#METABOLOMICS WORKBENCH xiehuijing_20230707_012117 DATATRACK_ID:4149 STUDY_ID:ST002774 ANALYSIS_ID:AN004515 VERSION 1 CREATED_ON 09-19-2023 #PROJECT PR:PROJECT_TITLE Defective Branched-Chain Amino Acid Catabolism in Dorsal Root Ganglia PR:PROJECT_TITLE Contributes to Mechanical Pain PR:PROJECT_SUMMARY Impaired branched-chain amino acid (BCAA) catabolism has recently been PR:PROJECT_SUMMARY implicated in the development of mechanical pain, but the molecular mechanisms PR:PROJECT_SUMMARY are unclear. Here we report that defective BCAA catabolism in dorsal root PR:PROJECT_SUMMARY ganglia (DRG) neurons sensitizes mice to mechanical pain by increasing lactate PR:PROJECT_SUMMARY production and Piezo2 expression, a mechanotransduction channel. In high-fat PR:PROJECT_SUMMARY diet fed obese mice, we observed a downregulation of PP2Cm, a key regulator in PR:PROJECT_SUMMARY BCAA catabolic pathway, in DRG neurons. Mice with conditional knockout of PP2Cm PR:PROJECT_SUMMARY in DRG neurons (cKO) exhibited mechanical allodynia under normal or spare nerve PR:PROJECT_SUMMARY injury (SNI)-induced neuropathic injury conditions. Further, in patients with PR:PROJECT_SUMMARY peripheral neuropathic pain, we found that the Visual Analogue Scale (VAS) PR:PROJECT_SUMMARY scores were positively correlated with BCAA contents in plasma, highlighting a PR:PROJECT_SUMMARY link between peripheral neuropathic pain and dysregulated BCAA catabolism. PR:PROJECT_SUMMARY Mechanistically, defective BCAA catabolism promotes the production of lactate PR:PROJECT_SUMMARY through glycolysis in DRG neurons, which increases H3K18la modification and PR:PROJECT_SUMMARY drives Piezo2 expression. Inhibition of lactate production or silencing of PR:PROJECT_SUMMARY Piezo2 expression attenuated the pain phenotype of cKO to mechanical stimuli. PR:PROJECT_SUMMARY Therefore, our study demonstrates a casual role of defective BCAA catabolism in PR:PROJECT_SUMMARY mechanical pain by enhancing metabolite-mediated epigenetic regulation. PR:INSTITUTE West China Hospital of Sichuan University PR:LABORATORY Laboratory of Mitochondria and Metabolism PR:LAST_NAME Li PR:FIRST_NAME Tao PR:ADDRESS No. 37 Guoxue Road, Wuhou District, Chendu 610041, Sichuan, China PR:EMAIL 563234879@qq.com PR:PHONE +8628-85164154 PR:DOI http://dx.doi.org/10.21228/M8ZD8R #STUDY ST:STUDY_TITLE Metabolomics studies on L4-5 Dorsal Root Ganglia of Ctrl and cKO mice ST:STUDY_SUMMARY Metabolomics studies on Dorsal Root Ganglia (DRGs) (Ctrl and cKO).The specific ST:STUDY_SUMMARY knockout of PP2Cm in the DRG was achieved by intracellular injection of ST:STUDY_SUMMARY AAV9-Pirt-Cre (1.0×1013 vg/ml) or AAV9-Pirt (1.0×1013 vg/ml)) as control. Mice ST:STUDY_SUMMARY were used for experiments at 4 weeks after the injection. ST:INSTITUTE West China Hospital of Sichuan University ST:LAST_NAME Li ST:FIRST_NAME Tao ST:ADDRESS No. 37 Guoxue Road, Wuhou District, Chendu 610041, Sichuan, China ST:EMAIL 563234879@qq.com ST:PHONE +8628-85164154 ST:SUBMIT_DATE 2023-07-07 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS cKO1 cKO1 Genotype:cKO RAW_FILE_NAME=XHJ_DRG NEG/POS-20221214-XHJ_DRG_aqu_KO1.mzML SUBJECT_SAMPLE_FACTORS cKO2 cKO2 Genotype:cKO RAW_FILE_NAME=XHJ_DRG NEG/POS-20221214-XHJ_DRG_aqu_KO2.mzML SUBJECT_SAMPLE_FACTORS cKO3 cKO3 Genotype:cKO RAW_FILE_NAME=XHJ_DRG NEG/POS-20221214-XHJ_DRG_aqu_KO3.mzML SUBJECT_SAMPLE_FACTORS cKO4 cKO4 Genotype:cKO RAW_FILE_NAME=XHJ_DRG NEG/POS-20221214-XHJ_DRG_aqu_KO4.mzML SUBJECT_SAMPLE_FACTORS cKO5 cKO5 Genotype:cKO RAW_FILE_NAME=XHJ_DRG NEG/POS-20221214-XHJ_DRG_aqu_KO5.mzML SUBJECT_SAMPLE_FACTORS cKO6 cKO6 Genotype:cKO RAW_FILE_NAME=XHJ_DRG NEG/POS-20221214-XHJ_DRG_aqu_KO6.mzML SUBJECT_SAMPLE_FACTORS Ctrl1 Ctrl1 Genotype:Ctrl RAW_FILE_NAME=XHJ_DRG NEG/POS-20221214-XHJ_DRG_aqu_C1.mzML SUBJECT_SAMPLE_FACTORS Ctrl2 Ctrl2 Genotype:Ctrl RAW_FILE_NAME=XHJ_DRG NEG/POS-20221214-XHJ_DRG_aqu_C2.mzML SUBJECT_SAMPLE_FACTORS Ctrl3 Ctrl3 Genotype:Ctrl RAW_FILE_NAME=XHJ_DRG NEG/POS-20221214-XHJ_DRG_aqu_C3.mzML SUBJECT_SAMPLE_FACTORS Ctrl4 Ctrl4 Genotype:Ctrl RAW_FILE_NAME=XHJ_DRG NEG/POS-20221214-XHJ_DRG_aqu_C4.mzML SUBJECT_SAMPLE_FACTORS Ctrl5 Ctrl5 Genotype:Ctrl RAW_FILE_NAME=XHJ_DRG NEG/POS-20221214-XHJ_DRG_aqu_C5.mzML SUBJECT_SAMPLE_FACTORS Ctrl6 Ctrl6 Genotype:Ctrl RAW_FILE_NAME=XHJ_DRG NEG/POS-20221214-XHJ_DRG_aqu_C6.mzML #COLLECTION CO:COLLECTION_SUMMARY Mice were anaesthetized with volatile anesthetic isoflurane (3-4% for induction, CO:COLLECTION_SUMMARY 1-2% for maintenance, in O2≈ 2 L/min). DRG (left L4–L5) were surgically CO:COLLECTION_SUMMARY exposed and diessected. Fresh DRG samples were frozen in liquid nitrogen.5-6 CO:COLLECTION_SUMMARY DRGs were collected as one sample CO:SAMPLE_TYPE DRG #TREATMENT TR:TREATMENT_SUMMARY No treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Fresh tissue samples were frozen in liquid nitrogen. After thawing on ice, 10 mg SP:SAMPLEPREP_SUMMARY tissue was homogenized in -80°C precooled labeled-solution (MeOH: H2O=4:1, v/v) SP:SAMPLEPREP_SUMMARY for 30 s, 4 cycles, and placed at -80°C for 30 minutes. In ice water bath, SP:SAMPLEPREP_SUMMARY ultrasonic wave was used for 10 min and centrifuged at 14000 rpm. The SP:SAMPLEPREP_SUMMARY precipitated protein was dissolved in no labeled-liquid (MeOH: H2O=4:1), SP:SAMPLEPREP_SUMMARY vortexed and centrifuged. After derivatization with SP:SAMPLEPREP_SUMMARY N-methyl-N-(trimethylsilyl)tri-fluoroacetamide for 30 min, the supernatant was SP:SAMPLEPREP_SUMMARY detected by GC-MS. Metabolites were identified by means of KEGG Metabolic SP:SAMPLEPREP_SUMMARY pathways. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Nexera UHPLC LC-30A  CH:COLUMN_NAME Waters BEH Amide (2.1 X 100mm, 1.7μm) CH:COLUMN_TEMPERATURE 40℃ CH:FLOW_GRADIENT 0～1.5min,90%B.1.5~5min,90-45%B.5~10min,45%B.10~11min,45~90B.11~23min,90B CH:FLOW_RATE 0.3ml/min CH:SOLVENT_A 10% acetonitrile/90% water; 0.2% acetic acid CH:SOLVENT_B 90% acetonitrile/10% water; 0.2% acetic acic CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Nexera UHPLC LC-30A  MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:MS_COMMENTS - MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS pmoles/l MS_METABOLITE_DATA_START Samples cKO1 cKO2 cKO3 cKO4 cKO5 cKO6 Ctrl1 Ctrl2 Ctrl3 Ctrl4 Ctrl5 Ctrl6 Factors Genotype:cKO Genotype:cKO Genotype:cKO Genotype:cKO Genotype:cKO Genotype:cKO Genotype:Ctrl Genotype:Ctrl Genotype:Ctrl Genotype:Ctrl Genotype:Ctrl Genotype:Ctrl 1-Methylhistamine 3236.0000 3033.7500 3032.7500 3842.1250 2425.6250 3-Nitrotyrosine 4-Hydroxyphenylpyruvic acid 12125.3750 4-Pyridoxic acid 23421.0000 12124.1250 26453.7040 16532.9770 14604.1250 15155.8750 23285.2270 22321.1250 11847.6250 16947.3750 3167.8750 23146.8750 5-Hydroxy-L-tryptophan 6267.6835 3639.6250 2829.8750 2223.5000 3842.1250 4449.0000 5-Hydroxymethyl-2^-deoxyuridine 3638.3750 3234.7500 5-Methyltetrahydrofolic 2756.2500 5-Thymidylic acid 6-Gluconolactone 9185.8505 12314.2630 21680.5205 19839.0645 7900.6365 8453.5070 8083.9015 14143.6835 13958.2270 4778.2350 33990.3610 8-Hydroxy-deoxyguanosine 3033.7500 3438.2500 5865.2500 8-Hydroxyguanosine 4044.0000 3234.5000 3033.0000 2629.2500 Acetoacetic acid 3581.5000 5511.2500 4409.3750 Aconitic acid 2755.0000 2755.0000 Alpha-Linolenic acid 85353.6912 73038.3610 70960.9880 110251.1335 57602.8970 140555.9745 80202.8615 66112.2817 59809.7035 81513.2426 187685.3155 96427.1355 Aminoadipic acid 267781.6470 213774.7495 206845.9830 245704.3275 147814.6185 274437.2630 202493.0250 278218.2355 252150.6640 168230.1280 217151.1725 228295.4180 Azelaic acid 141872.0335 294415.2050 157044.5700 182568.9630 147254.7050 122425.8955 153598.4905 204087.2925 167106.6040 106973.4030 163124.0275 184950.1175 Cadaverine 27703.5590 9503.2500 20625.0590 13345.5000 9503.5000 32961.4925 19109.1840 27501.0585 6269.0000 14357.3750 9098.5000 27300.1175 cGAMP 4247.2500 cGMP Cytidine monophosphate Cytidine triphosphate dCMP Deoxyuridine 3842.7500 3235.1250 3033.5000 1820.2500 4246.3750 D-Glucosamine 7176.4335 6068.1840 11527.3680 22859.1040 25078.8605 17694.3675 19621.1700 Dihydrofolic acid DL-Acetylcarnitine 2022.5000 1820.2500 2831.2500 DL-Homocysteine 1820.2500 9504.3750 4449.5000 Dopamine 10610.6025 7440.7040 17087.0565 6615.0795 15436.1820 4410.8525 7855.6025 5511.9770 9922.9545 7305.8300 29236.4454 Folic acid Gamma-Aminobutyric acid Gibberellin A3 Glyceraldehyde Glycerol Glycocholic acid Heptadecanoic acid 4684.3750 Histamine 44092.4710 Indole 40246.7870 50763.8385 25886.1690 32159.4125 28920.4780 32763.1695 30538.3605 23863.9855 38627.0950 40250.3980 40953.9710 31953.1100 Indoleacetic acid 8292.2500 6673.7500 6269.7500 8494.5000 3842.7500 11527.1250 5764.1250 4449.5000 8898.0000 10212.3750 5966.1250 7684.3750 Isobutyric acid 12677.0000 11574.8750 8129.0000 5786.5000 6475.3750 15982.7500 10195.7500 9368.7500 9369.1250 Isovaleric acid L-Ascorbic acid(Vit. C) L-Cysteine L-Cysteinylglycine 2628.0000 2224.5000 3640.3750 L-Cystine 4247.2500 3033.6250 2224.6250 L-Galactose 30861.5000 16119.8520 47124.7045 Linoleic acid 53194.0550 251809.4820 240772.2340 112184.3495 132858.0600 138791.6745 216109.3710 L-Proline 6007883.0250 5320591.0180 5710486.8070 5674570.3880 4100576.3100 6262856.7330 6750719.4240 6049151.9560 3726929.0850 4621220.8800 4589086.9780 6593024.8200 Melatonin 6876.5000 4247.2500 4651.7500 Methylmalonic acid 13230.1585 20675.7160 7303.8295 6615.2045 21226.9430 Myristic acid 12126.2275 8542.3520 13502.3750 4408.3750 N2_N2-Dimethylguanosine 4852.5000 7483.2500 2831.5000 9304.3090 5258.5000 4247.2500 3640.5000 3236.0000 N6-Methyladenosine 15774.9340 27908.2350 10312.5000 9809.9340 5862.5000 16178.3750 9099.7500 15368.6250 11727.5000 11730.6840 17189.5590 13146.1840 N-Acetylneuraminic acid 15982.7500 8266.2500 13227.1250 18464.7045 6888.7500 19016.2045 6888.2500 NAD NADP NADPH Normetanephrine 10110.6250 4447.0000 2831.5000 7481.1835 5662.6250 23055.4930 4449.1250 13044.2500 5662.0000 7076.7500 8089.4340 8090.0000 Orotidylic acid(OMP) oxaloacetate 82418.6015 41206.3230 77730.8505 50578.0095 63667.2085 112470.0895 34316.2950 122944.9410 52519.4730 30316.3395 118245.0100 110249.7040 Phenylpyruvic acidPA Quinolinic acid 11573.5000 7715.0000 10471.2500 4685.0000 6337.5000 5786.7500 10470.7500 4685.6250 12676.0000 Propionic acid Prostaglandin E2 Putrescine 4346.5000 8290.5000 6978.4340 6775.3090 14761.0000 3032.5000 4045.0000 5259.3090 4650.4335 11325.2500 15371.1840 2629.3750 Quinolinic acid Salicyluric acid 2830.1250 2426.0000 Spermidine 2223.3750 4650.7500 4651.7500 3032.5000 2224.7500 Taurocholic acid Tetrahydrofolic acid MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant 1-Methylhistamine 3-Nitrotyrosine 4-Hydroxyphenylpyruvic acid 4-Pyridoxic acid 5-Hydroxy-L-tryptophan 5-Hydroxymethyl-2'-deoxyuridine 5-Methyltetrahydrofolic 5-Thymidylic acid 6-Gluconolactone 8-Hydroxy-deoxyguanosine 8-Hydroxyguanosine Acetoacetic acid Aconitic acid Alpha-Linolenic acid Aminoadipic acid Azelaic acid Cadaverine cGAMP cGMP Cytidine monophosphate Cytidine triphosphate dCMP Deoxyuridine D-Glucosamine Dihydrofolic acid DL-Acetylcarnitine DL-Homocysteine Dopamine Folic acid Gamma-Aminobutyric acid Gibberellin A3 Glyceraldehyde Glycerol Glycocholic acid Heptadecanoic acid Histamine Indole Indoleacetic acid Isobutyric acid Isovaleric acid L-Ascorbic acid(Vit. C) L-Cysteine L-Cysteinylglycine L-Cystine L-Galactose Linoleic acid L-Proline Melatonin Methylmalonic acid Myristic acid N2,N2-Dimethylguanosine N6-Methyladenosine N-Acetylneuraminic acid NAD NADP NADPH Normetanephrine Orotidylic acid(OMP) oxaloacetate Phenylpyruvic acidPA Quinolinic acid Propionic acid Prostaglandin E2 Putrescine Quinolinic acid Salicyluric acid Spermidine Taurocholic acid Tetrahydrofolic acid METABOLITES_END #END