Papers - Furukawa, Yoshiaki

Does wild-type Cu/Zn-superoxide dismutase have pathogenic roles in amyotrophic lateral sclerosis?

Yoshiaki Furukawa, Eiichi Tokuda

Translational Neurodegeneration (Springer Science and Business Media LLC)  9 ( 1 )  2020.08

Accepted

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Amyotrophic lateral sclerosis (ALS) is characterized by adult-onset progressive degeneration of upper and lower motor neurons. Increasing numbers of genes are found to be associated with ALS; among those, the first identified gene, SOD1 coding a Cu/Zn-superoxide dismutase protein (SOD1), has been regarded as the gold standard in the research on a pathomechanism of ALS. Abnormal accumulation of misfolded SOD1 in affected spinal motor neurons has been established as a pathological hallmark of ALS caused by mutations in SOD1 (SOD1-ALS). Nonetheless, involvement of wild-type SOD1 remains quite controversial in the pathology of ALS with no SOD1 mutations (non-SOD1 ALS), which occupies more than 90% of total ALS cases. In vitro studies have revealed post-translationally controlled misfolding and aggregation of wild-type as well as of mutant SOD1 proteins; therefore, SOD1 proteins could be a therapeutic target not only in SOD1-ALS but also in more prevailing cases, non-SOD1 ALS. In order to search for evidence on misfolding and aggregation of wild-type SOD1 in vivo, we reviewed pathological studies using mouse models and patients and then summarized arguments for and against possible involvement of wild-type SOD1 in non-SOD1 ALS as well as in SOD1-ALS.

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Oxidative misfolding of Cu/Zn-superoxide dismutase triggered by non-canonical intramolecular disulfide formation.

Itsuki Anzai, Eiichi Tokuda, Sumika Handa, Hidemi Misawa, Shuji Akiyama, Yoshiaki Furukawa

Free radical biology & medicine (Free Radical Biology and Medicine)  147   187 - 199 2020.02

Accepted,  ISSN  08915849

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Misfolded Cu/Zn-superoxide dismutase (SOD1) is a pathological species in a subset of amyotrophic lateral sclerosis (ALS). Oxidative stress is known to increase in affected spinal cords of ALS and is thus considered to cause damages on SOD1 leading to the misfolding and aggregation. Despite this, it still remains elusive what triggers misfolding of SOD1 under oxidizing environment. Here, we show that a thiol group of Cys111 in SOD1 is oxidized to a sulfenic acid with hydrogen peroxide and reveal that further dissociation of the bound metal ions from the oxidized SOD1 allows another free Cys residue (Cys6) to nucleophilically attack the sulfenylated Cys111. As a result, an intra-molecular disulfide bond forms between Cys6 and Cys111. Such an abnormal SOD1 with the non-canonical disulfide bond was conformationally extended with significant cytotoxicity as well as high propensity to aggregate. Taken together, we propose a new model of SOD1 misfolding under oxidizing environment, in which formation of the non-canonical intramolecular disulfide bond plays a pivotal role.

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Wild-type Cu/Zn-superoxide dismutase is misfolded in cerebrospinal fluid of sporadic amyotrophic lateral sclerosis.

Eiichi Tokuda, Yo-Ichi Takei, Shinji Ohara, Noriko Fujiwara, Isao Hozumi, Yoshiaki Furukawa

Molecular neurodegeneration (Molecular Neurodegeneration)  14 ( 1 ) 42 - 42 2019.11

Accepted

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BACKGROUND: A subset of familial forms of amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene coding Cu/Zn-superoxide dismutase (SOD1). Mutant SOD1 proteins are susceptible to misfolding and abnormally accumulated in spinal cord, which is most severely affected in ALS. It, however, remains quite controversial whether misfolding of wild-type SOD1 is involved in more prevalent sporadic ALS (sALS) cases without SOD1 mutations. METHODS: Cerebrospinal fluid (CSF) from patients including sALS as well as several other neurodegenerative diseases and non-neurodegenerative diseases was examined with an immunoprecipitation assay and a sandwich ELISA using antibodies specifically recognizing misfolded SOD1. RESULTS: We found that wild-type SOD1 was misfolded in CSF from all sALS cases examined in this study. The misfolded SOD1 was also detected in CSF from a subset of Parkinson's disease and progressive supranuclear palsy, albeit with smaller amounts than those in sALS. Furthermore, the CSF samples containing the misfolded SOD1 exhibited significant toxicity toward motor neuron-like NSC-34 cells, which was ameliorated by removal of the misfolded wild-type SOD1 with immunoprecipitation. CONCLUSIONS: Taken together, we propose that misfolding of wild-type SOD1 in CSF is a common pathological process of ALS cases regardless of SOD1 mutations.

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Sequence- and seed-structure-dependent polymorphic fibrils of alpha-synuclein.

Tanaka G, Yamanaka T, Furukawa Y, Kajimura N, Mitsuoka K, Nukina N

Biochimica et biophysica acta. Molecular basis of disease (Biochimica et Biophysica Acta - Molecular Basis of Disease)  1865 ( 6 ) 1410 - 1420 2019.06

Accepted,  ISSN  09254439

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Synucleinopathies comprise a diverse group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. These share a common pathological feature, the deposition of alpha-synuclein (a-syn) in neurons or oligodendroglia. A-syn is highly conserved in vertebrates, but the primary sequence of mouse a-syn differs from that of human at seven positions. However, structural differences of their aggregates remain to be fully characterized. In this study, we found that human and mouse a-syn aggregated in vitro formed morphologically distinct amyloid fibrils exhibiting twisted and straight structures, respectively. Furthermore, we identified different protease-resistant core regions, long and short, in human and mouse a-syn aggregates. Interestingly, among the seven unconserved amino acids, only A53T substitution, one of the familial PD mutations, was responsible for structural conversion to the straight-type. Finally, we checked whether the structural differences are transmissible by seeding and found that human a-syn seeded with A53T aggregates formed straight-type fibrils with short protease-resistant cores. These results suggest that a-syn aggregates form sequence-dependent polymorphic fibrils upon spontaneous aggregation but become seed structure-dependent upon seeding.

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Biochemical and morphological classification of disease-associated alpha-synuclein mutants aggregates.

Tanaka G, Yamanaka T, Furukawa Y, Kajimura N, Mitsuoka K, Nukina N

Biochemical and biophysical research communications (Biochemical and Biophysical Research Communications)  508 ( 3 ) 729 - 734 2019.01

Accepted,  ISSN  0006291X

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Alpha-synuclein (a-syn) aggregation in brain is implicated in several synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Until date, at least six disease-associated mutations in a-syn (namely A30P, E46K, H50Q, G51D, A53T, and A53E) are known to cause dominantly inherited familial forms of synucleinopathies. Previous studies using recombinant proteins have reported that a subset of disease-associated mutants show higher aggregation propensities and form spectroscopically distinguishable aggregates compared to wild-type (WT). However, morphological and biochemical comparison of the aggregates for all disease-associated a-syn mutants have not yet been performed. In this study, we performed electron microscopic examination, guanidinium hydrochloride (GdnHCl) denaturation, and protease digestion to classify the aggregates from their respective point mutations. Using electron microscopy we observed variations of amyloid fibrillar morphologies among the aggregates of a-syn mutants, mainly categorized into two groups: twisted fibrils observed for both WT and E46K while straight fibrils for the other mutants. GdnHCl denaturation experiments revealed the a-syn mutants except for E46K were more resistant than WT against the denaturation. Mass spectrometry analysis of protease-treated aggregates showed a variety of protease-resistant cores, which may correspond to their morphological properties. The difference of their properties could be implicated in the clinicopathological difference of synucleinopathies with those mutations.

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Prion-like properties of misfolded Cu/Zn-superoxide dismutase in amyotrophic lateral sclerosis: Update and perspectives

Tokuda E., Marklund S., Furukawa Y.

Yakugaku Zasshi (Yakugaku Zasshi)  139 ( 7 ) 1015 - 1019 2019

ISSN  00316903

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© 2019 The Pharmaceutical Society of Japan. Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease that is characterized by the loss of motor neurons, which results in progressive muscle atrophy. The pathology spreads from the initial site of onset to contiguous anatomic regions. Mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1) have been identified in a dominantly inherited form of ALS (ALS-SOD1). A major hallmark of ALS-SOD1 is the abnormal accumulation of conformationally aberrant SOD1 protein (i.e., misfolded SOD1) within motor neurons. Emerging experimental evidence has suggested that misfolded proteins associated with neurodegenerative diseases exhibit prion-like properties, i.e., misfolded proteins act as conformational templates that convert normal proteins into a pathogenic form. Possibly as a result of this prion-like self-propagation property, misfolded forms of pathological proteins are considered to accumulate in the central nervous system and cause neurodegeneration. In this article, we review recent evidence for the role of prion-like mechanisms in ALS-SOD1. In particular, we discuss the propensity of misfolded SOD1 to act as a pathological seed, spread between cells, and propagate neuroanatomically.

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Elimination of TDP-43 inclusions linked to amyotrophic lateral sclerosis by a misfolding-specific intrabody with dual proteolytic signals

Yoshitaka Tamaki, Akemi Shodai, Toshifumi Morimura, Ryota Hikiami, Sumio Minamiyama, Takashi Ayaki, Ikuo Tooyama, Yoshiaki Furukawa, Ryosuke Takahashi, Makoto Urushitani

Scientific Reports (Scientific Reports)  8 ( 1 ) 6030 2018.12

Accepted,  ISSN  2045-2322

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© 2018 The Author(s). Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is implicated in the pathogenesis of sporadic and certain familial forms of amyotrophic lateral sclerosis (ALS), suggesting elimination of TDP-43 aggregates as a possible therapeutic strategy. Here we generated and investigated a single-chain variable fragment (scFv) derived from the 3B12A monoclonal antibody (MAb) that recognises D247 of the TDP-43 nuclear export signal, an epitope masked in the physiological state. In transfected HEK293A cells, 3B12A scFv recapitulated the affinity of the full-length MAb to mislocalised TDP-43 with a defective nuclear localising signal and to a TDP-43 inclusion mimic with cysteine-to-serine substitution at RRM1. Moreover, 3B12A scFv accelerated proteasome-mediated degradation of aggregated TDP-43, likely due to an endogenous PEST-like proteolytic signal sequence in the VH domain CDR2 region. Addition of the chaperone-mediated autophagy (CMA)-related signal to 3B12A scFv induced HSP70 transcription, further enhancing TDP-43 aggregate clearance and cell viability. The 3B12A scFv also reduced TDP-43 aggregates in embryonic mouse brain following in utero electroporation while causing no overt postnatal brain pathology or developmental anomalies. These results suggest that a misfolding-specific intrabody prone to synergistic proteolysis by proteasomal and autophagic pathways is a promising strategy for mitigation of TDP-43 proteinopathy in ALS.

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Cu/Zn-superoxide dismutase forms fibrillar hydrogels in a pH-dependent manner via a water-rich extended intermediate state.

Fujiwara N, Wagatsuma M, Oba N, Yoshihara D, Tokuda E, Sakiyama H, Eguchi H, Ichihashi M, Furukawa Y, Inoue T, Suzuki K

PloS one (Public Library of Science (PLoS))  13 ( 10 ) e0205090 - e0205090 2018.10

Accepted

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© 2018 Fujiwara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins.

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Rapid dissemination of alpha-synuclein seeds through neural circuits in an in-vivo prion-like seeding experiment.

Ayami Okuzumi, Masaru Kurosawa, Taku Hatano, Masashi Takanashi, Shuuko Nojiri, Takeshi Fukuhara, Tomoyuki Yamanaka, Haruko Miyazaki, Saki Yoshinaga, Yoshiaki Furukawa, Tomomi Shimogori, Nobutaka Hattori, Nobuyuki Nukina

Acta neuropathologica communications (Acta neuropathologica communications)  6 ( 1 ) 96 - 96 2018.09

Accepted

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Accumulating evidence suggests that the lesions of Parkinson's disease (PD) expand due to transneuronal spreading of fibrils composed of misfolded alpha-synuclein (a-syn), over the course of 5-10 years. However, the precise mechanisms and the processes underlying the spread of these fibril seeds have not been clarified in vivo. Here, we investigated the speed of a-syn transmission, which has not been a focus of previous a-syn transmission experiments, and whether a-syn pathologies spread in a neural circuit-dependent manner in the mouse brain. We injected a-syn preformed fibrils (PFFs), which are seeds for the propagation of a-syn deposits, either before or after callosotomy, to disconnect bilateral hemispheric connections. In mice that underwent callosotomy before the injection, the propagation of a-syn pathology to the contralateral hemisphere was clearly reduced. In contrast, mice that underwent callosotomy 24 h after a-syn PFFs injection showed a-syn pathology similar to that seen in mice without callosotomy. These results suggest that a-syn seeds are rapidly disseminated through neuronal circuits immediately after seed injection, in a prion-like seeding experiment in vivo, although it is believed that clinical a-syn pathologies take years to spread throughout the brain. In addition, we found that botulinum toxin B blocked the transsynaptic transmission of a-syn seeds by specifically inactivating the synaptic vesicle fusion machinery. This study offers a novel concept regarding a-syn propagation, based on the Braak hypothesis, and also cautions that experimental transmission systems may be examining a unique type of transmission, which differs from the clinical disease state.

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Identification of a novel zinc-binding protein, C1orf123, as an interactor with a heavy metal-associated domain.

Furukawa Y, Lim C, Tosha T, Yoshida K, Hagai T, Akiyama S, Watanabe S, Nakagome K, Shiro Y

PloS one (PLoS ONE)  13 ( 9 ) e0204355 2018.09

Accepted

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© 2018 Furukawa et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Heavy metal-associated (HMA) domains bind metal ions at its Cys-x-x-Cys (CxxC) motif and constitute an intracellular network for trafficking of metal ions for utilization and detoxification. We thus expect that novel metalloproteins can be identified by screening proteins interacting with a HMA domain. In this study, we performed yeast two-hybrid screening of the human proteome and found an uncharacterized protein encoded as open reading frame 123 in chromosome 1 (C1orf123) that can interact specifically with the HMA domain of a copper chaperone for superoxide dismutase (CCSdI). Our X-ray structural analysis of C1orf123 further revealed that it binds a Zn 2+ ion in a tetrahedral coordination with four thiolate groups from two conserved CxxC motifs. For the interaction between C1orf123 and CCSdI, the CxxC motifs in both C1orf123 and CCSdI were required, implying metal-mediated interaction through the CxxC motifs. Notably, C1orf123 did not interact with several other HMA domains containing CxxC motifs, supporting high specificity in the interaction between C1orf123 and CCSdI. Based upon these results, we further discuss functional and structural significance of the interaction between C1orf123 and CCS.

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A copper-deficient form of mutant Cu/Zn-superoxide dismutase as an early pathological species in amyotrophic lateral sclerosis

Eiichi Tokuda, Takao Nomura, Shinji Ohara, Seiji Watanabe, Koji Yamanaka, Yuta Morisaki, Hidemi Misawa, Yoshiaki Furukawa

Biochimica et Biophysica Acta - Molecular Basis of Disease (Biochimica et Biophysica Acta - Molecular Basis of Disease)  1864 ( 6 ) 2119 - 2130 2018.06

Accepted,  ISSN  09254439

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© 2018 Elsevier B.V. Dominant mutations in the gene encoding copper and zinc-binding superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS). Abnormal accumulation of misfolded SOD1 proteins in spinal motoneurons is a major pathological hallmark in SOD1-related ALS. Dissociation of copper and/or zinc ions from SOD1 has been shown to trigger the protein aggregation/oligomerization in vitro, but the pathological contribution of such metal dissociation to the SOD1 misfolding still remains obscure. Here, we tested the relevance of the metal-deficient SOD1 in the misfolding in vivo by developing a novel antibody (anti-apoSOD), which exclusively recognized mutant SOD1 deficient in metal ions at its copper-binding site. Notably, anti-apoSOD-reactive species were detected specifically in the spinal cords of the ALS model mice only at their early pre-symptomatic stages but not at the end stage of the disease. The cerebrospinal fluid as well as the spinal cord homogenate of one SOD1-ALS patient also contained the anti-apoSOD-reactive species. Our results thus suggest that metal-deficiency in mutant SOD1 at its copper-binding site is one of the earliest pathological features in SOD1-ALS.

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Pathological roles of copper dynamics in amyotrophic lateral sclerosis

古川 良明

生化学 (日本生化学会)  90 ( 3 ) 311 - 319 2018

ISSN  00371017

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An essential role of N-terminal domain of copper chaperone in the enzymatic activation of Cu/Zn-superoxide dismutase

Mami Fukuoka, Eiichi Tokuda, Kenta Nakagome, Zhiliang Wu, Isao Nagano, Yoshiaki Furukawa

JOURNAL OF INORGANIC BIOCHEMISTRY 175   208 - 216 2017.10

Accepted,  ISSN  0162-0134

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Regulatory Role of RNA Chaperone TDP-43 for RNA Misfolding and Repeat-Associated Translation in SCA31

Taro Ishiguro, Nozomu Sato, Morio Ueyama, Nobuhiro Fujikake, Chantal Sellier, Akemi Kanegami, Eiichi Tokuda, Bita Zamiri, Terence Gall-Duncan, Mila Mirceta, Yoshiaki Furukawa, Takanori Yokota, Keiji Wada, J. Paul Taylor, Christopher E. Pearson, Nicolas Charlet-Berguerand, Hidehiro Mizusawa, Yoshitaka Nagai, Kinya Ishikawa

NEURON 94 ( 1 ) 108 - + 2017.04

Accepted,  ISSN  0896-6273

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A misfolded dimer of Cu/Zn-superoxide dismutase leading to pathological oligomerization in amyotrophic lateral sclerosis

Itsuki Anzai, Eiichi Tokuda, Atsushi Mukaiyama, Shuji Akiyama, Fumito Endo, Koji Yamanaka, Hidemi Misawa, Yoshiaki Furukawa

PROTEIN SCIENCE 26 ( 3 ) 484 - 496 2017.03

Accepted,  ISSN  0961-8368

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Immunochemical characterization on pathological oligomers of mutant Cu/Znsuperoxide dismutase in amyotrophic lateral sclerosis

Tokuda, Eiichi, Anzai, Itsuki, Nomura, Takao, Toichi, Keisuke, Watanabe, Masahiko, Ohara, Shinji, Watanabe, Seiji, Yamanaka, Koji, Morisaki, Yuta, Misawa, Hidemi, Furukawa, Yoshiaki

MOLECULAR NEURODEGENERATION 12 ( 1 ) 2 2017.01

Research paper (scientific journal), Accepted,  ISSN  1750-1326

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Aggregation of FET proteins as a pathological change in amyotrophic lateral sclerosis

Yoshiaki Furukawa, Eiichi Tokuda

Advances in Experimental Medicine and Biology (Springer New York LLC)  925   1 - 12 2017

Accepted,  ISSN  2214-8019

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Screening of drugs inhibiting in vitro oligomerization of Cu/Zn-superoxide dismutase with a mutation causing amyotrophic lateral sclerosis

Itsuki Anzai, Keisuke Toichi, Eiichi Tokuda, Atsushi Mukaiyama, Shuji Akiyama, Yoshiaki Furukawa

Frontiers in Molecular Biosciences (Frontiers Media S.A.)  3 ( AUG ) 40 2016.08

Accepted,  ISSN  2296-889X

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Copper Homeostasis as a Therapeutic Target in Amyotrophic Lateral Sclerosis with SOD1 Mutations

Tokuda, Eiichi, Furukawa, Yoshiaki

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 17 ( 5 ) 847 - 867 2016.05

Research paper (scientific journal), Accepted,  ISSN  1422-0067

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Conformational Disorder of the Most Immature Cu, Zn-Superoxide Dismutase Leading to Amyotrophic Lateral Sclerosis

Furukawa, Yoshiaki, Anzai, Itsuki, Akiyama, Shuji, Imai, Mizue, Cruz, Fatima Joy C., Saio, Tomohide, Nagasawa, Kenichi, Nomura, Takao, Ishimori, Koichiro

JOURNAL OF BIOLOGICAL CHEMISTRY 291 ( 8 ) 4144 - 4155 2016.02

Research paper (scientific journal),  ISSN  0021-9258

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