Abnormal migration behavior linked to Rac1 signaling contributes to primordial germ cell exhaustion in Fanconi anemia pathway-deficient Fancg-/- embryos.

Fanconi anemia (FA) is a rare human genetic disorder characterized by bone marrow failure, predisposition to cancer and developmental defects including hypogonadism. Reproductive defects leading to germ cell aplasia are the most consistent phenotypes seen in FA mouse models. We examined the role of the nuclear FA core complex gene Fancg in the development of primordial germ cells (PGCs), the embryonic precursors of adult gametes, during fetal development. PGC maintenance was severely impaired in Fancg-/- embryos. We observed a defect in the number of PGCs starting at E9.5 and a strong attrition at E11.5 and E13.5. Remarkably, we observed a mosaic pattern reflecting a portion of testicular cords devoid of PGCs in E13.5 fetal gonads. Our in vitro and in vivo data highlight a potential role of Fancg in the proliferation and in the intrinsic cell motility abilities of PGCs. The random migratory process is abnormally activated in Fancg-/- PGCs, altering the migration of cells. Increased cell death and PGC attrition observed in E11.5 Fancg-/- embryos are features consistent with delayed migration of PGCs along the migratory pathway to the genital ridges. Moreover, we show that an inhibitor of RAC1 mitigates the abnormal migratory pattern observed in Fancg-/- PGCs.

Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ.

Canonical non-homologous end-joining (cNHEJ) is the prominent mammalian DNA double-strand breaks (DSBs) repair pathway operative throughout the cell cycle. Phosphorylation of Ku70 at ser27-ser33 (pKu70) is induced by DNA DSBs and has been shown to regulate cNHEJ activity, but the underlying mechanism remained unknown. Here, we established that following DNA damage induction, Ku70 moves from nucleoli to the sites of damage, and once linked to DNA, it is phosphorylated. Notably, the novel emanating functions of pKu70 are evidenced through the recruitment of RNA Pol II and concomitant formation of phospho-53BP1 foci. Phosphorylation is also a prerequisite for the dynamic release of Ku70 from the repair complex through neddylation-dependent ubiquitylation. Although the non-phosphorylable ala-Ku70 form does not compromise the formation of the NHEJ core complex per se, cells expressing this form displayed constitutive and stress-inducible chromosomal instability. Consistently, upon targeted induction of DSBs by the I-SceI meganuclease into an intrachromosomal reporter substrate, cells expressing pKu70, rather than ala-Ku70, are protected against the joining of distal DNA ends. Collectively, our results underpin the essential role of pKu70 in the orchestration of DNA repair execution in living cells and substantiated the way it paves the maintenance of genome stability.

Following transforming DNA in Helicobacter pylori from uptake to expression.

Natural transformation is a potent driver for genetic diversification in bacterial populations. It involves exogenous DNA binding, uptake, transport and internalization into the cytoplasm, where DNA can be processed and integrated into the host chromosome. Direct visualisation of transforming DNA (tDNA) has been limited to its binding to the surface or, in the case of Gram-negative species, to its entrance into the periplasm. We present here for the first time the direct visualisation of tDNA entering the bacterial cytoplasm. We used as a model the Gram-negative pathogen Helicobacter pylori, characterised by a large intraspecies variability that results from high mutation rates and efficient horizontal gene transfer. Using fluorescently labelled DNA, we followed for up to 3 h the fate of tDNA foci formed in the periplasm and eventually internalised into the cytoplasm. By tracking at the single cell level the expression of a fluorescent protein coded by the tDNA, we show that up to 50% of the cells express the transforming phenotype. The overall transformation process in H. pylori, from tDNA uptake to expression of the recombinant gene, can take place in less than 1 h, without requiring a growth arrest, and prior to the replication of the chromosome.

Intradermal injection of an anti-Langerin-HIVGag fusion vaccine targets epidermal Langerhans cells in nonhuman primates and can be tracked in vivo.

The development of new immunization strategies requires a better understanding of early molecular and cellular events occurring at the site of injection. The skin is particularly rich in immune cells and represents an attractive site for vaccine administration. Here, we specifically targeted vaccine antigens to epidermal Langerhans cells (LCs) using a fusion protein composed of HIV antigens and a monoclonal antibody targeting Langerin. We developed a fluorescence imaging approach to visualize, in vivo, the vaccine-targeted cells. Studies were performed in nonhuman primates (NHPs) because of their relevance as a model to assess human vaccines. We directly demonstrated that in NHPs, intradermally injected anti-Langerin-HIVGag specifically targets epidermal LCs and induces rapid changes in the LC network, including LC activation and migration out of the epidermis. Vaccine targeting of LCs significantly improved anti-HIV immune response without requirement of an adjuvant. Although the co-injection of the TLR-7/8 synthetic ligand, R-848 (resiquimod), with the vaccine, did not enhance significantly the antibody response, it stimulated recruitment of HLA-DR+ inflammatory cells to the site of immunization. This study allowed us to characterize the dynamics of early local events following the injection of a vaccine-targeted epidermal LCs and R-848.

Distinct spatiotemporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair.

Single-strand break repair (SSBR) and base excision repair (BER) of modified bases and abasic sites share several players. Among them is XRCC1, an essential scaffold protein with no enzymatic activity, required for the coordination of both pathways. XRCC1 is recruited to SSBR by PARP-1, responsible for the initial recognition of the break. The recruitment of XRCC1 to BER is still poorly understood. Here we show by using both local and global induction of oxidative DNA base damage that XRCC1 participation in BER complexes can be distinguished from that in SSBR by several criteria. We show first that XRCC1 recruitment to BER is independent of PARP. Second, unlike SSBR complexes that are assembled within minutes after global damage induction, XRCC1 is detected later in BER patches, with kinetics consistent with the repair of oxidized bases. Third, while XRCC1-containing foci associated with SSBR are formed both in eu- and heterochromatin domains, BER complexes are assembled in patches that are essentially excluded from heterochromatin and where the oxidized bases are detected.

Loss of CD24 promotes radiation­ and chemo­resistance by inducing stemness properties associated with a hybrid E/M state in breast cancer cells.

Cancer stem cells (CSCs) serve an essential role in failure of conventional antitumor therapy. In breast cancer, CD24­/low/CD44+ phenotype and high aldehyde dehydrogenase activity are associated with CSC subtypes. Furthermore, CD24­/low/CD44+ pattern is also characteristic of mesenchymal cells generated by epithelial­mesenchymal transition (EMT). CD24 is a surface marker expressed in numerous types of tumor, however, its biological functions and role in cancer progression and treatment resistance remain poorly documented. Loss of CD24 expression in breast cancer cells is associated with radiation resistance and control of oxidative stress. Reactive oxygen species (ROS) mediate the effects of anticancer drugs as well as ionizing radiation; therefore, the present study investigated if CD24 mediates radiation­ and chemo­resistance of breast cancer cells. Using a HMLE breast cancer cell model, CD24 expression has been artificially modulated and it was observed that loss of CD24 expression induced stemness properties associated with acquisition of a hybrid E/M phenotype. CD24­/low cells were more radiation­ and chemo­resistant than CD24+ cells. The resistance was associated with lower levels of ROS; CD24 controlled ROS levels via regulation of mitochondrial function independently of antioxidant activity. Together, these results suggested a key role of CD24 in de­differentiation of breast cancer cells and promoting acquisition of therapeutic resistance properties.

XLF/Cernunnos loss impairs mouse brain development by altering symmetric proliferative divisions of neural progenitors.

XLF/Cernunnos is a component of the ligation complex used in classical non-homologous end-joining (cNHEJ), a major DNA double-strand break (DSB) repair pathway. We report neurodevelopmental delays and significant behavioral alterations associated with microcephaly in Xlf-/- mice. This phenotype, reminiscent of clinical and neuropathologic features in humans deficient in cNHEJ, is associated with a low level of apoptosis of neural cells and premature neurogenesis, which consists of an early shift of neural progenitors from proliferative to neurogenic divisions during brain development. We show that premature neurogenesis is related to an increase in chromatid breaks affecting mitotic spindle orientation, highlighting a direct link between asymmetric chromosome segregation and asymmetric neurogenic divisions. This study reveals thus that XLF is required for maintaining symmetric proliferative divisions of neural progenitors during brain development and shows that premature neurogenesis may play a major role in neurodevelopmental pathologies caused by NHEJ deficiency and/or genotoxic stress.

Unveiling novel RecO distant orthologues involved in homologous recombination.

The generation of a RecA filament on single-stranded DNA is a critical step in homologous recombination. Two main pathways leading to the formation of the nucleofilament have been identified in bacteria, based on the protein complexes mediating RecA loading: RecBCD (AddAB) and RecFOR. Many bacterial species seem to lack some of the components involved in these complexes. The current annotation of the Helicobacter pylori genome suggests that this highly diverse bacterial pathogen has a reduced set of recombination mediator proteins. While it is now clear that homologous recombination plays a critical role in generating H. pylori diversity by allowing genomic DNA rearrangements and integration through transformation of exogenous DNA into the chromosome, no complete mediator complex is deduced from the sequence of its genome. Here we show by bioinformatics analysis the presence of a RecO remote orthologue that allowed the identification of a new set of RecO proteins present in all bacterial species where a RecR but not RecO was previously identified. HpRecO shares less than 15% identity with previously characterized homologues. Genetic dissection of recombination pathways shows that this novel RecO and the remote RecB homologue present in H. pylori are functional in repair and in RecA-dependent intrachromosomal recombination, defining two initiation pathways with little overlap. We found, however, that neither RecOR nor RecB contributes to transformation, suggesting the presence of a third, specialized, RecA-dependent pathway responsible for the integration of transforming DNA into the chromosome of this naturally competent bacteria. These results provide insight into the mechanisms that this successful pathogen uses to generate genetic diversity and adapt to changing environments and new hosts.

Lamin B1 sequesters 53BP1 to control its recruitment to DNA damage.

Double-strand breaks (DSBs) are harmful lesions and a major cause of genome instability. Studies have suggested a link between the nuclear envelope and the DNA damage response. Here, we show that lamin B1, a major component of the nuclear envelope, interacts directly with 53BP1 protein, which plays a pivotal role in the DSB repair. This interaction is dissociated after DNA damage. Lamin B1 overexpression impedes 53BP1 recruitment to DNA damage sites and leads to a persistence of DNA damage, a defect in nonhomologous end joining and an increased sensitivity to DSBs. The identification of interactions domains between lamin B1 and 53BP1 allows us to demonstrate that the defect of 53BP1 recruitment and the DSB persistence upon lamin B1 overexpression are due to sequestration of 53BP1 by lamin B1. This study highlights lamin B1 as a factor controlling the recruitment of 53BP1 to DNA damage sites upon injury.

Syndecan-1 Stimulates Adult Neurogenesis in the Mouse Ventricular-Subventricular Zone after Injury.

The production of neurons from neural stem cells (NSCs) persists throughout life in the mouse ventricular-subventricular zone (V-SVZ). We have previously reported that NSCs from adult V-SVZ are contained in cell populations expressing the carbohydrate SSEA-1/LeX, which exhibit either characteristics of quiescent NSCs (qNSCs) or of actively dividing NSCs (aNSCs) based on the absence or the presence of EGF-receptor, respectively. Using the fluorescence ubiquitination cell cycle indicator-Cdt1 transgenic mice to mark cells in G0/G1 phase of the cell cycle, we uncovered a subpopulation of qNSCs which were primed to enter the cell cycle in vitro. Besides, we found that treatment with Syndecan-1, a heparan sulfate proteoglycan involved in NSC proliferation, hastened the division of qNSCs and increased proliferation of aNSCs shortening their G1 phase in vitro. Furthermore, administration of Syndecan-1 ameliorated the recovery of neurogenic populations in the V-SVZ after radiation-induced injury providing potential cure for neurogenesis decline during brain aging or after injury.

Higher chromosome stability in embryonic neural stem and progenitor cells than in fibroblasts in response to acute or chronic genotoxic stress.

High fidelity of genetic transmission in neural stem and progenitor cells (NSPCs) has been long time considered to be crucial for brain development and homeostasis. However, recent studies have identified recurrent DSB clusters in dividing NSPCs, which may underlie the diversity of neuronal cell types. This raised the interest in understanding how NSPCs sense and repair DSBs and how this mechanism could be altered by environmental genotoxic stress caused by pollutants or ionizing radiation. Here, we show that embryonic mouse neural stem and progenitor cells (NSPCs) have significantly higher capacity than mouse embryonic fibroblasts (MEFs) to maintain their chromosome stability in response to acute (γ-radiation) and chronic (tritiated thymidine -3H-T- incorporation into DNA) genotoxic stress. Cells deficient for XLF/Cernunnos, which is involved in non-homologous end joining DNA (NHEJ) repair, highlighted important variations in fidelity of DNA repair pathways between the two cell types. Strikingly, a progressive and generalized chromosome instability was observed in MEFs cultured with 3H-T at long-term, whereas NSPCs cultured in the same conditions, preserved their chromosome stability thanks to higher DNA repair activity further enhanced by an adaptive response and also to the elimination of damaged cells by apoptosis. This specific DNA damage response of NSPCs may rely on the necessity for preservation of their genome stability together with their possible function in creating neuronal genetic diversity.

Electroporation as a vaccine delivery system and a natural adjuvant to intradermal administration of plasmid DNA in macaques.

In vivo electroporation (EP) is used to enhance the uptake of nucleic acids and its association with DNA vaccination greatly stimulates immune responses to vaccine antigens delivered through the skin. However, the effect of EP on cutaneous cell behavior, the dynamics of immune cell recruitment and local inflammatory factors, have not been fully described. Here, we show that intradermal DNA vaccination combined with EP extends antigen expression to the epidermis and the subcutaneous skin muscle in non-human primates. In vivo fibered confocal microscopy and dynamic ex vivo imaging revealed that EP promotes the mobility of Langerhans cells (LC) and their interactions with transfected cells prior to their migration from the epidermis. At the peak of vaccine expression, we detected antigen in damaged keratinocyte areas in the epidermis and we characterized recruited immune cells in the skin, the hypodermis and the subcutaneous muscle. EP alone was sufficient to induce the production of pro-inflammatory cytokines in the skin and significantly increased local concentrations of Transforming Growth Factor (TGF)-alpha and IL-12. Our results show the kinetics of inflammatory processes in response to EP of the skin, and reveal its potential as a vaccine adjuvant.

ComB proteins expression levels determine Helicobacter pylori competence capacity.

Helicobacter pylori chronically colonises half of the world's human population and is the main cause of ulcers and gastric cancers. Its prevalence and the increase in antibiotic resistance observed recently reflect the high genetic adaptability of this pathogen. Together with high mutation rates and an efficient DNA recombination system, horizontal gene transfer through natural competence makes of H. pylori one of the most genetically diverse bacteria. We show here that transformation capacity is enhanced in strains defective for recN, extending previous work with other homologous recombination genes. However, inactivation of either mutY or polA has no effect on DNA transformation, suggesting that natural competence can be boosted in H. pylori by the persistence of DNA breaks but not by enhanced mutagenesis. The transformation efficiency of the different DNA repair impaired strains correlates with the number of transforming DNA foci formed on the cell surface and with the expression of comB8 and comB10 competence genes. Overexpression of the comB6-B10 operon is sufficient to increase the transformation capacity of a wild type strain, indicating that the ComB complex, present in the bacterial wall and essential for DNA uptake, can be a limiting factor for transformation efficiency.

Novel microarray-based method for estimating exposure to ionizing radiation.

Accurate estimation of the dose of ionizing radiation to which individuals have been exposed is critical for therapeutic treatment. We investigated whether gene expression profiles could be used to evaluate the dose received, thereby serving as a biological dosimeter. We used cDNA microarrays to monitor changes in gene expression profiles induced by ionizing radiation in mouse total blood. The subsets of genes best characterizing each dose were identified by resampling the original data set and calculating the intersection of the dose signatures. This analytical strategy minimizes the impact of potential genetic/epigenetic variation between mice and overcomes the bias in gene selection inherent to microarray technology. The significance of the identified signatures was evaluated by monitoring the type I error rate by in silico negative control simulation. Based on the distribution of the mean ratios of the selected probes, we were able to identify transcription profiles giving 83% to 100% correct estimation of the dose received by test mice, demonstrating that the selected probes could be used to determine the dose of radiation to which the animals had been exposed. This method could potentially be generalized to determine the level of exposure to other toxins and could be used to develop new related clinical applications.

Interaction with OGG1 is required for efficient recruitment of XRCC1 to base excision repair and maintenance of genetic stability after exposure to oxidative stress.

XRCC1 is an essential protein required for the maintenance of genomic stability through its implication in DNA repair. The main function of XRCC1 is associated with its role in the single-strand break (SSB) and base excision repair (BER) pathways that share several enzymatic steps. We show here that the polymorphic XRCC1 variant R194W presents a defect in its interaction with the DNA glycosylase OGG1 after oxidative stress. While proficient for single-strand break repair (SSBR), this variant does not colocalize with OGG1, reflecting a defect in its involvement in BER. Consistent with a role of XRCC1 in the coordination of the BER pathway, induction of oxidative base damage in XRCC1-deficient cells complemented with the R194W variant results in increased genetic instability as revealed by the accumulation of micronuclei. These data identify a specific molecular role for the XRCC1-OGG1 interaction in BER and provide a model for the effects of the R194W variant identified in molecular cancer epidemiology studies.

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

Specific uptake and genotoxicity induced by polystyrene nanobeads with distinct surface chemistry on human lung epithelial cells and macrophages.

Nanoparticle surface chemistry is known to play a crucial role in interactions with cells and their related cytotoxic effects. As inhalation is a major route of exposure to nanoparticles, we studied specific uptake and damages of well-characterized fluorescent 50 nm polystyrene (PS) nanobeads harboring different functionalized surfaces (non-functionalized, carboxylated and aminated) on pulmonary epithelial cells and macrophages (Calu-3 and THP-1 cell lines respectively). Cytotoxicity of in mass dye-labeled functionalized PS nanobeads was assessed by xCELLigence system and alamarBlue viability assay. Nanobeads-cells interactions were studied by video-microscopy, flow cytometry and also confocal microscopy. Finally ROS generation was assessed by glutathione depletion dosages and genotoxicity was assessed by γ-H2Ax foci detection, which is considered as the most sensitive technique for studying DNA double strand breaks. The uptake kinetic was different for each cell line. All nanobeads were partly adsorbed and internalized, then released by Calu-3 cells, while THP-1 macrophages quickly incorporated all nanobeads which were located in the cytoplasm rather than in the nuclei. In parallel, the genotoxicity study reported that only aminated nanobeads significantly increased DNA damages in association with a strong depletion of reduced glutathione in both cell lines. We showed that for similar nanoparticle concentrations and sizes, aminated polystyrene nanobeads were more cytotoxic and genotoxic than unmodified and carboxylated ones on both cell lines. Interestingly, aminated polystyrene nanobeads induced similar cytotoxic and genotoxic effects on Calu-3 epithelial cells and THP-1 macrophages, for all levels of intracellular nanoparticles tested. Our results strongly support the primordial role of nanoparticles surface chemistry on cellular uptake and related biological effects. Moreover our data clearly show that nanoparticle internalization and observed adverse effects are not necessarily associated.

Assessment of an in vitro model of pulmonary barrier to study the translocation of nanoparticles.

As the lung is one of the main routes of exposure to manufactured nanoparticles, we developed an in vitro model resembling the alveolo-capillary barrier for the study of nanoparticle translocation. In order to provide a relevant and ethical in vitro model, cost effective and easy-to-implement human cell lines were used. Pulmonary epithelial cells (Calu-3 cell line) and macrophages (THP-1 differentiated cells) were cultivated on the apical side and pulmonary endothelial cells (HPMEC-ST1.6R cell line) on the basal side of a microporous polyester membrane (Transwell®). Translocation of non-functionalized (51 and 110 nm) and aminated (52 nm) fluorescent polystyrene (PS) nanobeads was studied in this system. The use of Calu-3 cells allowed high transepithelial electrical resistance (TEER) values (>1000 Ω cm2) in co-cultures with or without macrophages. After 24 h of exposure to non-cytotoxic concentrations of non-functionalized PS nanobeads, the relative TEER values (%/t0) were significantly decreased in co-cultures. Epithelial cells and macrophages were able to internalize PS nanobeads. Regarding translocation, Transwell® membranes per se limit the passage of nanoparticles between apical and basal side. However, small non-functionalized PS nanobeads (51 nm) were able to translocate as they were detected in the basal side of co-cultures. Altogether, these results show that this co-culture model present good barrier properties allowing the study of nanoparticle translocation but research effort need to be done to improve the neutrality of the porous membrane delimitating apical and basal sides of the model.

The Notch Delta-4 ligand helps to maintain the quiescence and the short-term reconstitutive potential of haematopoietic progenitor cells through activation of a key gene network.

Understanding the role of Notch and its ligands within the different bone marrow niches could shed light on the mechanisms regulating haematopoietic progenitor cells (HPCs) maintenance and self-renewal. Here, we report that murine bone marrow HPCs activation by the vascular Notch Delta-4 ligand maintains a significant proportion of cells specifically in the G0 state. Furthermore, Delta-4/Notch pathway limits significantly the loss of the in vivo short-term reconstitutive potential upon transplantation of Delta-4 activated HPCs into lethally irradiated recipient mice. Both effects are directly correlated with the decrease of cell cycle genes transcription such as CYCLIN-D1, -D2, and -D3, and the upregulation of stemness related genes transcription such as BMI1, GATA2, HOXB4 and C-MYC. In addition, the transcriptional screening also highlights new downstream post-transcriptional factors, named PUMILIO1 and -2, as part of the stem signature associated with the Delta-4/Notch signalling pathway.

Cell cooperation and role of the P2X7 receptor in pulmonary inflammation induced by nanoparticles.

Macrophages and alveolar epithelial cells are the first targets of inhaled nanoparticles (NPs) reaching the alveoli. Mono- or co-cultures of lung epithelial (A549 or NCI-H441) and macrophage (THP-1) cell lines were used to study the cell cooperation and the involvement of the P2X7 cell death receptor during the inflammation caused by SiO2 and TiO2 NPs. Here we show that, secretion of pro-inflammatory cytokines (IL-1ß, IL-6 and IL-8) in response to NPs exposure was higher in co-cultures than in mono-cultures. A functional P2X7 receptor was found in all the cell lines studied. Its involvement in IL-1ß secretion in co-cultures was demonstrated using a specific antagonist, the brilliant blue G. Furthermore, mono and co-cultures exhibited distinct secretion patterns of pro-inflammatory cytokines in response to NPs exposure, and we provide the first evidence that the P2X7 receptor is involved in the inflammation triggered by SiO2 and TiO2 NPs, by increasing IL-1ß secretion, and likely through the inflammasome pathway. Altogether, our data indicate that cell co-cultures used in this study represent valid models to study the inflammatory mechanisms of NPs within the alveoli.