#METABOLOMICS WORKBENCH yehe_20250421_114234 DATATRACK_ID:5856 STUDY_ID:ST003875 ANALYSIS_ID:AN006367 PROJECT_ID:PR002429 VERSION 1 CREATED_ON April 22, 2025, 10:30 pm #PROJECT PR:PROJECT_TITLE Glia-derived secretory fatty acid binding protein Obp44a regulates lipid storage PR:PROJECT_TITLE and efflux in the developing Drosophila brain PR:PROJECT_SUMMARY Glia derived secretory factors play diverse roles in supporting the development, PR:PROJECT_SUMMARY physiology, and stress responses of the central nervous system (CNS). Through PR:PROJECT_SUMMARY transcriptomics and imaging analyses, we have identified Obp44a as one of the PR:PROJECT_SUMMARY most abundantly produced secretory proteins from Drosophila CNS glia. Protein PR:PROJECT_SUMMARY structure homology modeling and Nuclear Magnetic Resonance (NMR) experiments PR:PROJECT_SUMMARY reveal Obp44a as a fatty acid binding protein (FABP) with a high affinity PR:PROJECT_SUMMARY towards long-chain fatty acids in both native and oxidized forms. Further PR:PROJECT_SUMMARY analyses demonstrate that Obp44a effectively infiltrates the neuropil, traffics PR:PROJECT_SUMMARY between neuron and glia, and is secreted into hemolymph, acting as a lipid PR:PROJECT_SUMMARY chaperone and scavenger to regulate lipid and redox homeostasis in the PR:PROJECT_SUMMARY developing brain. In agreement with this essential role, deficiency of Obp44a PR:PROJECT_SUMMARY leads to anatomical and behavioral deficits in adult animals and elevated PR:PROJECT_SUMMARY oxidized lipid levels. Collectively, our findings unveil the crucial involvement PR:PROJECT_SUMMARY of a noncanonical lipid chaperone to shuttle fatty acids within and outside the PR:PROJECT_SUMMARY brain, as needed to maintain a healthy brain lipid environment. These findings PR:PROJECT_SUMMARY could inspire the design of novel approaches to restore lipid homeostasis that PR:PROJECT_SUMMARY is dysregulated in CNS diseases. PR:INSTITUTE Advanced Science Research Center - CUNY PR:DEPARTMENT Neuroscience PR:LABORATORY He Lab 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 #STUDY ST:STUDY_TITLE Glia-derived secretory fatty acid binding protein Obp44a regulates lipid storage ST:STUDY_TITLE and efflux in the developing Drosophila brain ST:STUDY_SUMMARY Glia derived secretory factors play diverse roles in supporting the development, ST:STUDY_SUMMARY physiology, and stress responses of the central nervous system (CNS). Through ST:STUDY_SUMMARY transcriptomics and imaging analyses, we have identified Obp44a as one of the ST:STUDY_SUMMARY most abundantly produced secretory proteins from Drosophila CNS glia. Protein ST:STUDY_SUMMARY structure homology modeling and Nuclear Magnetic Resonance (NMR) experiments ST:STUDY_SUMMARY reveal Obp44a as a fatty acid binding protein (FABP) with a high affinity ST:STUDY_SUMMARY towards long-chain fatty acids in both native and oxidized forms. Further ST:STUDY_SUMMARY analyses demonstrate that Obp44a effectively infiltrates the neuropil, traffics ST:STUDY_SUMMARY between neuron and glia, and is secreted into hemolymph, acting as a lipid ST:STUDY_SUMMARY chaperone and scavenger to regulate lipid and redox homeostasis in the ST:STUDY_SUMMARY developing brain. In agreement with this essential role, deficiency of Obp44a ST:STUDY_SUMMARY leads to anatomical and behavioral deficits in adult animals and elevated ST:STUDY_SUMMARY oxidized lipid levels. Collectively, our findings unveil the crucial involvement ST:STUDY_SUMMARY of a noncanonical lipid chaperone to shuttle fatty acids within and outside the ST:STUDY_SUMMARY brain, as needed to maintain a healthy brain lipid environment. These findings ST:STUDY_SUMMARY could inspire the design of novel approaches to restore lipid homeostasis that ST:STUDY_SUMMARY is dysregulated in CNS diseases. ST:INSTITUTE Advanced Science Research Center - CUNY ST:DEPARTMENT Neuroscience ST:LABORATORY He Lab ST:LAST_NAME He ST:FIRST_NAME Ye ST:ADDRESS 85 St. Nicholas Terrace, Room ST:EMAIL yhe1@gc.cuny.edu ST:PHONE 12124133182 #SUBJECT SU:SUBJECT_TYPE Insect SU:SUBJECT_SPECIES Drosophila melanogaster SU:TAXONOMY_ID 7227 #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 - KO1_001 Sample source:flyhead | genotype:Obp44a_KO RAW_FILE_NAME(raw_file_name)=KO1_001.mzML SUBJECT_SAMPLE_FACTORS - KO1_002 Sample source:flyhead | genotype:Obp44a_KO RAW_FILE_NAME(raw_file_name)=KO1_002.mzML SUBJECT_SAMPLE_FACTORS - KO1_003 Sample source:flyhead | genotype:Obp44a_KO RAW_FILE_NAME(raw_file_name)=KO1_003.mzML SUBJECT_SAMPLE_FACTORS - KO2_001 Sample source:flyhead | genotype:Obp44a_KO RAW_FILE_NAME(raw_file_name)=KO2_001.mzML SUBJECT_SAMPLE_FACTORS - KO2_002 Sample source:flyhead | genotype:Obp44a_KO RAW_FILE_NAME(raw_file_name)=KO2_002.mzML SUBJECT_SAMPLE_FACTORS - KO2_003 Sample source:flyhead | genotype:Obp44a_KO RAW_FILE_NAME(raw_file_name)=KO2_003.mzML SUBJECT_SAMPLE_FACTORS - KO3_001 Sample source:flyhead | genotype:Obp44a_KO RAW_FILE_NAME(raw_file_name)=KO3_001.mzML SUBJECT_SAMPLE_FACTORS - KO3_002 Sample source:flyhead | genotype:Obp44a_KO RAW_FILE_NAME(raw_file_name)=KO3_002.mzML SUBJECT_SAMPLE_FACTORS - KO3_003 Sample source:flyhead | genotype:Obp44a_KO RAW_FILE_NAME(raw_file_name)=KO3_003.mzML SUBJECT_SAMPLE_FACTORS - WT1_001 Sample source:flyhead | genotype:Wild-type RAW_FILE_NAME(raw_file_name)=WT1_001.mzML SUBJECT_SAMPLE_FACTORS - WT1_002 Sample source:flyhead | genotype:Wild-type RAW_FILE_NAME(raw_file_name)=WT1_002.mzML SUBJECT_SAMPLE_FACTORS - WT1_003 Sample source:flyhead | genotype:Wild-type RAW_FILE_NAME(raw_file_name)=WT1_003.mzML SUBJECT_SAMPLE_FACTORS - WT2_001 Sample source:flyhead | genotype:Wild-type RAW_FILE_NAME(raw_file_name)=WT2_001.mzML SUBJECT_SAMPLE_FACTORS - WT2_002 Sample source:flyhead | genotype:Wild-type RAW_FILE_NAME(raw_file_name)=WT2_002.mzML SUBJECT_SAMPLE_FACTORS - WT2_003 Sample source:flyhead | genotype:Wild-type RAW_FILE_NAME(raw_file_name)=WT2_003.mzML SUBJECT_SAMPLE_FACTORS - WT3_001 Sample source:flyhead | genotype:Wild-type RAW_FILE_NAME(raw_file_name)=WT3_001.mzML SUBJECT_SAMPLE_FACTORS - WT3_002 Sample source:flyhead | genotype:Wild-type RAW_FILE_NAME(raw_file_name)=WT3_002.mzML SUBJECT_SAMPLE_FACTORS - WT3_003 Sample source:flyhead | genotype:Wild-type RAW_FILE_NAME(raw_file_name)=WT3_003.mzML #COLLECTION CO:COLLECTION_SUMMARY For each biological replicate, a pool of 100 brains from third instar larvae of CO:COLLECTION_SUMMARY either wild type (Canton-S) or Obp44a-/- was dissected and combined in PBS CO:COLLECTION_SUMMARY buffer. CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY For each biological replicate, a pool of 100 brains from third instar larvae of TR:TREATMENT_SUMMARY either genotype was dissected and combined in PBS buffer. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To extract metabolites, the brain samples were homogenized in 200 μl of cold SP:SAMPLEPREP_SUMMARY Methanol/Water solution (80/20, v/v) and subjected to gentle sonication using a SP:SAMPLEPREP_SUMMARY Bioruptor instrument (30 s on, 30 s off, 10 cycles) at 4°C. Subsequently, the SP:SAMPLEPREP_SUMMARY lysates were centrifuged at 10,000 x g for 10 minutes at 4°C, and the resulting SP:SAMPLEPREP_SUMMARY supernatants were collected for LC-MS/MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME SeQuant ZIC-HILIC (100 x 2.1mm,3.5um) CH:SOLVENT_A 97% acetonitrile/3% water/7mM ammonium acetate CH:SOLVENT_B 97% water/3% acetonitrile/7mM ammonium acetate 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:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker Daltonics maXis-II MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE 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:MS_RESULTS_FILE ST003875_AN006367_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END