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.

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.

Increase in lamin B1 promotes telomere instability by disrupting the shelterin complex in human cells.

Telomere maintenance is essential to preserve genomic stability and involves telomere-specific proteins, DNA replication and repair proteins. Lamins are key components of the nuclear envelope and play numerous roles, including maintenance of the nuclear integrity, regulation of transcription, and DNA replication. Elevated levels of lamin B1, one of the major lamins, have been observed in some human pathologies and several cancers. Yet, the effect of lamin B1 dysregulation on telomere maintenance remains unknown. Here, we unveil that lamin B1 overexpression drives telomere instability through the disruption of the shelterin complex. Indeed, lamin B1 dysregulation leads to an increase in telomere dysfunction-induced foci, telomeric fusions and telomere losses in human cells. Telomere aberrations were preceded by mislocalizations of TRF2 and its binding partner RAP1. Interestingly, we identified new interactions between lamin B1 and these shelterin proteins, which are strongly enhanced at the nuclear periphery upon lamin B1 overexpression. Importantly, chromosomal fusions induced by lamin B1 in excess were rescued by TRF2 overexpression. These data indicated that lamin B1 overexpression triggers telomere instability through a mislocalization of TRF2. Altogether our results point to lamin B1 as a new interacting partner of TRF2, that is involved in telomere stability.

An XRCC4 mutant mouse, a model for human X4 syndrome, reveals interplays with Xlf, PAXX, and ATM in lymphoid development.

We developed an Xrcc4M61R separation of function mouse line to overcome the embryonic lethality of Xrcc4-deficient mice. XRCC4M61R protein does not interact with Xlf, thus obliterating XRCC4-Xlf filament formation while preserving the ability to stabilize DNA ligase IV. X4M61R mice, which are DNA repair deficient, phenocopy the Nhej1-/- (known as Xlf -/-) setting with a minor impact on the development of the adaptive immune system. The core non-homologous end-joining (NHEJ) DNA repair factor XRCC4 is therefore not mandatory for V(D)J recombination aside from its role in stabilizing DNA ligase IV. In contrast, Xrcc4M61R mice crossed on Paxx-/-, Nhej1-/-, or Atm-/- backgrounds are severely immunocompromised, owing to aborted V(D)J recombination as in Xlf-Paxx and Xlf-Atm double Knock Out (DKO) settings. Furthermore, massive apoptosis of post-mitotic neurons causes embryonic lethality of Xrcc4M61R -Nhej1-/- double mutants. These in vivo results reveal new functional interplays between XRCC4 and PAXX, ATM and Xlf in mouse development and provide new insights into the understanding of the clinical manifestations of human XRCC4-deficient condition, in particular its absence of immune deficiency.

Cognitive effects of low dose of ionizing radiation - Lessons learned and research gaps from epidemiological and biological studies.

The last decades have seen increased concern about the possible effects of low to moderate doses of ionizing radiation (IR) exposure on cognitive function. An interdisciplinary group of experts (biologists, epidemiologists, dosimetrists and clinicians) in this field gathered together in the framework of the European MELODI workshop on non-cancer effects of IR to summarise the state of knowledge on the topic and elaborate research recommendations for future studies in this area. Overall, there is evidence of cognitive effects from low IR doses both from biology and epidemiology, though a better characterization of effects and understanding of mechanisms is needed. There is a need to better describe the specific cognitive function or diseases that may be affected by radiation exposure. Such cognitive deficit characterization should consider the human life span, as effects might differ with age at exposure and at outcome assessment. Measurements of biomarkers, including imaging, will likely help our understanding on the mechanism of cognitive-related radiation induced deficit. The identification of loci of individual genetic susceptibility and the study of gene expression may help identify individuals at higher risk. The mechanisms behind the radiation induced cognitive effects are not clear and are likely to involve several biological pathways and different cell types. Well conducted research in large epidemiological cohorts and experimental studies in appropriate animal models are needed to improve the understanding of radiation-induced cognitive effects. Results may then be translated into recommendations for clinical radiation oncology and imaging decision making processes.

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.

The HIF1α/JMY pathway promotes glioblastoma stem-like cell invasiveness after irradiation.

Human glioblastoma (GBM) is the most common primary malignant brain tumor. A minor subpopulation of cancer cells, known as glioma stem-like cells (GSCs), are thought to play a major role in tumor relapse due to their stem cell-like properties, their high resistance to conventional treatments and their high invasion capacity. We show that ionizing radiation specifically enhances the motility and invasiveness of human GSCs through the stabilization and nuclear accumulation of the hypoxia-inducible factor 1α (HIF1α), which in turn transcriptionally activates the Junction-mediating and regulatory protein (JMY). Finally, JMY accumulates in the cytoplasm where it stimulates GSC migration via its actin nucleation-promoting activity. Targeting JMY could thus open the way to the development of new therapeutic strategies to improve the efficacy of radiotherapy and prevent glioma recurrence.

AsiDNA Is a Radiosensitizer with no Added Toxicity in Medulloblastoma Pediatric Models.

PURPOSE: Medulloblastoma is an important cause of mortality and morbidity in pediatric oncology. Here, we investigated whether the DNA repair inhibitor, AsiDNA, could help address a significant unmet clinical need in medulloblastoma care, by improving radiotherapy efficacy without increasing radiation-associated toxicity. EXPERIMENTAL DESIGN: To evaluate the brain permeability of AsiDNA upon systemic delivery, we intraperitoneally injected a fluorescence form of AsiDNA in models harboring brain tumors and in models still in development. Studies evaluated toxicity associated with combination of AsiDNA with radiation in the treatment of young developing animals at subacute levels, related to growth and development, and at chronic levels, related to brain organization and cognitive skills. Efficacy of the combination of AsiDNA with radiation was tested in two different preclinical xenografted models of high-risk medulloblastoma and in a panel of medulloblastoma cell lines from different molecular subgroups and TP53 status. Role of TP53 on the AsiDNA-mediated radiosensitization was analyzed by RNA-sequencing, DNA repair recruitment, and cell death assays. RESULTS: Capable of penetrating young brain tissues, AsiDNA showed no added toxicity to radiation. Combination of AsiDNA with radiotherapy improved the survival of animal models more efficiently than increasing radiation doses. Medulloblastoma radiosensitization by AsiDNA was not restricted to a specific molecular group or status of TP53. Molecular mechanisms of AsiDNA, previously observed in adult malignancies, were conserved in pediatric models and resembled dose increase when combined with irradiation. CONCLUSIONS: Our results suggest that AsiDNA is an attractive candidate to improve radiotherapy in medulloblastoma, with no indication of additional toxicity in developing brain tissues.

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.

ALT cancer cells are specifically sensitive to lysine acetyl transferase inhibition.

Some cancer cells elongate their telomeres through the ALT (alternative lengthening of telomeres) pathway, which is based on homologous recombination for the addition of telomere repeats without telomerase activity. General control non-derepressible 5 (GCN5) and P300/CBP-associated factor (PCAF), two homologous lysine acetyltransferases, exert opposite effects on the ALT pathway, inhibiting or favoring it respectively. Here we show that ALT cells are particularly sensitive to the inhibition of acetyltransferases activities using Anacardic Acid (AA). AA treatment recapitulates the effect of PCAF knockdown on several ALT features, suggesting that AA decreased the ALT mechanism through the inhibition of lysine transferase activity of PCAF, but not that of GCN5. Furthermore, AA specifically sensitizes human ALT cells to radiation as compared to telomerase-positive cells suggesting that the inhibition of lysine acetyltransferases activity may be used to increase the radiotherapy efficiency against ALT cancers.

Distinct Molecular Signatures of Quiescent and Activated Adult Neural Stem Cells Reveal Specific Interactions with Their Microenvironment.

Deciphering the mechanisms that regulate the quiescence of adult neural stem cells (NSCs) is crucial for the development of therapeutic strategies based on the stimulation of their endogenous regenerative potential in the damaged brain. We show that LeXbright cells sorted from the adult mouse subventricular zone exhibit all the characteristic features of quiescent NSCs. Indeed, they constitute a subpopulation of slowly dividing cells that is able to enter the cell cycle to regenerate the irradiated niche. Comparative transcriptomic analyses showed that they express hallmarks of NSCs but display a distinct molecular signature from activated NSCs (LeX+EGFR+ cells). Particularly, numerous membrane receptors are expressed on quiescent NSCs. We further revealed a different expression pattern of Syndecan-1 between quiescent and activated NSCs and demonstrated its role in the proliferation of activated NSCs. Our data highlight the central role of the stem cell microenvironment in the regulation of quiescence in adult neurogenic niches.

Longitudinal Study of Irradiation-Induced Brain Microstructural Alterations With S-Index, a Diffusion MRI Biomarker, and MR Spectroscopy.

PURPOSE: Radiation therapy is widely used for the treatment of brain tumors, but it may lead to severe cognitive impairments. Previous studies have shown that ionizing irradiation induces demyelination, blood-brain barrier alterations, and impaired neurogenesis in animal models. Hence, noninvasive and sensitive biomarkers of irradiation injury are needed to investigate these effects in patients and improve radiation therapy protocols. METHODS AND MATERIALS: The heads of 3-month-old male C57BL/6RJ mice (15 control mice and 15 irradiated mice) were exposed to radiation doses of 3 fractions of 5 Gy from a 60Co source with a medical irradiator. A longitudinal study was performed to investigate cranial irradiation-induced (3 fractions of 5 Gy) microstructural tissue alterations using water diffusion magnetic resonance imaging and magnetic resonance spectroscopy in different areas of the mouse brain (cortex, thalamus, striatum, olfactory bulbs [OBs], hippocampus, and subventricular zone [SVZ]). In addition to the quantification of standard non-Gaussian diffusion parameters, apparent diffusion coefficient (ADC0) and kurtosis (K), we evaluated a new composite diffusion metric, designated the S-index (ie, "signature index"). RESULTS: We observed a significant decrease in the S-index in the SVZ from 1 month to 8 months after brain irradiation (P < .05). An interesting finding was that, along with a decrease in taurine levels (up to -15% at 2 months, P < .01), a delayed S-index drop was observed in the OBs from 4 months after irradiation and maintained until the end of our experiment (P < .0001). These observations suggest that S-index variations revealed the irradiation-induced decline of neurogenesis that was further confirmed by a decrease in neural stem cells in the SVZ and in newborn neurons in the OBs of irradiated animals. CONCLUSIONS: This study demonstrates that diffusion magnetic resonance imaging, especially through the S-index approach, is a relevant imaging modality to monitor brain irradiation injury and probe microstructural changes underlying irradiation-induced cognitive deficits.

PAXX and Xlf interplay revealed by impaired CNS development and immunodeficiency of double KO mice.

The repair of DNA double-stranded breaks (DNAdsb) through non-homologous end joining (NHEJ) is a prerequisite for the proper development of the central nervous system and the adaptive immune system. Yet, mice with Xlf or PAXX loss of function are viable and present with very mild immune phenotypes, although their lymphoid cells are sensitive to ionizing radiation attesting for the role of these factors in NHEJ. In contrast, we show here that mice defective for both Xlf and PAXX are embryonically lethal owing to a massive apoptosis of post-mitotic neurons, a situation reminiscent to XRCC4 or DNA Ligase IV KO conditions. The development of the adaptive immune system in Xlf-/-PAXX-/- E18.5 embryos is severely affected with the block of B- and T-cell maturation at the stage of IgH and TCRß gene rearrangements, respectively. This damaging phenotype highlights the functional nexus between Xlf and PAXX, which is critical for the completion of NHEJ-dependent mechanisms during mouse development.

Isolation of Neural Stem and Progenitor Cells from the Adult Brain and Live Imaging of Their Cell Cycle with the FUCCI System.

Neural stem cells (NSCs) enter quiescence in early embryonic stages to create a reservoir of dormant NSCs able to enter proliferation and produce neuronal precursors in the adult mammalian brain. Various approaches of fluorescent-activated cell sorting (FACS) have emerged to allow the distinction between quiescent NSCs (qNSCs), their activated counterpart (aNSCs), and the resulting progeny. In this article, we review two FACS techniques that can be used alternatively. We also show that their association with transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice allows an unprecedented overlook on the cell cycle dynamics of adult NSCs.

Association of a Platinum Complex to a G-Quadruplex Ligand Enhances Telomere Disruption.

Telomeres protect the ends of chromosomes against illegitimate recombination and repair. They can be targets for G-quadruplex ligands and platinum complexes due to their repeated G-rich sequences. Protection of telomeres is ensured by a complex of six proteins, including TRF2, which inhibits the DNA damage response pathway. We analyzed telomere modifications induced in cancer cells by the experimental hybrid platinum complex, Pt-MPQ, comprising both an ethylene diamine monofunctional platinum complex and a G-quadruplex recognition moiety (MPQ). Pt-MPQ promotes the displacement of two telomeric proteins (TRF2 and TRF1) from telomeres, as well as the formation of telomere damage and telomere sister losses, whereas the control compound MPQ does not. This suggests that the platinum moiety potentiates the targeting of the G-quadruplex ligand to telomeres, opening a new perspective for telomere biology and anticancer therapy. Interestingly, the chemotherapy drug cisplatin, which has no specific affinity for G-quadruplex structures, partially induces the TRF2 delocalization from telomeres but produces less telomeric DNA damage, suggesting that this TRF2 displacement could be independent of G-quadruplex recognition.

Opposite effects of GCN5 and PCAF knockdowns on the alternative mechanism of telomere maintenance.

Cancer cells can use a telomerase-independent mechanism, known as alternative lengthening of telomeres (ALT), to elongate their telomeres. General control non-derepressible 5 (GCN5) and P300/CBP-associated factor (PCAF) are two homologous acetyltransferases that are mutually exclusive subunits in SAGA-like complexes. Here, we reveal that down regulation of GCN5 and PCAF had differential effects on some phenotypic characteristics of ALT cells. Our results suggest that GCN5 is present at telomeres and opposes telomere recombination, in contrast to PCAF that may indirectly favour them in ALT cells.

Hedgehog Controls Quiescence and Activation of Neural Stem Cells in the Adult Ventricular-Subventricular Zone.

Identifying the mechanisms controlling quiescence and activation of neural stem cells (NSCs) is crucial for understanding brain repair. Here, we demonstrate that Hedgehog (Hh) signaling actively regulates different pools of quiescent and proliferative NSCs in the adult ventricular-subventricular zone (V-SVZ), one of the main brain neurogenic niches. Specific deletion of the Hh receptor Patched in NSCs during adulthood upregulated Hh signaling in quiescent NSCs, progressively leading to a large accumulation of these cells in the V-SVZ. The pool of non-neurogenic astrocytes was not modified, whereas the activated NSC pool increased after a short period, before progressively becoming exhausted. We also showed that Sonic Hedgehog regulates proliferation of activated NSCs in vivo and shortens both their G1 and S-G2/M phases in culture. These data demonstrate that Hh orchestrates the balance between quiescent and activated NSCs, with important implications for understanding adult neurogenesis under normal homeostatic conditions or during injury.

Age-related neurogenesis decline in the subventricular zone is associated with specific cell cycle regulation changes in activated neural stem cells.

Although neural stem cells (NSCs) sustain continuous neurogenesis throughout the adult lifespan of mammals, they progressively exhibit proliferation defects that contribute to a sharp reduction in subventricular neurogenesis during aging. However, little is known regarding the early age-related events in neurogenic niches. Using a fluorescence-activated cell sorting technique that allows for the prospective purification of the main neurogenic populations from the subventricular zone (SVZ), we demonstrated an early decline in adult neurogenesis with a dramatic loss of progenitor cells in 4 month-old young adult mice. Whereas the activated and quiescent NSC pools remained stable up to 12 months, the proliferative status of activated NSCs was already altered by 6 months, with an overall extension of the cell cycle resulting from a specific lengthening of G1. Whole genome analysis of activated NSCs from 2- and 6-month-old mice further revealed distinct transcriptomic and molecular signatures, as well as a modulation of the TGFß signalling pathway. Our microarray study constitutes a cogent identification of new molecular players and signalling pathways regulating adult neurogenesis and its early modifications.

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.