TGFß lengthens the G1 phase of stem cells in aged mouse brain.

Neurogenesis decreases during aging causing a progressive cognitive decline but it is still controversial whether proliferation defects in neurogenic niches result from a loss of neural stem cells or from an impairment of their progression through the cell cycle. Using an accurate fluorescence-activated cell sorting technique, we show that the pool of neural stem cells is maintained in the subventricular zone of middle-aged mice while they have a reduced proliferative potential eventually leading to the subsequent decrease of their progeny. In addition, we demonstrate that the G1 phase is lengthened during aging specifically in activated stem cells, but not in transit-amplifying cells, and directly impacts on neurogenesis. Finally, we report that inhibition of TGFß signaling restores cell cycle progression defects in stem cells. Our data highlight the significance of cell cycle dysregulation in stem cells in the aged brain and provide an attractive foundation for the development of anti-TGFß regenerative therapies based on stimulating endogenous neural stem cells.

Lack of a p21waf1/cip -dependent G1/S checkpoint in neural stem and progenitor cells after DNA damage in vivo.

The cyclin-dependent kinase inhibitor p21(waf1/cip) mediates the p53-dependent G1/S checkpoint, which is generally considered to be a critical requirement to maintain genomic stability after DNA damage. We used staggered 5-ethynyl-2'deoxyuridine/5-bromo-2'-deoxyuridine double-labeling in vivo to investigate the cell cycle progression and the role of p21(waf1/cip) in the DNA damage response of neural stem and progenitor cells (NSPCs) after exposure of the developing mouse cortex to ionizing radiation. We observed a radiation-induced p21-dependent apoptotic response in migrating postmitotic cortical cells. However, neural stem and progenitor cells (NSPCs) did not initiate a p21(waf1/cip1) -dependent G1/S block and continued to enter S-phase at a similar rate to the non-irradiated controls. The G1/S checkpoint is not involved in the mechanisms underlying the faithful transmission of the NSPC genome and/or the elimination of critically damaged cells. These processes typically involve intra-S and G2/M checkpoints that are rapidly activated after irradiation. p21 is normally repressed in neural cells during brain development except at the G1 to G0 transition. Lack of activation of a G1/S checkpoint and apoptosis of postmitotic migrating cells after DNA damage appear to depend on the expression of p21 in neural cells, since substantial cell-to-cell variations are found in the irradiated cortex. This suggests that repression of p21 during brain development prevents the induction of the G1/S checkpoint after DNA damage.

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.

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.

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.

Quiescent neural stem cells exit dormancy upon alteration of GABAAR signaling following radiation damage.

Quiescent neural stem cells (NSCs) are considered the reservoir for adult neurogenesis, generating new neurons throughout life. Until now, their isolation has not been reported, which has hampered studies of their regulatory mechanisms. We sorted by FACS quiescent NSCs and their progeny from the subventricular zone (SVZ) of adult mice according to the expression of the NSC marker LeX/CD15, the EGF receptor (EGFR) and the CD24 in combination with the vital DNA marker Hoechst 33342. Characterization of sorted cells showed that the LeX(bright)/EGFR-negative population was enriched in quiescent cells having an NSC phenotype. In contrast to proliferating NSCs and progenitors, the LeX(bright)/EGFR-negative cells, i.e. quiescent NSCs, resisted to a moderate dose of gamma-radiation (4Gy), entered the cell cycle two days after irradiation prior to EGFR acquisition and ultimately repopulated the SVZ. We further show that the GABAAR signaling regulates their cell cycle entry by using specific GABAAR agonists/antagonists and that the radiation-induced depletion of neuroblasts, the major GABA source, provoked their proliferation in the irradiated SVZ. Our study demonstrates that quiescent NSCs are specifically enriched in the LeX(bright)/EGFR-negative population, and identifies the GABAAR signaling as a regulator of the SVZ niche size by modulating the quiescence of NSCs.

Vascular-derived TGF-ß increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain.

Neurogenesis decreases during aging and following cranial radiotherapy, causing a progressive cognitive decline that is currently untreatable. However, functional neural stem cells remained present in the subventricular zone of high dose-irradiated and aged mouse brains. We therefore investigated whether alterations in the neurogenic niches are perhaps responsible for the neurogenesis decline. This hypothesis was supported by the absence of proliferation of neural stem cells that were engrafted into the vascular niches of irradiated host brains. Moreover, we observed a marked increase in TGF-ß1 production by endothelial cells in the stem cell niche in both middle-aged and irradiated mice. In co-cultures, irradiated brain endothelial cells induced the apoptosis of neural stem/progenitor cells via TGF-ß/Smad3 signalling. Strikingly, the blockade of TGF-ß signalling in vivo using a neutralizing antibody or the selective inhibitor SB-505124 significantly improved neurogenesis in aged and irradiated mice, prevented apoptosis and increased the proliferation of neural stem/progenitor cells. These findings suggest that anti-TGF-ß-based therapy may be used for future interventions to prevent neurogenic collapse following radiotherapy or during aging.

Similarities and differences in the in vivo response of mouse neonatal gonocytes and spermatogonia to genotoxic stress.

Neonatal gonocytes are the precursors of both spermatogonial stem cells and spermatogonia; thus, any persistent DNA damage in these cells may lead to heritable mutations. We investigated the response of male mouse neonatal germ cells to ionizing radiation. Both gonocytes and spermatogonia died in large numbers by apoptosis. However, we found that the gonocytes were significantly more sensitive than spermatogonia and somatic cells to radiation-induced double-strand breaks (DSBs), as assayed by the number of gamma-H2AFX foci. In contrast, gonocytes irradiated in G2 phase seemed to repair DSBs faster than spermatogonia. Moreover, when irradiated in S phase, gonocytes arrested their cell cycle at the G1/S phase transition, whereas spermatogonia were mostly blocked in G2/M phase. Despite these differences, both cell types expressed high levels of proteins involved in DSB signaling and repair. Within the first hours after irradiation, the expression of Atr, Mre11a, H2afx, Xrcc6, and Xrcc4 was downregulated in neonatal spermatogonia, whereas, in gonocytes, most gene expression was unaffected. Together, these results suggest that the response of neonatal testis to genotoxic stress is regulated by different mechanisms according to the cell type and the differentiation status.

Characterization of Spo11-dependent and independent phospho-H2AX foci during meiotic prophase I in the male mouse.

Meiotic DNA double strand breaks (DSBs) are indicated at leptotene by the phosphorylated form of histone H2AX (gamma-H2AX). In contrast to previous studies, we identified on both zygotene and pachytene chromosomes two distinct types of gamma-H2AX foci: multiple small (S) foci located along autosomal synaptonemal complexes (SCs) and larger signals on chromatin loops (L-foci). The S-foci number gradually declined throughout pachytene, in parallel with the repair of DSBs monitored by repair proteins suggesting that S-foci mark DSB repair events. We validated this interpretation by showing the absence of S-foci in Spo11(-/-) spermatocytes. By contrast, the L-foci number was very low through pachytene. Based on the analysis of gamma-H2AX labeling after irradiation of spermatocytes, the formation of DSBs clearly induced L-foci formation. Upon DSB repair, these foci appear to be processed and lead to the above mentioned S-foci. The presence of L-foci in wild-type pachytene and diplotene could therefore reflect delayed or unregulated DSB repair events. Interestingly, their distribution was different in Spo11(+/-) spermatocytes compared with Spo11(+/+) spermatocytes, where DSB repair might be differently regulated as a response to homeostatic control of crossing-over. The presence of these L-foci in Spo11(-/-) spermatocytes raises the interesting possibility of yet uncharacterized alterations in DNA or chromosome structure in Spo11(-/-) cells.

Poly(ADP-ribose) polymerase-2 contributes to the fidelity of male meiosis I and spermiogenesis.

Besides the established central role of poly(ADP-ribose) polymerase-1 (Parp-1) and Parp-2 in the maintenance of genomic integrity, accumulating evidence indicates that poly(ADP-ribosyl)ation may modulate epigenetic modifications under physiological conditions. Here, we provide in vivo evidence for the pleiotropic involvement of Parp-2 in both meiotic and postmeiotic processes. We show that Parp-2-deficient mice exhibit severely impaired spermatogenesis, with a defect in prophase of meiosis I characterized by massive apoptosis at pachytene and metaphase I stages. Although Parp-2(-/-) spermatocytes exhibit normal telomere dynamics and normal chromosome synapsis, they display defective meiotic sex chromosome inactivation associated with derailed regulation of histone acetylation and methylation and up-regulated X- and Y-linked gene expression. Furthermore, a drastically reduced number of crossover-associated Mlh1 foci are associated with chromosome missegregation at metaphase I. Moreover, Parp-2(-/-) spermatids are severely compromised in differentiation and exhibit a marked delay in nuclear elongation. Altogether, our findings indicate that, in addition to its well known role in DNA repair, Parp-2 exerts essential functions during meiosis I and haploid gamete differentiation.

Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.

BACKGROUND: Spermatogenesis in adult is a complex stepwise process leading to terminally differentiated spermatozoa. The cellular heterogeneity of testis renders complex the studies on molecular aspects of this differentiation process. Analysis of the regulation of adult spermatogenesis would undoubtedly benefit from the development of techniques to characterize each germinal differentiation step. METHODS: Hoechst 33342 staining of mouse testicular cells allows characterization of an enriched population in germinal stem cell and spermatogonia, called side population. In this study, we examined the definition of the various germinal populations stained by Hoechst 33342, notably meiotic and postmeiotic cells. RESULTS: Preleptotene spermatocytes, spermatocyte I, spermatocyte II, and round and elongated spermatids were discriminated by Hoechst 33342 staining. In addition, we associated differentiation of spermatocyte I through leptotene to diplotene with changes in Hoechst 33342 red fluorescence pattern. CONCLUSIONS: Hoechst 33342 staining of viable germinal cells constitutes a valuable tool to study normal and impaired mouse adult spermatogenesis or to isolate viable cells from various differentiation stages for studies of molecular mechanisms regulating spermatogenesis.