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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001820 |
| Study Title | WT neurons treated with APOE3/3 and APOE4/4 ACM |
| Study Summary | We 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 Name | Nuriel |
| First Name | Tal |
| Address | 630 W 168th St., P&S 12-430 |
| tn2283@cumc.columbia.edu | |
| Phone | 2123045683 |
| Submit Date | 2021-06-02 |
| Analysis Type Detail | LC-MS |
| Release Date | 2021-06-10 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
|---|---|---|
| SA169105 | 8 | APOE3/3 ACM |
| SA169106 | 9 | APOE3/3 ACM |
| SA169107 | 12 | APOE3/3 ACM |
| SA169108 | 7 | APOE3/3 ACM |
| SA169109 | 11 | APOE3/3 ACM |
| SA169110 | 10 | APOE3/3 ACM |
| SA169111 | 15 | APOE4/4 ACM |
| SA169112 | 16 | APOE4/4 ACM |
| SA169113 | 17 | APOE4/4 ACM |
| SA169114 | 18 | APOE4/4 ACM |
| SA169115 | 14 | APOE4/4 ACM |
| SA169116 | 13 | APOE4/4 ACM |
| SA169117 | 5 | No ACM |
| SA169118 | 2 | No ACM |
| SA169119 | 1 | No ACM |
| SA169120 | 4 | No ACM |
| SA169121 | 3 | No ACM |
| SA169122 | 6 | No 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 | HILIC |
| 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: | HILIC |
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 |
