH are independent of their effects on diabetes. Genetically obese melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet are a murine model that sequentially develops hepatic steatosis, NASH, and HCC in the presence of obesity and insulin resistance. In this study, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor anagliptin on NASH and HCC development in MC4R-KO mice. Anagliptin treatment effectively prevented inflammation, fibrosis, and carcinogenesis in the liver of MC4R-KO mice. Interestingly, anagliptin only marginally affected body weight, systemic glucose and lipid metabolism, and hepatic steatosis. Histological data and gene expression analysis suggest that anagliptin treatment targets macrophage activation in the liver during the progression from simple steatosis to NASH. As a molecular mechanism underlying anagliptin action, we showed that glucagon-like peptide-1 suppressed proinflammatory and profibrotic phenotypes of macrophages in vitro. This study highlights the glucose metabolism-independent effects of anagliptin on NASH and HCC development.

DOI： 10.1038/s41598-020-57935-6

Novel Oxindole-Curcumin Hybrid Compound for Antioxidative Stress and Neuroprotection Reviewed

Yoko Hirata, Yuki Ito, Madoka Takashima, Kazuya Yagyu, Kentaro Oh-Hashi, Hiromi Suzuki, Kenji Ono, Kyoji Furuta, Makoto Sawada

ACS Chem Neurosci . Vol. 11 ( 1 ) page： 76-85 2020.1

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

Oxidative stress plays an important role in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. The oxindole compound GIF-2165X-G1 is a hybrid molecule composed of the oxindole skeleton of the neuroprotective compound GIF-0726-r and the polyphenolic skeleton of the antioxidant curcumin. We previously reported that novel oxindole derivatives such as GIF-0726-r and GIF-2165X-G1 prevent endogenous oxidative stress-induced cell death in mouse hippocampal HT22 cells. In this study, we present a detailed investigation of the effect of GIF-2165X-G1 on endogenous oxidative stress in HT22 cells in comparison with GIF-0726-r and curcumin. GIF-2165X-G1 exhibited more potent neuroprotective activity than GIF-0726-r or curcumin and had less cytotoxicity than that observed with curcumin. Both GIF-0726-r and GIF-2165X-G1 were found to have ferrous ion chelating activity similar to that exhibited by curcumin. GIF-2165 X-G1 and curcumin induced comparable antioxidant response element transcriptional activity. Although the induction of heme oxygenase-1, an antioxidant response element-regulated gene product, was much stronger in curcumin-treated cells than in GIF-2165X-G1-treated cells, it turned out that the induction of heme oxygenase-1 is dispensable for neuroprotection. These results demonstrate that the introduction of the polyphenol skeleton of curcumin to the oxindole GIF-0726-r improves neuroprotective features. Furthermore, intrastriatal injection of GIF-2165X-G1 alleviated apomorphine-induced rotation and prevented dopaminergic neuronal loss in a 6-hydroxydopamine mouse model of Parkinson's diseases. Collectively, our novel findings indicate that the novel oxindole compound GIF-2165X-G1 serves to delay the progression of Parkinson's disease by suppressing oxidative stress.

DOI： 10.1021/acschemneuro.9b00619.

Novel oxindole derivatives prevent oxidative stress-induced cell death in mouse hippocampal HT22 cells. Reviewed

Hirata Y, Yamada C, Ito Y, Yamamoto S, Nagase H, Oh-Hashi K, Kiuchi K, Suzuki H, Sawada M, Furuta K

Neuropharmacology Vol. 135 page： 242-252 2018.3

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

The current medical and surgical therapies for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease offer symptomatic relief but do not provide a cure. Thus, small synthetic compounds that protect neuronal cells from degeneration are critically needed to prevent and treat these. Oxidative stress has been implicated in various pathophysiological conditions, including neurodegenerative diseases. In a search for neuroprotective agents against oxidative stress using the murine hippocampal HT22 cell line, we found a novel oxindole compound, GIF-0726-r, which prevented oxidative stress-induced cell death, including glutamate-induced oxytosis and erastin-induced ferroptosis. This compound also exerted a protective effect on tunicamycin-induced ER stress to a lesser extent but had no effect on campthothecin-, etoposide- or staurosporine-induced apoptosis. In addition, GIF-0726-r was also found to be effective after the occurrence of oxidative stress. GIF-0726-r was capable of inhibiting reactive oxygen species accumulation and Ca2+ influx, a presumed executor in cell death, and was capable of activating the antioxidant response element, which is a cis-acting regulatory element in promoter regions of several genes encoding phase II detoxification enzymes and antioxidant proteins. These results suggest that GIF-0726-r is a low-molecular-weight compound that prevents neuronal cell death through attenuation of oxidative stress. Among the more than 200 derivatives of the GIF-0726-r synthesized, we identified the 11 most potent activators of the antioxidant response element and characterized their neuroprotective activity in HT22 cells.

DOI： 10.1016/j.neuropharm.2018.03.015

Inducible nitric oxide synthase during the late phase of sepsis is associated with hypothermia and immune cell migration. Reviewed

Takatani Y, Ono K, Suzuki H, Inaba M, Sawada M, Matsuda N

Laboratory investigation Vol. 98 ( 5 ) page： 629 - 639 2018.2

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

Hypothermia is a significant sign of sepsis, which is associated with poor prognosis, but few mechanisms underlying the regulation of hypothermia are known. Inducible nitric oxide synthase (iNOS) is a key inflammatory mediator of sepsis. However, the therapeutic benefit of iNOS inhibition in sepsis is still controversial, and requires elucidation in an accurate model system. In this study, wild-type (WT) mice showed temperature drops in a biphasic manner at the early and late phase of sepsis, and all mice died within 48 h of sepsis. In contrast, iNOS-knockout (KO) mice never showed the second temperature drop and exhibited improved mortality. Plasma nitric oxide (NO) levels of WT mice increased in the late phase of sepsis and correlated to hypothermia. The results indicate that iNOS-derived NO during the late phase of sepsis caused vasodilation-induced hypothermia and a lethal hypodynamic state. The expression of the iNOS mRNA was high in the lung of WT mice with sepsis, which reflects the pathology of acute respiratory distress syndrome (ARDS). We obtained the results in a modified keyhole-type cecal ligation and puncture model of septic shock induced by minimally invasive surgery. In this accurate and reproducible model system, we transplanted the bone marrow cells of GFP transgenic mice into WT and iNOS-KO mice, and evaluated the role of increased pulmonary iNOS expression in cell migration during the late phase of sepsis. We also investigated the quantity and type of bone marrow-derived cells (BMDCs) in the lung. The number of BMDCs in the lung of iNOS-KO mice was less than that in the lung of WT mice. The major BMDCs populations were CD11b-positive, iNOS-negative cells in WT mice, and Gr-1-positive cells in iNOS-KO mice that expressed iNOS. These results suggest that sustained hypothermia may be a beneficial guide for future iNOS-targeted therapy of sepsis, and that iNOS modulated the migratory efficiency and cell type of BMDCs in septic ARDS.

DOI： 10.1038/s41374-018-0021-z

Visualization of Arc promoter-driven neuronal activity by magnetic resonance imaging Reviewed

Qi Wu, Kenji Ono, Hiromi Suzuki, Megumi Eguchi, Shun Yamaguchi, Makoto Sawada

Neuroscience Letters Vol. 666 page： 92 - 97 2018.2

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier Ireland Ltd

Visualization of direct neuronal activity to understand brain function is one of the most important challenges in neuroscience. We have previously demonstrated that in vivo and in vitro gene expression of the ferritin reporter system could be detected by magnetic resonance imaging (MRI). In addition, increased neuronal activity induces Arc, an immediate early gene, and insertion of a destabilized fluorescent reporter dVenus under Arc promoter control has been used for monitoring neuronal activities in the brain by optical imaging. In this study, to visualize Arc promoter-driven neuronal activities directly, we generated transgenic mice and cell lines that express a destabilized fusion reporter ferritin-mKate2 under Arc promoter control. When transgenic mice and cell lines were treated with pilocarpine, a non-selective muscarinic agonist, an increase in T2-weighted image signal was successfully found in neuronal cells. There was a difference in peak time between MRI and fluorescence imaging, which might result from the binding process of iron with ferritin. Visualization of Arc promoter-driven neuronal activity is essential to understand neural mechanisms underlying cognitive processes and complex behaviors, and could be a useful tool for therapeutic approaches in the brain by MRI.

DOI： 10.1016/j.neulet.2017.12.041

Optogenetic control of cell differentiation in channelrhodopsin-2-expressing OS3, a bipotential glial progenitor cell line

Ono K, Suzuki H, Yamamoto R, Sahashi H, Takido Y, Sawada M.

Neurochemistry International Vol. 104 page： 49-63 2017.3

Radiosynthesis and in vivo evaluation of two imidazopyridineacetamides, [11C]CB184 and [ 11C]CB190, as a PET tracer for 18 kDa translocator protein: direct comparison with [ 11C](R)-PK11195. Invited Reviewed

Hatano K, Sekimata K, Yamada T, Abe J, Ito K, Ogawa M, Magata Y, Toyohara J, Ishiwata K, Biggio G, Serra M, Laquintana V, Denora N, Latrofa A, Trapani G, Liso G, Suzuki H, Sawada M, Nomura M, Toyama H.

Annals of nuclear medicine Vol. 29 ( 4 ) page： 325 - 35 2015.5

Protective effect of INI-0602, a gap junction inhibitor, on dopaminergic neurodegeneration of mice with unilateral 6-hydroxydopamine injection

SUZUKI Hiromi,ONO Kenji,SAWADA Makoto

Journal of Neural Transmission Vol. 121 ( 11 ) page： 1349-1355 2014.11

Glutamate release from astrocyte cell-line GL261 via alterations in the intracellular ion environment Reviewed

Kenji Ono, Hiromi Suzuki, Madoka Higa, Kaori Tabata, Makoto Sawada

JOURNAL OF NEURAL TRANSMISSION Vol. 121 ( 3 ) page： 245 - 257 2014.3

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

Astrocytes modify and maintain neural activity and functions via gliotransmitter release such as, glutamate. They also change their properties and functions in response to alterations of ion environment resulting from neurotransmission; however, the direct evidence for whether intracellular ion alteration in astrocytes triggers gliotransmitter release is not indicated. Recent studies have reported that channelrhodopsin-2 (ChR2) is useful for alteration of intracellular ion environment in several types of cells with blue light exposure. Here, we show that ChR2-expressing GL261 (GLChR2) cells, clonal astrocytes, change their properties by photo-activation. Increased intracellular sodium and calcium ion concentrations and an altered membrane potential were observed in GLChR2 cells with blue light exposure. Alterations in the intracellular ion environment caused intracellular acidification and the inhibition of proliferation. In addition, it triggered glutamate release from GLChR2 cells. Glutamate from GLChR2 cells acted on N18 cells, clonal neuronal cells, as both a transmitter and neurotoxin depending on photo-activation. Our results show that the properties of ChR2-expressing astrocytes can be controlled by blue light exposure, and cation influx through photo-activated ChR2 might trigger functional cation influx via endogenous channels and result in the increase of glutamate release. Further, our results suggest that ChR2-expressing glial cells could become a useful tool in understanding the roles of glial cell activation and neural communication in the regulation of brain functions.

DOI： 10.1007/s00702-013-1096-8

New Fluorescent Probes Targeting the Mitochondrial-Located Translocator Protein 18 kDa (TSPO) as Activated Microglia Imaging Agents Reviewed

Detection of cytotoxic activation of microglia in a rat neuroinflammation model using a novel translocator protein ligand, [18F]FEPPA Invited Reviewed

Nomura, M; Toyama, H; Suzuki, H; Yamada, T; Ota, S; Ichise, M; Wilson, A; Sawada, M; Ito, K; Hatano, K

JOURNAL OF NUCLEAR MEDICINE Vol. 53 2012.5

Detection of cytotoxic activation of microglia in a rat neuroinflammation model using a novel translocator protein ligand, [18F]FEPPA Reviewed

Nomura,M, Toyama,H, Suzuki,H, Yamada,T, Ota,S, Ichise,M, Wilson,AA, Sawada,M, Ito,K, Hatano,K

J Nucl Med Vol. 53(Supplement 1) page： 1884 2012

New fluorescent probes targeting the mitochondrial-located translocator protein 18 kDa (TSPO) as activated microglia imaging agents. Reviewed

DENORA N, LAQUINTANA V, TRAPANI A, SUZUKI H, SAWADA M, TRAPANI G

Pharmaceutical Research Vol. 28 ( 11 ) page： 2820-2832 2011.11

Nunzio Denora, Valentino Laquintana, Adriana Trapani, Hiromi Suzuki, Makoto Sawada, Giuseppe Trapani

PHARMACEUTICAL RESEARCH Vol. 28 ( 11 ) page： 2820 - 2832 2011.11

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER/PLENUM PUBLISHERS

To evaluate the utility of new Translocator protein 18 kDa (TSPO)-targeted fluorescent probes for in vivo molecular imaging of activated microglia.
Compounds 2-4 were synthesized; their stability and affinity for TSPO were determined. Compounds 2-4 were incubated both with Ra2 cells in the presence of LPS, a potent activator of microglia, and with tissue sections of normal and chemically injured brains. Compounds 2-4 were injected into carotid artery or directly in striatum of mice. Cells and tissue sections from these in vitro and in vivo studies were observed by fluorescence microscopy after histochemical treatments.
Compounds 2-4 are stable in both buffer and physiological medium and showed high affinity for TSPO and were found to stain live Ra2 microglial cells effectively. Double staining with Mito Tracker Red suggested that binding sites of compounds 2 and 3 may exist on mitochondria. In vivo studies showed that compounds 2-4 may penetrate in part into brain; moreover, cells in mouse striatum were stained with compounds 2-4 and microglial marker CD11b.
Compounds 2-4 can fluorescently label activated microglia in vitro and in vivo.

DOI： 10.1007/s11095-011-0552-0