title: A novel unbiased proteomic approach to detect the reactivity of cerebrospinal fluid in neurological diseases.
abstract: Neurodegenerative diseases, such as multiple sclerosis represent global health issues. Accordingly, there is an urgent need to understand the pathogenesis of this and other central nervous system disorders, so that more effective therapeutics can be developed. Cerebrospinal fluid is a potential source of important reporter molecules released from various cell types as a result of central nervous system pathology. Here, we report the development of an unbiased approach for the detection of reactive cerebrospinal fluid molecules and target brain proteins from patients with multiple sclerosis. To help identify molecules that may serve as clinical biomarkers for multiple sclerosis, we have biotinylated proteins present in the cerebrospinal fluid and tested their reactivity against brain homogenate as well as myelin and myelin-axolemmal complexes. Proteins were separated by two-dimensional gel electrophoresis, blotted onto membranes and probed separately with biotinylated unprocessed cerebrospinal fluid samples. Protein spots that reacted to two or more multiple sclerosis-cerebrospinal fluids were further analyzed by matrix assisted laser desorption ionization-time-of-flight time-of-flight mass spectrometry. In addition to previously reported proteins found in multiple sclerosis cerebrospinal fluid, such as αβ crystallin, enolase, and 14-3-3-protein, we have identified several additional molecules involved in mitochondrial and energy metabolism, myelin gene expression and/or cytoskeletal organization. These include aspartate aminotransferase, cyclophilin-A, quaking protein, collapsin response mediator protein-2, ubiquitin carboxy-terminal hydrolase L1, and cofilin. To further validate these findings, the cellular expression pattern of collapsin response mediator protein-2 and ubiquitin carboxy-terminal hydrolase L1 were investigated in human chronic-active MS lesions by immunohistochemistry. The observation that in multiple sclerosis lesions phosphorylated collapsin response mediator protein-2 was increased, whereas Ubiquitin carboxy-terminal hydrolase L1 was down-regulated, not only highlights the importance of these molecules in the pathology of this disease, but also illustrates the use of our approach in attempting to decipher the complex pathological processes leading to multiple sclerosis and other neurodegenerative diseases.
authors.affiliation: Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia. krishnakumarmenon@aims.amrita.edu
authors.forename: Krishnakumar N
authors.initials: KN
authors.lastname: Menon
authors.forename: David L
authors.initials: DL
authors.lastname: Steer
authors.forename: Martin
authors.initials: M
authors.lastname: Short
authors.forename: Steven
authors.initials: S
authors.lastname: Petratos
authors.forename: Ian
authors.initials: I
authors.lastname: Smith
authors.forename: Claude C A
authors.initials: CC
authors.lastname: Bernard
mesh_terms: D000328:Adult; D000368:Aged; D001369:Axons / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western; D001921:Brain / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D002556:Cerebrospinal Fluid Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004734:Energy Metabolism; D005260:Female; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009103:Multiple Sclerosis / Q000134:cerebrospinal fluid* / Q000276:immunology / Q000378:metabolism; D009186:Myelin Sheath / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D040901:Proteomics; D043222:Ubiquitin Thiolesterase / Q000378:metabolism
chemical_list: D002556:Cerebrospinal Fluid Proteins; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; C507533:Ubiquitin carboxyl-Terminal Hydrolase L-1, human; C095114:collapsin response mediator protein-2; D043222:Ubiquitin Thiolesterase