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. ...

CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, UMR 967

INSERM, UMR 967

Université Paris Diderot, Sorbonne Paris Cité, UMR 967

Université Paris Sud, UMR 967

Gene expression in aging kidney and pituitary was determined by subtractive hybridization, DNA microarrays and RT-PCR. Kidneys and pituitary were removed from 10- and 30-month-old female WAG/Rij rats, which were free from chronic progressive nephrosis and had a low incidence of pituitary tumors with age. From 350 cDNA fragments isolated by subtractive hybridization, just one showed a more than twofold change in expression between 10 and 30 months. The use of a specific microarray with 4050 rodent genes also failed to detect downregulation lower than 0.5 or upregulation larger than 2.0 in aging rat kidney. Similarly, mRNA content for vasopressin V2 and V1 receptors, aquaporin 2 and 3, and adenylyl cyclase type VI was not significantly modified with age as determined by RT-PCR. In contrast, microarray analysis of pituitary mRNA expression showed upregulation of 11 genes with ratios equal to or greater than 2.0 and downregulation of 6 genes with ratios equal to or less than 0.5. Two cDNA sequences of unknown genes from the kidney subtractive library were part of the age-related up- and downregulated genes of the pituitary. Other genes were mainly related to cell differentiation, control of homeostasis, cellular signaling, endoplasmic reticulum trafficking and metabolism. These data indicated that mRNA expression is barely modified in aging kidney free from chronic progressive nephrosis, at least in the 0.5-2.0 range, in contrast to pituitary. They also suggest that the downregulation of proteins reported in aging kidneys free from gross disease is related to post-transcriptional changes.

Gene expression in aging kidney and pituitary.

Our knowledge of the molecular mechanisms that regulate hematopoiesis in physiologic and pathologic conditions is limited. Using a molecular approach based on cDNA microarrays, we demonstrated the emergence of an alternative pathway for mature bone marrow cell recovery after the programmed and reversible eradication of CD41+ cells in transgenic mice expressing a conditional toxigene targeted by the platelet alphaIIb promoter. The expression profile of the newly produced CD41+ cells showed high levels of transcripts encoding Ezh2, TdT, Rag2, and various immunoglobulin (Ig) heavy chains. In this context, we identified and characterized a novel population of Lin-Sca-1hi c-Kit- cells, with a lymphoid-like expression pattern, potentially involved in the reconstitution process. Our study revealed novel transcriptional cross talk between myeloid and lymphoid lineages and identified gene expression modifications that occur in vivo under these particular stress conditions, opening important prospects for therapeutic applications.

Novel pathway for megakaryocyte production after in vivo conditional eradication of integrin alphaIIb-expressing cells.

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.

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

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.

Unveiling novel RecO distant orthologues involved in homologous recombination.

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-1β, 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-1β 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-1β 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.

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

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.

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

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.

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.

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.

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

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.

Thierry Kortulewski

CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, UMR 967

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