LRP1 regulates peroxisome biogenesis and cholesterol homeostasis in oligodendrocytes and is required for proper CNS myelin development and repair
- University of Michigan School of Medicine, United States
- University of Michigan Medical School, United States
- University of Rochester Medical Center, United States
Figures
Figure 1 with 2 supplements
In adult mice, global and OL-lineage selective ablation of Lrp1 attenuates white matter repair.
(a) Timeline in weeks indicating when Lrp1 ablation was induced (Lrp1flox/flox;CAG-CreERTM, Lrp1 iKO), lysolecithin (LPC) injected, and animals sacrificed. (b) Cartoon showing unilateral injection …
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Figure 1—source data 1
Raw data and detail statistical analysis report.
- https://doi.org/10.7554/eLife.30498.005
Figure 1—figure supplement 1
Generation of Lrp1 global iKO mice.
(a) Lrp1 wildtype (wt) and conditional, LoxP flanked (floxed), alleles. The location of PCR primers used for genotyping, the neomycin cassette (Neo), and LoxP sites are shown. (b) For global …
Figure 1—figure supplement 2
LPC injection into the corpus callosum leads to focal white matter damage and upregulation of myelin-associated gene products.
(a) Coronal forebrain sections of adult Lrp1 control (naive) mice and mice subjected to stereotaxic injection of PBS into the corpus callosum. At 10 days post injection (10 DPI) and 21 DPI of PBS, …
Figure 2 with 2 supplements
Lrp1 ablation in the OL-lineage leads to hypomyelination and nodal defects.
(a) Ultrastructural images of optic nerve cross-sections from P10, P21, and P56 control and Lrp1flox/flox;Olig2-Cre conditional knockout mice (Lrp1 cKOOL). Scale bar = 1 µm. (b) Quantification of …
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Figure 2—source data 1
Raw data and detail statistical analysis report.
- https://doi.org/10.7554/eLife.30498.009
Figure 2—figure supplement 1
Lrp1 ablation in the OL lineage leads to CNS hypomyelination.
(a) Lrp1flox/flox mice were crossed with Olig2-Cre mice to conditionally ablate Lrp1 in OL lineage (cKOOL). (b) To ensure that the presence of the Olig2-Cre allele does not lead to altered …
Figure 2—figure supplement 2
Loss of Lrp1 in the OL lineage leads to faulty optic nerve conduction.
(a) Scheme depicting the orientation of an optic nerve prepared for compound action potential (CAP) recordings. Positions of the stimulating electrode, the recording electrode, and artifact …
Figure 3 with 1 supplement
Loss of Lrp1 in the OL-lineage attenuates OL differentiation.
(a) Cross-sections of Lrp1 control and cKOOL optic nerves stained with anti-PDGFRα (OPC marker), anti-Olig2 (pan-OL marker), anti-CC1 (mature OL marker), and Hoechst dye33342. Scale bar = 100 µm. (b)…
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Figure 3—source data 1
Raw data and detail statistical analysis report.
- https://doi.org/10.7554/eLife.30498.012
Figure 3—figure supplement 1
Loss of Lrp1 does not alter optic nerve size and OPC proliferation.
(a) Quantification of optic nerve diameter in Lrp1 control (n = 5) and cKO (n = 5) mice. (b) To ensure that the presence of the Olig2-Cre allele does not lead to altered expression of …
Figure 4 with 1 supplement
Free cholesterol is reduced in OPCs deficient for Lrp1.
(a) OPCs were isolated from P8 brains by anti-PDGFRα immunopanning, sonicated and subjected to measurement of cholesterol (Chol). (b and c) Quantification of free Chol (b) and total Chol (Chol and …
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Figure 4—source data 1
Raw data and detail statistical analysis report.
- https://doi.org/10.7554/eLife.30498.015
Figure 4—figure supplement 1
Lrp1-deficient OLs are sensitive to statin treatment but not to bath applied mevalonate.
(a) Primary OPCs were isolated by anti-PDGFRα immunopanning from Lrp1flox/+ and Lrp1flox/+;Olig2-Cre pups and cultured for 3 days in differentiation medium (D3 in DM). Cells were lysed and subjected …
Figure 5 with 1 supplement
In primary OLs, peroxisome density and distribution is regulated by Lrp1.
(a) Primary OLs prepared from Lrp1 control and cKOOL OL pups, cultured for 5 days in DM were stained with anti-MBP and anti-PMP70. Scale bar = 10 µm. (b–d) Quantification of PMP70 labeling intensity …
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Figure 5—source data 1
Raw data and detail statistical analysis report.
- https://doi.org/10.7554/eLife.30498.018
Figure 5—figure supplement 1
Gene ontology (GO) analysis of Lrp1-deficient OPCs revealed enrichment of peroxisomal genes.
Acutely isolated OPCs from Lrp1+/+ and Lrp1flox/flox;Olig2-Cre mouse pups were subjected to microarray analysis. (a) GO structure of biological process module related to peroxisome function. Each …
Figure 6
In Lrp1-deficient OPCs, PPARγ activation increases peroxisome density but does not promote cell differentiation.
(a) Timeline in days showing when growth medium (GM) or differentiation medium (DM) with pioglitazone (Pio) were supplied and cells were harvested for analysis. (b) Immunoblots of OL lysates …
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Figure 6—source data 1
Raw data and detail statistical analysis report.
- https://doi.org/10.7554/eLife.30498.020
Figure 7
The combined treatment with cholesterol and pioglitazone rescues the differentiation block of Lrp1 deficient OPCs.
(a) Timeline in days showing when growth medium (GM) or differentiation medium (DM) with pioglitazone (Pio) and cholesterol (Chol) was supplied and cells were harvested for analysis. (b) …
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Figure 7—source data 1
Raw data and detail statistical analysis report.
- https://doi.org/10.7554/eLife.30498.022
Figure 8
Working model of LRP1 regulated pathways in developing OLs.
(a) LRP1 in the OL-lineage is necessary for proper CNS myelin development and the timely repair of a chemically induced focal white matter lesion. In OPCs, Lrp1 deficiency leads to dysregulation of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Lrp1flox/flox | PMID:9634821 | RRID:IMSR_JAX:012604 | |
| Genetic reagent (Mus musculus) | Olig2-Cre | PMID:18691547 | RRID:MMRRC_011103-UCD | |
| Genetic reagent (Mus musculus) | CAG-CreERTM | PMID:11944939 | RRID:IMSR_JAX:004682 | |
| Genetic reagent (Mus musculus) | Pdgfra-CreERTM | PMID: 21092857 | RRID:IMSR_JAX:018280 | |
| Sequence-based reagent (cRNA) | Pdgfra cRNA | PMID:24948802 | ||
| Sequence-based reagent (cRNA) | Plp1 cRNA | PMID:24948802 | ||
| Sequence-based reagent (cRNA) | Mag cRNA | PMID:22131434 | ||
| Sequence-based reagent (cRNA) | Mbp cRNA | this study | Based on Allen Brain Atlas | |
| Antibody | anti-Digoxigenin-AP antibody | Roche | #11093274910 | |
| Antibody | rabbit anti-Olig2 | Millipore | #AB9610, RRID:AB_570666 | |
| Antibody | rat anti-PDGFRα | BD Pharmingen | #558774, RRID:AB_397117 | |
| Antibody | rabbit anti-GFAP | DAKO | # A 0334, RRID:AB_10013482 | |
| Antibody | mouse anti-APC | Calbiochem | #OP80, Clone CC1, RRID:AB_2057371 | |
| Antibody | rabbit anti-Caspr | PMID: 9118959 | RRID:AB_2572297 | |
| Antibody | mouse anti-Na Channel | PMID: 10460258 | K58/35 | |
| Antibody | rabbit anti-CNPase | Aves Labs | #27490 R12-2096 | |
| Antibody | mouse anti-MAG | Millipore | #MAB1567, RRID:AB_2137847 | |
| Antibody | rat anti-MBP | Millipore | #MAB386, RRID:AB_94975 | |
| Antibody | chicken anti-PLP | Aves Labs | #27592 | |
| Antibody | mouse anti-GFAP | Sigma | #G3893, RRID:AB_477010 | |
| Antibody | chicken anti-GFAP | Aves Labs | #GFAP | |
| Antibody | rabbit anti-NG2 | Millipore | #AB5320, RRID:AB_91789 | |
| Antibody | rabbit anti-LRP1β | Abcam | #ab92544, RRID:AB_2234877 | |
| Antibody | rabbit anti-PMP70 | Thermo | #PA1-650, RRID:AB_2219912 | |
| Antibody | mouse anti-βIII tubulin | Promega | #G7121, RRID:AB_430874 | |
| Antibody | mouse anti-β-actin | Sigma | #AC-15 A5441, RRID:AB_476744 | |
| Antibody | rabbit anti-MAG | PMID: 27008179 | ||
| Antibody | rabbit anti-PLP | Abcam | #ab28486, RRID:AB_776593 | |
| Antibody | rat anti-PLP/DM20 | PMID: 27008179 | AA3 hybridoma | Wendy Macklin |
| Antibody | mouse anti-CNPase | Abcam | #ab6319, RRID:AB_2082593 | |
| Antibody | rabbit anti-PXMP3 (PEX2) | One world lab | #AP9179c | |
| Antibody | rabbit anti-SREBP2 | One world lab | #7855 | |
| Commercial assay or kit | Cholesterol/Cholesteryl Ester Quantitation Kit | Chemicon | #428901 | |
| Commercial assay or kit | DCTM Protein Assay | Bio-Rad | #5000112 | |
| Commercial assay or kit | RNeasy Micro Kit | Qiagen | #74004 | |
| Chemical compound, drug | 10 mM dNTP mix | Promega | #C1141 | |
| Chemical compound, drug | 5X Green GoTaq Buffer | Promega | #M791A | |
| Chemical compound, drug | GoTaq DNA polymerase | Promega | #M3005 | |
| Chemical compound, drug | L-α-Lysophosphatidylcholine | Sigma | #L4129 | |
| Chemical compound, drug | Hoechst dye 33342 | Life technology | #H3570 | |
| Chemical compound, drug | ProLong Gold antifade reagent | Life technology | #P36930 | |
| Chemical compound, drug | Fluoromyelin-Green | Life technology | #F34651 | |
| Chemical compound, drug | PDGF-AA | Peprotech | #100-13A | |
| Chemical compound, drug | Forskolin | Sigma | #F6886 | |
| Chemical compound, drug | CNTF | Peprotech | #450–02 | |
| Chemical compound, drug | NT-3 | Peprotech | #450–03 | |
| Chemical compound, drug | T3 | Sigma | #T6397 | |
| Chemical compound, drug | Cholesterol | Sigma | #C8667 | |
| Chemical compound, drug | Pioglitazone | Sigma | #E6910 | |
| Chemical compound, drug | Simvastatin | Sigma | #S6196 | |
| Chemical compound, drug | GW9662 | Sigma | #M6191 | |
| Chemical compound, drug | Filipin | Sigma | #F9765 | |
| Chemical compound, drug | Super Signal West Pico substrate | Thermo | #34080 | |
| Chemical compound, drug | WesternSure PREMIUM Chemiluminescent Substrate | LI-COR Biosciences | #926–95000 | |
| Chemical compound, drug | Super Signal West Femto substrate | Thermo | #34095 | |
| Software, algorithm | Fiji | PMID: 22743772 | ||
| Software, algorithm | Axon pAlamp10.3 software | Molecular Devices | ||
| Software, algorithm | Origin9.1 software | Origin Lab | ||
| Software, algorithm | Image Studio Lite Western Blot Analysis Software | LI-COR Biosciences | ||
| Other | C-DiGit blot scanner | LI-COR Biosciences | #P/N 3600–00 | |
| Other | Multimode Plate Reader | Molecular Devices | #SpectraMax M5e | |
| Other | Stoelting stereotaxic instrument | Stoelting | #51730D | |
| Other | Stoelting quintessential stereotaxic injector | Stoelting | #53311 |
Additional files
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Source code 1
ANOVA source code.
- https://doi.org/10.7554/eLife.30498.024
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Transparent reporting form
- https://doi.org/10.7554/eLife.30498.025
