Eiichi Tokuda

Dysregulation of intracellular copper homeostasis is common to transgenic mice expressing human mutant superoxide dismutase-1s regardless of their copper-binding abilities

Over 170 mutations in superoxide dismutase-1 (SOD1) have been linked to amyotrophic lateral sclerosis (ALS). The properties of SOD1 mutants differ considerably including copper-binding abilities. Nevertheless, they cause the same disease phenotype, suggesting a common neurotoxic pathway. We have previously reported that copper homeostasis is disturbed in spinal cords of SOD1(G93A) mice. However, it is unknown whether copper dyshomeostasis is induced by other SOD1 mutants. Using the additional mouse strains SOD1(G127insTGGG), SOD1(G85R), and SOD1(D90A), which express SOD1 mutants with different copper-binding abilities, we show that copper dyshomeostasis is common to SOD1 mutants. The SOD1 mutants shifted the copper trafficking systems toward copper accumulation in spinal cords of the mice. Copper contents bound to the SOD1 active site varied considerably between SOD1 mutants. Still, copper bound to other ligands in the spinal cord were markedly increased in all. Zinc was also increased, whereas there were no changes in magnesium, calcium, aluminum, manganese and iron. Further support for a role of copper dyshomeostasis in ALS was gained from results of pharmacological intervention. Ammonium tetrathiomolybdate (TTM), a copper chelating agent, prolonged survival and slowed the disease progression of SOD1(G93A) mice, even when the treatment was started after the disease onset. TTM markedly attenuated pathology, including the loss of motor neurons and axons, and atrophy of skeletal muscles. Additionally, TTM decreased amounts of SOD1 aggregates. We propose that pharmacological agents that are capable of modulating copper dyshomeostasis, such as TTM, might be beneficial for the treatment of ALS caused by SOD1 mutations.

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

Microsatellite expansion disorders are pathologically characterized by RNA foci formation and repeat-associated non-AUG (RAN) translation. However, their underlying pathomechanisms and regulation of RAN translation remain unknown. We report that expression of expanded UGGAA (UGGAAexp) repeats, responsible for spinocerebellar ataxia type 31 (SCA31) in Drosophila, causes neurodegeneration accompanied by accumulation of UGGAAexp RNA foci and translation of repeat-associated pentapeptide repeat (PPR) proteins, consistent with observations in SCA31 patient brains. We revealed that motor-neuron disease (MND)-linked RNA-binding proteins (RBPs), TDP-43, FUS, and hnRNPA2B1, bind to and induce structural alteration of UGGAAexp. These RBPs suppress UGGAAexp-mediated toxicity in Drosophila by functioning as RNA chaperones for proper UGGAAexp folding and regulation of PPR translation. Furthermore, nontoxic short UGGAA repeat RNA suppressed mutated RBP aggregation and toxicity in MND Drosophila models. Thus, functional crosstalk of the RNA/RBP network regulates their own quality and balance, suggesting convergence of pathomechanisms in microsatellite expansion disorders and RBP proteinopathies.

Low autophagy capacity implicated in motor system vulnerability to mutant superoxide dismutase

IntroductionThe motor system is selectively vulnerable to mutations in the ubiquitously expressed aggregation-prone enzyme superoxide dismutase-1 (SOD1).ResultsAutophagy clears aggregates, and factors involved in the process were analyzed in multiple areas of the CNS from human control subjects (n = 10) and amyotrophic lateral sclerosis (ALS) patients (n = 18) with or without SOD1 mutations. In control subjects, the key regulatory protein Beclin 1 and downstream factors were remarkably scarce in spinal motor areas. In ALS patients, there was evidence of moderate autophagy activation and also dysregulation. These changes were largest in SOD1 mutation carriers. To explore consequences of low autophagy capacity, effects of a heterozygous deletion of Beclin 1 were examined in ALS mouse models expressing mutant SOD1s. This caused earlier SOD1 aggregation, onset of symptoms, motor neuron loss, and a markedly shortened survival. In contrast, the levels of soluble misfolded SOD1 species were reduced.ConclusionsThe findings suggest that an inherent low autophagy capacity might cause the vulnerability of the motor system, and that SOD1 aggregation plays a crucial role in the pathogenesis.

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

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease affecting both upper and lower motor neurons, and currently, there is no cure or effective treatment. Mutations in a gene encoding a ubiquitous antioxidant enzyme, Cu,Zn-superoxide dismutase (SOD1), have been first identified as a cause of familial forms of ALS. It is widely accepted that mutant SOD1 proteins cause the disease through a gain in toxicity but not through a loss of its physiological function. SOD1 is a major copper-binding protein and regulates copper homeostasis in the cell; therefore, a toxicity of mutant SOD1 could arise from the disruption of copper homeostasis. In this review, we will briefly review recent studies implying roles of copper homeostasis in the pathogenesis of SOD1-ALS and highlight the therapeutic interventions focusing on pharmacological as well as genetic regulations of copper homeostasis to modify the pathological process in SOD1-ALS.

Screening of Drugs Inhibiting In vitro Oligomerization of Cu/Zn-Superoxide Dismutase with a Mutation Causing Amyotrophic Lateral Sclerosis

Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene have been shown to cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS). A major pathological hallmark of this disease 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. Recently, we have reproduced the formation of SOD1 oligomers abnormally cross-linked via disulfide bonds in a test tube. Using our in vitro model of SOD1 oligomerization, therefore, 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.

Immunochemical characterization on pathological oligomers of mutant Cu/Zn-superoxide dismutase in amyotrophic lateral sclerosis.

BackgroundDominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS) with accumulation of misfolded SOD1 proteins as intracellular inclusions in spinal motor neurons. Oligomerization of SOD1 via abnormal disulfide crosslinks has been proposed as one of the misfolding pathways occurring in mutant SOD1; however, the pathological relevance of such oligomerization in the SOD1-ALS cases still remains obscure.MethodsWe prepared antibodies exclusively recognizing the SOD1 oligomers cross-linked via disulfide bonds in vitro. By using those antibodies, immunohistochemical examination and ELISA were mainly performed on the tissue samples of transgenic mice expressing mutant SOD1 proteins and also of human SOD1-ALS cases.ResultsWe showed the recognition specificity of our antibodies exclusively toward the disulfide-crosslinked SOD1 oligomers by ELISA using various forms of purified SOD1 proteins in conformationally distinct states in vitro. Furthermore, the epitope of those antibodies was buried and inaccessible in the natively folded structure of SOD1. The antibodies were then found to specifically detect the pathological SOD1 species in the spinal motor neurons of the SOD1-ALS patients as well as the transgenic model mice.ConclusionsOur findings here suggest that the SOD1 oligomerization through the disulfide-crosslinking associates with exposure of the SOD1 structural interior and is a pathological process occurring in the SOD1-ALS cases.

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

Cu/Zn-superoxide dismutase (SOD1) is an enzyme that disproportionates superoxide anion into hydrogen peroxide and molecular oxygen. The enzymatic activity of SOD1 requires the binding of copper and zinc ions and also the formation of a conserved intramolecular disulfide bond. In a eukaryotic cell, a copper chaperone for SOD1 (CCS) has been known to supply a copper ion and also introduce the disulfide bond into SOD1; however, a mechanism controlling the CCS-dependent activation of SOD1 remains obscure. Here, we characterized CCS isolated from a human liver fluke, Clonorchis sinensis, and found that an N-terminal domain of CCS was essential in supplying a copper ion in SOD1. Regardless of the presence and absence of the N-terminal domain, CCS was able to bind a cuprous ion at the CxC motif of its C-terminal domain with quite high affinity (Kd~10-17). The copper-bound form of full-length CCS successfully activated C. sinensis SOD1, but that of CCS lacking the N-terminal domain did not. Nonetheless, the N-terminally truncated CCS with the bound copper ion was found to correctly introduce the disulfide bond into SOD1. Based upon these results, we propose that the N-terminal domain of CCS has roles in the release of the copper ion bound at the C-terminal domain of CCS to SOD1.

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

Misfolding of mutant Cu/Zn‐superoxide dismutase (SOD1) is a pathological hallmark in a familial form of amyotrophic lateral sclerosis. Pathogenic mutations have been proposed to monomerize SOD1 normally adopting a homodimeric configuration and then trigger abnormal oligomerization of SOD1 proteins. Despite this, a misfolded conformation of SOD1 leading to the oligomerization at physiological conditions still remains ambiguous. Here, we show that, around the body temperature (∼37°C), mutant SOD1 maintains a dimeric configuration but lacks most of its secondary structures. Also, such an abnormal SOD1 dimer with significant structural disorder was prone to irreversibly forming the oligomers crosslinked via disulfide bonds. The disulfide‐crosslinked oligomers of SOD1 were detected in the spinal cords of the diseased mice expressing mutant SOD1. We hence propose an alternative pathway of mutant SOD1 misfolding that is responsible for oligomerization in the pathologies of the disease.

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

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.

Abnormal protein oligomers for neurodegeneration

Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease) is a neurodegenerative disease that causes the death of motor neurons controlling voluntary muscle movement. After the appearance of early symptoms such as muscle weakness/stiffness, patients with ALS suffer from progressive muscular paralysis and usually die from respiratory failure within 2 to 5 years. Riluzole and Edaravone are the FDA-approved drugs that could prolong ALS survival; however, there is still no known effective cure/prevention for this devastating disease. Most of the ALS cases are sporadic without any family histories, but an increasing number of genetic factors has been recently identified in the inherited form of the disease and contributes to our understanding on the pathomechanism of ALS [1]. Among those factors, SOD1 encoding the protein, Cu/Zn-superoxide dismutase, is the first identified gene causative for inherited ALS [2]. Transgenic rodents expressing human SOD1 with mutations are well known to recapitulate the progressive and selective degeneration of motor neurons with ALSlike symptoms [3]. Experimental results in vitro and in vivo have hence been significantly accumulated on pathogenic roles of mutant SOD1 proteins, based upon which mutant SOD1 proteins are proposed to exert toxicities through their “misfolding” into the non-native conformations. Despite such extensive research on SOD1 and ALS, however, it still remains obscure how mutant SOD1 becomes misfolded in the pathological conditions in vivo. Native SOD1 binds copper and zinc ions and also forms an intramolecular disulfide bond (Figure 1A), and these post-translational factors bestow incredibly high structural stability on the natively folded conformation of the protein (Tm ~ 90 oC). In contrast, ALS-causing mutations significantly destabilize the folded structure of SOD1 and facilitate its misfolding into non-native conformations [4, 5]. Among various non-native conformations that depend upon distinct experimental conditions, we have thus far proposed that the SOD1 oligomers cross-linked via disulfide bonds (called S-S oligomers) play pathological roles in SOD1-related ALS [6, 7]. SOD1 has four Cys residues of total, among which Cys57 and Cys146 usually form the intramolecular disulfide bond (Figure 1A). Mutant SOD1, in contrast, loses the natively folded structure at physiological temperatures (~37 oC), which we have found facilitates the “shuffling” of the disulfide bond [4, 7]. Namely, the remaining two Cys residues (Cys6 and Cys111) are located away from the Cys57-Cys146 disulfide bond in the folded conformation, but the mutation-induced structural disorder allows Cys6/111 to nucleophilically attack either Cys57 or Cys146, resulting in the disulfide shuffling among the four Cys residues in SOD1 (Figure 1B). We have then considered that the S-S oligomers form when the disulfide shuffling occurs between SOD1 molecules. Formation of the S-S oligomers was well reproduced in a test tube, and also, we previously detected the S-S oligomers specifically in the spinal cords of symptomatic ALS model mice [6]. We thus attempted to further test if the S-S oligomers do form in the human SOD1-related ALS cases. Unlike in the transgenic mice overexpressing mutant SOD1 proteins, nonetheless, it is expected to be difficult to biochemically characterize the S-S oligomers in human tissues, mainly because most of the spinal motor neurons in ALS patients are degenerated and usually absent at autopsies. In order to detect a trace amount, if any, of the S-S oligomers in ALS patients, therefore, we developed Editorial