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 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 beta 5 (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 beta5i (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.
Allen
Simon
S
Academic Unit of Neurology, Division of Genomic Medicine, University of Sheffield, S10 2RX, United Kingdom. simon.allen@sheffield.ac.uk
Heath
Paul Roy
PR
Kirby
Janine
J
Wharton
Stephen Barrie
SB
Cookson
Mark Robert
MR
Menzies
Fiona Mhairi
FM
Banks
Rosamonde Elizabeth
RE
Shaw
Pamela Jean
PJ
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002999:Clone Cells; D003600:Cytosol / Q000378:metabolism*; D017931:DNA Primers; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D008954:Models, Biological; D016472:Motor Neuron Disease / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000096:biosynthesis*; D020543:Proteome / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D020543:Proteome; D011994:Recombinant Proteins; D009569:Nitric Oxide; D013482:Superoxide Dismutase; D005978:Glutathione