Cellular senescence is triggered by various distinct stresses and characterized by a permanent cell cycle arrest. Senescent cells secrete a variety of inflammatory factors, collectively referred to as the senescence-associated secretory phenotype (SASP). The mechanism(s) underlying the regulation of the SASP remains incompletely understood. Here we define a role for innate DNA sensing in the regulation of senescence and the SASP. We find that cyclic GMP-AMP synthase (cGAS) recognizes cytosolic chromatin fragments (CCFs) in senescent cells. The activation of cGAS, in turn triggers the production of SASP factors via Stimulator of interferon genes (STING), thereby promoting paracrine senescence. We demonstrate that diverse stimuli of cellular senescence engage the cGAS-STING pathway in vitro and we show cGAS-dependent regulation of senescence upon irradiation and oncogene activation in vivo. Our findings provide insights into the mechanisms underlying cellular senescence by establishing the cGAS-STING pathway as a crucial regulator of senescence and the SASP.

histones in situ, which were partially lost upon aclarubicin treatment (Extended Data Fig. 1c, d). Thus, histones appear to dynamically engage cGAS in the nucleus.Consistent with prior work [13], functional analysis of cGAS in vitro enzymatic activity revealed that mononucleosomes (hereafter nucleosomes) inhibited DNA-induced cGAMP synthesis (Extended Data Fig. 1e). Likewise, compact chromatin fibres (12-mer nucleosome arrays) suppressed cGAS activity (Extended Data Fig. 1e). H2A-H2B dimers also had an inhibitory effect, but neither H2A or H2B monomers nor H3 or H4 monomers, respectively (Extended Data Fig. 1f, g). Thus, H2A-H2B dimers on their own can suppress cGAS (Extended Data Fig. 1h), albeit with weaker overall potency compared to fullassembled nucleosomes with additional features of nucleosomes in chromatin being necessary to exert maximal inhibition. Overall structure of the cGAS-NCP complexTo determine how cGAS interacts with nucleosomes, we pursued structural studies. A 1.5:1 molar mixture of human cGAS (residues 155 to 522) with a 147 bp 601 DNA nucleosome core particle (NCP) resulted in heterogenous particle distributions (Extended Data Fig. 2ad). To select for and stabilize more homogenous cGAS-NCP complexes, we combined gradient centrifugation with chemical crosslinking (GraFix) [15]. Both WT cGAS and cGAS K394E, a mutant impaired in dsDNA-mediated cGAS dimerisation [16], were used for structure determination. For the cGAS K394E mutant, we obtained a 4.1 Å reconstruction revealing two NCPs organized in a NCP 1 -cGAS 1 -cGAS 2 -NCP 2 sandwich arrangement with an expected molecular weight around 560 kDa, consistent with the most prominent peak fraction in multi-angle light scattering (MALS) (Fig. 1a, b, Extended Data Fig. 3, Supplementary Video 1, 2, and Extended Data Table 1a). The two individual nucleosomes are held together by two cGAS protomers. While the first cGAS protomer and its corresponding NCP (designated cGAS 1 and NCP 1 ) are well-resolved, the second nucleosome/cGAS pair (NCP 2 and cGAS 2 ) is less ordered (Extended Data Fig. 3e). In the dimeric NCP 1 -cGAS 1 -cGAS 2 -NCP 2 arrangement, each cGAS protomer interacts with the histone octamer of one NCP through histones H2A and H2B and the nucleosomal DNA (e.g. cGAS 1 and NCP 1 ), while contacting the second nucleosome (e.g. cGAS 1 and NCP 2 ) primarily through interactions with the nucleosomal DNA (Fig. 1a, b). In the WT cGAS structure, we observed a similar overall structural arrangement, with the NCP 1 -cGAS 1 -cGAS 2 -NCP 2 complex at 5.1Å and the focused NCP 1 -cGAS 1 structure at 4.7Å resolution (Extended Data

Low-grade inflammation is a hallmark of old age and a central driver of ageing-associated impairment and disease1. Multiple factors can contribute to ageing-associated inflammation2; however, the molecular pathways that transduce aberrant inflammatory signalling and their impact in natural ageing remain unclear. Here we show that the cGAS–STING signalling pathway, which mediates immune sensing of DNA3, is a critical driver of chronic inflammation and functional decline during ageing. Blockade of STING suppresses the inflammatory phenotypes of senescent human cells and tissues, attenuates ageing-related inflammation in multiple peripheral organs and the brain in mice, and leads to an improvement in tissue function. Focusing on the ageing brain, we reveal that activation of STING triggers reactive microglial transcriptional states, neurodegeneration and cognitive decline. Cytosolic DNA released from perturbed mitochondria elicits cGAS activity in old microglia, defining a mechanism by which cGAS–STING signalling is engaged in the ageing brain. Single-nucleus RNA-sequencing analysis of microglia and hippocampi of a cGAS gain-of-function mouse model demonstrates that engagement of cGAS in microglia is sufficient to direct ageing-associated transcriptional microglial states leading to bystander cell inflammation, neurotoxicity and impaired memory capacity. Our findings establish the cGAS–STING pathway as a driver of ageing-related inflammation in peripheral organs and the brain, and reveal blockade of cGAS–STING signalling as a potential strategy to halt neurodegenerative processes during old age.

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In infected cells rotavirus (RV) replicates in viroplasms, cytosolic structures that require a stabilized microtubule (MT) network for their assembly, maintenance of the structure and perinuclear localization. Therefore, we hypothesized that RV could interfere with the MT-breakdown that takes place in mitosis during cell division. Using synchronized RV-permissive cells, we show that RV infection arrests the cell cycle in S/G2 phase, thus favoring replication by improving viroplasms formation, viral protein translation, and viral assembly. The arrest in S/G2 phase is independent of the host or viral strain and relies on active RV replication. RV infection causes cyclin B1 down-regulation, consistent with blocking entry into mitosis. With the aid of chemical inhibitors, the cytoskeleton network was linked to specific signaling pathways of the RV-induced cell cycle arrest. We found that upon RV infection Eg5 kinesin was delocalized from the pericentriolar region to the viroplasms. We used a MA104-Fucci system to identify three RV proteins (NSP3, NSP5, and VP2) involved in cell cycle arrest in the S-phase. Our data indicate that there is a strong correlation between the cell cycle arrest and RV replication.