Proteome Analysis Of Biomarkers In The Cerebrospinal Fluid Of Central Nervous System Demyelinative Diseases

CNS demyelinative diseases are autoimmune diseases of the central nervous system(CNS) with the inflamation and mulifocality demyelination.They are characterized by recurrent attacks and relapsing-remitting.In multiple sclerosis(MS), the demyelination is primarily involved in the cerebrum,brainstem and cerebellum. However,in neuromyelitis optica(NMO),the demyelination predominantly affects optic nerves and spinal cord.MS,the most common autoimmune disease involving the CNS,is a chronic illness affecting CNS pathways and leading to progressive neurological dysfunction. The pathological hallmark of MS is the demyelinated plaque found throughout the brain and spinal cord,with CNS perivascular infiltration of inflammatory cells, demyelination,astrogliosis,and axonal injury.Symptoms are believed to result primarily from axonal demyelination that inhibits or blocks conduction.Amelioration of symptoms has been attributed to both partial remyelination and resolution of inflammation.NMO,also termed Devic's syndrome,is an idiopathic inflammatory demyelinating disease of the CNS predominantly affecting optic nerves and spinal cord,but unlike MS,does not affect the brain.The common affections are optic nerves and optic chiasma,sometimes involves the optic tract.It is a frequently disabling,occasionally life-threatening disease.Furthermore,the prognosis of NMO is often poor and more than half of patients will develop severe bilateral visual impairment and even visual loss in at least one eye and/or inability to ambulate without assistance within 5 years of disease onset.CNS demyelinative diseases severely affect human health.Up to now,the diagnosis of them are challenging for lack of specific biomarkers.Cerebrospinal fluid (CSF) is a promising source of biomarkers for disorders of the central nervous system, since the CSF compartment is in close contact with the brain interstitial fluid where biochemical changes related to the underlying disease are often reflected.Abnormal CSF findings are one of the characteristics of CNS demyelinative diseases. Proteomics is the study of identifying the entire protein components(proteome) of a cell,tissue or fluid at a given point in time.Since proteins play a central role in the life of an organism,the study of proteome of the organism can provide insight into the mechanisms of diseases,which is helpful in discovery of biomarkers of the diseases. Now it is well accepted that CSF proteomics is a powerful technique to describe the expression of proteins in CSF.Moreover,it can provide valuable information about the pathophysiological mechanisms underlying the diseases in CNS.In this study,by means of the proteomic analysis with two-dimensional electrophoresis(2-DE),followed by matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS) and database searching,in combination with the validation of the ELISA,Western blot test,and the establishment of the protein network,we studied the CSF protein alterations beween the CNS demyelinative diseases(MS and NMO) and the control groups,found the candidated biomarkers in CNS demyelinative diseases,expected to investigated their pathogenesy,diagnosis,prognosis and treatment on the molecular level. Objective:Comparative proteomics and biological signaling network analysis were carried out in the cerebrospinal fluid(CSF) of the patients with CNS demyelinative diseases to investigate the relations both among the potiental biomarkers and between the biomarkers and the diseases.These candidated biomarkers may be give information for studying the pathophysiological process,the clinical diagnosis and treatment of the diseases.Methods:CSF were extracted from patients with CNS demyelinative diseases and the controls respectively,and the proteins were precipitated with ice-cold acetone. Then the two-dimensional electrophoresis(2-DE) were applied and the differential expression protein spots were selected with the ImageMaster 2D-gel software.The peptides mass fingerprint(PMF) was got by the matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS) and searched in SWISSPROT database to identify differential proteins.The expressed obviously proteins were selected to be confirmed by subsequent ELISA and Western blot test. Finally,the correlations of these proteins were analized by MetaCore and PathwayStudio software.Results:2-DE maps of CSF with MS and the controls group were got.There were eight protein spots expressed differentially on the 2-DE maps,in which four up-regulated and four down-regulated protein spots were identified in MS.Cystatin C is down-regulated and further confirmed by the ELISA assay(4.36±1.22 mg/L, 6.00±1.68mg/L,P＜0.01).A map of signaling network about these proteins were built by MetaCore integrated software.By using 2-DE and MALDI-TOF MS we identified 13 different protein spots in NMO group.The CSF expression of four protein spots was enhanced,whereas the expression of nine protein spots was reduced in the NMO group compared with the control group.One of these proteins,vitamin D binding protein(DBP) was confirmed by the Western blot test(p＜0.01).Moreover,protein network of 11 proteins was partially got,which showed some biological interaction.Conclusions:The differential expressions and biological signaling network researches of the proteins were assistant to explore the pathogenesis,differential diagnosis and drug targets of CNS demyelinative diseases on molecular level.These disease-related proteins may be as candidate biomarkers for CNS demyelinative diseases.