Summary of study ST001820

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 PR001150. The data can be accessed directly via it's Project DOI: 10.21228/M82690 This work is supported by NIH grant, U2C- DK119886.

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Study IDST001820
Study TitleWT neurons treated with APOE3/3 and APOE4/4 ACM
Study SummaryWe performed a targeted lipidomic analysis on WT neurons treated with astrocyte conditioned media (ACM) from APOE3/3 or APOE4/4 astrocytes.
Institute
Columbia University
Last NameNuriel
First NameTal
Address630 W 168th St., P&S 12-430
Emailtn2283@cumc.columbia.edu
Phone2123045683
Submit Date2021-06-02
Analysis Type DetailLC-MS
Release Date2021-06-10
Release Version1
Tal Nuriel Tal Nuriel
https://dx.doi.org/10.21228/M82690
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001150
Project DOI:doi: 10.21228/M82690
Project Title:APOE4-associated differences in lipidomics signatures in mouse brain and cultured neurons
Project Type:Lipidomics analysis
Project Summary:Apolipoprotein E ε4 (APOE4) is the primary genetic risk factor for the late-onset form of Alzheimer's disease (AD). Although the reason for this association is not completely understood, researchers have uncovered numerous effects of APOE4 expression on AD-relevant brain processes, including amyloid beta (Aβ) accumulation, lipid metabolism, endosomal-lysosomal processing and bioenergetics. In this study, we aimed to determine the effect of APOE4 allelic dosage on regional brain lipid composition in aged mice, as well as in cultured neurons. We performed a targeted lipidomic analysis on the entorhinal cortex (EC) and primary visual cortex (PVC) from 14–15 month-old APOE3/3, APOE3/4, and APOE4/4 targeted replacement mice, as well as on WT neurons cultured with conditioned media from APOE3/3 or APOE4/4 astrocytes. Our results reveal that the EC possesses increased susceptibility to APOE4-associated lipid alterations compared to the PVC. In the EC, APOE4 expression showed a dominant effect in decreasing diacylglycerol (DAG) levels, and a semi-dominant additive effect in the upregulation of multiple ceramide, glycosylated sphingolipid and bis(monoacylglycerol)phosphate (BMP) species, lipids known to accumulate as a result of endosomal-lysosomal dysfunction and defective lysosomal clearance. Neurons treated with conditioned media from APOE4 vs. APOE3 astrocytes also showed similar alterations of DAG and BMP species as those observed in the mouse EC. Our results suggest that APOE4 expression differentially modulates regional and neuronal lipid signatures, which may underlie the increased susceptibility of EC-localized neurons to AD pathology.
Institute:Columbia University
Last Name:Nuriel
First Name:Tal
Address:630 W 168th St., P&S 12-430
Email:tn2283@cumc.columbia.edu
Phone:2123045683

Subject:

Subject ID:SU001897
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Not applicable

Factors:

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

mb_sample_id local_sample_id Condition
SA1691058APOE3/3 ACM
SA1691069APOE3/3 ACM
SA16910712APOE3/3 ACM
SA1691087APOE3/3 ACM
SA16910911APOE3/3 ACM
SA16911010APOE3/3 ACM
SA16911115APOE4/4 ACM
SA16911216APOE4/4 ACM
SA16911317APOE4/4 ACM
SA16911418APOE4/4 ACM
SA16911514APOE4/4 ACM
SA16911613APOE4/4 ACM
SA1691175No ACM
SA1691182No ACM
SA1691191No ACM
SA1691204No ACM
SA1691213No ACM
SA1691226No ACM
Showing results 1 to 18 of 18

Collection:

Collection ID:CO001890
Collection Summary:Astrocyte conditioned media (ACM) was obtained from immortalized astrocyte cell lines (a gift from Dr. David Holtzman) that were originally generated from primary astrocytes from P1-2 pups of APOE targeted replacement mice (42). The immortalized astrocytes were conditioned with Neurobasal media supplemented with B27, Glutamax-I, Normocin and 1% penicillin/streptomycin for 24 hours. This ACM was then collected, stored at -80ºC, and thawed prior to use. WT primary cortical neuronal cultures were obtained from embryonic day 17 (E17) C57Bl/6 embryos. Briefly, pregnant mice were euthanized by cervical dislocation and the embryo brains extracted. The meninges were carefully stripped off and the cortices dissected out. The cortices were then enzymatically dissociated in 0.25% Trypsin-EDTA and resuspended in Neurobasal media supplemented with B27, Glutamax-I, Normocin and 1% penicillin/streptomycin. Dissociated cells were counted and 300K neurons per well were plated directly into ACM in poly-D-lysine (PDL)-coated 6-well plates. ACM-treated neurons were then incubated at 37⁰C for 7 days in a humidified chamber with 5% CO2, with 50% media exchange (with newly thawed ACM) once every 3 days.
Sample Type:Neurons

Treatment:

Treatment ID:TR001910
Treatment Summary:WT neurons were incubated for 7 days in either no ACM, APOE3/3 ACM, or APOE4/4 ACM.

Sample Preparation:

Sampleprep ID:SP001903
Sampleprep Summary:Lipid and small-molecule metabolite extraction was performed using a methyl tert-butyl ether (MTBE)/methanol extraction protocol. Briefly, harvested neurons were homogenized in ice-cold methanol. Following homogenization, samples were incubated in 1200 ul of MTBE for 1 hr at room temperature to separate organic-soluble lipids from aqueous-soluble lipids and other small-molecules. Finally, 360 ul of ultrapure water was added (for a final ratio of 3:1:0.9 MTBE:methanol:water) to resolve the two liquid phases, and each samples were centrifuged at 10,000 x g for 10 min. For this experiment, the upper organic phase was collected from each sample and stored in a separate tube, and the remaining protein pellets were resuspended in 25 mM ammonium bicarbonate, pH 8, with 2.5% SDS. A BCA protein assay was performed on each protein fraction, and the organic phase was normalized to their protein concentration equivalent with 100% methanol. All samples were then stored at -80°C prior to analysis.

Combined analysis:

Analysis ID AN002954 AN002955 AN002956
Analysis type MS MS MS
Chromatography type Reversed phase Reversed phase Normal phase
Chromatography system Agilent 1260 Agilent 1260 Agilent 1260
Column Agilent Eclipse XDB-C18 (100 x 3.0mm) Agilent Eclipse XDB-C18 (100 x 3.0mm) Phenomenex Luna NH2 (150 x 2.1mm, 3um)
MS Type ESI ESI ESI
MS instrument type Triple quadrupole Triple quadrupole Triple quadrupole
MS instrument name Agilent 6490 QQQ Agilent 6490 QQQ Agilent 6490 QQQ
Ion Mode NEGATIVE POSITIVE POSITIVE
Units area under the curve area under the curve area under the curve

Chromatography:

Chromatography ID:CH002188
Chromatography Summary:Reverse phase
Instrument Name:Agilent 1260
Column Name:Agilent Eclipse XDB-C18 (100 x 3.0mm)
Chromatography Type:Reversed phase
  
Chromatography ID:CH002189
Chromatography Summary:Normal phase
Instrument Name:Agilent 1260
Column Name:Phenomenex Luna NH2 (150 x 2.1mm, 3um)
Chromatography Type:Normal phase

MS:

MS ID:MS002744
Analysis ID:AN002954
Instrument Name:Agilent 6490 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Lipid profiling was performed using an Agilent 1260 HPLC coupled to an Agilent 6490 triple quadrupole (QQQ) mass spectrometer (41). Each sample was run through three separate chromatographic conditions (reverse-phase negative mode, reverse-phase positive mode and normal-phase positive mode) for the effective quantification of over 337 distinct lipids from over 28 lipid subclasses, as previously described (41). Individual lipid species were measured by multiple reaction monitoring transitions.
Ion Mode:NEGATIVE
  
MS ID:MS002745
Analysis ID:AN002955
Instrument Name:Agilent 6490 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Lipid profiling was performed using an Agilent 1260 HPLC coupled to an Agilent 6490 triple quadrupole (QQQ) mass spectrometer (41). Each sample was run through three separate chromatographic conditions (reverse-phase negative mode, reverse-phase positive mode and normal-phase positive mode) for the effective quantification of over 337 distinct lipids from over 28 lipid subclasses, as previously described (41). Individual lipid species were measured by multiple reaction monitoring transitions.
Ion Mode:POSITIVE
  
MS ID:MS002746
Analysis ID:AN002956
Instrument Name:Agilent 6490 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
MS Comments:Lipid profiling was performed using an Agilent 1260 HPLC coupled to an Agilent 6490 triple quadrupole (QQQ) mass spectrometer (41). Each sample was run through three separate chromatographic conditions (reverse-phase negative mode, reverse-phase positive mode and normal-phase positive mode) for the effective quantification of over 337 distinct lipids from over 28 lipid subclasses, as previously described (41). Individual lipid species were measured by multiple reaction monitoring transitions.
Ion Mode:POSITIVE
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