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Specific ablation of Nampt in adult neural stem cells recapitulates their functional defects during aging

2014; Springer Nature; Linguagem: Inglês

10.1002/embj.201386917

1460-2075

Liana R. Stein, Shin‐ichiro Imai,

Signaling Pathways in Disease

Article8 May 2014free access Specific ablation of Nampt in adult neural stem cells recapitulates their functional defects during aging Liana R Stein Liana R Stein Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO USA Search for more papers by this author Shin-ichiro Imai Corresponding Author Shin-ichiro Imai Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO USA Search for more papers by this author Liana R Stein Liana R Stein Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO USA Search for more papers by this author Shin-ichiro Imai Corresponding Author Shin-ichiro Imai Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO USA Search for more papers by this author Author Information Liana R Stein1 and Shin-ichiro Imai 1 1Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO USA *Corresponding author. Tel: +1 314 362 7228; Fax: +1 314 362 7058; E-mail: [email protected] The EMBO Journal (2014)33:1321-1340https://doi.org/10.1002/embj.201386917 See also: C Wiley & J Campisi (June 2014) 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 ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Neural stem/progenitor cell (NSPC) proliferation and self-renewal, as well as insult-induced differentiation, decrease markedly with age. The molecular mechanisms responsible for these declines remain unclear. Here, we show that levels of NAD+ and nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in mammalian NAD+ biosynthesis, decrease with age in the hippocampus. Ablation of Nampt in adult NSPCs reduced their pool and proliferation in vivo. The decrease in the NSPC pool during aging can be rescued by enhancing hippocampal NAD+ levels. Nampt is the main source of NSPC NAD+ levels and required for G1/S progression of the NSPC cell cycle. Nampt is also critical in oligodendrocytic lineage fate decisions through a mechanism mediated redundantly by Sirt1 and Sirt2. Ablation of Nampt in the adult NSPCs in vivo reduced NSPC-mediated oligodendrogenesis upon insult. These phenotypes recapitulate defects in NSPCs during aging, giving rise to the possibility that Nampt-mediated NAD+ biosynthesis is a mediator of age-associated functional declines in NSPCs. Synopsis Levels of NAD+ and the NAD+ biosynthetic enzyme Nampt decline in the hippocampus during aging. Nampt ablation in adult neural stem/progenitor cells reduces self-renewal, proliferation, and oligodendrogenesis. Age-associated decreases in the stem cell pool are reversed by the administration of nicotinamide mononucleotide, a key NAD+ intermediate. Nampt is expressed in adult neural/progenitor stem cells and is the main source of their NAD+ levels. Loss of Nampt reduces proliferation by stalling progression of the neural stem/progenitor cell cycle at G0/G1. The NAD+-dependent sirtuins Sirt1 and Sirt2 redundantly mediate neural stem/progenitor cell fate decisions into oligodendrocytes. Loss of Nampt reduces neural stem/progenitor cell-derived oligodendrocyte regeneration after cuprizone-induced demyelination. Introduction Aging manifests as a physiological breakdown of robustness, leading to a significant functional decline, increased vulnerability to environmental insults, and many different diseases in a variety of tissues and organs. Cognitive decline is one such example, and cognitive impairment, even without dementia, occurs in 22% of people over age 71 in the United States (Plassman et al, 2008). With this prevalence, efforts to understand the mechanisms underlying age-associated cognitive decline and to develop therapeutic strategies to prevent it have become particularly poignant. With these goals in mind, the regulation of adult neurogenesis has been an intense focus of research. Throughout the lives of all mammals, including humans, two distinct populations of neural stem cells (NSCs) are maintained in the brain: one in the subgranular zone (SGZ) of the dentate gyrus (DG) and the other in the subventricular zone (SVZ) bordering the lateral ventricles. NSCs have the ability to self-renew and to differentiate into transient amplifying progenitors (collectively referred to as neural stem/progenitor cells or NSPCs), which in turn undergo finite, lineage-restricted cell divisions to produce neurons, oligodendrocytes, or astrocytes (Deng et al, 2010; Artegiani & Calegari, 2012; Jadasz et al, 2012). Aging is one of the strongest negative regulators of adult NSPC proliferation (Artegiani & Calegari, 2012). In mice, the number of proliferating NSPCs in the SGZ declines exponentially in the first 9 months of life (Ben Abdallah et al, 2010), and they are almost completely absent by 24 months of age (Jin et al, 2003; Lugert et al, 2010; Encinas et al, 2011). Aging is also associated with impaired oligodendrocyte differentiation and remyelination in response to insult (Sim et al, 2002; Franklin & Ffrench-Constant, 2008). Given the potential of NSPCs to contribute to cognitive function and their ability to proliferate and differentiate into the major cell types of the brain (Deng et al, 2010; Artegiani & Calegari, 2012; Jadasz et al, 2012), it is of great importance to understand key signaling pathways regulating the NSPC decisions of proliferation versus quiescence and self-renewal versus terminal differentiation. NSPCs can be reactivated in the aged brain (Decker et al, 2002; Jin et al, 2003; Lugert et al, 2010). Therefore, elucidation of the key signaling pathways that are involved in age-associated decline in NSPC functionality will provide critical insight into preventing age-associated cognitive decline. We have previously found that aging significantly reduces levels of the essential cofactor nicotinamide adenine dinucleotide (NAD+) in multiple peripheral tissues (Yoshino et al, 2011). This age-associated decrease in NAD+ levels is due to a decline in protein levels of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the biosynthetic pathway of NAD+ from nicotinamide (Fig 1A) (Revollo et al, 2004; Yoshino et al, 2011). Nampt converts nicotinamide, a major precursor in mammalian NAD+ biosynthesis, and 5′-phosphoribosyl-1-pyrophosphate to nicotinamide mononucleotide (NMN), a key NAD+ intermediate. NMN is then converted to NAD+ by nicotinamide/nicotinic acid mononucleotide adenylyltransferase (Nampt) (Revollo et al, 2004, 2007). We and others have previously reported that the expression of Nampt in the brain is extremely low compared to peripheral tissues (Revollo et al, 2007; Friebe et al, 2011). However, Nampt has uniquely strong expression in the hippocampus (Zhang et al, 2010; Wang et al, 2011a). Because recent studies show that the energetic demands of stem cell proliferation and lineage specification require distinct metabolic programs (Folmes et al, 2012), we hypothesized that NSPCs would be particularly sensitive to changes in NAD+ levels and accordingly alter their proliferation, self-renewal, and differentiation. Figure 1. Hippocampal NAD+ levels and Nampt expression decline with age A. NAD+ biosynthesis from nicotinamide. Nicotinamide phosphoribosyltransferase (Nampt) converts nicotinamide and 5′-phosphoribosyl-1-pyrophosphate (PRPP) to nicotinamide mononucleotide (NMN). Nicotinamide/nicotinic acid mononucleotide adenylyltransferase (Nampt) converts NMN and adenosine-5′-triphosphate (ATP) to NAD+. While NAD+ is commonly used in redox reactions, cells primarily require NAD+ as a co-substrate for several families of enzymes, one of which is the sirtuin family of protein deacetylases. The sirtuin family includes Sirt1 and Sirt2, which cleave NAD+ at its glycosidic bond, releasing nicotinamide (Stein & Imai, 2012). Red text indicates inhibitors used in subsequent experiments. B. HPLC analysis of NAD+ levels in hippocampal extracts (1 month, n = 5; 3–4 months, n = 16; 6 months, n = 10; 10–12 months, n = 28). C, D. Quantification of immunofluorescence for Nampt in the subgranular zone (SGZ). Measurement of thresholded levels of Nampt immunoreactivity (C) and the number of highly immunoreactive Nampt+ cells (D) along the SGZ (n = 5). E. Representative images of immunofluorescence for Dapi (blue) and Nampt (red) in the SGZ in young (6-month-old) and old (18-month-old) mice. Dotted lines denote the SGZ. Scale bars denote 20 μm. Data information: Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Here, we show that hippocampal NAD+ and Nampt levels decrease with age. Nampt mediates NSPC NAD+ biosynthesis. Pharmacological inhibition and genetic ablation of Nampt-mediated NAD+ biosynthesis in NSPCs impair NSPC proliferation, self-renewal, and formation of oligodendrocytes in vivo and in vitro. Furthermore, augmentation of NAD+ levels during aging with NMN administration maintains the NSPC pool. Thus, enhancing NAD+ levels in NSPCs may be an effective intervention to preserve the endogenous NSPC population to repair the aged, diseased, or damaged brain. Results Hippocampal NAD+ levels and Nampt expression decline with age Since aging significantly reduces levels of Nampt and NAD+ in multiple peripheral tissues (Yoshino et al, 2011), we hypothesized that aging also reduces Nampt-mediated NAD+ biosynthesis in the brain, particularly in the hippocampus, affecting the function of NSPCs. To address this hypothesis, we first measured NAD+ levels in hippocampi isolated from 1-, 3- to 4-, 6-, and 10- to 12-month-old C57Bl6 mice. NAD+ levels gradually decreased with age, reaching 63% in 10- to 12-month-old mice compared to that of 1-month-old mice (Fig 1B). Consistent with this finding, quantifying Nampt immunoreactivity in the SGZ of the DG by both a thresholded level of Nampt intensity and a count of the number of thresholded Nampt+ cells demonstrated that 18-month-old mice exhibit 52–66% of the Nampt immunoreactivity present in 6-month-old mice (Fig 1C–E). These results suggest that Nampt-mediated NAD+ biosynthesis in the hippocampus declines with age at a time course similar to that of NSPC proliferation (Ben Abdallah et al, 2010). Nampt is expressed in a subpopulation of SGZ NSPCs Previously, it was reported that Nampt is predominantly expressed in hippocampal neurons, but not in stellate astrocytes (Zhang et al, 2010; Wang et al, 2011a). Consistent with this finding, immunohistochemistry for Nampt and cell type-specific markers revealed that almost all NeuN+ neurons in the granule layer of the DG expressed Nampt, while almost no S100β+ glial cells did (Supplementary Fig S1A–E). However, we also noticed that many intensely Nampt immunoreactive cells along the SGZ of the DG did not express NeuN (Supplementary Fig S1B and E). Since NSPCs engage in the energetically costly tasks of proliferation and differentiation (Folmes et al, 2012), we performed co-immunohistochemistry for NSPC markers (Sox2+, radial Gfap+) and found that a majority of NSPCs expressed Nampt (Fig 2A–D). To assess in vivo colocalization between Nampt and Nestin, we crossed mice expressing Cre recombinase under the Nestin promoter (Nestin-CreERT2) (Lagace et al, 2007) to a GFP reporter mouse strain that expresses a loxP-flanked STOP cassette that prevents transcription of the downstream enhanced GFP (see the Materials and Methods section), generating iNSPC-GFP mice. Nampt also colocalized with GFP driven by the Nestin promoter (NestinGFP, Fig 2C and D) (Lagace et al, 2007). Quantification of these observations revealed that along the SGZ, 32% of Sox2+ cells, 55% of radial Gfap+ cells, and 78% of NestinGFP+ cells expressed Nampt (Fig 2D, Supplementary Fig S1F). Additionally, Ki67+ and Olig2+ cells along the SGZ also expressed Nampt (Supplementary Fig S1G–I). To confirm that Nampt is highly expressed in NSPCs, we cultured NSPCs from the hippocampi of postnatal pups as neurospheres. Neurospheres showed 22 or 32% higher expression levels of Nampt than did whole hippocampal extracts taken from postnatal (P12) or adult mice (2.5–4.5 months), respectively (Fig 2E), indicating that NSPCs have higher expression levels of Nampt compared to other hippocampal cell types. Together, these results suggest that Nampt is expressed in a large subpopulation of NSPCs. Figure 2. Nampt is expressed in a subpopulation of SGZ NSPCs A–C. Representative images of immunofluorescence for Dapi (blue), Nampt (red), and NSPC markers (Sox2, Gfap, and NestinGFP 3 days post-tamoxifen injection; green) in the SGZ. Dotted lines denote the SGZ. Single arrows indicate examples of colocalization. Double arrows indicate examples of non-colocalization. Scale bars denote 10 μm. D. Quantification of the percentages of NSPC marker-positive cells in the SGZ that also express Nampt in 3- to 6-month-old mice. At least 350 cells from 7 to 14 mice were assessed per group. E. A representative immunoblot and quantification of immunoblots for Nampt normalized by actin in neurospheres cultured from postnatal mice (n = 6 independent samples, 16 replicates), as well as hippocampal tissue extracts (HC) isolated from either postnatal (n = 12) or adult mice (n = 12). F. Nampt immunoreactivity was thresholded and the number of highly immunoreactive Nampt + cells along the SGZ was assessed for colocalization with the neuronal marker NeuN or the NSPC marker Sox2 in the subgranular zone (SGZ, n = 5). Data information: Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Having found that Nampt expression is present in both neurons and NSPCs, we next asked which cell populations lose Nampt expression with age. To address this question, we thresholded Nampt immunoreactivity and assessed the thresholded Nampt+ cells for colocalization with the neuronal marker NeuN and the NSPC marker Sox2. With age, the percentage of intensely Nampt immunoreactive cells that colocalized with NeuN increased slightly, whereas the percentage of intensely Nampt immunoreactive cells that colocalized with Sox2 decreased from 21 to 4% (Fig 2F). Similarly, in the SGZ, the percentage of NeuN+ that expressed Nampt increased with age, while the percentage of Sox2+ cells that expressed Nampt decreased (Supplementary Fig S1E and F). Thus, at least part of the decrease in Nampt expression in the SGZ with age is due to the loss of expression from Sox2+ NSPCs. Adult NSPC-specific deletion of Nampt impairs NSPC self-renewal in vivo Having seen enrichment of Nampt in NSPCs, we next asked whether inactivating Nampt specifically in adult NSPCs could recapitulate age-associated phenotypic changes in NSPC functionality in vivo. To address this question, we generated adult NSPC-specific inducible Nampt-knockout mice by crossing Namptflox/flox mice (Rongvaux et al, 2008) with Nestin-CreERT2 mice (iNSPC-Nampt-KO mice). To trace the progeny of adult NSPCs in which Nampt was inactivated and to confirm the specificity and magnitude of the deletion induced by tamoxifen, we also crossed iNSPC-Nampt-KO mice to the aforementioned iNSPC-GFP mice. After tamoxifen injection, these mice expressed NestinGFP in the SGZ and SVZ but not in non-neurogenic regions of the brain such as the corpus callosum or cortex (Supplementary Fig S2A and B). Immunohistochemistry and recombination PCR for NestinGFP confirmed that there was undetectable recombination present in vehicle-injected mice (Supplementary Fig S2A and C). To verify that the NestinGFP+ population consisted of NSPCs, we costained for the NSPC markers Sox2 and Gfap. 61% of Sox2+ cells and 34% of radial Gfap+ cells coexpressed NestinGFP 7 days post-tamoxifen (Supplementary Fig S2D). We also verified Nampt deletion efficiency by quantifying the percentage of NestinGFP+ cells that expressed Nampt 3 and 7 days post-tamoxifen injection. At 3 days post-tamoxifen injection, the percentage of NestinGFP+ cells that expressed Nampt in iNSPC-Nampt-KO mice was 40% less than littermate controls, and at 7 days post-tamoxifen injection, the percentage of NestinGFP+ cells that expressed Nampt was reduced by 62% (Supplementary Fig S2E). To assess the cumulative effect of loss of Nampt on NSPC proliferation, we deleted Nampt in iNSPC-Nampt-KO mice at 6 weeks of age with three rounds of five consecutive days of tamoxifen injections, separated by 6 weeks (Fig 3A). We then assessed control and iNSPC-Nampt-KO mice for the expression of lineage-specific markers by immunohistochemistry (Fig 3B). In iNSPC-Nampt-KO mice, we found that the Nestin+ NSPC pool was significantly decreased by 49% in the DG (Fig 3C). Indeed, incorporation of BrdU and the population of proliferating cells [Ki67+ (von Bohlen und Halbach, 2011)] were also decreased by 22% (P = 0.019) and 35% (P = 0.064), respectively (Fig 3D and E). Consistent with this defect in the NSPC pool and proliferation, the pool of newborn neurons [doublecortin, Dcx+ (von Bohlen und Halbach, 2011)] was also significantly decreased by 26% (Fig 3F and G). In contrast, we did not observe any significant difference in the maturation of newborn neurons, as assessed by categorization of Dcx+ cells with no or horizontal projections as immature neurons and Dcx+ cells with vertical projections spanning the granule cell layer as mature neurons (Supplementary Fig S2F). We next assessed NSPC/daughter cell survival by immunostaining for activated caspase-3. Only rare activated caspase-3+ cells were observed in both neurogenic and non-neurogenic regions of the brain (Supplementary Fig S2A and B), and these rare activated caspase-3+ cells were never observed in GFP+ cells in iNSPC-Nampt-KO DG, providing evidence against a potential contribution of cell death to the observed effects. Figure 3. Adult NSPC-specific deletion of Nampt impairs NSPC proliferation and self-renewal in vivo A. To assess proliferation, iNSPC-Nampt-KO mice and littermate controls were subjected to three rounds of five tamoxifen (TAM) injections (one injection per day, 6 weeks apart). Sacrifice was performed at 6 months of age. B. A scheme for the specificity of the markers assessed. C–F. Quantification of radial Nestin+ NSPCs (n = 15–16 mice) (C), BrdU+ proliferating cells (n = 14–16 mice) (D), Ki67+ proliferating cells (n = 7 mice) (E), and newborn neurons (Dcx+, n = 15–20 mice) (F), per unit area of the dentate gyrus (DG) in control and iNSPC-Nampt-KO mice. For BrdU labeling, four injections of BrdU at 100 mg/kg body weight were given intraperitoneally over 48 h. G. Representative images of immunofluorescence for Gfap (blue), Dcx (green), and BrdU (red) in the subgranular zone (SGZ). Scale bar denotes 200 μm. H. To assess differentiation, control littermates and iNSPC-Nampt-KO mice were subjected to four total TAM injections (two injections on the first day coupled with BrdU at 100 mg/kg body weight as well as two total injections on the subsequent 2 days). I. Quantification of the percentage of BrdU+ cells in the DG that also express markers of NSPCs (Gfap+, Nestin+), newborn neurons (Dcx+), and OPCs/oligodendrocytes (Olig2+) (n = 6–13 mice). J. Quantification of radial Nestin+ NSPCs in 6- and 18-month-old C57Bl6 mice and 18-month-old C57Bl6 mice treated with 100 or 300 mg/kg body weight NMN in their drinking water for 12 months (n = 5 mice). Data information: Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant. Download figure Download PowerPoint To assess the acute effect of loss of Nampt on NSPC fate decisions, we induced deletion of Nampt at 6 weeks of age with four total tamoxifen injections followed by sacrifice 72 h after the first injection (Fig 3H). To facilitate assessment of differentiation, we labeled dividing cells by injecting the mice with BrdU concurrently with the first day of tamoxifen treatment. iNSPC-Nampt-KO mice displayed significantly reduced levels of colocalization of BrdU with radial Nestin+ cells (Fig 3I), suggesting decreased self-renewal decisions. However, iNSPC-Nampt-KO mice exhibited normal levels of BrdU colocalization with neuronal (Dcx+), astrocytic (Gfap+, also marks NSPCs), and oligodendrocytic (Olig2+) markers, indicating that alterations in differentiated cell lineage decisions were undetectable under basal conditions. The lack of increase in colocalization of BrdU with cell type-specific markers may imply that a larger percentage of BrdU+ cells have failed to differentiate in iNSPC-Nampt-KO mice. iNSPC-Nampt-KO NSPCs could have stalled during differentiation after losing Nestin expression. While NSPC proliferation declines exponentially throughout life (Artegiani & Calegari, 2012), quiescent NSPCs can be reactivated in the aged murine hippocampus by multiple environmental stimuli (Decker et al, 2002; Jin et al, 2003; Lugert et al, 2010). If NSPCs quiesce due to reduced NAD+ levels, systemic administration of NMN may be able to correct age-associated defects in NSPC functionality. Intraperitoneal injection of NMN (500 mg/kg body weight) increased hippocampal NAD+ levels 34–39% within 15 min, strongly suggesting that NMN can cross the blood–brain barrier (Supplementary Fig S2G). To see whether NMN supplementation can maintain NSPC proliferation and self-renewal with age, we treated 6-month-old mice with NMN at the daily dose of 100 or 300 mg/kg body weight in their drinking water until 18 months of age. The number of Nestin+ cells along the SGZ was significantly lower in the 18-month-old control mice relative to 6-month-old mice, as previously reported (Encinas et al, 2011) (Fig 3J). Remarkably, mice treated with 300 mg/kg body weight NMN showed improved maintenance of the type 1 (radial Nestin+) population with age. However, the population of proliferating cells (Ki67+) remained similar to controls (Supplementary Fig S2H). While not statistically significant, the population of newborn neurons (Dcx+) tended to increase (Supplementary Fig S2I). The age-related depletion of the NSPC pool is thought to be caused by an increase in terminal fate decisions relative to self-renewal fate decisions (Encinas et al, 2011). Thus, it is possible that NMN administration maintains the NSPC pool by preventing the age-associated increase in terminal fate decisions. Inhibition of Nampt in NSPCs in vitro impairs NAD+ biosynthesis and proliferation Having shown that NAD+ levels in the hippocampus and Nampt expression in the SGZ decreased with age, we next asked whether Nampt mediates NSPC-specific NAD+ biosynthesis by using hippocampal neurospheres as the in vitro NSPC culture model. We treated neurospheres with a highly specific Nampt inhibitor, FK866, at a dosage and duration (10 nM, 48 h) that has little to no effect on cellular viability (Hasmann & Schemainda, 2003). Strikingly, FK866 reduced NAD+ levels in neurospheres to 4% of controls, a decrease completely rescued by concurrent NMN treatment ((Fig 4A, Supplementary Fig S3A), strongly suggesting that Nampt activity is the predominant source of NAD+ biosynthesis in NSPCs. Figure 4. Inhibition of Nampt in NSPCs in vitro impairs NAD+ biosynthesis and proliferationNeurospheres were cultured with the Nampt-specific inhibitor FK866 (10 nM) with or without NMN (100 μM) for 48 h. HPLC analysis of NAD+ levels (n = 6). Quantification of the fold increase of cell number in neurospheres (n = 6–30). Representative bright-field image of neurospheres. Scale bars denote 10 μm. Cell cycle-related pathways among the top 50 biological pathways downregulated by FK866. Parametric analysis of gene enrichment (PAGE) was conducted based on microarray analyses. See the Materials and Methods section. Quantitative RT–PCR results for mRNA expression of cyclin E2 (Ccne2), cyclin E1 (Ccne1), cyclin A2 (Ccna2), and E2F1 (n = 3). FACS analysis of FK866-treated NSPCs (n = 8). Data information: Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant. Download figure Download PowerPoint We next examined how inhibition of Nampt affects neurosphere proliferation. Consistent with the decreases in the NSPC pool and in NSPC proliferation in iNSPC-Nampt-KO mice, FK866 reduced NSPC number by 61% after 48 h, but not 24 h, of treatment (Fig 4B and C, Supplementary Fig S3B). To distinguish whether this decrease in cell number was due to an inhibition of proliferation or enhancement of death, we analyzed the protein levels of markers of proliferation, apoptosis, and autophagy. Expression of the proliferation markers Ki67 and PCNA decreased 87 and 43%, respectively (Supplementary Fig S3C–E), whereas levels of activated caspase-3 became detectable and levels of the autophagy marker, glycosylated LC3B, were unchanged. Consistent with these observations, parametric analysis of gene set enrichment (PAGE) of a microarray performed on neurospheres treated with FK866 showed that out of the top 50 downregulated pathways, 13 of them were related to the cell cycle, while none of the top 50 upregulated pathways were involved in cell death (Fig 4D, Supplementary Fig S3F and G). Analysis of specific gene changes by qRT–PCR revealed that cyclins E and A, the two cyclins required for cellular progression from G1 to S, as well as their upstream transcriptional regulator E2F1 (Wong et al, 2011), were the primary cell cycle factors affected by this treatment (Fig 4E). These alterations in gene expression indicated that reducing Nampt activity stalls NSPCs at G0/G1. Supporting this notion, FACS analysis of neurospheres demonstrated that FK866 treatment increased the proportion of NSPCs in G0/G1 and decreased the proportion in S phase (Fig 4F). Genetic ablation of Nampt in NSPCs in vitro impairs NAD+ biosynthesis, proliferation, and differentiation To assess the effect of chronic Nampt ablation on NSPC functionality, we genetically ablated Nampt by infecting neurospheres from Namptflox/flox mice with Cre recombinase- or LacZ-expressing (control) adenoviruses. Neurospheres infected with Cre recombinase (Nampt Ad-Cre) at passage 1 exhibited a 94% reduction in Nampt mRNA expression 3 days post-deletion, and the corresponding decreases in Nampt protein expression and NAD+ levels appeared 6 days post-deletion (Supplementary Fig S4A–E). Eight days post-deletion, NSPCs exhibited a 73% reduction in NAD+ levels that was rescued by concurrent NMN administration, further supporting the notion that Nampt activity is the predominant source of NSPCs NAD+ levels (Fig 5A). Figure 5. Genetic ablation of Nampt in NSPCs in vitro impairs NAD+ biosynthesis, proliferation, and differentiationNeurospheres were isolated from Namptflox/flox mice and infected with a Cre recombinase-expressing adenovirus (Nampt AD-Cre) or a control adenovirus expressing LacZ (Nampt AD-LacZ). A. HPLC analysis of NAD+ levels with and without NMN (100 μM, 48 h) (n = 10–22). B, C. Quantification of the fold increase in cell number (B) (n = 13–50) and neurosphere diameter (C) (n = 9 independent samples, 57–96 neurospheres). D. Representative images of neurospheres 7 days after dissociation. Scale bars denote 10 μm. E. The number of neurospheres formed 7 days after plating dissociated cells at 100 cells/ml, 0.5 ml/well in 24-well plates (n = 8 independent samples, 48–84 wells). F, G. Nampt Ad-Cre- and Nampt AD-LacZ-infected neurospheres were cultured without NMN until Nampt Ad-Cre-infected neurospheres exhibited a growth defect. Cultures were then passaged and plated at equal density with or without NMN (200 μM). Fold increases in cell number (F) (n = 6) and the percentages of total Dapi+ cells that express Ki67+ cells were quantified (G) (n = 3 independent samples, 9 fields of view). H–L. The percentages of total Dapi+ cells that express the indicated cell type-specific markers (H) by immunofluorescence after 6–7 days of differentiation: O4 (I), Gfap (J), and B-III-tubulin (K) (n = 3–6 independent samples, 23–43 fields of view). The effect of NMN was also examined for O4, S100β, TUNEL, and Nestin (L) (n = 3–6 independent samples, 10–26 fields of view). *, ^, and # indicate statistical significance between Nampt AD-LacZ and Nampt AD-Cre, Nampt AD-LacZ and Nampt AD-LacZ+NMN, and Nampt AD-Cre and Nampt AD-Cre+NMN, respectively. Data information: Data are presented as mean ± SEM. *, ^, #P < 0.05; **P < 0.01; ***, ###P < 0.001. Download figure Download PowerPoint Like FK866-treated cultures, proliferating Nampt Ad-Cre-infected NSPCs displayed reduced cell number (Fig 5B). Remarkably, Nampt Ad-Cre NSPCs were unable to increase their cell number between 24 and 144 h of culture. In contrast, Nampt AD-LacZ-infected cells were able to exponentially increase their cell number over 13-fold in this time frame. Consistent with this finding, Nampt Ad-Cre-infected NSPCs also showed a 49% reduction in diameter relative to Nampt AD-LacZ-infected NSPCs, indicative of reduced proliferation (Fig 5C and D). Since NSPC self-renewal decisions can also contribute to cell number, we assessed secondary neurosphere formation, an assay that quantifies the ability of neurosphere inhabitant cells to reformulate neurospheres upon dissociation. Nampt Ad-Cre-infected cells generated 63% fewer secondary neurospheres than did Nampt AD-LacZ-infected cells (Fig 5E). Nampt AD-LacZ and Nampt Ad-Cre NSPCs exhibited no difference in the percentages of TUNEL- or activated caspase-3-positive cells as well