#METABOLOMICS WORKBENCH yehe_20230620_132206 DATATRACK_ID:4105 STUDY_ID:ST002739 ANALYSIS_ID:AN004442 VERSION 1 CREATED_ON 08-09-2023 #PROJECT PR:PROJECT_TITLE Metabolic effect of Lamin A/C in oligodendrocyte on brain function PR:PROJECT_TYPE LC-MS/MS metabolomics of cell type specific Lmna conditional knockout and PR:PROJECT_TYPE wildtype mice brains at 26 weeks PR:PROJECT_SUMMARY Oligodendrocytes are specialized cells which insulate and support axons with PR:PROJECT_SUMMARY their myelin membrane, allowing proper brain function. Here, we identify Lamin PR:PROJECT_SUMMARY A/C (LMNA/C) as essential for transcriptional and functional stability of PR:PROJECT_SUMMARY myelinating oligodendrocytes. We show that LMNA/C levels increase with PR:PROJECT_SUMMARY differentiation of progenitors and that loss of Lmna in differentiated PR:PROJECT_SUMMARY oligodendrocytes profoundly alters their chromatin accessibility and PR:PROJECT_SUMMARY transcriptional signature. Lmna deletion in myelinating glia is compatible with PR:PROJECT_SUMMARY normal developmental myelination. However, altered chromatin accessibility is PR:PROJECT_SUMMARY detected in fully differentiated oligodendrocytes together with increased PR:PROJECT_SUMMARY expression of progenitor genes and decreased levels of lipid-related PR:PROJECT_SUMMARY transcription factors and inner mitochondrial membrane transcripts. As mice age, PR:PROJECT_SUMMARY they start to develop myelin-thinning and progressively worsening motor PR:PROJECT_SUMMARY phenotype. To address the metabolic effect of LMNA/C in oligodendrocyte on brain PR:PROJECT_SUMMARY function, we carried out LC-MS/MS metabolomic study of myelinating glia cell PR:PROJECT_SUMMARY specific Lmna conditional knockout and wildtype mice brains at 26 weeks. Each PR:PROJECT_SUMMARY LC-MS/MS experiment was performed with 3 biological replicates and 4 technical PR:PROJECT_SUMMARY replicates per genotype. Overall, our data identify LMNA/C as essential for PR:PROJECT_SUMMARY maintaining the transcriptional and functional stability of myelinating PR:PROJECT_SUMMARY oligodendrocytes. PR:INSTITUTE Advanced Science Research Center - CUNY PR:DEPARTMENT Neuroscience PR:LABORATORY Casaccia lab, He lab, MALDI and MS core. PR:LAST_NAME He PR:FIRST_NAME Ye PR:ADDRESS 85 St. Nicholas Terrace, New York, New York, 10031, USA PR:EMAIL yhe1@gc.cuny.edu PR:PHONE 2124133182 PR:PUBLICATIONS Pruvost M, Patzig J, Yattah C, Selcen I, Hernandez M, Park HJ, Moyon S, Liu S, PR:PUBLICATIONS Morioka MS, Shopland L, Al-Dalahmah O, Bendl J, Fullard JF, Roussos P, Goldman PR:PUBLICATIONS J, He Y, Dupree JL, Casaccia P. The stability of the myelinating oligodendrocyte PR:PUBLICATIONS transcriptome is regulated by the nuclear lamina. Cell Rep. 2023 Jul PR:PUBLICATIONS 27;42(8):112848. doi: 10.1016/j.celrep.2023.112848. Epub ahead of print. PMID: PR:PUBLICATIONS 37515770. (https://www.cell.com/cell-reports/fulltext/S2211-1247(23)00859-8) PR:DOI http://dx.doi.org/10.21228/M8FM85 #STUDY ST:STUDY_TITLE Metabolic effect of Lamin A/C in oligodendrocyte on brain function ST:STUDY_TYPE LC-MS/MS metabolomics of cell type specific Lmna conditional knockout and ST:STUDY_TYPE wildtype mice brains at 26 weeks ST:STUDY_SUMMARY Oligodendrocytes are specialized cells which insulate and support axons with ST:STUDY_SUMMARY their myelin membrane, allowing proper brain function. Here, we identify Lamin ST:STUDY_SUMMARY A/C (LMNA/C) as essential for transcriptional and functional stability of ST:STUDY_SUMMARY myelinating oligodendrocytes. We show that LMNA/C levels increase with ST:STUDY_SUMMARY differentiation of progenitors and that loss of Lmna in differentiated ST:STUDY_SUMMARY oligodendrocytes profoundly alters their chromatin accessibility and ST:STUDY_SUMMARY transcriptional signature. Lmna deletion in myelinating glia is compatible with ST:STUDY_SUMMARY normal developmental myelination. However, altered chromatin accessibility is ST:STUDY_SUMMARY detected in fully differentiated oligodendrocytes together with increased ST:STUDY_SUMMARY expression of progenitor genes and decreased levels of lipid-related ST:STUDY_SUMMARY transcription factors and inner mitochondrial membrane transcripts. As mice age, ST:STUDY_SUMMARY they start to develop myelin-thinning and progressively worsening motor ST:STUDY_SUMMARY phenotype. To address the metabolic effect of LMNA/C in oligodendrocyte on brain ST:STUDY_SUMMARY function, we carried out LC-MS/MS metabolomic study of myelinating glia cell ST:STUDY_SUMMARY specific Lmna conditional knockout and wildtype mice brains at 26 weeks. Each ST:STUDY_SUMMARY LC-MS/MS experiment was performed with 3 biological replicates and 4 technical ST:STUDY_SUMMARY replicates per genotype. Overall, our data identify LMNA/C as essential for ST:STUDY_SUMMARY maintaining the transcriptional and functional stability of myelinating ST:STUDY_SUMMARY oligodendrocytes. ST:INSTITUTE Advanced Science Research Center - CUNY ST:DEPARTMENT Neuroscience ST:LABORATORY Casaccia lab, He lab, MALDI and MS core. ST:LAST_NAME He ST:FIRST_NAME Ye ST:ADDRESS 85 St. Nicholas Terrace, New York, New York, 10031, USA ST:EMAIL yhe1@gc.cuny.edu ST:PHONE 2124133182 ST:SUBMIT_DATE 2023-06-20 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE 26 weeks SU:GENDER Female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - KO1_001 genotype:Lmna Knock-Out RAW_FILE_NAME=KO1_001_RB4_1_6141.mzML SUBJECT_SAMPLE_FACTORS - KO1_002 genotype:Lmna Knock-Out RAW_FILE_NAME=KO1_002_RB4_1_6149.mzML SUBJECT_SAMPLE_FACTORS - KO1_003 genotype:Lmna Knock-Out RAW_FILE_NAME=KO1_003_RB4_1_6157.mzML SUBJECT_SAMPLE_FACTORS - KO1_004 genotype:Lmna Knock-Out RAW_FILE_NAME=KO1_004_RB4_1_6165.mzML SUBJECT_SAMPLE_FACTORS - KO2_001 genotype:Lmna Knock-Out RAW_FILE_NAME=KO2_001_RB5_1_6142.mzML SUBJECT_SAMPLE_FACTORS - KO2_002 genotype:Lmna Knock-Out RAW_FILE_NAME=KO2_002_RB5_1_6150.mzML SUBJECT_SAMPLE_FACTORS - KO2_003 genotype:Lmna Knock-Out RAW_FILE_NAME=KO2_003_RB5_1_6158.mzML SUBJECT_SAMPLE_FACTORS - KO2_004 genotype:Lmna Knock-Out RAW_FILE_NAME=KO2_004_RB5_1_6166.mzML SUBJECT_SAMPLE_FACTORS - KO3_001 genotype:Lmna Knock-Out RAW_FILE_NAME=KO3_001_RB6_1_6143.mzML SUBJECT_SAMPLE_FACTORS - KO3_002 genotype:Lmna Knock-Out RAW_FILE_NAME=KO3_002_RB6_1_6151.mzML SUBJECT_SAMPLE_FACTORS - KO3_003 genotype:Lmna Knock-Out RAW_FILE_NAME=KO3_003_RB6_1_6159.mzML SUBJECT_SAMPLE_FACTORS - KO3_004 genotype:Lmna Knock-Out RAW_FILE_NAME=KO3_004_RB6_1_6167.mzML SUBJECT_SAMPLE_FACTORS - WT1_001 genotype:Wild-type RAW_FILE_NAME=WT1_001_RB1_1_6137.mzML SUBJECT_SAMPLE_FACTORS - WT1_002 genotype:Wild-type RAW_FILE_NAME=WT1_002_RB1_1_6145.mzML SUBJECT_SAMPLE_FACTORS - WT1_003 genotype:Wild-type RAW_FILE_NAME=WT1_003_RB1_1_6153.mzML SUBJECT_SAMPLE_FACTORS - WT1_004 genotype:Wild-type RAW_FILE_NAME=WT1_004_RB1_1_6161.mzML SUBJECT_SAMPLE_FACTORS - WT2_001 genotype:Wild-type RAW_FILE_NAME=WT2_001_RB2_1_6138.mzML SUBJECT_SAMPLE_FACTORS - WT2_002 genotype:Wild-type RAW_FILE_NAME=WT2_002_RB2_1_6146.mzML SUBJECT_SAMPLE_FACTORS - WT2_003 genotype:Wild-type RAW_FILE_NAME=WT2_003_RB2_1_6154.mzML SUBJECT_SAMPLE_FACTORS - WT2_004 genotype:Wild-type RAW_FILE_NAME=WT2_004_RB2_1_6162.mzML SUBJECT_SAMPLE_FACTORS - WT3_001 genotype:Wild-type RAW_FILE_NAME=WT3_001_RB3_1_6139.mzML SUBJECT_SAMPLE_FACTORS - WT3_002 genotype:Wild-type RAW_FILE_NAME=WT3_002_RB3_1_6147.mzML SUBJECT_SAMPLE_FACTORS - WT3_003 genotype:Wild-type RAW_FILE_NAME=WT3_003_RB3_1_6155.mzML SUBJECT_SAMPLE_FACTORS - WT3_004 genotype:Wild-type RAW_FILE_NAME=WT3_004_RB3_1_6163.mzML #COLLECTION CO:COLLECTION_SUMMARY Murine brains were harvested at 26 weeks and were snap-frozen for 5 min on an CO:COLLECTION_SUMMARY aluminum boat floating on liquid nitrogen. CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY The experimental group was composed of three 26-week-old female mice with TR:TREATMENT_SUMMARY conditional ablation of Lmna in the oligodendrocytes (CnpCre/+;Lmnafl/fl). The TR:TREATMENT_SUMMARY control group consisted of three wildtype 26-week-old female mice #SAMPLEPREP SP:SAMPLEPREP_SUMMARY A coronal slice of brain (approximate 15 mg) at (-0.88 mm to -1.335mm from SP:SAMPLEPREP_SUMMARY Bregma) plane was sectioned and homogenized in cold Methanol/Water (80/20, v/v) SP:SAMPLEPREP_SUMMARY to a final concentration of 30mg/ml. Following 20 min of gentle sonication in SP:SAMPLEPREP_SUMMARY Bioruptor (30 s on, 30 s off, 20 cycles) at 4 °C, samples were centrifuged for SP:SAMPLEPREP_SUMMARY 10 min at 10,000xg at 4 °C and were processed for LC-MS/MS #CHROMATOGRAPHY CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME SeQuant ZIC-HILIC (100 x 2.1mm,3.5um) CH:COLUMN_TEMPERATURE 30 CH:FLOW_GRADIENT 0.15mL/min; 0-5.0 min; 2.0% B, 5.0-28.0 min; 2.0-60.0% B, 28.0-38.0 min; 60.0% CH:FLOW_GRADIENT B, 38.0-39.0 min; 60.0-2.0% B, 39.0-48.0 min, 2.0% B. CH:FLOW_RATE 0.15mL/min CH:SOLVENT_A 97% acetonitrile/3% water/7mM ammonium acetate CH:SOLVENT_B 97% water/3% acetonitrile/7mM ammonium acetate CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker maXis-II MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS MS spectra were obtained using a Bruker maXis-II-ETD UHR-ESI-QTOF, witb Ultra MS:MS_COMMENTS High Resolution QTOF (UHR) technology in addition to Electron-Transfer MS:MS_COMMENTS Dissociation (ETD). Compound identification and descriptive statistical analysis MS:MS_COMMENTS of the LC-MS/MS data were performed through Metaboscape and XCMSPlus software. MS:MS_COMMENTS Bruker MetaboBase Personal 3.0, MoNA, MSDIAL, METLIN, and HMDB metabolomic MS:MS_COMMENTS libraries were used in compound identification. Ultimately, both accurate MS:MS_COMMENTS mass-measurements (with less than 5 pmm accuracy) and fragmentation spectra (or MS:MS_COMMENTS simply MS/MS spectra) were used for confident identification of metabolites and MS:MS_COMMENTS lipids. MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST002739_AN004442_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END