C9ORF72 suppresses JAK-STAT mediated inflammation

Summary Hexanucleotide repeat expansion in the gene C9ORF72 is a leading cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). C9ORF72 deficiency leads to severe inflammatory phenotypes in mice, but exactly how C9ORF72 regulates inflammation remains to be fully elucidated. Here, we report that loss of C9ORF72 leads to the hyperactivation of the JAK-STAT pathway and an increase in the protein levels of STING, a transmembrane adaptor protein involved in immune signaling in response to cytosolic DNA. Treatment with a JAK inhibitor rescues the enhanced inflammatory phenotypes caused by C9ORF72 deficiency in cell culture and mice. Furthermore, we showed that the ablation of C9ORF72 results in compromised lysosome integrity, which could contribute to the activation of the JAK/STAT-dependent inflammatory responses. In summary, our study identifies a mechanism by which C9ORF72 regulates inflammation, which might facilitate therapeutic development for ALS/FTLD with C9ORF72 mutations.


INTRODUCTION
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two devastating neurodegenerative diseases that belong to the same disease spectrum. These two diseases have overlapping clinical, pathological, and genetic features. 1,2 One of the main genetic causes of both ALS and FTLD is hexanucleotide repeat (GGGGCC (G 4 C 2 )) expansion (HRE) in the first intron of the C9ORF72 gene, 3,4 which results in disease phenotypes via both gains of toxicity of RNA repeats and dipeptides and loss of function of the C9ORF72 protein. [5][6][7][8][9] While the physiological functions of C9ORF72 remain to be fully elucidated, the most striking phenotype of C9orf72 knockout mice is enhanced inflammatory responses, resulting in age-dependent lymphadenopathy and splenomegaly. [10][11][12][13] An increased level of inflammatory cytokines, including TNF-a, IL-1b, IL-6, and IL-10 has been detected in the spleen and serum of C9orf72 deficient mice. 11,12,14,15 Patients with C9ORF72 mutations also have an increased propensity to autoimmune diseases due to the constant, uncontrolled production of inflammatory cytokines. 16 These studies all suggest that C9ORF72 directly or indirectly regulates one or more inflammatory pathways. Recently, the endosomal toll-like receptor (TLR) and the cGAS-STING signaling pathway have been shown to get upregulated under C9ORF72 deficient conditions. 17,18 However, the mechanism that leads to the upregulation of these two innate immune signaling pathways is still unclear.
To explore the mechanism of how the loss of C9ORF72 upregulates inflammatory responses in vitro and in vivo, we examined inflammatory responses in C9orf72 À/À macrophages and mice. Here, we confirmed that C9ORF72 deficiency leads to an increase in the protein levels of STING and enhanced inflammatory responses. The inhibition of JAK activities rescues the inflammatory phenotypes of C9ORF72 deficient cells and mice. In addition, we demonstrate that lysosome integrity is compromised under C9ORF72 deficient conditions, which leads to JAK-dependent inflammatory activation.  (1 mM). Data represent the mean G SEM. Statistical significance was analyzed by two-way ANOVA (n = 3). Groups that exhibit non-significant differences with two-way ANOVA were re-analyzed using unpaired two-tailed Student's t test, and the results are indicated in parentheses. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) RT-qPCR measurement of mRNA levels of cytokines Cxcl10 and Il1b in control and C9orf72 À/À RAW264.7 cells without and with 16h of DMXAA (100 mg/mL) treatment. Data represent the mean G SEM. Statistical significance was analyzed by two-way ANOVA (n = 3), ns = not significant, *p < 0.05, ****p < 0.0001. (C) RT-qPCR measurement of mRNA level of Irf3 in control and C9orf72 À/À RAW264.7 cells before and after DMXAA treatment. Data represent the mean G SEM. Statistical significance was analyzed by two-way ANOVA (n = 3). ns = not significant, *p < 0.05, **p < 0.01. (D) Western blot analysis of IRF3 and p-IRF3 levels in control and C9orf72 À/À RAW264.7 cells before and after DMXAA treatment. Data represent the mean G SEM. Statistical significance was analyzed by two-way ANOVA (n = 3). Groups that exhibit non-significant differences with two-way ANOVA were re-analyzed using unpaired two-tailed Student's t test, and the results are indicated in parentheses. ns = not significant, *p < 0.05. iScience Article C9orf72 À/À cells after DMXAA treatment ( Figure 1D), supporting the hyperactivation of the STING pathway under C9ORF72 deficient conditions.

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Since C9ORF72 has been shown to regulate the activities of ARF and RAB GTPases, 21-24 key regulators of membrane trafficking, we hypothesized the protein turnover rate of TLRs and STINGs are affected by C9ORF72. Unfortunately, we failed to detect the endogenous TLR proteins in RAW264.7 cells using the commercially available TLR antibodies via western blot or immunostaining. On the other hand, we found that the protein levels of STING are significantly elevated in C9orf72 À/À cells compared to control RAW264.7 cells (Figure 2A). This increase was also observed in the spleen lysates derived from C9orf72 À/À mice ( Figure 2B). The increase in STING protein levels under C9ORF72 deficient condition could be due to decreased lysosome degradation, enhanced transcription, and/or trafficking defect(s) of STING. STING is a membrane protein normally localized in the endoplasmic reticulum (ER) and gets Figure 2. C9ORF72 does not affect STING protein degradation or trafficking (A) Western blot analysis of the protein levels of STING in control and C9orf72 À/À RAW264.7 cells. Data represent the mean G SEM. Statistical significance was analyzed by unpaired two-tail Student's t test (n = 4), **p < 0.01. (B) Western blot analysis of the protein levels of STING in spleen lysates from 6-month-old WT and C9orf72 À/À mice. Mixed male and female mice were used. Data represent the mean G SEM. Statistical significance was analyzed by unpaired two-tail Student's t test (n = 3), *p < 0.05. (C) Control and C9orf72 À/À RAW264.7 cells treated with DMXAA for 0, 1, or 2h and STING levels are quantified by western blot. Experiments were repeated three times and a representative western blot was shown. Data represent the mean G SEM.
(D) Control and C9orf72 À/À RAW264.7 cells were untreated, treated with DMXAA for 2h or treated with DMXAA for 2h and allowed to recover for 1 h. Cells were fixed and stained with antibodies against STING and Golgi marker ACBD3 (scale bar = 10 mm). Experiments were repeated three times and representative images were shown. iScience Article translocated to the Golgi apparatus upon activation. Activated STING is then sent to the lysosome for degradation. 25 To examine whether the degradation of STING has been affected, we treated control and C9orf72 À/À RAW264.7 cells with DMXAA at various time points to induce STING activation and degradation. 26 Under normal conditions, C9orf72 À/À cells exhibited a 1.6-fold increase in STING levels compared to control cells. The levels of STING decreased significantly after 1 h of DMXAA treatment in both C9orf72 À/À and control cells. After 2 h of treatment, the STING levels were reduced to a similar extent in C9orf72 À/À and control cells ( Figure 2C), indicating that STING degradation is not affected by C9ORF72 deficiency. To determine whether C9ORF72 affects STING trafficking, we performed immunostaining to examine the localization of STING in control and C9orf72 À/À RAW264.7 cells. STING shows similar localization in the ER under normal conditions and translocates to the Golgi compartment upon DMXAA treatment in both control and C9orf72 À/À cells ( Figure 2D). This Golgi enrichment of STING disappears in both control and C9orf72 À/À cells 1 h after DMXAA removal ( Figure 2D). Thus, C9ORF72 deficiency does not have any obvious effect on STING localization and trafficking.
The JAK-STAT pathway is upregulated under C9ORF72 deficient condition Since the degradation and trafficking of STING are not affected in C9orf72 À/À cells, next we examined whether STING transcription is altered by C9ORF72 deficiency. We found that STING transcription is significantly upregulated in C9orf72 À/À cells after DMXAA treatment ( Figure 3A), indicating that C9ORF72 deficiency results in alterations in the pathway(s) upstream of STING instead of affecting STING signaling directly. Since STING transcription is partially regulated by the transcriptional factor STAT1, 27 we next investigated alterations in STAT1 levels under C9ORF72 deficient conditions. A significant increase in the protein levels of STAT1 was found in lysates derived from spleens of C9orf72 À/À mice ( Figure 3B) and C9orf72 À/À RAW264.7 cells ( Figure 3C). In addition, a more dramatic increase in the levels of phosphorylated STAT1 (p-STAT1) was observed in C9orf72 À/À RAW264.7 cells after DMXAA treatment compared to the increase in STAT1 levels ( Figure 3C), indicating that the JAK activities are hyper upregulated in the absence of C9ORF72 to mediate STAT1 phosphorylation. Moreover, RT-qPCR analysis revealed a significant increase in the mRNA levels of Stat1 in C9orf72 deficient RAW264.7 cells in response to DMXAA ( Figure 3D), indicating that C9ORF72 might affect Stat1 transcription in response to STING activation.
It is well known that the JAK-STAT pathway is activated by type-I IFNs 28 and DMXAA is a type-I IFN inducer. 29 Increased levels of STAT1 protein and mRNA in C9orf72 À/À cells with DMXAA treatment led us to hypothesize that type-I IFN levels might be altered in C9orf72 À/À cells. Indeed, we observed a significant increase in the levels of secreted IFN-b in C9orf72 À/À RAW264.7 cells after DMXAA treatment ( Figure 3E), suggesting that C9ORF72 deficiency results in elevated type-I IFN production, which could lead to increased STAT1 levels and JAK/STAT signaling.

Inhibition of JAK signaling rescues inflammatory phenotypes associated with C9ORF72 loss
To confirm whether the increase in inflammatory responses under C9ORF72 deficient conditions is due to the activation of the JAK-STAT pathway, we treated C9ORF72 deficient cells with ruxolitinib, a widely used inhibitor of JAK1/2 kinase activities. 30 Ruxolitinib treatment significantly rescued the increase in mRNA levels of Cxcl10 ( Figure 4A), Stat1 ( Figure 4B), and Sting ( Figure 4C) in C9orf72 À/À cells after DMXAA treatment. These results support that the hyperactive JAK-STAT pathway causes elevated inflammatory responses under C9ORF72 deficient conditions. It should be noted that ruxolitinib treatment does not fully rescue the increases in mRNA levels of Cxcl10 and Sting in C9orf72 À/À RAW264.7 cells in response to DMXAA ( Figures 4A and 4C), consistent with the complicated regulatory system of STING transcription, in which other transcription factors besides STAT1, such as CREB or c-Myc are involved. 31 In addition, we found that C9ORF72 deficiency results in exacerbated inflammatory responses to IFNa, a type-I IFN, as shown by elevated mRNA levels of Stat1, Sting, and Cxcl10 ( Figures 4D-4F). Inhibition of JAK activities with ruxolitinib fully rescues the increase in Stat1 levels and partially rescues the increase in Cxcl10 and Sting levels ( Figures 4D-4F), indicating that the JAK-STAT signaling is elevated under C9ORF72 deficient condition in response to type-I IFN stimulation.
We then investigated whether ruxolitinib can rescue the hyperactivated JAK-STAT pathway in response to C9ORF72 loss in vivo. C9orf72 À/À mice were treated with ruxolitinib for 2 weeks or 3 weeks and inflammatory phenotypes were analyzed. Remarkably, splenomegaly and lymphadenopathy in C9orf72 À/À mice were greatly reduced after 2 weeks of ruxolitinib treatment and significantly rescued after 3 weeks of ll iScience Article ruxolitinib treatment ( Figure 5A). The protein level of STAT1 in the spleen lysate of C9orf72 À/À mice also decreased significantly with ruxolitinib treatment compared to the DMSO control ( Figure 5B). In addition, a significant correlation between spleen size and STAT1 level has been observed in C9orf72 À/À mice, despite a relatively low R 2 between spleen size and STAT1 level due to variabilities ( Figure 5C).
Taken together, our results suggest that C9ORF72 deficiency causes elevated JAK-STAT signaling, resulting in increased STAT1 levels and IFN production, which further elevates inflammatory signaling via a positive feedback loop under C9ORF72 deficient conditions.

C9ORF72 deficiency causes impairment of lysosome integrity
Lysosome dysregulation was found to be one of the causes of inflammation. Undegraded substrates such as DNA or lysosome hydrolases leaked out from damaged lysosomes have been shown to induce Data represent the mean G SEM. Statistical significance was analyzed by two-way ANOVA (n = 3), ns = not significant, *p < 0.05, **p < 0.01. (B) Protein levels of STAT1 in spleen lysates from 6-month-old WT and C9orf72 À/À mice were analyzed using western blot. Mixed male and female mice were used. Data represent the mean G SEM. Statistical significance was analyzed by unpaired one-tail Student's t test (n = 4), ns = not significant, *p < 0.05. (C) Protein levels of STAT1 and p-STAT1 in control or C9orf72 À/À RAW264.7 cells untreated or treated with DMXAA for 2h were analyzed using western blot and normalized to GAPDH. Data represent the mean G SEM. Statistical significance was analyzed by unpaired two-tail Student's t test (n = 3), ns = not significant, *p < 0.05. (D) RT-qPCR analysis of Stat1 levels in control and C9orf72 À/À RAW264.7 cells untreated or treated with DMXAA for 16h. Data represent the mean G SEM. Statistical significance was analyzed by two-way ANOVA (n = 3), ns = not significant, *p < 0.05, ****p < 0.0001. (E) IFN-b levels in control and C9orf72 À/À RAW264.7 cells untreated or treated with DMXAA for 16h were measured using ELISA. Data represent the mean G SEM. Statistical significance was analyzed by two-way ANOVA (n = 3), ns = not significant, **p < 0.01. iScience Article inflammatory responses. 32,33 Previous studies have reported lysosome deficits under C9ORF72 deficient conditions. 10,13,18,34 We observed an increased number of enlarged lysosomes in C9orf72 À/À RAW264.7 cells after IFNa treatment ( Figures 6A and 6B). Enlarged lysosomes could be a consequence of accumulated undigested materials, 35 which can induce cell stress and/or inflammation if released into the cytosol. Moreover, we found that lysosomes in C9orf72 À/À RAW264.7 cells are more sensitive to lysosome permeabilization compared to control cells. In response to treatment with L-Leucyl-L-Leucine methyl ester (LLOME), a lysosome damaging reagent. C9orf72 À/À RAW264.7 cells showed an increased number of cells with lysosomes labeled by Galectin 3 (Gal3): a lectin recruited to lysosomes due to the exposure of lysosomal glycoproteins caused by lysosomal membrane permeabilization ( Figures 6C  and 6D).

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To confirm whether lysosome leakage could lead to inflammation in C9orf72 À/À cells, RT-qPCR was used to examine changes in the mRNA levels of Stat1 and Cxcl10 in control or C9orf72 À/À RAW264.7 cells with or without LLOME treatment. LLOME treatment leads to a significant elevation of Stat1 ( Figure 6E) and Cxcl10 ( Figure 6F) mRNA levels in C9orf72 À/À cells. This increase is rescued by ruxolitinib treatment, indicating that lysosome damage could cause JAK-dependent inflammatory response under C9ORF72 deficient conditions.

DISCUSSION
In this study, we found an interesting link between C9ORF72, lysosomal dysfunction, and inflammatory signaling (

C9ORF72 deficiency results in the hyperactivation of the JAK-STAT pathway
While exploring the inflammatory pathways regulated by C9ORF72, we demonstrated that C9ORF72 deficiency leads to the hyperactivation of the JAK-STAT pathway, in addition to the endosomal TLR signaling pathways and the cGAS-STING signaling pathway previously reported. 17,18 A significant increase in STAT1 levels was observed in C9ORF72 deficient cells and tissues (Figure 3), which may result in enhanced signal transduction involving STAT1. For example, activation of the JAK-STAT pathway via IFNa treatment results in enhanced mRNA levels of Stat1, Sting, and Cxcl10 in C9orf72 À/À cells, which can be fully or partially rescued by inhibiting JAK1/2 kinase activities ( Figures 4D-4F). . JAK1 inhibition rescues spleen and lymph node enlargement in C9orf72 À/À mice (A) 2.6-2.8 month-old WT mice were treated with DMSO (n = 4) and C9orf72 À/À mice were treated with either DMSO (n = 6), or with ruxolitinib (90 mg/kg daily) for 2 weeks (n = 3), or 3 weeks (n = 5). Representative image of lymph nodes (scale bar = 2 mm) and spleens (scale bar = 1 cm) from 2.6-2.8 month-old DMSO-treated WT mice or DMSO, or ruxolitinib (3 weeks) treated C9orf72 À/À mice are shown (left). The spleen weight (mg) over body weight (g) is quantified and shown on the right. Data represent the mean G SEM. Statistical significance was analyzed by one-way ANOVA, n = 4-6. Groups that exhibit non-significant differences with two-way ANOVA were re-analyzed using unpaired two-tailed Student's t test, and the results are indicated in parentheses. ns = not significant, *p < 0.05, **p < 0.01. (B) Western blot analysis of STAT1 protein levels in spleen samples for experiments in (A). Samples from C9orf72 À/À mice treated with ruxolitinib for 3 weeks were used. Data represent the mean G SEM. Statistical significance was analyzed by one-way ANOVA, ns = not significant, n = 4-6, *p < 0.05. (C) Correlation graph between spleen weight and STAT1 protein levels in DMSO or ruxolitinib treated C9orf72 À/À mice. Pearson correlation coefficient (r) and significance (p) were calculated. n = 14.

OPEN ACCESS
iScience 26, 106579, May 19, 2023 7 iScience Article Figure 6. Impairment of lysosome integrity due to C9ORF72 loss results in increased JAK-STAT-mediated inflammation (A and B) Immunostaining of lysosome marker LAMP1 in control or C9orf72 À/À RAW264.7 cells without and with 16h of IFNa treatment (scale bar = 10 mm). The number of lysosomes with a diameter larger than 0.75 mm in each cell was counted and normalized to untreated control RAW264.7 cells. 106-142 cells from three independent experiments (142 (Ctrl); 178 (Ctrl IFNa); 106 (C9orf72 À/À Ctrl); 117 (C9orf72 À/À IFNa)) were quantified for the experiment in (A). Data represent the mean G SEM. Statistical significance was analyzed by two-way ANOVA. Groups that exhibit non-significant differences with two-way ANOVA were re-analyzed using unpaired two-tailed Student's t test, and the results are indicated in parentheses. ns = not significant, **p < 0.01. (C and D) Immunostaining of Galectin 3 and LAMP1 in control or C9orf72 À/À RAW264.7 cells without and with 2h LLOME (1 mM) treatment (scale bar = 10 mm). The number of cells with Galectin-3 puncta was quantified and normalized to the total number of cells in each condition. Total number of cells from four independent experiments analyzed: Ctrl 1h LLOME n = 525, C9orf72 À/À 1h LLOME n = 492, Ctrl 2h LLOME n = 319, C9orf72 À/À 2h LLOME n = 294. Data represent the mean G SEM. Statistical significance was analyzed by two-way ANOVA. Groups that exhibit non-significant differences with twoway ANOVA were re-analyzed using unpaired two-tailed Student's t test, and the results are indicated in parentheses. ns = not significant, *p < 0.05. (E and F) Control or C9orf72 À/À RAW264.7 cells are either untreated (Ctrl), or treated with 4hrs of LLOME then recover for 12hrs in normal cell medium (LLOME), or treated with ruxolitinib for 16hrs before 4 h of LLOME treatment and then iScience Article In addition, Cxcl10 levels are increased in C9orf72 À/À cells without any treatment, and this increase is rescued by ruxolitinib treatment, indicating that the JAK-STAT pathway is activated in C9orf72 À/À cells without any stimuli (Figures 4A and 4D). More importantly, ruxolitinib treatment rescues splenomegaly and lymphadenopathy observed in C9orf72 À/À mice ( Figure 5), confirming the importance of the JAK-STAT activation in the inflammatory responses under C9ORF72 deficient conditions. It should be noted that ruxolitinib inhibits both JAK1 and JAK2 and also alleviates inflammatory responses in patients with STAT1 or STAT3 gain-of-function variants. 36 Thus, the strong anti-inflammatory effect of ruxolitinib that we observed under the C9ORF72 deficient condition may not be solely due to its inhibition of JAK-STAT1 and other JAK-STAT pathways might be affected by C9ORF72 deficiency. Nevertheless, JAK and/or STAT dysregulations are found to cause multiple autoimmune diseases such as rheumatoid arthritis, and inhibitors of the JAK-STAT pathway are used to treat these diseases. 37 Interestingly, a newly developed machine learning framework, DRIAD (Drug Repurposing In Alzheimer's Disease), identified multiple drugs that inhibit one or more proteins in the JAK family as top-scoring drugs for treating Alzheimer's disease. 38 In addition, ALS/FTLD patients with C9ORF72 HRE expansion were found to harbor cytoplasmic double-stranded RNA (dsRNA), an established trigger of innate immunity, and inhibition of JAK/STAT signaling by ruxolitinib was shown to reverse cell death triggered by dsRNA in cultured human neurons. 39 Based on these studies, baricitinib, an FDA-approved JAK inhibitor, is currently in the clinical trial for people with mild cognitive impairment, ALS, or ALS patients carrying C9ORF72 mutant (NCT05189106). Our demonstration of JAK-STAT hyperactivation under C9ORF72 deficient conditions may further explain the relatively high occurrence of autoimmune diseases in ALS/FTLD patients with C9ORF72 mutations 16 and support the inhibition of the JAK/STAT pathway as a valid therapeutic approach to treat these patients.

Regulation of STING levels and signaling by C9ORF72
Consistent with a previous report, we have found an increase in STING protein levels under C9ORF72 deficient conditions. 17 However, in contrast to the previous report, we did not observe a defect in STING degradation in C9ORF72 deficient cells ( Figure 2C). We explored whether the increase in the levels of STING proteins could be due to transcriptional upregulation. However, Sting mRNA levels do not seem to be significantly altered in C9ORF72 deficient cells under normal conditions, although there is a trend of increase under some conditions ( Figures 3A, 4C, and 4F). In contrast, Sting mRNA levels are significantly increased in C9orf72 À/À cells in response to DMXAA treatment ( Figures 3A and 4C), consistent with a profound increase in p-STAT1 levels in these cells with DMXAA ( Figure 3C). Interestingly, inhibition of JAK signaling with ruxolitinib treatment only partially suppresses this increase in Sting transcription ( Figures 4C and 4F), indicating that there might be other factors, such as CREB or c-Myc involved in regulating Sting mRNA levels. 31 In addition, since both Sting mRNA levels ( Figures 3A, 4C, and 4F) and STING protein degradation ( Figure 2C) are not significantly altered by C9ORF72 deficiency under normal conditions, the mechanisms leading to increased STING protein levels in C9ORF72 deficient cells remain to be determined.

Regulation of IRF3 levels by C9ORF72
Our study also showed a significant increase in the mRNA and protein levels of the transcription factor IRF3 in C9ORF72 deficient Raw264.7 cells (Figures 1C and 1D). Interestingly, a recent study identified several small GTPases, including ARF1 and ARF6, that can stimulate IRF3 phosphorylation. 40 C9ORF72 is known to form a complex with two other cytosolic proteins, Smith-Magenis Chromosome Regions 8 (SMCR8) and WD repeat-containing protein (WDR41) 10,23,34,41 to function as a GTPase accelerating protein (GAP) for ARF1 and ARF6 GTPases. 21 Thus, the increased levels of p-IRF3 in C9orf72 À/À cells ( Figure 1D) could be due to the enhanced levels of active Arf1 and/or Arf6 GTPases caused by loss of C9ORF72. IRF3 is a also cofactor for the induction of interferon-stimulated genes (ISGs). 42 Upon phosphorylation and nuclear translocation, IRF3 can formenhanceosome with other transcription factors to induce IFN-b transcription. 43 It should be noted that several other IRF family members, including IRF7 and 9, have also been shown to get upregulated under C9ORF72 deficient conditions and in ALS patients with C9ORF72 HRE. 17,44 IRF7 activation and dimerization can also induce IFN-a transcription. 43 Figure 6. Continued recover for 12hrs in normal cell medium with ruxolitinib (LLOME+rux). The mRNA level of Stat1 and Cxcl10 was analyzed by RT-qPCR. Data represent the mean G SEM. Statistical significance was analyzed by two-way ANOVA (n = 3). Groups that exhibit non-significant differences with two-way ANOVA were re-analyzed using unpaired two-tailed Student's t test, and the results are indicated in parentheses. ns = not significant, *p < 0.05, **p < 0.01. iScience Article In addition, type-I IFN treatment leads to the upregulation of STAT1 levels ( Figure 4E). STAT1 is known to upregulate its own transcription once activated. 28 Furthermore, STAT1 together with STAT2 and IRF9 forms Interferon-stimulated gene factor 3 (ISGF3), leading to the transcription of IRF7 and other ISGs. 45 STING and IRF3 have also been identified as ISGs, thus their expression may also be elevated by interferons. 27,46 Additionally, there are many cross-talks between different immune signaling pathways. For example, IRF3 also functions downstream of TLR signaling and cytosolic DNA/RNA sensing utilizing STING and other receptors. 47 It is conceivable that the increase in IRF levels together with the increase in STAT1 levels greatly sensitizes the C9ORF72 deficient cells to inflammatory responses to many different stimuli.

Impaired lysosomal integrity in C9ORF72 deficient cells
C9ORF72 deficiency has long been associated with various lysosomal defects including lysosome enlargement, poor lysosome acidification, and a decrease in autophagosome-lysosome fusion, and so forth. 10,13,18,34 Here, we show that loss of C9ORF72 results in impaired lysosome membrane integrity under stress conditions. Since the lysosome is the degradation center of the cell, maintaining lysosomal membrane integrity is vital to prevent leakage of undegraded materials and lysosomal hydrolases that are toxic to the cells. The increased lysosome membrane permeabilization after LLOME treatment in C9ORF72 deficient cells suggests that C9ORF72 is critical for the maintenance of lysosomal membrane integrity. Although the exact mechanism is still unknown, it may be related to the function of the C9ORF72/SMCR8/WDR41 complex as a GAP and/or GEF of small GTPases ARF and/or RAB. 48 The C9ORF72 complex is recruited to the lysosome under amino acid-deprived conditions by interacting with PQLC2, a transporter on the lysosomal membrane. 49 This recruitment may act as a switch to switch on or off activities of C9ORF72 toward one or more small GTPase. For example, the C9ORF72 complex has been shown to function as a GAP for ARF1 GTPase, 21 -thus by recruiting the complex to the lysosome, its GAP activity toward ARF1 is inhibited because ARF1 is localized to the Golgi. On the other hand, the lysosomelocalized C9ORF72 complex might affect the activities of RAB7 GTPase, which iScience Article localizes to the lysosome 50 and interacts with C9ORF72 51,52 . Future work is needed to identify the GTPase(s) and mechanisms regulated by the C9ORF72 complex to affect lysosomal membrane integrity.
Another question that remains to be answered is the exact mechanism of how lysosome permeabilization leads to the activation of the JAK/STAT pathway under the C9ORF72 deficient condition. In this regard, it has been shown that lysosome substrate overload and/or lysosome permeabilization can lead to the generation of reactive oxygen species (ROS) 53,54 and elevated levels of ROS can lead to the activation of the JAK/STAT pathway. 55 For example, STAT3 can be activated by oxidative stress caused by the deficiency of AEP, a lysosomal asparagine endopeptidase, and STAT3 activation can in turn promote lysosomal hydrolase expression. 53 Interestingly, C9ORF72 deficiency has been shown to increase ROS levels in the iPSC-derived motor neurons from multiple ALS/FTLD patients with C9ORF72 mutations 56 and in C9orf72 À/À bone marrow-derived primary macrophages after zymosan ingestion. 11 Thus, the elevated ROS levels due to lysosome leakage could lead to the activation of the JAK-STAT pathway under the C9ORF72 deficient condition.

Limitations of the study
While our studies show a critical role of C9ORF72 in suppressing JAK/STAT mediated inflammation, the exact mechanism of how C9ORF72 deficiency leads to JAK-STAT1 activation awaits further investigation. One possibility is that JAK/STAT is activated by ROS and undegraded substrates from permeabilized lysosomes in C9orf72 À/À cells since we as well as others have observed the lysosome defects and decreased lysosomal membrane integrity in C9orf72 À/À cells. Nevertheless, the precise mechanisms connecting lysosome defects and JAK-STAT1 activation, as well as the function of the C9ORF72 complex in maintaining proper lysosomal function and lysosomal membrane integrity, are still elusive. Another potential avenue through which C9ORF72 may affect inflammation is via IRF3, as we have observed increased levels of IRF3 and phospho-IRF3 under C9ORF72 deficient conditions. However, the mechanisms by which C9ORF72 regulates IRF3 levels remain to be investigated. Considering that ARF1 and ARF6 have been shown to stimulate IRF3 phosphorylation 40 and the C9ORF72 complex functions as a GAP for ARF1 and ARF6 GTPases, 21 we propose that C9ORF72 might regulate IRF3 signaling via ARF1 and ARF6. However, the mechanism by which how ARF1/6 GTPases regulate IRF3 phosphorylation, and whether ARF1/6 GTPases are responsible for increased IRF3 levels in C9orf72 À/À cells, need to be determined. Moreover, the exact cause of the increase in STING protein level under C9ORF72 deficient conditions remains unclear. Addressing these missing links will provide further insights into the molecular and cellular mechanisms by which C9ORF72 regulates inflammation.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: iScience Article iScience Article RT-PCR RNA was purified from RAW264.7 cells using TRIzol Reagent (Invitrogen). One microgram of total RNA was reverse transcribed using a poly(T) primer and SuperScript III Reverse Transcriptase (Invitrogen). qPCR was performed on a LightCycler 480 (Roche Applied Science), and transcript levels were calculated using efficiency-adjusted DD-CT. All transcripts were normalized to Tbp.

Image analysis
For the quantitative analysis of enlarged lysosomes, the number of lysosomes with a diameter larger than 0.75 mm was counted manually. At least 50 cells were analyzed in each treatment per experiment and the experiment was independently repeated four times. For the quantitative analysis of cells with Galectin 3 puncta, the total number of cells and the number of cells with Galectin3 puncta were counted manually. Around 100 cells were counted in each treatment per experiment and the experiment was independently repeated three times.

Statistical analysis
The data were presented as mean G SEM. Two-group analysis was performed using the Student's t test. Two-way ANOVA followed by Bonferroni's multiple comparison tests was used for multiple-group comparison. All statistical analyses were performed using GraphPad Prism version 9 software (GraphPad Software, San Diego, CA). p-values <0.05 were considered statistically significant.