Analysis of the Cytosolic Proteome in a Cell Culture Model of Familial Amyotrophic Lateral Sclerosis Reveals Alterations to the Proteasome, Antioxidant Defenses, and Nitric Oxide Synthetic Pathways

Injury to motor neurons associated with mutant Cu,Zn-superoxide dismutase (SOD1)-related familial amyotrophic lateral sclerosis (FALS) results from a toxic gain-of-function of the enzyme. The mechanisms by which alterations to SOD1 elicit neuronal death remain uncertain despite intensive research ef...

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Published in:	The Journal of biological chemistry Vol. 278; no. 8; pp. 6371 - 6383
Main Authors:	Allen, Simon, Heath, Paul Roy, Kirby, Janine, Wharton, Stephen Barrie, Cookson, Mark Robert, Menzies, Fiona Mhairi, Banks, Rosamonde Elizabeth, Shaw, Pamela Jean
Format:	Journal Article
Language:	English
Published:	United States American Society for Biochemistry and Molecular Biology 21-02-2003
Subjects:	Amino Acid Substitution Animals Base Sequence Cell Line Clone Cells Cytosol - metabolism DNA Primers Glutathione - metabolism Humans Kinetics Mice Mice, Transgenic Models, Biological Motor Neuron Disease - enzymology Motor Neuron Disease - genetics Mutagenesis, Site-Directed Nitric Oxide - biosynthesis Proteome - metabolism Recombinant Proteins - metabolism Reverse Transcriptase Polymerase Chain Reaction Superoxide Dismutase - genetics Superoxide Dismutase - metabolism Transfection
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Summary:	Injury to motor neurons associated with mutant Cu,Zn-superoxide dismutase (SOD1)-related familial amyotrophic lateral sclerosis (FALS) results from a toxic gain-of-function of the enzyme. The mechanisms by which alterations to SOD1 elicit neuronal death remain uncertain despite intensive research effort. Analysis of the cellular proteins that are differentially expressed in the presence of mutant SOD1 represents a novel approach to investigate further this toxic gain-of-function. By using the motor neuron-like cell line NSC34 stably transfected with wild-type, G93A, or G37R mutant human SOD1, we investigated the effects of mutant human SOD1 on protein expression using proteomic approaches. Seven up-regulated proteins were identified as argininosuccinate synthase, argininosuccinate lyase, neuronal nitric-oxide synthase, RNA-binding motif protein 3, peroxiredoxin I, proteasome subunit Î²5 (X), and glutathione S -transferase (GST) Alpha 2. Seven down-regulated proteins were identified as GST Mu 1, GST Mu 2, GST Mu 5, a hypothetical GST Mu, GST Pi B, leukotriene B 4 12-hydroxydehydrogenase, and proteasome subunit Î²5i (LMP7). GST assays demonstrated a significant reduction in the total GST activity of cells expressing mutant human SOD1. Proteasome assays demonstrated significant reductions in chymotrypsin-like, trypsin-like, and post-glutamylhydrolase proteasome activities. Laser capture microdissection of spinal cord motor neurons from human FALS cases, in conjunction with reverse transcriptase-PCR, demonstrated decreased levels of mRNA encoding GST Mu 1, leukotriene B 4 12-hydroxydehydrogenase, and LMP7. These combined approaches provide further evidence for involvement of alterations in antioxidant defenses, proteasome function, and nitric oxide metabolism in the pathophysiology of FALS.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23
ISSN:	0021-9258 1083-351X
DOI:	10.1074/jbc.M209915200