Screening Of Biomarkers In Amyotrophic Lateral Sclerosis

Objective:Amyotrophic lateral sclerosis(ALS)is a neurodegenerative disease that is heterogeneous regarding genetics,phenotype and pathophysiology.There are limited therapeutic options to date.Biomarkers are of great significance for prompt diagnosis,disease progression evaluation,prognostic prediction,pathophysiology understanding and trial outcome measures in subsequent trial access.The first part of this study aims to determine the serum and cerebrospinal fluid(CSF)levels of neurofilament light chain(NFL)and phosphorylated neurofilament heavy chain(p NFH)in ALS patients,to analyze their clinical relevance associated with numerous clinical characteristics,and to verify their potential role as a diagnostic and prognostic biomarker in ALS.The second part aims to screen the differentially expressed proteins of inflammatory cytokines in ALS patients using protein microarray.Bioinformatics analyses were performed to explore reliable inflammatory cytokines as potential biomarkers for ALS.Methods:1.In this study,52 patients with ALS and 30 controls with non-inflammatory neurological diseases were included.Serum and CSF samples were obtained.2.NFL and p NFH levels in serum and CSF were measured by enzyme-linked immunosorbent assay.The discriminatory performance was evaluated by a receiver operating characteristic curve(ROC)analysis.Correlation analysis with several clinical characteristics were performed.3.Human inflammatory cytokines arrays were used to screen differentially expressed proteins in serum and CSF of ALS patients and controls.Bioinformatic analysis was carried out including hierarchical clustering,functional enrichment,protein-protein network construction and drug-gene interaction.Results:1.Compared with the control group,levels of NFL and p NFH in the serum and CSF of ALS patients were significantly increased.2.The levels of NFL and pNFH in serum and CSF were not significantly correlated with the clinical onset.These values were negatively correlated with ALSFRS-r score and disease duration(except CSF-NFL with disease duration),and were positively correlated with disease progression rate(DPR)and UMN score.3.The optimal cut-off values were obtained in according with the ROC curve analysis to discriminate ALS from controls.4.Using high throughput protein chip technology,eight up-regulated differentially expressed proteins were screened in CSF,namely IL-6,IL-1α,IFNγ,IL-8,MCP-1,TNFα,IL-10,IL-4.One up-regulated differentially expressed protein was screened in serum,i.e.MCP-1.5.GO pathway analysis demonstrated that in terms of biological process,differentially expressed proteins were mostly enriched in positive regulation of cell-cell adhesion,cytokine biosynthetic process and cytokine metabolic process;in terms of molecular function,differentially expressed proteins were mainly abundant in cytokine activity,cytokine receptor binding and receptor ligand activity.KEGG pathway analysis presented that cytokine-cytokine receptor interaction was highly enriched.6.A PPI network consisting of 8 nodes and 23 edges was constructed,of which 23 interaction pairs had combined score > 0.9 and MCP-1 was the most highly connected.7.Drug-gene interaction analysis predicted a total of 134 interaction pairs for 8differential proteins,including numerous anti-inflammatory drugs and immunomodulatory drugs.Conclusion:The levels of NFL and pNFH in serum and CSF can be used as potential biomarkers in ALS.They tend to play a vital role in the prompt diagnosis,disease severity and progression prediction.Eight differentially expressed proteins of inflammatory cytokines in ALS patients were screened using protein microarray and bioinformatics analysis,including IL-6,IL-1α,IFNγ,IL-8,MCP-1,TNFα,IL-10 and IL-4.Among them,MCP-1can be used as a potential biomarker with significance and functional diversity,providing a candidate drug target for the treatment of ALS.