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

Abstract

C9ORF72 mutations are found in a significant fraction of patients suffering from amyotrophic lateral sclerosis and frontotemporal dementia, yet the function of the C9ORF72 gene product remains poorly understood. We show that mice harboring loss-of-function mutations in the ortholog of C9ORF72 develop splenomegaly, neutrophilia, thrombocytopenia, increased expression of inflammatory cytokines, and severe autoimmunity, ultimately leading to a high mortality rate. Transplantation of mutant mouse bone marrow into wild-type recipients was sufficient to recapitulate the phenotypes observed in the mutant animals, including autoimmunity and premature mortality. Reciprocally, transplantation of wild-type mouse bone marrow into mutant mice improved their phenotype. We conclude that C9ORF72 serves an important function within the hematopoietic system to restrict inflammation and the development of autoimmunity.

Fig. 1. A loss of function mutation in the C9orf72 ortholog

(A) The schematic illustrates replacement of exons 2-6 to generate the KOMP allele and crosses with Sox2-cre mice to generate the Neo-deleted allele. (B) Frequency of genotypes born from KOMP +/- crosses. (C) C9orf72 expression in KOMP whole blood by quantitative RT-PCR. **p<0.01 Tukey's multiple comparisons. ns not significant. (D) Western blotting of cortical tissue from the 3 KOMP genotypes using anti-C9ORF72 antibodies. (E) Quantification of Western blot bands. *p<0.05 **p<0.01 Tukey's multiple comparisons. (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's 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 C57BL/6 background (KOMP) and those outcrossed with Sox2-cre expressing mice to remove the neomycin cassette (Neo deleted), were aged for survival studies. Kaplan Meier survival curves for (A) KOMP and (B) Neo deleted mice. *p<0.05, **p<0.01 Generalized Wilcoxon test. 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 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 & eosin staining of spleens from Neo deleted mice at day 300. Scale bar (i) 500 micron and (ii) 50 micron. (C, D) Quantification of spleen weight in (C) end stage KOMP and Neo deleted animals and (D) aged Neo deleted animals. C,E,F *p<0.05, **p<0.01 Tukey's multiple comparisons. (D) *p<0.05, **p<0.01 Student t-test. ns not significant. (E) Splenocyte counts from day 200 Neo deleted mice. (F) Quantification of splenocyte subsets in day 200 Neo deleted mice. (G) PCR analysis of T cell and B cell clonality in the spleens of day 400 Neo deleted mice. LN2 and LN3 represent ESCs generated by nuclear transfer from lymph node-derived T cells that harbor monoclonal TCRβ rearrangements.

Fig. 4. Mice with C9orf72 mutations develop hematological phenotypes

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

(A, 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's multiple comparisons. ns not significant. (C, D) Anti-double stranded (ds)DNA antibody reactivity in plasma of (C) day 300+ KOMP and Neo deleted animals and (D) aged Neo deleted animals. (E, 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 or C9orf72 deficient animals were lethally irradiated at day 110 and reconstituted with wild type or mutant bone marrow. Recipient mice were regularly weighed, monitored for survival and bled for whole blood counts and plasma analyses and necropsied at end stage. (B, C) Quantification of flow cytometry-based assessment of 17 week post-transplant peripheral blood reconstitution. (B,C, F-K) Each dot represents one mouse. (D) Survival curves for transplanted mice. *p<0.05 Generalized Wilcoxon test. (E) Average body weight ± SEM. *p<0.05 Dunnett's multiple comparisons. (F) End-stage spleen weight. (F-K) *p<0.05, **p<0.01 Tukey's multiple comparisons. ns not significant. (G) End-stage peripheral blood neutrophil counts. (H) End-stage peripheral blood platelet counts. (I) End-stage hematocrit. (J-K) Plasma anti-dsDNA antibody activity in animals at (J) day 230 and (K) end-stage.

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

(A) Schematic showing the CRISPR/Cas9 targeting strategy to cause DNA double strand breaks in exon 4 of C9orf72, resulting in several distinct mutations. (B) DNA sequences from CRISPR/Cas9-targeted mice indicated that 23/24 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 age-matched C57BL/6 control. (E) Spleen weights for CRISPR/Cas9 mutant and KOMP mice. (E-G) Each dot represents one mouse. *p<0.05 **p<0.05 Dunn's multiple comparisons. (F) Hematocrit for day 300+ CRISPR/Cas9-targeted animals. (G) Plasma anti-dsDNA antibody activity in day 300+ CRISPR/Cas9 targeted animals. (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's multiple comparisons. ns not significant. (J) Proposed model for how C9orf72 may act in bone marrow-derived cells to limit fatal immune deregulation.