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Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells
Published on: October 4, 2017
Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS
Jiou Wang1, George W Farr, Caroline J Zeiss
1Howard Hughes Medical Institute, Department of Genetics, Section of Comparative Medicine, and Department of Neurosurgery, Yale University School of Medicine, New Haven, CT 06510, USA.
Abstract:
Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially "compensated" to maintain the misfolded protein in a soluble state, become progressively unable to do so.
Insights
Amyotrophic lateral sclerosis (ALS) linked to superoxide dismutase 1 (SOD1) involves protein misfolding. Transgenic mice expressing misfolding-prone SOD1 developed motor neuron disease, showing protein aggregation and insolubility over time.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to mutations in superoxide dismutase 1 (SOD1).
- Misfolding and aggregation of mutant SOD1 are implicated in motor neuron degeneration.
- Understanding the cellular fate of misfolded SOD1 is crucial for ALS pathogenesis research.
Purpose of the Study:
- To investigate the expression, aggregation, and cellular fate of a misfolding-prone mutant human SOD1 (G85R) in a transgenic mouse model.
- To determine the temporal progression of G85R SOD1 misfolding, aggregation, and associated motor symptoms.
- To explore chaperone interactions with misfolded SOD1 during disease progression.
Main Methods:
- Generation of transgenic mice expressing human SOD1 G85R fused to YFP.
- In situ RNA hybridization to track SOD1 expression in spinal cords.
- Biochemical analyses (solubility, gel filtration) of spinal cord extracts.
- Immunoaffinity capture followed by mass spectrometry to identify protein interactions.
Main Results:
- Transgenic mice expressing G85R SOD1-YFP developed lethal motor symptoms, unlike wild-type SOD1-YFP transgenics.
- G85R SOD1-YFP misfolded as a monomer, progressively became insoluble, and formed aggregates in motor neurons.
- Misfolded G85R SOD1-YFP interacted with cytosolic chaperones Hsc70 and Hsp110, but insoluble inclusions still formed.
Conclusions:
- Motor neurons initially compensate for misfolded SOD1 but eventually fail to maintain its solubility.
- Progressive accumulation of misfolded SOD1 leads to motor neuron dysfunction and paralysis.
- The findings highlight the role of protein misfolding and aggregation in SOD1-linked ALS pathogenesis.
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