CNS myelination and remyelination depend on fatty acid synthesis by oligodendrocytes

  1. Penelope Dimas
  2. Laura Montani  Is a corresponding author
  3. Jorge A Pereira
  4. Daniel Moreno
  5. Martin Trötzmüller
  6. Joanne Gerber
  7. Clay F Semenkovich
  8. Harald C Köfeler
  9. Ueli Suter  Is a corresponding author
  1. Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, ETH Zürich, Switzerland
  2. Medical University of Graz, Austria
  3. Washington University Medical School, United States
9 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
Generation of mutant mice lacking FASN in the oligodendrocyte lineage.

(a) Experimental strategy of conditional Fasn allele inactivation upon Olig2-driven Cre expression in vivo. (b) Schematic of thoracic spinal cord. Square insert ‘c’: White matter (WM) where …

https://doi.org/10.7554/eLife.44702.002
Figure 1—figure supplement 1
Recombination efficiency in spinal cord white matter.

(a) Representative immunostaining of cross-sectioned thoracic spinal cord white matter from P14 CT and cMU mice, n = 5 mice for each, CT and cMU. Cre-dependent expression of the reporter Rosa26-loxPs…

https://doi.org/10.7554/eLife.44702.003
Figure 1—figure supplement 2
FASN expression in gray matter oligodendrocytes of CT but not in cMU mice.

(a) Schematic of the thoracic spinal cord. Square insert ‘b’: Gray matter (GM) area where subsequent immunostaining images were acquired. WM = white matter. (b) Representative immunostaining of …

https://doi.org/10.7554/eLife.44702.004
Figure 2 with 1 supplement
De novo fatty acid synthesis is not required to achieve correct numbers of oligodendrocyte lineage cells during development.

(a) Representative immunostaining of ventral white matter in cross-sectioned spinal cords of P4 and P10 mice identifying proliferating (EdU+) OLs (Olig2+; examples indicated by arrowheads), n = 3 …

https://doi.org/10.7554/eLife.44702.005
Figure 2—figure supplement 1
FASN expression in oligodendrocyte progenitors is marginal and dispensable for their early maintenance.

(a) Representative immunostaining of ventral white matter in cross-sections of lumbar spinal cords from P14 CT mice depicting marginal (if any) FASN expression in OPCs (PDGFRα+; examples indicated …

https://doi.org/10.7554/eLife.44702.006
Figure 3 with 3 supplements
De novo fatty acid synthesis by oligodendrocytes is essential to achieve accurate myelination in the spinal cord.

(a) Representative EM images of P14 control (CT, a’ and a’’) and conditional mutant (cMU, b’ and b’’) white matter from the ventral funiculi of lumbar spinal cords. cMUs show more naked axons, …

https://doi.org/10.7554/eLife.44702.007
Figure 3—figure supplement 1
Recombination efficiency in the optic nerve.

(a) Representative immunostaining of cross-sectioned optic nerves from P14 CT and cMU mice, n = 5 mice for each, CT and cMU. Cre-dependent expression of the reporter Rosa26-loxPstoploxP-YFP allele …

https://doi.org/10.7554/eLife.44702.008
Figure 3—figure supplement 2
De novo fatty acid synthesis by oligodendrocytes is essential to achieve accurate myelination of the optic nerve.

(a) Representative EM images of optic nerves from P14 control (CT) and conditional mutant (cMU) mice. Note the increased number of axons not-yet enwrapped by myelin in cMUs when compared to CTs. …

https://doi.org/10.7554/eLife.44702.009
Figure 3—figure supplement 3
De novo fatty acid synthesis by oligodendrocytes is essential to achieve accurate myelination of the corpus callosum.

(a) Representative EM images of corpus callosum from P180 CT and cMU mice. cMUs show slightly more axons not-yet enwrapped by myelin compared to CTs. cMU axons are also covered by thinner myelin …

https://doi.org/10.7554/eLife.44702.010
Figure 4 with 2 supplements
De novo fatty acid synthesis by oligodendrocytes is required to maintain structural stability of myelinated axons.

(a) Exemplary EM images of aberrant myelin-axon profiles showing vacuolation (examples indicated by arrowheads) in the ventral white matter of spinal cords of P14 cMUs. Where identifiable, axons …

https://doi.org/10.7554/eLife.44702.011
Figure 4—figure supplement 1
De novo fatty acid synthesis by oligodendrocytes supports structural stability of myelinated axons in the optic nerve.

(a) EM images of aberrant myelinated axon profiles (examples indicated by arrowheads) in the optic nerve of P14 cMU mice. Scale bars: I = 2 µm, II = 1 µm, III = 2 µm, IV = 1 µm, V = 1 µm, VI = 1 µm. …

https://doi.org/10.7554/eLife.44702.012
Figure 4—figure supplement 2
Lipid profiles of spinal cord myelin of adult control and FASN mutant mice.

Lipids extracted from myelin isolated from spinal cords of control (CT) and mutant (cMU) mice were identified and quantified by liquid chromatography-mass spectrometry. Individual species of 10 …

https://doi.org/10.7554/eLife.44702.013
Figure 5 with 1 supplement
Transcriptome analysis of optic nerves of Fasn mutant mice reveal oligodendrocyte defects in late stages of maturation, including myelination.

(a, b) GeneOntology analysis depicting biological processes affected by down-regulated (a) and up-regulated (b) transcripts in P14 optic nerves of mutant (cMU) mice. Metacore Pathway analysis …

https://doi.org/10.7554/eLife.44702.014
Figure 5—source data 1

Expressed transcripts in optic nerves from control and cMU mice at P14.

https://doi.org/10.7554/eLife.44702.016
Figure 5—figure supplement 1
FASN deficiency is correlated with molecular signs of defective oligodendrocyte maturation.

Graph of qRT-PCR analysis of Sox10, Myrf and MBP in optic nerves of P14 CT and cMU mice. Data are normalized to β-actin and to the mean of CTs (unpaired two-tailed two sample Student’s t-test; …

https://doi.org/10.7554/eLife.44702.015
Increasing dietary lipid intake can partially compensate defects in radial myelin growth caused by lack of endogenous fatty acid synthesis in oligodendrocytes.

(a) Scheme of the experimental design. (b) Exemplary EM micrographs of the ventral funiculus of the lumbar spinal cord from mutant (cMU) and control (CT) mice that were fed a standard (STD) or a …

https://doi.org/10.7554/eLife.44702.017
Figure 7 with 1 supplement
Generation of tamoxifen-inducible PdgfrαCreERT2:FASN-floxed conditionally mutant mice.

(a) Tamoxifen-mediated nuclear translocation of Pdgfrα-driven CreERT2 induces conditional Fasn allele inactivation (i-cMU) and expression of yellow fluorescent protein (YFP). (b) Timeline of …

https://doi.org/10.7554/eLife.44702.018
Figure 7—figure supplement 1
Recombination efficiency in spinal cords of PdgfrαCreERT2:Rosa26-loxPstoploxP-YFP control animals induced with tamoxifen.

(a) Exemplary immunostaining of cross-sectioned thoracic spinal cord white matter from 5 weeks post tamoxifen, n = 3 CT mice. Cre-dependent expression of the reporter Rosa26-loxPstoploxP-YFP allele …

https://doi.org/10.7554/eLife.44702.019
Figure 8 with 3 supplements
De novo fatty acid synthesis is required for maintaining the adult oligodendrocyte progenitor-derived oligodendrocyte population during remyelination.

(a) Representative immunostaining of lesions of control (CT) and inducible conditional mutant (i-cMU) mice on cross sections of ventral white matter of the spinal cord at 7, 10, 14 and 21 days …

https://doi.org/10.7554/eLife.44702.020
Figure 8—figure supplement 1
FASN expression in adult oligodendrocyte progenitors is marginal and largely dispensable for their response following demyelination.

(a) Representative immunostaining of the lesion area in cross sections of the spinal cord of induced control (CT) mice at 7 days post-lysolecithin injection (dpl). Images depict marginal expression …

https://doi.org/10.7554/eLife.44702.021
Figure 8—figure supplement 2
FASN expression is dispensable for the proliferation of oligodendrocytes lineage cells in remyelination.

(a) Representative immunostaining in the lesion area of cross-sectioned thoracic spinal cords of control (CT) and inducible conditional mutant (i-cMU) mice at 7 dpl. n = 5 mice for each, CT and …

https://doi.org/10.7554/eLife.44702.022
Figure 8—figure supplement 3
FASN is critical to sustain adult oligodendrocyte progenitor-derived oligodendrocytes during remyelination.

(a) Representative immunostaining of the lesion area in cross sections of the thoracic spinal cord at 12 days post-lysolecithin injection (dpl) from control (CT) and inducible conditional mutant …

https://doi.org/10.7554/eLife.44702.023
De novo fatty acid synthesis is essential to achieve efficient remyelination in the spinal cord.

(a) Representative EM images of lesions in the spinal cord of control (CT) and inducible conditional mutant (i-cMU) mice during remyelination at 14, 21 and 56 days post-lysolecithin injection (dpl). …

https://doi.org/10.7554/eLife.44702.024

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent
(M. musculus)
Fasnlox/loxPMID:16054078MGI:3765070Dr. Clay F.
Semenkovich, Washington University, St. Louis USA
Genetic reagent (M. musculus)PdgfraCreERT2PMID:18849983MGI:3832569Dr. William D. Richardson,
University College London, London UK
Genetic reagent (M. musculus)Olig2CreJackson LaboratoryStock#:011103; MGI:3810299PMID:18691547
Genetic reagent (M. musculus)Rosa26-loxPstoploxP-YFPJackson LaboratoryStock#:006148; MGI:3621481PMID:11299042
Sequence-based reagentGenotyping
primer: Fasn forward
PMID:294340295'-GGATAGCTGTGTAGTGTAACCAT-3’
Sequence-based reagentGenotyping primer: Fasn reversePMID:294340295’-GGTCACCCAGCAGGAAAGGGC- 3’
Sequence-based reagentGenotyping primer: Cre forwardPMID: 294340295’-TTCCCGCAGAACCTGAAGATGTTCG-3’
Sequence-based reagentGenotyping primer: Cre reversePMID: 294340295’-GGGTGTTATAAGCAATCCCCAGAAATG-3’
Sequence-based reagentGenotyping primer: Rosa26-loxPstoploxP-YFP forwardPMID: 285225365’-AAAGTCGCTCTGAGTTGTTAT-3’
Sequence-based reagentGenotyping primer: Rosa26-loxPstoploxP-YFP reverse transgenicPMID: 285225365’-GCGAAGAGTTTGTCCTCAACC-3’
Sequence-based reagentGenotyping primer: Rosa26-loxPstoploxP-YFP reverse wildtypePMID: 285225365’-GGAGCGGGAGAAATGGATATG-3’
Sequence-
based reagent
qRT-PCR primer: SOX10 ForwardThis paper5’-CCGACCAGTACCCTCACCT-3’
Sequence-based reagentqRT-PCR primer: SOX10 ReverseThis paper5’- TCAATGAAGGGGCGCTTGT-3’
Sequence-based reagentqRT-PCR primer: MYRF ForwardThis paper5’-ATGGAGGTGGTGGACGAGAC-3’
Sequence-based reagentqRT-PCR primer:MYRF ReverseThis paper5’-GGCGTCCTCTTTGCCAATGT-3’
Sequence-
based reagent
qRT-PCR primer: MBP ForwardThis paper5’-ACACGAGAACTACCCATTATGGC-3’
Sequence-
based reagent
qRT-PCR primer:
MBP Reverse
This paper5’-CCAGCTAAATCTGCTGAGGGGA-3’
Sequence-
based reagent
qRT-PCR primer:
Actin Forward
PMID: 288801495’-GTCCACACCCGCCACC-3’
Sequence-
based reagent
qRT-PCR primer: Actin ReversePMID: 288801495’-GGCCTCGTCACCCACATAG-3’
AntibodyRabbit polyclonal anti-cleaved Caspase 3Cell signaling
Technology, Danvers, MA, USA
Cat# 9661; RRID:AB_23411881:500 dilution
AntibodyMouse monoclonal anti-CC1Merck Millipore, Billerica, MA, USACat; # OP80; RRID:AB_2057371Development: 1:200 dilution;
Remyelination 1:300 dilution
Antibodyrabbit polyclonal anti-FASNAbcam, UKCat# Ab22759;
RRID:AB_732316
1:200 dilution
AntibodyChicken polyclonal anti-GFPAbcam, UKCat# Ab13970; RRID:AB_3007981:1000 dilution
Antibodymouse monoclonal anti-ki67Dako Agilent,
Santa Clara, CA, USA
Cat# m7249; clone MIB-5; RRID:AB_22505031:200 dilution
Antibodyrat monoclonal anti-MBPSerotec/BioRad
Laboratories, Hercules, CA, USA
Cat# MCA409S; RRID:AB_325004Development: 1:200 dilution; Remyelination 1:300 dilution
AntibodyGoat polyclonal anti-Olig2R and D Systems,
Minneapolis, MN, USA
Cat# AF2418; RRID:AB_21575541:25 dilution
Antibodymouse monoclonal anti-Olig2Merck Millipore, Billerica, MA, USACat# MABN50; clone 211F1.1; RRID:AB_108074101:1000 dilution
Antibodyrabbit polyclonal anti-Olig2Merck Millipore, Billerica, MA, USACat# AB9610; RRID:AB_570666Development: 1:500 dilution; Remyelination 1:400 dilution
AntibodyRabbit monoclonal anti-PDGFRαCell signaling Technology, Danvers, MA, USACat# 3174; RRID:AB_21623451:500 dilution
Commercial assay or kitClick-iT EdU
Assay
Thermo Fisher
Scientific, Waltham, MA, USA
Cat# C10337
Commercial assay or kitMouse blocking reagentVector Laboratories, Burlingame, CA, USACat# MKB-2213
Commercial assay or kitStreptavidin/Biotin blocking KitVector Laboratories, Burlingame, CA, USACat# SP-2002
Commercial assay or kitQiagen MiniKit (RNeasy Mini Kit)Qiagen, Hilden, GermanyCat# 74104
Commercial assay or kitTruSeq Stranded mRNA Sample Prep KitIllumina, San Diego, CA, USACat# 20020594
Commercial
assay or kit
Maxima RT-KitThermo Fisher Scientific, Waltham, MA, USACat# K1641
Chemical compound, drugLysolecithinSigma-Aldrich, Sant Louis, MO, USACat# L4129
Chemical compound, drugTamoxifenSigma-Aldrich, Sant Louis, MO, USACat# T5648
Chemical compound, drugLipid standardsAvanti Polar Lipids, Alabaster, AL, USA
Software, algorithmLipid Data Analyzer softwarePMID: 29058722
Software, algorithmPhotoshop CS5 or CS6Adobe
Software,
algorithm
FIJIImageJ (http://imagej.nih.gov/ij/)
Software, algorithmSTAR Aligner(v2.5.1b)PMID: 23104886
Software, algorithmRSEM (v1.2.22)PMID: 21816040
Software, algorithmEdgeRPMID: 19910308
Software, algorithmMetacore (vs6.33)Thomson Reuters
OtherStandard diet (STD)KLIBA NAFAG, Provimi KLIBA, SwitzerlandCat# 3437
OtherHigh fat diet (HFD)KLIBA NAFAG, Provimi KLIBA, SwitzerlandCat# 2127

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