Applications Of Metabolomics In Molecular Mechanism Study And Biomarker Discovery Of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis（ALS）is a fatal neurodegenerative disease characterized by progressive loss of upper and lower motor neurons,with progressive muscle atrophy,weakness,and limb paralysis as the primary clinical symptoms.The exact etiology and pathogenesis of ALS are still unknown due to a complex interplay of genetic factor,environmental factor and other exogenous influences.With the rise of metabolomics,the connection between metabolic reprogramming and pathogenesis of diseases have received increasing attention.There is growing evidence suggesting significant metabolic changes before and after the onset of ALS.However,due to the involvement of complex metabolic network regulation and the limited research on metabolic reprogramming in ALS both domestically and internationally,the comprehensive metabolic changes remain unclear.Moreover,the clinical manifestation of ALS is heterogeneous.The lack of reliable biochemical and imaging diagnostic indicators further limits early diagnosis and intervention in ALS.Therefore,in this dissertation,we utilized untargeted metabolomics analysis to explore metabolic changes and identified the most critical metabolic pathways in ALS,and sought novel biomarkers that could potentially provide a strong aid in clinical diagnosis.Additionally,by integrating proteomics data,we tried to further elucidate metabolic network regulation changes in ALS.The main contents of the dissertation are as follows:1.Due to the unclear pathogenesis and lack of reliable biomarkers for clinical diagnosis of ALS,plasma samples from ALS patients and controls were collected and subjected to both gas chromatography-mass spectrometry（GC-MS）and liquid chromatography-mass spectrometry（LC-MS）-based untargeted metabolomics analysis,to explore changes in metabolic profile of plasma and metabolic pathways in ALS patients,and identify potential biomarkers to assist clinical diagnosis.It was found that differential metabolites in the plasma of ALS patients were mainly glycerophospholipids,fatty acyls,and amino acids.Disturbances in amino acid metabolic pathways such as aminoacyl-t RNA biosynthesis,phenylalanine,tyrosine,and tryptophan biosynthesis were significant in ALS.Receiver operating characteristic（ROC）curve analysis identified hypoxanthine as a potential biomarker with the best discriminative power for ALS compared to other metabolites.2.To investigate whether metabolic reprogramming occurs before pathological changes in ALS and further elucidate key metabolic pathways involved in disease progression,we collected plasma,motor cortex,and lumbar spinal cord from SOD1^G93Amice and age-matched wildtype（WT）littermates in the presymptomatic and symptomatic stages for LC-MS-based untargeted metabolomics analysis.We found that metabolic reprogramming of the SOD1^G93A mice appeared in the pre-symptomatic stage,and was more prominent and complicated in the symptomatic stage.Dysregulation of purine nucleotides and their degradation products were found in the plasma,spinal cord,and cortex of SOD1^G93A mice since the presymptomatic stage,and became more prominent in the symptomatic stage.In the presymptomatic stage,purine metabolism abnormalities in the spinal cord of SOD1^G93A mice were characterized by increased purine nucleotides synthesis and decreased degradation,while in the symptomatic stage,the degradation of purine nucleotides was intensified,suggesting that the dysregulation of purine metabolism is a key event in the progression of ALS.3.To elucidate the potential mechanisms underlying the metabolic changes in ALS and clarify the changes in metabolic network regulation,the spinal cord samples from symptomatic SOD1^G93A mice and WT littermates were subjected to proteomics analysis to screen differentially expressed proteins associated with ALS.The results showed that differentially expressed proteins were mainly involved in the regulation of metabolic pathways such as oxidative phosphorylation,retrograde endocannabinoid signaling,and fatty acid metabolism.The results of joint pathway analysis of proteomics and metabolomics suggested that the abnormalities of purine metabolism,fatty acid metabolism,TCA cycle,and folate-mediated one-carbon metabolism are crucial events in ALS pathophysiology.Abnormalities in fatty acid metabolism in ALS might be the result of impaired fatty acid degradation caused by the downregulation of fatty acid transport enzymes.Intensified purine nucleotide degradation induced by excessive upregulation of purine metabolizing enzymes might account for the imbalance of purine metabolism in both ALS patients and mice model.In summary,to elucidate the molecular mechanisms of ALS from a metabolic perspective and seek biomarkers,this dissertation investigated the metabolic alteration of ALS patients and animal models and identified the most critical metabolic pathways using mass spectrometry-based metabolomics approaches combined with proteomics analysis.It reveals molecular changes and regulatory features during ALS progression and identifies potential diagnostic biomarkers with great discriminative power.The results of this dissertation provide new insights into the underlying molecular mechanisms and targets of ALS,as well as potential new approaches for the clinical diagnosis of ALS.