{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002450","ANALYSIS_ID":"AN003998","VERSION":"1","CREATED_ON":"01-23-2023"},

"PROJECT":{"PROJECT_TITLE":"APOE modulates microglial immunometabolism in response to age, amyloid pathology, and inflammatory challenge","PROJECT_SUMMARY":"The E4 allele of Apolipoprotein E (APOE) is associated with both metabolic dysfunction and a heightened pro-inflammatory response – two findings that may be intrinsically linked through the concept of immunometabolism. Here, we combined bulk, single-cell, and spatial transcriptomics with cell-specific and spatially resolved metabolic analyses to systematically address the role of APOE across age, neuroinflammation, and AD pathology. RNAseq highlighted immunometabolic changes across the APOE4 glial transcriptome, specifically in subsets of metabolically distinct microglia enriched in the E4 brain during aging or following an inflammatory challenge. E4 microglia display increased Hif1α expression, a disrupted TCA cycle, and are inherently pro-glycolytic, while spatial transcriptomics and MALDI mass spectrometry imaging highlight an E4-specific response to amyloid that is characterized by widespread alterations in lipid metabolism. Taken together, our findings emphasize a central role for APOE in regulating microglial immunometabolism.","INSTITUTE":"University of Kentucky, Department of Physiology","LAST_NAME":"Devanney","FIRST_NAME":"Nicholas","ADDRESS":"Physiology, 760 Press Ave, Healthy Kentucky Research Bldg, Rm152, Lexington, Kentucky, 40508, USA","EMAIL":"Nicholas.Devanney@uky.edu","PHONE":"8593238083","PROJECT_COMMENTS":"Part 2 of 3","DOI":"http://dx.doi.org/10.21228/M8G711"},

"STUDY":{"STUDY_TITLE":"APOE modulates microglial immunometabolism in response to age, amyloid pathology, and inflammatory challenge (Part 1 of 3)","STUDY_SUMMARY":"The E4 allele of Apolipoprotein E (APOE) is associated with both metabolic dysfunction and a heightened pro-inflammatory response – two findings that may be intrinsically linked through the concept of immunometabolism. Here, we combined bulk, single-cell, and spatial transcriptomics with cell-specific and spatially resolved metabolic analyses to systematically address the role of APOE across age, neuroinflammation, and AD pathology. RNAseq highlighted immunometabolic changes across the APOE4 glial transcriptome, specifically in subsets of metabolically distinct microglia enriched in the E4 brain during aging or following an inflammatory challenge. E4 microglia display increased Hif1α expression, a disrupted TCA cycle, and are inherently pro-glycolytic, while spatial transcriptomics and MALDI mass spectrometry imaging highlight an E4-specific response to amyloid that is characterized by widespread alterations in lipid metabolism. Taken together, our findings emphasize a central role for APOE in regulating microglial immunometabolism.","INSTITUTE":"University of Kentucky, Department of Physiology","LAST_NAME":"Devanney","FIRST_NAME":"Nicholas","ADDRESS":"Physiology, 760 Press Ave, Healthy Kentucky Research Bldg, Rm152, Lexington, Kentucky, 40508, USA","EMAIL":"Nicholas.Devanney@uky.edu","PHONE":"8593238083","SUBMIT_DATE":"2022-09-20"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"Primary microglia isolated from APOE-targeted replacement mice homozygous for human E3 (B6.129P2-Apoe^tm2(APOE*3)Mae N8, Taconic #1548-F) or human E4 (B6.129P2- Apoe^tm3(APOE*4)Mae N8, Taconic #1549-F) alleles","AGE_OR_AGE_RANGE":"P0-P3","GENDER":"Pooled"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"15_3_2",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"15_3_2.CDF","Protein Amount (BCA)":"725.828"}
},
{
"Subject ID":"-",
"Sample ID":"15_3_3",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"15_3_3.CDF","Protein Amount (BCA)":"1044.435"}
},
{
"Subject ID":"-",
"Sample ID":"16_3_2",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"16_3_2.CDF","Protein Amount (BCA)":"1534.876"}
},
{
"Subject ID":"-",
"Sample ID":"16_3_3_1",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"16_3_3_1.CDF","Protein Amount (BCA)":"791.458"}
},
{
"Subject ID":"-",
"Sample ID":"16_3_3_2",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"16_3_3_2.CDF","Protein Amount (BCA)":"1050.401"}
},
{
"Subject ID":"-",
"Sample ID":"16_3_4",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"16_3_4.CDF","Protein Amount (BCA)":"727.021"}
},
{
"Subject ID":"-",
"Sample ID":"16_3_5",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"16_3_5.CDF","Protein Amount (BCA)":"742.534"}
},
{
"Subject ID":"-",
"Sample ID":"16_3_6",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"16_3_6.CDF","Protein Amount (BCA)":"834.417"}
},
{
"Subject ID":"-",
"Sample ID":"3_3",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"3_3.CDF","Protein Amount (BCA)":"1361.61"}
},
{
"Subject ID":"-",
"Sample ID":"3_4",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"3_4.CDF","Protein Amount (BCA)":"1174.855"}
},
{
"Subject ID":"-",
"Sample ID":"3_5",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"3_5.CDF","Protein Amount (BCA)":"894.722"}
},
{
"Subject ID":"-",
"Sample ID":"3_6",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"3_6.CDF","Protein Amount (BCA)":"1162.522"}
},
{
"Subject ID":"-",
"Sample ID":"3_7",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"3_7.CDF","Protein Amount (BCA)":"1305.231"}
},
{
"Subject ID":"-",
"Sample ID":"3_8",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"3_8.CDF","Protein Amount (BCA)":"1078.834"}
},
{
"Subject ID":"-",
"Sample ID":"3_9",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"3_9.CDF","Protein Amount (BCA)":"762.435"}
},
{
"Subject ID":"-",
"Sample ID":"MG14_1",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"MG14_1.CDF","Protein Amount (BCA)":"767.228"}
},
{
"Subject ID":"-",
"Sample ID":"MG14_2",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"MG14_2.CDF","Protein Amount (BCA)":"687.234"}
},
{
"Subject ID":"-",
"Sample ID":"MG15_2",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"MG15_2.CDF","Protein Amount (BCA)":"395.081"}
},
{
"Subject ID":"-",
"Sample ID":"MG15_3",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"MG15_3.CDF","Protein Amount (BCA)":"424.644"}
},
{
"Subject ID":"-",
"Sample ID":"MG16_E3_1",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"MG16_E3_1.CDF","Protein Amount (BCA)":"312.99"}
},
{
"Subject ID":"-",
"Sample ID":"MG16_E3_1_1",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"MG16_E3_1_1.CDF","Protein Amount (BCA)":"380.592"}
},
{
"Subject ID":"-",
"Sample ID":"MG16_E3_2",
"Factors":{"Genotype":"E3"},
"Additional sample data":{"RAW_FILE_NAME":"MG16_E3_2.CDF","Protein Amount (BCA)":"435.457"}
},
{
"Subject ID":"-",
"Sample ID":"15_4_4",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"15_4_4.CDF","Protein Amount (BCA)":"693.609"}
},
{
"Subject ID":"-",
"Sample ID":"15_4_5",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"15_4_5.CDF","Protein Amount (BCA)":"795.038"}
},
{
"Subject ID":"-",
"Sample ID":"16_4_3",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"16_4_3.CDF","Protein Amount (BCA)":"914.367"}
},
{
"Subject ID":"-",
"Sample ID":"16_4_4",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"16_4_4.CDF","Protein Amount (BCA)":"828.45"}
},
{
"Subject ID":"-",
"Sample ID":"16_4_5_1",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"16_4_5_1.CDF","Protein Amount (BCA)":"1150.638"}
},
{
"Subject ID":"-",
"Sample ID":"16_4_5_2",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"16_4_5_2.CDF","Protein Amount (BCA)":"815.324"}
},
{
"Subject ID":"-",
"Sample ID":"16_4_6_1",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"16_4_6_1.CDF","Protein Amount (BCA)":"742.534"}
},
{
"Subject ID":"-",
"Sample ID":"16_4_6_2",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"16_4_6_2.CDF","Protein Amount (BCA)":"976.418"}
},
{
"Subject ID":"-",
"Sample ID":"4_1",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"4_1.CDF","Protein Amount (BCA)":"1678.742"}
},
{
"Subject ID":"-",
"Sample ID":"4_2",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"4_2.CDF","Protein Amount (BCA)":"1381.871"}
},
{
"Subject ID":"-",
"Sample ID":"4_3",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"4_3.CDF","Protein Amount (BCA)":"968.719"}
},
{
"Subject ID":"-",
"Sample ID":"4_4",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"4_4.CDF","Protein Amount (BCA)":"959.029"}
},
{
"Subject ID":"-",
"Sample ID":"4_5",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"4_5.CDF","Protein Amount (BCA)":"1005.718"}
},
{
"Subject ID":"-",
"Sample ID":"4_6",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"4_6.CDF","Protein Amount (BCA)":"1177.497"}
},
{
"Subject ID":"-",
"Sample ID":"4_7",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"4_7.CDF","Protein Amount (BCA)":"1149.308"}
},
{
"Subject ID":"-",
"Sample ID":"4_8",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"4_8.CDF","Protein Amount (BCA)":"1336.944"}
},
{
"Subject ID":"-",
"Sample ID":"MG15_4",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"MG15_4.CDF","Protein Amount (BCA)":"254.222"}
},
{
"Subject ID":"-",
"Sample ID":"MG15_5",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"MG15_5.CDF","Protein Amount (BCA)":"389.864"}
},
{
"Subject ID":"-",
"Sample ID":"MG16_E4_1",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"MG16_E4_1.CDF","Protein Amount (BCA)":"300.254"}
},
{
"Subject ID":"-",
"Sample ID":"MG16_E4_2",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"MG16_E4_2.CDF","Protein Amount (BCA)":"338.463"}
},
{
"Subject ID":"-",
"Sample ID":"MG16_E4_4",
"Factors":{"Genotype":"E4"},
"Additional sample data":{"RAW_FILE_NAME":"MG16_E4_4.CDF","Protein Amount (BCA)":"468.768"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Primary microglia were plated at 7x106 cells/well in 6-well plates (VWR #10062-894) and incubated at 5% CO2 37°C. Upon reaching confluence, cells were removed from the incubator washed with warm 0.9% NaCl solution. Culture plates were placed on a bed of crushed dry ice and 1mL of ice cold 50% methanol (HPLC-grade, Sigma #A456-4) was added to quench cellular metabolic activity followed by a 10 minute incubation at -80°C to ensure cell lysis. After removing from the freezer, cells were detached with a cell scraper (VWR #10062-906) and the entire contents collected into a microcentrifuge tube, vortexed briefly, and placed on ice until all samples were collected. The tubes were then placed on a Disruptor Genie Cell Disruptor Homogenizer (Scientific Industries) for 5 min at 3,000 rpm. Tubes were then centrifuged at 20,000 x g for 10 min at 4 °C. The supernatant containing polar metabolites was isolated to a new tube and stored at -80C, and the resulting pellet was briefly dried at 10-3 mbar using a CentriVap vacuum concentrator (LabConco) to evaporate remaining methanol, followed by determination of protein content via BCA assay (ThermoFisher #23225) to normalize metabolite concentrations to total protein amount of each sample.","SAMPLE_TYPE":"Cultured cells","COLLECTION_METHOD":"Ice-cold methanol was added to primary microglia cultures in 6-well plates after which cells were harvested using a cell scraper","COLLECTION_LOCATION":"University of Kentucky","VOLUMEORAMOUNT_COLLECTED":"1ml","STORAGE_CONDITIONS":"-80℃","COLLECTION_TUBE_TEMP":"-80"},

"TREATMENT":{"TREATMENT_SUMMARY":"Cells were not treated, as we were just comparing APOE3/3 vs. APOE4/4 genotypes","CELL_GROWTH_CONTAINER":"6-well plate","CELL_MEDIA":"DMEM/F12 with 10% FBS, 10% LCCM supplement (see full methods in associated publication for preparation details), and 1% Penicillin/Streptomycin","CELL_ENVIR_COND":"37C, 5% CO2","CELL_HARVESTING":"Cell scraper","CELL_PCT_CONFLUENCE":"100","CELL_MEDIA_LASTCHANGED":"2-4 days prior to harvest"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The supernatant fraction containing polar metabolites was thawed gently on ice and dried at 10-3 mbar using a CentriVap vacuum concentrator (LabConco) to evaporate methanol. The dried polar metabolite pellet was derivatized by a two-step methoxyamine protocol first by addition of 70µL methoxyamine HCl (Sigma-Aldrich #226904-5G) in pyridine (20 mg/mL; Sigma-Aldrich #TS25730) to each pellet followed by 90 min dry heat incubation at 30°C. Samples were then centrifuged at 20,000 x g for 10 minutes after which 50µL of each sample was transferred to an amber V-shaped glass chromatography vial (Agilent #5184-3554) containing 80µL N-methyl-trimethylsilyl-trifluoroacetamide (MSTFA; ThermoFisher #TS48915) and gently vortexed followed by 30 min dry heat incubation at 37°C.","EXTRACTION_METHOD":"50% methanol","EXTRACT_STORAGE":"-80℃","SAMPLE_DERIVATIZATION":"MSTFA"},

"CHROMATOGRAPHY":{"INSTRUMENT_NAME":"Agilent 8890","COLUMN_NAME":"Agilent HP5-MS (30m x 0.25mm, 0.25 um)","CHROMATOGRAPHY_TYPE":"GC"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","DATA_FORMAT":"CDF"},

"MS":{"INSTRUMENT_NAME":"Agilent 5977","INSTRUMENT_TYPE":"Single quadrupole","MS_TYPE":"EI","MS_COMMENTS":"Metabolites were identified and quantified using DExSI v1.11 (https://github.com/DExSI/DExSI)","ION_MODE":"POSITIVE"},

"MS_METABOLITE_DATA":{
"Units":"Relative Abundance",

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}