Maria Teresa Carrì

Expression of a Cu,Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis induces mitochondrial alteration and increase of cytosolic Ca2+ concentration in transfected neuroblastoma SH-SY5Y cells

We have set up a model system for familial amyotrophic lateral sclerosis (FALS) by transfecting human neuroblastoma cell line SH‐SY5Y with plasmids directing constitutive expression of either wild‐type human Cu,Zn superoxide dismutase (Cu,ZnSOD) or a mutant of this enzyme (G93A) associated with FALS. We have tested mitochondrial function and determined cytosolic Ca2+ concentration in control cells (untransfected) and in cells expressing either wild‐type Cu,ZnSOD or G93A. We report that G93A induces a significant loss of mitochondrial membrane potential, an increased sensitivity toward valinomycin and a parallel increase in cytosolic Ca2+ concentration. The above phenomena are not related to total Cu,ZnSOD content and activity in the cell.

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.

Neurodegeneration in amyotrophic lateral sclerosis: the role of oxidative stress and altered homeostasis of metals

Amyotrophic lateral sclerosis is one of the most common neurodegenerative disorders, with an incidence of about 1/100,000. One of the typical features of this progressive, lethal disease, occurring both sporadically and as a familial disorder, is degeneration of cortical and spinal motor neurones. Present evidence indicates that loss of neurones in patients results from a complex interplay among oxidative injury, excitotoxic stimulation, dysfunction of critical proteins and genetic factors. This review focuses on existing evidence that oxidative stress is a major culprit in the pathogenesis of amyotrophic lateral sclerosis. An increase in reactive oxygen species and in products of oxidation has been observed both in post-mortem samples and in experimental models for ALS. This increase may be consequent to altered metabolism of copper and iron ions, that share the property to undergo redox cycling and generate reactive oxygen species. Metal-mediated oxidative stress would lead to several intracellular alterations and contribute to the induction of cell death pathways.

Amyotrophic lateral sclerosis: from current developments in the laboratory to clinical implications

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive degeneration of motor neurons occurring both as a sporadic and a familial disease. The etiology of ALS remains unknown, but one fifth of instances are due to specific gene defects, the best characterized of which is point mutations in the gene coding for Cu/Zn superoxide dismutase (SOD1). Because sporadic and familial ALS affect the same neurons with similar pathology, it is hoped that understanding these gene defects will help in devising therapies effective in both forms. A wealth of evidence has been collected in rodents made transgenic for mutant SOD1, which represent the best available models for familial ALS. Mutant SOD1 likely induces selective vulnerability of motor neurons through a combination of several mechanisms, including protein misfolding, mitochondrial dysfunction, oxidative damage, cytoskeletal abnormalities and defective axonal transport, excitotoxicity, inadequate growth factor signaling, and inflammation. Damage within motor neurons is enhanced by noxious signals originating from nonneuronal neighboring cells, where mutant SOD1 induces an inflammatory response that accelerates disease progression. The clinical implication of these findings is that promising therapeutic approaches can be derived from multidrug treatments aimed at the simultaneous interception of damage in both motor neurons and nonmotor neuronal cells.

Mitochondrial dysfunction in ALS

In the present article, we review the many facets of mitochondrial dysfunction in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease due to loss of upper motor neurons in cerebral cortex and lower motor neurons in brainstem and spinal cord. Accumulating evidence from recent studies suggests that the many, interconnected facets of mitochondrial dysfunction may play a more significant role in the etiopathogenesis of this disorder than previously thought. This notion stems from our expanding knowledge of the complex physiology of mitochondria and of alteration of their properties that might confer an intrinsic susceptibility to long-lived, post-mitotic motor neurons to energy deficit, calcium mishandling and oxidative stress. The wealth of evidence implicating mitochondrial dysfunction as a major event in the pathology of ALS has prompted new studies aimed to the development of new mitochondria-targeted therapies. However, it is now clear that drugs targeting more than one aspect of mitochondrial dysfunction are needed to fight this devastating disease.

Treatment with lithium carbonate does not improve disease progression in two different strains of SOD1 mutant mice

It has been shown that chronic treatment with lithium carbonate (Li2CO3) in presymptomatic SOD1G93A transgenic male mice, a model of ALS, was able to remarkably increase their lifespan through the activation of autophagy and the promotion of mitochondriogenesis and neurogenesis. This prompted us to test the lithium effect also in female SOD1G93A mice with two phenotypes of different disease severity. Female SOD1G93A mice of C57BL/6J or 129S2/Sv genetic background were treated daily with Li2CO3 37 mg/kg (1 mEq/kg) i.p. starting from age 75 days until death. Grip strength, latency to fall on rotarod and body weight were monitored twice weekly. At the time of death the spinal cord was removed to assess the number of motor neurons and to measure the expression of a marker of autophagy (LCII) and the activity of mitochondrial complex IV. We observed a significant anticipation of the onset and reduced survival in 129Sv/G93A and no effect in C57/G93A mice treated with lithium compared to vehicle treated mice. Moreover, lithium neither exerted neuroprotective effects nor increased the expression of LCII and the activity of mitochondrial complex IV in the spinal cord. The present study does not identify any therapeutic or neuroprotective effect of lithium in SOD1G93A female mice.

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.

Neuroprotective and neuritogenic activities of novel multimodal iron-chelating drugs in motor-neuron-like NSC-34 cells and transgenic mouse model of amyotrophic lateral sclerosis

Novel therapeutic approaches for the treatment of neurodegenerative disorders comprise drug candidates designed specifically to act on multiple central nervous system targets. We have recently synthesized multifunctional, nontoxic, brain‐permeable iron‐chelating drugs, M30 and HLA20, possessing the A‐propargylamine neuroprotective moiety of rasagiline (Azilect) and the iron‐chelating moiety of VK28. The present study demonstrates that M30 and HLA20 possess a wide range of pharmacological activities in mouse NSC‐34 motor neuron cells, including neuroprotective effects against hydrogen peroxide‐ and 3‐morpholinosydnonimine‐induced neurotoxicity, induction of differentiation, and up‐regulation of hypoxia‐inducible factor (HIF)‐la and HIF‐target genes (enolasel and vascular endothe‐lial growth factor). Both compounds induced NSC‐34 neuritogenesis, accompanied by a marked increase in the expression of brain‐derived neurotrophic factor and growth‐associated protein‐43, which was inhibited by PD98059 and GF109203X, indicating the involvement of mitogen‐activated protein kinase and protein kinase C pathways. A major finding was the ability of M30 to significantly extend the survival of G93A‐SOD1 amyotrophic lateral sclerosis mice and delay the onset of the disease. These properties of the novel multimodal iron‐chelating drugs possessing neuroprotective/ neuritogenic activities may offer future therapeutic possibilities for motor neurodegenerative diseases.— Kupershmidt, L., Weinreb, O., Amit, T., Mandel, S., Carri, M. T., Youdim, M. B. H. Neuroprotective and neuritogenic activities of novel multimodal iron‐chelating drugs in motor‐neuron‐like NSC‐34 cells and transgenic mouse model of amyotrophic lateral sclerosis. FASEBJ. 23, 3766‐3779 (2009). www.fasebj.org

Evidence for co-regulation of Cu,Zn superoxide dismutase and metallothionein gene expression in yeast through transcriptional control by copper via the ACE 1 factor.

Saccharomyces cerevisiae mutant strain DTY26, lacking ACE1, the protein mediator for the induction or metallothionein gene expression, is unable to increase Cu,Zn superoxide dismutase mRNA in response to copper. In the wild‐type strain DTY22 transcription or both Cu,Zn superoxide dismutase and metallothionein genes is induced by copper and silver, as expected on the basis of previous results indicating that ACE1 binds only Ag(I) besides Cu(I). We conclude that at the transcriptional level Cu,ZnSOD is co‐regulation with metallothionein. Furthermore, structural similarities between the two promoters were found, which could explain the co‐regulation effect and the quantitative differences in the response of the two genes to copper.

Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN) in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS) and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD) imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS.ResultsTo gain insights into the molecular mechanisms of SOD1G93A associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage – dependent cytosolic Ca2+ elevations and mitochondria – controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca2+ elevations in WT (SH-SY5YWT) and G93A (SH-SY5YG93A) expressing cells were observed. In contrast, mitochondrial intracellular Ca2+ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation.ConclusionIn this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca2+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells an established cellular model of ALS. The primary goals of this paper are (1) method development, and (2) screening for deficits in mutant cells on the single cell level. On the technological level, our method promises to serve as a valuable tool to identify mitochondrial and Ca2+-related defects during G93A-mediated MN degeneration. In addition, our experiments support a model where a specialized interplay between cytosolic calcium profiles and mitochondrial mechanisms contribute to the selective degeneration of neurons in ALS.