metallothionein and Amyotrophic-Lateral-Sclerosis

Lou Gehrig's disease, a synonym of amyotrophic lateral sclerosis, is an adult-onset lethal neurodegenerative disorder. Irrespective of extensive efforts to elucidate the pathogenesis of the disease and searches for therapies, no favorable pharmacotherapeutic strategies have yet to be proposed. In a popular rodent model of ALS, G93A SOD1 strain of mouse, intracellular copper conditions were geared toward copper accumulation inside cells, resulting in an acceleration of oxidative stress and apoptotic process. Disruption of intracellular copper homeostasis was common to transgenic mice expressing human mutant SOD1s. In this review, the novel hypothesis that disruption of intracellular copper homeostasis could be involved in the development of the disease was introduced. Based upon the hypothesis, therapeutic outcomes of agents that are capable of correcting and/or modifying intracellular copper homeostasis are described. Administration of ammonium tetrathiomolybdate, a selective intracellular copper chelator, delayed onset, slowed progression, and prolonged survival of a rodent model of the disease (G93A SOD1 mice). Metallothionein is a low molecular weight, cysteine-rich, metal-binding cytoplasmic protein that has beneficial properties in detoxification of toxic heavy metals, homeostatic regulation of intracellular essential trace elements, including copper, antioxidant, and antiapoptotic roles. In animal experiments of the G93A SOD1 mice, an increase of metallothionein proteins by means of induction by exercise or dexamethasone, genetic overexpression, or intraperitoneal administration, all resulted in a preferable outcome. The therapeutic effects were not inferior to those of approved drugs for ALS in humans. These observations suggest that metallothionein could be worth investigating the therapeutic potential in clinical use.

Topics: Amyotrophic Lateral Sclerosis; Animals; Antioxidants; Chelating Agents; Humans; Metallothionein; Oxidative Stress

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease that selectively affects motor neurons. Reactive oxygen species (ROS) are assumed to be involved in the pathogenesis of ALS. Metallothioneins (MTs) are self-protective, multifunctional proteins that scavenge ROS. In particular, metallothionein-III (MT-III) has a strong scavenging effect on hydroxyl radicals. MTs have been suggested to have important roles in the pathophysiology of ALS. Therefore we investigated single nucleotide polymorphisms (SNPs) of the MT-III and the metallothionein-IIA (MT-IIA) promoter region in 37 Japanese SALS cases and 206 sex-matched healthy controls using polymerase chain reaction (PCR)-direct sequencing or PCR-temporal temperature gradient gel electrophoresis (TTGE). We detected no SNPs of the MT-III gene in SALS cases and controls, and no detectable association between SALS phenotypes and a SNP of the MT-IIA promoter region. We conclude that gene polymorphisms of MT-IIA promoter region and MT-III gene are not associated with SALS phenotypes in a Japanese population.

Topics: Amyotrophic Lateral Sclerosis; Female; Genetic Predisposition to Disease; Genetic Testing; Humans; Incidence; Japan; Male; Metallothionein; Metallothionein 3; Middle Aged; Nerve Tissue Proteins; Polymorphism, Genetic; Polymorphism, Single Nucleotide; Risk Assessment; Risk Factors; Statistics as Topic

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised by the degeneration of motor neurons innervating skeletal muscle. The mechanisms underlying neurodegeneration in ALS are not yet fully elucidated, and with current therapeutics only able to extend lifespan by a matter of months there is a clear need for novel therapies to increase lifespan and patient quality of life. Here, we evaluated whether moderate-intensity treadmill exercise and/or treatment with metallothionein-2 (MT2), a neuroprotective protein, could improve survival, behavioural or neuropathological outcomes in SOD1

Topics: Amyotrophic Lateral Sclerosis; Animals; Disease Models, Animal; Female; Metallothionein; Mice; Mice, Transgenic; Physical Conditioning, Animal; Quality of Life; Superoxide Dismutase-1

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), an incurable motor neuron disease. The pathogenesis of the disease is poorly understood, but intracellular copper dyshomeostasis has been implicated as a key process in the disease. We recently observed that metallothioneins (MTs) are an excellent target for the modification of copper dyshomeostasis in a mouse model of ALS (SOD1(G93A)). Here, we offer a therapeutic strategy designed to increase the level of endogenous MTs. The upregulation of endogenous MTs by dexamethasone, a synthetic glucocorticoid, significantly improved the disease course and rescued motor neurons in SOD1(G93A) mice, even if the induction was initiated when peak body weight had decreased by 10%. Neuroprotection was associated with the normalization of copper dyshomeostasis, as well as with decreased levels of SOD1(G93A) aggregates. Importantly, these benefits were clearly mediated in a MT-dependent manner, as dexamethasone did not provide any protection when endogenous MTs were abolished from SOD1(G93A) mice. In conclusion, the upregulation of endogenous MTs represents a promising strategy for the treatment of ALS linked to mutant SOD1.

Topics: Age Factors; Amyotrophic Lateral Sclerosis; Animals; Anti-Inflammatory Agents; CD11b Antigen; Copper; Dexamethasone; Disease Models, Animal; Glial Fibrillary Acidic Protein; Humans; Lipid Peroxides; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phosphopyruvate Hydratase; Protein Isoforms; Spinal Cord; Superoxide Dismutase; Time Factors; Up-Regulation

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.

Topics: Amyotrophic Lateral Sclerosis; Animals; Astrocytes; Caspase 3; Copper; Disease Models, Animal; Disease Progression; Female; Gene Expression; Humans; Intracellular Space; Longevity; Male; Metallothionein; Mice; Motor Neurons; Mutation; Neuroglia; Phenotype; Proteolysis; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1

Amyotrophic lateral sclerosis (ALS) is a late-onset fatal neurodegenerative disease reflecting degeneration of upper and lower motoneurons (MNs). The cause of ALS and the mechanisms of neuronal death are still largely obscure, thus impairing the establishment of efficacious therapies. Glutamate (Glu)-mediated excitotoxicity plays a major role in MN degeneration in ALS. We recently demonstrated that the activation of Group I metabotropic Glu autoreceptors, belonging to both type 1 and type 5 receptors (mGluR1 and mGluR5), at glutamatergic spinal cord nerve terminals, produces excessive Glu release in mice over-expressing human superoxide-dismutase carrying the G93A point mutation (SOD1(G93A)), a widely used animal model of human ALS. To establish whether these receptors are implicated in ALS, we generated mice expressing half dosage of mGluR1 in the SOD1(G93A) background (SOD1(G93A)Grm1(crv4/+)), by crossing the SOD1(G93A) mutant mouse with the Grm1(crv4/+) mouse, lacking mGluR1 because of a spontaneous recessive mutation. SOD1(G93A)Grm1(crv4/+) mice showed prolonged survival probability, delayed pathology onset, slower disease progression and improved motor performances compared to SOD1(G93A) mice. These effects were associated to reduction of mGluR5 expression, enhanced number of MNs, decreased astrocyte and microglia activation, normalization of metallothionein and catalase mRNA expression, reduced mitochondrial damage, and decrease of abnormal Glu release in spinal cord of SOD1(G93A)Grm1(crv4/+)compared to SOD1(G93A) mice. These results demonstrate that a lower constitutive level of mGluR1 has a significant positive impact on mice with experimental ALS, thus providing the rationale for future pharmacological approaches to ALS by selectively blocking Group I metabotropic Glu receptors.

Topics: Amyotrophic Lateral Sclerosis; Animals; Astrocytes; Catalase; Disease Progression; Excitatory Amino Acid Transporter 2; Glutamic Acid; Metallothionein; Mice; Mice, Transgenic; Microglia; Mitochondria; Motor Activity; Motor Neurons; Point Mutation; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; RNA, Messenger; Severity of Illness Index; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; Survival Analysis

Amyotrophic lateral sclerosis (ALS) affects anterior horn cells of the spinal cord causing an indolent slow and steady deterioration of muscle strength leading inevitably to death in respiratory failure. ALS is a model condition for neurodegenerative disorders. Exposure to different agents dispersed in the environment has been suggested to cause neurodegeneration but no convincing evidence for such a link has yet been presented. Respiratory exposure to metallic mercury (Hg(0)) from different sources may be suspected. Body distribution of metallic mercury is fast and depends on solubility properties. Routes of transport, metabolism, excretion and biological half-life determine the overall toxic effects. Inhalation experiments were performed in 1984 where small marmoset monkeys (Callithrix jacchus) were exposed to (203) Hg(0 vapour) mixed into the breathing air (4-5 μg/l). After 1 hr of exposure, they were killed and whole body autoradiograms prepared to study the distribution of mercury within organs. Autoradiograms showed that Hg was deposited inside the spinal cord. Areas of enhanced accumulation anatomically corresponding to motor nuclei could be observed. This study describes a reinvestigation, with new emphasis on the spinal cord, of these classical metal exposure data in a primate, focusing on their relevance for the causation of neurodegenerative disorders. A comparison with more recent rodent experiments with similar findings is included. The hypothesis that long-time low-dose respiratory exposure to metals, for example, Hg, contributes to neurodegenerative disorders is forwarded and discussed.

Topics: Amyotrophic Lateral Sclerosis; Animals; Anterior Horn Cells; Autoradiography; Callithrix; Dose-Response Relationship, Drug; Half-Life; Inhalation Exposure; Male; Mercury; Metallothionein; Mice; Mice, 129 Strain; Spinal Cord

Regular exercise has displayed a beneficial effect on the progression of amyotrophic lateral sclerosis (ALS). However, the mechanism is poorly understood. We here present that regular exercise on a treadmill induces metallothioneins (MTs: MT-1, MT-2, and MT-3) in spinal cords of mice. As MTs are strong scavengers of reactive oxygen species and have some neurotrophic activities, exercise may have some beneficial effects on spinal motor neurons in patients with ALS owing to the induction of MTs. The running exercise on a treadmill for 30 min/day increased the mRNA expression levels of MT-1, MT-2, and MT-3 up to 193%, 298%, and 196%, respectively, of the control value 12 h after the start of exercise. After two weeks of daily exercise, Western blotting of the MTs proteins showed that the expression levels of MT-1/2 and MT-3 reached 173% and 146%, respectively, compared with those in sedentary mice. Running exercise on a treadmill for 2 weeks led to the gradual accumulation of MT proteins in the spinal cords of the mice. In addition, MT-1/2 and MT-3 immunoreactivities were enhanced in astrocytes particularly in the gray matter of the spinal cord. We revealed that regular exercise induced transient increases in the expression levels of MT mRNAs and resulted in accumulation of MT proteins in the spinal cords of the normal mice.

Topics: Amyotrophic Lateral Sclerosis; Animals; Astrocytes; Cell Survival; Cytoprotection; Exercise Test; Exercise Therapy; Free Radical Scavengers; Male; Metallothionein; Metallothionein 3; Mice; Mice, Inbred C57BL; Motor Neurons; Nerve Tissue Proteins; Oxidative Stress; Physical Conditioning, Animal; Reactive Oxygen Species; RNA, Messenger; Spinal Cord; Up-Regulation

We analyzed the expression of MTs using immunohistochemistry on the spinal cords of patients with ALS (n =12) and controls (n =12). The immunoreactivities of both MT-1/2 and MT-3 stained dominantly in glial cells and were decreased in the spinal cords of patients with ALS, particularly in patients on respirators. The immunoreactivity of MT-1/2 in the ALS groups was significantly reduced compared with controls. In addition, a statistical analysis revealed that the immunoreactivity of MT-3 in astrocytes in the gray matter of the lumbar spinal cord was negatively correlated with the duration of ALS. Both MT-1/2 and MT-3 immunoreactivities were detected mainly in the glias and also detected in some neurons in both control patients and patients with ALS. Interestingly, the patients with MT-3-positive neurons showed definite MT-3-immunoreactive glial reaction around neurons. Previous studies have reported that familial ALS (FALS) model mice (G93A SOD1) crossed with MT-1/2 or MT-3 knock-out mice had accelerated expression of ALS. Judged from these findings, both MT-1/2 and MT-3 play important roles in the progression of ALS. MTs are defensive proteins that can scavenge free radicals; therefore, manipulation of their expression has a strong therapeutic potential for ALS patients.

Topics: Adult; Aged; Amyotrophic Lateral Sclerosis; Animals; Autopsy; Female; Humans; Immunohistochemistry; Male; Metallothionein; Metallothionein 3; Mice; Middle Aged; Neuroglia; Protein Isoforms; Spinal Cord

It has been hypothesized that copper-mediated oxidative stress contributes to the pathogenesis of familial amyotrophic lateral sclerosis (ALS), a fatal motor neuron disease in humans. To verify this hypothesis, we examined the copper and zinc concentrations and the amounts of lipid peroxides, together with that of the expression of metallothionein (MT) isoforms in a mouse model [superoxide dismutase1 transgenic (SOD1 Tg) mouse] of ALS. The expression of MT-I and MT-II (MT-I/II) isoforms were measured together with Western blotting, copper level, and lipid peroxides amounts increased in an age-dependent manner in the spinal cord, the region responsible for motor paralysis. A significant increase was already seen as early as 8-week-old SOD1 Tg mice, at which time the mice had not yet exhibited motor paralysis, and showed a further increase at 16 weeks of age, when paralysis was evident. Inversely, the spinal zinc level had significantly decreased at both 8 and 16 weeks of age. The third isoform, the MT-III level, remained at the same level as an 8-week-old wild-type mouse, finally increasing to a significant level at 16 weeks of age. It has been believed that a mutant SOD1 protein, encoded by a mutant SOD1, gains a novel cytotoxic function while maintaining its original enzymatic activity, and causes motor neuron death (gain-of-toxic function). Copper-mediated oxidative stress seems to be a probable underlying pathogenesis of gain-of-toxic function. Taking the above current concepts and the classic functions of MT into account, MTs could have a disease modifying property: the MT-I/II isoform for attenuating the gain-of-toxic function at the early stage of the disease, and the MT-III isoform at an advanced stage.

Topics: Age Factors; Amyotrophic Lateral Sclerosis; Animals; Blotting, Western; Cerebellum; Copper; Disease Models, Animal; Female; Humans; Lipid Peroxides; Male; Metallothionein; Mice; Mice, Transgenic; Mutation; Polymerase Chain Reaction; Protein Isoforms; Spinal Cord; Superoxide Dismutase; Time Factors; Zinc

Sporadic amyotrophic lateral sclerosis (SALS) results from the death of motor neurons in the brain and spinal cord. Environmental exposure to heavy metals has been implicated in SALS and impaired detoxification of these metals may cause susceptibility to the disease. The metallothionein (MT) family of proteins are the primary detoxification mechanism for heavy metals and MT-Ia and MT-IIa are the most common human isoforms. Inappropriate methylation at the promoters of these genes could lead to silencing of transcription and reduce the availability of MTs. We therefore measured the level of methylation in the promoters of MT-Ia and MT-IIa in 25 leukocyte and six brain DNA samples from SALS patients and compared these with controls. No promoter methylation was evident in any SALS or control samples. In conclusion, it is unlikely that methylation at these gene promoters is a common cause of SALS.

Topics: Amyotrophic Lateral Sclerosis; Brain; CpG Islands; DNA Methylation; Female; Gene Silencing; Humans; Leukocytes; Male; Metallothionein; Middle Aged; Promoter Regions, Genetic

Environmental toxicants such as heavy metals, pesticides, and chemicals appear to be risk factors for sporadic amyotrophic lateral sclerosis (SALS). An impaired ability to break down these toxicants because of differences in detoxification genes could underlie some cases of this disease. We therefore examined the frequencies of single nucleotide polymorphisms (SNPs) in 186 SALS patients and 186 controls at the allele, genotype, and haplotype levels for the metallothionein (MT) family of genes, metal transcription factor-1 (MTF-1), and glutathione synthetase (GSS). Exposure to heavy metals, solvents/chemicals, and pesticides/herbicides was assessed by questionnaire, and gene-toxicant interactions were analyzed. An intronic SNP upstream of MT-Ie differed in SALS patients and controls at the allele and genotype levels. Haplotypes covering MT-I isoforms also differed between the two groups. Alleles and genotypes of one MTF-1 SNP differed in female SALS patients. One GSS haplotype interacted with both metals and solvents/chemicals to increase the risk of the disease. Differences in genes involved in handling toxicants, and interactions between toxicants and these genes, appear to be present in some patients with SALS. This suggests that impaired detoxification mechanisms play a role in SALS.

Topics: Aged; Alleles; Amyotrophic Lateral Sclerosis; Case-Control Studies; DNA-Binding Proteins; Female; Genetic Predisposition to Disease; Genotype; Glutathione Synthase; Haplotypes; Hazardous Substances; Humans; Male; Metallothionein; Middle Aged; Polymorphism, Single Nucleotide; Protein Isoforms; Risk Factors; Transcription Factor MTF-1; Transcription Factors

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.

Topics: Amyotrophic Lateral Sclerosis; Animals; Cerebellum; Disease Models, Animal; Gene Expression Regulation; Kidney; Liver; Metallothionein; Metallothionein 3; Mice; Mice, Transgenic; Motor Activity; Nerve Tissue Proteins; RNA, Messenger; Spinal Cord; Spleen; Up-Regulation

Gene expression analysis is a powerful tool that has been used to define the pathological processes underlying many diseases. Several laboratories, including our own, have used this approach to identify molecular abnormalities in the G93A/SOD1 mouse, an animal model of amyotrophic lateral sclerosis (ALS). Here, we report the results of analysis of an expanded panel of genes throughout the entire lifetime in the spinal cord of these animals. In addition to upregulation of microglia/neuroinflammatory genes identified previously, we observed upregulation of metallothionein-I and -II (MT-I, MT-II). MT-I and MT-II play an important role in disposition of zinc ion, and other studies have also indicated their levels are altered in development of motor neuron disease in these animals. We also analyzed the effect on these expression profiles of several candidate drugs that have been shown to have neuroprotective effects in vivo or in vitro. That is, we asked whether administration to the G93A/SOD1 mice of any of these drugs could reverse the alterations in gene expression patterns that occur as the animals develop. The mice were given daily doses of these drugs when they were 9-11 weeks old, at a stage early in development of motor neuron disease, continuing for 5 weeks, at which time they were sacrificed. Treatment of the mice with l-carnosine, a dipeptide that scavenges free radicals and chelates zinc, did not affect expression of any of the genes altered in these animals. However, it did upregulate 3 genes unaffected by the presence of the G93A/SOD1 mutation: glial fibrillary acidic protein (GFAP), stroma-derived factor-1 (SDF-1), and excitatory amino acid transporter-2 (EAAT2). In contrast, metallothionein-III (MT-III) was downregulated. Treatment of the animals with baicalein, an herbal extract with anti-inflammatory and numerous other effects, downregulated the microglia markers CD68, CD80, and CD86, all of which were upregulated in untreated mutant animals. Baicalein treatment also downregulated tumor necrosis factor receptor (TNFRp55) and upregulated noninducible nitric oxide synthase (nNOS) and glutamine synthase (GS). These 3 genes were unaffected by the presence of the G93A mutation. We discuss the implication of these results for testing the effects of these and other candidate drugs in mutant SOD1 mice.

Topics: Amyotrophic Lateral Sclerosis; Animals; Antioxidants; Apoptosis; Carnosine; Disease Models, Animal; Drug Evaluation, Preclinical; Flavanones; Gene Expression; Gene Expression Regulation; Genetic Markers; Inflammation; Metallothionein; Mice; Mice, Knockout; Neuroprotective Agents; Neurotoxins; Oxidative Stress; RNA; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1

Mutations in Cu/Zn-superoxide dismutase 1 (SOD1) are responsible for a familial form of amyotrophic lateral sclerosis (FALS). It has been proposed that oxidative stress and abnormal metal homeostasis contribute to death of motor neurons in this disease. Also, inability of motor neurons to upregulate protective proteins under stress may contribute to their preferential vulnerability to toxicity. Metallothioneins (MT) are low molecular weight, metal-binding proteins with established antioxidant capabilities. This study investigated the ability of motor neurons to upregulate MT isoforms in response to expression of mutant SOD1(G93A) or exposure to other neurotoxicants, and the ability of MT-I gene transfer to protect motor neurons from these stresses. MT isoform-I and -II were expressed constitutively in astrocytes and other non-neuronal cells of dissociated spinal cord cultures, but not in motor neurons. MT-I/II was upregulated in astrocytes, but not motor neurons, following treatment with ZnCl(2) or excitotoxic concentrations of glutamate. MT-III expression was restricted to neurons and was unaffected by treatment with ZnCl(2), paraquat, or glutamate. Overexpression of MT-I in motor neurons by gene transfer reduced the toxicity of ZnCl(2) and paraquat, but failed to protect them against glutamate or SOD1(G93A). These data are evidence against metal-catalyzed, oxidative stress being the primary mechanisms of toxicity conferred by disease-causing mutations in SOD1.

Topics: Amyotrophic Lateral Sclerosis; Animals; Blotting, Western; Cell Survival; Cells, Cultured; Chlorides; Genetic Vectors; Herbicides; Immunohistochemistry; Inclusion Bodies; Isoenzymes; Isomerism; Metallothionein; Mice; Motor Neurons; Mutation; Oxidative Stress; Paraquat; Plasmids; Superoxide Dismutase; Zinc Compounds

Transgenic mice overexpressing the human mutated form (G93A) of Cu,Zn-superoxide dismutase (mSOD1) develop motor neuron degeneration resembling amyotrophic lateral sclerosis. In vitro studies have shown that mSOD1-induced, reactive oxygen species-mediated apoptosis of motor neurons depends on the presence of copper and the relative absence of zinc. Oral intake of zinc sulphate induces the expression of metallothioneins, enzymes that decrease oxidative stress, and leads to higher serum zinc, and lower copper levels. We therefore tested the effect of chronic oral administration of zinc sulfate (0.075 and 0.375 g/kg) on disease onset and survival of mSOD1 transgenic mice. We observed that zinc sulfate, rather than prolonging survival, decreased survival. We conclude that zinc sulfate amplifies the mSOD1 transgenic mouse phenotype. This finding may shed more light on the role of zinc in mSOD1-mediated toxicity.

Topics: Amyotrophic Lateral Sclerosis; Animals; Disease Models, Animal; Female; Humans; Male; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Transgenic; Superoxide Dismutase; Zinc Sulfate

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene cause one form of familial amyotrophic lateral sclerosis, a progressive disorder of motor neurons leading to weakness and death of affected individuals. Experiments using both transgenic mice expressing mutant SOD1 and SOD1 knock-out mice have demonstrated that disease is caused by a toxic gain of function and not by a loss of normal SOD1 activity. Precise mechanisms underlying mutant SOD1 toxicity are unclear but may involve abnormal interactions between zinc and SOD1. The metallothioneins (MTs) represent a family of zinc binding proteins that can function as zinc chaperones for apo-SOD1 in vitro. We hypothesized that manipulation of metallothioneins in vivo might alter the disease phenotype of transgenic mice expressing G93A SOD1 and therefore crossed this line with MT-I and MT-II or MT-III knock-out mice. G93A SOD1 mice deficient of either MT-I and MT-II or MT-III exhibited significant reductions in survival compared with G93A SOD1 mice. In addition, motor dysfunction was markedly accelerated in G93A SOD1 mice deficient in metallothioneins with regard to onset (MT-I and MT-II) or progression (MT-III). These results indicate that the disease course in G93A SOD1 mice is dependent on levels of metallothionein expression. Because MT-I and MT-II are expressed in glia whereas MT-III is found in neurons, these results also indicate that primary changes within non-neuronal cells can affect mutant SOD1-induced disease and do so in ways distinct from primary neuronal changes.

Topics: Amyotrophic Lateral Sclerosis; Animals; Blotting, Western; Cell Count; Crosses, Genetic; Disease Models, Animal; Disease Progression; Glial Fibrillary Acidic Protein; Humans; Metallothionein; Metallothionein 3; Mice; Mice, Transgenic; Motor Activity; Nerve Tissue Proteins; Neuroglia; Neurons; Phenotype; Spinal Cord; Superoxide Dismutase; Survival Analysis; Zinc

We previously reported that abnormal copper release from mutated Cu, Zn-superoxide dismutase (SOD1) proteins might be a common toxic gain-of-function in the pathogenesis of familial amyotrophic lateral sclerosis (FALS) [Ogawa et al. (1997) Biochem. Biophys. Res. Commun., 241, 251-257.]. In the present study, we first examined metallothioneins (MTs), known to bind copper ions and decrease oxidative toxicity, and found a twofold increase in MTs in the spinal cord of the SOD1 transgenic mice with a FALS-linked mutation (G93A), but not in the spinal cord of wild-type SOD1 transgenic mice. We then investigated whether the clinical course of FALS mice could be modified by the reduced expression of MTs, by crossing the FALS mice with MT-I- and MT-II-deficient mice. FALS mice clearly reached the onset of clinical signs and death significantly earlier in response to the reduction of protein expression. These results indicated that the copper-mediated free radical generation derived from mutant SOD1 might be related to the degeneration of motor neurons in FALS and that MTs might play a protective role against the expression of the disease.

Topics: Amyotrophic Lateral Sclerosis; Animals; Copper; Disease Models, Animal; Gene Dosage; Gene Expression Regulation, Enzymologic; Humans; Hydroxyl Radical; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Transgenic; Oxidative Stress; Spinal Cord; Superoxide Dismutase

Transgenic mice carrying familial amyotrophic lateral sclerosis (FALS)-linked mutant Cu/Zn superoxide dismutase (SOD1) genes such as G93A (G93A-mice) and G85R (G85R-mice) genes develop limb paresis. Introduction of human wild type SOD1 (hWT-SOD1) gene, which does not cause motor impairment by itself, into different FALS mice resulted in different effects on their clinical courses, from no effect in G85R-mice to acceleration of disease progression in G93A-mice. However, the molecular mechanism which causes the observed difference, has not been clarified. We hypothesized that the difference might be caused by the stability of mutant SOD1 proteins. Using a combination of mass spectrometry and enzyme-linked immunosorbent assay, we found that the concentration of G93A-SOD1 protein was markedly elevated in tissues of transgenic mice carrying both G93A- and hWT-SOD1 genes (G93A/hWT-mice) compared to that in G93A-mice, and also found that the concentration of G93A-SOD1 protein had a close relation to the disease duration. The concentration of metallothionein-I/II in the spinal cord, reflecting the degree of copper-mediated oxidative stress, was highest in G93A/hWT-mice, second in G93A-mice, and normal in the mice carrying hWT-SOD1 gene. These results indicated that the increase of G93A-SOD1 protein was responsible for the increase of oxidative stress and disease acceleration in G93A/hWT-mice. We speculate that coexpression of hWT-SOD1 protein is deleterious to transgenic mice carrying a stable mutant such as G93A-SOD1, because this mutant protein is stabilized by hWT-SOD1 protein, but not to transgenic mice carrying an unstable mutant such as G85R-SOD1, because this mutant protein is not stabilized by hWT-SOD1.

Topics: Amyotrophic Lateral Sclerosis; Animals; Female; Free Radicals; Gene Expression Regulation, Enzymologic; Humans; Male; Metallothionein; Mice; Mice, Neurologic Mutants; Mice, Transgenic; Mutation; Oxidative Stress; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; Up-Regulation

Missense mutations in the gene encoding copper zinc superoxide dismutase (SOD1) have been found to cause one form of familial amyotrophic lateral sclerosis (FALS). Although the exact mechanism of disease is unknown, abnormalities in the ability of mutant SOD1 to bind zinc or copper ions may be crucial in the pathogenesis of disease. Because members of the metallothionein (MT) family of zinc and copper binding proteins function as important cellular regulators of metal ion bioavailability in the central nervous system, we used in situ hybridization and immunohistochemistry to study the expression pattern of these molecules in a transgenic mouse model of familial ALS. In adult wild-type mouse spinal cord, expression of MT-I and MT-II is restricted to ependymal cells and a subset of astrocytes located in white matter tracts, while MT-III synthesis is limited to neurons within gray matter. Compared to wild-type littermates, transgenic mice carrying the G93A SOD1 mutation demonstrate markedly increased expression of MT-I and MT-II within astrocytes in both white and gray matter as weakness develops. MT-III synthesis in neurons is also greatly upregulated as G93A SOD1 animals age, with glial cell expression of MT-III evident by later stages of the disease. Changes in MT expression occur before the onset of motor deficits or significant motor neuron pathology in G93A SOD1 mice and remarkably extend beyond ventral horn populations of neurons and glia. These results are consistent with the hypothesis that metallothioneins may serve an early and important protective function in FALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Astrocytes; Disease Models, Animal; Gene Expression; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; In Situ Hybridization; Inclusion Bodies; Metallothionein; Mice; Mice, Transgenic; Motor Neurons; Neurofilament Proteins; RNA, Messenger; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; Ubiquitins; Zinc

Mutations to Cu/Zn superoxide dismutase (SOD) linked to familial amyotrophic lateral sclerosis (ALS) enhance an unknown toxic reaction that leads to the selective degeneration of motor neurons. However, the question of how >50 different missense mutations produce a common toxic phenotype remains perplexing. We found that the zinc affinity of four ALS-associated SOD mutants was decreased up to 30-fold compared to wild-type SOD but that both mutants and wild-type SOD retained copper with similar affinity. Neurofilament-L (NF-L), one of the most abundant proteins in motor neurons, bound multiple zinc atoms with sufficient affinity to potentially remove zinc from both wild-type and mutant SOD while having a lower affinity for copper. The loss of zinc from wild-type SOD approximately doubled its efficiency for catalyzing peroxynitrite-mediated tyrosine nitration, suggesting that one gained function by SOD in ALS may be an indirect consequence of zinc loss. Nitration of protein-bound tyrosines is a permanent modification that can adversely affect protein function. Thus, the toxicity of ALS-associated SOD mutants may be related to enhanced catalysis of protein nitration subsequent to zinc loss. By acting as a high-capacity zinc sink, NF-L could foster the formation of zinc-deficient SOD within motor neurons.

Topics: Amyotrophic Lateral Sclerosis; Animals; Binding Sites; Catalysis; Cloning, Molecular; Humans; Kinetics; Liver; Metallothionein; Models, Structural; Mutagenesis, Site-Directed; Mutation; Nitrates; Protein Conformation; Rabbits; Recombinant Proteins; Superoxide Dismutase; Tyrosine; Zinc

In earlier studies of sporadic amyotrophic lateral sclerosis (ALS), a disease of unknown etiology, the amount of metallothioneins (MTs), a group of small (6-7 kDa) metal-binding proteins, appeared higher in liver, kidney and spinal cord from patients than from non-neurologic controls. Immunohistochemically, the expression of MT in the central nervous system appeared limited to glia. Since the highly conserved MTs isotypes share antigenic epitopes, they could not be distinguished by immunological methods. It thus proved necessary to estimate the expression of each individual MT messenger ribonucleic acid (mRNA) by performing reverse transcriptase polymerase chain reaction (RT-PCR)-mediated analysis of tissue samples. Tissues selected included liver, motor cortex and cervical cord at C6; MT mRNAs analyzed included MT1A, 1B, 1E, 1F, 1G, 2A, and 3. Also, special care was taken to avoid interference by amplification of the 6 MT pseudogenes. Except of MT3, already known as brain-specific, and MT1B which was not expressed in any tissue, mRNA levels of the other MT genes tended to be higher in ALS than in control liver samples, but the differences did not attain statistical significance. In the nervous system, the diverse MT genes were expressed over a greater range in ALS than in controls, but exhibited no change in a consistent direction. At the motor cortex, changes seemed to be less pronounced than at C6. MT3 was expressed in the motor cortex and the cord. The results provide no evidence for either the induction of a specific MT repertoire, or for the inability of glia to express any MT gene in ALS. Because the semi-quantitative RT-PCR technique does not permit detailed comparisons between the subtypes of MT expressed in the various tissues, the question whether a single inductor may be held responsible for the elevation of MT in the ALS liver and nervous system remains open. In conclusion, ALS tissue remains capable of expressing all the major MT genes. MT, present in protoplasmic glia, arises locally and is not secondary to increases of hepatic or renal MT. Because MT3 is also expressed by the normal and ALS spinal cord, it is a central nervous system-specific and not only a brain-specific protein. Thus, the excess of MT in ALS liver seems to be an effect of slower catabolism rather than faster synthesis of protein.

Topics: Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; DNA Primers; Gene Expression; Humans; Liver; Metallothionein; Middle Aged; Motor Cortex; Polymerase Chain Reaction; RNA, Messenger; Spinal Cord

Topics: Amyotrophic Lateral Sclerosis; Brain; Brain Chemistry; Humans; Mercury; Metallothionein; Motor Neurons; Spinal Cord

Sections of the spinal cord from 10 patients with classic amyotrophic lateral sclerosis and from 10 control cases were examined by immunocytochemical methods to localize metallothionein. Metallothionein immunoreactivity was seen in the nucleus and cytoplasm of a subset of astrocytes, largely confined to the gray matter. Also, diffuse gray matter staining was observed, probably representing small glial fibers. Astrocytic metallothionein immunoreactivity (P less than 0.01) and strong gray matter matrix staining (P less than 0.03) was increased in the spinal cords from patients with amyotrophic lateral sclerosis. Although compatible with induction by metals, increased metallothionein expression in the spinal cords from patients with amyotrophic lateral sclerosis may also have resulted from inflammation or gliosis.

Topics: Amyotrophic Lateral Sclerosis; Antibodies, Monoclonal; Astrocytes; Cell Nucleus; Cytoplasm; Humans; Immunohistochemistry; Metallothionein; Spinal Cord

To evaluate the putative role of metals and trace elements in the pathogenesis of classic amyotrophic lateral sclerosis, we studied the metallothionein levels in liver and kidney samples obtained at autopsy from 24 patients with amyotrophic lateral sclerosis and 18 controls. To assay metallothioneins and copper, cadmium, and zinc bound to metallothioneins, we used high-performance liquid chromatography directly coupled to flame atomic absorption spectrometry. Total cadmium, zinc, and copper concentrations were determined separately with the use of graphite furnace atomic absorption spectrometry with Zeeman background correction. The median liver metallothionein level was 60.3 mg/kg (range, 9 to 318 mg/kg) in the patients with amyotrophic lateral sclerosis and 12.6 mg/kg (range, 0 to 104.5 mg/kg) in the controls. In the kidney, median metallothionein levels were 126.9 mg/kg (range, 44 to 387 mg/kg) in the patients with amyotrophic lateral sclerosis and 64 mg/kg (range, 13.1 to 187 mg/kg) in the controls. Total zinc, cadmium, and copper concentrations, as measured by atomic absorption spectrometry, were not significantly different in patients vs controls. Our finding of elevated metallothionein levels in organs from patients with amyotrophic lateral sclerosis may indicate an increased exposure to metals.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Cadmium; Copper; Female; Humans; Kidney; Liver; Male; Metallothionein; Middle Aged; Zinc