Familial Amyotrophic Lateral Sclerosis Mutant Research Articles

The Copper Chaperone CCS Directly Interacts with Copper/Zinc Superoxide Dismutase

Dominantly inherited mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) result in the fatal motor neuron disease familial amyotrophic lateral sclerosis (FALS). These mutations confer a gain-of-function to SOD1 with neuronal degeneration resulting from enhanced free radical generating activity of the copper present in the mutant enzyme. The delivery of copper to SOD1 is mediated through a soluble factor identified as the copper chaperone for SOD1 (CCS). Amino acid sequence alignment of SOD1 and CCS reveals a striking homology with conservation of the amino acids essential for mediating SOD1 homodimerization. Here we demonstrate that CCS and SOD1 directly interact in vitro and in vivo and that this interaction is mediated via the homologous domains in each protein. Importantly, CCS interacts not only with wild-type SOD1 but also with SOD1 containing the common missense mutations resulting in FALS. Our findings therefore reveal a common mechanism whereby different SOD1 FALS mutants may result in neuronal injury and suggest a novel therapeutic approach in patients affected by this fatal disease.

Axonal Transport of Mutant Superoxide Dismutase 1 and Focal Axonal Abnormalities in the Proximal Axons of Transgenic Mice

Superoxide dismutase 1 (SOD1), a ubiquitously expressed enzyme, detoxifies superoxide radicals and participates in copper homeostasis. Mutations in this enzyme have been linked to a subset of autosomal dominant cases of familial amyotrophic lateral sclerosis (FALS), a disorder characterized by selective degeneration of motor neurons. Transgenic mice expressing FALS mutant human (Hu) SOD1 at high levels develop a motor neuron disease, indicating that mutant Hu SOD1 gains properties that are particularly toxic to motor neurons. In this report, we demonstrate that transgenic mice expressing Hu SOD1 with the G37R FALS mutation, but not mice expressing wild-type enzyme, develop focal increases in immunoreactivity in the proximal axons of spinal motor neurons. This SOD1 immunoreactivity and immunoreactivity to hypophosphorylated neurofilament H epitopes are found adjacent to small vacuoles in axons. Using metabolic radiolabeling methods, we show that mutant G37R HuSOD1 as well as endogenous mouse SOD1 are transported anterograde in slow componentbin motor and sensory axons of the sciatic nerve. Together, these findings suggest that anterogradely transported mutant SOD1 may act locally to damage motor axons.

The use of transgenic mouse models of amyotrophic lateral sclerosis in preclinical drug studies

The discovery of mutations in the human SOD1 gene encoding Cu,Zn superoxide dismutase (Cu,Zn SOD) in patients with familial amyotrophic lateral sclerosis (ALS) has made possible the development of etiological models of the disease. Expression of mutant SOD1 genes in transgenic mice causes a progressive paralytic disease whose general features resemble ALS in humans. We have used the transgenic model to explore etiological mechanisms and to screen potential therapeutics. Our results and those of others show that familial ALS mutations cause a gain-of-function in Cu,Zn SOD that enhances the generation of damaging oxygen radicals. This may render motor neurons sensitive to the excitotoxic effects of ambient glutamate, as a putative glutamatergic inhibitor such as riluzole has therapeutic efficacy both in the transgenic model and in human ALS. This finding highlights the utility of the SOD1-G93A transgenic mouse model for preclinical drug studies in ALS.

Proteasome inhibition enhances the stability of mouse [formula omitted] superoxide dismutase with mutations linked to familial amyotrophic lateral sclerosis

Point mutations occurring within the Cu Zn superoxide dismutase ( SOD1) gene have been implicated in the etiology of some cases of familial amyotrophic lateral sclerosis (FALS). In order to better understand the functional consequences of these mutations, we have introduced FALS mutations into the mouse SOD1 gene and studied the expression of the mutant templates in stably transformed cell lines. Pulse-chase analyses of lysates derived from cell lines stably expressing the Cu Zn SOD isoforms indicate that the FALS mutant Cu Zn SOD proteins are turned over more rapidly than wild-type SOD. Protease inhibitors specific for the major intracellular proteolytic activities were used to characterize the degradative pathways involved in the turnover of mutant Cu Zn SOD. Inhibition of the chymotrypsin-like activity of the proteasome (also known as multicatalytic proteinase or ubiquitin, ATP-dependent proteinase) by a synthetic dipeptide aldehyde led to a significant increase in levels of the mutant Cu Zn SOD implicating this proteolytic pathway in the turnover of the FALS mutant SOD proteins.

A gain-of-function of an amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutant: An enhancement of free radical formation due to a decrease in Km for hydrogen peroxide.

Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS). Transgenic mice that express a mutant Cu,Zn-SOD, Gly-93--> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms. We cloned the FALS mutant, G93A, and wild-type cDNA of human Cu,Zn-SOD, overexpressed them in Sf9 insect cells, purified the proteins, and studied their enzymic activities for catalyzing the dismutation of superoxide anions and the generation of free radicals with H2O2 as substrate. Our results showed that both enzymes contain one copper ion per subunit and have identical dismutation activity. However, the free radical-generating function of the G93A mutant, as measured by the spin trapping method, is enhanced relative to that of the wild-type enzyme, particularly at lower H2O2 concentrations. This is due to a small, but reproducible, decrease in the value of Km for H2O2 for the G93A mutant, while the kcat is identical for both enzymes. Thus, the ALS symptoms observed in G93A transgenic mice are not caused by the reduction of Cu,Zn-SOD activity with the mutant enzyme; rather, it is induced by a gain-of-function, an enhancement of the free radical-generating function. This is consistent with the x-ray crystallographic studies showing the active channel of the FALS mutant is slightly larger than that of the wild-type enzyme; thus, it is more accessible to H2O2. This gain-of-function, in part, may provide an explanation for the association between ALS and Cu,Zn-SOD mutants.

Mutations associated with amyotrophic lateral sclerosis convert superoxide dismutase from an antiapoptotic gene to a proapoptotic gene: studies in yeast and neural cells.

Familial amyotrophic lateral sclerosis (FALS) is associated with mutations in SOD1, the gene encoding copper/zinc superoxide dismutase (CuZnSOD). However, the mechanism by which these mutations lead to amyotrophic lateral sclerosis is unknown. We report that FALS mutant SODs expressed in yeast lacking CuZnSOD are enzymatically active and restore the yeast to the wild-type phenotype. In mammalian neural cells, the overexpression of wild-type SOD1 inhibits apoptosis induced by serum and growth factor withdrawal or calcium ionophore. In contrast, FALS-associated SOD1 mutants promote, rather than inhibit, neural apoptosis, in a dominant fashion, despite the fact that these mutants retain enzymatic SOD activity both in yeast and in mammalian neural cells. The results dissociate the SOD activity of FALS-associated mutants from the induction of neural cell death, suggesting that FALS associated with mutations in SOD1 may not be simply the result of a decrease in the enzymatic function of CuZnSOD. Furthermore, the results provide an in vitro model that may help to define the mechanism by which FALS-associated SOD1 mutations lead to neural cell death.

Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity.

Familial amyotrophic lateral sclerosis (FALS) has been linked to mutations in the homodimeric enzyme Cu/Zn superoxide dismutase 1 (SOD1). Assay by transient expression in primate cells of six FALS mutant enzymes revealed a continuum of enzymatic activity bounded by the enzyme carrying the mutation Gly-85-->Arg, which was inactive, and mutant enzyme G37R carrying the Gly-37-->Arg change, which retained full specific activity but displayed a 2-fold reduction in polypeptide stability. The G37R mutant displayed similar properties in transformed lymphocytes from an individual heterozygous for the G37R and wild-type SOD1 genes; heterodimeric enzymes composed of mutant and wild-type subunits were detected, but there was no measurable diminution in the stability and activity of the wild-type subunits. Thus, for mutants such as G37R, either surprisingly modest losses in activity (involving only the mutant subunit) can yield motor neuron death, or alternatively, mutant SOD1 may acquire properties that injure motor neurons by one or more mechanisms unrelated to the metabolism of oxygen radicals.

Rapid Communication: Cu/Zn Superoxide Dismutase Activity in Familial and Sporadic Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease that is inherited as an autosomal dominant trait in approximately 10% of cases. Recently we and others identified several single-base mutations in the Cu/Zn superoxide dismutase (SOD1) gene in patients with familial ALS (FALS). Using single-strand conformational polymorphism, we studied the C to G mutation in exon 2 of the SOD1 gene (resulting in a leucine to valine substitution in position 38) in affected and unaffected members of a large Belgian family with FALS. We measured the SOD1 activity in red blood cell lysates in 14 members of this family, including the only surviving clinically affected patient. SOD1 activity of the family members carrying the mutation was less than half that of members without the mutation. In addition, in 11 patients with sporadic ALS and 11 age- and sex-matched controls, red blood cell SOD1 activity was normal. These studies indicate that SOD1 activity is reduced in these FALS patients but not in sporadic ALS patients. Moreover, this SOD1 enzyme abnormality is detectable years before onset of clinical ALS in carriers of this FALS mutation.

Amyotrophic Lateral Ssclerosis and Structural Defects in Cu,Zn Superoxide Dismutase

Single-site mutants in the Cu,Zn superoxide dismutase (SOD) gene (SOD1) occur in patients with the fatal neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). Complete screening of the SOD1 coding region revealed that the mutation Ala4 to Val in exon 1 was the most frequent one; mutations were identified in exons 2, 4, and 5 but not in the active site region formed by exon 3. The 2.4 A crystal structure of human SOD, along with two other SOD structures, established that all 12 observed FALS mutant sites alter conserved interactions critical to the beta-barrel fold and dimer contact, rather than catalysis. Red cells from heterozygotes had less than 50 percent normal SOD activity, consistent with a structurally defective SOD dimer. Thus, defective SOD is linked to motor neuron death and carries implications for understanding and possible treatment of FALS.