Structural properties and neuronal toxicity of amyotrophic lateral sclerosis-associated Cu/Zn superoxide dismutase 1 aggregates

The appearance of protein aggregates is a characteristic of protein misfolding disorders including familial amyotrophic lateral sclerosis, a neurodegenerative disease caused by inherited mutations in Cu/Zn superoxide dismutase 1 (SOD1). Here, we use live cell imaging of neuronal and nonneuronal cells to show that SOD1 mutants (G85R and G93A) form an aggregate structure consisting of immobile scaffolds, through which noninteracting cellular proteins can diffuse. Hsp70 transiently interacts, in a chaperone activity-dependent manner, with these mutant SOD1 aggregate structures. In contrast, the proteasome is sequestered within the aggregate structure, an event associated with decreased degradation of a proteasomal substrate. Through the use of time-lapse microscopy of individual cells, we show that nearly all (90%) aggregate-containing cells express higher levels of mutant SOD1 and died within 48 h, whereas 70% of cells expressing a soluble mutant SOD1 survived. Our results demonstrate that SOD1 G85R and G93A mutants form a distinct class of aggregate structures in cells destined for neuronal cell death.     

Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis

Accumulating evidence indicates that abnormal conformation of mutant superoxide dismutase 1 (SOD1) is an essential feature underlying the pathogenesis of mutant SOD1-linked familial amyotrophic lateral sclerosis (ALS). Here we investigated the role of ubiquitin-proteasome pathway in the mutant SOD1-related cell death and the effect of oxidative stress on the misfolding of mutant SOD1. Transient overexpression of ubiquitin with human SOD1 (wild-type, ala4val, gly85arg, gly93ala) in Neuro2A cells decreased the amount of mutant SOD1, but not of wild-type, while only mutants were co-immunoprecipitated with poly-ubiquitin. Proteasome inhibition by lactacystin augmented accumulation of mutant SOD1 in the non-ionic detergent-insoluble fraction. The spinal cord lysates from mutant SOD1 transgenic mice showed multiple carbonylated proteins, including mutant SOD1 with SDS-resistant dimer formation. Furthermore, the treatment of hSOD1-expressing cells with hydrogen peroxide promoted the oligomerization, and detergent-insolubility of mutant SOD1 alone, and the oxidized mutant SOD1 proteins were more heavily poly-ubiquitinated. In Neuro2A cells stably expressing human SOD1 protein, the proteasome function measured by chymotrypsin-like activity, was decreased over time without a quantitative alteration of the 20S proteasomal component. Finally, primary motor neurons from the mouse embryonic spinal cord were more vulnerable to lactacystin than non-motor neurons. These results indicate that the sustained expression of mutant SOD1 leads to proteasomal inhibition and motor neuronal death, which in part explains the pathogenesis of mutant SOD1-linked ALS.     

Mutant SOD1(G93A) microglia are more neurotoxic relative to wild-type microglia

Recent studies suggest that microglia over-expressing mutant human superoxide dismutase (mSOD1(G93A)) may contribute to motoneuron death in a transgenic mouse model of familial amyotrophic lateral sclerosis. To further assess the relative neurotoxicity of wild-type microglia, mSOD1(G93A) microglia, and microglia over-expressing wild-type human SOD1, we used primary cultures of microglia and motoneurons in the presence and absence of lipopolysaccharide stimulation. Following activation with lipopolysaccharide, mSOD1(G93A) microglia released more nitric oxide, more superoxide, and less insulin-like growth factor-1 than wild-type microglia. In microglia/motoneuron co-cultures, mSOD1(G93A) microglia induced more motoneuron death and decreased neurite numbers and length compared with wild-type microglia. Mutant SOD1(G93A) microglia also induced more motoneuron injury than microglia over-expressing wild-type human SOD1 in microglia/motoneuron co-cultures. Motoneuron survival was inversely correlated with nitrate + nitrite concentrations in mSOD1(G93A) co-cultures, suggesting the important role of nitric oxide in microglia-induced motoneuron injury. Thus, relative to wild-type microglia, mSOD1(G93A) microglia were more neurotoxic and induced more motoneuron injury than similarly treated wild-type microglia.     

Increased SOD1 association with chromatin,DNA damage,p53 activation,and apoptosis in a cellular model of SOD1-linked ALS

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multi-factorial and remain unclear. Here we examined DNA damage, p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly⁹³ → Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis.     

Proteolytic degradation of tyrosine nitrated proteins

Tyrosine nitration is a covalent posttranslational protein modification that has been detected under several pathological conditions. This study reports that nitrated proteins are degraded by chymotrypsin and that protein nitration enhances susceptibility to degradation by the proteasome. Chymotrypsin cleaved the peptide bond between nitrated-tyrosine 108 and serine 109 in bovine Cu,Zn superoxide dismutase. However, the rate of chymotryptic cleavage of nitrated peptides was considerably slower than control. In contrast, nitrated bovine Cu,Zn superoxide dismutase was degraded at a rate 1. 8-fold faster than that of control by a gradient-purified 20S/26S proteasome fraction from bovine retina. Exposure of PC12 cells to a nitrating agent resulted in the nitration of tyrosine hydroxylase and a 58 +/- 12.5% decline in the steady-state levels of the protein 4 h after nitration. The steady-state levels of tyrosine hydroxylase were restored by selective inhibition of the proteasome activity with lactacystin. These data indicate that nitration of tyrosine residue(s) in proteins is sufficient to induce an accelerated degradation of the modified proteins by the proteasome and that the proteasome may be critical for the removal of nitrated proteins in vivo.     

Inactivation of cytochrome c oxidase by mutant SOD1s in mouse motoneuronal NSC-34 cells is independent from copper availability but is because of nitric oxide

The copper-enzyme cytochrome c oxidase (Cytox) has been indicated as a primary molecular target of mutant copper, zinc superoxide dismutase (SOD1) in familial amyotrophic lateral sclerosis (fALS); however, the mechanism underlying its inactivation is still unclear. As the toxicity of mutant SOD1s could arise from their selective recruitment to mitochondria, it is conceivable that they might compete with Cytox for the mitochondrial copper pool causing Cytox inactivation. To investigate this issue, we used mouse motoneuronal neuroblastoma × spinal cord cell line-34, stably transfected for the inducible expression of low amounts of wild-type or mutant (G93A, H46R, and H80R) human SOD1s and compared the effects observed on Cytox with those obtained by copper depletion. We demonstrated that all mutants analyzed induced cell death and decreased the Cytox activity, but not the protein content of the Cytox subunit II, at difference with copper depletion that also affected subunit II protein. Copper supplementation did not counteract mutant hSOD1s toxicity. Otherwise, the treatment of neuroblastoma × spinal cord cell line-34 expressing G93A, H46R, or H80R hSOD1 mutants, and showing constitutive expression of iNOS and nNOS, with either a NO scavenger, or NOS inhibitors prevented the inhibition of Cytox activity and rescued cell viability. These results support the involvement of NO in mutant SOD1s-induced Cytox damage, and mitochondrial toxicity.     

Antioxidant enzymatic systems in neuronal and glial cell-enriched fractions of rat brain during aging

The activities of Cu,Zn superoxide dismutase, glutathione peroxidase, catalase and glutathione reductase in neuronal and glial cell-enriched fractions obtained from the cerebral cortex of rat brain during aging (15, 30, 90, 350, 750 days of age) were assayed. Our results showed that glutathione peroxidase, catalase and glutathione reductase activities varied little during the examined periods. Only the Cu,Zn superoxide dismutase activity decreased notably from 15th to 750th day of age in both neuronal and glial cells, moreover the activities of all enzymes studied were always detected at lower levels in neuronal cells with respect to glial cells. In agreement with diminished SOD activity, the lipid peroxidation showed an elevated increase with aging; this fact is more evident in neuronal than in glial cells. In conclusion our data show that Cu,Zn superoxide dismutase is the most affected antioxidant enzymatic system of brain aging and it could be responsible for the increased lipid peroxidation in both cell types examined.A preliminary report of these results was presented at the 19th Meeting F.E.B.S. Rome July 2–7, 1989.     

A mutation in dynein rescues axonal transport defects and extends the life span of ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by motoneuron degeneration and muscle paralysis. Although the precise pathogenesis of ALS remains unclear, mutations in Cu/Zn superoxide dismutase (SOD1) account for approximately 20-25% of familial ALS cases, and transgenic mice overexpressing human mutant SOD1 develop an ALS-like phenotype. Evidence suggests that defects in axonal transport play an important role in neurodegeneration. In Legs at odd angles (Loa) mice, mutations in the motor protein dynein are associated with axonal transport defects and motoneuron degeneration. Here, we show that retrograde axonal transport defects are already present in motoneurons of SOD1(G93A) mice during embryonic development. Surprisingly, crossing SOD1(G93A) mice with Loa/+ mice delays disease progression and significantly increases life span in Loa/SOD1(G93A) mice. Moreover, there is a complete recovery in axonal transport deficits in motoneurons of these mice, which may be responsible for the amelioration of disease. We propose that impaired axonal transport is a prime cause of neuronal death in neurodegenerative disorders such as ALS.     

Glutathione and copper, zinc superoxide dismutase are modulated by overexpression of neuronal nitric oxide synthase

This study examines the effects of neuronal nitric oxide overexpression (nNOS) in neuronal and non-neuronal cell lines. The up-regulation of nNOS causes an increase in the intracellular concentration of glutathione (GSH) that was mandatory for counteracting NO-mediated cytotoxicity. Indeed, inhibition of GSH synthesis by buthionine sulfoximine (BSO) significantly enhances NO toxicity. nNOS increase also mediates a down-regulation of copper, zinc superoxide dismutase (SOD1) in terms of mRNA production, protein and activity levels. The nNOS inhibitor (7-Ni), while restores the GSH content, does not recover the SOD1 level, suggesting that NO is not directly involved in SOD1 modulation. SOD1 reduction is most probably due to an increased DNA binding capacity of AP-1, which seems to play a negative role in the capacity of Sp1 to bind to the sod1 gene promoter. Actually, this study demonstrates that nNOS directly interacts with Sp1, both in the cytosol as well as in the nucleus, forming a stable heterocomplex that could have an important physiological role in the modulation of Sp1 activity.     

Oxidative inactivation of calcineurin by Cu,Zn superoxide dismutase G93A, a mutant typical of familial amyotrophic lateral sclerosis

Calcineurin is a serine/threonine phosphatase involved in a wide range of cellular responses to calcium mobilizing signals. Previous evidence supports the notion of the existence of a redox regulation of this enzyme, which might be relevant for neurodegenerative processes, where an imbalance between generation and removal of reactive oxygen species could occur. In a recent work, we have observed that calcineurin activity is depressed in two models for familial amyotrophic lateral sclerosis (FALS) associated with mutations of the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1), namely in neuroblastoma cells expressing either SOD1 mutant G93A or mutant H46R and in brain areas from G93A transgenic mice. In this work we report that while wild-type SOD1 has a protective effect, calcineurin is oxidatively inactivated by mutant SOD1s in vitro; this inactivation is mediated by reactive oxygen species and can be reverted by addition of reducing agents. Furthermore, we show that calcineurin is sensitive to oxidation only when it is in an 'open', calcium-activated conformation, and that G93A-SOD1 must have its redox-active copper site available to substrates in order to exert its pro-oxidant properties on calcineurin. These findings demonstrate that both wild-type and mutant SOD1s can interfere directly with calcineurin activity and further support the possibility of a relevant role for calcineurin-regulated biochemical pathways in the pathogenesis of FALS.     

Overexpression of Abeta is associated with acceleration of onset of motor impairment and superoxide dismutase 1 aggregation in an amyotrophic lateral sclerosis mouse model

Transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) recapitulate the motor impairment of human amyotrophic lateral sclerosis (ALS). The amyloid-beta (Abeta) peptide associated with Alzheimer's disease is neurotoxic. To investigate the potential role of Abeta in ALS development, we generated a double transgenic mouse line that overexpresses SOD1(G93A) and amyloid precursor protein (APP)-C100. The transgenic mouse C100.SOD1(G93A) overexpresses Abeta and shows earlier onset of motor impairment but has the same lifespan as the single transgenic SOD1(G93A) mouse. To determine the mechanism associated with this early-onset phenotype, we measured copper and zinc levels in brain and spinal cord and found both significantly elevated in the single and double transgenic mice compared with their littermate control mice. Increased glial fibrillary acidic protein and decreased APP levels in the spinal cord of C100.SOD1(G93A) mice compared with the SOD1(G93A) mice agree with the neuronal damage observed by immunohistochemical analysis. In the spinal cords of C100.SOD1(G93A) double transgenic mice, soluble Abeta was elevated in mice at end-stage disease compared with the pre-symptomatic stage. Buffer-insoluble SOD1 aggregates were significantly elevated in the pre-symptomatic mice of C100.SOD1(G93A) compared with the age-matched SOD1(G93A) mice, correlating with the earlier onset of motor impairment in the C100.SOD1(G93A) mice. This study supports abnormal SOD1 protein aggregation as the pathogenic mechanism in ALS, and implicates a potential role for Abeta in the development of ALS by exacerbating SOD1(G93A) aggregation.     

Dietary Zn deficiency does not influence systemic blood pressure and vascular nitric oxide signaling in normotensive rats

Because zinc (Zn) is an important component for cell protection against certain oxygen species, it has been suggested that Zn deficiency impairs the potent oxidant defense capacity, which is constitutively provided in the vascular system. However, the influence of dietary Zn deficiency on systemic blood pressure and vascular system is controversial and unclear. We therefore examine the effect of dietary Zn deficiency on systemic blood pressure, a potent superoxide scavenger, aortic Cu/Zn superoxide dismutase (SOD) activity, a most representative synthase of the endothelium-derived relaxing factor, and aortic endothelial nitric oxide synthase (eNOS) expression. Furthermore, the direct effects of intravenous administration of NOS inhibitor, N ^ω-nitro-l-arginine methyl ester (l-NAME), and a SOD mimetic compound, tempol, in normotensives were tested in Wistar-Kyoto (WKY) rats. A Zn-deficient diet (4 wk) contributed to growth retardation, the decrease in thymus weight, and the lower levels of serum Zn compared with the standard diet group. However, no significant difference in conscious systolic and diastolic blood pressure was found in the Zn-deficiency group. The administration of l-NAME caused an increase in the mean arterial pressure (MAP) levels in the two groups of rats and the involvement of the vasodilator nitric oxide (NO) in the regulation of systemic BP in the normotensive state. On the other hand, administration of the superoxide scavenger, tempol, led to a decrease in MAP levels in the two groups of rats, indicating the participation of the oxygen free radical, superoxide, in the maintenance of the systemic BP in a normotensive state. There were no significant differences between the Zn-deficient diet group and the standard diet group in the normotensive state. eNOS expression and Cu/Zn SOD activity in the aorta were also intact in Zn-deficient normotensive rats. These findings suggest that the 4 wk of Zn deficiency was inadequate to alter systemic blood pressure and focal NO signaling in the normotensive state. Long-term Zn deficiency affects the neuronal, immune, and hematopoietic systems, which contribute to systemic and/or local circulation. However, Zn deficiency alone does not cause hypertension and local vascular dysfunction in the normotensive state.     

Superoxide dismutase 1 modulates expression of transferrin receptor

Copper–zinc superoxide dismutase (SOD1) plays a protective role against the toxicity of superoxide, and studies in Saccharomyces cerevisiae and in Drosophila have suggested an additional role for SOD1 in iron metabolism. We have studied the effect of the modulation of SOD1 levels on iron metabolism in a cultured human glial cell line and in a mouse motoneuronal cell line. We observed that levels of the transferrin receptor and the iron regulatory protein 1 were modulated in response to altered intracellular levels of superoxide dismutase activity, carried either by wild-type SOD1 or by an SOD-active amyotrophic lateral sclerosis (ALS) mutant enzyme, G93A-SOD1, but not by a superoxide dismutase inactive ALS mutant, H46R-SOD1. Ferritin expression was also increased by wild-type SOD1 overexpression, but not by mutant SOD1s. We propose that changes in superoxide levels due to alteration of SOD1 activity affect iron metabolism in glial and neuronal cells from higher eukaryotes and that this may be relevant to diseases of the nervous system.     

Increased affinity for copper mediated by cysteine 111 in forms of mutant superoxide dismutase 1 linked to amyotrophic lateral sclerosis

Mutations in Cu,Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (ALS). It has been proposed that neuronal cell death might occur due to inappropriately increased Cu interaction with mutant SOD1. Using Cu immobilized metal-affinity chromatography (IMAC), we showed that mutant SOD1 (A4V, G85R, and G93A) expressed in transfected COS7 cells, transgenic mouse spinal cord tissue, and transformed yeast possessed higher affinity for Cu than wild-type SOD1. Serine substitution for cysteine at the Cys111 residue in mutant SOD1 abolished the Cu interaction on IMAC. C111S substitution reversed the accelerated degradation of mutant SOD1 in transfected cells, suggesting that the Cys111 residue is critical for the stability of mutant SOD1. Aberrant Cu binding at the Cys111 residue may be a significant factor in altering mutant SOD1 behavior and may explain the benefit of controlling Cu access to mutant SOD1 in models of familial ALS.     

Coexpression of yeast copper chaperone (yCCS) and CuZn-superoxide dismutases in Escherichia coli yields protein with high copper contents

To fully understand the function of the Cu- and Zn-containing superoxide dismutases in normal and disordered cells, it is essential to study protein variants with full metal contents. We describe the use of an Escherichia coli-based expression system for the overproduction of human intracellular wild type CuZn-superoxide dismutase (SOD), the CuZnSOD variant F50E/G51E (monomeric), two amyotrophic lateral sclerosis-related mutant CuZnSOD variants (D90A and G93A), and PseudoEC-SOD, all with high Cu contents. This system is based on coexpression of the SOD variants with the yeast copper chaperone yCCS during growth in a medium supplemented with Cu(2+) and Zn(2+). The recombinant SOD enzymes were all found in the cytosol and represented 30-50% of the total bacterial protein. The enzymes were purified to homogeneity and active enzymes were obtained in high yield. The resulting proteins were characterized through immunochemical reactivity and specific activity analyses, in conjunction with mass-, photo-, and atomic absorption-spectroscopy.     

Mutant SOD1-induced neuronal toxicity is mediated by increased mitochondrial superoxide levels

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease in adults, is characterized by the selective degeneration and death of motor neurons leading to progressive paralysis and eventually death. Approximately 20% of familial ALS cases are associated with mutations in SOD1, the gene encoding Cu/Zn-superoxide dismutase (CuZnSOD). Previously, we reported that overexpression of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD or SOD2) attenuates cytotoxicity induced by expression of the G37R-SOD1 mutant in a human neuroblastoma cell culture model of ALS. In the present study, we extended these earlier findings using several different SOD1 mutants (G93C, G85R, and I113T). Additionally, we tested the hypothesis that mutant SOD1 increases mitochondrial-produced superoxide (O(2) (*)) levels and that SOD2 overexpression protects neurons from mutant SOD1-induced toxicity by reducing O(2) (*) levels in mitochondria. In the present study, we demonstrate that SOD2 overexpression markedly attenuates the neuronal toxicity induced by adenovirus-mediated expression of all four SOD1 mutants (G37R, G93C, G85R, or I113T) tested. Utilizing the mitochondrial-targeted O(2) (*)-sensitive fluorogenic probe MitoSOX Red, we observed a significant increase in mitochondrial O(2) (*) levels in neural cells expressing mutant SOD1. These elevated O(2) (*) levels in mitochondria were significantly diminished by the overexpression of SOD2. These data suggest that mitochondrial-produced O(2) (*) radicals play a critical role in mutant SOD1-mediated neuronal toxicity and implicate mitochondrial-produced free radicals as potential therapeutic targets in ALS.     

Dynamic properties of the G93A mutant of copper-zinc superoxide dismutase as detected by NMR spectroscopy: implications for the pathology of familial amyotrophic lateral sclerosis

The backbone assignment of the copper-zinc superoxide dismutase amyotrophic lateral sclerosis G93A mutant was performed on an (15)N-enriched protein sample. (15)N R(1), R(2), and R(1)(rho) and (15)N-(1)H NOE experiments were then carried out at 600 MHz on G93A Cu(2)Zn(2)SOD and the values compared to the dynamics data for the "wild-type" protein. In addition, (15)N and (1)H chemical shift comparisons between wild-type Cu(2)Zn(2)SOD and its G93A mutant were also made. G93A exhibits a higher mobility than wild-type Cu(2)Zn(2)SOD, particularly in loops III and V, on a time scale faster than the rate of protein tumbling. There are also distinct chemical shift and NOE differences in residues 35-42 and 92-95, which comprise these loops. These two regions of Cu(2)Zn(2)SOD form the end of the beta-barrel termed the "beta-barrel plug" [Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982) J. Mol. Biol. 160, 181-217]. The increased mobility and reduction of the number of observed NOEs in this region indicate an opening of the beta-barrel that may lead to amyloid fibrillogenesis. Alternatively, a motor neuron-specific substrate may bind this region of the protein, leading to deleterious modifications and/or reactions.     

Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has altered redox activities and is toxic to motor neurons in vitro. Using bovine SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding activity six times faster than superoxide dismutase activity, whereas inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine. Although urate, xanthine, and ascorbate can protect superoxide dismutase activity of Cu,Zn SOD from inactivation, they were not effective at protecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydrogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible.