C9orf72 is required for proper macrophage and microglial function in mice

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Science  18 Mar 2016:
Vol. 351, Issue 6279, pp. 1324-1329
DOI: 10.1126/science.aaf1064

Linking neurodegeneration and immune cells

The expansion of a repetitive DNA sequence in the C9orf72 gene is the major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Although the expansion decreases C9orf72 expression, most research has focused on the toxic RNA and protein products it creates in neurons. O'Rourke et al. found that C9orf72 unexpectedly plays a key role in innate immune cells. Loss of C9orf72 in mice led to macrophage and microglial dysfunction and age-related neuroinflammation. This raises the possibility of a “dual-effect” disease mechanism, in which toxic byproducts in neurons are combined with microglial dysfunction from decreased C9orf72 expression, together promoting neurodegeneration.

Science, this issue p. 1324


Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Decreased expression of C9orf72 is seen in expansion carriers, suggesting that loss of function may play a role in disease. We found that two independent mouse lines lacking the C9orf72 ortholog (3110043O21Rik) in all tissues developed normally and aged without motor neuron disease. Instead, C9orf72 null mice developed progressive splenomegaly and lymphadenopathy with accumulation of engorged macrophage-like cells. C9orf72 expression was highest in myeloid cells, and the loss of C9orf72 led to lysosomal accumulation and altered immune responses in macrophages and microglia, with age-related neuroinflammation similar to C9orf72 ALS but not sporadic ALS human patient tissue. Thus, C9orf72 is required for the normal function of myeloid cells, and altered microglial function may contribute to neurodegeneration in C9orf72 expansion carriers.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders with overlapping clinical presentations, pathology, and genetic origins (1, 2). Expansions of a GGGGCC hexanucleotide repeat in the first intron/promoter of the C9orf72 gene are the most commonly identified genetic cause of ALS/FTD (3, 4) and are found in other neurodegenerative diseases (5). Microglial dysfunction is strongly tied to ALS/FTD pathogenesis (6), with mutations in progranulin causing FTD (7, 8) and variants in the microglial expressed genes TREM2 and TBK1 implicated in ALS (911). However, no connection has been made between microglial function and C9orf72, where focus instead has been on its role in neurons (12, 13). Although the repeat expansion leads to decreased C9orf72 expression in human patient tissues, most research has focused on gain-of-function toxicity as the primary mechanism in disease rather than loss of function (1418).

To investigate the function of the mouse ortholog of C9orf72 (3110043O21Rik, referred to as C9orf72 below), we analyzed two independent loss-of-function alleles in mice (figs. S1 and S2). C9orf72+/− and C9orf72−/− mice showed normal weight gain and life span; had normal sensorimotor coordination, limb strength, femoral motor and sensory axon counts, and muscle electrophysiology; and showed no evidence of neurodegeneration on histology through advanced age (17 months) (figs. S1 to S3). The only histologic abnormalities in the nervous system were rare chromatolytic structures seen with hemotoxylin and eosin (H&E) staining, found in both gray and white matter of the spinal cord, that did not increase with age or show reactive gliosis (fig. S3). All studies were performed using the Knockout Mouse Project line except where specified.

C9orf72−/− mice from both lines developed visibly enlarged cervical lymph nodes and spleens (Fig. 1, A and B), detectable as early as 1 month after birth, that slowly enlarged with age (Fig. 1C and fig. S2). No gross or histological defects were observed in other organs at 5 months of age. Histology of lymph nodes and the white pulp of the spleen showed disruption of the normal follicular structure by enlarged debris-filled cells (Fig. 1D) that expressed CD11b and contained ubiquitin- and p62-positive vacuoles consistent with macrophages (Fig. 2A and fig. S4). Immunoblotting confirmed increased amounts of p62 and LC3 proteins, indicating an increase in components of the autophagy machinery in homozygote spleens (Fig. 2B). Massive up-regulation of Trem2 expression was observed in C9orf72−/− spleens, a cell surface receptor expressed by macrophages and monocytes, as were inflammatory cytokines, including IL-1β, IL-6, and IL-10 (Fig. 2C). Despite the altered follicular architecture, there were no differences in the proportions of B cells, T cells, or CD11b+ myeloid cells (Fig. 2D and fig. S2J). However, flow cytometry revealed changes in myeloid subsets, including the emergence of a CD11b+Ly6CLy6Gint population unique to C9orf72−/− mice, and a decrease in F4/80+ red pulp macrophages, indicating that C9orf72 deficiency has a selective effect on myeloid populations in the spleen (Fig. 2, E to G). Complete blood counts and flow cytometry of bone marrow were normal in C9orf72−/− mice at 5 months (fig. S5), supporting the idea that splenic enlargement was not related to deficient hematopoiesis in bone marrow.

Fig. 1 Generation of C9orf72 (3110043O21Rik) null mice.

(A) Gross images of cervical lymphadenopathy (arrows) in C9orf72−/− mice (9 months of age). Wt, wild-type; homo, homozygote. (B) Gross images of splenomegaly (12 months of age). (C) Spleen weights (in milligrams) normalized to body weight (in grams) at indicated ages [***P = 0.0008, ****P < 0.0001, two-way analysis of variance (ANOVA)]. (D) H&E staining of wild-type and homozygote lymph nodes and spleens at 5 months (top; scale bar = 3 mm) showing disruption of follicular architecture in null mice by large cells with swollen cytoplasm. Higher-magnification images shown in bottom panels; scale bars = 100 μm and 10 μm (lymph node) and 300 μm and 10 μm (spleen).

Fig. 2 C9orf72 null mice develop progressive splenomegaly with engorged macrophages, altered monocyte populations, and inflammation.

(A) Enlarged cells in homozygote spleens (5 months) stained for CD11b and containing p62 and ubiquitin (Ub) accumulations. Scale bars = 100 μm and 20 μm. (B) Immunoblot of spleen lysates showed an increase in p62 and LC3 in C9orf72−/− mice (n = 3; 14 months). (C) qRT-PCR analysis of spleens (14 months) showed an increase in macrophage marker Trem2 (**P = 0.008), and cytokines IL-10 (*P = 0.035), IL-6 (****P < 0.0001), and IL-1β (****P < 0.0001; one-way ANOVA). (D) Immunostains of wild-type and C9orf72−/− spleens (5 months) for CD20 (B cells), CD3 (T cells), and F4/80 (red pulp macrophages). The dashed outline highlights the region of abnormal CD11b+ cells in the C9orf72−/− spleens. Scale bars = 1 mm and 300 μm. (E) FACS analysis of spleens (5 months). (F) Dot plots and (G) bar graphs showed a unique population of CD11b+ Ly6C-Ly6Gint cells in C9orf72−/− spleens and a decrease in F4/80+ red pulp macrophages as compared to wild-type mice or hemizygotes (n = 4; 5 months) (**P = 0.01, one-way ANOVA).

Given the progressive splenomegaly with altered myeloid cells, and the buildup of engorged macrophages with accumulations of LC3 and p62 in the spleens of C9orf72−/− mice, we hypothesized that C9orf72 protein is important for endosomal trafficking in macrophages. We first examined the expression of C9orf72 by fluorescence-activated cell sorting (FACS) of different populations from wild-type mouse spleens and found that C9orf72 was expressed at high levels in CD11b+ (myeloid cell), as compared to CD3+ (T cell) and CD19+ (B cell) populations (Fig. 3A). Query of the immunological genome project ( confirmed that the expression of C9orf72 was highest in macrophages and dendritic cells as compared to other immune cells (fig. S6, A and B). Pathway analysis (19) of the 35 genes in the C9orf72 constellation was significant for only one pathway, lysosomal function (Bonferroni P = 2.32−6) (fig. S6C). To examine whether C9orf72 is necessary for macrophage function, we differentiated bone marrow–derived macrophages (BMDMs) from C9orf72−/− mice and stained them for endosomal markers. BMDMs from C9orf72−/− mice showed marked accumulation of LysoTracker- and Lamp1-positive vesicles, indicating a defect in late endosome/lysosomal trafficking (Fig. 3, B and C). No changes in the early or late endosomal markers Rab5 or Rab7 were observed (figs. S7 and S8). The accumulation of LysoTracker- and Lamp1-positive vesicles was rescued by viral expression of human C9orf72, indicating that this defect was due to the loss of C9orf72 (Fig. 3, D and E). C9orf72−/− BMDMs showed normal initial phagocytosis of zymosan particles (Fig. 3F); however, BMDMs from both C9orf72−/− and to a lesser extent C9orf72+/− mice showed enhanced production of phagocyte oxidase-derived reactive oxygen species (ROS) after feeding with zymosan particles (Fig. 3G), which has been reported in cells with defective fusion of phagosomes to lysosomes (20). BMDMs from C9orf72−/− and C9orf72+/− mice also showed enhanced cytokine production in response to several immune stimuli, including those sensed in endosomal/lysosomal compartments such as peptidoglycan, CpG, and silica (Fig. 3, H and I). Thus, C9orf72 is critical for the proper function of macrophages, and the loss of C9orf72 leads to a pro-inflammatory state that probably drives the splenic and lymph node hyperplasia. Although hemizygous mice did not have a phenotype at the tissue level, haploinsufficiency of C9orf72 led to altered inflammatory responses in macrophages at the cellular level, which could lead to a physiological phenotype when the system is stressed.

Fig. 3 Analysis of macrophages and microglia from C9orf72-deficient mice.

(A) qRT-PCR analysis from B cells, T cells, and CD11b+ cells FAC-sorted from wild-type mouse spleens (n = 2). (B and C) BMDMs from C9orf72−/− mice showed accumulation of LysoTracker- and Lamp1-stained vesicles as compared to wild-type mice. Scale bars = 50 μm and 20 μm. (D) C9orf72−/− BMDMs treated with lentivirus encoding either human C9orf72 isoform 1-IRES-GFP (hC9-iso1) or isoform 2-IRES-GFP (hC9-iso2). LysoTracker (top panel) or Lamp1 (bottom panel) accumulation was rescued by either hC9-iso1 or hC9-iso2 (top panel). Arrow, hC9-iso1 infected cell; asterisk, uninfected cell. (E) Quantitation of LysoTracker accumulation in BMDMs of the indicated genotype or homozygotes treated with hC9-iso1 and hC9-iso2 lentivirus (***P = 0.0002, **P = 0.0018, one-way ANOVA). (F) BMDMs fed with fluorescent zymosan particles for 15 min and then analyzed by FACS analysis. (G) ROS production by BMDMs after zymosan ingestion in indicated genotypes (****P = <0.0001, two-way ANOVA). (H) C9orf72+/− and C9orf72−/− BMDMs showed increased TNF-α production after stimulation with Pam3CSK4 (Pam), peptidoglycan (PGN), and CpG, but not lipopolysaccharide (LPS) (****P < 0.0001, ***P = 0.0002, two-way ANOVA; N.D., not detected). (I) IL-1β production after stimulation with silica (*P < 0.05, two-way ANOVA). (J) RNA-seq of C9orf72 in indicated cell types from the cerebral cortex (21). (K) qRT-PCR of C9orf72 from neurons and microglia isolated from the adult mouse brain. (L) Microglia purified from C9orf72−/− mice showed accumulation of LysoTracker- and Lamp1-positive enlarged vesicles. (M) Quantification of percentage of microglia with enlarged LysoTracker-positive vesicles (*P = 0.027, one-tailed t test).

The defects in C9orf72−/− BMDMs raised the possibility that other myeloid cells, including resident microglia in the brain, also require C9orf72 for normal function. Although an earlier report suggested that microglia express low levels of C9orf72 (12), we observed that microglia showed the highest levels of C9orf72 expression of any cell type in the brain in published data sets (2123) (Fig. 3J) and in quantitative reverse transcription polymerase chain reaction (qRT-PCR) of cells isolated from adult mouse brains (Fig. 3K). Microglia from C9orf72−/− mice showed accumulation of LysoTracker- and Lamp1-positive structures, similar to BMDMs (Fig. 3, L and M), whereas primary cortical neurons did not (fig. S9). To probe the functional state of microglia lacking C9orf72, we performed qRT-PCR on spinal cord microglia isolated from C9orf72−/− mice and found increased levels of cytokines IL-6 and IL-1b, supporting the idea that the altered lysosomal function leads to a proinflammatory state (Fig. 4A) similar to that observed in BMDMs.

Fig. 4 Neuroinflammation in C9orf72−/− mice and C9orf72 expansion human patient tissue.

(A) qRT-PCR of inflammatory cytokines (IL-6 and IL-1β) in microglia isolated from C9orf72−/− mice (***P = 0.0007; **** P = <0.0001, one-way ANOVA). (B) Tables showing the number of up- and down-regulated pathways on GSEA (FDR < 0.05) of RNA-seq from 3- and 17-month-old lumbar spinal cords. (C) Table of up-regulated pathways in C9orf72−/− versus C9orf72+/− and wild-type mouse spinal cords (FDR < 0.05) at 17 months. Pathways up-regulated in both C9orf72−/− mice and human C9-ALS brain tissue are highlighted in red. (D) (Top) Venn diagrams showing overlap between the 19 up-regulated pathways in C9orf72−/− mice from (C) and those up-regulated in the cortex or cerebellum of sporadic ALS (left) or C9orf72 ALS (right). (Bottom) Venn diagrams for the immune pathways from (C). (E) Human motor cortex and spinal cord tissue from C9-ALS and sALS cases double-labeled with Iba1 (red) to identify microglia and Lamp1 (green). Large accumulations of Lamp1 immunoreactivity (white arrows) were detected in activated microglia of C9-ALS but not sALS tissue.

Although we did not see overt neurodegeneration in C9orf72−/− mice, given the pro-inflammatory phenotype in isolated microglia, we used transcriptional profiling to investigate C9orf72-deficient nervous tissue in greater detail. Gene set enrichment analysis (GSEA) on RNA sequencing (RNA-seq) of spinal cords from young animals (3 months) showed little difference between genotypes. In contrast, in aged animals (17 months), a large number of pathways were altered in C9orf72−/− versus C9orf72+/− or wild-type animals [false discovery rate (FDR) < 0.05] (Fig. 4B). We focused on the 19 pathways that were up-regulated in C9orf72−/− versus C9orf72+/− and control animals for further analysis (fig. S10). Of these 19 pathways, almost a third [6 out of 19 (6/19)] were related to inflammation (Fig. 4C). To determine whether similar changes are observed in C9orf72 ALS (C9-ALS) tissue, we analyzed a recent RNA-seq data set that includes normal controls, sporadic ALS (sALS), and C9-ALS cases (24). Of the 19 up-regulated pathways in C9orf72−/− mice, there was little overlap (1/19) with pathways up-regulated in sporadic ALS brain tissue (frontal cortex or cerebellum; Fig. 4D). In contrast, the majority (10/19) of pathways up-regulated in C9orf72−/− mice were also up-regulated in C9-ALS human patient brains, including nearly all of the immune pathways (5/6). A direct comparison showed a significant increase in inflammatory pathways in C9-ALS versus sALS cases (fig. S11). Finally, we performed immunostaining for Iba1 and Lamp1 on motor cortex and spinal cord tissue from C9-ALS (n = 3) and sALS (n = 3) cases. Although frequent reactive microglia were present in all ALS cases, microglia containing large accumulations of Lamp1-positive material were only observed in the C9-ALS cases (Fig. 4E and fig. S11). Thus, both transcriptome and histologic analyses of C9-ALS patient tissue are consistent with the idea that the decreased C9orf72 expression in C9-ALS leads to altered microglial function and neuroinflammation.

In summary, the loss of C9orf72 in mice led to age-related inflammation in the spleen and nervous system, with defects in lysosomal trafficking and immune responses in macrophages and microglia. The disruption of lysosomal function in macrophages is consistent with the idea that C9orf72 is a member of the DENN family of Rab-GEFs involved in late endosomal trafficking and autophagy (2527). Our data support a model where C9orf72 regulates the maturation of phagosomes to lysosomes in macrophages, because we observed both altered responses to immune stimuli, including those sensed in endosomal/lysosomal compartments (PGN, CpG, and silica) in BMDMs lacking C9orf72. Furthermore, loss of C9orf72 function could affect neurodegeneration in C9-ALS and FTD by diminishing the ability of microglia to clear aggregated proteins and/or altering their immune responses. Our findings of altered immune responses in haploinsufficient macrophages indicate that even this partial decrease in C9orf72 levels could affect microglial function (3, 2830). These data raise the possibility of a dual-effect mechanism for the pathogenesis of a single gene defect: that gain-of-function manifestations of C9orf72 expansion (RNA foci and RAN dipeptides) in neurons are coupled with “primed” and dysfunctional microglia, which ultimately results in neurodegeneration (31). Given that many ALS genes are involved in late endosomal trafficking and lysosome function (TBK1, TMEM106B, OPTN, SQSTM1, UBQLN2, VCP, CHMP2B, and PGRN) (32) and are expressed in both neurons and microglia, the concept of a dual-effect mechanism may generalize to other forms of inherited ALS.

Finally, our findings raise important considerations about therapeutic knockdown of C9orf72 in the nervous system. Although these approaches effectively target gain-of-function manifestations in neurons, they could exacerbate microglial dysfunction by further suppressing C9orf72, unless they specifically target repeat-containing transcripts (33). An initial report of C9orf72 knockdown in mice using ASOs revealed up-regulation of immune markers in the nervous system, including Trem2 and Tyrobp (34), suggesting that innate immune function should be monitored when performing C9orf72 knockdown strategies in humans.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S11

Data Tables S1 and S2


Acknowledgments: We thank V. Funari for assistance with RNA sequencing, A. Cammack for assisting with patient tissue, and A. Koehne for assisting with pathology evaluation. This work was supported by NIH grants NS069669 (R.H.B), NS087351 (C.M.L), GM085796 (D.M.U.), NS078398 (T.M.M.), and UL1TR000124; the Robert and Louise Schwab family; the Cedars-Sinai ALS Research Fund (R.H.B.); and the Cedars-Sinai Board of Governors Regenerative Medicine Institute. T.M.M. has served on medical advisory boards for Ionis Pharmaceuticals and Biogen Idec. Mouse line F12 is available through the Jackson Repository, no. 27068, C57BL/6J-3110043O21Rik<em5Lutzy>/J. RNA-seq data are located in the Gene Expression Omnibus, accession number GSE77681.
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