Circulating platelets modulate oligodendrocyte progenitor cell differentiation during remyelination

  1. Amber R Philp
  2. Carolina R Reyes
  3. Josselyne Mansilla
  4. Amar Sharma
  5. Chao Zhao
  6. Carlos Valenzuela-Krugmann
  7. Khalil S Rawji
  8. Ginez A Gonzalez Martinez
  9. Penelope Dimas
  10. Bryan Hinrichsen
  11. César Ulloa-Leal
  12. Amie K Waller
  13. Diana M Bessa de Sousa
  14. Maite A Castro
  15. Ludwig Aigner
  16. Pamela Ehrenfeld
  17. Maria Elena Silva
  18. Ilias Kazanis
  19. Cedric Ghevaert
  20. Robin JM Franklin
  21. Francisco J Rivera  Is a corresponding author
  1. Laboratory of Stem Cells and Neuroregeneration, Institute of Anatomy, Histology and Pathology, Faculty of Medicine, Universidad Austral de Chile, Chile
  2. Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Chile
  3. Wellcome-MRC Cambridge Stem Cell Institute & Department of Clinical Neurosciences, University of Cambridge, United Kingdom
  4. Translational Regenerative Neurobiology Group (TReN), Molecular and Integrative Biosciences Research Programme (MIBS), Faculty of Biological and Environmental Sciences, University of Helsinki, Finland
  5. Escuela de Ciencias Agrícolas y Veterinarias, Universidad Viña del Mar, Chile
  6. Department of Haematology and NHS Blood and Transplant, University of Cambridge, United Kingdom
  7. Institute of Molecular Regenerative Medicine, Paracelsus Medical University, Austria
  8. Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Chile
  9. Laboratory of Cellular Pathology, Institute of Anatomy, Histology & Pathology, Faculty of Medicine, Universidad Austral de Chile, Chile
  10. School of Life Sciences, University of Westminster, United Kingdom
4 figures, 1 table and 1 additional file

Figures

Platelets accumulate in response to demyelination.

(A) LPC induced demyelinating lesions in spinal cord white matter of WT mice at 1, 3, 7, and 14 dpl, stained for platelets (CD41+). Scale bar 100 μm. (B) Quantification of CD41+ signal within the demyelinated lesion at 1 (n=6), 3 (n=5), 5 (n=5), 7 (n=6), 10 (n=4), and 14 dpl (n=4), and in NAWM (n=3). (C) Platelet staining (CD41+) in spinal cord white matter injected with PBS/DAPI. Scale bar 50 μm. (D) Upper left panel: localization of platelets within blood vessels (ColIV+) and in close proximity with OPCs (Olig2+) at 5 dpl. Upper right panel: IMARIS 3D projection shows the spatial distribution of platelets. Scale bar 10 μm. Lower panels: magnification of the IMARIS projection showing platelet aggregation within the blood (left panel) and penetration into the parenchyma (right panel). Scale bars: 5 μm (left panel) and 7 μm (right panel). Data were analysed using a Kruskal Wallis test. Data represent the mean ± SD. ** p<0.01; ns (not significant), p>0.05.

Figure 2 with 2 supplements
Platelet depletion impairs remyelination in vivo.

(A) Schematic representation of the LPC-induced demyelination model coupled with platelet depletion using anti-CD42b. (B) Quantitative analysis of CD41+ signal at 5 dpl in untreated (n=5) and platelet depleted mice (n=3). (C) Representative images of immunofluorescence staining of oligodendroglial lineage cells in untreated and platelet depleted mice at 7 dpl using Olig2+ (upper panels) and mature oligodendrocytes using Olig2+/CC1+ (lower panels). Boxed areas represent high magnification images. (D–F) Quantitative analysis of oligodendroglia at 7 dpl in untreated (n=3) and platelet depleted mice (n=5). (G) Representative images of toluidine blue staining of remyelination in untreated (n=6) and platelet depleted mice (n=3) at 14 dpl and (H–I) its quantification by relative ranking analysis. Data were analysed using an Unpaired Student’s t-test or Mann-Whitney U test. Data represent mean ± SD. * p<0.05; ** p<0.01; ns (not significant), p>0.05. Scale bars, 100 μm.

Figure 2—figure supplement 1
Platelet depletion does not alter BBB permeability.

(A) Representative immunofluorescence images of neutrophils (NIMP-R14+) and (B) number of neutrophils in LPC-induced white matter spinal cord lesion at 7 dpl in untreated (n=3) and platelet depleted mice (n=5). (C) Representative immunofluorescence images of Fibrinogen and (D) Quantification of Fibrinogen signal within the demyelinated lesion at 7 dpl in untreated and platelet depleted mice (n=4). Scale bar 50 μm. Data were analysed using an unpaired t-test. Data represent the mean ± SD. ns (not significant), p>0.05.

Figure 2—figure supplement 2
Changes in circulating platelet numbers does not alter the macrophage/microglia population during remyelination.

(A) Platelets (CD41+) are located in close proximity to the macrophage/microglia population (IBA-1+) at 5 dpl. Scale bar 10 μm. (B) Total macrophage/microglia population (IBA-1+), M1 (CD16/32+) and M2 (Arg-1+) cell subpopulations at 10 dpl. Scale bar 50 μm. (C–E) Quantitative analysis of total, M1 and M2 cell subpopulations in untreated (n=6), platelet depleted (n=3), and Calr+/- mice (n=4). (F) Representative Oil-Red O staining of myelin debris at 10 dpl. (G) Quantification of Oil-Red O (ORO) staining in untreated (n=6) and platelet depleted mice (n=3). Scale bar 100 μm. Data were analysed using an ordinary one-way ANOVA and or an unpaired t-test. Data represent the mean ± SD. ns (not significant), p>0.05.

Prolonged exposure to platelets suppresses their ability to enhance OPC differentiation.

(A) Representative fluorescence images of OPCs co-cultured with 1 (n=6), 5 (n=6), and 10% (n=6) washed platelets (WP) for 3 days in vitro (DIV), followed by WP removal for an additional 3 DIV (Pulse). Additionally, OPCs were co-cultured in the presence of 10% WP for 6 DIV (n=5) (Sustained). Vehicle treated OPCs represents the control condition (n=6). (B) Graph represents the percentage of Olig2+MBP+ oligodendrocytes within the total Olig2 population (quantitative analysis of OPC differentiation). (C) Representative images of OPCs exposed to 1% platelet lysate (PL) (n=5) for 6 DIV. Vehicle treated OPCs represents the control condition (n=5). (D) Graph represents the quantitative analysis of OPC differentiation as in B. (E) Representative images of OPCs exposed to either PL for 9 DIV (Sustained) (n=5) or 6 DIV with PL followed by its removal for an additional 3 more DIV (Withdrawn) (n=5). Vehicle-treated OPCs represents the control condition (n=5). (F) Graph shows the quantitative analysis of OPC differentiation as in B and D. Data were analysed using one-way ANOVA followed by Tukey’s post-hoc test, a Mann-Whitney U test, or Kruskal-Wallis test. Data represent the mean ± SD. * p<0.05; *** p<0.001; **** p<0.0001; ns (not significant), p>0.05. Scale bars, 50 μm.

A sustained increase in circulating platelets impairs remyelination in-vivo.

(A) Representative fluorescence images of platelets (CD41+) in LPC induced demyelinating lesions of spinal cord white matter of WT and Calr +/-mice at 5 and 10 dpl. Scale bar 50 μm. (B) Quantification of circulating platelets in WT vs Calr +/-mice at 5 (n=4 and n=5, respectively) and 10 dpl (n=5 and n=6, respectively). (C) Quantification of CD41+ signal in demyelinated lesions of WT vs Calr +/-mice at 5 dpl (n=5 and n=5, respectively) and 10 dpl (n=4 and n=4, respectively). (D) Representative immunofluorescence staining of oligodendroglial lineage cells in untreated and platelet depleted mice at 10 dpl using Olig2+ (upper panels) and mature oligodendrocytes using Olig2+/CC1+ (lower panels) (n=4). Scale bar 100 μm. (E–G) Quantitative analysis of oligodendroglia at 10 dpl. (H) Correlation between the circulating platelet number with the number of Olig2+/CC1+ cells within the demyelinated lesion. Data were analysed using a two-way ANOVA followed by Bonferroni’s post-hoc test, an unpaired t-test, Welch’s t-test, a Mann-Whitney U test, or Pearson’s correlation coefficient analysis. Data represent the mean ± SD. * p<0.05; ** p<0.01; ns (not significant), p>0.05.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyCD41 rat monoclonalAbcamCat# ab33661;
RRID:AB_726487
Working dilution (1:200)
AntibodyCD16/32
rat monoclonal
BD BiosciencesCat # BD 553142
RRID:AB_394656
Working dilution (1:200)
AntibodyIba-1 rabbit polyclonalWAKOCat#
019–19741;
RRID:AB_839504
Working dilution (1:500)
AntibodyArg-1 goat polyclonalSanta CruzCat# sc-18351;
RRID:AB_2258542
Working dilution (1:200)
AntibodyNIMP-R14 rat monoclonalAbcamCat# ab2557;
RRID:AB_303154
Working dilution (1:200)
AntibodyOlig2 rabbit monoclonalAbcamCat# Ab109186;
RRID:AB_10861310
Working dilution
(1:200 in vivo)
(1:500 in vitro)
AntibodyCC1 mouse monoclonalMilliporeCat# OP80;
RRID:AB_2057371
Working dilution (1:1000)
AntibodyMBP rat monoclonalBio-radCat# MCA409S;
RRID:AB_325004
Working dilution (1:500)
AntibodyCollagen IV (ColIV) goat polyclonalMilliporeCAT# AB769;
RRID:AB_92262
Working dilution (1:100)
AntibodyFibrinogen rabbit polyclonalAbcamCat # ab34269
RRID:AB_732367
Working dilution (1:200)
Chemical compound, drugL-α-lysophosphatidylcholineSigma-AldrichCat # L1381Demyelinating agent, Working concentration 1%
Chemical compound, drugCD42b (mixture of rat monoclonal antibodies)Emfret Analytics; 
Evans et al., 2021
Cat #R300
RRID:AB_2721041
Platelet depletion antibody, Working concentration 0.6 μg/g
Strain, strain background (Mus musculus)Mouse: C7BL/6Charles River LaboratoriesRRID:SCR_003792
Strain, strain background (Mus musculus)Mouse: Calrfl/+:Vav1-Cre miceLi et al., 2018
Strain, strain background (Rattus norvegicus)Rat:
Sprague Dawley
Charles River LaboratoriesRRID:SCR_003792

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  1. Amber R Philp
  2. Carolina R Reyes
  3. Josselyne Mansilla
  4. Amar Sharma
  5. Chao Zhao
  6. Carlos Valenzuela-Krugmann
  7. Khalil S Rawji
  8. Ginez A Gonzalez Martinez
  9. Penelope Dimas
  10. Bryan Hinrichsen
  11. César Ulloa-Leal
  12. Amie K Waller
  13. Diana M Bessa de Sousa
  14. Maite A Castro
  15. Ludwig Aigner
  16. Pamela Ehrenfeld
  17. Maria Elena Silva
  18. Ilias Kazanis
  19. Cedric Ghevaert
  20. Robin JM Franklin
  21. Francisco J Rivera
(2024)
Circulating platelets modulate oligodendrocyte progenitor cell differentiation during remyelination
eLife 12:RP91757.
https://doi.org/10.7554/eLife.91757.3