Original Paper 31
Anzai I, Toichi K, Tokuda E, Mukaiyama A, AKiyama S, and Furukawa Y*
“Screening of drugs inhibiting in vitro oligomerization of Cu/Zn-superoxide dismutase with a mutation causing amyotrophic lateral sclerosis”
Frontiers in Molecular Biosciences 2016 Vol.3 Article 40

A major pathological hallmark of SOD1-ALS is abnormal accumulation of mutant SOD1 oligomers in the affected spinal motor neurons. While no effective therapeutics for SOD1-ALS is currently available, SOD1 oligomerization will be a good target for developing cures of this disease. In this paper, we screened 640 FDA-approved drugs for inhibiting the oligomerization of SOD1 proteins, and three effective classes of chemical compounds were identified. Those hit compounds will provide valuable information on the chemical structures for developing a novel drug candidate suppressing the abnormal oligomerization of mutant SOD1 and possibly curing the disease.


Review Paper 5
Tokuda E and Furukawa Y
“Copper Homeostasis as a Therapeutic Target in Amyotrophic Lateral Sclerosis with SOD1 Mutations”
International Journal of Molecular Sciences 2016 Vol.17 636 

It has long been suggested that intracellular metabolism of copper ions is important in understanding the pathomechanism of ALS, because one of the causative gene for ALS is coding Cu/Zn-superoxide dismutase, a copper binding protein.  Dr. Tokuda has recently characterized abnormal accumulation of copper ions in spinal cord of mice expressing mutant SOD1.  Besides, administration of a certain form of copper complex has been reported to be ameliorating the disease phenotype.  We thus review in this report the current status of the pathological and pharmacological relationship between copper and SOD1-related ALS.


Original Paper 30
Furukawa Y*, Anzai I, Akiyama S, Imai M, Cruz FJC, Saio T, Nagasawa K, Nomura T, and Ishimori K
“Conformational Disorder of the Most Immature Cu,Zn-Superoxide Dismutase Leading to Amyotrophic Lateral Sclerosis”
The Journal of Biological Chemistry 2016 Vol.291 pp.4144-4155

In this study, which was a nice collaborative work with Prof. Akiyama group in IMS, we examined the conformation(s) of misfolded SOD1 by small-angle X-ray scattering technique as well as several spectroscopic methods. The misfolded conformations were quite distinct from those of the native one and characterized by significant disorder at the loop regions. Based upon the results of this study, the drugs suppressing the loop disorder will be developed and tested for their efficacy curing ALS.

(SOD1タンパク質のミスフォールディング (構造異常)は神経変性疾患ALSに見られる主要な病理学的変化ですが、どのように構造が異常となるのかは議論の的です。私たちは、分子科学研究所の秋山さんとの共同研究で、X線小角散乱という実験手法を用いて、ミスフォールド型 SOD1の構造的特徴を捉えました。天然構造とは大きく異なり、特に、ループと呼ばれる構造部位が大きく異なっていることを明らかにしました。)

Original Paper 29
Furukawa Y*, Suzuki Y, Fukuoka M, Nagasawa K, Nakagome K, Shimizu H, Mukaiyama A, and Akiyama S 
“A Molecular Mechanism Realizing Sequence-specific Recognition of Nucleic Acids by TDP-43”
Scientific Reports 2016 Vol.6 20576 

In this study, a molecular mechanism of how TDP-43 protein recognizes nucleotides with specific sequences has been proposed.  TDP-43 is a DNA/RNA-binding protein, and its misfolding has been reported in ALS. TDP-43 has two RNA-recognition motifs (RRMs) in its sequence.  We have found that the spatial arrangement of those two RRMs is required for sequence-specific binding of RNA.  Misfolding of TDP-43 affecting the arrangement of RRMs will be pathogenic for ALS. 

(TDP-43と呼ばれるRNA/DNA結合 タンパク質は、SOD1と同様に、神経変性疾患ALSの原因となりうるタンパク質として知られています。 TDP-43は、ある特定の塩基配列のRNAだけを認識することが知られていますが、なぜ、認識配列に特異性が出てくるのか明らかではありませんでし た。私たちは、TDP-43が有する二つのRNA結合部位に着目し、それらの空間的な配置が特異性の発現を制御していることを明らかにしました。)

Original Paper 28
Ogawa M, Shidara H, Oka K, Kurosawa M, Nukina N, and Furukawa Y
“Cysteine residues in Cu,Zn-superoxide dismutase are essential to toxicity in Caenorhabditis elegans model of amyotrophic lateral sclerosis”
Biochemical and Biophysical Research Communications 2015 Vol.463 pp.1196-1202 

Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) cause a familial form of amyotrophic lateral sclerosis (ALS). A pathological hallmark of the familial ALS is the formation of mutant SOD1 aggregates, leading to the proposal that SOD1 gains toxicities through protein misfolding triggered by mutations. Nevertheless, molecular requirements for mutant SOD1 to acquire pathogenicity still remain obscure. Here, we show that Cys residues in SOD1 are essential to exerting toxicities of SOD1 in a Caenorhabditis elegans model. Exogenous expression of wild-type as well as pathogenic mutant SOD1 fused with a fluorescent protein in C. elegans resulted in the accumulation of disulfide-reduced SOD1 and retarded the worm’s motility. In contrast, little effects of exogenously expressed SOD1 on the motility were observed when all four Cys residues in SOD1 were replaced with Ser. Taken together, we propose that deregulation of Cys chemistry in SOD1 proteins is involved in the pathogenesis of SOD1-related ALS.

(私たちの主要な研究テーマでもある筋萎縮性側索硬化症ALSでは、一部の家族性ALSがSOD1をコードする遺伝子の変異によって引き起こされます。変異型SOD1タンパク質がどのようにALSを発症させるのか、未だに明らかとなっていません。しかし、変異に伴うSOD1の構造異常が神経細胞に毒性をもたらすのではないかと考えられています。特に、私たちの研究室では、SOD1が持っているシステイン残基に着目して、SOD1の構造異常とALS発症の関係を明らかにしようとしています。本研究では、SOD1が持っている4つ全てのシステイン残基をセリンに置換したCys-less SOD1を線虫の神経細胞に発現させて、その毒性を評価しました。システイン残基を持っている通常のSOD1を過剰に発現させると、線虫の運動性が低下するのに対して、 Cys-less SOD1を発現させても線虫の運動性は低下しませんでした。つまり、今回の研究から、SOD1のCys残基が神経細胞に対する毒性の発現に重要な役割を果たしていることがわかりました。) 

Original Paper 27
Sakurai Y, Anzai I, and Furukawa Y
“A Primary Role for Disulfide Formation in the Productive Folding of Prokaryotic Cu,Zn-superoxide dismutase”
The Journal of Biological Chemistry 2014 Vol.289 pp.20139-20149 

SOD1, a main target of our research, is an antioxidant enzyme that can remove a reactive oxygen species, superoxide anion. Given its pathological roles in neurodegenerative diseases, human SOD1 have been extensively studied by many groups including us. Also notably, bacteria have SOD1 proteins to cope with their oxidatively stressful environment. For example, bacteria that attempt to infect our bodies will be recognized by macrophages, and macrophages generate superoxide anions to kill the infecting bacteria. In the absence of SOD1 activity, therefore, bacteria will succumb to the macrophage attack. In this study, we have proposed that the antioxidant activity of SOD1 is up-regulated under the oxidative environment through formation of the disulfide bond in SOD1. 


Review Paper 4
Ogawa M and Furukawa Y
“A seeded propagation of Cu,Zn-superoxide dismutase aggregates in amyotrophic lateral sclerosis”
Frontiers in CELLULAR NEUROSCIENCE 2014 Vol.8 Article 83

Protein fibrillar aggregates exhibit a self-perpetuating property, which can convert a soluble native protein into insoluble fibrillar aggregates. Such “seeding reaction” of protein fibrils can accelerate the aggregation significantly and would contribute to the spread of inclusion pathologies from an affected cell to its neighboring cells in neurodegenerative diseases. In ALS, a pathological change first occurs at the site of disease onset and then propagates throughout the affected tissues in a time-dependent manner; therefore, it can be assumed that seeded aggregation may be the key factor of disease progression in ALS. In this mini review, we will briefly summarize recent studies on possible roles of a seeded aggregation of SOD1 in pathomechanism of ALS. 

(タンパク質の線維状凝集体には「シード」機能を有していることがあります。 シードとは、大きな結晶を作るときに予め加えておく小さな結晶のことで、いわゆる種結晶のことを指します。 タンパク質が構造変化を起こして線維状に凝集すると、それらが「シード」として機能することで、溶液中の残りのタンパク質がどんどん線維化してしまうことがあります。 このようなタンパク質の線維化促進現象のことをシーディングと呼んだりしますが、近年では、このシーディングが各種の神経変性疾患の症状進行を制御しているのではないかと考えられています。 そこで、神経変性疾患の一種である筋萎縮性側索硬化症(ALS)に焦点を当て、ALSにおいて見られるタンパク質凝集のシーディングに関する研究をまとめてみました。)

Original Paper 26
Nomura T, Watanabe S, Kaneko K, Yamanaka K, Nukina N, and Furukawa Y*
“Intranuclear aggregation of mutant FUS/TLS as a molecular pathomechanism of amyotrophic lateral sclerosis”
The Journal of Biological Chemistry 2014 Vol.289 pp.1192-1202 

Dominant mutations in FUS/TLS cause a familial form of amyotrophic lateral sclerosis (fALS), where abnormal accumulation of mutant FUS proteins in cytoplasm has been observed as a major pathological change. Many of pathogenic mutations have been shown to deteriorate the nuclear localization signal in FUS and thereby facilitate cytoplasmic mislocalization of mutant proteins. Several other mutations, however, exhibit no effects on the nuclear localization of FUS in cultured cells, and their roles in the pathomechanism of fALS remain obscure. Here, we show that a pathogenic mutation, G156E, significantly increases the propensities for aggregation of FUS and propose that intranuclear aggregation of FUS triggered by a subset of pathogenic mutations is an alternative pathomechanism of FUS-related fALS diseases.

(私たちの研究室での主役であるSOD1以外にも、家族性の筋萎縮性側索硬化症(ALS)の原因タンパク質がいくつか同定されており、FUS/TLSもその一つです。FUS/TLSは核に存在するDNA/RNA結合タンパク質ですが、ALSの原因となるようなアミノ酸変異が生じると核に移行することができず、その機能を発揮できなくなると言われています。しかし、ALS変異のなかには、FUS/TLSの核移行に全く影響を及ぼさないものもあり、なぜALS につながるのか明らかではありませんでした。私たちは、核移行に影響を及ぼさないものの、FUS/TLSの凝集性を増大させるALS変異があることを見いだしました。変異型FUS/TLSが核内で凝集するために、その生理機能がうまく発揮できなくなって病気になるのかもしれません。)