Summary of Study ST003523

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 PR002167. The data can be accessed directly via it's Project DOI: 10.21228/M8HJ9G 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	ST003523
Study Title	Hypermetabolism induced pantothenate depletion in MT-ATP6 neuropathy
Study Summary	Mitochondrial DNA mutations frequently result in mitochondrial dysfunction and energy deficits, compromising cellular and tissue functions. However, in some cases, bioenergetic deficits triggers a futile cycle of energy production and consumption to maintain cellular function, a phenomenon termed hypermetabolism. In this study, we used patient iPSC-derived motor neurons to investigate a truncating heteroplasmic mutation in the MT-ATP6 gene, which encodes a subunit of the proton pore of the ATP synthase. We successfully generated motor neurons with 50% mutation heteroplasmy, despite adverse effects on ATP synthase assembly. Through a combination of respirometry, metabolomic tracing, and proteomic analysis, we observed increased metabolic activity in mutant motor neurons, indicative of a hypermetabolic phenotype. This is the first study to explore hypermetabolism in neurons, revealing new significant implications of this state. This hypermetabolic state Hypermetabolism led to the reprioritization of the core metabolite acetyl-CoA and depletion of its precursor, resulting in the downregulation . We also observed the downregulation of epigenetic and SUMOylation pathways, likely as compensatory and adaptive mechanisms. Our findings suggest a link between mitochondrial dysfunction and SUMOylation, highlighting a potential new contributor to the mitochondrial hypermetabolic phenotype.
Institute	University of Helsinki
Last Name	Torregrosa-Muñumer
First Name	Rubén
Address	Biomedicum Helsinki, Haartmaninkatu 8, 00290 Helsinki, Finland
Email	ruben.torregrosa@helsinki.fi
Phone	+358294125800
Submit Date	2024-10-08
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-10-22
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002167
Project DOI:	doi: 10.21228/M8HJ9G
Project Title:	Hypermetabolism induced pantothenate depletion in MT-ATP6 neuropathy
Project Summary:	Mitochondrial DNA mutations frequently result in mitochondrial dysfunction and energy deficits, compromising cellular and tissue functions. However, in some cases, bioenergetic deficits triggers a futile cycle of energy production and consumption to maintain cellular function, a phenomenon termed hypermetabolism. In this study, we used patient iPSC-derived motor neurons to investigate a truncating heteroplasmic mutation in the MT-ATP6 gene, which encodes a subunit of the proton pore of the ATP synthase. We successfully generated motor neurons with 50% mutation heteroplasmy, despite adverse effects on ATP synthase assembly. Through a combination of respirometry, metabolomic tracing, and proteomic analysis, we observed increased metabolic activity in mutant motor neurons, indicative of a hypermetabolic phenotype. This is the first study to explore hypermetabolism in neurons, revealing new significant implications of this state. This hypermetabolic state Hypermetabolism led to the reprioritization of the core metabolite acetyl-CoA and depletion of its precursor, resulting in the downregulation . We also observed the downregulation of epigenetic and SUMOylation pathways, likely as compensatory and adaptive mechanisms. Our findings suggest a link between mitochondrial dysfunction and SUMOylation, highlighting a potential new contributor to the mitochondrial hypermetabolic phenotype.
Institute:	University of Helsinki
Department:	Faculty of Medicine
Laboratory:	Tyynismaa lab
Last Name:	Torregrosa-Muñumer
First Name:	Rubén
Address:	Biomedicum Helsinki, Haartmaninkatu 8, 00290 Helsinki, Finland
Email:	ruben.torregrosa@helsinki.fi
Phone:	+358294125800
Funding Source:	Academy of Finland Centre of Excellence on Stem Cell Metabolism (MetaStem), Academy of Finland Postdoctoral fellowship to RTM, and Sigrid Juselius Foundation.
Contributors:	Rubén Torregrosa-Muñumer, Jeremi Turkia, Jouni Kvist, Jana Pennonen, Emilia Kuuluvainen, Pekka Katajisto, Ville Hietakangas, Emil Ylikallio, Henna Tyynismaa

Subject:

Subject ID:	SU003652
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Not applicable
Species Group:	Mammals

Factors:

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

mb_sample_id	local_sample_id	Genotype	Heteroplasmy level	Time
SA386804	MUT.49%_80_12h_1	MUT	49%	12h
SA386805	MUT.49%_80_12h_6	MUT	49%	12h
SA386806	MUT.49%_80_12h_5	MUT	49%	12h
SA386807	MUT.49%_80_12h_3	MUT	49%	12h
SA386808	MUT.49%_80_12h_2	MUT	49%	12h
SA386809	MUT.49%_80_12h_4	MUT	49%	12h
SA386810	MUT.49%_80_1h_6	MUT	49%	1h
SA386811	MUT.49%_80_1h_1	MUT	49%	1h
SA386812	MUT.49%_80_1h_2	MUT	49%	1h
SA386813	MUT.49%_80_1h_3	MUT	49%	1h
SA386814	MUT.49%_80_1h_4	MUT	49%	1h
SA386815	MUT.49%_80_1h_5	MUT	49%	1h
SA386816	MUT.49%_80_48h_2	MUT	49%	48h
SA386817	MUT.49%_80_48h_6	MUT	49%	48h
SA386818	MUT.49%_80_48h_5	MUT	49%	48h
SA386819	MUT.49%_80_48h_4	MUT	49%	48h
SA386820	MUT.49%_80_48h_3	MUT	49%	48h
SA386821	MUT.49%_80_48h_1	MUT	49%	48h
SA386822	MUT.52%_56_12h_5	MUT	52%	12h
SA386823	MUT.52%_56_12h_4	MUT	52%	12h
SA386824	MUT.52%_56_12h_1	MUT	52%	12h
SA386825	MUT.52%_56_12h_3	MUT	52%	12h
SA386826	MUT.52%_56_12h_2	MUT	52%	12h
SA386827	MUT.52%_56_12h_6	MUT	52%	12h
SA386828	MUT.52%_56_1h_2	MUT	52%	1h
SA386829	MUT.52%_56_1h_6	MUT	52%	1h
SA386830	MUT.52%_56_1h_5	MUT	52%	1h
SA386831	MUT.52%_56_1h_4	MUT	52%	1h
SA386832	MUT.52%_56_1h_3	MUT	52%	1h
SA386833	MUT.52%_56_1h_1	MUT	52%	1h
SA386834	MUT.52%_56_48h_3	MUT	52%	48h
SA386835	MUT.52%_56_48h_6	MUT	52%	48h
SA386836	MUT.52%_56_48h_1	MUT	52%	48h
SA386837	MUT.52%_56_48h_2	MUT	52%	48h
SA386838	MUT.52%_56_48h_5	MUT	52%	48h
SA386839	MUT.52%_56_48h_4	MUT	52%	48h
SA386840	MUT.54%_84_12h_1	MUT	54%	12h
SA386841	MUT.54%_84_12h_6	MUT	54%	12h
SA386842	MUT.54%_84_12h_5	MUT	54%	12h
SA386843	MUT.54%_84_12h_3	MUT	54%	12h
SA386844	MUT.54%_84_12h_4	MUT	54%	12h
SA386845	MUT.54%_84_12h_2	MUT	54%	12h
SA386846	MUT.54%_84_1h_6	MUT	54%	1h
SA386847	MUT.54%_84_1h_5	MUT	54%	1h
SA386848	MUT.54%_84_1h_4	MUT	54%	1h
SA386849	MUT.54%_84_1h_1	MUT	54%	1h
SA386850	MUT.54%_84_1h_3	MUT	54%	1h
SA386851	MUT.54%_84_1h_2	MUT	54%	1h
SA386852	MUT.54%_84_48h_1	MUT	54%	48h
SA386853	MUT.54%_84_48h_2	MUT	54%	48h
SA386854	MUT.54%_84_48h_3	MUT	54%	48h
SA386855	MUT.54%_84_48h_4	MUT	54%	48h
SA386856	MUT.54%_84_48h_5	MUT	54%	48h
SA386857	MUT.54%_84_48h_6	MUT	54%	48h
SA386858	WT_73_12h_2	WT	0%	12h
SA386859	WT_73_12h_3	WT	0%	12h
SA386860	WT_89_12h_6	WT	0%	12h
SA386861	WT_73_12h_4	WT	0%	12h
SA386862	WT_73_12h_5	WT	0%	12h
SA386863	WT_73_12h_6	WT	0%	12h
SA386864	WT_89_12h_1	WT	0%	12h
SA386865	WT_89_12h_3	WT	0%	12h
SA386866	WT_89_12h_4	WT	0%	12h
SA386867	WT_89_12h_5	WT	0%	12h
SA386868	WT_89_12h_2	WT	0%	12h
SA386869	WT_73_12h_1	WT	0%	12h
SA386870	WT_73_1h_1	WT	0%	1h
SA386871	WT_89_1h_5	WT	0%	1h
SA386872	WT_73_1h_4	WT	0%	1h
SA386873	WT_73_1h_2	WT	0%	1h
SA386874	WT_73_1h_5	WT	0%	1h
SA386875	WT_89_1h_1	WT	0%	1h
SA386876	WT_89_1h_2	WT	0%	1h
SA386877	WT_89_1h_4	WT	0%	1h
SA386878	WT_89_1h_3	WT	0%	1h
SA386879	WT_89_1h_6	WT	0%	1h
SA386880	WT_73_1h_6	WT	0%	1h
SA386881	WT_73_1h_3	WT	0%	1h
SA386882	WT_89_48h_2	WT	0%	48h
SA386883	WT_89_48h_6	WT	0%	48h
SA386884	WT_73_48h_6	WT	0%	48h
SA386885	WT_73_48h_5	WT	0%	48h
SA386886	WT_73_48h_4	WT	0%	48h
SA386887	WT_73_48h_3	WT	0%	48h
SA386888	WT_73_48h_2	WT	0%	48h
SA386889	WT_73_48h_1	WT	0%	48h
SA386890	WT_89_48h_5	WT	0%	48h
SA386891	WT_89_48h_4	WT	0%	48h
SA386892	WT_89_48h_1	WT	0%	48h
SA386893	WT_89_48h_3	WT	0%	48h

Showing results 1 to 90 of 90

Showing results 1 to 90 of 90

Collection:

Collection ID:	CO003645
Collection Summary:	Human iPSC derived from a patient carrying the MT-ATP6 mutation were generated previously (Kenvin et al., 2022, DOI: 10.1093/hmg/ddab299). Two isogenic controls with 0% heteroplasmy and three mutants with 49%, 52% and 54% heteroplasmy were differentiated into motor neurons. Each genotype was incubated with 13C glucose for 1, 12, or 48 hours at day 30 of differentiation. 6 replicates per cell line per time point were collected for metabolomic profiling. Cell lines numbers are in-house identifiers.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR003661
Treatment Summary:	iPSCs were differentiated into iPSC-MN following a protocol described elsewhere (Guo et al., 2017, DOI: 10.1038/s41467-017-00911-y & Maury et al., 2015, DOI: 10.1038/nbt.3049) with slight modifications. Neuronal basal medium (vol:vol DMEM/F-12 (Gibco, #31331-028) and Neurobasal (Gibco, #21103-049)) was supplemented with N2 (Gibco, #17502048), B-27 (Gibco, #17504044), 0.1mM L-ascorbic acid (Santa Cruz, #sc-394304), 50µg/mL Uridine (Sigma, #U3003) and 100µg/mL primocin (InvivoGen, #ant-pm-05). For neural induction and caudalization to obtain neuroepithelial progenitor (NEP) cells, iPSCs were dissociated using EDTA and 10⁵ cells / cm2 were plated on ultra-low attachment dishes. On day 0, neuronal basal medium was supplemented with 3 μM Chir-99021 (Selleckchem, #S1263), 40 μM SB431542 (Millipore, #616461), 0,2 μM LDN-193189 (Sigma, #SML0559) and 5 μM Y-27632 (Selleckhem, #S6390). The medium was replaced the following day. From day 2 to 6, media was changed to neuronal basal medium supplemented with 0.1 μM retinoic acid (ThermoFisher, #044540-04) and 0.5 μM SAG (Millipore, #566660). On day 7, medium was replaced with neuronal basal medium supplemented with retinoic acid and SAG as previously, plus 10ng/ml BDNF (Peprotech, #450-02) and GDNF 10ng/ml (Peprotech, #450-10). On day 9, medium was replaced as previously, and 20 μM DAPT (Sigma, #D5942) was added to the fresh media. On day 10, the motor neuron spheres were dissociated into single cells with Accumax (Invitrogen, #00-4666-56), and motor neuron progenitors were plated on 50 μg/ml poly-D-lysine (Millipore, #633307) and 10 μg/ml laminin (Sigma, #L2020) coated plates at 60 000 cells / cm2. On day 11, fresh medium was added to the wells. On day 14, half of the medium was replaced with neuronal basal medium supplemented only with DAPT, BDNF and GDNF. From day 16 on, we used neuronal basal medium supplemented with 10 ng/ml BDNF, GDNF and CNTF (Peprotech, #450-13) and replaced half of the culture medium every other/third day. All the experiments were performed on day 30 unless otherwise indicated. Occasionally, "flat cells" (other neuronal types and progenitors) appear in the motor neuron culture. However, we only used dishes containing pure motor neurons for our studies to avoid confounding results.

Treatment ID:

TR003661

Treatment Summary:

iPSCs were differentiated into iPSC-MN following a protocol described elsewhere (Guo et al., 2017, DOI: 10.1038/s41467-017-00911-y & Maury et al., 2015, DOI: 10.1038/nbt.3049) with slight modifications. Neuronal basal medium (vol:vol DMEM/F-12 (Gibco, #31331-028) and Neurobasal (Gibco, #21103-049)) was supplemented with N2 (Gibco, #17502048), B-27 (Gibco, #17504044), 0.1mM L-ascorbic acid (Santa Cruz, #sc-394304), 50µg/mL Uridine (Sigma, #U3003) and 100µg/mL primocin (InvivoGen, #ant-pm-05). For neural induction and caudalization to obtain neuroepithelial progenitor (NEP) cells, iPSCs were dissociated using EDTA and 10⁵ cells / cm2 were plated on ultra-low attachment dishes. On day 0, neuronal basal medium was supplemented with 3 μM Chir-99021 (Selleckchem, #S1263), 40 μM SB431542 (Millipore, #616461), 0,2 μM LDN-193189 (Sigma, #SML0559) and 5 μM Y-27632 (Selleckhem, #S6390). The medium was replaced the following day. From day 2 to 6, media was changed to neuronal basal medium supplemented with 0.1 μM retinoic acid (ThermoFisher, #044540-04) and 0.5 μM SAG (Millipore, #566660). On day 7, medium was replaced with neuronal basal medium supplemented with retinoic acid and SAG as previously, plus 10ng/ml BDNF (Peprotech, #450-02) and GDNF 10ng/ml (Peprotech, #450-10). On day 9, medium was replaced as previously, and 20 μM DAPT (Sigma, #D5942) was added to the fresh media. On day 10, the motor neuron spheres were dissociated into single cells with Accumax (Invitrogen, #00-4666-56), and motor neuron progenitors were plated on 50 μg/ml poly-D-lysine (Millipore, #633307) and 10 μg/ml laminin (Sigma, #L2020) coated plates at 60 000 cells / cm2. On day 11, fresh medium was added to the wells. On day 14, half of the medium was replaced with neuronal basal medium supplemented only with DAPT, BDNF and GDNF. From day 16 on, we used neuronal basal medium supplemented with 10 ng/ml BDNF, GDNF and CNTF (Peprotech, #450-13) and replaced half of the culture medium every other/third day. All the experiments were performed on day 30 unless otherwise indicated. Occasionally, "flat cells" (other neuronal types and progenitors) appear in the motor neuron culture. However, we only used dishes containing pure motor neurons for our studies to avoid confounding results.

Sample Preparation:

Sampleprep ID:	SP003659
Sampleprep Summary:	Control and mutant iPSC-derived motor neurons were incubated with 21.25 mM U-13C glucose (Cambridge Isotope Laboratories) and 2mM glutamine in 50% DMEM-F12 (w/o glucose, Biowest, #L0091) + 50% Neurobasal®-A (w/o glucose, Gibco, #A24775-01) for 1,12, or 48 hours starting from day 30. Concentration of glucose and glutamine were comparable to normal culture conditions. Then metabolites were immediately extracted: cells were washed once with PBS and 150 µl of ice-cold H2O/acetonitrile (20:80) was added. The cells were scraped, collected, and vortexed for 5 seconds. Finally, the extracted samples were then centrifuged at 13,000g for 10 minutes at 4°C, and the supernatant was stored at -80°C until analysis. For each cell line, 6 separated wells were used as technical replicates.

Sampleprep ID:

SP003659

Sampleprep Summary:

Control and mutant iPSC-derived motor neurons were incubated with 21.25 mM U-13C glucose (Cambridge Isotope Laboratories) and 2mM glutamine in 50% DMEM-F12 (w/o glucose, Biowest, #L0091) + 50% Neurobasal®-A (w/o glucose, Gibco, #A24775-01) for 1,12, or 48 hours starting from day 30. Concentration of glucose and glutamine were comparable to normal culture conditions. Then metabolites were immediately extracted: cells were washed once with PBS and 150 µl of ice-cold H2O/acetonitrile (20:80) was added. The cells were scraped, collected, and vortexed for 5 seconds. Finally, the extracted samples were then centrifuged at 13,000g for 10 minutes at 4°C, and the supernatant was stored at -80°C until analysis. For each cell line, 6 separated wells were used as technical replicates.

Chromatography:

Chromatography ID:	CH004391
Chromatography Summary:	Samples were analyzed on a Thermo Q Exactive Focus Quadrupole Orbitrap mass spectrometer coupled with a Thermo Dionex UltiMate 3000 HPLC system (Thermo Fisher Scientific). The HPLC was equipped with a hydrophilic ZIC-pHILIC column (150 × 2.1 mm, 5 μm) with a ZIC-pHILIC guard column (20 × 2.1 mm, 5 μm, Merck Sequant). A total of 5 µl sample was injected into the LC–MS instrument after quality controls in randomized order having every tenth samples as blank. A linear solvent gradient was applied in decreasing organic solvent (80–35%, 16 min) at 0.15 ml min–1 flow rate and 45 ◦C column oven temperature. Mobile phases were aqueous 200 mmol per litre ammonium bicarbonate solution (pH 9.3, adjusted with 25% ammonium hydroxide), 100% acetonitrile and 100% water. Ammonium bicarbonate solution was kept at 10% throughout the run, resulting in a steady 20 mmol per litre concentration.
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
Column Temperature:	45
Flow Gradient:	A linear solvent gradient was applied in decreasing organic solvent (Solvent A), 80–35% in 16 min at 0.15 mL/min flow rate
Flow Rate:	0.15 mL/min
Solvent A:	100% acetonitrile; 200 mM ammonium bicarbonate; 0.01% ammonium hydroxide
Solvent B:	100% water; 200 mM ammonium bicarbonate; 0.01% ammonium hydroxide
Chromatography Type:	HILIC

Analysis:

Analysis ID:	AN005785
Analysis Type:	MS
Chromatography ID:	CH004391
Num Factors:	12
Num Metabolites:	300
Units:	Peak area

Analysis ID:	AN005786
Analysis Type:	MS
Chromatography ID:	CH004391
Num Factors:	12
Num Metabolites:	406
Units:	Peak area