Summary of Study ST003749

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 PR002333. The data can be accessed directly via it's Project DOI: 10.21228/M82K0C This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST003749
Study Title	Phosphatidylinositol remodeling by lysophospholipid acyltransferase 11 ensures embryonic radial glial cell integrity - part 3
Study Summary	To further examine whether PI(4,5)P2 is needed for the proper morphology of the Golgi apparatus in RGCs, we treated E12.5 hemispheres with UNC3230, a PIPKIγ inhibitor. UNC3230 significantly reduced the total amount of PI(4,5)P2, primarily affecting PI(4,5)P2 species containing arachidonic acid and its analogous fatty acid, Mead acid (20:3), while exerting minimal effect on other PI(4,5)P2 molecular species. UNC3230 treatment caused rounding of the Golgi apparatus, as observed in Mboat7 KO mice. We then administered UNC3230 into the lateral ventricle at E12.5 and immunostained E13.5 cortices with antibodies against PI(4,5)P2, GM130, E-cadherin, Sox2, and p-H3. UNC3230 administration caused a decreased level of PI(4,5)P2, a rounding of the Golgi apparatus, and reduced apical expression of E-cadherin in the ventricular zone, whereas the detachment of RGCs from the ventricular wall was not apparent. These results suggest that reduced PI(4,5)P2 levels cause rounding of the Golgi apparatus and decreased E-cadherin expression at the apical surface. We also treated E12.5 Mboat7 heterozygous hemispheres with Sevenin-1, an LPLAT11 inhibitor. Sevenin-1 treatment caused rounding of the Golgi apparatus in the Mboat7 heterozygous hemispheres, whereas it had no effect on the morphology of the Golgi apparatus in the Mboat7 KO hemispheres, highlighting the importance of LPLAT11 activity. SFC-MS analysis confirmed that Sevenin-1 decreased the amount of PI with arachidonic acid and the total amount of PI(4,5)P2.
Institute	University of Tokyo
Last Name	Nozomu
First Name	Kono
Address	Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, JAPAN
Email	nozomu@mol.f.u-tokyo.ac.jp
Phone	81-3-5841-4723
Submit Date	2025-01-25
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2025-03-14
Release Version	1

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Project:

Project ID:	PR002333
Project DOI:	doi: 10.21228/M82K0C
Project Title:	Phosphatidylinositol remodeling by lysophospholipid acyltransferase 11 ensures embryonic radial glial cell integrity
Project Summary:	Arachidonic acid, a vital polyunsaturated fatty acid in brain development, is enriched in phosphatidylinositol (PI). The arachidonic acyl chain in PI is introduced by lysophospholipid acyltransferase 11 (LPLAT11)/membrane-bound O-acyltransferase 7 (MBOAT7), the loss of which causes microcephaly in humans and mice. Here, we show that LPLAT11 deficiency impaired indirect neurogenesis in the developing neocortex, resulting in fewer layer II-V neurons. LPLAT11-deficient radial glial cells had defects in differentiation into intermediate progenitor cells and increased apoptosis. Prior to these anomalies, LPLAT11 deficiency caused a rounding of the Golgi apparatus, accompanied by impaired apical trafficking of E-cadherin, and deregulated apical detachment. Moreover, impaired PI acyl chain remodeling led to a decreased amount of PI(4,5)P2, leading to Golgi apparatus rounding. Thus, these results clarify the underlying mechanism of microcephaly by LPLAT11 deficiency and highlight the critical role of arachidonic acid in PI in the integrity of radial glial cells.
Institute:	University of Tokyo
Department:	Graduate School of Pharmaceutical Sciences
Laboratory:	Department of Health Chemistry
Last Name:	Kono
First Name:	Nozomu
Address:	Hongo 7-3-1,, Bunyo-ku, Tokyo, 113-0033, Japan
Email:	nozomu@mol.f.u-tokyo.ac.jp
Phone:	81-3-5841-4723

Subject:

Subject ID:	SU003882
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

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

mb_sample_id	local_sample_id	Gestational ages	Treatment
SA408346	DMSO (UNC3230 control)_1	E12.5	DMSO
SA408347	DMSO (UNC3230 control)_6	E12.5	DMSO
SA408348	DMSO (UNC3230 control)_5	E12.5	DMSO
SA408349	DMSO (UNC3230 control)_4	E12.5	DMSO
SA408350	DMSO (UNC3230 control)_3	E12.5	DMSO
SA408351	DMSO(Sevenin-1 control)_2	E12.5	DMSO
SA408352	DMSO(Sevenin-1 control)_1	E12.5	DMSO
SA408353	DMSO(Sevenin-1 control)_4	E12.5	DMSO
SA408354	DMSO(Sevenin-1 control)_5	E12.5	DMSO
SA408355	DMSO(Sevenin-1 control)_3	E12.5	DMSO
SA408356	DMSO (UNC3230 control)_2	E12.5	DMSO
SA408357	Sevenin-1_4	E12.5	Sevenin-1
SA408358	Sevenin-1_5	E12.5	Sevenin-1
SA408359	Sevenin-1_3	E12.5	Sevenin-1
SA408360	Sevenin-1_2	E12.5	Sevenin-1
SA408361	Sevenin-1_1	E12.5	Sevenin-1
SA408362	UNC3230_1	E12.5	UNC3230
SA408363	UNC3230_2	E12.5	UNC3230
SA408364	UNC3230_3	E12.5	UNC3230
SA408365	UNC3230_4	E12.5	UNC3230
SA408366	UNC3230_5	E12.5	UNC3230
SA408367	UNC3230_6	E12.5	UNC3230

Showing results 1 to 22 of 22

Showing results 1 to 22 of 22

Collection:

Collection ID:	CO003875
Collection Summary:	The brains of embryos were dissected in individual 35 mm dishes containing culture medium (Opti-MEM I with 20 mM glucose, 55 µM 2-mercaptoethanol, and 1% penicillin/streptomycin). Separate hemispheres were transferred to a new 35m dish containing culture medium using a P1000 Pipetman with a cut pipette tip and kept on ice until every dissection was completed. Hemispheres were transferred to an individual well of a 12-well plate containing 2 ml of culture medium. Plates were then placed on a nutator inside a 5% CO2 incubator for 24 h.
Sample Type:	Cultured hemisphere

Treatment:

Treatment ID:	TR003891
Treatment Summary:	N-(2-(Bicyclo[2.2.1]heptanyl)ethyl)-4-(2-(N-methylsulfamoyl)phenyl)piperazine-1-carboxamide (Sevenin-1) was added at 10 µM final concentration in the culture medium. UNC3230 was added at 0.25 µM final concentration in the culture medium.

Sample Preparation:

Sampleprep ID:	SP003888
Sampleprep Summary:	Cultured hemispheres were collected into a safe-lock poly-propylene tube (2ml) with 750 µl of ice-cold quench mix and sonicated. 170 µl of H2O was mixed and stored at -80 ºC before lipid extraction. The single-phase sample (a mixture of 170 µl of an aqueous sample, 10 ng of internal standards [17:0/20:4 PI, 17:0/20:4 PI4P, and 17:0/20:4 PI(4,5)P2], and 750 µl of quench mix) were mixed with 725 µl of CHCl3 and 170 µl of 2 M HCl, followed by vortex-mixing and centrifugation (1,500 g, 5 min at room temperature). The lower organic phase was collected into a new 2 ml safe-lock poly-propylene tube and mixed with 708 µl of pre-derivatization wash solution, followed by vortex-mixing and centrifugation (1,500 g, 3 min at room temperature). The lower phase was collected into another fresh tube and subjected to derivatization. Fifty µl trimethylsilyl diazomethane in hexane (2 M solution) was added to the lipid extracts (~1 ml), and after leaving to stand 10 min at room temperature, the reaction was quenched with 6 µl of acetic acid. Next, 700 µl post-derivatization wash solution was added to the mixture, followed by centrifugation (1,500 g, 3 min). The resultant lower phase was collected and rewashed with a 700 µl post-derivatization wash solution. Samples were temporarily stored at -80 ºC. Ninety µl of methanol and 10 µl of H2O were added to the final collected lower phase. The samples were dried up with a centrifugal evaporator. Samples were dissolved in 80 µl methanol, sonicated for 1 min, and 20 µl H2O was added. After centrifugation at 15,000 g for 30 min at 4 ºC, the supernatants were collected. To avoid degradation, the samples were stored at -80 ºC until use.

Sampleprep ID:

SP003888

Sampleprep Summary:

Cultured hemispheres were collected into a safe-lock poly-propylene tube (2ml) with 750 µl of ice-cold quench mix and sonicated. 170 µl of H2O was mixed and stored at -80 ºC before lipid extraction. The single-phase sample (a mixture of 170 µl of an aqueous sample, 10 ng of internal standards [17:0/20:4 PI, 17:0/20:4 PI4P, and 17:0/20:4 PI(4,5)P2], and 750 µl of quench mix) were mixed with 725 µl of CHCl3 and 170 µl of 2 M HCl, followed by vortex-mixing and centrifugation (1,500 g, 5 min at room temperature). The lower organic phase was collected into a new 2 ml safe-lock poly-propylene tube and mixed with 708 µl of pre-derivatization wash solution, followed by vortex-mixing and centrifugation (1,500 g, 3 min at room temperature). The lower phase was collected into another fresh tube and subjected to derivatization. Fifty µl trimethylsilyl diazomethane in hexane (2 M solution) was added to the lipid extracts (~1 ml), and after leaving to stand 10 min at room temperature, the reaction was quenched with 6 µl of acetic acid. Next, 700 µl post-derivatization wash solution was added to the mixture, followed by centrifugation (1,500 g, 3 min). The resultant lower phase was collected and rewashed with a 700 µl post-derivatization wash solution. Samples were temporarily stored at -80 ºC. Ninety µl of methanol and 10 µl of H2O were added to the final collected lower phase. The samples were dried up with a centrifugal evaporator. Samples were dissolved in 80 µl methanol, sonicated for 1 min, and 20 µl H2O was added. After centrifugation at 15,000 g for 30 min at 4 ºC, the supernatants were collected. To avoid degradation, the samples were stored at -80 ºC until use.

Combined analysis:

Analysis ID	AN006156
Chromatography ID	CH004676
MS ID	MS005861
Analysis type	MS
Chromatography type	Supercritical fluid chromatography
Chromatography system	Shimadzu Nexera UC/s
Column	ULTRON AF-HILIC-CD (250 x 2.0mm, 5.0um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex 4500 QTrap
Ion Mode	POSITIVE
Units	pmol/hemisphere

Chromatography:

Chromatography ID:	CH004676
Instrument Name:	Shimadzu Nexera UC/s
Column Name:	ULTRON AF-HILIC-CD (250 x 2.0mm, 5.0um)
Column Temperature:	10 ºC
Flow Gradient:	0-16 min: 5% B to 20% B; 16.01-18 min: 40% B; 18.01-22 min: 5% B
Flow Rate:	1.5 mL/min
Solvent A:	99.99% supercritical carbon dioxide
Solvent B:	2.5% water/97.5% methanol; 0.1% formic acid
Chromatography Type:	Supercritical fluid chromatography

MS:

MS ID:	MS005861
Analysis ID:	AN006156
Instrument Name:	ABI Sciex 4500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	The instrument parameters of QTRAP4500 for positive ion mode were as follows: curtain gas, 20 psi; ionspray voltage, 4500 V; temperature, 300 ºC; ion source gas 1, 18 psi; ion source gas 2, 20 psi. The specific detection of PI and phosphoinositide species was performed using MRM. 17:0/20:4 PI, 17:0/20:4 PI4P, and 17:0/20:4 PI4,5P2 ware used as internal standards. Analyst (SCIEX) was used for data acquisition and processing. MultiQuant (SCIEX) was used for data evaluation. Gaussian smoothing width was 2.0 points. Retention time of PI and PI(4,5)P2 is confirmed by measuring standards [17:0/20:4 PI, PI(3,4)P2, PI(3,5)P2, and PI(4,5)P2] before measuring samples. Peak areas of PI and PI(4,5)P2 were integrated from the ion chromatogram of PI and of PIP2, respectively, based on retention time, and isotopic correction was applied. The peak area of the lipid species was divided by the corresponding internal standard area, and the total amount of phosphoinositides per hemisphere was calculated from the ratio of injection volume to total sample volume.
Ion Mode:	POSITIVE