Vascular‐derived TGF‐β increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain
2013; Springer Nature; Volume: 5; Issue: 4 Linguagem: Inglês
10.1002/emmm.201202197
ISSN1757-4684
AutoresJosé Ramón Pineda, Mathieu Daynac, Alexandra Chicheportiche, Arantxa Cebrián‐Silla, Karine Sii-Felice, José Manuel García‐Verdugo, François D. Boussin, Marc‐André Mouthon,
Tópico(s)TGF-β signaling in diseases
ResumoResearch Article25 March 2013Open Access Source Data Vascular-derived TGF-β increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain Jose R. Pineda Jose R. Pineda CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Fontenay-aux-Roses, France Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France Université Paris Sud, UMR 967, Fontenay-aux-Roses, France Search for more papers by this author Mathieu Daynac Mathieu Daynac CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Fontenay-aux-Roses, France Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France Université Paris Sud, UMR 967, Fontenay-aux-Roses, France Search for more papers by this author Alexandra Chicheportiche Alexandra Chicheportiche CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Fontenay-aux-Roses, France Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France Université Paris Sud, UMR 967, Fontenay-aux-Roses, France Search for more papers by this author Arantxa Cebrian-Silla Arantxa Cebrian-Silla Laboratorio de Neurobiología Comparada, Instituto Cavanilles, Universidad de Valencia, Valencia, CIBERNED, Spain Search for more papers by this author Karine Sii Felice Karine Sii Felice CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Fontenay-aux-Roses, France Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France Université Paris Sud, UMR 967, Fontenay-aux-Roses, France Search for more papers by this author Jose Manuel Garcia-Verdugo Jose Manuel Garcia-Verdugo Laboratorio de Neurobiología Comparada, Instituto Cavanilles, Universidad de Valencia, Valencia, CIBERNED, Spain Search for more papers by this author François D. Boussin Corresponding Author François D. Boussin [email protected] CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Fontenay-aux-Roses, France Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France Université Paris Sud, UMR 967, Fontenay-aux-Roses, France Search for more papers by this author Marc-André Mouthon Corresponding Author Marc-André Mouthon [email protected] CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Fontenay-aux-Roses, France Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France Université Paris Sud, UMR 967, Fontenay-aux-Roses, France Search for more papers by this author Jose R. Pineda Jose R. Pineda CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Fontenay-aux-Roses, France Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France Université Paris Sud, UMR 967, Fontenay-aux-Roses, France Search for more papers by this author Mathieu Daynac Mathieu Daynac CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Fontenay-aux-Roses, France Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France Université Paris Sud, UMR 967, Fontenay-aux-Roses, France Search for more papers by this author Alexandra Chicheportiche Alexandra Chicheportiche CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Fontenay-aux-Roses, France Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France Université Paris Sud, UMR 967, Fontenay-aux-Roses, France Search for more papers by this author Arantxa Cebrian-Silla Arantxa Cebrian-Silla Laboratorio de Neurobiología Comparada, Instituto Cavanilles, Universidad de Valencia, Valencia, CIBERNED, Spain Search for more papers by this author Karine Sii Felice Karine Sii Felice CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Fontenay-aux-Roses, France Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France Université Paris Sud, UMR 967, Fontenay-aux-Roses, France Search for more papers by this author Jose Manuel Garcia-Verdugo Jose Manuel Garcia-Verdugo Laboratorio de Neurobiología Comparada, Instituto Cavanilles, Universidad de Valencia, Valencia, CIBERNED, Spain Search for more papers by this author François D. Boussin Corresponding Author François D. Boussin [email protected] CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Fontenay-aux-Roses, France Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France Université Paris Sud, UMR 967, Fontenay-aux-Roses, France Search for more papers by this author Marc-André Mouthon Corresponding Author Marc-André Mouthon [email protected] CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Fontenay-aux-Roses, France Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France Université Paris Sud, UMR 967, Fontenay-aux-Roses, France Search for more papers by this author Author Information Jose R. Pineda1,2,3,4, Mathieu Daynac1,2,3,4, Alexandra Chicheportiche1,2,3,4, Arantxa Cebrian-Silla5, Karine Sii Felice1,2,3,4, Jose Manuel Garcia-Verdugo5, François D. Boussin *,1,2,3,4,† and Marc-André Mouthon *,1,2,3,4,† 1CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France 2INSERM, U967, Fontenay-aux-Roses, France 3Université Paris Diderot, Sorbonne Paris Cité, UMR 967, Fontenay-aux-Roses, France 4Université Paris Sud, UMR 967, Fontenay-aux-Roses, France 5Laboratorio de Neurobiología Comparada, Instituto Cavanilles, Universidad de Valencia, Valencia, CIBERNED, Spain † These authors contributed equally to this work. *Tel: +33 1 46 54 94 61; Fax: +33 1 46 54 91 80 EMBO Mol Med (2013)5:548-562https://doi.org/10.1002/emmm.201202197 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract 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-β1 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-β/Smad3 signalling. Strikingly, the blockade of TGF-β 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-β-based therapy may be used for future interventions to prevent neurogenic collapse following radiotherapy or during aging. The paper explained PROBLEM: A progressive cognitive decline occurs during aging and following cranial radiotherapy and is currently untreatable. A key element of this decline is the decreased production of new neurons by neural stem cells. However, mechanisms that underlie changes that occur in both stem cells and neurogenesis are poorly understood. RESULTS: One of our major findings is that a deficit in neurogenesis that is observed following high-dose radiation and during aging is due to alterations in the microenvironment that regulates stem cell fate rather than to a direct effect on the stem cells. Moreover, we have elucidated the molecular mechanism by which the increased synthesis of TGF-β1 by brain endothelial cells in the stem cell niche provokes stem cell dormancy and increases their susceptibility to apoptosis. The second important finding is that the pharmacological blockade of TGF-β signalling restored the production of new neurons and their integration into the olfactory bulbs of irradiated and elderly mice. IMPACT: Our newly discovered mechanism should encourage the development of anti-TGF-β therapies for (i) limiting brain injury that is caused by radiotherapy or (ii) rejuvenating neurogenesis in elderly individuals with cognitive decline. INTRODUCTION Decreased adult neurogenesis following cranial irradiation, which is a central adjuvant treatment for brain tumours in both paediatric and adult patients, is believed to contribute to cognitive decline (Monje & Palmer, 2003). We and others have reported that exposure of the brain to 15 Gy is accompanied by the perturbation of olfactory memory and is associated with decreased neurogenesis in mice (Lazarini et al, 2009; Valley et al, 2009). Neural stem cells (NSCs) are located in the adult subventricular zone (SVZ) and are involved in neurogenesis during adulthood (Doetsch et al, 1999). Adult NSCs successively give rise to transit amplifying progenitors (TAPs) and then to neuroblasts, which migrate in chains to the olfactory bulbs (OBs), where they differentiate into neurons (Alvarez-Buylla & Lim, 2004). Dividing NSCs and TAPs establish intimate interactions with blood vessels at sites that lack pericyte coverage to form vascular niches within the adult SVZ (Mirzadeh et al, 2008; Shen et al, 2008; Tavazoie et al, 2008). Increasing evidence has revealed the importance of growth factors that are synthesized by brain endothelial cells (BECs) of the vascular niche in the regulation of neurogenesis, including NSC proliferation (Ramirez-Castillejo et al, 2006). Moreover, molecular cross-talk between NSCs and BECs includes signals that act on both cell types. These signals include members of the vascular endothelial growth factor family (Calvo et al, 2011). Irradiation provokes apoptosis in proliferating cells in the SVZ and a clear dose-dependent impairment of neurogenesis that is permanent for doses exceeding 10 Gy in rodents (Tada et al, 1999). Whereas some NSCs have been reported to survive after 10 Gy irradiation, they lack the ability to give rise to new neurons (Achanta et al, 2012). Apart from a reduction in the number of resident NSCs, irradiation may also generate a hostile microenvironment. In particular, this treatment may lessen NSC proliferation and differentiation in vivo. Indeed, microglial inflammation that accompanies radiation injury has been implicated in neurogenic collapse and NSC dysfunction in the hippocampus (Monje et al, 2003); however, the mechanisms of neurogenesis alteration in the SVZ remain elusive. Studies indicate that physiological aging is also associated with a progressive reduction in proliferating cells and in doublecortin-positive neuroblasts in the SVZ and OBs of rodents (Enwere et al, 2004; Maslov et al, 2004; Tropepe et al, 1997). A significant decline in neural stem/progenitor cells is apparent by 6 months of age in the SVZ, ultimately resulting in a dramatic reduction in the number of these cells in elderly mice (Enwere et al, 2004). A premature decrease in the NSC pool owing to aging suggests that these NSCs have no self-renewal capacity and/or are programmed to complete only a limited number of divisions (Sii-Felice et al, 2008). However, when the SVZ from aged mice were cultured in vitro, NSCs retain their capacity to proliferate and to differentiate into functional neurons, similar to the NSCs in young adult mice, albeit with lower efficiency (Ahlenius et al, 2009; Tropepe et al, 1997). Furthermore, the neurogenesis decline that is observed during ageing in the hippocampus has been attributed in part to changes in the systemic milieu (Villeda et al, 2011). TGF-β has been widely recognized as an injury-related cytokine, as its levels are strongly and rapidly upregulated in the brain following different forms of injuries (Gomes et al, 2005) and during aging (Werry et al, 2010). The chronic elevation of TGF-β1 triggers accumulation of basement proteins and results in Alzheimer's disease-like cerebrovascular amyloidosis and microvascular degeneration (Wyss-Coray et al, 2000). Although TGF-β promotes the survival of adult neurons (Boche et al, 2003; Schober et al, 2007), it also has an apoptotic effect on proliferating neural-crest-derived multipotent progenitor cells (Hagedorn et al, 2000). Furthermore, TGF-β1 inhibits the proliferation of adult NSCs, although both positive and negative effects of TGF-β1 have been reported on adult neurogenesis (Battista et al, 2006; Buckwalter et al, 2006; Wachs et al, 2006). This study explores whether the decline in SVZ neurogenesis during aging or following irradiation is merely a function of NSC depletion or reflects more profound changes in the NSC vascular niche. We demonstrate that TGF-β pathway activation was persistently increased in the SVZ niches of irradiated or aged mice. We also report that the selective inhibition of this pathway significantly improved neurogenesis. RESULTS High-dose radiation decreases neurogenesis but spares NSCs A mouse model of whole-brain irradiation with a total radiation dose of 15 Gy, divided into three doses of 5 Gy that were delivered at 48 h intervals, was used to explore the effects of radiation on adult neurogenesis in the SVZ. This 15 Gy split-dose irradiation paradigm did not provoke the mobilization or the activation of microglial cells with respect to the number and the resting morphology of CD68+ and Iba1+ cells in the SVZ/striatum (Supporting Information Fig S1). As estimated by Ki67-positivity, proliferation was dramatically decreased in the SVZ 4 months following radiation exposure, and the total number of nuclei was also reduced (Supporting Information Fig S2). Despite this reduced proliferation capacity, the survival of NSCs was indicated by the presence of Nestin+GFAP+ double-positive cells lining the lateral ventricle that were negative for S100β (Supporting Information Fig S3). We previously reported that this irradiation regimen reduces the number of neuroblasts in the SVZ and decreases their arrival at the OBs, inducing olfactory memory deficits in mice (Lazarini et al, 2009). The drastic decrease in neuroblasts/type A and TAPs/type C cells was also observed 1 year following irradiation (Fig 1B and C). Although their absolute number was decreased, half of the type B cells persisted for 1 year following exposure (Fig 1B and C). Figure 1. NSCs resisted high radiation exposure but presented proliferation defects. A,B.. Electron microscopy revealed the persistence of type B/NSCs 1 year following irradiation in the SVZ, whereas type C/TAPs and type A/neuroblasts were nearly completely lost. Scale bar: 10 µm. C–F.. Ependymal cell (Epend), neuron (Neu) and microglial cell (MG) numbers were unaltered. FACS analysis of SVZ populations (percentage in D–F) and their proliferating fraction (DNA >2N in D′–F′): neuroblasts (CD24+ in D and D′), TAPs (EGFR+ in E and E′) and activated NSCs (LeX+EGFR+ in F and F′). The p-value was determined using the Mann–Whitney U-test. G.. The quantification of NSCs and TAPs in the N-CFCA. The mean ± SD of two to five independent experiments is shown (the number of mice is indicated within the bars). The p-value was determined using the Mann–Whitney U-test. H.. NSC-derived clones were subcultured to confirm their self-renewal capacity. The Kaplan–Meier's analysis is shown. Download figure Download PowerPoint We further examined the content of NSCs and their progeny at 4 months by FACS analysis on freshly dissociated microdissected SVZs. In agreement with the reduction in proliferation described above, the total number of cells in the dissociated SVZ decreased to 25 ± 5 × 103 cells/SVZ 4 months following irradiation compared to 47 ± 7 × 103 cells in young non-irradiated control mice (p = 0.019). The total number of SVZ cells also significantly decreased in 12-month-old (i.e. middle-aged) mice, reaching 32 ± 4 × 103 cells/SVZ (p = 0.032). Given that LeX is expressed in the SVZ on GFAP-positive cells that have NSC features (Capela & Temple, 2002), an anti-LeX antibody was used in combination with CD24 and an EGF fluorescent ligand to label neuroblasts and activated NSCs. According to a previous report (Pastrana et al, 2009), we defined the following three populations: (i) CD24−LeX+EGFR+ activated NSCs, (ii) CD24−LeX−EGFR+ TAPs and (iii) CD24+ neuroblasts. The purity of these sorted SVZ populations was confirmed using qRT-PCR for the mRNA expression levels of specific NSC, TAP and neuroblast markers (Supporting Information Fig S4). As was expected from our previous findings (Lazarini et al, 2009), a decrease in CD24+ neuroblasts was observed in irradiated mice (Fig 1D). The percentage of TAPs (EGFR+LeX−) diminished in the SVZ of both middle-aged and irradiated mice (Fig 1E), whereas the relative number of NSCs (LeX+EGFR+) was unaltered (Fig 1F). FACS analysis using GLAST, which is another NSC marker that is expressed on nearly all LeX-positive cells (Supporting Information Fig S4D), confirmed that the percentage of CD24−GLAST+ cells, i.e. the population that contained NSCs, was maintained in the SVZ following irradiation and during aging (Supporting Information Fig S5). However, as the total number of cells was reduced in the SVZ, the absolute number of NSCs was decreased following irradiation and during aging; however, this decrease was less pronounced than for their progeny. Moreover, all of these populations exhibited a diminution in their proliferation status (DNA content >2N), being reduced for NSCs by 72% and 47% during aging and following irradiation, respectively (Fig 1D′–F′). These findings led us to analyse the capacity of SVZ cells from irradiated mice to form neurospheres in the presence of EGF and FGF2 using the neural colony forming cell assay (N-CFCA), which enables NSCs to be discriminated from TAPs based on neurosphere size (Louis et al, 2008). When SVZ cells from irradiated mice were cultured in N-CFCA, small neurospheres that were initiated by TAPs were reduced in number following irradiation, an effect that was observed at both 10 days and 4 months (Fig 1G). In contrast, larger neurospheres that were derived from NSCs were generated with the same efficacy as control mice, suggesting that NSCs resisted this radiation regimen and preserved their capacity to proliferate in vitro (Fig 1G). These neurospheres were individually subcultured in neurosphere medium; half of these neurospheres exhibited a capacity for self-renewal for three subsequent passages, with similar efficiencies for the irradiated mice and the controls (Fig 1H). Therefore, our data demonstrate that radiation induced a dramatic decrease in neurogenesis despite the persistence of functional NSCs, as was previously reported in aged mice (Ahlenius et al, 2009). The NSC niche is altered following irradiation In light of these results, we reasoned that the dramatic decline in neurogenesis may be rooted in an alteration in the NSC microenvironment rather than the intrinsic loss of the NSCs. To test this hypothesis, we used a transplantation model in which neural stem/progenitor cells were grafted into the SVZ of irradiated hosts or control C57Bl6 mice. Antibodies to clusters of differentiation markers were used to remove endothelial cells (CD31), microglial/blood cells (CD45) as well as ependymal and neuroblasts (CD24) from young mouse SVZs. Freshly sorted CD24−CD31−CD45− triple negative cells were enriched in neural stem/progenitor cells (58% GFAP+, 51% Sox2+ and 26% LeX+) and nearly devoid of neuroblasts ( 2N). K–N.. The phosphorylation of Smad3 was observed in the majority of SVZ cells in irradiated and aged mice, including cells with a GFAP+ type B/NSC phenotype. Download figure Download PowerPoint Two TGF-β receptor chains, TβRI and TβRII, are required for TGF-β1 binding and signalling through Smad2/3 phosphorylation and for the latter's subsequent translocation into the nucleus (Massague, 2012). TβRII expression has been observed on nestin-positive adult neural progenitors (Wachs et al, 2006). We analysed the expression of TβRI and TβRII using immunofluorescence on freshly dissociated SVZ cells from control young adult mice. The expression of both receptors was associated with NSCs and TAPs (GFAP+LeX+ and Mash1+), whereas both receptors were scarcely expressed in Dcx+ neuroblasts (Fig 4F–I). We further examined the binding of biotinylated TGF-β1 on SVZ cells from young mice and observed that it bound to nearly 50% of activated NSCs but to only 17% of TAPs and 3% of neuroblasts (Fig 4J and Supporting Information Table 1). This result confirmed the preferential expression of TGF-β receptors on NSCs. A significant increase in TGF-β1 binding was observed on cycling SVZ cells (DNA content >2N) during aging and following irradiation (Fig 4J). Smad2 phosphorylation was undetected in the SVZ, even following irradiation or during aging (Supporting Information Fig S7). However, strong Smad3 phosphorylation was observed in the nucleus of the majority of SVZ cells from irradiated mice as well as in most SVZ cells from aged mice. In contrast, Smad3 staining was barely detectable in SVZ cells from young adult mice (Fig 4K–N). Interestingly, GFAP+ cells that were localized beneath the ependymal layer exhibited Smad3 phosphorylation, suggesting that the activation of TGF-β signalling occurred
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