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Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells
Published on: October 4, 2017
Superoxide dismutase-1 and other proteins in inclusions from transgenic amyotrophic lateral sclerosis model mice
Daniel Bergemalm1, Karin Forsberg, Vaibhav Srivastava
1Department of Medical Biosciences, Clinical Chemistry, Umeå University, Umeå, Sweden.
Abstract:
Mutant superoxide dismutase-1 (SOD1) causes amyotrophic lateral sclerosis (ALS) through a cytotoxic mechanism of unknown nature. A hallmark in ALS patients and transgenic mouse models carrying human SOD1 (hSOD1) mutations are hSOD1-immunoreactive inclusions in spinal cord ventral horns. The hSOD1 inclusions may block essential cellular functions or cause toxicity through sequestering of other proteins. Inclusions from four different transgenic mouse models were examined after density gradient ultracentrifugation. The inclusions are complex structures with heterogeneous densities and are disrupted by detergents. The aggregated hSOD1 was mainly composed of subunits that lacked the native stabilizing intra-subunit disulfide bond. A proportion of subunits formed hSOD1 oligomers or was bound to other proteins through disulfide bonds. Dense inclusions could be isolated and the protein composition was analyzed using proteomic techniques. Mutant hSOD1 accounted for half of the protein. Ten other proteins were identified. Two were cytoplasmic chaperones, four were cytoskeletal proteins, and 4 were proteins that normally reside in the endoplasmic reticulum (ER). The presence of ER proteins in inclusions containing the primarily cytosolic hSOD1 further supports the notion that ER stress is involved in ALS.
Insights
Mutant superoxide dismutase-1 (SOD1) aggregates in amyotrophic lateral sclerosis (ALS) form complex inclusions. These inclusions contain SOD1, chaperones, cytoskeletal proteins, and endoplasmic reticulum proteins, suggesting ER stress in ALS.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Mutant superoxide dismutase-1 (SOD1) is a known cause of amyotrophic lateral sclerosis (ALS).
- SOD1 inclusions are a pathological hallmark in ALS patients and transgenic mouse models.
- The exact cytotoxic mechanism of mutant SOD1 in ALS remains unclear.
Purpose of the Study:
- To investigate the composition and structure of mutant human SOD1 (hSOD1) inclusions.
- To identify proteins associated with hSOD1 aggregates in ALS.
- To explore the potential role of endoplasmic reticulum (ER) stress in ALS pathogenesis.
Main Methods:
- Density gradient ultracentrifugation was used to isolate inclusions from four transgenic mouse models.
- Proteomic techniques were employed to analyze the protein composition of isolated inclusions.
- Detergent disruption was used to assess inclusion structure.
Main Results:
- hSOD1 inclusions are complex structures with heterogeneous densities, disrupted by detergents.
- Aggregated hSOD1 subunits primarily lacked the native disulfide bond and formed oligomers or disulfide-bound complexes.
- Proteomic analysis revealed mutant hSOD1 constituted half the inclusion mass, with identified proteins including chaperones, cytoskeletal elements, and ER-resident proteins.
Conclusions:
- Mutant hSOD1 inclusions are intricate molecular assemblies.
- The presence of ER proteins within cytosolic hSOD1 inclusions supports a role for ER stress in ALS.
- Understanding inclusion composition may offer insights into ALS pathogenesis and therapeutic targets.
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