Summary of Study ST001957

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001244. The data can be accessed directly via it's Project DOI: 10.21228/M8X40N This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001957
Study TitleUntargeted Mass Spectrometry Metabolomic Profiles of iPSC-derived Dopaminergic Neurons from Clinically Discordant Brothers with Identical PRKN Deletions
Study Typeuntargeted metabolomics analysis
Study SummaryWe have previously reported on two brothers, PM and SM, who carry identical compound heterozygous PRKN mutations but present with very different clinical Parkinson’s disease (PD) phenotypes, with PM, but not SM having been diagnosed with early onset disease. The occurrence of juvenile cases demonstrates that PD is not necessarily an age-associated disease, indeed evidence is accumulating that there is a developmental component to PD pathogenesis. We hypothesize that additional genetic modifiers, potentially including genetic loci relevant to mesencephalic dopamine neuron development may play a role. We differentiated human-induced pluripotent stem cells (hiPSCs) derived from SM and PM into mitotically active mesencephalic neural precursor cells and early postmitotic dopaminergic neurons and performed whole exome sequencing, transcriptomic- and metabolomic analyses. No significant differences in canonical markers of differentiation were observed between SM and PM. Yet our transcriptomic analysis revealed a significant down regulation of three neurodevelopmentally relevant cell adhesion molecules, CNTN6, CNTN4 and CHL1 in PM- compared to SM cultures on days 11 and 25 of differentiation. In addition, several HLA genes, known to play a role in neurodevelopment, independent of their well-established function in immunity, were differentially regulated in PM and SM developing dopamine neurons. EN2, a transcription factor crucial for mesencephalic dopamine neuron development, was also differentially regulated. We further observed differences in cellular processes relevant to dopamine homeostasis. Lastly, our whole exome sequencing, transcriptomics and metabolomics data of SM and PM neurons revealed differences in GSH homeostasis, the dysregulation of which has been associated with PD.
Institute
Vanderbilt University
DepartmentChemistry
LaboratoryCenter for Innovative Technology
Last NameCODREANU
First NameSIMONA
Address1234 STEVENSON CENTER LANE
EmailSIMONA.CODREANU@VANDERBILT.EDU
Phone6158758422
Submit Date2021-10-13
Raw Data AvailableYes
Raw Data File Type(s)raw(Waters)
Analysis Type DetailLC-MS
Release Date2022-01-17
Release Version1
SIMONA CODREANU SIMONA CODREANU
https://dx.doi.org/10.21228/M8X40N
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001244
Project DOI:doi: 10.21228/M8X40N
Project Title:Untargeted Mass Spectrometry Metabolomic Profiles of iPSC-derived Dopaminergic Neurons from Clinically Discordant Brothers with Identical PRKN Deletions
Project Type:Untargeted Metabolomics analysis
Project Summary:We have previously reported on two brothers, PM and SM, who carry identical compound heterozygous PRKN mutations but present with very different clinical Parkinson’s disease (PD) phenotypes, with PM, but not SM having been diagnosed with early onset disease. The occurrence of juvenile cases demonstrates that PD is not necessarily an age-associated disease, indeed evidence is accumulating that there is a developmental component to PD pathogenesis. We hypothesize that additional genetic modifiers, potentially including genetic loci relevant to mesencephalic dopamine neuron development may play a role. We differentiated human-induced pluripotent stem cells (hiPSCs) derived from SM and PM into mitotically active mesencephalic neural precursor cells and early post mitotic dopaminergic neurons and performed whole exome sequencing, transcriptomic- and metabolomic analyses. No significant differences in canonical markers of differentiation were observed between SM and PM. Yet our transcriptomic analysis revealed a significant down regulation of three neurodevelopmentally relevant cell adhesion molecules, CNTN6, CNTN4 and CHL1 in PM - compared to SM cultures on days 11 and 25 of differentiation. In addition, several HLA genes, known to play a role in neurodevelopment, independent of their well-established function in immunity, were differentially regulated in PM and SM developing dopamine neurons. EN2, a transcription factor crucial for mesencephalic dopamine neuron development, was also differentially regulated. We further observed differences in cellular processes relevant to dopamine homeostasis. Lastly, our whole exome sequencing, transcriptomics and metabolomics data of SM and PM neurons revealed differences in GSH homeostasis, the dysregulation of which has been associated with PD.
Institute:VANDERBILT UNIVERSITY
Department:Chemistry
Laboratory:Center for Innovative Technology
Last Name:CODREANU
First Name:SIMONA
Address:1234 STEVENSON CENTER LANE, NASHVILLE, TN, 37235, USA
Email:SIMONA.CODREANU@VANDERBILT.EDU
Phone:6158758422

Subject:

Subject ID:SU002037
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Genotype Strain:PRKN mutations (PD)
Gender:Male

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id genotype Mn treatment (uM)
SA184428HILICn_44_SM3_200_2PRKN_no_PD 200
SA184429HILICn_53_SM5_200_2PRKN_no_PD 200
SA184430HILICn_54_SM5_200_3PRKN_no_PD 200
SA184431HILICn_52_SM5_200_1PRKN_no_PD 200
SA184432HILICn_45_SM3_200_3PRKN_no_PD 200
SA184433HILICn_43_SM3_200_1PRKN_no_PD 200
SA184434RPLCp_35_SM14_200_TH1PRKN_no_PD 200
SA184435RPLCp_36_SM14_200_TH2PRKN_no_PD 200
SA184436RPLCp_41_SM15_200_TH1PRKN_no_PD 200
SA184437RPLCp_42_SM15_200_TH2PRKN_no_PD 200
SA184438HILICp_44_SM3_200_2PRKN_no_PD 200
SA184439HILICp_43_SM3_200_1PRKN_no_PD 200
SA184440HILICp_54_SM5_200_3PRKN_no_PD 200
SA184441HILICp_18_SM14_200PRKN_no_PD 200
SA184442HILICp_45_SM3_200_3PRKN_no_PD 200
SA184443HILICp_21_SM15_200PRKN_no_PD 200
SA184444HILICp_52_SM5_200_1PRKN_no_PD 200
SA184445HILICp_53_SM5_200_2PRKN_no_PD 200
SA184446HILICn_49_SM5_50_1PRKN_no_PD 50
SA184447HILICp_20_SM15_50PRKN_no_PD 50
SA184448HILICp_41_SM3_50_2PRKN_no_PD 50
SA184449HILICn_41_SM3_50_2PRKN_no_PD 50
SA184450HILICp_42_SM3_50_3PRKN_no_PD 50
SA184451HILICp_40_SM3_50_1PRKN_no_PD 50
SA184452HILICp_17_SM14_50PRKN_no_PD 50
SA184453RPLCp_39_SM15_50_TH1PRKN_no_PD 50
SA184454HILICn_40_SM3_50_1PRKN_no_PD 50
SA184455HILICp_50_SM5_50_2PRKN_no_PD 50
SA184456HILICp_51_SM5_50_3PRKN_no_PD 50
SA184457RPLCp_33_SM14_50_TH1PRKN_no_PD 50
SA184458HILICn_42_SM3_50_3PRKN_no_PD 50
SA184459HILICn_50_SM5_50_2PRKN_no_PD 50
SA184460HILICn_51_SM5_50_3PRKN_no_PD 50
SA184461RPLCp_34_SM14_50_TH2PRKN_no_PD 50
SA184462HILICp_49_SM5_50_1PRKN_no_PD 50
SA184463RPLCp_40_SM15_50_TH2PRKN_no_PD 50
SA184464HILICn_48_SM5_0_3PRKN_no_PD Control
SA184465HILICn_47_SM5_0_2PRKN_no_PD Control
SA184466HILICn_46_SM5_0_1PRKN_no_PD Control
SA184467HILICn_38_SM3_0_2PRKN_no_PD Control
SA184468HILICp_16_SM14_0PRKN_no_PD Control
SA184469HILICn_39_SM3_0_3PRKN_no_PD Control
SA184470HILICn_37_SM3_0_1PRKN_no_PD Control
SA184471HILICp_37_SM3_0_1PRKN_no_PD Control
SA184472RPLCp_32_SM14_0_TH2PRKN_no_PD Control
SA184473RPLCp_31_SM14_0_TH1PRKN_no_PD Control
SA184474HILICp_19_SM15_0PRKN_no_PD Control
SA184475RPLCp_37_SM15_0_TH1PRKN_no_PD Control
SA184476RPLCp_38_SM15_0_TH2PRKN_no_PD Control
SA184477HILICp_48_SM5_0_3PRKN_no_PD Control
SA184478HILICp_47_SM5_0_2PRKN_no_PD Control
SA184479HILICp_46_SM5_0_1PRKN_no_PD Control
SA184480HILICp_38_SM3_0_2PRKN_no_PD Control
SA184481HILICp_39_SM3_0_3PRKN_no_PD Control
SA184374HILICp_62_PM1_200_2PRKN_PD 200
SA184375HILICp_63_PM1_200_3PRKN_PD 200
SA184376HILICn_61_PM1_200_1PRKN_PD 200
SA184377HILICn_62_PM1_200_2PRKN_PD 200
SA184378HILICn_63_PM1_200_3PRKN_PD 200
SA184379RPLCp_23_PM12_200_TH1PRKN_PD 200
SA184380RPLCp_29_PM17_200_TH1PRKN_PD 200
SA184381HILICp_71_PM18_200_2PRKN_PD 200
SA184382HILICp_70_PM18_200_1PRKN_PD 200
SA184383HILICp_72_PM18_200_3PRKN_PD 200
SA184384RPLCp_30_PM17_200_TH2PRKN_PD 200
SA184385HILICp_12_PM12_200PRKN_PD 200
SA184386RPLCp_24_PM12_200_TH2PRKN_PD 200
SA184387HILICp_61_PM1_200_1PRKN_PD 200
SA184388HILICn_70_PM18_200_1PRKN_PD 200
SA184389HILICn_72_PM18_200_3PRKN_PD 200
SA184390HILICn_71_PM18_200_2PRKN_PD 200
SA184391HILICp_15_PM17_200PRKN_PD 200
SA184392RPLCp_28_PM17_50_TH2PRKN_PD 50
SA184393RPLCp_27_PM17_50_TH1PRKN_PD 50
SA184394HILICn_67_PM18_50_1PRKN_PD 50
SA184395HILICn_68_PM18_50_2PRKN_PD 50
SA184396RPLCp_22_PM12_50_TH2PRKN_PD 50
SA184397HILICn_69_PM18_50_3PRKN_PD 50
SA184398HILICp_69_PM18_50_3PRKN_PD 50
SA184399HILICp_58_PM1_50_1PRKN_PD 50
SA184400HILICp_67_PM18_50_1PRKN_PD 50
SA184401HILICp_68_PM18_50_2PRKN_PD 50
SA184402RPLCp_21_PM12_50_TH1PRKN_PD 50
SA184403HILICp_60_PM1_50_3PRKN_PD 50
SA184404HILICp_14_PM17_50PRKN_PD 50
SA184405HILICn_60_PM1_50_3PRKN_PD 50
SA184406HILICn_59_PM1_50_2PRKN_PD 50
SA184407HILICp_59_PM1_50_2PRKN_PD 50
SA184408HILICn_58_PM1_50_1PRKN_PD 50
SA184409HILICp_11_PM12_50PRKN_PD 50
SA184410HILICp_10_PM12_0PRKN_PD Control
SA184411HILICp_66_PM18_0_3PRKN_PD Control
SA184412HILICp_55_PM1_0_1PRKN_PD Control
SA184413HILICp_64_PM18_0_1PRKN_PD Control
SA184414HILICp_65_PM18_0_2PRKN_PD Control
SA184415HILICp_57_PM1_0_3PRKN_PD Control
SA184416HILICn_56_PM1_0_2PRKN_PD Control
SA184417HILICn_66_PM18_0_3PRKN_PD Control
SA184418HILICp_13_PM17_0PRKN_PD Control
SA184419RPLCp_20_PM12_0_TH2PRKN_PD Control
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Collection:

Collection ID:CO002030
Collection Summary:iPSC lines were derived from dermal fibroblasts from two human subjects, PM and SM, and four independent clones from each patient were used in this study (PM1, PM12, PM17 and PM18; SM3, SM5, SM14 and SM15).Fibroblasts were obtained by skin biopsy after the appropriate patient consent/assent under the guidelines of an approved Internal Review Board (IRB) protocol at Vanderbilt University (#080369). Three of the iPSC lines (PM1, SM3, SM5) were generated by transducing the fibroblasts with a lentivirus as previously described. The other five lines (PM12, PM17, PM18, SM14, SM15) were reprogrammed by electroporation with CXLE plasmid vectors (Addgene) using the Neon Transfection System (Life Technologies) following published methods.
Sample Type:Skin Biopsy
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002049
Treatment Summary:Differentiation of the iPSCs to a mesencephalic dopaminergic lineage was performed as previously described 39,40,126. In brief, iPSCs were differentiated in a first stage into floor plate cells (mesencephalic neural precursors) (days 0-11) via dual SMAD inhibition combined with ventral midbrain patterning. In a second stage, these floor plate cells were further differentiated (days 11-25) into early post mitotic mesencephalic dopamine neurons. Cells were treated in culture with either 0, 50uM or 200uM Mn.
Treatment Compound:Mn
Treatment Dose:0uM, 50uM, 200uM

Sample Preparation:

Sampleprep ID:SP002043
Sampleprep Summary:For method #1 (RPLC pos/ HILIC pos) day 11 neural dopaminergic precursor cells (floor plate cells of one well of a 6 well plate) were harvested into 500 µl of ice-cold methanol, flash frozen and then stored at -80oC. To extract the metabolites, the 500 µl methanol cell suspensions were thawed and 100 µl of H2O added. Then the samples were frozen on dry ice for 3 min, defrosted in ice over a 10 min period, and sonicated with 10 pulses using a probe sonicator at 30% power. The freeze-thaw-sonication sequence was repeated three times. The proteins were precipitated by placing the lysates at -80°C overnight and then pelleted by centrifugation at 15,000 rpm for 15 minutes. Cleared supernatants containing the metabolites were placed in clean Eppendorf tubes, dried in a vacuum concentrator and stored frozen at -80°C. For reverse phase liquid chromatography (RPLC)-positive (pos) mode mass spectrometry analysis the dried extracts were reconstituted in 60 μl of RPLC buffer (acetonitrile/water with 0.1% formic acid, 2:98, v/v). Samples were vortexed rigorously to solubilize the metabolites, cleared by centrifugation for 5 min at 15,000 rpm, and the supernatants were injected twice (5μl/injection) randomly. For method #2 (HILIC pos/neg) the cells were washed three times with 2.5 ml of an ammonium formate buffer (50 mM, pH 6.8), scraped into the same buffer, centrifugated at 200 x for 5 min, the cell pellets flash frozen in liquid nitrogen and stored at -80o C. To extract the metabolites, cell pellets were lysed in 200 µl ice-cold lysis buffer (1:1:2, Acetonitrile:MeOH:Ammonium Bicarbonate 0.1M, pH 8.0, LC-MS grade) and sonicated once as described above. The protein concentration was determined (BCA assay, Thermo Fisher Scientific) and adjusted to 1 mg/ml. Isotopically labeled standard molecules, Phenylalanine-D8 and Biotin-D2 were added to the 200 µl cell lysates, the protein precipitated by addition of 800 µl of ice-cold methanol and stored at -80°C overnight. Upon thawing, the precipitated proteins were pelleted by centrifugation at 9300 x g for 10 min, the supernatants transferred into two clean Eppendorf tubes, dried down in vacuo and stored at -80°C.
Sampleprep Protocol Filename:Materials_and_Methods_DN.pdf
Processing Storage Conditions:-80℃
Extract Storage:On ice

Combined analysis:

Analysis ID AN003189 AN003190 AN003191 AN003192
Analysis type MS MS MS MS
Chromatography type Reversed phase HILIC HILIC HILIC
Chromatography system Thermo Vanquish Thermo Vanquish Thermo Vanquish Thermo Vanquish
Column Thermo Hypersil Gold (100 x 2. mm,1.9um) EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um) EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um) EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um)
MS Type ESI ESI ESI ESI
MS instrument type QTOF Orbitrap Orbitrap Orbitrap
MS instrument name Waters Synapt G2 Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE POSITIVE NEGATIVE POSITIVE
Units peak area peak area peak area peak area

Chromatography:

Chromatography ID:CH002357
Chromatography Summary:Method#1_RPLC_positive
Methods Filename:Materials_and_Methods_DN.pdf
Instrument Name:Thermo Vanquish
Column Name:Thermo Hypersil Gold (100 x 2. mm,1.9um)
Column Temperature:40
Flow Rate:0.25 mL/min
Injection Temperature:8
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile, 0.1% formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH002358
Chromatography Summary:Method#1_HILIC_positive
Methods Filename:Materials_and_Methods_DN.pdf
Instrument Name:Thermo Vanquish
Column Name:EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um)
Column Temperature:40
Flow Rate:0.20 mL/min
Injection Temperature:8
Solvent A:90% acetonitrile/10% water; 5 mM ammonium formate
Solvent B:10% water/90% acetonitrile; 5 mM ammonium formate
Chromatography Type:HILIC
  
Chromatography ID:CH002359
Chromatography Summary:Method#2_HILIC_positive
Methods Filename:Materials_and_Methods_DN.pdf
Instrument Name:Thermo Vanquish
Column Name:EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um)
Column Temperature:40
Flow Rate:0.20 mL/min
Injection Temperature:8
Solvent A:90% acetonitrile/10% water; 5 mM ammonium formate
Solvent B:10% water/90% acetonitrile; 5 mM ammonium formate
Chromatography Type:HILIC
  
Chromatography ID:CH002360
Chromatography Summary:Method#2_HILIC_negative
Methods Filename:Materials_and_Methods_DN.pdf
Instrument Name:Thermo Vanquish
Column Name:EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um)
Column Temperature:40
Flow Rate:0.20 mL/min
Injection Temperature:8
Solvent A:90% acetonitrile/10% water; 5 mM ammonium formate
Solvent B:10% water/90% acetonitrile; 5 mM ammonium formate
Chromatography Type:HILIC

MS:

MS ID:MS002967
Analysis ID:AN003189
Instrument Name:Waters Synapt G2
Instrument Type:QTOF
MS Type:ESI
MS Comments:Progenesis QI
Ion Mode:POSITIVE
Analysis Protocol File:Materials_and_Methods_DN.pdf
  
MS ID:MS002968
Analysis ID:AN003190
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Progenesis QI
Ion Mode:POSITIVE
Analysis Protocol File:Materials_and_Methods_DN.pdf
  
MS ID:MS002969
Analysis ID:AN003191
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Progenesis QI
Ion Mode:NEGATIVE
Analysis Protocol File:Materials_and_Methods_DN.pdf
  
MS ID:MS002970
Analysis ID:AN003192
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Progenesis QI
Ion Mode:POSITIVE
Analysis Protocol File:Materials_and_Methods_DN.pdf
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