Shin Ichi Ono

Ammonium tetrathiomolybdate delays onset, prolongs survival, and slows progression of disease in a mouse model for amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) cause a form of familial amyotrophic lateral sclerosis (ALS). The pathogenesis of familial ALS may be associated with aberrant copper chemistry through a cysteine residue in mutant SOD1. Ammonium tetrathiomolybdate (TTM) is a copper-chelating drug that is capable of removing a copper ion from copper-thiolate clusters, such as SOD1. We found that TTM exerted therapeutic benefits in a mouse model of familial ALS (SOD1(G93A)). TTM treatment significantly delayed disease onset, slowed disease progression and prolonged survival by approximately 20%, 42% and 25%, respectively. TTM also effectively depressed the spinal copper ion level and inhibited lipid peroxidation, with a significant suppression of SOD1 enzymatic activity in SOD1(G93A). These results support the hypothesis that aberrant copper chemistry through a cysteine residue plays a critical role in mutant SOD1 toxicity and that TTM may be a promising therapy for familial ALS with SOD1 mutants.

Effects of gamma radiation on levels of brain metallothionein and lipid peroxidation in transgenic mice

The induction of metallothionein (MT) synthesis in the brain was investigated in MT-I isoform-overexpressing transgenic (MT-I*) and control mice after exposure to increasing doses of 2 to 20 Gy of whole-body gamma radiation. Although the MT-I isoform was the major isoform of MT in this transgenic mouse, the other isoforms, MT-II and MT-III, were also present in the brain. The total concentration of MT in the brain was measured by a cadmium-binding assay, while zinc and lipid peroxides were measured by atomic absorption spectrophotometry and by the thiobarbituric acid method, respectively. In MT-I* mice at 24 h after radiation exposure, the level of MT in the brain was increased from a basal level of 44.4 +/- 4.0 microg/g to a maximum level of 91.0 +/- 9.0 microg/g after 5 Gy and remained high after 10 and 20 Gy. In a time-course experiment with 5 Gy, the concentration of MT in the brain of MT-I* mice increased at 3 h and reached a maximum of 175.3 +/- 15.3 microg/g at 6 h. This high level of MT remained unchanged for 48 h after radiation exposure. Metallothionein was not induced markedly in the brains of control mice either at 24 h after exposure to 2-20 Gy gamma radiation or at different times after exposure to 5 Gy gamma radiation. In both strains of mice, the total concentration of zinc in the brain decreased with increasing radiation dose. No differences in lipid peroxide levels were seen in control mice exposed to 5 Gy at 6 and 12 h or after exposure to three other doses (2, 10 and 20 Gy) at 24 h. Slight increases (1.35 and 1.22, respectively) in lipid peroxide levels were observed in control mice at 24 and 48 h after exposure to 5 Gy. Lipid peroxide levels in the brain were not changed in irradiated MT-I* mice. The results show a marked increase in the levels of MT in the brain of transgenic mice after exposure to gamma radiation. The induced synthesis of MT may be only one of several mechanisms that prevent the induction of lipid peroxidation in the brain by gamma radiation.

Regional distribution of metallothionein, zinc, and copper in the brain of different strains of rats

The regional brain distribution of metallothionein (MT), zinc, and copper in the brain was determined in nine anatomical regions (olfactory bulb, cortex, corpus striatum, hippocampus, thalamus plus hypothalamus, pons plus medulla oblongata, cerebellum, midbrain, and white matter) and was compared between two different strains of rat (Sprague-Dawley [SD] and Lewis). No significant difference was observed in the whole-brain MT level between the two strains (17.8 ± 3.4 μg/g in SD rats and 20.3 ± 2.3 μg/g in Lewis rats). In SD rats, however, MT was more highly expressed in the white matter than in the other regions studied. In contrast, MT concentration was highest in the cortex and lowest in the olfactory bulb in Lewis rats. The MT levels in the cortex, corpus striatum, hippocampus, and thalamus plus hypothalamus were significantly lower in SD rats than in Lewis rats. In both strains, the olfactory bulb contained markedly higher levels of both zinc and copper than the other regions (27.9 ±6.8 μg/g zinc in SD rats and 27.6 ± 6.9 μg/g zinc in Lewis rats, and 5.2 ± 1.5 μg/g copper in SD rats and 11.1 ± 4.8 μg/g copper in Lewis rats). The next high-est zinc levels were seen in the hippocampus, whereas the next highest copper levels were in the corpus striatum in both SD and Lewis rats. The high levels of zinc and copper in the olfactory bulb were not accompanied by concomitant high MT concentrations. These results indicate that the strain of rat as well as the anatomical brain region should be taken into account in MT and metal distribution studies. However, the highest concentrations of zinc and copper in olfactory bulb were common to both SD and Lewis rats. The discrepancy between MT and the metal levels in olfactory bulb suggests a role for other proteins in addition to MT in the homeostatic control of zinc and copper.

Dysregulation of intracellular copper trafficking pathway in a mouse model of mutant copper/zinc superoxide dismutase‐linked familial amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) are responsible for 20% of familial amyotrophic lateral sclerosis through a gain‐of‐toxic function. We have recently shown that ammonium tetrathiomolybdate, an intracellular copper‐chelating reagent, has an excellent therapeutic benefit in a mouse model for amyotrophic lateral sclerosis. This finding suggests that mutant SOD1 might disrupt intracellular copper homeostasis. In this study, we investigated the effects of mutant SOD1 on the components of the copper trafficking pathway, which regulate intracellular copper homeostasis. We found that mutant, but not wild‐type, SOD1 shifts intracellular copper homeostasis toward copper accumulation in the spinal cord during disease progression: copper influx increases, copper chaperones are up‐regulated, and copper efflux decreases. This dysregulation was observed within spinal motor neurons and was proportionally associated with an age‐dependent increase in spinal copper ion levels. We also found that a subset of the copper trafficking pathway constituents co‐aggregated with mutant SOD1. These results indicate that the nature of mutant SOD1 toxicity might involve the dysregulation of the copper trafficking pathway, resulting in the disruption of intracellular copper homeostasis.

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.

Regional brain distribution of metallothionein, zinc and copper in toxic milk mutant and transgenic mice

The regional distribution of metallothionein (MT), zinc and copper was measured in brains of transgenic MT-I overexpressor (MT-I*) mice, MT-I/MT-II gene knockout (MT-I/MT-II null) mice, and in brains of control C57BL/6J mice with normal MT expression. Toxic milk (tx) mutant mice with abnormally high MT and copper accumulation were also assessed. Although there were significant differences in MT levels (assessed by a cadmium-binding assay) in whole brain of MT-I/MT-II null and control mice (16.5 +/- 2.9 microg/g vs 25.6 +/- 7.4 microg/g), different regions of the brain (cerebral cortex, corpus striatum, hippocampus, thalamus plus hypothalamus, cerebellum, and brain stem) contained similar amounts of MT. Male MT-I* mice had significantly higher whole brain MT level than controls (35.5 +/- 8.1 microg/g vs 25.6 +/- 7.4 microg/g), and had a 2-fold higher MT level in cerebellum, but not in other brain regions. Female MT-I* mice had significantly increased MT levels in all brain regions, with the highest increase in cerebellum (3.5-fold), and the lowest increase in cortex (2-fold). MT level in whole brain of female MT-I* mice was also significantly higher than that of male MT-I* (75.2 +/- 8.0 microg/g vs 35.4 +/- 8.1 microg/g). Toxic milk mice had significantly higher MT levels in all brain regions compared to age-matched controls (51.8 +/- 10.8 microg/g vs 30.3 +/- 5.8 microg/g), while no specific region of tx mouse brain showed a preferential increase in MT. In MT-I* and MT-I/MT-II null mice, altered MT levels did not always result in altered zinc and/or copper concentrations. However, all mouse strains exhibited region-specific accumulation of zinc, with the highest level in hippocampus. In control, MT-I/MT-II null, and male MT-I* mice, the hippocampus accumulated the highest level of copper. However, MT-I/MT-II null and both male and female MT-I* mice had similar levels of copper, compared to control mice. Toxic milk mice, on the other hand, had significantly higher copper levels in cerebral cortex, corpus striatum, thalamus/hypothalamus, and brain stem, compared to control mice. Zinc levels in corpus striatum, hippocampus, and cerebellum were also significantly increased. These data indicate that, in normal control and MT-I/MT-II null mice, MT is expressed uniformly in different regions of the brain. MT-I* mice, on the other hand, exhibit regional and gender-associated change in brain MT, and tx mice have markedly increased MT, copper, and zinc levels in most brain regions. These mouse strains will be useful models in elucidating the role of MT in the pathological effects of altered zinc and copper in brain.

Overexpression of metallothionein-I, a copper-regulating protein, attenuates intracellular copper dyshomeostasis and extends lifespan in a mouse model of amyotrophic lateral sclerosis caused by mutant superoxide dismutase-1

Over 170 mutations in superoxide dismutase-1 (SOD1) cause familial amyotrophic lateral sclerosis (ALS), a lethal motor neuron disease. Although the molecular properties of SOD1 mutants differ considerably, we have recently shown that intracellular copper dyshomeostasis is a common pathogenic feature of different SOD1 mutants. Thus, the potentiation of endogenous copper regulation could be a therapeutic strategy. In this study, we investigated the effects of the overexpression of metallothionein-I (MT-I), a major copper-regulating protein, on the disease course of a mouse model of ALS (SOD1(G93A)). Using double transgenic techniques, we found that the overexpression of MT-I in SOD1(G93A) mice significantly extended the lifespan and slowed disease progression, but the effects on disease onset were modest. Genetically induced MT-I normalized copper dyshomeostasis in the spinal cord without influencing SOD1 enzymatic activity. The overexpression of MT-I in SOD1(G93A) mice markedly attenuated the pathological features of the mice, including the death of motor neurons, the degeneration of ventral root axons, the atrophy of skeletal muscles, and the activation of glial cells. Double transgenic mice also showed a decreased level of SOD1 aggregates within the glial cells of the spinal cord. Furthermore, the overexpression of MT-I in SOD1(G93A) mice reduced the number of spheroid-shaped astrocytes cleaved by active caspase-3. We concluded that therapeutic strategies aimed at the potentiation of copper regulation by MT-I could be of benefit in cases of ALS caused by SOD1 mutations.

Dysequilibrium between caspases and their inhibitors in a mouse model for amyotrophic lateral sclerosis.

Mutations in copper/zinc superoxide dismutase (SOD1) have been implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 protein likely gains a novel cytotoxic property, leading to the death of motor neurons. We therefore investigated whether caspase-mediated apoptosis is associated with novel cytotoxic properties in a rodent model for familial ALS (G93A SOD1 transgenic mice). Caspase-9 (an effecter in the mitochondrial apoptotic pathway), caspase-8 (an effecter in the Fas apoptotic pathway), and caspase-3 (an executioner of both pathways) proteins were all present in nonactive forms in the spinal cords of wild-type mice during the early stage of the disease (8 weeks), at which time the mice had not yet exhibited motor paralysis. In transgenic mice, however, these proteins were present in their active forms, and their mRNA levels were significantly upregulated in the represent to this conversion from nonactive to active forms. During the advanced stage of the disease (16 weeks), when paralysis was evident, the active caspase levels were further elevated. On the other hand, the mRNA and protein levels of survivin, a counteraction protein against caspases, were significantly suppressed during the early stage, and sharply increased during the advanced stage. Although the mRNA and protein levels of X-linked inhibitor of apoptosis protein (XIAP) remained at the same levels as those seen in the control (wild-type mice) during the early stage, they were significantly depressed at an age of 16 weeks. These findings were observed exclusively in the spinal cord, the region responsible for the disease, and not in the cerebellum, a non-responsible region. We conclude that conditions facilitating the apoptotic process during the early stage of the disease play causative roles in the pathogenesis of ALS and that the suppression of XIAP levels during the advanced stage could contribute to disease expression and/or progression.

Comparative study of hydrogen peroxide- and 4-hydroxy-2-nonenal-induced cell death in HT22 cells

Several studies have indicated that lipid peroxidation often occurs in response to oxidative stress, and that many aldehydic products including 4-hydroxy-2-nonenal (HNE) are formed when lipid hydroperoxides break down. In order to clarify the mechanism of oxidative stress-induced neuronal death in the nervous system, we investigated H(2)O(2)- and HNE-induced cell death pathways in HT22 cells, a mouse hippocampal cell line, under the same experimental conditions. Treatment with H(2)O(2) and HNE decreased the viability of these cells in a time- and concentration-dependent manner. In the cells treated with H(2)O(2), significant increases in the immunoreactivities of DJ-1 and nuclear factor-kappaB (NF-kappaB) subunits (p65 and p50) were observed in the nuclear fraction. H(2)O(2) also induced an increase in the intracellular concentration of Ca(2+), and cobalt chloride (CoCl(2)), a Ca(2+) channel inhibitor, suppressed the H(2)O(2)-induced cell death. In HNE-treated cells, none of these phenomena were observed; however, HNE adduct proteins were formed after exposure to HNE, but not to H(2)O(2). N-Acetyl-L-cysteine (NAC) suppressed both HNE-induced cell death and HNE-induced expression of HNE adduct proteins, whereas H(2)O(2)-induced cell death was not affected. These findings suggest that the mechanisms of cell death induced by H(2)O(2) different from those induced by HNE in HT22 cells, and that HNE adduct proteins play an important role in HNE-induced cell death. It is also suggested that the pathway for H(2)O(2)-induced cell death in HT22 cells does not involve HNE production.

Upregulation of metallothionein-I mRNA expression in a rodent model for amyotrophic lateral sclerosis.

Metallothionein (MT) mRNA expression was investigated in a rodent model (G93A SOD1 transgenic mouse) for a lethal motor neuron disease, amyotrophic lateral sclerosis (ALS). In 8-wk-old mice that did not yet exhibit motor paralysis, MT-I mRNA expression was already significantly upregulated in the region of the spinal cord responsible for motor paralysis. The expression of another isoform, MT-III, was not changed. In the cerebellum, which is not responsible for motor paralysis in ALS, neither the expression profiles of MT-I nor MT-III were altered. In 16-wk-old mice exhibiting motor paralysis, the expression of MT-I mRNA remained upregulated and the MT-III level tended to be elevated. Although no significant differences were found in the levels of both isoforms in the liver or kidney of 8-wk-old mice, the MT-I mRNA expression level was significantly upregulated in the kidney and liver of 16-wk-old mice. These results indicated that the MT-I isoform, but not the MT-III isoform, is associated with motor neuron death in ALS and suggested that the disease might be a systemic disorder to which the spinal cord is particularly susceptible.