Research ArticleALS

Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease

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Science Translational Medicine  13 Jul 2016:
Vol. 8, Issue 347, pp. 347ra93
DOI: 10.1126/scitranslmed.aaf6038
  • Fig. 1. A loss-of-function mutation in the C9orf72 ortholog.

    (A) The schematic illustrates replacement of exons 2 to 6 to generate the KOMP allele and crosses with Sox2-cre mice to generate the Neo-deleted allele. 3′ HA, 3′ homology arm; FRT, flippase recognition target. (B) Frequency of genotypes born from KOMP +/− crosses. (C) C9orf72 expression in KOMP whole blood by quantitative reverse transcription polymerase chain reaction (RT-PCR). **P < 0.01, Tukey multiple comparisons. (D) Western blotting of cortical tissue from the three KOMP genotypes using anti-C9ORF72 antibodies. (E) Quantification of Western blot bands. *P < 0.05, **P < 0.01, Tukey multiple comparisons. ns, not significant. Ab, antibody. (F) Frequency of genotypes born from Neo-deleted +/− crosses. (G) C9orf72 expression in Neo-deleted whole blood by quantitative RT-PCR. **P < 0.01, Tukey multiple comparisons.

  • Fig. 2. C9orf72 mutations lead to premature death of mice.

    Mice harboring loss-of-function mutations in C9orf72, generated by homologous recombination using a targeting vector in embryonic stem cells on a C57BL/6 background (KOMP) and those outcrossed with Sox2-cre–expressing mice to remove the neomycin cassette (Neo-deleted), were aged for survival studies. (A and B) Kaplan-Meier survival curves for (A) KOMP and (B) Neo-deleted mice. *P < 0.05, **P < 0.01, generalized Wilcoxon test. (C and D) Body weight of female (C) KOMP mice and (D) Neo-deleted animals over time. *P < 0.05, Dunnett’s multiple comparisons. (E) Cause of death or obligatory euthanasia in KOMP and Neo-deleted mice. “Days of decline” indicates the time between maximum body weight (onset) and death or obligatory euthanasia.

  • Fig. 3. Identity of cells within the enlarged mutant spleens of Neo-deleted mice.

    (A) Spleens from day 300 Neo-deleted animals. Scale bar, 1 cm. (B) Hematoxylin and eosin staining of spleens from Neo-deleted mice at day 300. Scale bar, 500 μm (i) and 50 μm (ii). (C and D) Quantification of spleen weight in (C) end-stage KOMP and Neo-deleted animals and (D) aged Neo-deleted animals. (E) Splenocyte counts from day 200 Neo-deleted mice. (F) Quantification of splenocyte subsets in day 200 Neo-deleted mice. NKs, natural killer cells. DCs, dendritic cells. (C, E, and F) *P < 0.05, **P < 0.01, Tukey multiple comparisons. (D) *P < 0.05, **P < 0.01, Student’s t test. ns, not significant. (G) PCR analysis of T and B cell clonality in the spleens of day 400 Neo-deleted mice. LN2 and LN3 represent embryonic stem cells generated by nuclear transfer from lymph node–derived T cells that harbor monoclonal TCRβ (T cell receptor β) rearrangements.

  • Fig. 4. Mice with C9orf72 mutations develop hematological phenotypes.

    (A to H) Peripheral blood counts assessed for day 300+ KOMP and Neo-deleted animals. *P < 0.05, **P < 0.01, Tukey multiple comparisons. ns, not significant. (A) White blood cells. (B) Neutrophils. (C) Lymphocytes. (D) Monocytes. (E) Platelets. (F) Red blood cells (RBCs). (G) Hematocrit. (H) Mean corpuscular volume. (I to K) Peripheral blood counts assessed for aged Neo-deleted animals. (I) White blood cells. (J) Neutrophils. (K) Platelets.

  • Fig. 5. Mice with C9orf72 mutations show increased cytokines, chemokines, and autoantibodies.

    (A and B) Analysis of 36 plasma cytokines and chemokines in day 300+ (A) KOMP and (B) Neo-deleted animals. *P < 0.05, **P < 0.01, Tukey multiple comparisons. (C and D) Anti-dsDNA antibody reactivity in plasma of (C) day 300+ KOMP and Neo-deleted animals and (D) aged Neo-deleted animals. *P < 0.05, **P < 0.01, Tukey multiple comparisons. ns, not significant. (E and F) Plasma from day 300+ Neo-deleted mice assessed for (E) IgM and (F) IgG reactivity against 124 self-antigens (26). Unsupervised hierarchical clustering grouped individual animals (x axis) and self-antigens (y axis).

  • Fig. 6. C9orf72 acts in bone marrow–derived cells to prevent autoimmunity.

    (A) Wild-type (WT) or C9orf72-deficient animals were lethally irradiated at day 110 and reconstituted with WT or mutant bone marrow. Recipient mice were regularly weighed, monitored for survival, and bled for whole-blood cell counts and plasma analyses, and animals were necropsied at end stage. (B and C) Quantification of flow cytometry–based assessment of 17-week posttransplant peripheral blood reconstitution. (B, C, and F to K) Each dot represents one mouse. (D) Survival curves for transplanted mice. *P < 0.05, generalized Wilcoxon test. IR, irradiation. (E) Average body weight ± SEM. *P < 0.05, Dunnett’s multiple comparisons. (F) End-stage spleen weight. (G) End-stage peripheral blood neutrophil counts. (H) End-stage peripheral blood platelet counts. (I) End-stage hematocrit. (J and K) Plasma anti-dsDNA antibody activity in animals at (J) day 230 (D230) and (K) end stage. (F to K) *P < 0.05, **P < 0.01, Tukey multiple comparisons. ns, not significant.

  • Fig. 7. CRISPR/Cas9-induced mutations in C9orf72 lead to autoimmunity.

    (A) Schematic showing the CRISPR/Cas9-targeting strategy that causes DNA double-strand breaks in exon 4 of C9orf72, resulting in several distinct mutations. (B) DNA sequences from CRISPR/Cas9-targeted mice indicated that 23 of 24 animals harbored a stop codon in exon 4 of the C9orf72 gene. (C) Survival of CRISPR/Cas9-targeted mice. (D) Spleens from a CRISPR/Cas9-targeted mouse at end stage and an age-matched C57BL/6 control. (E) Spleen weights for CRISPR/Cas9 mutant and KOMP mice. (F) Hematocrit for day 300+ CRISPR/Cas9-targeted animals. (G) Plasma anti-dsDNA antibody activity in day 300+ CRISPR/Cas9-targeted animals. (E to G) Each dot represents one mouse. *P < 0.05, **P < 0.05, Dunnett’s multiple comparisons. ns, not significant. (H) Mice harboring a 38-nucleotide deletion in exon 4 of C9orf72 were bred to heterozygosity (+/del) and homozygosity (del/del), as visualized by PCR of tail DNA using primers flanking the deletion site. (I) Plasma anti-dsDNA antibody reactivity in day 150 +/+, +/del, and del/del animals. **P < 0.01, Tukey multiple comparisons. (J) Proposed model for how C9orf72 may act in bone marrow–derived cells to limit fatal immune deregulation.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/347/347ra93/DC1

    Text

    Fig. S1. Validation of C9orf72 loss-of-function allele.

    Fig. S2. Analysis of spinal motor neurons.

    Fig. S3. Motor cortex histology.

    Fig. S4. Histology of thalamus, CA1, and cerebellum.

    Fig. S5. GFAP expression in the central nervous system.

    Fig. S6. Individual mouse weight curves after disease onset.

    Fig. S7. Splenocyte-gating scheme.

    Fig. S8. Bone marrow histology and cell counts.

    Fig. S9. Analysis of B and T cells in cervical lymph node.

    Fig. S10. Hematopoietic liver infiltrates occur in a subset of mutant animals.

    Fig. S11. Analysis of cytokines and chemokines.

    Fig. S12. Analysis of CD4+ CD25+ cells.

    Fig. S13. Cellular and organismal phenotypes after bone marrow transplant.

    Fig. S14. CRISPR/Cas9-targeted mutations in exon 4 of C9orf72.

    Table S1. Analysis of autoantibodies in Neo-deleted mice.

    Table S2. CRISPR/Cas9-induced mutations in C9orf72 exon 4.

    Video 1. End-stage −/− marrow donor to −/− recipient.

    Video 2. End-stage −/− marrow donor to WT recipient.

    Video 3. End-stage WT marrow donor to −/− recipient.

  • Supplementary Material for:

    Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease

    Aaron Burberry, Naoki Suzuki, Jin-Yuan Wang, Rob Moccia, Daniel A. Mordes, Morag H. Stewart, Satomi Suzuki-Uematsu, Sulagna Ghosh, Ajay Singh, Florian T. Merkle, Kathryn Koszka, Quan-Zhen Li, Leonard Zon, Derrick J. Rossi, Jennifer J. Trowbridge, Luigi D. Notarangelo, Kevin Eggan*

    *Corresponding author. Email: eggan{at}mcb.harvard.edu

    Published 13 July 2016, Sci. Transl. Med. 8, 347ra93 (2016)
    DOI: 10.1126/scitranslmed.aaf6038

    This PDF file includes:

    • Text
    • Fig. S1. Validation of C9orf72 loss-of-function allele.
    • Fig. S2. Analysis of spinal motor neurons.
    • Fig. S3. Motor cortex histology.
    • Fig. S4. Histology of thalamus, CA1, and cerebellum.
    • Fig. S5. GFAP expression in the central nervous system.
    • Fig. S6. Individual mouse weight curves after disease onset.
    • Fig. S7. Splenocyte-gating scheme.
    • Fig. S8. Bone marrow histology and cell counts.
    • Fig. S9. Analysis of B and T cells in cervical lymph node.
    • Fig. S10. Hematopoietic liver infiltrates occur in a subset of mutant animals.
    • Fig. S11. Analysis of cytokines and chemokines.
    • Fig. S12. Analysis of CD4+ CD25+ cells.
    • Fig. S13. Cellular and organismal phenotypes after bone marrow transplant.
    • Fig. S14. CRISPR/Cas9-targeted mutations in exon 4 of C9orf72.
    • Table S1. Analysis of autoantibodies in Neo-deleted mice.
    • Table S2. CRISPR/Cas9-induced mutations in C9orf72 exon 4.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Video 1 (.wmv format). End-stage −/− marrow donor to −/− recipient.
    • Video 2 (.wmv format). End-stage −/− marrow donor to WT recipient.
    • Video 3 (.wmv format). End-stage WT marrow donor to −/− recipient.

    [Download Videos]

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