#METABOLOMICS WORKBENCH mChu_20240710_120031 DATATRACK_ID:4996 STUDY_ID:ST003352 ANALYSIS_ID:AN005493 PROJECT_ID:PR001855 VERSION 1 CREATED_ON July 17, 2024, 11:19 am #PROJECT PR:PROJECT_TITLE Integrated multi-omics reveals mTOR-LPL-driven dysregulated lipid metabolism PR:PROJECT_TITLE induces neuronal hyperexcitability in human microglia of tuberous sclerosis PR:PROJECT_TITLE complex PR:PROJECT_SUMMARY Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder PR:PROJECT_SUMMARY caused by mutations in either TSC1 or TSC2. There's evidence suggests a PR:PROJECT_SUMMARY connection between microglia activation and epilepsy as well as cognitive PR:PROJECT_SUMMARY impairment in TSC patients. However, how the causal variants of TSC1/2 genes PR:PROJECT_SUMMARY identified in TSC patients affect human microglia and how they contribute to the PR:PROJECT_SUMMARY neurological manifestations remains unclear. This project is focus on this PR:PROJECT_SUMMARY problem using human microglia generated from induced pluripotent stem cells PR:PROJECT_SUMMARY (iPSCs) derived from a TSC patient. PR:INSTITUTE Stjude children research hospital PR:LAST_NAME Chu PR:FIRST_NAME Mengqi PR:ADDRESS 262 Danny Tomas Place, Memphis, Tennessee, 38104, USA PR:EMAIL Mengqi.Chu@stjude.org PR:PHONE 901-603-2366 #STUDY ST:STUDY_TITLE Untargeted lipidomics profiling of LPL-overexpression and LPL-knockdown ST:STUDY_TITLE microglia ST:STUDY_SUMMARY In this study, human microglia from induced pluripotent stem cells (iPSCs) ST:STUDY_SUMMARY derived from a TSC patient cohort were generated. Upregulated ST:STUDY_SUMMARY glycerophosphocholines and fatty acyls were found in TSC microglia, resulting in ST:STUDY_SUMMARY increased phagocytosis and inflammation. Strikingly, the dysregulated lipid ST:STUDY_SUMMARY metabolism in TSC microglia is driven by hyper-activation of mTOR-lipoprotein ST:STUDY_SUMMARY lipase (LPL) pathway. Furthermore, cellular and electrophysiological assessments ST:STUDY_SUMMARY of neuron/microglia co-cultures revealed that TSC microglia directly affect ST:STUDY_SUMMARY neuronal development and excitability as well as neuronal network activity, ST:STUDY_SUMMARY which could be largely ameliorated by mTOR/LPL inhibition. Through integrated ST:STUDY_SUMMARY multi-omics analysis, levels of glycerophosphocholines were found increased in ST:STUDY_SUMMARY the microglia of humans with tuberous sclerosis complex. To determine whether ST:STUDY_SUMMARY glycerophosphocholines are regulated by the LPL gene, we performed experiments ST:STUDY_SUMMARY with LPL overexpression in microglia and LPL knockdown in microglia. ST:INSTITUTE Stjude children research hospital ST:LAST_NAME Chu ST:FIRST_NAME Mengqi ST:ADDRESS 262 Danny Tomas Place, Memphis, Tennessee, 38104, USA ST:EMAIL Mengqi.Chu@stjude.org ST:PHONE 901-603-2366 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - 426_1 Genotype:WT | Treatment:Control for Overexpression | Sample source:iPSC RAW_FILE_NAME(Raw file name)=426_1.mzXML SUBJECT_SAMPLE_FACTORS - 426_2 Genotype:WT | Treatment:Control for Overexpression | Sample source:iPSC RAW_FILE_NAME(Raw file name)=426_2.mzXML SUBJECT_SAMPLE_FACTORS - 426_3 Genotype:WT | Treatment:Control for Overexpression | Sample source:iPSC RAW_FILE_NAME(Raw file name)=426_3.mzXML SUBJECT_SAMPLE_FACTORS - 426_4 Genotype:WT | Treatment:Control for Overexpression | Sample source:iPSC RAW_FILE_NAME(Raw file name)=426_4.mzXML SUBJECT_SAMPLE_FACTORS - 426_5 Genotype:WT | Treatment:Control for Overexpression | Sample source:iPSC RAW_FILE_NAME(Raw file name)=426_5.mzXML SUBJECT_SAMPLE_FACTORS - LPL_1 Genotype:WT | Treatment:LPL Overexpress | Sample source:iPSC RAW_FILE_NAME(Raw file name)=LPL_1.mzXML SUBJECT_SAMPLE_FACTORS - LPL_2 Genotype:WT | Treatment:LPL Overexpress | Sample source:iPSC RAW_FILE_NAME(Raw file name)=LPL_2.mzXML SUBJECT_SAMPLE_FACTORS - LPL_3 Genotype:WT | Treatment:LPL Overexpress | Sample source:iPSC RAW_FILE_NAME(Raw file name)=LPL_3.mzXML SUBJECT_SAMPLE_FACTORS - LPL_4 Genotype:WT | Treatment:LPL Overexpress | Sample source:iPSC RAW_FILE_NAME(Raw file name)=LPL_4.mzXML SUBJECT_SAMPLE_FACTORS - LPL_5 Genotype:WT | Treatment:LPL Overexpress | Sample source:iPSC RAW_FILE_NAME(Raw file name)=LPL_5.mzXML SUBJECT_SAMPLE_FACTORS - TSC_1 Genotype:TSC mutation | Treatment:Control for Knockdown | Sample source:iPSC RAW_FILE_NAME(Raw file name)=TSC_1.mzXML SUBJECT_SAMPLE_FACTORS - TSC_2 Genotype:TSC mutation | Treatment:Control for Knockdown | Sample source:iPSC RAW_FILE_NAME(Raw file name)=TSC_1.mzXML SUBJECT_SAMPLE_FACTORS - TSC_3 Genotype:TSC mutation | Treatment:Control for Knockdown | Sample source:iPSC RAW_FILE_NAME(Raw file name)=TSC_1.mzXML SUBJECT_SAMPLE_FACTORS - TSC_4 Genotype:TSC mutation | Treatment:Control for Knockdown | Sample source:iPSC RAW_FILE_NAME(Raw file name)=TSC_1.mzXML SUBJECT_SAMPLE_FACTORS - TSC_5 Genotype:TSC mutation | Treatment:Control for Knockdown | Sample source:iPSC RAW_FILE_NAME(Raw file name)=TSC_1.mzXML SUBJECT_SAMPLE_FACTORS - KD_1 Genotype:TSC mutation | Treatment:LPL knockdown | Sample source:iPSC RAW_FILE_NAME(Raw file name)=KD_1.mzXML SUBJECT_SAMPLE_FACTORS - KD_2 Genotype:TSC mutation | Treatment:LPL knockdown | Sample source:iPSC RAW_FILE_NAME(Raw file name)=KD_2.mzXML SUBJECT_SAMPLE_FACTORS - KD_3 Genotype:TSC mutation | Treatment:LPL knockdown | Sample source:iPSC RAW_FILE_NAME(Raw file name)=KD_3.mzXML SUBJECT_SAMPLE_FACTORS - KD_4 Genotype:TSC mutation | Treatment:LPL knockdown | Sample source:iPSC RAW_FILE_NAME(Raw file name)=KD_4.mzXML SUBJECT_SAMPLE_FACTORS - KD_5 Genotype:TSC mutation | Treatment:LPL knockdown | Sample source:iPSC RAW_FILE_NAME(Raw file name)=KD_5.mzXML #COLLECTION CO:COLLECTION_SUMMARY Cell line and sample collection were done at Emory University All iPSC lines CO:COLLECTION_SUMMARY have been fully characterized and cultured on Corning matrigel matrix (Corning, CO:COLLECTION_SUMMARY 354234) coated plates in human iPSC mTeSR™1 media (Stemcell, 85850). CO:COLLECTION_SUMMARY hiPSC-microglia were generated using microglia differentiation media and CO:COLLECTION_SUMMARY microglia mature media. 2 million microglia cells were collected for each sample CO:SAMPLE_TYPE Microglia #TREATMENT TR:TREATMENT_SUMMARY LPL overexpression and knockdown were achieved through lentivirus infection. LPL TR:TREATMENT_SUMMARY overexpression in a control cell line and knockdown in a TSC cell line were TR:TREATMENT_SUMMARY validated by qPCR and bulk RNA-RNAseq. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipids were extracted from cell by cold isopropanol (10:1 v/v, solvent to SP:SAMPLEPREP_SUMMARY sample). Extracted lipids were dried under nitrogen gas, resuspended in 65% SP:SAMPLEPREP_SUMMARY acetonitrile, 30% isopropanol and 5% water. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters NanoAcquity CH:COLUMN_NAME Ascentis Express C18 column, 2.7um, 90A, 15cm × 300µm CH:SOLVENT_A 60% water/40% acetonitrile; 10 mM ammonium formate; 0.1% formic acid CH:SOLVENT_B 90% isopropanol/10% acetonitrile; 10 mM ammonium formate; 0.1% formic acid CH:FLOW_GRADIENT 10-75%B in 34min CH:FLOW_RATE - CH:COLUMN_TEMPERATURE RT #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS MS1:120k resolution, 100-1200 m/z, 3e6 AGC, 50 ms maximal ion time MS2:top 20, MS:MS_COMMENTS 30k resolution, 2e5 AGC, 45 ms maximal ion time, stepped HCD NCE at 30, 75, 150 MS:MS_COMMENTS In-house JUMPm software and Shiny were used for data processing, feature MS:MS_COMMENTS assignment and statistic analysis MS:MS_RESULTS_FILE ST003352_AN005493_Results.txt UNITS:Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END