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 ␣␤ 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.

Opening Pandora‘s Jar: A Primer on the Putative Roles of Crmp2 In A Panoply of Neurodegenerative, Sensory and Motor Neuron, and Central Disorders

“…Increased CRMP2 levels were detected in cerebrospinal fluids from patients with MS. Phosphorylated CRMP2 levels were reported to be upregulated in human brain sections containing chronic, active MS lesions and plaques compared with the absence of such reactivity in normal brains post-mortem [157]. Using a well-established animal model of MS, experimental autoimmune encephalomyelitis, which mimics many of the pathophysiological hallmarks of MS, Petratos et al showed that CRMP2 is phosphorylated and hence inhibited during the progression of experimental autoimmune encephalomyelitis in degenerating axons [68].…”

Comparative Proteomics for the Evaluation of Protein Expression and Modifications in Neurodegenerative Diseases

“…The presence in the CSF of autoantibodies and the identification of the immunogenic antigens have been investigated with SERPA also in some neurological disease, including multiple sclerosis, in which several antigenic candidates have been identified (Almeras et al, 2004;Gini et al, 2008;Menon et al, 2011). An early inflammatory phase has been postulated in several movement disorders, such as PD, and in the hyperkinetic movement disorders (HMDs) characterized by involuntary movements (Sanger et al, 2010).…”

Cerebrospinal Fluid in Clinical Neurology

“…An alternative proteomics approach for CSF investigation might be the serological proteome analysis (SERPA) (De Monte et al 2008 ;Privitera et al 2013 ) that exploits the presence of autoantibodies in some neurological disease like multiple sclerosis (Menon et al 2011 ). In the SERPA approach, the autoantibodies present in the CSF of the patients are used to screen the proteome of the patient resolved by 2-dimensional electrophoresis in order to identify putative autoantigen/s.…”