#METABOLOMICS WORKBENCH lai_yunjia_20240623_154308 DATATRACK_ID:4952 STUDY_ID:ST003589 ANALYSIS_ID:AN005894 PROJECT_ID:PR002219 VERSION 1 CREATED_ON November 25, 2024, 5:33 pm #PROJECT PR:PROJECT_TITLE Biotin rescues manganese-induced parkinson's disease phenotypes and PR:PROJECT_TITLE neurotoxicity PR:PROJECT_TYPE untargeted metabolomics PR:PROJECT_SUMMARY Occupational exposure to manganese (Mn) induces manganism and has been widely PR:PROJECT_SUMMARY linked as a contributing environmental factor to Parkinson's disease (PD), PR:PROJECT_SUMMARY featuring dramatic signature overlaps between the two in motor symptoms and PR:PROJECT_SUMMARY clinical hallmarks. However, the molecular mechanism underlying this link PR:PROJECT_SUMMARY remains elusive, and for combating PD, effective mechanism-based therapies are PR:PROJECT_SUMMARY lacking. Here, we developed an adult Drosophila model of Mn toxicity to PR:PROJECT_SUMMARY recapitulate key Parkinsonian features, spanning behavioral deficits, neuronal PR:PROJECT_SUMMARY loss, and dysfunctions in lysosomes and mitochondria. We performed global PR:PROJECT_SUMMARY metabolomics on flies at an early toxicity stage and identified metabolism of PR:PROJECT_SUMMARY the B vitamin, biotin (vitamin B7), as a master pathway underpinning Mn toxicity PR:PROJECT_SUMMARY with systemic, body–brain increases in Mn-treated groups compared to the PR:PROJECT_SUMMARY controls. Using BtndRNAi mutant flies, we show that biotin depletion exacerbates PR:PROJECT_SUMMARY Mn-induced neurotoxicity, Parkinsonism, and mitochondrial dysfunction, while in PR:PROJECT_SUMMARY Mn-exposed wildtype flies, biotin feeding dramatically ameliorates these PR:PROJECT_SUMMARY pathophenotypes. We further show in human induced stem cells PR:PROJECT_SUMMARY (iPSCs)-differentiated midbrain dopaminergic neurons that the supplemented PR:PROJECT_SUMMARY biotin protects against Mn-induced neuronal loss, cytotoxicity, and PR:PROJECT_SUMMARY mitochondrial dysregulation. Finally, human data profiling biotin-related PR:PROJECT_SUMMARY proteins show for PD cases elevated levels of biotin transporters compared to PR:PROJECT_SUMMARY healthy controls, suggesting a potential role of biotin metabolism in PD. Taken PR:PROJECT_SUMMARY together, our findings identified the compensatory biotin pathway as a PR:PROJECT_SUMMARY convergent, systemic driver of Mn toxicity and Parkinsonian pathology, providing PR:PROJECT_SUMMARY a new basis for devising effective countermeasures against manganism and PD. PR:INSTITUTE Columbia University PR:DEPARTMENT Department of Environmental Health Sciences PR:LABORATORY The Miller Lab PR:LAST_NAME Lai PR:FIRST_NAME Yunjia PR:ADDRESS 630 West 168th Street, P&S 16-421, New York, NY 10032 PR:EMAIL yunjia.lai@outlook.com PR:PHONE 9194805489 PR:FUNDING_SOURCE National Institutes of Health PR:PUBLICATIONS Science Signaling #STUDY ST:STUDY_TITLE Metabolomics of Drosophila melanogaster under manganese (Mn) exposure (head) ST:STUDY_TYPE untargeted metabolomics ST:STUDY_SUMMARY Parkinson’s disease (PD) is the most common motor disorder, for which ST:STUDY_SUMMARY environmental factors such as manganese (Mn) exposure may play a major etiologic ST:STUDY_SUMMARY role. In this study, we aim to identify metabolic pathways related to Mn-induced ST:STUDY_SUMMARY parkinsonism and discover therapeutic targets for manganism and potentially for ST:STUDY_SUMMARY PD. We developed an in vivo model of acute Mn toxicity in male Drosophila ST:STUDY_SUMMARY melanogaster (0, 1, 10, and 30 mM of Mn2+ for 10 days) to recapitulate PD ST:STUDY_SUMMARY hallmarks, spanning behavior (climbing, locomotor assay), histology (H&E ST:STUDY_SUMMARY staining of neurons, MitoTracker assay), and mitochondrial function (Seahorse ST:STUDY_SUMMARY assay). High-coverage metabolomics of flies were conducted at an earlier point ST:STUDY_SUMMARY of dosage (Day 5), to identify early biochemical modulators before motor ST:STUDY_SUMMARY symptoms surfaced. Fly heads and bodies were extracted and analyzed using liquid ST:STUDY_SUMMARY chromatography–quadrupole-orbitrap mass spectrometry, following informatics ST:STUDY_SUMMARY approaches for compound identification and pathway analysis. The uploaded is the ST:STUDY_SUMMARY untargeted metabolomics component which contains raw data, alignment tables, and ST:STUDY_SUMMARY associated method details. ST:INSTITUTE Columbia University ST:DEPARTMENT Department of Environmental Health Sciences ST:LABORATORY The Miller Lab ST:LAST_NAME Lai ST:FIRST_NAME Yunjia ST:ADDRESS 630 West 168th Street, P&S 16-421, New York, NY 10032 ST:EMAIL yunjia.lai@outlook.com ST:PHONE 9194805489 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 150; five samples per group; 10 subjects per sample ST:NUM_MALES 150; five samples per group; 10 subjects per sample ST:PUBLICATIONS Science Signaling #SUBJECT SU:SUBJECT_TYPE Insect SU:SUBJECT_SPECIES Drosophila melanogaster SU:TAXONOMY_ID 7227 SU:GENOTYPE_STRAIN wild-type (red eye) SU:AGE_OR_AGE_RANGE all adult: 5-20 days, depending on the experiments SU:GENDER Male #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 - head_Pos_x1h1 Sample source:head | Treatment:Mn, 30 mM RAW_FILE_NAME(raw data file names)=head_Pos_x1h1.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x1h2 Sample source:head | Treatment:Mn, 30 mM RAW_FILE_NAME(raw data file names)=head_Pos_x1h2.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x1h3 Sample source:head | Treatment:Mn, 30 mM RAW_FILE_NAME(raw data file names)=head_Pos_x1h3.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x1h4 Sample source:head | Treatment:Mn, 30 mM RAW_FILE_NAME(raw data file names)=head_Pos_x1h4.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x1h5 Sample source:head | Treatment:Mn, 30 mM RAW_FILE_NAME(raw data file names)=head_Pos_x1h5.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x2h1 Sample source:head | Treatment:Vehicle RAW_FILE_NAME(raw data file names)=head_Pos_x2h1.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x2h2 Sample source:head | Treatment:Vehicle RAW_FILE_NAME(raw data file names)=head_Pos_x2h2.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x2h3 Sample source:head | Treatment:Vehicle RAW_FILE_NAME(raw data file names)=head_Pos_x2h3.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x2h4 Sample source:head | Treatment:Vehicle RAW_FILE_NAME(raw data file names)=head_Pos_x2h4.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x2h5 Sample source:head | Treatment:Vehicle RAW_FILE_NAME(raw data file names)=head_Pos_x2h5.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x3h1 Sample source:head | Treatment:Mn, 10 mM RAW_FILE_NAME(raw data file names)=head_Pos_x3h1.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x3h2 Sample source:head | Treatment:Mn, 10 mM RAW_FILE_NAME(raw data file names)=head_Pos_x3h2.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x3h3 Sample source:head | Treatment:Mn, 10 mM RAW_FILE_NAME(raw data file names)=head_Pos_x3h3.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x3h4 Sample source:head | Treatment:Mn, 10 mM RAW_FILE_NAME(raw data file names)=head_Pos_x3h4.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_x3h5 Sample source:head | Treatment:Mn, 10 mM RAW_FILE_NAME(raw data file names)=head_Pos_x3h5.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_poolinj1 Sample source:head | Treatment:Pooled QC RAW_FILE_NAME(raw data file names)=head_Pos_poolinj1.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_poolinj2 Sample source:head | Treatment:Pooled QC RAW_FILE_NAME(raw data file names)=head_Pos_poolinj2.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_poolinj3 Sample source:head | Treatment:Pooled QC RAW_FILE_NAME(raw data file names)=head_Pos_poolinj3.mzML SUBJECT_SAMPLE_FACTORS - head_Pos_poolinj4 Sample source:head | Treatment:Pooled QC RAW_FILE_NAME(raw data file names)=head_Pos_poolinj4.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x1h1 Sample source:head | Treatment:Mn, 30 mM RAW_FILE_NAME(raw data file names)=head_Neg_x1h1.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x1h2 Sample source:head | Treatment:Mn, 30 mM RAW_FILE_NAME(raw data file names)=head_Neg_x1h2.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x1h3 Sample source:head | Treatment:Mn, 30 mM RAW_FILE_NAME(raw data file names)=head_Neg_x1h3.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x1h4 Sample source:head | Treatment:Mn, 30 mM RAW_FILE_NAME(raw data file names)=head_Neg_x1h4.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x1h5 Sample source:head | Treatment:Mn, 30 mM RAW_FILE_NAME(raw data file names)=head_Neg_x1h5.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x2h1 Sample source:head | Treatment:Vehicle RAW_FILE_NAME(raw data file names)=head_Neg_x2h1.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x2h2 Sample source:head | Treatment:Vehicle RAW_FILE_NAME(raw data file names)=head_Neg_x2h2.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x2h3 Sample source:head | Treatment:Vehicle RAW_FILE_NAME(raw data file names)=head_Neg_x2h3.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x2h4 Sample source:head | Treatment:Vehicle RAW_FILE_NAME(raw data file names)=head_Neg_x2h4.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x2h5 Sample source:head | Treatment:Vehicle RAW_FILE_NAME(raw data file names)=head_Neg_x2h5.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x3h1 Sample source:head | Treatment:Mn, 10 mM RAW_FILE_NAME(raw data file names)=head_Neg_x3h1.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x3h2 Sample source:head | Treatment:Mn, 10 mM RAW_FILE_NAME(raw data file names)=head_Neg_x3h2.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x3h3 Sample source:head | Treatment:Mn, 10 mM RAW_FILE_NAME(raw data file names)=head_Neg_x3h3.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x3h4 Sample source:head | Treatment:Mn, 10 mM RAW_FILE_NAME(raw data file names)=head_Neg_x3h4.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_x3h5 Sample source:head | Treatment:Mn, 10 mM RAW_FILE_NAME(raw data file names)=head_Neg_x3h5.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_poolinj1 Sample source:head | Treatment:Pooled QC RAW_FILE_NAME(raw data file names)=head_Neg_poolinj1.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_poolinj2 Sample source:head | Treatment:Pooled QC RAW_FILE_NAME(raw data file names)=head_Neg_poolinj2.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_poolinj3 Sample source:head | Treatment:Pooled QC RAW_FILE_NAME(raw data file names)=head_Neg_poolinj3.mzML SUBJECT_SAMPLE_FACTORS - head_Neg_poolinj4 Sample source:head | Treatment:Pooled QC RAW_FILE_NAME(raw data file names)=head_Neg_poolinj4.mzML #COLLECTION CO:COLLECTION_SUMMARY To capture basic biochemical changes, flies were beheaded at 5-day of Mn CO:COLLECTION_SUMMARY exposure with heads and bodies collected and snap-frozen separately; each group CO:COLLECTION_SUMMARY had five sample replicates, and each replicate sample contained ten CO:COLLECTION_SUMMARY heads/bodies. CO:SAMPLE_TYPE fly head CO:COLLECTION_TUBE_TEMP -80 #TREATMENT TR:TREATMENT_SUMMARY Fly crosses were conducted in a 25 °C incubator and aged at 25 °C for 5-20 TR:TREATMENT_SUMMARY days, depending on the experiments. The pan-neuronal driver nSyb-GAL4 was used TR:TREATMENT_SUMMARY to mediate the genetic knockdown of the btnd gene. Both the stocks for nSyb-GAL4 TR:TREATMENT_SUMMARY and UAS-Btnd RNAi were obtained from Bloomington Drosophila Stock Center (BDSC) TR:TREATMENT_SUMMARY (HMC05012; Bloomington #60020). For Mn treatment, 0, 10, and 30 mM of MnCl2 TR:TREATMENT_SUMMARY (#7773-01-5; Santa Cruz Biotechnology, Dallas, TX) was mixed in 3 mL of water, TR:TREATMENT_SUMMARY and the instant food for flies was made using this water. Biotin supplementation TR:TREATMENT_SUMMARY was performed as previously described (Lohr et al., 2020). Biotin was added to TR:TREATMENT_SUMMARY instant fly food (Carolina Biological, Burlington, NC) at a final concentration TR:TREATMENT_SUMMARY of 30 mM. Unless noted otherwise, male flies were used throughout the study. TR:TREATMENT_SUMMARY Lohr KM, Frost B, Scherzer C, Feany MB. Biotin rescues mitochondrial dysfunction TR:TREATMENT_SUMMARY and neurotoxicity in a tauopathy model. Proc Natl Acad Sci U S A. 2020 Dec TR:TREATMENT_SUMMARY 29;117(52):33608-33618. doi: 10.1073/pnas.1922392117. Epub 2020 Dec 14. PMID: TR:TREATMENT_SUMMARY 33318181; PMCID: PMC7777082. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To capture basic biochemical changes, flies were beheaded at 5-day of Mn SP:SAMPLEPREP_SUMMARY exposure with heads and bodies collected and snap-frozen separately; each group SP:SAMPLEPREP_SUMMARY had five sample replicates, and each replicate sample contained ten SP:SAMPLEPREP_SUMMARY heads/bodies. To extract metabolites, thawed heads and bodies were added with SP:SAMPLEPREP_SUMMARY ice-cold methanol:H2O (2:1, v/v) solution pre-spiked with isotope-labeled SP:SAMPLEPREP_SUMMARY internal standards alongside zirconium oxide beads (0.5 mm i.d., Yittria SP:SAMPLEPREP_SUMMARY stabilized) (Next Advance, Troy, NY). Samples were touch vortexed for 30 sec, SP:SAMPLEPREP_SUMMARY manually ground using sterile, disposable pestles (DWK Life Sciences, Millville, SP:SAMPLEPREP_SUMMARY NJ), placed on a bead beater (Next Advance, Troy, NY) in a cold room for 5 mins SP:SAMPLEPREP_SUMMARY at the maximum speed, and finally centrifuged at 18,000 × g for 10 mins. The SP:SAMPLEPREP_SUMMARY supernatant was SpeedVac-dried and reconstituted into acetonitrile:H2O (2:98, SP:SAMPLEPREP_SUMMARY v/v) upon instrumental analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC HSS C18 (100 x 2.1mm,1.8um) CH:SOLVENT_A 100% Water; 0.1% formic acid CH:SOLVENT_B 100% Acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT a 15-min gradient: 2% B, 0–1 min; 2 to 15% B, 1–3 min; 15 to 50% B, 3–6 CH:FLOW_GRADIENT min; 50 to 98% B, 6–7.5 min; 98% B, 7.5–11.5 min; 98 to 2% B, 11.5–11.6 CH:FLOW_GRADIENT min; 2% B, 11.6–15 min CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 40°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 240 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Data in both ESI positive and negative modes were acquired. Quality assurance MS:MS_COMMENTS and quality control (QA/QC) procedures were implemented, spanning timely mass MS:MS_COMMENTS calibration, sample blindfolding, sample randomization, and internal MS:MS_COMMENTS standard-based monitoring.MS1 *.raw data were converted to *.abf, and processed MS:MS_COMMENTS in MS-DIAL 4.90 (Riken, Japan) to obtain peak alignment tables separately for MS:MS_COMMENTS the head (ESI+), head (ESI-), body (ESI+), and body (ESI-) modes of analysis; MS:MS_COMMENTS detailed settings for data processing can be found in the SI Appendix. Welch's MS:MS_COMMENTS t-test and one-way ANOVA were performed on each of the four datasets to screen MS:MS_COMMENTS for ion features of statistical significance; retrospectively, tandem MS/MS mass MS:MS_COMMENTS spectra were collected for these features correspondingly using pooled samples, MS:MS_COMMENTS separately for fly heads and bodies. MS:MS_RESULTS_FILE ST003589_AN005894_Results.txt UNITS:Peak intensity (area) Has m/z:Yes Has RT:Yes RT units:Minutes #END