Monica Nencini

Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials

Recent studies suggest that the toxicity of familial amyotrophic lateral sclerosis mutant Cu, Zn superoxide dismutase (SOD1) arises from its selective recruitment to mitochondria. Here we demonstrate that each of 12 different familial ALS-mutant SOD1s with widely differing biophysical properties are associated with mitochondria of motoneuronal cells to a much greater extent than wild-type SOD1, and that this effect may depend on the oxidation of Cys residues. We demonstrate further that mutant SOD1 proteins associated with the mitochondria tend to form cross-linked oligomers and that their presence causes a shift in the redox state of these organelles and results in impairment of respiratory complexes. The observation that such a diverse set of mutant SOD1 proteins behave so similarly in mitochondria of motoneuronal cells and so differently from wild-type SOD1 suggests that this behavior may explain the toxicity of ALS-mutant SOD1 proteins, which causes motor neurons to die.

Cysteine 111 affects aggregation and cytotoxicity of mutant CU/ZN superoxide dismutase associated with familial amyotrophic lateral sclerosis

Converging evidence indicates that aberrant aggregation of mutant Cu,Zn-superoxide dismutase (mutSOD1) is strongly implicated in familial amyotrophic lateral sclerosis (FALS). MutSOD1 forms high molecular weight oligomers, which disappear under reducing conditions, both in neural tissues of FALS transgenic mice and in transfected cultured cells, indicating a role for aberrant intermolecular disulfide cross-linking in the oligomerization and aggregation process. To study the contribution of specific cysteines in the mechanism of aggregation, we mutated human SOD1 in each of its four cysteine residues and, using a cell transfection assay, analyzed the solubility and aggregation of those SOD1s. Our results suggest that the formation of mutSOD1 aggregates are the consequence of covalent disulfide cross-linking and non-covalent interactions. In particular, we found that the removal of Cys-111 strongly reduces the ability of a range of different FALS-associated mutSOD1s to form aggregates and impair cell viability in cultured NSC-34 cells. Moreover, the removal of Cys-111 impairs the ability of mutSOD1s to form disulfide cross-linking. Treatments that deplete the cellular pool of GSH exacerbate mutSOD1s insolubility, whereas an overload of intracellular GSH or overexpression of glutaredoxin-1, which specifically catalyzes the reduction of protein-SSG-mixed disulfides, significantly rescues mutSOD1s solubility. These data are consistent with the view that the redox environment influences the oligomerization/aggregation pathway of mutSOD1 and point to Cys-111 as a key mediator of this process.

Glutaredoxin 2 prevents aggregation of mutant SOD1 in mitochondria and abolishes its toxicity

Vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS) arises from a combination of several mechanisms, including protein misfolding and aggregation, mitochondrial dysfunction and oxidative damage. Protein aggregates are found in motoneurons in models for ALS linked to a mutation in the gene coding for Cu,Zn superoxide dismutase (SOD1) and in ALS patients as well. Aggregation of mutant SOD1 in the cytoplasm and/or into mitochondria has been repeatedly proposed as a main culprit for the degeneration of motoneurons. It is, however, still debated whether SOD1 aggregates represent a cause, a correlate or a consequence of processes leading to cell death. We have exploited the ability of glutaredoxins (Grxs) to reduce mixed disulfides to protein thiols either in the cytoplasm and in the IMS (Grx1) or in the mitochondrial matrix (Grx2) as a tool for restoring a correct redox environment and preventing the aggregation of mutant SOD1. Here we show that the overexpression of Grx1 increases the solubility of mutant SOD1 in the cytosol but does not inhibit mitochondrial damage and apoptosis induced by mutant SOD1 in neuronal cells (SH-SY5Y) or in immortalized motoneurons (NSC-34). Conversely, the overexpression of Grx2 increases the solubility of mutant SOD1 in mitochondria, interferes with mitochondrial fragmentation by modifying the expression pattern of proteins involved in mitochondrial dynamics, preserves mitochondrial function and strongly protects neuronal cells from apoptosis. The toxicity of mutant SOD1, therefore, mostly arises from mitochondrial dysfunction and rescue of mitochondrial damage may represent a promising therapeutic strategy.

Cell death in amyotrophic lateral sclerosis: interplay between neuronal and glial cells

Mutations in the gene coding for the ubiquitous, anti‐oxidant enzyme Cu,Zn superoxide dismutase (SOD1) are associated with familial amyotrophic lateral sclerosis (fALS), a fatal disease characterized by selective loss of motor neurons. Expression of a mutant SOD1 typical of fALS patients restricted to either motor neurons or astrocytes is insufficient to generate a pathological phenotype in mouse models, suggesting that a deleterious interplay between different cell types is necessary for the pathogenesis of the disease. In this study, we demonstrate the actual role of a functional cross‐talk between glial and neuronal cells expressing fALS mutant G93A‐SOD1, where an increase in the production of reactive oxygen species occurs. We show that human glioblastoma cells expressing G93A‐SOD1 induce activation of caspase‐1, release of cytokines, and activation of apoptotic pathways in cocultured human neuroblastoma cells also expressing G93A‐SOD1. Activation of caspase‐1 and caspase‐3 is observed also in neuroblastoma lines expressing other fALS‐SOD1s (G37R, G85R, and I113T) cocultured with glioblastoma lines expressing the corresponding mutant enzymes. These effects are consequent to activation of inflammatory processes in G93A‐glioblastoma cells stimulated by cocultured G93A‐neuroblastoma. Furthermore, selective death of embryonal spinal motor neurons from G93A‐SOD1 transgenic mice is induced by coculture with G93A‐glioblastoma and prevented by inhibition of NO synthase. Proinflammatory cytokines, interferon‐‐γ and nitric oxide are among the molecular signals exchanged between glial and neuronal cells that generate a functional interplay between the two cell types. This cross‐talk may be crucial for the pathogenesis of SOD1‐linked fALS but also for the more common sporadic form of the disease, where markers of increased oxidative stress and of glial activation have been found.

Nuclear accumulation of mRNAs underlies G4C2 repeat-induced translational repression in a cellular model of C9orf72 ALS

A common feature of non‐coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic because they sequester RNA‐binding proteins, thus affecting various steps of post‐transcriptional gene regulation. However, the precise step that is affected by C9orf72 GGGGCC (G4C2) repeat expansion, the major genetic cause of amyotrophic lateral sclerosis (ALS), is still poorly defined. In this work, we set out to characterise these mechanisms by identifying proteins that bind to C9orf72 RNA. Sequestration of some of these factors into RNA foci was observed when a (G4C2)31 repeat was expressed in NSC34 and HeLa cells. Most notably, (G4C2)31 repeats widely affected the distribution of Pur‐alpha and its binding partner fragile X mental retardation protein 1 (FMRP, also known as FMR1), which accumulate in intra‐cytosolic granules that are positive for stress granules markers. Accordingly, translational repression is induced. Interestingly, this effect is associated with a marked accumulation of poly(A) mRNAs in cell nuclei. Thus, defective trafficking of mRNA, as a consequence of impaired nuclear mRNA export, might affect translation efficiency and contribute to the pathogenesis of C9orf72 ALS.

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.

Oxidative modulation of nuclear factor-κB in human cells expressing mutant fALS-typical superoxide dismutases

Previous evidence supports the notion of a redox regulation of protein phosphatase calcineurin that might be relevant for neurodegenerative processes where an imbalance between generation and removal of reactive oxygen species occurs. We have recently observed that calcineurin activity is depressed in human neuroblastoma cells expressing Cu,Zn superoxide dismutase (SOD1) mutant G93A and in brain areas from G93A transgenic mice, and that mutant G93A‐SOD1 oxidatively inactivates calcineurin in vitro. We have studied the possibility that, by interfering directly with calcineurin activity, mutant SOD1 can modulate pathways of signal transduction mediated by redox‐sensitive transcription factors. In this paper, we report a calcineurin‐dependent activation of nuclear factor‐κB (NF‐κB) induced by the expression of familial amyotrophic lateral sclerosis (fALS)‐SOD1s in human neuroblastoma cell lines. Alteration of the phosphorylation state of IκBα (the inhibitor of NF‐κB translocation into the nucleus) and induction of cyclooxygenase 2 are consistent with the up‐regulation of this transcription factor in this system. All of these modifications might be relevant to signaling pathways involved in the pathogenesis of fALS.

Activity of protein phosphatase calcineurin is decreased in sporadic and familial amyotrophic lateral sclerosispatients.

Calcineurin (CaN) is a Ser/Thr protein phosphatase involved in a wide range of cellular responses to calcium mobilizing signals. Previous evidence supports the notion that calcineurin is oxidatively inhibited by mutant Cu, Zn superoxide dismutase (SOD1) typical of familial ALS patients in vitro and in transgenic mice. We report that calcineurin activity is markedly inhibited in lymphocytes from 37 sporadic, eight familial ALS patients and an asymptomatic subject carrying an SOD1 mutation as compared to 28 healthy controls. Two other healthy subjects, heterozygous for the D90A mutation from a recessive pedigree, have normal calcineurin activity. Immunoreactive CaN protein, age, sex and riluzole treatment are not related to calcineurin activity in samples from patients. However, we have observed a marked increase in total protein oxidation in extracts from ALS lymphocytes, as compared to extracts from control subjects. These data confirm that modification of calcineurin activity and possibly of calcineurin‐mediated pathways of signal transduction (including modulation of apoptotic neuronal death) may contribute to the pathogenesis of ALS.

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

J. Neurochem. (2010) 112, 183–192.

Inflammatory cytokines increase mitochondrial damage in motoneuronal cells expressing mutant SOD1

Recent studies indicate that molecular signals from microglia determine disease progression in transgenic mice overexpressing mutant superoxide dismutase (mutSOD1) typical of amyotrophic lateral sclerosis patients and that toxicity of mutSOD1 in motor neurons descends from its tendency to associate with mitochondria. To assess whether the neurotoxicity of mutSOD1 is influenced by signals from glia, we challenged motoneuronal cells overexpressing either wild-type or mutant SOD1 with inflammatory cytokines. We have obtained evidence that combined treatment with tumor necrosis factor alpha and interferon gamma increases the fraction of both wtSOD1 and mutSOD1 associated with mitochondria, but these inflammatory cytokines dramatically alter morphological features and functionality of mitochondria only in cells expressing mutSOD1. As an effect downstream the increase in mitochondria-associated mutSOD1, the ratio between reduced and oxidized glutathione further shifts toward the oxidized form in this compartment and a clear death phenotype is evoked upon treatment with inflammatory cytokines. These results suggest that signals coming from non-neuronal cells contribute to death of motor neurons induced by mutSOD1 through reinforcement of mitochondrial damage.