RESUMO
Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1.
Assuntos
Esclerose Lateral Amiotrófica , Neurônios Motores/efeitos dos fármacos , Mutação/genética , Compostos Organometálicos/administração & dosagem , Superóxido Dismutase/genética , Tiossemicarbazonas/administração & dosagem , Administração Oral , Fatores Etários , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/mortalidade , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas de Transporte de Cátions/genética , Cromatografia em Gel , Complexos de Coordenação , Transportador de Cobre 1 , Modelos Animais de Doenças , Humanos , Locomoção/efeitos dos fármacos , Locomoção/genética , Camundongos , Camundongos Transgênicos , Fenótipo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1RESUMO
The final steps in the absorption and excretion of copper at the molecular level are accomplished by 2 closely related proteins that catalyze the ATP-dependent transport of copper across the plasma membrane. These proteins, ATP7A and ATP7B, are encoded by the genes affected in human genetic copper-transport disorders, namely, Menkes and Wilson diseases. We studied the effect of copper perfusion of an isolated segment of the jejunum of ATP7A transgenic mice on the intracellular distribution of ATP7A by immunofluorescence of frozen sections. Our results indicate that ATP7A is retained in the trans-Golgi network under copper-limiting conditions, but relocalized to a vesicular compartment adjacent to the basolateral membrane in intestines perfused with copper. The findings support the hypothesis that the basolateral transport of copper from the enterocyte into the portal blood may involve ATP7A pumping copper into a vesicular compartment followed by exocytosis to release the copper, rather than direct pumping of copper across the basolateral membrane.