The Screening And Analysis Of The Protein Signatures Associated With Signal Pathway In Amyotrophic Lateral Sclerosis

Objection: Amyotrophic Lateral Sclerosis (ALS) is a fatal degenerative disorder of thecentral nervous system, manifests as the progressive weakening and atrophy of muscles. Sofar the exact causes and pathogenesis of ALS are still unknown, for half of the ALS patients,the average survival time from onset was2.5years, and the5-year survival rate of the patentswas only28%. For ALS patients, lack of early diagnosis and effective treatment methods arethe current problem, therefore, searching for useful biomarkers as diagnosis and treatmenttarget are necessary. The purposes of our experiment are screening diagnostic and prognosticprotein targets for ALS and finding their molecular biological mechanisms, and identify theseproteins involved in signal transduction pathways.Methods: Total proteins were extracted from skeletal muscle samples of36ALS,17spinal muscular atrophy (SMA) and36normal individuals. The expression level of134proteins and phosphoproteins were analyzed using Protein Pathway Array (PPA) method.Differentially expressed proteins and phosphoproteins were found between ALS tissues andnormal control tissues; cluster analysis was used to identify these differentially expressedproteins. We performed proteomic-wide expression analysis using Ingenuity PathwayAnalysis (IPA) software and Exploratory Gene Association Networks (EGAN) software toinvestigate the significant protein and phosphoproteins expression in ALS signaling network.Compared these differentially expressed proteins in36cases of ALS with17cases of SMAskeletal muscle tissues and analysed the differentially expressed proteins further. We didhistochemistry staining in36cases of ALS include Hematoxylin and Eosin (HE) and Nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) staining and observedwith light microscopy. We got the relationship between different degree of muscle atrophyand protein expression, as well as the relationship between ALS protein expression profilingand the patient's clinical features like age, gender, ALS functional rating scale-revised(ALS-FRSr) score, muscle atrophy and site of onset, also the relationship between proteinexpression profiling and ALS diagnostic level. The overall survival of36cases of ALS wasanalysed to find risk factors affecting survival, and verified by the application of Western-Blotanalysis.Results: In our study, we found that17proteins were differentially expressed betweenALS and normal, with these17proteins,36ALS and36normal skeletal muscle tissues couldbe separated into two categories by hierarchical clustering analysis; IPA systems analysisresults showed that these proteins were closely related with cell growth and proliferation,gene expression, cell cycle and the free radical scavenging. EGAN systems analysis resultsshowed that these proteins were related with skeletal system morphogenesis, muscle tissue,structure development, skeletal system development, muscle cell differentiation, proliferation,regulation of muscle contraction. The proteins were also related with regulation of nervoussystem development, process, neuronal differentiation, migration, neuron death and apoptosis,signal transdution in response to DNA damage and oxidative stress. In the17proteins,9proteins were differentially expressed between ALS and SMA muscle tissues and suggestedthat these proteins may be associated with molecular biology mechanisms. Histochemicalstaining showed that36ALS had different degrees of muscle atrophy, target fibers and musclefiber grouping were showed in NADH-TR staining. Differential protein expressed at differentages, gender, ALS-FRSr score, atrophy, and site of onset of ALS patients. In this study wefound five proteins may be used as biomarkers to help diagnosis since they expresseddifferent in four different level of diagnostic group. Our results also show that Akt and FactorXIII B were independent prognostic risk factors, the low level expression of Akt and FactorXIII B correlated with unfavorable survival and the risk score calculated based on these two proteins predicted the survival of each individual patient, we used Western-Blot method toverify the protein expression and got results consistent with the above.Conclusion: Our research focused on ALS signaling pathway, we did protein screeningand PPA analysis with ALS, SMA and normal skeletal muscle tissue, compared the proteinexpression of ALS and normal controls and analysed the function of protein signaltransduction pathway in ALS in muscle cells and neurons proliferation, differentiation andapoptosis. Our data demonstrated abroad dysregulation of signaling proteins were exsit inALS. Nine proteins may be associated with molecular mechanisms of ALS,5proteins may beselected as biomarkers with pathological diagnosis,10proteins may related to the clinicalfeatures, particularly, Akt and Factor XIIIB were found to be promising biomarkers for theprognosis of ALS and an effective detection system could be established for prognosis. Thishigh-throughput detection of protein experimental model supports further investigation forALS in the protein levels.