Journal
JOURNAL OF NEUROCHEMISTRY
Volume 140, Issue 1, Pages 140-150Publisher
WILEY-BLACKWELL
DOI: 10.1111/jnc.13864
Keywords
aggregation; amyotrophic lateral sclerosis; live imaging; oxidation; superoxide dismutase 1
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Funding
- National Institutes of Neurological Disease and Stroke [P01 NS049134]
- UCSD/UCLA NIDDK Diabetes Research Center [P30 DK063491]
- Amyotrophic Lateral Sclerosis Association
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A common property of Cu/Zn superoxide dismutase 1 (SOD1), harboring mutations associated with amyotrophic lateral sclerosis, is a high propensity to misfold and form abnormal aggregates. The aggregation of mutant SOD1 has been demonstrated in vitro, with purified proteins, in mouse models, in human tissues, and in cultured cell models. In vitro translation studies have determined that SOD1 with amyotrophic lateral sclerosis mutations is slower to mature, and thus perhaps vulnerable to off-pathway folding that could generate aggregates. The aggregation of mutant SOD1 in living cells can be monitored by tagging the protein with fluorescent fluorophores. In this study, we have taken advantage of the Dendra2 fluorophore technology in which excitation can be used to switch the output color from green to red, thereby clearly creating a time stamp that distinguishes preexisting and newly made proteins. In cells that transiently overexpress the Ala 4 to Val variant of SOD1-Dendra2, we observed that newly made mutant SOD1 was rapidly captured by pathologic intracellular inclusions. In cell models of mutant SOD1 aggregation over-expressing untagged A4V-SOD1, we observed that immature forms of the protein, lacking a Cu cofactor and a normal intramolecular disulfide, persist for extended periods. Our findings fit with a model in which immature forms of mutant A4V-SOD1, including newly made protein, are prone to misfolding and aggregation.
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