A somatic mutation in erythro-myeloid progenitors causes neurodegenerative disease

Journal name:
Nature
Volume:
549,
Pages:
389–393
Date published:
DOI:
doi:10.1038/nature23672
Received
Accepted
Published online

The pathophysiology of neurodegenerative diseases is poorly understood and there are few therapeutic options. Neurodegenerative diseases are characterized by progressive neuronal dysfunction and loss, and chronic glial activation1. Whether microglial activation, which is generally viewed as a secondary process, is harmful or protective in neurodegeneration remains unclear1, 2, 3, 4, 5, 6, 7, 8. Late-onset neurodegenerative disease observed in patients with histiocytoses9, 10, 11, 12, which are clonal myeloid diseases associated with somatic mutations in the RAS–MEK–ERK pathway such as BRAF(V600E)13, 14, 15, 16, 17, suggests a possible role of somatic mutations in myeloid cells in neurodegeneration. Yet the expression of BRAF(V600E) in the haematopoietic stem cell lineage causes leukaemic and tumoural diseases but not neurodegenerative disease18, 19. Microglia belong to a lineage of adult tissue-resident myeloid cells that develop during organogenesis from yolk-sac erythro-myeloid progenitors (EMPs) distinct from haematopoietic stem cells20, 21, 22, 23. We therefore hypothesized that a somatic BRAF(V600E) mutation in the EMP lineage may cause neurodegeneration. Here we show that mosaic expression of BRAF(V600E) in mouse EMPs results in clonal expansion of tissue-resident macrophages and a severe late-onset neurodegenerative disorder. This is associated with accumulation of ERK-activated amoeboid microglia in mice, and is also observed in human patients with histiocytoses. In the mouse model, neurobehavioural signs, astrogliosis, deposition of amyloid precursor protein, synaptic loss and neuronal death were driven by ERK-activated microglia and were preventable by BRAF inhibition. These results identify the fetal precursors of tissue-resident macrophages as a potential cell-of-origin for histiocytoses and demonstrate that a somatic mutation in the EMP lineage in mice can drive late-onset neurodegeneration. Moreover, these data identify activation of the MAP kinase pathway in microglia as a cause of neurodegeneration and this offers opportunities for therapeutic intervention aimed at the prevention of neuronal death in neurodegenerative diseases.

At a glance

Figures

  1. Targeting BRAF(V600E) in tissue-resident macrophages.
    Figure 1: Targeting BRAF(V600E) in tissue-resident macrophages.

    a, b, Breeding scheme for experimental mice and genotype distribution (n = 342). 4-OHT, 4-hydroxytamoxifen. E8.5, embryonic day 8.5. c, d, YFP expression in bone marrow lineage, Sca-1+, Kit+ (LSK) blood leukocytes and microglia from one-month-old mice, representative of n = 5 per group. e, Proportion of YFP+F4/80+ cells in tissues from one-month-old mice. AM, alveolar macrophages; IM, interstitial macrophages. Circles represent individual mice, red lines are means. Unpaired two-tailed t-test. f, A > T transversion encoding Braf V600E in YFP+ Kupffer cells at the Braf locus. Red and blue bars indicate forward and reverse strands. g, Ki-67 and cleaved caspase-3 (Casp3) expression in YFP+ microglia from one-month-old mouse brains. n = 5 per group. Unpaired two-tailed t-test. h, Gene set enrichment analysis of differentially expressed genes in YFP+ microglia from Braf VE mice (n = 3) and littermate controls (n = 2). Q value <0.01. i, Heatmap representation of selected genes from h, values are displayed as z scores. See also Extended Data Fig. 1.

  2. Neurodegenerative disease in Braf VE mice.
    Figure 2: Neurodegenerative disease in Braf VE mice.

    a, Footprint assays. n = 20 mice per group. b, Limb-clasping reflexes in 6–8-month old mice. n = 10 per group. c, Cumulative incidence of behavioural abnormalities in Braf  VE mice and controls. log-rank (Mantel–Cox) test. d, Overlap distance and stride length of mice that are fed a control or PLX4270 (PLX) diet from one month (1 m) or three months (3 m) of age. Mean ± s.d. for each group, two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. e, Disease progression in mice from d, average score excluding mice euthanized owing to paralysis (†). Two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. f, Cumulative incidence of behavioural abnormalities in mice from d. log-rank (Mantel–Cox) test. g, Scheme depicting microglia (IBA1) and neuronal (NeuN) densities in brain regions from Braf VE and Braf WT (n = 4 per group). h, IBA1 staining and quantitative analysis of microglial accumulation (IBA1+), phagocytosis (IBA1+LAMP2+), astrogliosis (GFAP+), relative synapse density (determined by synaptophysin and homer1), neuronal loss (NeuN) and amyloid precursor protein (APP) expression in brainstems of 5–9-month-old Braf VE mice on a control diet (n = 4), Braf VE mice on a PLX diet (n = 4–6) and Braf WT (n = 4). One-way ANOVA. See also Extended Data Figs 4, 6.

  3. ERK activation in BRAF(V600E) microglia.
    Figure 3: ERK activation in BRAF(V600E) microglia.

    a, CD68, YFP and pERK staining in spinal cord from 7-month-old mice. Scale bars, 500 μm and 10 μm (insets). n = 4 per group. b, pERK+ microglia in the brainstem. Circles represent individual mice. One-way ANOVA. c, ERK phosphorylation in spinal cords and brains from 6–9-month-old mice. Top, representative western blot; bottom, pERK:ERK ratio, n = 5 per group. One-way ANOVA. d, pERK expression in YFP+ microglia from Braf VE mice. n = 5 per group. Scale bars, 5 μm. e, Number of microglia from 5–9-month-old mice. Circles represent individual mice. One-way ANOVA. f, Heatmap of cell frequency among CD45+ cells in the brain. n = 3 per group. g, Ki-67+ and cleaved caspase-3+ (Casp3+) expression in YFP+ microglia from 5–9-month-old Braf VE mice, n = 6 per group. Unpaired two-tailed t-test. See also Extended Data Fig. 7.

  4. Molecular features of ERK-activated microglia and their presence in patients with histiocytoses.
    Figure 4: Molecular features of ERK-activated microglia and their presence in patients with histiocytoses.

    a, Gene set enrichment analysis of differentially expressed genes in YFP+ microglia from 6–7-month-old littermates. Q value <0.05. EMT, epithelial–mesenchymal transition. b, Heatmap of selected differentially expressed genes, values are displayed as z scores. c, Validation of gene expression in YFP+ microglia. n = 5 per group. FMO, fluorescence minus one. d, IL-1b expression in spinal cords from 6–7-month-old mice. n = 3 per group. Scale bars, 10 μm. e, Bioplex analysis of IL-1b and IL-17a in spinal cords from 6–9-month-old mice. Circles represent individual mice. Unpaired two-tailed t-test. f, Collagen IV and collagen VI expression in spinal cords from mice described in d. n = 3 per group. Scale bars, 10 μm. g, CD163, pERK and BRAF(V600E) expression in brain tissues from patients with ECD. H&E, haematoxylin and eosin. Top, 400× objective; bottom, scale bar, 10 μm. h, Quantification of pERK microglia in brains from controls (n = 6) and patients with ECD (n = 3). Mann–Whitney U-test. (i) Heatmap of selected genes from RNA-seq analysis of brain tissue from five controls and two patients with histiocytoses (JXG and LCH), values are displayed as z scores. Q value <0.01. See also Extended Data Fig. 9.

  5. Analysis of one-month-old Csf1r MeriCreMer;Braf LSL-V600E;Rosa26 LSL-YFP mice.
    Extended Data Fig. 1: Analysis of one-month-old Csf1r MeriCreMer;Braf LSL-V600E;Rosa26 LSL-YFP mice.

    a, The percentage of mice born from the cross depicted in Fig. 1a according to their genotype (n = 42), but without injection of hydroxy-tamoxifen (4-OHT) to test for adverse effects of 4-OHT administration. b, Flow cytometry analysis of YFP expression in blood leukocytes. Representative of n = 8 mice per genotype. c, Flow cytometry analysis of YFP+ cells in the liver. YFP+ cells, present only in Csf1r MeriCreMer+ (Cre+) mice (top), were gated as F4/80+CD11b+ Kupffer cells (bottom). Representative of n = 8 mice per genotype. d, YFP expression by immunofluorescence in the liver of Braf VE and Braf WT mice. YFP+ cells are F4/80+ Kupffer cells. Representative of n = 6 mice per genotype. Scale bars, 200 μm and 5 μm (insets). e, Total tissue-resident macrophage cell numbers per gram (g) of tissue were analysed by flow cytometry in Braf VE mice (n = 4) and Braf WT (n = 6). Circles represent individual mice. Unpaired two-tailed t-test. f, In situ analysis of phospho-histone H3 (pHis3) staining in YFP+ cells from brains of Braf VE and Braf WT mice. Circles represent individual mice (n = 3). Unpaired two-tailed t-test. g, RNA-seq analysis, heatmap of MAPK target genes in YFP+ microglia from Braf VE (n = 3) and Braf WT (n = 2) mice, values are displayed as z scores. h, Histological analysis of liver, lung, kidney and spleen in Braf VE and Braf WT mice. HE, haematoxylin and eosin. Representative of n = 4 mice per genotype. Scale bars, 200 μm and 10 μm (insets).

  6. Effect of constitutive Braf V600E expression in Csf1r-expressing cells.
    Extended Data Fig. 2: Effect of constitutive Braf V600E expression in Csf1r-expressing cells.

    a, Breeding scheme. b, Embryonic lethality of Csf1r iCre+;Braf  LSL-V600E;Rosa26LSL-YFP mice, bars represent the percentage of mice born from the cross depicted in a according to their genotype (n = 39). c, Bright field (top) and epifluorescence microscopy (bottom) of Csf1r iCre+Braf  VE and Csf1r iCre+;Braf  WT embryos showing haemorrhagic foci in the liver (arrow) and accumulation of YFP+ cells in the fetal liver. A dead embryo is indicated by a dagger (†). Pictures are representative of n = 3 per genotype. d, The number of mouse embryos found alive during different developmental stages. Csf1r iCre+;Braf LSL-V600E;Rosa26LSL-YFP mice are associated with 100% lethality beyond E14.5. e, Liver weight of E12.5 embryos. Circles represent individual mice. n = 8 for WT;cre, n = 14 for VE;cre, n = 16 for VE;cre, n = 12 for VE;cre+. One-way ANOVA. f, Flow cytometry analysis of LinKit+ blast, erythroid cell (Ter119) and haematopoietic stem cell numbers (LSK CD150+CD48 and CD150CD48) in the E12.5 fetal liver and of E12.5 tissue-resident macrophages in the limbs, head and liver. Circles represent individual mice. n = 4 for Braf WT and n = 6 for Braf VE. Unpaired two-tailed t-test.

  7. Analysis of the CD11cCre;Braf V600E mouse model.
    Extended Data Fig. 3: Analysis of the CD11cCre;Braf V600E mouse model.

    a, Kaplan–Meier survival curve of Braf VE (n = 16) and Braf WT (n = 66) controls. Log-rank (Mantel–Cox) test. b, Representative photographs of lung and spleen from Braf VE mice at time of death with representative Braf WT control organs. c, d, Haematoxylin and eosin staining of lung tissue from Braf VE and littermate controls. e, CD68 immunohistochemistry of Braf VE lung tissue. f, Haematoxylin and eosin staining of liver tissue from Braf VE and littermate controls with magnified image of granuloma in the Braf VE liver. g, Haematoxylin and eosin staining of bone marrow (BM) of Braf VE and littermate controls with CD68 immunohistochemistry of Braf VE mouse tissue. All images for bg are representative of n = 5 per genotype.

  8. Longitudinal study and PLX treatment of the Csf1r MeriCreMer;Braf V600E;Rosa26 LSL-YFP mice.
    Extended Data Fig. 4: Longitudinal study and PLX treatment of the Csf1r MeriCreMer;Braf V600E;Rosa26 LSL-YFP mice.

    a, b, Latency to fall in the rotarod assay and footprint assay quantification for Braf VE mice (n = 7) and Braf WT littermates (n = 8). a, Rotarod assay at 1–4 months of age. Values are mean ± s.d. b, Rotarod and footprint assay at 4 months of age displaying single values. Mice that are score 1 are labelled in red. c, Footprint assay quantification of Braf VE mice at score 1 and littermate controls. Circles represent individual mice. n = 10 for Braf WT and n = 11 for Braf VE. d, Representative weight curves of Braf WT and Braf VE mice on control or PLX4720 diet. e, PLX4720 concentration in serum (ng ml−1), liver and brain (ng g−1) of 7–9-month-old Braf WT (n = 9) and Braf VE mice placed on the diet at 1 (n = 8) or at 3 months (n = 3) of age. Circles represent individual mice. f, Footprint assay quantification from Braf VE mice on PLX4720 diet at 1 month (n = 8) or at 3 months (n = 6) and control (Ctrl) diet (n = 13) and Braf WT (n = 32, black). Mice reaching paralysis were excluded from further analysis. See also g, where the dagger (†) indicates when Braf VE animals were euthanized. Values are mean ± s.d. Two-way ANOVA comparing treated and untreated Braf VE mice. *P < 0.05. g, Disease progression of Braf VE mice on control or PLX4720 diet. †Animal euthanasia owing to paralysis.

  9. Microglia activation in the brain starts at early, preclinical stages.
    Extended Data Fig. 5: Microglia activation in the brain starts at early, preclinical stages.

    a, Histological analysis by haematoxylin and eosin (HE) and luxol fast blue–PAS (LFB–PAS) and immunohistochemistry analysis of T cells (CD3), B cells (B220) and astrocyte activation (GFAP) in one-month-old Braf VE mice and Braf WT littermates. Representative of n = 5 Braf WT and n = 4 Braf VE mice. b, Immunohistochemistry analysis and quantification of IBA1+ cell density, cortical neurons (NeuN) and expression of amyloid precursor protein (APP), a positive signal for neurodegeneration in one-month-old Braf VE mice and Braf WT. Representative of n = 5 Braf WT and n = 4 Braf VE mice. Circles represent individual mice. Scale bars, 100 μm and 10 μm (insets). Unpaired two-tailed t-test.

  10. The neurodegenerative process in Braf VE mice.
    Extended Data Fig. 6: The neurodegenerative process in Braf VE mice.

    a, IBA1 and GFAP immunohistochemistry of brain and spinal cord from six-month-old Braf VE and Braf WT mice. Anatomical regions of insets are indicated. Representative of n = 5 Braf WT and n = 4 Braf VE mice. Scale bars, 500 μm (spinal cords), 1 mm (brains) and 50 μm (insets). b, Immunohistochemistry and immunofluorescence as used for quantification in Fig. 2h of brain stem for NeuN (neurons), APP (amyloid precursor protein) and GFAP (astrocytes), IBA1+LAMP2+ cells (phagocytosis), synaptophysin (Syn) and homer1 (synapse density) and staining with LFB–PAS. Scale bars, 100 μm and 10 μm (insets); 25 μm (IBA1 LAMP2); 10 μm (Syn Homer1). Representative of 6–9-month old Braf WT (n = 5), Braf VE (n = 4) mice and Braf VE mice on the PLX4720 diet (n = 4–6). c, LFB staining of spinal cord samples from a. Scale bar, 100 μm. d, Immunohistochemistry of brain stem for B220 (B cells) from Braf VE on control and PLX4720 diet. Representative of n = 4 mice per genotype. Scale bars, 100 μm and 10 μm (insets).

  11. Microglia and T-cell phenotype in Braf VE mice.
    Extended Data Fig. 7: Microglia and T-cell phenotype in Braf VE mice.

    a, Representative pERK staining in IBA1+ microglia as used for the quantification in Fig. 3b in brain stems of 5–9-month-old Braf WT and Braf VE mice on control or PLX4720 diet. Scale bar, 50 μm. b, Representative t-SNE analysis of flow cytometry staining of CD45+ cells from the brain of paralyzed Braf VE mice and littermate controls. Arrow indicates expansion of F4/80+YFP+ cells. Representative of n = 3 mice per genotype. c, FSC profile of YFP+ and YFP microglia from b obtained from Braf VE and Braf WT mice indicates an increase in YFP+ microglia cell size. Representative of n = 3 mice per genotype. d, Proportion of YFP+F4/80+ cells in indicated organs analysed by flow cytometry. The proportion of YFP+ among F4/80+ cells from Braf WTcre+ on control diet was normalized and set to one. Analysis was performed on 5–8-month-old Braf VE mice (n = 5–6) and Braf WT mice (n = 6) on control diet, and 7–9-month-old Braf VE mice (n = 6) and Braf WT mice (n = 4) on PLX4720 diet. Circles represent values for individual mice. One-way ANOVA. *P < 0.05, **P < 0.01. e, CD3 immunohistochemistry of brain and spinal cord of 6-month-old Braf VE and Braf WT mice. Anatomical regions of insets are indicated. Representative of n = 5 Braf WT and n = 4 Braf VE mice. Scale bars, 500 μm (spinal cords), 1 mm (brains) and 50 μm (insets). f, g, Analysis of CD8+, CD4+ and Foxp3+ T-cell numbers (f) and proliferation (g) in brain and spinal cord by flow cytometry in 5–8-month-old Braf VE (n = 4) and Braf WT (n = 6) mice on control diet, and 7–9-month-old Braf VE (n = 6) and Braf WT (n = 5) mice on PLX diet. Circles represent values for individual mice. One-way ANOVA.

  12. Analysis of Braf VE mice outside the central nervous system.
    Extended Data Fig. 8: Analysis of Braf VE mice outside the central nervous system.

    a, Proportion of YFP+F4/80+ cells in indicated organs analysed by flow cytometry. The proportion of YFP+ cells among F4/80+ cells from Braf WTcre+ (n = 6) was normalized and set to one (dotted line). Circles represent values for individual Braf VE mice (n = 7). Unpaired two-tailed t-test. b, Analysis of liver Kupffer cells as in a was performed on tissues from 5–8-month-old Braf VE (n = 5) and Braf WT (n = 4) mice on control diet, and 7–9-month-old Braf VE (n = 6) and Braf WT (n = 4) mice on PLX4720 diet. Circles represent values for individual mice. One-way ANOVA. *P < 0.05. c, Immunofluorescence analysis of pERK in F4/80+ Kupffer cells from 5–8-month-old Braf VE mice. Results are representative of n = 3. d, Serum analysis of Braf VE mice (score 1, n = 6) and littermate controls (n = 6). ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase. e, Gross liver, lung, kidney and spleen structure (haematoxylin and eosin, Trichrome) of paralyzed Braf VE and Braf WT mice. Representative of n = 7 mice per genotype. Scale bars, 200 μm and 10 μm (insets). f, Liver and spleen gross organs from paralyzed Braf VE and Braf WT mice. Representative of n = 5 mice per genotype.

  13. Patients with ECD.
    Extended Data Fig. 9: Patients with ECD.

    a, Table summarizing observed pathological and molecular findings in brain tissue of three patients with ECD with neurologic presentations. BRAF status was determined by immunohistochemical analysis and by sequencing. Neuronal loss and demyelination was determined by immunohistochemistry of neurofilament and myelin basic protein (MBP). RF, Rosenthal fibre. n/a, not applicable/no tissue available for further analysis. b, Immunohistochemistry and immmunofluorescence analysis of brain tissue from a patient with ECD for CD163, pERK and BRAF(V600E) (anti-BRAF-VE1 antibody). Scale bars, 200 μm (top) and 5 μm (bottom). c, Immunohistochemistry analysis of brain tissue from a patient with ECD for neurofilament and MBP shows areas of myelin deficits with preserved axons in the same region. Scale bar, 200 μm.

Videos

  1. Video 1: Hind limb paresis, 7-month-old BRAFVE mouse
    Video 1: Video 1: Hind limb paresis, 7-month-old BRAFVE mouse
    This video shows hind limb paresis in a 7-month-old BRAFVE mouse.
  2. Video 2: Axial rolling, 4-month-old BRAFVE mouse
    Video 2: Video 2: Axial rolling, 4-month-old BRAFVE mouse
    This video shows axial rolling in 4-month-old BRAFVE mouse (labelled by the black spot).
  3. Video 3: Hind limb paralysis of an 8-month-old BRAFVE mouse.
    Video 3: Video 3: Hind limb paralysis of an 8-month-old BRAFVE mouse.
    This video shows hind limb paralysis of an 8-month-old BRAFVE mouse.

Accession codes

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Gene Expression Omnibus

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Author information

  1. These authors contributed equally to this work.

    • Christian E. Jacome-Galarza &
    • Thomas Blank
  2. These authors jointly supervised this work.

    • Marco Prinz,
    • Omar Abdel-Wahab &
    • Frederic Geissmann

Affiliations

  1. Immunology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Elvira Mass,
    • Christian E. Jacome-Galarza,
    • Tomi Lazarov &
    • Frederic Geissmann
  2. Institute of Neuropathology, Faculty of Medicine, BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany

    • Thomas Blank,
    • Marius Schwabenland &
    • Marco Prinz
  3. Human Oncology and Pathogenesis Program, Leukemia Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Benjamin H. Durham,
    • Alessandro Pastore,
    • Young Rock Chung &
    • Omar Abdel-Wahab
  4. Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Neval Ozkaya &
    • Marc K. Rosenblum
  5. Centre for Molecular and Cellular Biology of Inflammation (CMCBI), King’s College London, London SE1 1UL, UK

    • Frederic Geissmann

Contributions

F.G. and E.M. designed the study, analysed data and wrote the manuscript. O.A.-W. and M.P. participated in study design and analysis. E.M. performed and analysed mouse experiments, cell sorting, flow cytometry, confocal microscopy of mouse and human samples, western blotting and behavioural assays with the help of C.E.J.-G. and T.L. T.B. and M.S. performed neuropathological analysis of mouse (Csf1r MeriCreMer;Braf LSL-V600E;Rosa26 LSL-YFP mice) and human brain and spinal cords. B.H.D. performed pathological examination of CD11ccre;Braf LSL-V600E and Csf1r MeriCreMer;Braf LSL-V600E;Rosa26LSL-YFP mice. M.K.R. and N.O. diagnosed and performed morphologic and immunophenotypic evaluation of brain biopsies from patients with ECD. A.P. performed primary and differential analysis of the RNA-seq data. Y.R.C. helped with mouse handling. All authors contributed to the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Analysis of one-month-old Csf1r MeriCreMer;Braf LSL-V600E;Rosa26 LSL-YFP mice. (781 KB)

    a, The percentage of mice born from the cross depicted in Fig. 1a according to their genotype (n = 42), but without injection of hydroxy-tamoxifen (4-OHT) to test for adverse effects of 4-OHT administration. b, Flow cytometry analysis of YFP expression in blood leukocytes. Representative of n = 8 mice per genotype. c, Flow cytometry analysis of YFP+ cells in the liver. YFP+ cells, present only in Csf1r MeriCreMer+ (Cre+) mice (top), were gated as F4/80+CD11b+ Kupffer cells (bottom). Representative of n = 8 mice per genotype. d, YFP expression by immunofluorescence in the liver of Braf VE and Braf WT mice. YFP+ cells are F4/80+ Kupffer cells. Representative of n = 6 mice per genotype. Scale bars, 200 μm and 5 μm (insets). e, Total tissue-resident macrophage cell numbers per gram (g) of tissue were analysed by flow cytometry in Braf VE mice (n = 4) and Braf WT (n = 6). Circles represent individual mice. Unpaired two-tailed t-test. f, In situ analysis of phospho-histone H3 (pHis3) staining in YFP+ cells from brains of Braf VE and Braf WT mice. Circles represent individual mice (n = 3). Unpaired two-tailed t-test. g, RNA-seq analysis, heatmap of MAPK target genes in YFP+ microglia from Braf VE (n = 3) and Braf WT (n = 2) mice, values are displayed as z scores. h, Histological analysis of liver, lung, kidney and spleen in Braf VE and Braf WT mice. HE, haematoxylin and eosin. Representative of n = 4 mice per genotype. Scale bars, 200 μm and 10 μm (insets).

  2. Extended Data Figure 2: Effect of constitutive Braf V600E expression in Csf1r-expressing cells. (299 KB)

    a, Breeding scheme. b, Embryonic lethality of Csf1r iCre+;Braf  LSL-V600E;Rosa26LSL-YFP mice, bars represent the percentage of mice born from the cross depicted in a according to their genotype (n = 39). c, Bright field (top) and epifluorescence microscopy (bottom) of Csf1r iCre+Braf  VE and Csf1r iCre+;Braf  WT embryos showing haemorrhagic foci in the liver (arrow) and accumulation of YFP+ cells in the fetal liver. A dead embryo is indicated by a dagger (†). Pictures are representative of n = 3 per genotype. d, The number of mouse embryos found alive during different developmental stages. Csf1r iCre+;Braf LSL-V600E;Rosa26LSL-YFP mice are associated with 100% lethality beyond E14.5. e, Liver weight of E12.5 embryos. Circles represent individual mice. n = 8 for WT;cre, n = 14 for VE;cre, n = 16 for VE;cre, n = 12 for VE;cre+. One-way ANOVA. f, Flow cytometry analysis of LinKit+ blast, erythroid cell (Ter119) and haematopoietic stem cell numbers (LSK CD150+CD48 and CD150CD48) in the E12.5 fetal liver and of E12.5 tissue-resident macrophages in the limbs, head and liver. Circles represent individual mice. n = 4 for Braf WT and n = 6 for Braf VE. Unpaired two-tailed t-test.

  3. Extended Data Figure 3: Analysis of the CD11cCre;Braf V600E mouse model. (929 KB)

    a, Kaplan–Meier survival curve of Braf VE (n = 16) and Braf WT (n = 66) controls. Log-rank (Mantel–Cox) test. b, Representative photographs of lung and spleen from Braf VE mice at time of death with representative Braf WT control organs. c, d, Haematoxylin and eosin staining of lung tissue from Braf VE and littermate controls. e, CD68 immunohistochemistry of Braf VE lung tissue. f, Haematoxylin and eosin staining of liver tissue from Braf VE and littermate controls with magnified image of granuloma in the Braf VE liver. g, Haematoxylin and eosin staining of bone marrow (BM) of Braf VE and littermate controls with CD68 immunohistochemistry of Braf VE mouse tissue. All images for bg are representative of n = 5 per genotype.

  4. Extended Data Figure 4: Longitudinal study and PLX treatment of the Csf1r MeriCreMer;Braf V600E;Rosa26 LSL-YFP mice. (508 KB)

    a, b, Latency to fall in the rotarod assay and footprint assay quantification for Braf VE mice (n = 7) and Braf WT littermates (n = 8). a, Rotarod assay at 1–4 months of age. Values are mean ± s.d. b, Rotarod and footprint assay at 4 months of age displaying single values. Mice that are score 1 are labelled in red. c, Footprint assay quantification of Braf VE mice at score 1 and littermate controls. Circles represent individual mice. n = 10 for Braf WT and n = 11 for Braf VE. d, Representative weight curves of Braf WT and Braf VE mice on control or PLX4720 diet. e, PLX4720 concentration in serum (ng ml−1), liver and brain (ng g−1) of 7–9-month-old Braf WT (n = 9) and Braf VE mice placed on the diet at 1 (n = 8) or at 3 months (n = 3) of age. Circles represent individual mice. f, Footprint assay quantification from Braf VE mice on PLX4720 diet at 1 month (n = 8) or at 3 months (n = 6) and control (Ctrl) diet (n = 13) and Braf WT (n = 32, black). Mice reaching paralysis were excluded from further analysis. See also g, where the dagger (†) indicates when Braf VE animals were euthanized. Values are mean ± s.d. Two-way ANOVA comparing treated and untreated Braf VE mice. *P < 0.05. g, Disease progression of Braf VE mice on control or PLX4720 diet. †Animal euthanasia owing to paralysis.

  5. Extended Data Figure 5: Microglia activation in the brain starts at early, preclinical stages. (765 KB)

    a, Histological analysis by haematoxylin and eosin (HE) and luxol fast blue–PAS (LFB–PAS) and immunohistochemistry analysis of T cells (CD3), B cells (B220) and astrocyte activation (GFAP) in one-month-old Braf VE mice and Braf WT littermates. Representative of n = 5 Braf WT and n = 4 Braf VE mice. b, Immunohistochemistry analysis and quantification of IBA1+ cell density, cortical neurons (NeuN) and expression of amyloid precursor protein (APP), a positive signal for neurodegeneration in one-month-old Braf VE mice and Braf WT. Representative of n = 5 Braf WT and n = 4 Braf VE mice. Circles represent individual mice. Scale bars, 100 μm and 10 μm (insets). Unpaired two-tailed t-test.

  6. Extended Data Figure 6: The neurodegenerative process in Braf VE mice. (682 KB)

    a, IBA1 and GFAP immunohistochemistry of brain and spinal cord from six-month-old Braf VE and Braf WT mice. Anatomical regions of insets are indicated. Representative of n = 5 Braf WT and n = 4 Braf VE mice. Scale bars, 500 μm (spinal cords), 1 mm (brains) and 50 μm (insets). b, Immunohistochemistry and immunofluorescence as used for quantification in Fig. 2h of brain stem for NeuN (neurons), APP (amyloid precursor protein) and GFAP (astrocytes), IBA1+LAMP2+ cells (phagocytosis), synaptophysin (Syn) and homer1 (synapse density) and staining with LFB–PAS. Scale bars, 100 μm and 10 μm (insets); 25 μm (IBA1 LAMP2); 10 μm (Syn Homer1). Representative of 6–9-month old Braf WT (n = 5), Braf VE (n = 4) mice and Braf VE mice on the PLX4720 diet (n = 4–6). c, LFB staining of spinal cord samples from a. Scale bar, 100 μm. d, Immunohistochemistry of brain stem for B220 (B cells) from Braf VE on control and PLX4720 diet. Representative of n = 4 mice per genotype. Scale bars, 100 μm and 10 μm (insets).

  7. Extended Data Figure 7: Microglia and T-cell phenotype in Braf VE mice. (411 KB)

    a, Representative pERK staining in IBA1+ microglia as used for the quantification in Fig. 3b in brain stems of 5–9-month-old Braf WT and Braf VE mice on control or PLX4720 diet. Scale bar, 50 μm. b, Representative t-SNE analysis of flow cytometry staining of CD45+ cells from the brain of paralyzed Braf VE mice and littermate controls. Arrow indicates expansion of F4/80+YFP+ cells. Representative of n = 3 mice per genotype. c, FSC profile of YFP+ and YFP microglia from b obtained from Braf VE and Braf WT mice indicates an increase in YFP+ microglia cell size. Representative of n = 3 mice per genotype. d, Proportion of YFP+F4/80+ cells in indicated organs analysed by flow cytometry. The proportion of YFP+ among F4/80+ cells from Braf WTcre+ on control diet was normalized and set to one. Analysis was performed on 5–8-month-old Braf VE mice (n = 5–6) and Braf WT mice (n = 6) on control diet, and 7–9-month-old Braf VE mice (n = 6) and Braf WT mice (n = 4) on PLX4720 diet. Circles represent values for individual mice. One-way ANOVA. *P < 0.05, **P < 0.01. e, CD3 immunohistochemistry of brain and spinal cord of 6-month-old Braf VE and Braf WT mice. Anatomical regions of insets are indicated. Representative of n = 5 Braf WT and n = 4 Braf VE mice. Scale bars, 500 μm (spinal cords), 1 mm (brains) and 50 μm (insets). f, g, Analysis of CD8+, CD4+ and Foxp3+ T-cell numbers (f) and proliferation (g) in brain and spinal cord by flow cytometry in 5–8-month-old Braf VE (n = 4) and Braf WT (n = 6) mice on control diet, and 7–9-month-old Braf VE (n = 6) and Braf WT (n = 5) mice on PLX diet. Circles represent values for individual mice. One-way ANOVA.

  8. Extended Data Figure 8: Analysis of Braf VE mice outside the central nervous system. (801 KB)

    a, Proportion of YFP+F4/80+ cells in indicated organs analysed by flow cytometry. The proportion of YFP+ cells among F4/80+ cells from Braf WTcre+ (n = 6) was normalized and set to one (dotted line). Circles represent values for individual Braf VE mice (n = 7). Unpaired two-tailed t-test. b, Analysis of liver Kupffer cells as in a was performed on tissues from 5–8-month-old Braf VE (n = 5) and Braf WT (n = 4) mice on control diet, and 7–9-month-old Braf VE (n = 6) and Braf WT (n = 4) mice on PLX4720 diet. Circles represent values for individual mice. One-way ANOVA. *P < 0.05. c, Immunofluorescence analysis of pERK in F4/80+ Kupffer cells from 5–8-month-old Braf VE mice. Results are representative of n = 3. d, Serum analysis of Braf VE mice (score 1, n = 6) and littermate controls (n = 6). ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase. e, Gross liver, lung, kidney and spleen structure (haematoxylin and eosin, Trichrome) of paralyzed Braf VE and Braf WT mice. Representative of n = 7 mice per genotype. Scale bars, 200 μm and 10 μm (insets). f, Liver and spleen gross organs from paralyzed Braf VE and Braf WT mice. Representative of n = 5 mice per genotype.

  9. Extended Data Figure 9: Patients with ECD. (248 KB)

    a, Table summarizing observed pathological and molecular findings in brain tissue of three patients with ECD with neurologic presentations. BRAF status was determined by immunohistochemical analysis and by sequencing. Neuronal loss and demyelination was determined by immunohistochemistry of neurofilament and myelin basic protein (MBP). RF, Rosenthal fibre. n/a, not applicable/no tissue available for further analysis. b, Immunohistochemistry and immmunofluorescence analysis of brain tissue from a patient with ECD for CD163, pERK and BRAF(V600E) (anti-BRAF-VE1 antibody). Scale bars, 200 μm (top) and 5 μm (bottom). c, Immunohistochemistry analysis of brain tissue from a patient with ECD for neurofilament and MBP shows areas of myelin deficits with preserved axons in the same region. Scale bar, 200 μm.

Supplementary information

Video

  1. Video 1: Video 1: Hind limb paresis, 7-month-old BRAFVE mouse (3.81 MB, Download)
    This video shows hind limb paresis in a 7-month-old BRAFVE mouse.
  2. Video 2: Video 2: Axial rolling, 4-month-old BRAFVE mouse (5.28 MB, Download)
    This video shows axial rolling in 4-month-old BRAFVE mouse (labelled by the black spot).
  3. Video 3: Video 3: Hind limb paralysis of an 8-month-old BRAFVE mouse. (3.94 MB, Download)
    This video shows hind limb paralysis of an 8-month-old BRAFVE mouse.

PDF files

  1. Supplementary Information (2.8 MB)

    This file contains Supplementary Figure 1 (gel source data) and Supplementary Figure 2 which shows the gating strategy for different tissues and cell types.

  2. Reporting Summary (76 KB)

Excel files

  1. Supplementary Table 1 (399 KB)

    RNA-seq analysis, complement to figure 1: list of genes differentially expressed in macrophages from one-month old BRAFVE and BRAFWT littermates (FACS-sorted microglia and Kupffer cells).

  2. Supplementary Table 2 (125 KB)

    RNA-seq analysis, complement to figure 1: GSEA, GO, KEGG and REACTOME analysis of differentially expressed genes in microglia and Kupffer cells in one-month old BRAFVE and BRAFWT littermates.

  3. Supplementary Table 3 (1.2 MB)

    RNA-seq analysis, complement to figure 4: list of genes differentially expressed and GSEA and KEGG analysis in YFP+ microglia from 7 months-old BRAFVE and BRAFWT littermates.

  4. Supplementary Table 4 (2.3 MB)

    Differentially expressed genes from RNA-seq of brain tissue from control and histiocytosis patients.

  5. Supplementary Table 5 (40 KB)

    A list of antibodies used for flow cytometry.

  6. Supplementary Table 6 (10 KB)

    Clinical and pathological characteristics of human tissue samples from ECD patients and age-matched controls

Additional data