EK39-EPOSTER-GIORDANO-ANNA-MARIA-SOLE - Multiple endogenous nucleic acid sources contribute to spontaneous activation of inflammatory and DNA damage programs in Aicardi-Goutières Syndrome astrocytes leading to neurotoxicity
Multiple endogenous nucleic acid sources contribute to spontaneous activation of inflammatory and DNA damage programs in Aicardi-Goutières Syndrome astrocytes leading to neurotoxicity Anna Maria Sole Giordano 1,2, Francesca Gatto 1, Marco Luciani 1,2, Chiara Beghè 6, Lucrezia Della Volpe 1,2, Alessandro Migliara 1,2, Sara Valsoni 1, Martin Reijns 3, Marco Genua 1, Giacomo Frati 1, Silvia Giliani 4, Simona Orcesi 5, Elisa Fazzi 4, Renato Ostuni 1, Andrew Jackson 3, Raffaella Di Micco1, Ivan Merelli 1, Natalia Gromak 6, Angelo Lombardo 1, Angela Gritti 1, Anna Kajaste-Rudnitski 1* 1 San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy, 2Vita-Salute San Raffaele University, School of Medicine, Milan, Italy, 3The University of Edinburgh, Edinburgh, UK, 4 University of Brescia, Italy, 5 C. Mondino National Neurological Institute, Pavia, Italy, 6 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK. * Address correspondence to: Anna Kajaste-Rudnitski, San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), and Division of Regenerative Medicine, Stem Cells and Gene Therapy, Via Olgettina 58, Milan, Italy. Phone: +39-0226435007; Fax: +39-0226434668/4621; E-mail: email@example.com Aberrant sensing of nucleic acids (NA) deriving from endogenous retro-elements or accumulating DNA damage and the consequent induction of type I IFN have been suggested to be a primary driver of Aicardi-Goutières Syndrome (AGS) pathogenesis. However, the molecular mechanisms triggering disease remain elusive and few studies have explored this in the context of the human central nervous system. Here, we generated isogenic iPSC clones knock-out (KO) for two AGS-causing genes TREX1 or RNaseH2b to model AGS gene defects in a neurological context in vitro and analyzed disease-associated phenotypes at the single cell level in AGS-patient derived neuronal cultures. No phenotypic differences between the AGS and WT cells were observed at the pluripotent iPSC and multipotent Neural Stem Cells (NSC) stages while differentiation of KO NSC towards pro-inflammatory astrocyte lead to spontaneous immune activation. Interestingly, only TREX1 KO astrocytes harbored the expected type I IFN signature, while pro-inflammatory and DNA damage responses were activated in both TREX1 and RNaseH2b KO cells. Importantly, these observations were confirmed in AGS patient iNSC-derived astrocytes at single cell level. RNaseH2b deficient astrocytes showed accumulation of R-loops and increased numbers of micronuclei were observed in both TREX1 and RNaseH2B defective cells. Moreover, AGS astrocytes expressed more LINE-1, potentially contributing to the pool of endogenous triggers in these cells. Importantly, WT neurons exposed to conditioned medium from KO and patient-derived astrocytes showed significant signs of toxicity that could be rescued by a combined inhibition of pro-apoptotic or inflammatory cascades in KO or patient-derived astrocytes. Together, these results highlight the key role of astrocytes in AGS neurotoxicity, provide temporal indications on the emergence of disease-associated phenotypes, shed light on the endogenous triggers leading to AGS, reveal genotype-specific pathological mechanisms of disease and pave the way for the development of novel therapeutic strategies to counteract astrocyte-mediated neurotoxicity in AGS.