title: Metal-deficient aggregates and diminished copper found in cells expressing SOD1 mutations that cause ALS.
abstract: Disruptions in metal ion homeostasis have been described in association with amyotrophic lateral sclerosis (ALS) for a number of years but the precise mechanism of involvement is poorly understood. Metal ions are especially important to familial ALS cases caused by mutations in the metalloenzyme copper-zinc superoxide dismutase (SOD1). To investigate the role of metals in aggregation of mutant SOD1, we have examined the localization of metal ions in a cell culture model of overexpression. Chinese hamster ovary cells (CHO-K1) were transfected to overexpress SOD1 fused to yellow fluorescent protein (YFP) to readily identify the transfected cells and the intracellular aggregates that develop in the cells expressing mutant or wild-type (WT) SOD1. The concentration and distribution of iron, copper, and zinc were determined for four SOD1 mutants (A4V, G37R, H80R, and D125H) as well as a WT SOD1 using X-ray fluorescence microscopy (XFM). Results demonstrated that the SOD1 aggregates were metal-deficient within the cells, which is consistent with recent in vitro studies. In addition, all SOD1 mutants showed significantly decreased copper content compared to the WT SOD1 cells, regardless of the mutant's ability to bind copper. These results suggest that SOD1 overexpression creates an unmet demand on the cell for copper. This is particularly true for the SOD1 mutants where copper delivery may also be impaired. Hence, the SOD1 mutants are less stable than WT SOD1 and if copper is limited, aggregate formation of the metal-deficient, mutant SOD1 protein occurs.
authors: Department of Chemistry, Stony Brook University Stony Brook, NY, USA.
authors: Megan W
authors: MW
authors: Bourassa
authors: Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, Santa Fe Health Care Alzheimer's Disease Research Center, McKnight Brain Institute, University of Florida Gainesville, FL, USA.
authors: Hilda H
authors: HH
authors: Brown
authors: Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, Santa Fe Health Care Alzheimer's Disease Research Center, McKnight Brain Institute, University of Florida Gainesville, FL, USA.
authors: David R
authors: DR
authors: Borchelt
authors: Advanced Photon Source, Argonne National Laboratory Argonne, IL, USA.
authors: Stefan
authors: S
authors: Vogt
authors: Department of Chemistry, Stony Brook University Stony Brook, NY, USA ; Photon Sciences Directorate, Brookhaven National Laboratory Upton, NY, USA.
authors: Lisa M
authors: LM
authors: Miller
keywords: X-ray fluorescence microscopy; amyotrophic lateral sclerosis; superoxide dismutase; synchrotron