Summary of Study ST002450

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 PR001580. The data can be accessed directly via it's Project DOI: 10.21228/M8G711 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 IDST002450
Study TitleAPOE modulates microglial immunometabolism in response to age, amyloid pathology, and inflammatory challenge (Part 1 of 3)
Study SummaryThe E4 allele of Apolipoprotein E (APOE) is associated with both metabolic dysfunction and a heightened pro-inflammatory response – two findings that may be intrinsically linked through the concept of immunometabolism. Here, we combined bulk, single-cell, and spatial transcriptomics with cell-specific and spatially resolved metabolic analyses to systematically address the role of APOE across age, neuroinflammation, and AD pathology. RNAseq highlighted immunometabolic changes across the APOE4 glial transcriptome, specifically in subsets of metabolically distinct microglia enriched in the E4 brain during aging or following an inflammatory challenge. E4 microglia display increased Hif1α expression, a disrupted TCA cycle, and are inherently pro-glycolytic, while spatial transcriptomics and MALDI mass spectrometry imaging highlight an E4-specific response to amyloid that is characterized by widespread alterations in lipid metabolism. Taken together, our findings emphasize a central role for APOE in regulating microglial immunometabolism.
Institute
University of Kentucky, Department of Physiology
Last NameDevanney
First NameNicholas
AddressPhysiology, 760 Press Ave, Healthy Kentucky Research Bldg, Rm152, Lexington, Kentucky, 40508, USA
EmailNicholas.Devanney@uky.edu
Phone8593238083
Submit Date2022-09-20
Raw Data AvailableYes
Raw Data File Type(s)cdf
Analysis Type DetailGC-MS
Release Date2023-01-25
Release Version1
Nicholas Devanney Nicholas Devanney
https://dx.doi.org/10.21228/M8G711
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001580
Project DOI:doi: 10.21228/M8G711
Project Title:APOE modulates microglial immunometabolism in response to age, amyloid pathology, and inflammatory challenge
Project Summary:The E4 allele of Apolipoprotein E (APOE) is associated with both metabolic dysfunction and a heightened pro-inflammatory response – two findings that may be intrinsically linked through the concept of immunometabolism. Here, we combined bulk, single-cell, and spatial transcriptomics with cell-specific and spatially resolved metabolic analyses to systematically address the role of APOE across age, neuroinflammation, and AD pathology. RNAseq highlighted immunometabolic changes across the APOE4 glial transcriptome, specifically in subsets of metabolically distinct microglia enriched in the E4 brain during aging or following an inflammatory challenge. E4 microglia display increased Hif1α expression, a disrupted TCA cycle, and are inherently pro-glycolytic, while spatial transcriptomics and MALDI mass spectrometry imaging highlight an E4-specific response to amyloid that is characterized by widespread alterations in lipid metabolism. Taken together, our findings emphasize a central role for APOE in regulating microglial immunometabolism.
Institute:University of Kentucky, Department of Physiology
Last Name:Devanney
First Name:Nicholas
Address:Physiology, 760 Press Ave, Healthy Kentucky Research Bldg, Rm152, Lexington, Kentucky, 40508, USA
Email:Nicholas.Devanney@uky.edu
Phone:8593238083
Project Comments:Part 2 of 3

Subject:

Subject ID:SU002539
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:Primary microglia isolated from APOE-targeted replacement mice homozygous for human E3 (B6.129P2-Apoe^tm2(APOE*3)Mae N8, Taconic #1548-F) or human E4 (B6.129P2- Apoe^tm3(APOE*4)Mae N8, Taconic #1549-F) alleles
Age Or Age Range:P0-P3
Gender:Pooled

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype
SA24501516_3_5E3
SA24501616_3_6E3
SA24501716_3_4E3
SA24501816_3_3_1E3
SA24501916_3_2E3
SA245020MG14_1E3
SA2450213_3E3
SA245022MG14_2E3
SA245023MG16_E3_1_1E3
SA245024MG16_E3_2E3
SA245025MG16_E3_1E3
SA245026MG15_3E3
SA245027MG15_2E3
SA24502815_3_3E3
SA24502916_3_3_2E3
SA2450303_7E3
SA2450313_8E3
SA2450323_9E3
SA24503315_3_2E3
SA2450343_5E3
SA2450353_6E3
SA2450363_4E3
SA245037MG16_E4_4E4
SA2450384_1E4
SA245039MG16_E4_2E4
SA245040MG15_4E4
SA245041MG16_E4_1E4
SA245042MG15_5E4
SA2450434_2E4
SA24504416_4_6_1E4
SA2450454_7E4
SA2450464_6E4
SA2450474_8E4
SA24504815_4_5E4
SA24504915_4_4E4
SA2450504_5E4
SA2450514_4E4
SA24505216_4_5_1E4
SA24505316_4_5_2E4
SA24505416_4_4E4
SA24505516_4_3E4
SA2450564_3E4
SA24505716_4_6_2E4
Showing results 1 to 43 of 43

Collection:

Collection ID:CO002532
Collection Summary:Primary microglia were plated at 7x106 cells/well in 6-well plates (VWR #10062-894) and incubated at 5% CO2 37°C. Upon reaching confluence, cells were removed from the incubator washed with warm 0.9% NaCl solution. Culture plates were placed on a bed of crushed dry ice and 1mL of ice cold 50% methanol (HPLC-grade, Sigma #A456-4) was added to quench cellular metabolic activity followed by a 10 minute incubation at -80°C to ensure cell lysis. After removing from the freezer, cells were detached with a cell scraper (VWR #10062-906) and the entire contents collected into a microcentrifuge tube, vortexed briefly, and placed on ice until all samples were collected. The tubes were then placed on a Disruptor Genie Cell Disruptor Homogenizer (Scientific Industries) for 5 min at 3,000 rpm. Tubes were then centrifuged at 20,000 x g for 10 min at 4 °C. The supernatant containing polar metabolites was isolated to a new tube and stored at -80C, and the resulting pellet was briefly dried at 10-3 mbar using a CentriVap vacuum concentrator (LabConco) to evaporate remaining methanol, followed by determination of protein content via BCA assay (ThermoFisher #23225) to normalize metabolite concentrations to total protein amount of each sample.
Sample Type:Cultured cells
Collection Method:Ice-cold methanol was added to primary microglia cultures in 6-well plates after which cells were harvested using a cell scraper
Collection Location:University of Kentucky
Volumeoramount Collected:1ml
Storage Conditions:-80℃
Collection Tube Temp:-80

Treatment:

Treatment ID:TR002551
Treatment Summary:Cells were not treated, as we were just comparing APOE3/3 vs. APOE4/4 genotypes
Cell Growth Container:6-well plate
Cell Media:DMEM/F12 with 10% FBS, 10% LCCM supplement (see full methods in associated publication for preparation details), and 1% Penicillin/Streptomycin
Cell Envir Cond:37C, 5% CO2
Cell Harvesting:Cell scraper
Cell Pct Confluence:100
Cell Media Lastchanged:2-4 days prior to harvest

Sample Preparation:

Sampleprep ID:SP002545
Sampleprep Summary:The supernatant fraction containing polar metabolites was thawed gently on ice and dried at 10-3 mbar using a CentriVap vacuum concentrator (LabConco) to evaporate methanol. The dried polar metabolite pellet was derivatized by a two-step methoxyamine protocol first by addition of 70µL methoxyamine HCl (Sigma-Aldrich #226904-5G) in pyridine (20 mg/mL; Sigma-Aldrich #TS25730) to each pellet followed by 90 min dry heat incubation at 30°C. Samples were then centrifuged at 20,000 x g for 10 minutes after which 50µL of each sample was transferred to an amber V-shaped glass chromatography vial (Agilent #5184-3554) containing 80µL N-methyl-trimethylsilyl-trifluoroacetamide (MSTFA; ThermoFisher #TS48915) and gently vortexed followed by 30 min dry heat incubation at 37°C.
Extraction Method:50% methanol
Extract Storage:-80℃
Sample Derivatization:MSTFA

Combined analysis:

Analysis ID AN003998
Analysis type MS
Chromatography type GC
Chromatography system Agilent 8890
Column Agilent HP5-MS (30m x 0.25mm, 0.25 um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Agilent 5977
Ion Mode POSITIVE
Units Relative Abundance

Chromatography:

Chromatography ID:CH002952
Instrument Name:Agilent 8890
Column Name:Agilent HP5-MS (30m x 0.25mm, 0.25 um)
Chromatography Type:GC

MS:

MS ID:MS003746
Analysis ID:AN003998
Instrument Name:Agilent 5977
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:Metabolites were identified and quantified using DExSI v1.11 (https://github.com/DExSI/DExSI)
Ion Mode:POSITIVE
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