Summary of Study ST003737
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 PR002323. The data can be accessed directly via it's Project DOI: 10.21228/M8C24H 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 ID | ST003737 |
| Study Title | PILRA regulates microglial neuroinflammation and lipid metabolism as a candidate therapeutic target for Alzheimer’s disease |
| Study Type | In vitro study |
| Study Summary | The Alzheimer’s disease (AD) human genetic landscape identified microglia as a key disease-modifying brain cell type. A common loss-of-function coding variant in paired immunoglobulin-like type 2 receptor alpha (PILRA) associated with reduced AD risk, was found enriched in a cohort of healthy centenarians and rescued APOE4 AD risk, however the mechanisms underlying protection are undefined. Here we identify biological functions of PILRA, an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor, in human iPSC-derived microglia (iMG) and chimeric AD mice. CRISPR-mediated PILRA knockout (KO) in iMG rescued ApoE4-induced immunometabolic deficits. Moreover, loss of PILRA confers a signature of metabolic resilience in microglia with increased mitochondrial capacity in tandem with elevated antioxidants, reduced ROS and toxic lipid species. Additionally, PILRA KO iMG exhibit improved lysosomal degradation, enhanced migration, and attenuated cytokine responses. We show PPAR and STAT1/3 act as master regulators that mediate downstream signaling and regulate PILRA-dependent microglial functions. Importantly, AD mice transplanted with human PILRA KO microglia showed reduced amyloid pathology and rescued levels of synaptic markers. Finally, we identify a high-affinity human PILRA-specific antagonist antibody that phenocopies PILRA KO. Together, these findings suggest a therapeutic approach to modulate microglial immunometabolism by inhibiting PILRA, thus identifying a pharmacologically tractable target for AD. |
| Institute | Denali Therapeutics |
| Last Name | Suh |
| First Name | Jung |
| Address | 161 Oyster Point Blvd, South San Francisco, CA 94080 |
| suh@dnli.com | |
| Phone | +1 06507973837 |
| Submit Date | 2025-02-14 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-03-10 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002323 |
| Project DOI: | doi: 10.21228/M8C24H |
| Project Title: | PILRA regulates microglial neuroinflammation and lipid metabolism as a candidate therapeutic target for Alzheimer’s disease |
| Project Type: | Cellular studies |
| Project Summary: | The Alzheimer’s disease (AD) human genetic landscape identified microglia as a key disease-modifying brain cell type. A common loss-of-function coding variant in paired immunoglobulin-like type 2 receptor alpha (PILRA) associated with reduced AD risk, was found enriched in a cohort of healthy centenarians and rescued APOE4 AD risk, however the mechanisms underlying protection are undefined. Here we identify biological functions of PILRA, an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor, in human iPSC-derived microglia (iMG) and chimeric AD mice. CRISPR-mediated PILRA knockout (KO) in iMG rescued ApoE4-induced immunometabolic deficits. Moreover, loss of PILRA confers a signature of metabolic resilience in microglia with increased mitochondrial capacity in tandem with elevated antioxidants, reduced ROS and toxic lipid species. Additionally, PILRA KO iMG exhibit improved lysosomal degradation, enhanced migration, and attenuated cytokine responses. We show PPAR and STAT1/3 act as master regulators that mediate downstream signaling and regulate PILRA-dependent microglial functions. Importantly, AD mice transplanted with human PILRA KO microglia showed reduced amyloid pathology and rescued levels of synaptic markers. Finally, we identify a high-affinity human PILRA-specific antagonist antibody that phenocopies PILRA KO. Together, these findings suggest a therapeutic approach to modulate microglial immunometabolism by inhibiting PILRA, thus identifying a pharmacologically tractable target for AD. |
| Institute: | Denali Therapeutics |
| Last Name: | Suh |
| First Name: | Jung |
| Address: | 161 Oyster Point Blvd, South San Francisco, CA 94080 |
| Email: | suh@dnli.com |
| Phone: | +1 6507973837 |
Subject:
| Subject ID: | SU003869 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | WT, PILRA KO |
| Cell Biosource Or Supplier: | University of California Irvine |
| Cell Strain Details: | Induced pluripotent stem cells derived microglia cells |
| Cell Primary Immortalized: | no |
| Cell Counts: | 500,000 |
| Species Group: | human |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | genotype | treatment | Sample source |
|---|---|---|---|---|
| SA407796 | HSA-000033326 | PILRAKO | LPS | iPSC microglia |
| SA407797 | HSA-000033338 | PILRAKO | LPS | iPSC microglia |
| SA407798 | HSA-000033328 | PILRAKO | LPS | iPSC microglia |
| SA407799 | HSA-000033339 | PILRAKO | LPS | iPSC microglia |
| SA407800 | HSA-000033340 | PILRAKO | LPS | iPSC microglia |
| SA407801 | HSA-000033327 | PILRAKO | LPS | iPSC microglia |
| SA407802 | HSA-000033335 | PILRAKO | Vehicle | iPSC microglia |
| SA407803 | HSA-000033324 | PILRAKO | Vehicle | iPSC microglia |
| SA407804 | HSA-000033336 | PILRAKO | Vehicle | iPSC microglia |
| SA407805 | HSA-000033323 | PILRAKO | Vehicle | iPSC microglia |
| SA407806 | HSA-000033337 | PILRAKO | Vehicle | iPSC microglia |
| SA407807 | HSA-000033325 | PILRAKO | Vehicle | iPSC microglia |
| SA407808 | HSA-000033320 | WT | LPS | iPSC microglia |
| SA407809 | HSA-000033333 | WT | LPS | iPSC microglia |
| SA407810 | HSA-000033321 | WT | LPS | iPSC microglia |
| SA407811 | HSA-000033332 | WT | LPS | iPSC microglia |
| SA407812 | HSA-000033334 | WT | LPS | iPSC microglia |
| SA407813 | HSA-000033322 | WT | LPS | iPSC microglia |
| SA407814 | HSA-000033330 | WT | Vehicle | iPSC microglia |
| SA407815 | HSA-000033329 | WT | Vehicle | iPSC microglia |
| SA407816 | HSA-000033317 | WT | Vehicle | iPSC microglia |
| SA407817 | HSA-000033319 | WT | Vehicle | iPSC microglia |
| SA407818 | HSA-000033318 | WT | Vehicle | iPSC microglia |
| SA407819 | HSA-000033331 | WT | Vehicle | iPSC microglia |
| Showing results 1 to 24 of 24 |
Collection:
| Collection ID: | CO003862 |
| Collection Summary: | WT and PILRA KO microglia cells (500,000) were pelleted, and washed 1x with 0.9% sodium chloride and collected in collected in Lobind 1.5 mL Eppendorf tubes and snap frozen. |
| Sample Type: | iPSC cells |
| Storage Conditions: | -80℃ |
| Collection Vials: | Lobind 1.5 mL Eppendorf tubes |
| Storage Vials: | Lobind 1.5 mL Eppendorf tubes |
Treatment:
| Treatment ID: | TR003878 |
| Treatment Summary: | WT and PILRA KO microglia cells were cultured in iPSC-complete media without TGFBeta and plated on matrigel-coated 12-well plate. Wells were supplemented with PBS (vehicle) or endotoxin (LPS) (100ng/ml, Sigma-Aldrich, 055-B5) for 48 hours and harvested for metabolite extraction. |
| Treatment Compound: | endotoxin (LPS; Sigma-Aldrich, 055-B5) |
| Treatment Dose: | 100 ng/mL |
| Treatment Doseduration: | 48 hours |
| Treatment Vehicle: | PBS |
Sample Preparation:
| Sampleprep ID: | SP003875 |
| Sampleprep Summary: | On the day of lipid and metabolite extraction, cell pellets were reconstituted with 400 μL MS grade methanol spiked with internal standards.Samples were vortexed and volumes were adjusted to 800 μL with MS grade H2O, vortexed again, then 600 μL tert-butyl methyl ether (MTBE) was added, vortexed, then centrifuged at 21,000 g for 10 min at 4C. The two phases generated by centrifugation were separated. Each phase (top: non-polar lipids, bottom: polar metabolites) was transferred to glass vials and dried overnight using a Genevac EZ3. Non-polar lipids were resuspended MS grade methanol and polar metabolites were resuspended in 90% methanol:water mixture. |
| Processing Storage Conditions: | On ice |
| Extract Storage: | -20℃ |
Combined analysis:
| Analysis ID | AN006132 | AN006133 | AN006134 | AN006135 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | HILIC | Ion exchange | Reversed phase | Reversed phase |
| Chromatography system | Agilent 1290 Infinity II | Agilent 1290 Infinity II | Agilent 1290 Infinity II | Agilent 1290 Infinity II |
| Column | Waters ACQUITY UPLC BEH Amide (150 x 2.1 mm, 1.7 μm) | Imtakt Intrada Organic Acid (150 x 2 mm, 3 μm) | Waters ACQUITY UPLC BEH Amide (100 x 2.1 mm, 1.7 μm) | Waters ACQUITY UPLC BEH Amide (100 x 2.1 mm, 1.7 μm) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | Triple quadrupole | Triple quadrupole | Triple quadrupole | Triple quadrupole |
| MS instrument name | ABI Sciex Triple Quad 6500+ | ABI Sciex 6500+ QTrap | ABI Sciex 6500+ QTrap | ABI Sciex 6500+ QTrap |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE | NEGATIVE |
| Units | area | area | area | area |
Chromatography:
| Chromatography ID: | CH004656 |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Waters ACQUITY UPLC BEH Amide (150 x 2.1 mm, 1.7 μm) |
| Column Temperature: | 55°C |
| Flow Gradient: | 0.0-8.0 min from 45% B to 99% B, 8.0-9.0 min at 99% B, 9.0-9.1 min to 45% B, and 9.1-10.0 min at 45% B. |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 60:40 acetonitrile/water (v/v) with 10 mM ammonium formate + 0.1% formic acid |
| Solvent B: | 90:10 isopropyl alcohol/acetonitrile (v/v) with 10 mM ammonium formate + 0.1% formic acid |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH004657 |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Imtakt Intrada Organic Acid (150 x 2 mm, 3 μm) |
| Column Temperature: | 60°C |
| Flow Gradient: | 0.0–1.0 min at 0% B; 1.0–7.0 min to 100% B; 7.1 at 0% B; and 7.1-10 min at 0% B. |
| Flow Rate: | 0.2 mL/min |
| Solvent A: | 10% acetonitrile/90% water; 0.1% formic acid |
| Solvent B: | 10% acetonitrile/90% water; 100mM ammonium formate |
| Chromatography Type: | Ion exchange |
| Chromatography ID: | CH004658 |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Waters ACQUITY UPLC BEH Amide (100 x 2.1 mm, 1.7 μm) |
| Column Temperature: | 55°C |
| Flow Gradient: | 0.0-8.0 min from 45% B to 99% B, 8.0-9.0 min at 99% B, 9.0-9.1 min to 45% B, and 9.1-10.0 min at 45% B. |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 60% acetonitrile/40% water; 10 mM ammonium formate; 0.1% formic acid |
| Solvent B: | 90% isopropyl alcohol/10% acetonitrile; 10 mM ammonium formate; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH004659 |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Waters ACQUITY UPLC BEH Amide (100 x 2.1 mm, 1.7 μm) |
| Column Temperature: | 55°C |
| Flow Gradient: | 0.0-8.0 min from 45% B to 99% B, 8.0-9.0 min at 99% B, 9.0-9.1 min to 45% B, and 9.1-10.0 min at 45% B |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 60% acetonitrile/40% water; 10 mM ammonium acetate; 0.1% acetic acid |
| Solvent B: | 90% isopropyl alcohol/10% acetonitrile; 10 mM ammonium acetate; 0.1% acetic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS005838 |
| Analysis ID: | AN006132 |
| Instrument Name: | ABI Sciex Triple Quad 6500+ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | curtain gas at 30 psi; collision gas was set at medium; ion spray voltage at 5500 V; temperature at 600°C; ion source Gas 1 at 50 psi; ion source Gas 2 at 60 psi; entrance potential at 10 V; and collision cell exit potential at 12.5 V. |
| Ion Mode: | POSITIVE |
| MS ID: | MS005839 |
| Analysis ID: | AN006133 |
| Instrument Name: | ABI Sciex 6500+ QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | curtain gas at 40 V; collision gas was set at medium; ion spray voltage at -4500 V; temperature at 600°C; ion source Gas 1 at 50 psi; ion source Gas 2 at 60 psi; entrance potential at -10 V; and collision cell exit potential at -15.0 V. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS005840 |
| Analysis ID: | AN006134 |
| Instrument Name: | ABI Sciex 6500+ QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Curtain gas at 40 psi (positive mode); collision gas was set at medium; ion spray voltage at 5500 V ; temperature at 250°C ; ion source Gas 1 at 55 psi; ion source Gas 2 at 60 psi; entrance potential at 10 V ; and collision cell exit potential at 12.5 V. Quantification was performed using MultiQuant 3.02 (Sciex). |
| Ion Mode: | POSITIVE |
| MS ID: | MS005841 |
| Analysis ID: | AN006135 |
| Instrument Name: | ABI Sciex 6500+ QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Curtain gas at 40 psi (positive mode); collision gas was set at medium; ion spray voltage at -4500 V ; temperature at 250°C ; ion source Gas 1 at 55 psi; ion source Gas 2 at 60 psi; entrance potential at - 10 V; and collision cell exit potential at -15 V. Quantification was performed using MultiQuant 3.02 (Sciex). |
| Ion Mode: | NEGATIVE |
