#METABOLOMICS WORKBENCH FendtLab_20251003_074113 DATATRACK_ID:6524 STUDY_ID:ST004268 ANALYSIS_ID:AN007104 PROJECT_ID:PR002694 VERSION 1 CREATED_ON October 9, 2025, 12:42 pm #PROJECT PR:PROJECT_TITLE Histone deacetylase SIRT6 regulates tryptophan catabolism and prevents PR:PROJECT_TITLE metabolite imbalance associated with neurodegeneration PR:PROJECT_SUMMARY In the brain, tryptophan byproducts are involved in the biosynthesis of PR:PROJECT_SUMMARY proteins, energy-rich molecules (e.g., NAD+), and neurotransmitters (serotonin PR:PROJECT_SUMMARY and melatonin). Impaired tryptophand catabolism, seen in aging, PR:PROJECT_SUMMARY neurodegeneration and psychiatric diseases affects mood, learning, and sleep; PR:PROJECT_SUMMARY however, the reasons for those impairments in elder and these ailments remain PR:PROJECT_SUMMARY unknown. Our results from cellular, Drosophila melanogaster, and mouse models PR:PROJECT_SUMMARY indicate that SIRT6 regulates tryptophan catabolism by balancing its usage. PR:PROJECT_SUMMARY Mechanistically, SIRT6 regulates tryptophan and sleep quality through changes in PR:PROJECT_SUMMARY gene expression of key genes (e.g., TDO2, AANAT), which results in elevated PR:PROJECT_SUMMARY concentration of neurotoxic metabolites from the kynurenic pathway at the PR:PROJECT_SUMMARY expense of serotonin and melatonin production. Such neurotoxic metabolites can PR:PROJECT_SUMMARY affect various processes in the brain. However, by redirecting tryptophan PR:PROJECT_SUMMARY through TDO2 inhibition in our new SIRT6-KO D. melanogaster model, the PR:PROJECT_SUMMARY impairments in neuromotor behavior and vacuolar formation - parameters of PR:PROJECT_SUMMARY neurodegeneration - could be significantly reversed. PR:INSTITUTE VIB-KU Leuven Center for Cancer Biology PR:LAST_NAME Fernández-García PR:FIRST_NAME Juan PR:ADDRESS Campus Gasthuisberg, O&N4, Herestraat 49, box 912, 3000 Leuven, Belgium PR:EMAIL juan.fernandezgarcia@kuleuven.be PR:PHONE +32 16 37 32 61 #STUDY ST:STUDY_TITLE Histone deacetylase SIRT6 regulates tryptophan catabolism and prevents ST:STUDY_TITLE metabolite imbalance associated with neurodegeneration - Drosophila ST:STUDY_SUMMARY In the brain, tryptophan byproducts are involved in the biosynthesis of ST:STUDY_SUMMARY proteins, energy-rich molecules (e.g., NAD+), and neurotransmitters (serotonin ST:STUDY_SUMMARY and melatonin). Impaired tryptophan catabolism, seen in aging, neurodegeneration ST:STUDY_SUMMARY and psychiatric diseases affects mood, learning, and sleep; however, the reasons ST:STUDY_SUMMARY for those impairments in elder and these ailments remain unknown. Our results ST:STUDY_SUMMARY from Drosophila melanogaster models show how the SIRT6-regulated accumulation of ST:STUDY_SUMMARY neurotoxic metabolites from the kynurenic pathway, at the expense of serotonin ST:STUDY_SUMMARY and melatonin, can affect various processes in the brain, leading to behavioral ST:STUDY_SUMMARY defects. However, by redirecting tryptophan through TDO2 inhibition in our new ST:STUDY_SUMMARY SIRT6-KO D. melanogaster model, the impairments in neuromotor behavior and ST:STUDY_SUMMARY vacuolar formation - parameters of neurodegeneration - could be significantly ST:STUDY_SUMMARY reversed. ST:INSTITUTE VIB-KU Leuven Center for Cancer Biology ST:LAST_NAME Fernández-García ST:FIRST_NAME Juan ST:ADDRESS Campus Gasthuisberg, O&N4, Herestraat 49, box 912, 3000 Leuven, Belgium ST:EMAIL juan.fernandezgarcia@kuleuven.be ST:PHONE +32 16 37 32 61 #SUBJECT SU:SUBJECT_TYPE Insect SU:SUBJECT_SPECIES Drosophila melanogaster SU:TAXONOMY_ID 7227 SU:GENDER Male and female #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 SIRT6 KO Female SKO-F1 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Female | Batch:1 RAW_FILE_NAME=SKO-F1.d SUBJECT_SAMPLE_FACTORS SIRT6 KO Female SKO-F2 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Female | Batch:1 RAW_FILE_NAME=SKO-F2.d SUBJECT_SAMPLE_FACTORS SIRT6 KO Female SKO-F3 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Female | Batch:1 RAW_FILE_NAME=SKO-F3.d SUBJECT_SAMPLE_FACTORS SIRT6 KO Female SKO-F4 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Female | Batch:2 RAW_FILE_NAME=SKO-F4.d SUBJECT_SAMPLE_FACTORS SIRT6 KO Female SKO-F5 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Female | Batch:2 RAW_FILE_NAME=SKO-F5.d SUBJECT_SAMPLE_FACTORS SIRT6 KO Female SKO-F6 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Female | Batch:2 RAW_FILE_NAME=SKO-F6.d SUBJECT_SAMPLE_FACTORS SIRT6 KO Male SKO-M1 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Male | Batch:1 RAW_FILE_NAME=SKO-M1.d SUBJECT_SAMPLE_FACTORS SIRT6 KO Male SKO-M2 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Male | Batch:1 RAW_FILE_NAME=SKO-M2.d SUBJECT_SAMPLE_FACTORS SIRT6 KO Male SKO-M3 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Male | Batch:1 RAW_FILE_NAME=SKO-M3.d SUBJECT_SAMPLE_FACTORS SIRT6 KO Male SKO-M4 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Male | Batch:2 RAW_FILE_NAME=SKO-M4.d SUBJECT_SAMPLE_FACTORS SIRT6 KO Male SKO-M5 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Male | Batch:2 RAW_FILE_NAME=SKO-M5.d SUBJECT_SAMPLE_FACTORS SIRT6 KO Male SKO-M6 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Male | Batch:2 RAW_FILE_NAME=SKO-M6.d SUBJECT_SAMPLE_FACTORS Wild type Female WT-F1 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Female | Batch:1 RAW_FILE_NAME=WT-F1.d SUBJECT_SAMPLE_FACTORS Wild type Female WT-F2 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Female | Batch:1 RAW_FILE_NAME=WT-F2.d SUBJECT_SAMPLE_FACTORS Wild type Female WT-F3 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Female | Batch:1 RAW_FILE_NAME=WT-F3.d SUBJECT_SAMPLE_FACTORS Wild type Female WT-F4 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Female | Batch:2 RAW_FILE_NAME=WT-F4.d SUBJECT_SAMPLE_FACTORS Wild type Female WT-F5 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Female | Batch:2 RAW_FILE_NAME=WT-F5.d SUBJECT_SAMPLE_FACTORS Wild type Female WT-F6 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Female | Batch:2 RAW_FILE_NAME=WT-F6.d SUBJECT_SAMPLE_FACTORS Wild type Male WT-M1 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Male | Batch:1 RAW_FILE_NAME=WT-M1.d SUBJECT_SAMPLE_FACTORS Wild type Male WT-M2 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Male | Batch:1 RAW_FILE_NAME=WT-M2.d SUBJECT_SAMPLE_FACTORS Wild type Male WT-M3 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Male | Batch:1 RAW_FILE_NAME=WT-M3.d SUBJECT_SAMPLE_FACTORS Wild type Male WT-M4 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Male | Batch:2 RAW_FILE_NAME=WT-M4.d SUBJECT_SAMPLE_FACTORS Wild type Male WT-M5 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Male | Batch:2 RAW_FILE_NAME=WT-M5.d SUBJECT_SAMPLE_FACTORS Wild type Male WT-M6 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Male | Batch:2 RAW_FILE_NAME=WT-M6.d SUBJECT_SAMPLE_FACTORS Mock Mock03 Sample source:Mock | Genotype:NA | Sex:NA | Batch:1 RAW_FILE_NAME=Mock03.d SUBJECT_SAMPLE_FACTORS Mock Mock04 Sample source:Mock | Genotype:NA | Sex:NA | Batch:1 RAW_FILE_NAME=Mock04.d SUBJECT_SAMPLE_FACTORS Mock Mock05 Sample source:Mock | Genotype:NA | Sex:NA | Batch:2 RAW_FILE_NAME=Mock05.d SUBJECT_SAMPLE_FACTORS Mock Mock06 Sample source:Mock | Genotype:NA | Sex:NA | Batch:2 RAW_FILE_NAME=Mock06.d #COLLECTION CO:COLLECTION_SUMMARY Wild type D. melanogaster flies, strain w1118, were kindly donated by Dr David CO:COLLECTION_SUMMARY Ben-Menahem (Ben-Gurion University, Israel). All flies used in the study were CO:COLLECTION_SUMMARY backcrossed to w1118 for 8 generations. SIRT6 (#39) D. melanogaster mutant CO:COLLECTION_SUMMARY strain was a kind gift from Dr Alena Bruce Krejci from the University of South CO:COLLECTION_SUMMARY Bohemia in the Czech Republic. Flies were maintained under non-crowding CO:COLLECTION_SUMMARY conditions on standard cornmeal diet 5g dextrose, 2.5% yeast extract, 8.6% CO:COLLECTION_SUMMARY cornmeal, 2% agar, 0.1% ortho-phosphoric acid, and 0.1% propionic acid) and CO:COLLECTION_SUMMARY grown at 25oC with 12h light/dark cycle. Age-matched flies were used for all CO:COLLECTION_SUMMARY experiments. CO:SAMPLE_TYPE Whole animals #TREATMENT TR:TREATMENT_SUMMARY The SIRT6 knockout flies were created by crossing transgenic Sirt6 gRNA flies TR:TREATMENT_SUMMARY with UAS-Cas9 stock, selecting the Sirt6-deficient allele and back-crossing the TR:TREATMENT_SUMMARY stock five times to the w1118 background. The resulting homozygous stock TR:TREATMENT_SUMMARY contained Sirt6 alleles with four nucleotide deletions at position 96-99 after TR:TREATMENT_SUMMARY ORF, causing a translational shift after the first 31 amino acids of Sirt6. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Drosophila melanogaster metabolite extraction was performed in a mixture of SP:SAMPLEPREP_SUMMARY ice/dry ice, by a cold two-phase methanol–water–chloroform solution. The SP:SAMPLEPREP_SUMMARY samples were resuspended in 800 μl of precooled methanol/water (5:3 v/v). SP:SAMPLEPREP_SUMMARY Afterwards, 500 μl of precooled chloroform was added to each sample. Samples SP:SAMPLEPREP_SUMMARY were vortexed for 10 min at 4 °C and then centrifuged (max. speed, 10 min, 4 SP:SAMPLEPREP_SUMMARY °C). The methanol–water phase containing polar metabolites was separated and SP:SAMPLEPREP_SUMMARY dried using a vacuum concentrator at 4 °C overnight, and then stored at -80 °C SP:SAMPLEPREP_SUMMARY until sample preparation for LC-MS analysis. Polar metabolite extracts SP:SAMPLEPREP_SUMMARY previously stored at -80 °C were resuspended in 50 µL of water prior to LC-MS SP:SAMPLEPREP_SUMMARY analysis. SP:PROCESSING_STORAGE_CONDITIONS Described in summary SP:EXTRACT_STORAGE Described in summary #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY For the detection of tryptophan derivatives by LC–MS, we used an Infinity II CH:CHROMATOGRAPHY_SUMMARY 1290 liquid chromatography system (Agilent Technologies) with a thermal CH:CHROMATOGRAPHY_SUMMARY autosampler set at 4 °C, coupled to a Q-TOF 6546 mass spectrometer (Agilent CH:CHROMATOGRAPHY_SUMMARY Technologies). Samples were resuspended in 50 µL of water, and 20 µL of each CH:CHROMATOGRAPHY_SUMMARY sample were injected onto a C18 column (Acquity UPLC Premier HSS C18 1.8 μm 2.1 CH:CHROMATOGRAPHY_SUMMARY × 100 mm). Metabolite separation was achieved with a flow rate of 0.25 ml/min CH:CHROMATOGRAPHY_SUMMARY at 30 °C, and based on the following 40-minute gradient (solvent A = H2O, 0.1% CH:CHROMATOGRAPHY_SUMMARY Formic acid, 15 mM acetic acid; solvent B = Acetonitrile + 0.1% Formic acid): 0 CH:CHROMATOGRAPHY_SUMMARY min 8% B; 2 min 8% B; 14 min 90% B; 16 min 90% B; 17 min 8% B; 22 min 8% B. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 0.1% formic acid; 15 mM acetic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0 min 8% B; 2 min 8% B; 14 min 90% B; 16 min 90% B; 17 min 8% B; 22 min 8% B CH:FLOW_RATE 0.25 ml/min CH:COLUMN_TEMPERATURE 30 °C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6546 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Tryptophan derivatives were detected using a Q-TOF 6546 mass spectrometer MS:MS_COMMENTS (Agilent Technologies). LC-MS data peak identification and integration were MS:MS_COMMENTS performed with the MassHunter Qualitative and Profinder softwares (Agilent MS:MS_COMMENTS Technologies, version 10.0 in both cases), respectively. Integrated data were MS:MS_COMMENTS then analyzed using a home-made MATLAB (R2023b) script. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples WT-F1 WT-F2 WT-F3 WT-F4 WT-F5 WT-F6 SKO-F1 SKO-F2 SKO-F3 SKO-F4 SKO-F5 SKO-F6 WT-M1 WT-M2 WT-M3 WT-M4 WT-M5 WT-M6 SKO-M1 SKO-M2 SKO-M3 SKO-M4 SKO-M5 SKO-M6 Factors Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Female | Batch:1 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Female | Batch:1 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Female | Batch:1 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Female | Batch:2 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Female | Batch:2 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Female | Batch:2 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Female | Batch:1 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Female | Batch:1 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Female | Batch:1 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Female | Batch:2 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Female | Batch:2 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Female | Batch:2 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Male | Batch:1 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Male | Batch:1 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Male | Batch:1 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Male | Batch:2 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Male | Batch:2 Sample source:D. melanogaster whole animal | Genotype:Wild type | Sex:Male | Batch:2 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Male | Batch:1 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Male | Batch:1 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Male | Batch:1 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Male | Batch:2 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Male | Batch:2 Sample source:D. melanogaster whole animal | Genotype:SIRT6 KO | Sex:Male | Batch:2 Melatonin 454377 889349 198326 7834808 5032389 869634 122531 166089 169919 2864833 5216738 646740 NA 572258 412397 2384818 1295157 796791 NA 100727 290923 2032951 493909 701307 Kynurenic acid 2957734 4051727 2025177 1515214 2157106 2653444 4366630 3333311 3373702 1319396 4779885 6416883 716035 479725 584518 939822 1186144 1534019 2356005 1376044 1340925 2071653 4947561 1111982 Xanthurenic acid 1109077 1076983 882779 878800 1028028 1182880 846399 864392 861713 409355 807041 1107668 408946 388062 477006 438267 583740 631499 440563 352986 433218 380938 506005 295029 Kynurenine 364008310 330482349 321318032 310772495 292734291 292740439 445158642 409188881 421907539 228579193 345659601 379671365 40223518 30169020 43569695 39642702 69634002 45402251 86901431 59675772 73896248 99330413 116713396 43820379 Anthranilic acid 293746 334110 253293 221959 207082 224514 279566 286511 324183 63728 183830 151622 230414 187744 296791 260498 174140 232310 267393 231763 328127 241629 262098 228590 Serotonin 390271 460693 410000 570630 248805 644218 822375 184746 399448 321327 108312 213945 724772 621656 448274 410953 456715 469401 609702 381652 1073137 332728 746611 616486 Tryptophan 212506528 239703378 205523984 185635948 176528498 172900754 216414958 245825955 244350060 88093263 168989701 233024956 146435246 143205564 171931878 143749138 157787668 139686030 194400564 192080691 184470835 211439420 167327566 150751395 Dopamine 741851 655993 978937 547095 337366 496528 1177213 492285 922812 667619 381183 NA 868514 1288024 1061065 743069 566875 661750 677596 640626 1175650 464290 379983 460779 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem CID Retention Time (min) Melatonin 896 9.2 Kynurenic acid 3845 6.9 Xanthurenic acid 5699 6.4 Kynurenine 161166 3.6 Anthranilic acid 227 8.6 Serotonin 5202 2.9 Tryptophan 6305 6.5 Dopamine 681 2.2 METABOLITES_END #END