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Variants of the RNA-editing enzyme ADAR1 cause Aicardi-Goutières syndrome (AGS), in which severe inflammation occurs in the brain due to innate immune activation. Here, we analyze the RNA-editing status and innate immune activation in an AGS mouse model that carries the AdarP195A mutation in the N terminus of the ADAR1 p150 isoform, the equivalent of the P193A human Zα variant causal for disease. This mutation alone can cause interferon-stimulated gene (ISG) expression in the brain, especially in the periventricular areas, reflecting the pathologic feature of AGS. However, in these mice, ISG expression does not correlate with an overall decrease in RNA editing. Rather, the enhanced ISG expression in the brain due to the P195A mutant is dose dependent. Our findings indicate that ADAR1 can regulate innate immune responses through Z-RNA binding without changing overall RNA editing.

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Adenosine deaminase acting on RNA 1 (ADAR1) is an essential protein for embryonic liver development. ADAR1 loss is embryonically lethal because of severe liver damage. Although ADAR1 is required in adult livers to prevent liver cell death, as demonstrated by liver-specific conditional knockout (Alb-ADAR1KO) mice, the mechanism remains elusive. We systematically analyzed Alb-ADAR1KOmice for liver damage. Differentiation genes and inflammatory pathways were examined in hepatic tissues from Alb-ADAR1KOand littermate controls. Inducible ADAR1 KO mice were used to validate regulatory effects of ADAR1 on inflammatory cytokines. We found that Alb-ADAR1KOmice showed dramatic growth retardation and high mortality because of severe structural and functional damage to the liver, which showed overwhelming inflammation, cell death, fibrosis, fatty change, and compensatory regeneration. Simultaneously, Alb-ADAR1KOshowed altered expression of key differentiation genes and significantly higher levels of hepatic inflammatory cytokines, especially type I interferons, which was also verified by inducible ADAR1 knockdown in primary hepatocyte cultures. We conclude that ADAR1 is an essential molecule for maintaining adult liver homeostasis and, in turn, morphological and functional integrity. It inhibits the production of type I interferons and other inflammatory cytokines. Our findings may provide novel insight in the pathogenesis of liver diseases caused by excessive inflammatory responses, including autoimmune hepatitis.

In the soft energy regime < 1 keV, the Galactic background is a significant component of the celestial diffuse X-ray radiation and carries important information about hot gas in the disk and in the corona of the Galaxy. We have conducted a study of the Galactic diffuse soft X-ray background with the data collected with the Imaging Proportional Counter on board the Einstein X-ray Observatory. The high sensitivity and spatial resolution of the data enable us to detect and subtract discrete sources to a flux level < 1013erg cm-2s-1, to measure the background with minmized confusion between various components in the radiation, and to study arcmin-scale fluctuation of the background. The results reported here demonstrate the value of imaging data in studying the background. At high Galactic latitudes, the Galactic background, indicated by the global enhancement of the X-ray radiation intensity toward the Galactic center, could arise in the corona of the galaxy. We have measured the intensity of the radiation in the 0.2-0.8 keV band in the latitude interval 30 degrees < |b|< 70 degrees and have compared the distribution of the intensity with a simple emission model of the Galactic corona. This comparison suggests that the corona probably accounts for more than half of the radiation in the energy band. The estimated density and pressure in the coronal gas are consistent with both theoretical arguments and UV observations made with IUE, HST, and rocket experiments. The intensity and spectrum of the Galactic background at the Galactic plane are measured with a deep image of the plane. The spectrum in the 0.2-3.5 keV band can be characterized by the emission from optically-thin, thermal coronal gas of a temperature at 4 x 106K, and the M-band intensity is Ix(0.5 - 1 keV) 3 X 10-8erg cm-2s-1sr-1. An angular autocorrelation analysis of the imaging data leads to the conclusion that no known stellar population is likely responsible for the bulk of the background. A hot gas model seems consistent with the observation; but a satisfactory fit to the observed spectrum requires X-ray emission from hot gas beyond the 106K Local Bubble. We further point out that the X-ray background, enhanced in the M-band at the Galactic plane, is apparently not related to the Galactic ridge emission inferred from observations in the higher energy 2-10 keV regime.

No relevant conflicts of interest to declare.

Abstract 917

Adenosine Deaminases Acting on RNA (ADAR) are RNA-editing enzymes converting adenosine residues into inosine (A-to-I) in many double-stranded RNA substrates including coding and non-coding sequences as well as microRNAs. Disruption of the ADAR1 gene in mice results in fetal liver, but not yolk sac, defective erythropoiesis and death at E11.5 (Wang Q et al, Science 2000). Subsequently, a conditional knockout mouse model confirmed these findings and showed massively increased cell death in the affected organs (Wang Q et al, JBC 2004). However, the actual impact of ADAR1 absence on definitive or adult hematopoiesis has not been examined. To define the role of ADAR1 in adult hematopoiesis, we first examined the expression of ADAR1 in different hematopoietic stem/progenitor cell subsets isolated from bone marrow by real-time RT-PCR. ARAR1 was present in hematopoietic stem cells (HSCs) at relatively low level and increased in hematopoietic progenitor cells (HPCs). A series of functional hematopoietic assays were then undertaken. A conditional deletion of ADAR1 was achieved by transducing Lin− or Lin−cKit+ bone marrow cells from ADAR1-lox/lox mice with a MSCV retroviral vector co-expressing Cre and GFP. PCR analysis confirmed the complete deletion of ADAR1 in the transduced cells within 72 hours after the transduction. This system allowed us to evaluate the acute effect of ADAR1 deletion in a specific hematopoietic cell population. Following 4 days of in vitro culture after transduction, the absolute number of Lin− Sca1+ cells in the Cre transduced group was similar to the input number; however the differentiating Lin+ cells significantly decreased whereas both the Lin−Sca1+ and Lin+ cells in the vector (MSCV carrying GFP alone) transduced group increased during culture. Moreover, the colony forming cell (CFC) assay showed much fewer and smaller colonies that contained dead cells from the gene deleted group as compared to those from the control group (p<0.001). The TUNEL assay showed a dramatic increase of apoptosis in the Lin+ population but not in the Lin− cells. Given the mixed genetic background of the ADAR1-lox/lox mice, repopulation of the transduced hematopoietic cells in vivo was examined in immunodeficient mice. Sublethally irradiated (3.5 Gy) NOD/SCID-γcnull recipient were transplanted with either 1.5 × 105 Cre or vector transduced Lin− ADAR1-lox/lox cells. Multi-lineage engraftment in peripheral blood was monitored monthly. While the vector transduced cells were able to constitute more than 90% in multiple lineages of the peripheral blood at 1 to 3 months, Cre-transduced cells were virtually undetectable at all the time points (n=9 to 13, p<0.001). A similar result was found in the hematopoietic organs, including the bone marrow, spleen and thymus. Interestingly, however, the Lin−Sca1+cKit+ cell population was preserved in the Cre transduced group despite the very low level of total donor-derived cells in the bone marrow (n=6 to 7, p<0.01). Consistently, the single cell culture experiment demonstrated that there was no significant difference between ADAR−/− and wild-type HSCs in terms of survival and division during the first 3 days of culture. Taken together, our current study demonstrates nearly absolute requirement of ADAR1 for hematopoietic repopulation in adult mice and it is also suggested that ADAR1 has a preferential effect on the survival of differentiating progenitor cells over more primitive cells.

Adenosine Deaminases Acting on RNA (ADAR) are RNA-editing enzymes converting adenosine residues into inosine (A-to-I) in many double-stranded RNA substrates including coding and non-coding sequences as well as microRNAs. Disruption of the ADAR1 gene in mice results in fetal liver, but not yolk sac, defective erythropoiesis and death at E11.5 (Wang Q et al, Science 2000). Subsequently, a conditional knockout mouse model confirmed these findings and showed massively increased cell death in the affected organs (Wang Q et al, JBC 2004). However, the actual impact of ADAR1 absence on definitive or adult hematopoiesis has not been examined. To define the role of ADAR1 in adult hematopoiesis, we first examined the expression of ADAR1 in different hematopoietic stem/progenitor cell subsets isolated from bone marrow by real-time RT-PCR. ARAR1 was present in hematopoietic stem cells (HSCs) at relatively low level and increased in hematopoietic progenitor cells (HPCs). A series of functional hematopoietic assays were then undertaken. A conditional deletion of ADAR1 was achieved by transducing Lin−or Lin−cKit+bone marrow cells from ADAR1-lox/loxmice with a MSCV retroviral vector co-expressing Cre and GFP. PCR analysis confirmed the complete deletion of ADAR1 in the transduced cells within 72 hours after the transduction. This system allowed us to evaluate the acute effect of ADAR1 deletion in a specific hematopoietic cell population. Following 4 days of in vitroculture after transduction, the absolute number of Lin−Sca1+cells in the Cre transduced group was similar to the input number; however the differentiating Lin+cells significantly decreased whereas both the Lin−Sca1+and Lin+cells in the vector (MSCV carrying GFP alone) transduced group increased during culture. Moreover, the colony forming cell (CFC) assay showed much fewer and smaller colonies that contained dead cells from the gene deleted group as compared to those from the control group (p<0.001). The TUNEL assay showed a dramatic increase of apoptosis in the Lin+population but not in the Lin−cells. Given the mixed genetic background of the ADAR1-lox/loxmice, repopulation of the transduced hematopoietic cells in vivowas examined in immunodeficient mice. Sublethally irradiated (3.5 Gy) NOD/SCID-γcnullrecipient were transplanted with either 1.5 × 105Cre or vector transduced Lin−ADAR1-lox/loxcells. Multi-lineage engraftment in peripheral blood was monitored monthly. While the vector transduced cells were able to constitute more than 90% in multiple lineages of the peripheral blood at 1 to 3 months, Cre-transduced cells were virtually undetectable at all the time points (n=9 to 13, p<0.001). A similar result was found in the hematopoietic organs, including the bone marrow, spleen and thymus. Interestingly, however, the Lin−Sca1+cKit+cell population was preserved in the Cre transduced group despite the very low level of total donor-derived cells in the bone marrow (n=6 to 7, p<0.01). Consistently, the single cell culture experiment demonstrated that there was no significant difference between ADAR−/−and wild-type HSCs in terms of survival and division during the first 3 days of culture. Taken together, our current study demonstrates nearly absolute requirement of ADAR1 for hematopoietic repopulation in adult mice and it is also suggested that ADAR1 has a preferential effect on the survival of differentiating progenitor cells over more primitive cells.