Vascular‐derived TGF‐β increases in the stem cell niche and perturbs neurogenesis during aging and following irradiation in the adult mouse brain

• DOI: 10.1002/emmm.201202197
• Publication Date: 2013-03-25T06:56:55Z
• Authors: Jose R. Pineda; Mathieu Daynac; Alexandra Chichep...; Arantxa Cebrian‐S...; Karine Sii Felice; Jose Manuel Garci...; François D. Boussin; Marc‐André Mouthon
• Keywords: Male; Medicine (General); Aging; Neurogenesis; QH426-470; Mice; 03 medical and health sciences; R5-920; Neural Stem Cells; Transforming Growth Factor beta; TGF‐beta; Genetics; Animals; Humans; Stem Cell Niche; Research Articles; neural stem cells; Cell Proliferation; 0303 health sciences; irradiation; aging; Brain; Endothelial Cells; endothelial cells; Mice, Inbred C57BL; Signal Transduction

Research Article25 March 2013Open Access Source Data Vascular-derived TGF-β increases in the stem cell niche and perturbs neurogenesis during aging following irradiation adult mouse brain Jose R. Pineda CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, Fontenay-aux-Roses, France INSERM, U967, Université Paris Diderot, Sorbonne Cité, UMR 967, Sud, Search for more papers by this author Mathieu Daynac Alexandra Chicheportiche Arantxa Cebrian-Silla Laboratorio Neurobiología Comparada, Instituto Cavanilles, Universidad Valencia, CIBERNED, Spain Karine Sii Felice Manuel Garcia-Verdugo François D. Boussin Corresponding Author [email protected] Marc-André Mouthon Information Pineda1,2,3,4, Daynac1,2,3,4, Chicheportiche1,2,3,4, Cebrian-Silla5, Felice1,2,3,4, Garcia-Verdugo5, *,1,2,3,4,† 1CEA 2INSERM, 3Université 4Université 5Laboratorio † These authors contributed equally to work. *Tel: [phone]; Fax: 91 80 EMBO Mol Med (2013)5:548-562https://doi.org/10.1002/emmm.201202197 PDFDownload PDF of article text main figures. Peer ReviewDownload a summary editorial decision process including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Neurogenesis decreases cranial radiotherapy, causing progressive cognitive decline that is currently untreatable. However, functional neural cells remained present subventricular zone high dose-irradiated aged brains. We therefore investigated whether alterations neurogenic niches are perhaps responsible decline. This hypothesis was supported absence proliferation were engrafted into vascular irradiated host Moreover, we observed marked increase TGF-β1 production endothelial both middle-aged mice. In co-cultures, induced apoptosis stem/progenitor via TGF-β/Smad3 signalling. Strikingly, blockade signalling vivo using neutralizing antibody or selective inhibitor SB-505124 significantly improved mice, prevented increased cells. findings suggest anti-TGF-β-based therapy may be used future interventions prevent collapse radiotherapy aging. The paper explained PROBLEM: A occurs key element decreased new neurons mechanisms underlie changes occur poorly understood. RESULTS: One our major deficit high-dose radiation due microenvironment regulates fate rather than direct effect on have elucidated molecular mechanism which synthesis provokes dormancy their susceptibility apoptosis. second important finding pharmacological restored integration olfactory bulbs elderly IMPACT: Our newly discovered should encourage development anti-TGF-β therapies (i) limiting injury caused (ii) rejuvenating individuals with INTRODUCTION Decreased irradiation, central adjuvant treatment tumours paediatric patients, believed contribute (Monje Palmer, 2003). others reported exposure 15 Gy accompanied perturbation memory associated mice (Lazarini et al, 2009; Valley 2009). Neural (NSCs) located (SVZ) involved adulthood (Doetsch 1999). Adult NSCs successively give rise transit amplifying progenitors (TAPs) then neuroblasts, migrate chains (OBs), where they differentiate (Alvarez-Buylla Lim, 2004). Dividing TAPs establish intimate interactions blood vessels at sites lack pericyte coverage form within SVZ (Mirzadeh 2008; Shen Tavazoie 2008). Increasing evidence has revealed importance growth factors synthesized (BECs) regulation neurogenesis, NSC (Ramirez-Castillejo 2006). cross-talk between BECs includes signals act types. include members factor family (Calvo 2011). Irradiation proliferating clear dose-dependent impairment permanent doses exceeding 10 rodents (Tada Whereas some been survive after ability (Achanta 2012). Apart from reduction number resident NSCs, also generate hostile microenvironment. particular, lessen differentiation vivo. Indeed, microglial inflammation accompanies implicated dysfunction hippocampus 2003); however, alteration remain elusive. Studies indicate physiological doublecortin-positive neuroblasts OBs (Enwere 2004; Maslov Tropepe 1997). significant apparent 6 months age SVZ, ultimately resulting dramatic these premature decrease pool owing suggests no self-renewal capacity and/or programmed complete only limited divisions (Sii-Felice when cultured vitro, retain proliferate neurons, similar young albeit lower efficiency (Ahlenius Furthermore, ageing attributed part systemic milieu (Villeda widely recognized as an injury-related cytokine, its levels strongly rapidly upregulated different forms injuries (Gomes 2005) (Werry 2010). chronic elevation triggers accumulation basement proteins results Alzheimer's disease-like cerebrovascular amyloidosis microvascular degeneration (Wyss-Coray 2000). Although promotes survival (Boche 2003; Schober 2007), it apoptotic neural-crest-derived multipotent progenitor (Hagedorn inhibits although positive negative effects (Battista 2006; Buckwalter Wachs study explores merely function depletion reflects profound niche. demonstrate pathway activation persistently report inhibition neurogenesis. RESULTS High-dose but spares model whole-brain total dose Gy, divided three 5 delivered 48 h intervals, explore SVZ. split-dose paradigm did not provoke mobilization respect resting morphology CD68+ Iba1+ SVZ/striatum (Supporting Fig S1). As estimated Ki67-positivity, dramatically 4 exposure, nuclei reduced S2). Despite capacity, indicated presence Nestin+GFAP+ double-positive lining lateral ventricle S100β S3). previously regimen reduces arrival OBs, inducing deficits drastic neuroblasts/type TAPs/type C year (Fig 1B C). absolute decreased, half type B persisted Figure 1. resisted presented defects. A,B.. Electron microscopy persistence B/NSCs whereas C/TAPs A/neuroblasts nearly completely lost. Scale bar: µm. C–F.. Ependymal (Epend), neuron (Neu) (MG) numbers unaltered. FACS analysis populations (percentage D–F) fraction (DNA >2N D′–F′): (CD24+ D D′), (EGFR+ E E′) activated (LeX+EGFR+ F F′). p-value determined Mann–Whitney U-test. G.. quantification N-CFCA. mean ± SD two five independent experiments shown (the bars). H.. NSC-derived clones subcultured confirm capacity. Kaplan–Meier's shown. Download figure PowerPoint further examined content progeny freshly dissociated microdissected SVZs. agreement described above, 25 × 103 cells/SVZ compared 47 7 non-irradiated control (p = 0.019). 12-month-old (i.e. middle-aged) reaching 32 0.032). Given LeX expressed GFAP-positive features (Capela Temple, 2002), anti-LeX combination CD24 EGF fluorescent ligand label NSCs. According previous (Pastrana 2009), defined populations: CD24−LeX+EGFR+ CD24−LeX−EGFR+ (iii) CD24+ neuroblasts. purity sorted confirmed qRT-PCR mRNA expression specific NSC, TAP neuroblast markers S4). expected 1D). percentage (EGFR+LeX−) diminished 1E), relative (LeX+EGFR+) unaltered 1F). GLAST, another marker all LeX-positive S4D), CD24−GLAST+ cells, i.e. population contained maintained S5). aging; less pronounced progeny. exhibited diminution status >2N), being 72% 47% respectively 1D′–F′). led us analyse neurospheres FGF2 colony forming assay (N-CFCA), enables discriminated based neurosphere size (Louis When N-CFCA, small initiated days 1G). contrast, larger derived generated same efficacy suggesting preserved vitro individually medium; subsequent passages, efficiencies controls 1H). Therefore, data despite altered light results, reasoned rooted intrinsic loss To test hypothesis, transplantation grafted hosts C57Bl6 Antibodies clusters remove (CD31), microglial/blood (CD45) well ependymal (CD24) Freshly CD24−CD31−CD45− triple enriched (58% GFAP+, 51% Sox2+ 26% LeX+) devoid (<1% Dcx+). NSC/TAP-enriched GFP+ unilaterally transplanted near hosts. month transplantation, graft rejection, such phagocytosis S6A). composed Ki67-positive Dcx-positive 2A,C Table 1). third GFAP, S100β, indicating mature astrocytes 2B,D GFAP+ elongated cytoplasmic processes those distinguishing them typical stellate astrocytes. subset along rostral migratory stream integrated expressing neuronal-lineage markers, NeuN S6C). had characteristics dendritic spine contacted synapse cell, electron S6D). observations GFP+CD24−CD31−CD45− neurogenic. worth noting microscopic analyses demonstrated astrocyte-like close contact 2E), what normal endogenous (Tavazoie Thus, recapitulated niches. 2. ceased A–H.. GFP (A–E, n 4) (F–H′, 5). grafting hosts, grafts (B, inset), Dcx+ (C C′) (D D′). phenotype (E). inset shows GFP-immunogold labelling abundant filamentous components discontinue (F F′) still contain (G G′) (H H′). bars microscopy: 50 µm; µm Phenotype Mice (n) Cells/graft Ki67 (%) GFAP Dcx Ctrl (4) 5959 3090 26 38 23 3 (5) 4374 1301 0 13 28 p 0.274 0.008 0.200 0.170 calculated sharp survived proliferating, Ki-67 BrdU incorporation 2F Supporting S6B). Nonetheless, quarter differentiated 2G,H neuronal S6C D). modifications Neither nor mural microvessels speculate irradiation-induced would disturb thereby impact capillaries just beneath tangential layer analysed immunostainings laminin desmin component membrane intermediate filament pericytes, respectively. controls, staining partially covered 3A). Consistently, 30% capillary surface pericytes 3B), partial NG2/desmin 1.5 per 100 length 3C slight any 3A–C). 3. Pericyte U-test (B) Kruskal–Wallis (C). A.. immunostaining coverage, absent regions numerous (dashed line). B.. microscopy. C.. NG2/pericytes bars. D.. NG2/pericyte (arrowhead). somewhat different, 3A) 3C). exclude possibility Smad3 hypothesized factor(s) directly perturb prototypical anti-mitotic cytokine cytostatic critical homeostasis many epithelial tissues (Massague, animals 4B C), remarkably stronger 4D). undetectable 4A). indeed BECs, anti-CD31 whole brains irradiation. Blood excluded anti-CD45 antibody. Quantitative RT-PCR level twofold 4E). conclude 4. represented (*p 0.020; other values given 2). illustrations representative experiments, group. A–D.. (laminin-positive) E.. qPCR BECs. F–I.. TβRI TβRII GFAP+LeX+ Mash1+ J.. binding biotinylated (CD24+), (GLAST+CD24−) >2N). K–N.. phosphorylation majority B/NSC phenotype. Two receptor chains, TβRII, required through Smad2/3 latter's translocation nucleus nestin-positive (Wachs immunofluorescence receptors (GFAP+LeX+ Mash1+), scarcely 4F–I). bound 50% 17% 3% 4J result preferential cycling >2N) 4J). Smad2 undetected even S7). strong most barely detectable 4K–N). Interestingly, localized phosphorylation, occurred