Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS.
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.
Wang
Jiou
J
Howard Hughes Medical Institute, Department of Genetics, Section of Comparative Medicine, and Department of Neurosurgery, Yale University School of Medicine, New Haven, CT 06510, USA.
Farr
George W
GW
Zeiss
Caroline J
CJ
Rodriguez-Gil
Diego J
DJ
Wilson
Jean H
JH
Furtak
Krystyna
K
Rutkowski
D Thomas
DT
Kaufman
Randal J
RJ
Ruse
Cristian I
CI
Yates
John R
JR
Perrin
Steve
S
Feany
Mel B
MB
Horwich
Arthur L
AL
D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics*; D000818:Animals; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D018832:Molecular Chaperones / Q000378:metabolism*; D009046:Motor Neurons / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D012995:Solubility; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics*; D025801:Ubiquitin / Q000378:metabolism
D005904:Glial Fibrillary Acidic Protein; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D025801:Ubiquitin; D013482:Superoxide Dismutase