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.
| Study ID | ST001957 |
| Study Title | Untargeted Mass Spectrometry Metabolomic Profiles of iPSC-derived Dopaminergic Neurons from Clinically Discordant Brothers with Identical PRKN Deletions |
| Study Type | untargeted metabolomics analysis |
| Study 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 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 |
| Department | Chemistry |
| Laboratory | Center for Innovative Technology |
| Last Name | CODREANU |
| First Name | SIMONA |
| Address | 1234 STEVENSON CENTER LANE |
| SIMONA.CODREANU@VANDERBILT.EDU | |
| Phone | 6158758422 |
| Submit Date | 2021-10-13 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Waters) |
| Analysis Type Detail | LC-MS |
| Release Date | 2022-01-17 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
