Summary of Study ST001035

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 PR000693. The data can be accessed directly via it's Project DOI: 10.21228/M83H4H This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST001035
Study Title	TCA Isotopomers in Neuromyelitis Optica Patients (part - I)
Study Summary	Patients with an inflammatory disease of the central nervous system known as neuromyelitis optica (NMO) experience increased levels of depression. These patients have an antibody that recognizes a type of cell in the brain called astrocytes – binding of this antibody to astrocytes triggers a stress response in the cell that results in the development of brain lesions that cause disability and cognitive disturbances. We recently observed a change in the level of glutamate in a part of the brain involved in depression in patients with NMO. Glutamate is a chemical that is used in the brain for communication between neurons – reduced levels of glutamate are thought to trigger depression by reducing neuronal activity in specific circuits. Based on this observation and the known role of astrocytes in maintaining glutamate levels in the brain, we hypothesize that the NMO antibody disturbs metabolic activity in astrocytes and thereby reduces glutamate and triggers depression. We intend to trace the metabolic response induced in astrocytes by the NMO antibody using TCA isotopomers. It is our hope that we will not only learn something about the mechanisms of astrocyte dysregulation in neuromyelitis optica, but that we will learn something about the mechanisms of depression in general that may lead to new therapies for this disease.
Institute	Mayo Clinic
Last Name	Howe
First Name	Charles
Address	200 First St. SW, Rochester, Minnesota, 55905, USA
Email	howe@mayo.edu
Phone	507-284-9288
Submit Date	2018-08-13
Analysis Type Detail	GC-MS
Release Date	2020-08-20
Release Version	1

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Project:

Project ID:	PR000693
Project DOI:	doi: 10.21228/M83H4H
Project Title:	Mayo Pilot and Feasibility: The pathogenic NMO IgG dysregulates the astrocytic glutamine-glutamate cycle: a metabolic basis for depression in NMO patients
Project Summary:	Neuromyelitis optica (NMO) is a disabling central nervous system (CNS) inflammatory disorder that involves a pathogenic autoantibody (NMO IgG) directed against aquaporin-4, the major brain water channel, expressed on astrocytes. Astrocytes in brain tissue from patients with NMO exhibit a spectrum of abnormalities and pathologies ranging from sublytic gliosis and reactivity to outright destruction. Our current working model for NMO pathogenesis involves an early and robust NMO IgG-induced astrocytic stress response that drives metabolic dyshomeostasis and the production of pro-inflammatory cytokines and chemokines that amplify pathology by recruiting innate immune effector cells to the CNS. Notably, astrocytes are implicated in clinical depression and patients with NMO experience depression at levels that exceed the general population. Preliminary magnetic resonance spectroscopy data from our group indicates that glutamate levels are reduced in the prefrontal cortex of NMO patients, suggesting that unipolar depression in these individuals is a direct pathogenic effect of NMO IgG-induced astrocytic dysregulation. Because astrocytes are critical for glutamine-glutamate cycling in the CNS, we hypothesize that stimulation of primary astrocytes with patient-derived NMO IgG will drive a metabolic shift marked by alterations in cellular levels of glutamate and glutamine. In preliminary experiments using 1H-NMR to measure metabolic changes induced in astrocytes by stimulation with NMO IgG we observed variable glutamate and glutamine responses. To overcome issues of signal-to-noise and the high basal levels of glutamate and glutamine produced by astrocytes, we now propose to use isotopic tracing and 13C-NMR to quantify NMO IgG-induced metabolic dysregulation. Our ultimate goal is to use NMO IgG-induced dyshomeostasis as a microscope to reveal basic mechanisms of pathogenic glutamate-glutamine metabolism in astrocytes that may not only impact the health of patients with NMO but may also yield novel insights into the mechanisms of depression in general.
Institute:	Mayo Clinic
Last Name:	Howe
First Name:	Charles
Address:	200 First St. SW, Rochester, Minnesota, 55905, USA
Email:	howe@mayo.edu
Phone:	507-284-9288

Subject:

Subject ID:	SU001074
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

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

mb_sample_id	local_sample_id	Time	condition	matrix	Tracer
SA069228	ms6905-32	0h	BGP 50uMGt Gn	Fresh Media	Unlabelled
SA069230	ms6905-30	0h	BGPGt 0.5mM Gn	Fresh Media	Unlabelled
SA069229	ms6983-32	0h	BGPGt 0.5 mM Gn	Fresh med	Unlabelled
SA069232	ms6983-34	0h	BGPGt 50 mM Gn	Fresh med	Unlabelled
SA069233	ms6905-31	0h	BGPGt 5mM Gn	Fresh Media	Unlabelled
SA069231	ms6983-33	0h	BGPGt 5 mM Gn	Fresh med	Unlabelled
SA069234	ms6905-25	0h	E Med	Fresh Media	13C6
SA069235	ms6983-25	0h	E Med	Fresh med	13C6
SA069236	ms6983-35	0h	E Med	Fresh med	Unlabelled
SA069223	MS6735-41	0 hr	BGPGt - 0.5mM Gn	Fresh med	Unlabelled
SA069224	MS6735-40	0 hr	BGPGt - 0.5mM Gn	Fresh med	Unlabelled
SA069225	MS6735-39	0 hr	BGPGt	Fresh med	Unlabelled
SA069226	MS6735-26	0 hr	E Med	Fresh med	13C6
SA069227	MS6735-25	0 hr	E Med	Fresh med	Unlabelled
SA069238	ms6905-22	1h nonCO2	BGP50uMGt Gn	Spent Sup	13C6
SA069239	ms6905-24	1h nonCO2	BGP50uMGt Gn	Spent Sup	13C6
SA069240	ms6905-23	1h nonCO2	BGP50uMGt Gn	Spent Sup	13C6
SA069237	ms6905-21	1h nonCO2	BGP 50uMGt Gn	Spent Sup	Unlabelled
SA069241	MS6735-19	1h nonCO2	BGPGt - 0.5mM Gn	Spent Sup	13C6
SA069242	MS6735-20	1h nonCO2	BGPGt - 0.5mM Gn	Spent Sup	13C6
SA069243	MS6735-18	1h nonCO2	BGPGt - 0.5mM Gn	Spent Sup	13C6
SA069249	ms6983-16	1h nonCO2	BGPGt 0.5 mM Gn	Spent Sup	13C6
SA069250	ms6983-15	1h nonCO2	BGPGt 0.5 mM Gn	Spent Sup	13C6
SA069251	ms6983-14	1h nonCO2	BGPGt 0.5 mM Gn	Spent Sup	13C6
SA069253	ms6905-15	1h nonCO2	BGPGt 0.5mM Gn	Spent Sup	13C6
SA069254	ms6905-16	1h nonCO2	BGPGt 0.5mM Gn	Spent Sup	13C6
SA069255	ms6905-14	1h nonCO2	BGPGt 0.5mM Gn	Spent Sup	13C6
SA069244	MS6735-17	1h nonCO2	BGPGt - 0.5mM Gn	Spent Sup	Unlabelled
SA069252	ms6983-13	1h nonCO2	BGPGt 0.5 mM Gn	Spent Sup	Unlabelled
SA069256	ms6905-13	1h nonCO2	BGPGt 0.5mM Gn	Spent Sup	Unlabelled
SA069261	ms6983-22	1h nonCO2	BGPGt 50 mM Gn	Spent Sup	13C6
SA069262	ms6983-24	1h nonCO2	BGPGt 50 mM Gn	Spent Sup	13C6
SA069263	ms6983-23	1h nonCO2	BGPGt 50 mM Gn	Spent Sup	13C6
SA069264	ms6983-21	1h nonCO2	BGPGt 50 mM Gn	Spent Sup	Unlabelled
SA069245	MS6735-22	1h nonCO2	BGPGt - 5mM Gn	Spent Sup	13C6
SA069246	MS6735-24	1h nonCO2	BGPGt - 5mM Gn	Spent Sup	13C6
SA069247	MS6735-23	1h nonCO2	BGPGt - 5mM Gn	Spent Sup	13C6
SA069257	ms6983-18	1h nonCO2	BGPGt 5 mM Gn	Spent Sup	13C6
SA069258	ms6983-20	1h nonCO2	BGPGt 5 mM Gn	Spent Sup	13C6
SA069259	ms6983-19	1h nonCO2	BGPGt 5 mM Gn	Spent Sup	13C6
SA069265	ms6905-20	1h nonCO2	BGPGt 5mM Gn	Spent Sup	13C6
SA069266	ms6905-19	1h nonCO2	BGPGt 5mM Gn	Spent Sup	13C6
SA069267	ms6905-18	1h nonCO2	BGPGt 5mM Gn	Spent Sup	13C6
SA069248	MS6735-21	1h nonCO2	BGPGt - 5mM Gn	Spent Sup	Unlabelled
SA069260	ms6983-17	1h nonCO2	BGPGt 5 mM Gn	Spent Sup	Unlabelled
SA069268	ms6905-17	1h nonCO2	BGPGt 5mM Gn	Spent Sup	Unlabelled
SA069269	MS6735-14	1h nonCO2	BGPGt	Spent Sup	13C6
SA069270	MS6735-16	1h nonCO2	BGPGt	Spent Sup	13C6
SA069271	MS6735-15	1h nonCO2	BGPGt	Spent Sup	13C6
SA069272	MS6735-13	1h nonCO2	BGPGt	Spent Sup	Unlabelled
SA069298	ms6905-10	23h +1h nonCO2	BGP50uMGt Gn	pellet	13C6
SA069299	ms6905-12	23h +1h nonCO2	BGP50uMGt Gn	pellet	13C6
SA069300	ms6905-11	23h +1h nonCO2	BGP50uMGt Gn	pellet	13C6
SA069297	ms6905-9	23h +1h nonCO2	BGP 50uMGt Gn	pellet	Unlabelled
SA069273	MS6735-8	23h + 1h nonCO2	BGPGt - 0.5mM Gn	pellet	13C6
SA069274	MS6735-7	23h + 1h nonCO2	BGPGt - 0.5mM Gn	pellet	13C6
SA069275	MS6735-6	23h + 1h nonCO2	BGPGt - 0.5mM Gn	pellet	13C6
SA069281	ms6983-03	23h + 1h nonCO2	BGPGt 0.5 mM Gn	pellet	13C6
SA069282	ms6983-04	23h + 1h nonCO2	BGPGt 0.5 mM Gn	pellet	13C6
SA069283	ms6983-02	23h + 1h nonCO2	BGPGt 0.5 mM Gn	pellet	13C6
SA069301	ms6905-3	23h +1h nonCO2	BGPGt 0.5mM Gn	pellet	13C6
SA069302	ms6905-2	23h +1h nonCO2	BGPGt 0.5mM Gn	pellet	13C6
SA069303	ms6905-4	23h +1h nonCO2	BGPGt 0.5mM Gn	pellet	13C6
SA069276	MS6735-5	23h + 1h nonCO2	BGPGt - 0.5mM Gn	pellet	Unlabelled
SA069284	ms6983-01	23h + 1h nonCO2	BGPGt 0.5 mM Gn	pellet	Unlabelled
SA069304	ms6905-1	23h +1h nonCO2	BGPGt 0.5mM Gn	pellet	Unlabelled
SA069289	ms6983-10	23h + 1h nonCO2	BGPGt 50 mM Gn	pellet	13C6
SA069290	ms6983-11	23h + 1h nonCO2	BGPGt 50 mM Gn	pellet	13C6
SA069291	ms6983-12	23h + 1h nonCO2	BGPGt 50 mM Gn	pellet	13C6
SA069292	ms6983-09	23h + 1h nonCO2	BGPGt 50 mM Gn	pellet	Unlabelled
SA069277	MS6735-10	23h + 1h nonCO2	BGPGt - 5mM Gn	pellet	13C6
SA069278	MS6735-12	23h + 1h nonCO2	BGPGt - 5mM Gn	pellet	13C6
SA069279	MS6735-11	23h + 1h nonCO2	BGPGt - 5mM Gn	pellet	13C6
SA069285	ms6983-06	23h + 1h nonCO2	BGPGt 5 mM Gn	pellet	13C6
SA069286	ms6983-07	23h + 1h nonCO2	BGPGt 5 mM Gn	pellet	13C6
SA069287	ms6983-08	23h + 1h nonCO2	BGPGt 5 mM Gn	pellet	13C6
SA069305	ms6905-7	23h +1h nonCO2	BGPGt 5mM Gn	pellet	13C6
SA069306	ms6905-8	23h +1h nonCO2	BGPGt 5mM Gn	pellet	13C6
SA069307	ms6905-6	23h +1h nonCO2	BGPGt 5mM Gn	pellet	13C6
SA069280	MS6735-9	23h + 1h nonCO2	BGPGt - 5mM Gn	pellet	Unlabelled
SA069288	ms6983-05	23h + 1h nonCO2	BGPGt 5 mM Gn	pellet	Unlabelled
SA069308	ms6905-5	23h +1h nonCO2	BGPGt 5mM Gn	pellet	Unlabelled
SA069293	MS6735-3	23h + 1h nonCO2	BGPGt	pellet	13C6
SA069294	MS6735-2	23h + 1h nonCO2	BGPGt	pellet	13C6
SA069295	MS6735-4	23h + 1h nonCO2	BGPGt	pellet	13C6
SA069296	MS6735-1	23h + 1h nonCO2	BGPGt	pellet	Unlabelled
SA069309	ms6905-28	23h	E Med	Spent Sup	13C6
SA069310	ms6905-29	23h	E Med	Spent Sup	13C6
SA069311	ms6905-27	23h	E Med	Spent Sup	13C6
SA069312	ms6983-29	23h	E Med	Spent Sup	13C6
SA069313	MS6735-36	23h	E Med	Spent Sup	13C6
SA069314	MS6735-37	23h	E Med	Spent Sup	13C6
SA069315	ms6983-31	23h	E Med	Spent Sup	13C6
SA069316	MS6735-38	23h	E Med	Spent Sup	13C6
SA069317	MS6735-35	23h	E Med	Spent Sup	13C6
SA069318	MS6735-34	23h	E Med	Spent Sup	13C6
SA069319	ms6983-30	23h	E Med	Spent Sup	13C6
SA069320	MS6735-33	23h	E Med	Spent Sup	13C6
SA069321	ms6905-26	23h	E Med	Spent Sup	Unlabelled
SA069322	MS6735-32	23h	E Med	Spent Sup	Unlabelled

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Collection:

Collection ID:	CO001068
Collection Summary:	4,000,000 CLOD cells collected by methanol scraping. 1ml sup also collected. There was no tracer element added to the sample. Also methanol extarction was done in ice. After the extraction samples were kept in dry ice and delivered to core.
Sample Type:	astrocytes

Treatment:

Treatment ID:	TR001088
Treatment Summary:	Murine derived pure cortical astrocytes were plated at 4x10^6 density in 10cm petri dish and grown in astro growth medium (AGM) for 2-3 days until it reaches the 90% confluency. Day before the treatment with glutamine or glutamate AGM was changed to serum free “E” media. In case of isotomer analysis 40% of glucose was switched with U13C6 (glucose) and added to serum free “E” media. On the day of treatment first the spent media was collected and then washed with base media. After washing, astrocytes were exposed to different ratio of glutamate: glutamine for 1h under non-CO2 condition. 1h spent media was collected. After washing three times with phosphate buffer saline cell pellet was collected in ice cold 100% methanol by scrapping. All the samples were immediately transferred to -70C for future analysis. E media composition: DMEM (w/o phenol red)+ 1% PenStrep+ 10% FBS+ Glutamate+ Glucose Abbreviation: Gn-Glutamine Gt-Glutamate B- Base media G- Glucose P- Sodium pyruvate

Treatment ID:

TR001088

Treatment Summary:

Murine derived pure cortical astrocytes were plated at 4x10^6 density in 10cm petri dish and grown in astro growth medium (AGM) for 2-3 days until it reaches the 90% confluency. Day before the treatment with glutamine or glutamate AGM was changed to serum free “E” media. In case of isotomer analysis 40% of glucose was switched with U13C6 (glucose) and added to serum free “E” media. On the day of treatment first the spent media was collected and then washed with base media. After washing, astrocytes were exposed to different ratio of glutamate: glutamine for 1h under non-CO2 condition. 1h spent media was collected. After washing three times with phosphate buffer saline cell pellet was collected in ice cold 100% methanol by scrapping. All the samples were immediately transferred to -70C for future analysis. E media composition: DMEM (w/o phenol red)+ 1% PenStrep+ 10% FBS+ Glutamate+ Glucose Abbreviation: Gn-Glutamine Gt-Glutamate B- Base media G- Glucose P- Sodium pyruvate

Sample Preparation:

Sampleprep ID:	SP001081
Sampleprep Summary:	13C6 isotopomer analysis of Astrocyte We intend to test the experimental hypothesis that stimulation of primary murine astrocytes with patient-derived NMO IgG will drive a metabolic shift marked by alterations in cellular levels of glutamate and glutamine. To do so we will use isotopic tracing to measure glutamate and glutamine levels in cell extracts following stimulation with NMO IgG. The cell preparations, patient-derived antibody preps, isotopic tracer incubations, and cell stimulations will be performed in the PI’s lab.

Sampleprep ID:

SP001081

Sampleprep Summary:

13C6 isotopomer analysis of Astrocyte We intend to test the experimental hypothesis that stimulation of primary murine astrocytes with patient-derived NMO IgG will drive a metabolic shift marked by alterations in cellular levels of glutamate and glutamine. To do so we will use isotopic tracing to measure glutamate and glutamine levels in cell extracts following stimulation with NMO IgG. The cell preparations, patient-derived antibody preps, isotopic tracer incubations, and cell stimulations will be performed in the PI’s lab.

Combined analysis:

Analysis ID	AN001696
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890B
Column	Agilent HP5-MS (30m × 0.25mm, 0.25 um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5977A
Ion Mode	POSITIVE
Units	Enrichment

Chromatography:

Chromatography ID:	CH001195
Instrument Name:	Agilent 7890B
Column Name:	Agilent HP5-MS (30m × 0.25mm, 0.25 um)
Chromatography Type:	GC

MS:

MS ID:	MS001571
Analysis ID:	AN001696
Instrument Name:	Agilent 5977A
Instrument Type:	Single quadrupole
MS Type:	EI
Ion Mode:	POSITIVE