{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001035","ANALYSIS_ID":"AN001696","VERSION":"1","CREATED_ON":"August 15, 2018, 12:35 pm"},

"PROJECT":{"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"},

"STUDY":{"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"},

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{
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{
"Subject ID":"-",
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}
],
"COLLECTION":{"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_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"},

"SAMPLEPREP":{"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."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"GC","INSTRUMENT_NAME":"Agilent 7890B","COLUMN_NAME":"Agilent HP5-MS (30m × 0.25mm, 0.25 um)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Agilent 5977A","INSTRUMENT_TYPE":"Single quadrupole","MS_TYPE":"EI","ION_MODE":"POSITIVE"},

"MS_METABOLITE_DATA":{
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}