[SAMHD1 acts at stalled replication forks to prevent interferon induction]. / SAMHD1 agit sur les fourches de réplication bloquées pour empêcher l'induction d'interféron.

DNA replication is an extremely complex process, involving thousands of replication forks progressing along chromosomes. These forks are frequently slowed down or stopped by various obstacles, such as secondary DNA structures, chromatin-acting proteins or a lack of nucleotides. This slowing down, known as replicative stress, plays a central role in tumour development. Complex processes, which are not yet fully understood, are set up to respond to this stress. Certain nucleases, such as MRE11 and DNA2, degrade the neo-replicated DNA at the level of blocked forks, allowing the replication to restart. The interferon pathway is a defense mechanism against pathogens that detects the presence of foreign nucleic acids in the cytoplasm and activates the innate immune response. DNA fragments resulting from genomic DNA metabolism (repair, retrotransposition) can diffuse into the cytoplasm and activate this pathway. A pathological manifestation of this process is the Aicardi-Goutières syndrome, a rare disease characterized by chronic inflammation leading to neurodegenerative and developmental problems. In this encephalopathy, it has been suggested that DNA replication may generate cytosolic DNA fragments, but the mechanisms involved have not been characterized. SAMHD1 is frequently mutated in the Aicardi-Goutières syndrome as well as in some cancers, but its role in the etiology of these diseases was largely unknown. We show that cytosolic DNA accumulates in SAMHD1-deficient cells, particularly in the presence of replicative stress, activating the interferon response. SAMHD1 is important for DNA replication under normal conditions and for the processing of stopped forks, independent of its dNTPase activity. In addition, SAMHD1 stimulates the exonuclease activity of MRE11 in vitro. When SAMHD1 is absent, degradation of neosynthesized DNA is inhibited, which prevents activation of the replication checkpoint and leads to failure to restart the replication forks. Resection of the replication forks is performed by an alternative mechanism which releases DNA fragments into the cytosol, activating the interferon response. The results obtained show, for the first time, a direct link between the response to replication stress and the production of interferons. These results have important implications for our understanding of the Aicardi-Goutières syndrome and cancers related to SAMHD1. For example, we have shown that MRE11 and RECQ1 are responsible for the production of DNA fragments that trigger the inflammatory response in cells deficient for SAMHD1. We can therefore imagine that blocking the activity of these enzymes could decrease the production of DNA fragments and, ultimately, the activation of innate immunity in these cells. In addition, the interferon pathway plays an essential role in the therapeutic efficacy of irradiation and certain chemotherapeutic agents such as oxaliplatin. Modulating this response could therefore be of much wider interest in anti-tumour therapy.

La réplication de l'ADN est un processus extrêmement complexe, impliquant des milliers de fourches de réplication progressant le long des chromosomes. Ces fourches sont fréquemment ralenties ou arrêtées par différents obstacles, tels que des structures secondaires de l'ADN, des protéines agissant sur la chromatine ou encore un manque de nucléotides. Ce ralentissement, qualifié de stress réplicatif, joue un rôle central dans le développement tumoral. Des processus complexes, qui ne sont pas encore totalement connus, sont mis en place pour répondre à ce stress. Certaines nucléases, comme MRE11 et DNA2, dégradent l'ADN néorépliqué au niveau des fourches bloquées, ce qui permet le redémarrage des réplisomes. La voie interféron est un mécanisme de défense contre les agents pathogènes qui détecte la présence d'acides nucléiques étrangers dans le cytoplasme et active la réponse immunitaire innée. Des fragments d'ADN issus du métabolisme de l'ADN génomique (réparation, rétrotransposition) peuvent diffuser dans le cytoplasme et activer cette voie. Une manifestation pathologique de ce processus est le syndrome d'Aicardi-Goutières, une maladie rare caractérisée par une inflammation chronique générant des problèmes neurodégénératifs et développementaux. Dans le cadre de cette encéphalopathie, il a été suggéré que la réplication de l'ADN pouvait générer des fragments d'ADN cytosoliques, mais les mécanismes impliqués n'avaient pas été caractérisés. SAMHD1 est fréquemment muté dans le syndrome d'Aicardi-Goutières ainsi que dans certains cancers, mais son rôle dans l'étiologie de ces maladies était jusqu'à présent largement inconnu. Nous montrons que de l'ADN cytosolique s'accumule dans les cellules déficientes pour SAMHD1, particulièrement en présence de stress réplicatif, activant la réponse interféron. Par ailleurs, SAMHD1 est important pour la réplication de l'ADN en conditions normales et pour le processing des fourches arrêtées, indépendamment de son activité dNTPase. De plus, SAMHD1 stimule l'activité exonucléase de MRE11 in vitro. Lorsque SAMHD1 est absent, la dégradation de l'ADN néosynthétisé est inhibée, ce qui empêche l'activation du checkpoint de réplication et entraine un défaut de redémarrage des fourches de réplication. De plus, la résection des fourches de réplication est réalisée par un mécanisme alternatif qui libère des fragments d'ADN dans le cytosol, activant la réponse interféron. Les résultats obtenus montrent, pour la première fois, un lien direct entre la réponse au stress réplicatif et la production d'interférons. Ces résultats ont des conséquences importantes dans notre compréhension du syndrome d'Aicardi Goutières et des cancers liés à SAMHD1. Par exemple, nous avons démontré que MRE11 et RECQ1 sont responsables de la production des fragments d'ADN qui déclenchent la réponse inflammatoire dans les cellules déficientes pour SAMHD1. Nous pouvons donc imaginer que bloquer l'activité de ces enzymes pourrait diminuer la production des fragments d'ADN et, in fine, l'activation de l'immunité innée dans ces cellules. Par ailleurs, la voie interférons joue un rôle essentiel dans l'efficacité thérapeutique de l'irradiation et de certains agents chimiothérapiques comme l'oxaliplatine. Moduler cette réponse pourrait donc avoir un intérêt beaucoup plus large en thérapie anti-tumorale.

Strategy for anti-aquaporin-4 auto-antibody identification and quantification using a new cell-based assay.

NMO-IgG is a specific biomarker of neuromyelitis optica (NMO) that targets the aquaporin-4 (AQP4) water channel protein. The current gold standard for NMO-IgG identification is indirect immunofluorescence (IIF). Our aim in this study was to develop a new quantitative cell-based assay (CBA) and to propose a rational strategy for anti-AQP4 Ab identification and quantification. We observed an excellent correlation between the CBA and IIF for NMO-IgG/anti-AQP4 detection. The CBA appeared more sensitive than IIF but on the other hand, IIF allows the simultaneous detection of various auto-Abs, underlining the complementarity between both methods. In conclusion, we propose to use IIF for the screening of patients at diagnosis in order to identify auto-Abs targeting the central nervous system. A highly sensitive, AQP4 specific and quantitative assay such as our CBA could be used thereafter to specifically identify the target of the Ab and to monitor its serum concentration under treatment.

Rap1-suppressed tumorigenesis is concomitant with the interference in ras effector signaling.

Expression of Rap1 blocks epithelial growth factor-induced extracellular signal-regulated kinases (ERKs) activation. However, recent studies demonstrated that Rap1 mediates ERKs activation induced by nerve growth factor. The anti-oncogenic effect of Rap1 has been reported but its mechanism remains unclear. To evaluate the correlation between the anti-transforming effect and the activation of ERKs, we transfected rap1 cDNA into Hep3B cells and selected stable transfectants. The Rap1 transfectants completely lost their intrinsic tumorigenicity in Balb/c nude mice. Both insulin and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-stimulated ERK activations were also blocked. Our findings suggest that Rap1-suppressed tumorigenicity is concomitant with ERKs inhibition.

A naturally occurring sequence variation that creates a YY1 element is associated with increased cystic fibrosis transmembrane conductance regulator gene expression.

We have identified previously a novel complex mutant allele in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a patient affected with cystic fibrosis (CF). This allele contained a mutation in CFTR exon 11 known to cause CF (S549R(T>G)), associated with the first alteration described so far in the minimal CFTR promoter region (-102T>A). Studies on genotype-phenotype correlations revealed striking differences between patients carrying mutation (S549R(T>G)) alone, who had a severe disease, and patients carrying the complex allele (-102(T>A)+S549R(T>G)), who exhibited milder forms of CF. We thus postulated that the sequence change (-102T>A) may attenuate the effects of the severe (S549R(T>G)) mutation through regulation of CFTR expression. Analysis of transiently transfected cell lines with wild-type and -102A variant human CFTR-directed luciferase reporter genes demonstrates that constructs containing the -102A variant (which creates a Yin Yang 1 (YY1) core element) increases CFTR expression significantly. Electrophoretic mobility shift assays indicate that the -102 site is located in a region of multiple DNA-protein interactions and that the -102A allele recruits specifically an additional nuclear protein related to YY1. The finding that the YY1-binding allele causes a significant increase in CFTR expression in vitro may allow a better understanding of the milder phenotype observed in patients who carry a severe CF mutation within the same gene.

Complexes formation between insulin receptor and extracellular signal-regulated kinases ERKs.

A property of signal transduction pathways that might explain their efficiency and specificity is the formation of signaling complexes. The recent demonstration that adaptor proteins can interact with many components of the extracellular signal-regulated kinases (ERKs) signaling cascade leads us to investigate whether such complexes may include the transmembrane receptor. The present work shows that in human hepatoma Hep3B cells, insulin receptor (IR) can be coimmunoprecipitated with other components of the ERKs cascade: insulin receptor substrate (IRS), Raf-1, and ERKs. Furthermore, these complexes formed near the cytoplasmic membrane even prior to insulin stimulation.

Survival of newly postmitotic motoneurons is transiently independent of exogenous trophic support.

We compared the survival requirements of early- and late-born motoneurons from E5 chicken spinal cord. Density gradient centrifugation followed by immunopanning using SC1 antibody allowed us to purify two size classes of motoneuron. Large motoneurons retained by 6.8% metrizamide were shown by BrdU labeling in ovo to be born on average 1.5 d earlier than the small motoneurons recovered from the metrizamide pellet. Large motoneurons were both biochemically and functionally more mature: they expressed higher levels of choline acetyltransferase and low-affinity neurotrophin receptor, and had an acute requirement for trophic support from muscle-derived factors. After 24 hr in culture in basal medium, all early-born motoneurons died, whereas 60% of late-born motoneurons survived. Small motoneurons can develop into large motoneurons in ovo, suggesting that they represent a general transitional stage in motoneuron development. Our results suggest that a defined period elapses between birth of a motoneuron and its acquisition of trophic dependence, possibly corresponding to the time required for target innervation. This property may have important consequences for the timing and regulation of developmental motoneuron death.

Motoneuron survival factors: biological roles and therapeutic potential.

The existence of factors that promote motoneuron survival in the spinal cord at critical stages of development was first deduced 50 yr ago. The large amount of work that has been put into characterizing such factors reflects both their biological importance and the hope that such molecules may be used therapeutically to slow motoneuron death in pathologies such as the spinal muscular atrophies and amyotrophic lateral sclerosis. Since 1990, several factors have been shown to have in vitro and/or in vivo activities that suggest they play a role in regulating motoneuron survival. Their physiological functions during motoneuron development are probably different and complementary. Several of them seem reasonable candidates for preclinical development, but many crucial experiments remain to be done.

Embryonic wing and leg motoneurons have intrinsically different survival properties.

Although spinal motoneurons in the chick embryo are born in a rostro-caudal gradient, the timing of their naturally occurring cell death varies in the opposite sense: brachial motoneurons (MNs) die later than lumbar MNs. We used in vitro methods to determine whether this difference results from factors intrinsic or extrinsic to the MNs. Embryonic MNs were purified from E5 lumbar and brachial spinal cord by a method that enriches for MNs with heightened trophic requirements; they were cultured in serum-free medium. In the absence of muscle extract, death of both populations was rapid; similar results are obtained in ovo after limb ablation. In the presence of muscle extracts, however, lumbar MNs always died more rapidly than brachial MNs, as in the normal embryo. We tested the ability of wing and leg bud extracts to provide trophic factors for MNs in culture. MNs from a given level responded equally well to wing or leg extracts. However, dose-response studies showed that near-maximal survival of brachial MNs was obtained with lower concentrations of limb extract than those required for optimal survival of lumbar MNs. These results suggest that the delayed apoptosis in the brachial MN population is not a result of differing peripheral trophic support, but is intrinsically programmed, at least partially through a higher sensitivity to trophic factors.

Neurotrophins promote motor neuron survival and are present in embryonic limb bud.

Embryonic spinal motor neurons are thought to depend for survival on unidentified factors secreted both by their peripheral targets and by cells within the central nervous system. The neurotrophins are a family of polypeptides required for survival of discrete central and peripheral neuronal populations in vivo and in vitro. In spite of their ability to reduce motor neuron death in vivo, the known neurotrophins have been thought to be without direct effect on motor neurons. Here we show that picomolar concentrations of three of them, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-5, can prevent the death of cultured embryonic rat spinal motor neurons. Furthermore, messenger RNA coding for neurotrophins is present at appropriate stages in spinal cord and limb bud, and mRNA for their receptors is found in motor neurons. These neurotrophins may therefore be physiological motor neuron growth factors.

[Growth and survival factors of spinal motoneurons]. / Facteurs de croissance et de survie des motoneurones spinaux.

The development of motoneurons in the spinal cord is strongly dependent on their interactions with their target tissue, skeletal muscle, and with other cells of the central nervous system. The molecular nature of these interactions has remained obscure for many years. However, over the last few years, known growth factors have been shown to have biological activity on the survival of motoneurons, at least in culture. The factors that have been studied are members of the FGF family (fibroblast growth factors), the TGF-beta family (transforming growth factor-beta), CNTF (ciliary neurotrophic factor) and CDF-LIF (cholinergic development factor-leukaemia inhibitory factor). There are also strong reasons to suppose that at least one member of the neurotrophin family (the family that contains Nerve Growth Factor) is involved in motoneuron development. A more detailed analysis of the biological role of each of these factors should not only enlighten us as to the importance of cell-cell interactions in development of the motoneuron, but also open the way to attempts to slow motoneuron death in pathological situations, either in animals or in man.

Neurotrophic factors in development and plasticity of spinal neurons.

Factors affecting neuronal growth may be considered to fall into two major categories: those required for neuronal survival during development or following a lesion, and those which enhance growth or regeneration of axonal or dendritic processes. We briefly review here some recent studies on the former in spinal cord development and plasticity as an introduction to other papers in the session on Factors controlling Neural Growth, and then present in more detail work on factors affecting motoneuron development in vitro. The neurotrophins are a closely-related family of basic neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and neurotrophins -3, -4 and -5 that enhance neuronal survival by binding to surface receptors whose major components are the trk tyrosine kinases and p75NGF-R. Only the latter has been studied in the context of spinal cord neuroplasticity: its levels on motoneurons are up-regulated following central or peripheral trauma, although its function there remains unknown. Much evidence exists for the existence of 'motoneuron growth factors' involved in regulation of survival and development of spinal motoneurons. Following a critical comparison of techniques for their purification, we review results obtained in vitro and in vivo using known growth factors such as ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and transforming growth factor (TGF/ß1). Although none of them satisfies all the criteria for the embryonic 'motoneuron growth factor', CNTF is of potential interest for reducing motoneuron loss in pathological situations.

Drosophila Sgs3 TATA: effects of point mutations on expression in vivo and protein binding in vitro with staged nuclear extracts.

The Drosophila salivary gland secretion protein gene, Sgs3, has a consensus TATA sequence and gives rise to abundant stage and tissue-specific transcripts. Two TATA point mutations (TAAA and TAGA) reduce transcript levels approximately 50-fold when assayed in transgenic flies. This effect is reflected in vitro, in DNase I footprint and gel retardation assays where we observed TATA-probe-specific complexes that are not seen with TAAA, TAGA or non-specific probes. The binding patterns observed when using nuclear extracts from 0-2- and 0-20-h embryos (Sgs3 inactive) differ from those seen with extracts from third instar salivary glands (Sgs3 active). There are also differences in in vitro binding when using an hsp70 TATA fragment, previously shown to substitute in vivo for the Sgs3 TATA sequence, as probe. Together these observations suggest the possibility that more than one TATA box factor may be present in these extracts. We conclude that a wild-type TATA motif is crucial for the binding of a TATA box factor and all subsequent interactions with other factors bound to the proximal and distal regulatory sequences that are necessary for the normal expression of Sgs3.

Olfactory marker protein gene: its structure and olfactory neuron-specific expression in transgenic mice.

Olfactory marker protein (OMP) genomic clones were isolated from a Charon 4A phage lambda rat genomic library. A 16.5-kilobase (kb) fragment of the rat genome containing the gene was isolated and characterized. Sequence analysis of the gene showed the absence of introns and the lack of CAAT and TATA boxes in the 5' flanking region. The transcription initiation site was mapped, and two sites 55 and 58 base pairs upstream of the ATG were observed. The 5' flanking region is rich in G+C residues and contains a G+C-rich motif as well as direct and inverted repeats. Functional OMP regulatory sequences were demonstrated in transgenic mice. An 11-kb chimeric gene was constructed in which the coding region for OMP was replaced with that for Thy-1.1. In Thy-1.2 mice carrying this transgene, Thy-1.1 was expressed solely by olfactory receptor neurons and their axons and terminals in the olfactory bulb.

Regulatory elements and interactions in the Drosophila 68C glue gene cluster.

We reviewed studies on the developmental regulation of the 68C glue gene cluster of Drosophila melanogaster. Extensive transformation analyses of Sgs-3 have shown that four regions necessary for normal expression can be distinguished. The first(+10 to -50) contains the transcription start site and TATA motif. This region can be replaced functionally by corresponding sequences from the hsp70 gene, but it is sensitive to point mutations in the TATA sequence. The second region (-50 to -98) contains more than one upstream sequence that, in combination with the other elements, leads to stage and tissue-specific expression. The third region (centered at -600) contains an element that enhances transcript levels some 20-fold. The final region (between -1.65 and -2.35 kb) contains elements having modest (twofold to threefold) effects on expression, one of which is contained in the coding sequences of Sgs-7, a second member of the cluster.

Sps-3 transcript levels are determined by multiple remote sequence elements.

The region from 1.4 to 2.7 kb upstream of Drosophila melanogaster gene Sgs-3 is responsible for a 10-fold increase in Sgs-3 transcript levels in the third instar larval salivary gland. This region includes the related Sgs-7 gene from the 68C glue gene cluster as well as 400 bp of its 5' sequences. We show that two elements are involved, each contributing a modest 3-fold effect. One of these includes Sgs-7 transcribed sequences some 2.3 kb upstream of Sgs-3, although Sgs-7 transcription is not involved. Although important for the overall levels of Sgs-3 expression, they are clearly not strong, viral-like enhancer elements. We propose that many position effects observed in P element transformation studies are the consequence of insertion in the vicinity of similar elements dispersed throughout the genome and having modest effects on transcript levels.

The Drosophila sgs3 gene: an in-vivo test of intron function.

An intronless Drosophila melanogaster sgs3 "glue" gene from the Formosa strain was constructed and inserted by P element transformation into a strain carrying an sgs3 variant that produces a larger mRNA and protein. By comparing both the RNAs and proteins produced by the two alleles we show that the intron has no detectable effect on sgs3 expression.