#METABOLOMICS WORKBENCH Jung_DNLI_20250214_171815 DATATRACK_ID:5629 STUDY_ID:ST003737 ANALYSIS_ID:AN006133 PROJECT_ID:PR002323 VERSION 1 CREATED_ON February 16, 2025, 11:41 am #PROJECT PR:PROJECT_TITLE PILRA regulates microglial neuroinflammation and lipid metabolism as a candidate PR:PROJECT_TITLE therapeutic target for Alzheimer’s disease PR:PROJECT_TYPE Cellular studies PR:PROJECT_SUMMARY The Alzheimer’s disease (AD) human genetic landscape identified microglia as a PR:PROJECT_SUMMARY key disease-modifying brain cell type. A common loss-of-function coding variant PR:PROJECT_SUMMARY in paired immunoglobulin-like type 2 receptor alpha (PILRA) associated with PR:PROJECT_SUMMARY reduced AD risk, was found enriched in a cohort of healthy centenarians and PR:PROJECT_SUMMARY rescued APOE4 AD risk, however the mechanisms underlying protection are PR:PROJECT_SUMMARY undefined. Here we identify biological functions of PILRA, an immunoreceptor PR:PROJECT_SUMMARY tyrosine-based inhibitory motif (ITIM)-containing receptor, in human PR:PROJECT_SUMMARY iPSC-derived microglia (iMG) and chimeric AD mice. CRISPR-mediated PILRA PR:PROJECT_SUMMARY knockout (KO) in iMG rescued ApoE4-induced immunometabolic deficits. Moreover, PR:PROJECT_SUMMARY loss of PILRA confers a signature of metabolic resilience in microglia with PR:PROJECT_SUMMARY increased mitochondrial capacity in tandem with elevated antioxidants, reduced PR:PROJECT_SUMMARY ROS and toxic lipid species. Additionally, PILRA KO iMG exhibit improved PR:PROJECT_SUMMARY lysosomal degradation, enhanced migration, and attenuated cytokine responses. We PR:PROJECT_SUMMARY show PPAR and STAT1/3 act as master regulators that mediate downstream signaling PR:PROJECT_SUMMARY and regulate PILRA-dependent microglial functions. Importantly, AD mice PR:PROJECT_SUMMARY transplanted with human PILRA KO microglia showed reduced amyloid pathology and PR:PROJECT_SUMMARY rescued levels of synaptic markers. Finally, we identify a high-affinity human PR:PROJECT_SUMMARY PILRA-specific antagonist antibody that phenocopies PILRA KO. Together, these PR:PROJECT_SUMMARY findings suggest a therapeutic approach to modulate microglial immunometabolism PR:PROJECT_SUMMARY by inhibiting PILRA, thus identifying a pharmacologically tractable target for PR:PROJECT_SUMMARY AD. PR:INSTITUTE Denali Therapeutics PR:LAST_NAME Suh PR:FIRST_NAME Jung PR:ADDRESS 161 Oyster Point Blvd, South San Francisco, CA 94080 PR:EMAIL suh@dnli.com PR:PHONE +1 6507973837 #STUDY ST:STUDY_TITLE PILRA regulates microglial neuroinflammation and lipid metabolism as a candidate ST:STUDY_TITLE therapeutic target for Alzheimer’s disease ST:STUDY_TYPE In vitro study ST:STUDY_SUMMARY The Alzheimer’s disease (AD) human genetic landscape identified microglia as a ST:STUDY_SUMMARY key disease-modifying brain cell type. A common loss-of-function coding variant ST:STUDY_SUMMARY in paired immunoglobulin-like type 2 receptor alpha (PILRA) associated with ST:STUDY_SUMMARY reduced AD risk, was found enriched in a cohort of healthy centenarians and ST:STUDY_SUMMARY rescued APOE4 AD risk, however the mechanisms underlying protection are ST:STUDY_SUMMARY undefined. Here we identify biological functions of PILRA, an immunoreceptor ST:STUDY_SUMMARY tyrosine-based inhibitory motif (ITIM)-containing receptor, in human ST:STUDY_SUMMARY iPSC-derived microglia (iMG) and chimeric AD mice. CRISPR-mediated PILRA ST:STUDY_SUMMARY knockout (KO) in iMG rescued ApoE4-induced immunometabolic deficits. Moreover, ST:STUDY_SUMMARY loss of PILRA confers a signature of metabolic resilience in microglia with ST:STUDY_SUMMARY increased mitochondrial capacity in tandem with elevated antioxidants, reduced ST:STUDY_SUMMARY ROS and toxic lipid species. Additionally, PILRA KO iMG exhibit improved ST:STUDY_SUMMARY lysosomal degradation, enhanced migration, and attenuated cytokine responses. We ST:STUDY_SUMMARY show PPAR and STAT1/3 act as master regulators that mediate downstream signaling ST:STUDY_SUMMARY and regulate PILRA-dependent microglial functions. Importantly, AD mice ST:STUDY_SUMMARY transplanted with human PILRA KO microglia showed reduced amyloid pathology and ST:STUDY_SUMMARY rescued levels of synaptic markers. Finally, we identify a high-affinity human ST:STUDY_SUMMARY PILRA-specific antagonist antibody that phenocopies PILRA KO. Together, these ST:STUDY_SUMMARY findings suggest a therapeutic approach to modulate microglial immunometabolism ST:STUDY_SUMMARY by inhibiting PILRA, thus identifying a pharmacologically tractable target for ST:STUDY_SUMMARY AD. ST:INSTITUTE Denali Therapeutics ST:LAST_NAME Suh ST:FIRST_NAME Jung ST:ADDRESS 161 Oyster Point Blvd, South San Francisco, CA 94080 ST:EMAIL suh@dnli.com ST:PHONE +1 06507973837 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN WT, PILRA KO SU:CELL_BIOSOURCE_OR_SUPPLIER University of California Irvine SU:CELL_STRAIN_DETAILS Induced pluripotent stem cells derived microglia cells SU:CELL_PRIMARY_IMMORTALIZED no SU:CELL_COUNTS 500,000 SU:SPECIES_GROUP human #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 - HSA-000033340 genotype:PILRAKO | treatment:LPS | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_002.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_002.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_002.mzML; cell_line=Line 2 MBJ; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_002.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033323 genotype:PILRAKO | treatment:Vehicle | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_003.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_003.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_003.mzML; cell_line=Line 1 DNLI; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_003.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033319 genotype:WT | treatment:Vehicle | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_004.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_004.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_004.mzML; cell_line=Line 1 DNLI; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_004.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033335 genotype:PILRAKO | treatment:Vehicle | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_005.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_005.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_005.mzML; cell_line=Line 2 MBJ; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_005.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033332 genotype:WT | treatment:LPS | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_006.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_006.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_006.mzML; cell_line=Line 2 MBJ; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_006.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033327 genotype:PILRAKO | treatment:LPS | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_007.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_007.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_007.mzML; cell_line=Line 1 DNLI; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_007.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033331 genotype:WT | treatment:Vehicle | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_008.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_008.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_008.mzML; cell_line=Line 2 MBJ; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_008.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033329 genotype:WT | treatment:Vehicle | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_009.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_009.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_009.mzML; cell_line=Line 2 MBJ; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_009.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033326 genotype:PILRAKO | treatment:LPS | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_010.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_010.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_010.mzML; cell_line=Line 1 DNLI; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_010.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033334 genotype:WT | treatment:LPS | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_011.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_011.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_011.mzML; cell_line=Line 2 MBJ; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_011.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033324 genotype:PILRAKO | treatment:Vehicle | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_012.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_012.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_012.mzML; cell_line=Line 1 DNLI; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_012.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033322 genotype:WT | treatment:LPS | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_013.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_013.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_013.mzML; cell_line=Line 1 DNLI; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_013.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033320 genotype:WT | treatment:LPS | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_015.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_015.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_015.mzML; cell_line=Line 1 DNLI; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_015.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033337 genotype:PILRAKO | treatment:Vehicle | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_016.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_016.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_016.mzML; cell_line=Line 2 MBJ; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_016.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033317 genotype:WT | treatment:Vehicle | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_017.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_017.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_017.mzML; cell_line=Line 1 DNLI; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_017.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033339 genotype:PILRAKO | treatment:LPS | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_018.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_018.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_018.mzML; cell_line=Line 2 MBJ; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_018.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033330 genotype:WT | treatment:Vehicle | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_019.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_019.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_019.mzML; cell_line=Line 2 MBJ; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_019.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033328 genotype:PILRAKO | treatment:LPS | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_020.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_020.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_020.mzML; cell_line=Line 1 DNLI; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_020.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033325 genotype:PILRAKO | treatment:Vehicle | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_021.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_021.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_021.mzML; cell_line=Line 1 DNLI; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_021.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033321 genotype:WT | treatment:LPS | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_022.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_022.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_022.mzML; cell_line=Line 1 DNLI; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_022.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033336 genotype:PILRAKO | treatment:Vehicle | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_023.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_023.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_023.mzML; cell_line=Line 2 MBJ; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_023.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033338 genotype:PILRAKO | treatment:LPS | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_024.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_024.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_024.mzML; cell_line=Line 2 MBJ; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_024.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033318 genotype:WT | treatment:Vehicle | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_025.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_025.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_025.mzML; cell_line=Line 1 DNLI; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_025.mzML SUBJECT_SAMPLE_FACTORS - HSA-000033333 genotype:WT | treatment:LPS | Sample source:iPSC microglia RAW_FILE_NAME(Met_Neg)=20230911_TW_HEX-000666_Met_Neg_026.mzML; RAW_FILE_NAME(Lipid_Pos)=20230908_TW_HEX-000666_Lipid_Pos_026.mzML; RAW_FILE_NAME(Lipid_Neg)=20230909_TW_HEX-000666_Lipid_Neg_026.mzML; cell_line=Line 2 MBJ; RAW_FILE_NAME(Met_Pos)=20230909_TW_HEX-000666_Met_Pos_026.mzML #COLLECTION CO:COLLECTION_SUMMARY WT and PILRA KO microglia cells (500,000) were pelleted, and washed 1x with 0.9% CO:COLLECTION_SUMMARY sodium chloride and collected in collected in Lobind 1.5 mL Eppendorf tubes and CO:COLLECTION_SUMMARY snap frozen. CO:SAMPLE_TYPE iPSC cells CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_VIALS Lobind 1.5 mL Eppendorf tubes CO:STORAGE_VIALS Lobind 1.5 mL Eppendorf tubes #TREATMENT TR:TREATMENT_SUMMARY WT and PILRA KO microglia cells were cultured in iPSC-complete media without TR:TREATMENT_SUMMARY TGFBeta and plated on matrigel-coated 12-well plate. Wells were supplemented TR:TREATMENT_SUMMARY with PBS (vehicle) or endotoxin (LPS) (100ng/ml, Sigma-Aldrich, 055-B5) for 48 TR:TREATMENT_SUMMARY hours and harvested for metabolite extraction. TR:TREATMENT_COMPOUND endotoxin (LPS; Sigma-Aldrich, 055-B5) TR:TREATMENT_DOSE 100 ng/mL TR:TREATMENT_DOSEDURATION 48 hours TR:TREATMENT_VEHICLE PBS #SAMPLEPREP SP:SAMPLEPREP_SUMMARY On the day of lipid and metabolite extraction, cell pellets were reconstituted SP:SAMPLEPREP_SUMMARY with 400 μL MS grade methanol spiked with internal standards.Samples were SP:SAMPLEPREP_SUMMARY vortexed and volumes were adjusted to 800 μL with MS grade H2O, vortexed again, SP:SAMPLEPREP_SUMMARY then 600 μL tert-butyl methyl ether (MTBE) was added, vortexed, then centrifuged SP:SAMPLEPREP_SUMMARY at 21,000 g for 10 min at 4C. The two phases generated by centrifugation were SP:SAMPLEPREP_SUMMARY separated. Each phase (top: non-polar lipids, bottom: polar metabolites) was SP:SAMPLEPREP_SUMMARY transferred to glass vials and dried overnight using a Genevac EZ3. Non-polar SP:SAMPLEPREP_SUMMARY lipids were resuspended MS grade methanol and polar metabolites were resuspended SP:SAMPLEPREP_SUMMARY in 90% methanol:water mixture. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACT_STORAGE -20℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Ion exchange CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Imtakt Intrada Organic Acid (150 x 2 mm, 3 μm) CH:SOLVENT_A 10% acetonitrile/90% water; 0.1% formic acid CH:SOLVENT_B 10% acetonitrile/90% water; 100mM ammonium formate CH:FLOW_GRADIENT 0.0–1.0 min at 0% B; 1.0–7.0 min to 100% B; 7.1 at 0% B; and 7.1-10 min at CH:FLOW_GRADIENT 0% B. CH:FLOW_RATE 0.2 CH:COLUMN_TEMPERATURE 60°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 6500+ QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS curtain gas at 40 V; collision gas was set at medium; ion spray voltage at -4500 MS:MS_COMMENTS V; temperature at 600°C; ion source Gas 1 at 50 psi; ion source Gas 2 at 60 MS:MS_COMMENTS psi; entrance potential at -10 V; and collision cell exit potential at -15.0 MS:MS_COMMENTS V. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS area MS_METABOLITE_DATA_START Samples HSA-000033340 HSA-000033323 HSA-000033319 HSA-000033335 HSA-000033332 HSA-000033327 HSA-000033331 HSA-000033329 HSA-000033326 HSA-000033334 HSA-000033324 HSA-000033322 HSA-000033320 HSA-000033337 HSA-000033317 HSA-000033339 HSA-000033330 HSA-000033328 HSA-000033325 HSA-000033321 HSA-000033336 HSA-000033338 HSA-000033318 HSA-000033333 Factors genotype:PILRAKO | treatment:LPS | Sample source:iPSC microglia genotype:PILRAKO | treatment:Vehicle | Sample source:iPSC microglia genotype:WT | treatment:Vehicle | Sample source:iPSC microglia genotype:PILRAKO | treatment:Vehicle | Sample source:iPSC microglia genotype:WT | treatment:LPS | Sample source:iPSC microglia genotype:PILRAKO | treatment:LPS | Sample source:iPSC microglia genotype:WT | treatment:Vehicle | Sample source:iPSC microglia genotype:WT | treatment:Vehicle | Sample source:iPSC microglia genotype:PILRAKO | treatment:LPS | Sample source:iPSC microglia genotype:WT | treatment:LPS | Sample source:iPSC microglia genotype:PILRAKO | treatment:Vehicle | Sample source:iPSC microglia genotype:WT | treatment:LPS | Sample source:iPSC microglia genotype:WT | treatment:LPS | Sample source:iPSC microglia genotype:PILRAKO | treatment:Vehicle | Sample source:iPSC microglia genotype:WT | treatment:Vehicle | Sample source:iPSC microglia genotype:PILRAKO | treatment:LPS | Sample source:iPSC microglia genotype:WT | treatment:Vehicle | Sample source:iPSC microglia genotype:PILRAKO | treatment:LPS | Sample source:iPSC microglia genotype:PILRAKO | treatment:Vehicle | Sample source:iPSC microglia genotype:WT | treatment:LPS | Sample source:iPSC microglia genotype:PILRAKO | treatment:Vehicle | Sample source:iPSC microglia genotype:PILRAKO | treatment:LPS | Sample source:iPSC microglia genotype:WT | treatment:Vehicle | Sample source:iPSC microglia genotype:WT | treatment:LPS | Sample source:iPSC microglia 13C3-Citric acid 12574719.9 11381336.97 15110150.86 12476975.8 11134298.77 13071364.85 9148048.572 11180759.6 12717813.65 6508601.993 12712511.75 19349285.88 15669480.26 16447438.45 13856435.66 13218038.89 10059379.78 10100416.51 10942396.86 20957887.16 9757794.572 11032550.7 13493508.99 12558934.31 13C4-Fumaric acid 218282.3168 256375.9055 176121.4962 253095.9967 160121.1502 383357.1625 219986.725 380291.7945 292684.9681 128674.7166 451851.7654 417171.9698 255599.3037 320536.8749 291101.4918 220336.4038 362946.8835 151464.372 155371.3799 342253.5333 275389.8033 265907.0383 311179.4005 329111.8967 13C5-Itaconic acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 13C4-Malic acid 30092449.36 37423282.35 35661985.63 34097408.89 33963831.37 36023538.14 31041608.76 38631911.27 31480988.26 21124351.91 37301726.61 21390082.14 33364652.57 33867574.65 33839756.8 33015625.75 34164703.28 34958630.86 32034534.27 30892065.48 31400824.89 27311076.3 35942286.5 34589853.65 13C4-Succinic acid 49101122.09 43049620.7 41482035.39 40001950.2 40696419.25 43292840.94 38047296.87 42273504.54 48636709.32 32785894.19 44083957.19 48097302.04 43552065.02 40427784.45 48949922.17 44367604.74 39559127.36 46450582.69 43112091.7 39713129.43 46196691.68 34623248.77 39718125.77 38596390.98 13C5-Oxoglutaric acid 4290317.713 3994943.264 4804974.763 4159148.402 3661691.999 4871402.472 4111734.031 3705512.515 4338152.672 2253031.574 4628720.278 6634981.171 4314065.851 4900144.246 5447182.991 4297533.452 5057030.307 4262912.081 4122721.238 4176291.6 3998161.643 4631540.342 4227000.774 4948141.354 15N5-ADP 93562456.57 138603054.4 101216754.4 121358814.9 128930705.9 104412034 91331661.52 108053887 133241940.3 93342712.42 126849626.3 110964308.9 116021104.5 137912386 130770760.9 111133640.7 131455671.3 136843136.5 100737290.7 118533165.5 63747474.79 151590692.2 108152646.8 105615888.6 2-Hydroxyglutarate NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 2-Phosphoglyceric acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 3-Hydroxybutyric acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 3-Phosphoglyceric acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 6-Phosphogluconic acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Acetoacetic acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Adenosine monophosphate 179625136.6 152248610.3 228263993.2 140473452.5 109836431.4 162546283.3 150920020.9 106005841.1 111226347.4 98507493.63 163321608.8 194642758.7 154947321.2 176229998.1 148846363.8 149966188.7 119383303.7 165786625.1 172120819 167725616.4 167342868.9 116635224.7 162647856.9 136595812.4 Adenosine triphosphate NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Adenosine triphosphate-d4 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA ADP 56252236.61 102343665.1 94230894.52 94805308.96 100242871.2 101483138.9 52536588.79 78149888.49 104075875.2 45079243 85565378.61 111475429.1 139271911.9 64009811.96 119477613.1 79788589.54 42856948.69 86938906.46 48519142.41 136991239.7 44204840.53 68364588.92 125387225 64370976.11 Alanine NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Alanine-d4 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Arginine NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Arginine-d4C13 55115601.84 59278907.9 54807699.71 56506444.67 65205450.81 56172857.1 63338670.52 68853874.61 45385894.61 46191044.73 52639781.66 56758792.12 55938663.68 47586450.31 58973668.24 49027311 49412474.22 49842350.04 52029740.8 52485978.32 42962202.17 43485379.18 49296338.15 48277419.57 Asparagine NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Aspartic acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Aspartic acid-d3 58794575.99 69850328.69 54129202.26 71061169.93 79342077.68 71721470.8 75772803.92 75563637.6 53944316.77 33471673.93 67778299.44 66971853.81 57983647.04 38173003.8 65613886.97 55646651.04 40446585.66 61209376.09 63283291.02 53309924.05 66920613.76 43310884.54 84310487.72 40514611.42 cis-Aconitic acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Sum of Citric acid and Isocitric acid 31606664.31 33176756.15 57124058.39 34497867.69 36766090.56 52551926.29 28198092.35 30229016.85 40865010.96 18588077.79 41860756.01 81158784.79 80841109.75 52401134.35 66000926.14 41238648.87 36357750.73 37593075.51 41082794.16 95251960.32 38774733.83 30170478.46 66483511.4 46539395.43 Dihydroxyacetone phosphate 14950094.62 9643633.905 13352414.05 9422375.947 11567256.05 7958491.475 12104178.79 8216857.589 12597238.03 6646826.237 13590772.78 22067147.17 17822346.21 18491073.48 12288802.95 12595780.76 8834062.912 11594607.53 13677222.73 12717287.09 9030540.247 11548306.3 14585686.18 11756759.19 Erythose 4-phosphate 4372994.619 3781409.205 6711057.854 2860588.25 2846551.322 5242369.45 2583761.338 2114257.919 3370389.252 1021311.837 4737341.666 8910979.279 7188017.267 4467852.551 9103675.67 3339174.714 1753356.257 5573841.225 3215619.32 5916792.507 1450261.118 2569765.765 7361408.365 3271040.294 Fructose 1,6-bisphosphate 7969240.281 6147156.266 11098640.75 5745896.327 16799498.83 3550635.436 3352947.822 5680629.16 9036435.308 2030163.868 6617343.218 21836830.59 32537948.33 7277632.061 25437717.73 6904306.71 2936124.472 4415027.525 2885551.328 23831157.85 1356065.392 3619607.475 22716238.19 3313834.953 Fructose 6-phosphate NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Fumaric acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Glucose 1-phosphate NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Glucose 6-phosphate 50156766.22 57399185.27 67199339.79 40854968.98 42664884.1 58927827.11 37328265.39 34852664.81 50163918.26 27297213.28 68304271.34 88774024.07 77643280.52 66946329.93 75216515.67 57901299.56 36082633.99 68076338.76 54338571.64 81434443.76 45265646.37 49307824.34 74178454.09 59254925.14 Glutamic acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Glutamic acid-d3 52527408.25 35897079.46 47432379.34 49393136.06 47965353.52 46468146.82 52453313.86 53449212.46 38867686.85 31764185.92 39471143.51 30881831.67 25479840.6 27848790.29 31650755.37 31910807.95 38456107.13 36061119.7 36951306.07 28257319.84 25610907.34 27491937.35 26614040.92 27666410.47 Glutamine NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Glyceraldehyde 3-phosphate 12193871.63 6332502.489 11337549.51 8462280.396 10149440.07 6149230.346 10392217.54 7380209.45 10093865.14 4749212.618 10189848.04 19365583.25 14778096.43 15701571.03 11018639.5 9334401.077 7121644.234 9261723.183 10535236.07 5166609.003 7452955.819 11346951.55 10463209.05 9117148.034 Itaconic acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Lactic acid 53104476.71 42895019.09 53795554.27 47378312.27 44587856.8 51538272.03 41326484.76 37623632.63 43976858.75 25723915.76 40317888.53 58259582.58 65518622.34 51516089.39 38675496.07 45020532.17 37867354.65 40402779.86 39920709.43 56861210.26 45053187.12 39222577.77 42536278.62 37832719.57 Malic acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NAD 2198999.794 3000925.4 5055772.231 1373609.462 2217127.375 3751100.11 1633513.676 2110681.836 3845109.619 866898.9666 3240603.705 5499553.158 5741664.272 2063347.54 5370054.99 1636004.873 1240577.51 3509775.272 2314302.933 6003862.533 1480798.574 1573442.898 4915307.869 1510502.521 NADH NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NADP NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NADPH NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Oxalacetic acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Oxoglutaric acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Phosphocreatine NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Phosphoenolpyruvic acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Pyruvic acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Ribose 5-phosphate 41171871.91 48640576.49 54155850.43 33015678.72 29827327.64 50039221.3 27437855.09 22623664.75 39062525.96 11843276.84 55429544.03 75788821.98 72030890.92 56549102.75 59128700.46 40828279.84 25590317.81 55083790.31 49280536.99 62994887.73 39802857.09 36028421.94 60347239.21 38517701.71 Ribulose 5-phosphate NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Sedoheptulose 7-phosphate 14895604.24 12757188.42 22496377.05 6525677.272 11656429.8 15265826.72 9449562.934 7032667.842 11613111.29 3881647.551 15346295.21 31236752.3 24381561.95 11988881.54 22969318.38 10261907.06 5941541.198 15590972.58 12269382.63 24727066.2 6120925.85 6796040.775 23983824.18 9419916.523 Serine NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Succinic acid 52128310.89 44078193.34 51428757.17 54587741.06 48542398.95 51527865.78 50069884.12 50058613.11 48240886.79 45017827.39 45119856.34 58277695.34 42908674.47 38639266.51 52338084.67 42600888.41 33976416.33 46593080.38 51282041.9 44654704.16 41201647.32 35963195.11 40803701.29 35520387.99 U-13C10,U-15N5-Adenosine monophosphate 221496773.3 210862671.8 231930193.8 208593351 217666734.6 224998402.9 213300036.3 212223566.8 230971679.7 167057161 221902134.3 208838081.6 218980940.1 218530574.8 215004720.5 211556383.7 195791662.4 225046307 207586804.2 212366333.2 217368860.7 195129652.8 202727932.4 212717483.2 U-13C3-Pyruvic acid NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA U-13C6-Glucose 6-phosphate 26357414.24 21716067.15 23372448.98 21843623.94 23282356.36 21198595.57 22729619.31 23940635.32 24478895.58 15883782.05 25125710.5 26227504.02 24317503.97 25544450.15 27573594.67 22575773.23 19366751.11 23285523.98 21570340.19 23842066.36 19651205.17 22396657.64 25815979.74 20524744.32 Xylulose 5-phosphate NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Fructose 1-phosphate NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Expected RT MS1_mz MS2_mz 13C3-Citric acid 4.61 194.2 113 13C4-Fumaric acid 4.48 119.2 74.3 13C5-Itaconic acid 2.67 134 89.2 13C4-Malic acid 3.37 137.2 119.2 13C4-Succinic acid 2.32 121.3 76 13C5-Oxoglutaric acid 4.7 150.2 105.3 15N5-ADP 5.95 431.1 79 2-Hydroxyglutarate 2.9 145 101 2-Phosphoglyceric acid 5.66 185.5 97.1 3-Hydroxybutyric acid 2.17 103.2 59.2 3-Phosphoglyceric acid 5.67 185.5 97 6-Phosphogluconic acid 5.3 275 97 Acetoacetic acid 0.31 101 101 Adenosine monophosphate 3.9 346 79 Adenosine triphosphate 7 506.1 158.9 Adenosine triphosphate-d4 7 510 158.9 ADP 5.97 426.1 79 Alanine 2.18 88 88 Alanine-d4 2.07 92 92 Arginine 1.24 173 131 Arginine-d4C13 1.24 178 136 Asparagine 1.78 131 114 Aspartic acid 2.25 132 88 Aspartic acid-d3 2.25 135 91 cis-Aconitic acid 5.33 173 129 Sum of Citric acid and Isocitric acid 4.54 191.2 111 Dihydroxyacetone phosphate 4.55 169.1 79 Erythose 4-phosphate 4.36 199 97 Fructose 1,6-bisphosphate 5.51 339.1 79 Fructose 6-phosphate 4.5 259 97.1 Fumaric acid 4.7 115.2 71.3 Glucose 1-phosphate 4.5 259 241 Glucose 6-phosphate 4.5 259 97 Glutamic acid 1.9 146 128 Glutamic acid-d3 1.91 149 131 Glutamine 1.78 145 127 Glyceraldehyde 3-phosphate 4.55 169.1 97 Itaconic acid 2.67 129.2 85 Lactic acid 2.5 89.2 43.2 Malic acid 3.36 133.2 115.2 NAD 3.77 662.1 540.3 NADH 2.87 664.1 408.1 NADP 5.97 742.1 620 NADPH 4.73 744.1 408 Oxalacetic acid 3.63 131 131 Oxoglutaric acid 4.87 145.2 101.3 Phosphocreatine 3.53 210 79 Phosphoenolpyruvic acid 5.78 167 79 Pyruvic acid 4.64 87.2 43.2 Ribose 5-phosphate 4.54 229 97 Ribulose 5-phosphate 4.42 229 97.1 Sedoheptulose 7-phosphate 4.41 289 97 Serine 1.77 104 74 Succinic acid 2.28 117.2 73.2 U-13C10,U-15N5-Adenosine monophosphate 3.9 361 79 U-13C3-Pyruvic acid 4.64 90.2 45.2 U-13C6-Glucose 6-phosphate 4.4 265 97 Xylulose 5-phosphate 4.5 229.1 97 Fructose 1-phosphate 4.5 259.1 79 METABOLITES_END #END