Roberta Gabbianelli

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.

Aberrant Copper Chemistry as a Major Mediator of Oxidative Stress in a Human Cellular Model of Amyotrophic Lateral Sclerosis

Abstract : We have investigated the response to oxidative stress in a model system obtained by stable transfection of the human neuroblastoma cell line SH‐SY5Y with plasmids directing constitutive expression of either wild‐type human Cu,Zn superoxide dismutase or a mutant of this enzyme (H46R) associated with familial amyotrophic lateral sclerosis. We report that expression of mutant H46R Cu,Zn superoxide dismutase induces a selective increase in paraquat sensitivity that is reverted by addition of d‐penicillamine. Furthermore, expression of this mutant enzyme affects the activity of the endogenous wild‐type enzyme both in basal conditions and in copper overloading experiments. Our data indicate that aberrant metal chemistry of this mutant enzyme is the actual mediator of oxidative stress and that concurrent impairment of the activity of wild‐type endogenous enzyme compromises the cell’s ability to respond to oxidative stress.

Calcineurin Activity Is Regulated Both by Redox Compounds and by Mutant Familial Amyotrophic Lateral Sclerosis‐Superoxide Dismutase

Calcineurin (CN) is a protein phosphatase involved in a wide range of cellular responses to calcium‐mobilizing signals, and a role for this enzyme in neuropathology has been postulated. We have investigated the possibility that redox modulation of CN activity is relevant to neuropathological conditions where an imbalance in reactive oxygen species has been described. We have monitored CN activity in cultured human neuroblastoma SH‐SY5Y cells and obtained evidence that CN activity is promoted by treatment with ascorbate or dithiothreitol and impaired by oxidative stress. Evidence for the existence of a redox regulation of this enzyme has been also obtained by overexpression of wild‐type antioxidant Cu,Zn superoxide dismutase (SOD1) that promotes CN activity and protects it from oxidative inactivation. On the contrary, overexpression of mutant SOD1s associated with familial amyotrophic lateral sclerosis (FALS) impairs CN activity both in transfected human neuroblastoma cell lines and in the motor cortex of brain from FALS‐transgenic mice. These data suggest that CN might be a target in the pathogenesis of SOD1‐linked FALS.

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.

Voltage-activated sodium currents in a cell line expressing a Cu,Zn superoxide dismutase typical of familial ALS.

THE whole-cell configuration of the patch-clamp recording was used to study the voltage-dependent Na+ currents in a model system for the familial form of amyotrophic lateral sclerosis (ALS) associated with mutations in Cu, Zn superoxide dismutase. Here we report that the amplitude of voltage-gated Na+ currents is significantly reduced in cell lines expressing mutant Cu, Zn superoxide dismutase G93A when compared with the parental, untransfected cell line and to a cell line expressing the wild-type enzyme. This effect is associated with a shift toward positive values of the steady-state inactivation curve of the Na+ currents. These results indicate that expression of a Cu, Zn superoxide dismutase typical of patients affect with familial ALS influence the functionality of the voltage-dependent Na+channels; this effect may contribute to the pathogenesis of the disease.

Distinctive functional features in prokaryotic and eukaryotic Cu, Zn superoxide dismutases

Abstract Bacterial and eukaryotic Cu,Zn superoxide dismutases show remarkable differences in the active site region and in their quaternary structure organization. We report here a functional comparison between four Cu,Zn superoxide dismutases from Gram-negative bacteria and the eukaryotic bovine enzyme. Our data indicate that bacterial dimeric variants are characterized by catalytic rates higher than that of the bovine enzyme, probably due to the solvent accessibility of their active site. Prokaryotic Cu,Zn superoxide dismutases also show higher resistance to hydrogen peroxide inactivation and lower HCO3 --dependent peroxidative activity. Moreover, unlike the eukaryotic enzyme, all bacterial variants are susceptible to inactivation by chelating agents and show variable sensitivity to proteolytic attack, with the E. coli monomeric enzyme showing higher rates of inactivation by EDTA and proteinase K. We suggest that differences between individual bacterial variants could be due to the influence of modifications at the dimer interface on the enzyme conformational flexibility.

A study of the dual role of copper in superoxide dismutase as antioxidant and pro-oxidant in cellular models of amyotrophic lateral sclerosis

All living organisms require dietary copper for continued growth and development. This nutritional requirement arises from the essential role of the metal in the function of cuproenzymes, which play a critical role in the biochemistry of oxygen activation, not only for energy transduction (e.g. ATP synthesis by cytochrome oxidase or dopamine α-hydroxilation), but also in futile oxygen activation for chemical transformation of metabolic intermediates (e.g. H2O2-producing amine oxidase) (Linder and Hazegh-Azam, 1996). However, excess copper — as exemplified by non-physiological conditions such as experimental copper overload or by clinical conditions related to impaired metal transport as Wilson disease — mediates free radical production and direct oxidation of cellular components. Reduction of copper by physiological reductants triggers a series of radical reactions. Autoxidation of Cu+ yields, which dismutes to H2O2, reacting in turn with Cu+ with the ultimate production of OH, the actual agent of oxidative damage (Fridovich, 1995). In order to prevent and counterbalance such reactions, living organisms have evolved various mechanisms of defense. These involve, besides the direct sequestering of copper and other metal ions by metallothioneins, so that they never exist in the free form, a complex system of oxyradicals interception including several enzymatic and non-enzymatic scavengers. Among the species involved in oxyradicals interception, a key role is played by Cu,Zn Superoxide dismutase (SOD) where copper scavenges the primary source of damage (see above) at diffusion-limited rate.