Protein kinase R and the integrated stress response drive immunopathology caused by mutations in the RNA deaminase ADAR1

Abstract

The RNA deaminase ADAR1 is an essential negative regulator of the RNA sensor MDA5, and loss of ADAR1 function triggers inappropriate activation of MDA5 by self-RNAs. Mutations in ADAR, the gene that encodes ADAR1, cause human immune diseases, including Aicardi-Goutières syndrome (AGS). However, the mechanisms of MDA5-dependent disease pathogenesis in vivo remain unknown. Here we generated mice with a single amino acid change in ADAR1 that models the most common human ADAR AGS mutation. These Adar mutant mice developed lethal disease that required MDA5, the RIG-I-like receptor LGP2, type I interferons, and the eIF2α kinase PKR. A small-molecule inhibitor of the integrated stress response (ISR) that acts downstream of eIF2α phosphorylation prevented immunopathology and rescued the mice from mortality. These findings place PKR and the ISR as central components of immunopathology in vivo and identify therapeutic targets for treatment of human diseases associated with the ADAR1-MDA5 axis.

Keywords: ADAR1; Aicardi-Goutières syndrome; LGP2; MDA5; PKR; integrated stress response; interferons.

Figure 1.. Adar P195A mice model the most common Aicardi-Goutieres Syndrome mutation

(A) Schematic of the structure of ADAR1 protein, and the location of the P195A mutation. Zα, Zβ, = Z-DNA binding domains, NES = Nuclear export signal, NLS = nuclear localization signal. (B) Percentage of mice of the indicated genotype from intercrosses of Adar^P195A/+ mice. (n=140 pups) (C) Survival of Adar^+/+ (n=23), Adar^P195A/+ (n=47), Adar^P195A/P195A (n=48) mice. (D) Weights of mice at 23 days of age. Adar^+/+ (n= 6F,12M), Adar^P195A/+ (n= 13F,14M), Adar^P195A/P195A (n= 8F, 9M). Bar represents the mean. See also Figure S1.

Figure 2.. Recapitulation of AGS patient genotypes causes severe disease in Adar^P195A mice

(A-B) Survival of Ifihi1^+/+ mice of the indicated genotypes: Adar^r+/+ (n=14), Adar^+/− (n=21), Adar^P195A/Adar+ (n=41), Adar^{P195A/Adar−} (n=9); Adar p150^+/+ (n=15), Adar p150^+/− (n=6), Adar^P195A/p150+ (n=34), Adar^{P195A/p150−} (n=25). (C-D) Survival of Ifihi1^−/− mice of the indicated genotypes: Adar^+/+ (n=18), Adar^+/− (n=22), Adar^P195A/Adar+ (n=14), Adar^P195A/Adar- (n=19); Adar p150+/+ (n=17), Adar p150+/− (n=24), Adar^P195A/p150+ (n=25), Adar^P195A/p150- (n=28). (E-F). Weights of mice of the indicated genotypes, measured at 23 days. Bars represent mean. Male and female mice are pooled in (E) because there was no significant difference by sex. Adar^+/+ (n=2); Adar^+/− (n=3); Adar^P195A/Adar+ (n=4); Adar^{P195A/Adar−} (n=5); Adar p150^+/+ (n=5M, 6F); p150^+/− (n=5M, 5F); Adar^P195A/p150+ (n=11M, 10F); AdarP195A/p150− (n=6M, 4F); Adar^P195A/p150+ (n=9M, 8F); Adar^{P195A/p150−} (n=11M, 8F). See also Figure S2

Figure 3.. Adar^{P195A/p150−} mice develop organ-specific pathology

(A) Representative histology of kidney and liver from Adar^{P195A/p150−} mice measured at 23 days of age. (B) Representative histology of kidney and liver from Adar^{P195A/p150−}Ifih1^−/− mice measured at 23 days of age. (C) Representative histology of kidney, liver, and spleen from Adar^{P195A/p150−} mice measured at 147 days of age. Arrows indicate glomeruli in kidneys, asterisk indicates region of cytoplasmic vacuolation in liver with small arrow showing microvesicular vacuolation and large arrow showing macrovesicular vacuolation. (D-F) Histological scores in the kidney, liver, and spleen, measured at 23 days of age. Adar p150^+/+ (n=5); Adar^P195A/p150+ (n=5); Adar^{P195A/p150−} (n=6); Adar^P195A/p150+Ifih1^−/− (n=4); Adar^P195A/p150Ifih1^−/− (n=5). (G) Representative (of 3 of each genotype) immunofluorescence of the kidney for Adar^{P195A/p150−} and Adar^P195/p150+ mice. DAPI is blue. IgG is red. (H) Ifnb transcript measured by qRT-PCR in BMMs of the indicated genotypes, with and without 24 hours of treatment with recombinant mouse IFNβ. Adar^P195A/p150+ (n=4); Adar^{P195A/p150−} (n=4); Adar^P195A/p150+Ifih1^−/− (n=3); Adar^{P195A/p150−}Ifihi1^−/− (n=5).

Figure 4.. Adar^{P195A/p150−} mice have an MDA5-dependent interferon signature

(A-C) Expression data for ISGs, defined by the GO term ‘response to type I interferon,’ were evaluated in the liver, kidney, and cerebellum of 23 day old Adar^{P195A/p150−} mice, plotting the log₂ fold change over matched Adar^P195A/p150+ control mice. ISGs that were not significantly changed are shown in bright red; significant expression changes are shown in dark red. (D-F) Expression of ISGs identified in A-C, measured by TaqMan qPCR, in 23 day old mice of the indicated genotypes. Adar^P195A/p150+ (n=6); Adar^{P195A/p150−} (n=7); Adar^P195A/p150+Ifihi1^−/− (n=4); Adar ^{P195A/p150−} Ifih1^−/− (n=6).

Figure 5.. Identification of a genetic pathway linking the Adar^P195A mutation to disease

(A) Survival of Dhx58^−/− mice of the indicated genotype: Adar p150^+/+ (n=3), Adar p150^+/− (n=8), Adar^P195A/p150+ (n=5), Adar^{P195A/p150−} (n=12). (B) Weights, measured at 23 days, of Adar^{P195A/p150−}Dhx58^−/− mice (n=7), as a percentage of the average weight of age- and sex-matched Adar^P195A/p150+Dhx58^−/− (n=7) control mice. (C) Expression of the indicated ISGs measured by TaqMan qRT-PCR, normalized to HPRT, in the cerebellum, liver, or kidney, comparing Adar^{P195A/p150−}Dhx58^−/− mice (n=8) to Adar^P195A/p150+Dhx58^−/− controls (n=6). (D) Survival of Ifnar1^−/− mice of the indicated genotype: Adar p150^+/+ (n=3), Adar p150^+/− (n=7), Adar^P195A/p150+ (n=4), Adar^{P195A/p150−} (n=16). (E) Weights, measured at 23 days, of Adar^{P195A/p150−}Ifnar1^−/− (n=19) mice, as a percentage of the average weight of age- and sex-matched Adar^P195A/p150+Ifnar1^−/− (n=6) control mice. (F) Expression of the indicated ISGs measured by TaqMan qRT-PCR, normalized to HPRT, in the cerebellum, liver, or kidney, comparing Adar^{P195A/p150−}Ifnar1^−/−(n=8) mice to Adar^P195A/p150+Ifnar1^−/− (n=5) controls. (G) Survival of Eif2ak2^−/− mice of the indicated genotype: Adar^P195A/p150+ (n=4), or Adar^{P195A/p150−} (n=7). (H) Weights, measured at 23 days, of Adar^{P195A/p150−}Eif2ak2^−/− mice (n=6), as a percentage of the average weight of age- and sex-matched Adar^P195A/p150+Eif2ak2^−/− control (n=4)mice. (I) Expression of the indicated ISGs measured by TaqMan qRT-PCR, normalized to HPRT, in the cerebellum, liver, or kidney, comparing Adar^{P195A/p150−}Eif2ak2^−/− mice(n=4) to Adar^P195A/p150+Eif2ak2^−/− controls (n=4). (See also figures S3 and S4)

Figure 6.. An ISR gene expression signature is evident in Adar^{P195A/p150−} mice

(A-C) Expression data for ISR gene set genes in the liver, kidney, and cerebellum of 23 day old Adar^{P195A/p150−} mice (n=7), plotting the log2 fold change over control Adar^P195A/p150+ (n=5) mice. ISR genes that are not significantly changed are shown in bright red; significant expression changes are shown in dark red. (D-F) Expression of ISR transcripts identified in the livers of rescued mice, measured by TaqMan qRT-PCR, and compared among the indicated genotypes. Each data point represents an individual mouse. Adar^P195A/p150+ (n=6); Adar^{P195A/p150−} (n=7); Adar^P195A/p150+Ifihi1^−/− (n=4); Adar^{P195A/p150−}Ifih1^−/− (n=6); Adar^P195A/p150+Dhx58^−/− (n=7); Adar^{P195A/p150−}Dhx58^−/− (n=8); Adar^P195A/p150+Ifnar1^−/− (n=4); Adar^{P195A/p150−} Ifnar1^−/− (n=7); Adar^P195A/p150+Eif2ak2^−/− (n=4); Adar^{P195A/p150−}Eif2ak2^−/− mice (n=4).

Figure 7.. Pharmacological Inhibition of the ISR rescues Adar^{P195A/p150−} mice

(A) Survival of mice on control chow: Adar^P195A/p150+ (n=23), Adar^{P195A/p150−} (n=10); versus survival of mice on 2BAct chow: Adar^P195A/p150+ (n=17), Adar^{P195A/p150−} (n=17). (B) Weights of Adar^{P195A/p150−} mice on control chow Adar^P195A/p150+ (n=12), Adar^{P195A/p150−} (n=9) or 2BAct chow Adar^P195A/p150+ (n=16), Adar^{P195A/p150−} (n=7), as a percentage of average weight of age- and sex-matched Adar^P195A/p150+ mice on control chow. (C) Representative histology of kidney, liver, and spleen of untreated, Eif2ak2^−/−, and 2BAct-treated mice of the indicated genotypes. (D) Histological scores of the kidneys, livers, and spleens of 2BAct-treated mice at day 23. Adar^P195A/p150+ (n=7), Adar^{P195A/p150−} (n=8). Arrows indicate glomeruli, asterisk indicates region of cytoplasmic vacuolation, and oval indicates periarteriolar lymphoid sheath (white pulp of spleen). (E) ISG expression in the kidneys and livers of Adar^P195A/p150+ (n=5) and Adar^{P195A/p150−}(n=7) mice treated with 2BAct. (F) ISR transcript expression in the liver of Adar^P195A/p150+ (n=5) and Adar^{P195A/p150−} (n=7) mice treated with 2BAct. Bars represents the mean in all graphs for B-F. (G) Human A549 cells were transduced simultaneously with two lentiCRISPR constructs, each containing a distinct selectable marker. The first construct (puromycin-resistant) encoded gRNAs targeting just the p150 isoform of ADAR1, both isoforms of ADAR1, or an H1 nontargeting control. The second construct (blasticidin resistant) encoded either an H1 nontargeting control gRNA or a gRNA targeting the human EIFAK2 (PKR) gene. After selection in puromycin and blasticidin, the cells were treated with 1000U recombinant human IFNb for 72 hours, followed by measurement of the indicated genes by qRT-PCR. n=3 independent transduced populations for each group. Data are representative of 2 independent repeats.