PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC Feb 1, 2011.
Published in final edited form as:
PMCID: PMC2935844
NIHMSID: NIHMS225966
Notch1 is required for maintenance of the reservoir of adult hippocampal stem cells
Jessica L. Ables,1 Nathan A. DeCarolis,1 Madeleine A. Johnson,1 Phillip D. Rivera,1 Zhengliang Gao,2 Don C. Cooper,3 Freddy Radtke,4 Jenny Hsieh,2 and Amelia J. Eisch1
1 Department of Psychiatry, UT Southwestern Medical Center, Dallas, TX 75390, USA
2 Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA
3 Institute for Behavioral Genetics, University of Colorado at Boulder, Boulder, CO 80309 USA
4 Ecole Polytechnique Fédérale de Lusanne, Swiss Institute for Experimental Cancer Research, 1066 Epalinges, Switzerland
Corresponding author: Amelia J. Eisch, Department of Psychiatry, University of Texas, Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9070, amelia.eisch/at/utsouthwestern.edu
Notch1 regulates neural stem cell (NSC) number during development, but its role in adult neurogenesis is unclear. We generated nestin-CreERT2/R26R-YFP/Notch1loxP/loxP (Notch1 iKO) mice to allow tamoxifen (TAM)-inducible elimination of Notch1 and concomitant expression of yellow fluorescent protein (YFP) in nestin-expressing Type-1 NSCs and their progeny in the adult hippocampal subgranular zone (SGZ). Consistent with previous research, YFP+ cells in all stages of neurogenesis were evident in the subgranular zone (SGZ) of wild type mice (WT; nestin-CreERT2/R26R-YFP/Notch1wt/wt) after tamoxifen (post-TAM), producing adult-generated YFP+ dentate gyrus neurons. Compared to WT littermates, Notch1 iKO mice had similar numbers of total SGZ YFP+ cells 13 and 30 days post-TAM but had significantly fewer SGZ YFP+ cells 60 and 90 days post-TAM. Significantly fewer YFP+ Type-1 NSCs and transiently-amplifying progenitors (TAPs) resulted in generation of fewer YFP+ granule neurons in Notch1 iKO mice. Strikingly, 30 days of running rescued this deficit, as the total YFP+ cell number in Notch iKO mice was equivalent to WT levels. This was even more notable given the persistent deficits in the Type-1 NSC and TAP reservoirs. Our data show that Notch1 signaling is required to maintain a reservoir of undifferentiated cells and ensure continuity of adult hippocampal neurogenesis, but that alternative Notch1- and Type-1 NSC-independent pathways compensate in response to physical activity. These data shed light on the complex relationship between Type-1 NSCs, adult neurogenesis, the neurogenic niche, and environmental stimuli.
Keywords: transcription factor, adult neurogenesis, Nestin-CreERT2, dentate gyrus, exercise, subgranular zone
Neurogenesis occurs throughout adulthood in the subgranular zone (SGZ) of the mammalian dentate gyrus (Lagace et al., 2007; Imayoshi et al., 2008) and is linked with mood and hippocampal function (e.g. Doetsch and Hen, 2005; Ming and Song, 2005). Adult neurogenesis is dynamic and is thought to consist of a series of stages: proliferation of nestin-expressing neural stem cells (NSCs) and transiently amplifying progenitor cells (TAPs), maturation of doublecortin (DCX)-expressing neuroblasts, and survival of adult-born neurons that ultimately integrate into hippocampal circuitry (Kempermann et al., 2004; Duan et al., 2008). Each stage is discretely regulated by a variety of intrinsic and extrinsic factors, and modulation of adult SGZ neurogenesis by myriad stimuli (e.g. physical activity) is the focus of intense research (Eisch et al., 2008; Zhao et al., 2008). However, more information is needed about molecules that modulate the in vivo interaction between NSC, TAPs, and the neurogenic microenvironment - or ‘neurogenic niche’ (Basak and Taylor, 2009).
Notch1 is a membrane-tethered transcription factor ideally situated to integrate cues from the niche to regulate various stages of neurogenesis (Artavanis-Tsakonas et al., 1999; Radtke et al., 2005; Yoon and Gaiano, 2005; Androutsellis-Theotokis et al., 2006; Johnson et al., 2009). In response to signals presented on the surface of neighboring cells, Notch1 governs self-renewal and fate in embryonic NSCs (Yoon and Gaiano, 2005; Corbin et al., 2008). Notch1 also promotes radial glia-like identity and negatively regulates cell cycle exit and neuronal differentiation in GFAP+ NSCs in the postnatal brain (Breunig et al., 2007; Favaro et al., 2009). However, the long-term consequences of impaired Notch1 signaling in nestin+ Type-1 NSCs in the adult SGZ are unknown. Consistent with the idea that Notch signaling can also modulate neurogenesis in response to stimuli, recent studies suggest that ischemia-induced changes in neurogenesis are dependent on Notch1 (Carlen et al., 2009; Wang et al., 2009). A recent study using a Hes5-GFP reporter found that Notch-responsive stem cells respond differently to various stimuli (Lugert et al., 2010). However, there is no direct research into the causative links between Notch1, adult neurogenesis, and physical activity.
We hypothesized that Notch1 signaling is critical for both basal and exercise-induced SGZ neurogenesis. To address this, we generated nestin-CreERT2/R26R-YFP/Notch1loxP/loxP (Notch1 iKO) mice. Tamoxifen (TAM)-induced recombination allowed us to ablate Notch1 from nestin-expressing Type-1 NSCs and their progeny and to track the recombined cells via yellow fluorescent protein (YFP). We assessed YFP+ cell number, proliferation, differentiation, and cell death in the SGZ of adult wild type (WT) and Notch1 iKO mice over three months under both basal and running conditions. We find that Notch1 is required for maintenance of adult hippocampal stem and progenitor cells and for continuity of adult neurogenesis. We further show that physical activity normalizes deficits in neurogenesis, despite persistent loss of NSCs.
Notch1 iKO mice
Mice were housed in an ALAAC-approved facility at UT Southwestern on a 12-h light/dark cycle. All procedures and husbandry were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Nestin-CreERT2 and R26R-YFP mice (Lagace et al., 2007) maintained on a C57BL/6J background were crossed with floxed Notch1 mice (Radtke et al., 1999), maintained on an ICR (CD1) background to generate viable and developmentally normal adult nestin-CreERT2/R26R-YFP/Notchwt/wt (WT) and nestin-CreERT2/R26R-YFP/Notch1loxP/loxP (Notch iKO) littermates. Mice were genotyped as previously described (Radtke et al., 1999; Lagace et al., 2007). WT and Notch1 iKO mice (4-5 weeks old, male and female) received TAM daily for 6 days (180 mg/kg i.p., 30 mg/ml in 10% EtOH/sunflower oil, Sigma-Aldrich). Only F3 intercross offspring were examined to control for gene dosage from the different backgrounds and to ensure that all littermates were heterozygous for both cre and yfp and varied only for the floxed Notch1 allele (appropriate Mendelian ratios were observed). This was important given that running activity and neurogenesis are both highly strain dependent (Kempermann et al., 2006; Pietropaolo et al., 2008; Bednarczyk et al., 2009), and to minimize Cre and/or YFP-mediated toxicity (Imayoshi et al., 2006).
Voluntary physical activity
Mice were single-housed in modified cages with a locked (unable to turn) or open running wheel (Coulbourn Instruments). Revolutions were monitored, and activity was analyzed using ClockLab (ActiMetrics Software). There was no statistical difference between any measures from mice on a locked wheel (cage dimensions=13×31.8×13.7cm) and naïve group-housed mice (12.7×28.3×17.5cm), so data from these non-runner groups were combined and compared to the running group.
Tissue preparation and immunohistochemistry
Mice were killed and perfused 13, 30, 60 or 90 days after the last TAM injection, and brain sections were prepared as previously described (Donovan et al., 2006; Lagace et al., 2007). Staining was performed slide-mounted (SM) or free-floating (FF) (Lagace et al., 2007; Donovan et al., 2008) using the following primary antibodies: rabbit polyclonal anti-GFP (1:3000 SM; Invitrogen, Cat# A11122), chicken polyclonal anti-GFP (1:500 SM, 1:100,000 FF, Aves Labs, Cat# GFP-1020), rabbit monoclonal anti-Ki67 (1:500 SM, Lab Vision/NeoMarkers, Thermo Fisher Scientific, Cat# RM-9106-S), rabbit polyclonal anti-cleaved caspase-3 (AC3, 1:500 SM, Cell Signal, Cat# 9661), goat polyclonal anti-DCX (1:100 SM, 1:5000 FF; Santa Cruz Biotechnology, Cat # sc-8066), rabbit anti-S100β (1:2000 FF, Swant, Cat #37-a), mouse anti-GFAP (1:2500 FF, Millipore, Cat# MAB360), mouse anti-NeuN (1:1000 FF, Millipore, Cat# MAB377).
For SM IHC, sections were mounted on slides and incubated in 0.01M citric acid (pH 6.0, 100°C) for 15 min for antigen retrieval. For AC3 additional permeabilization in 0.1% trypsin in 0.1% CaCl2 in 0.1M Tris for 10 min was followed by 2N HCl in 1×TBS for 30 min. Slides were incubated with blocking solution (3% normal donkey serum, 0.3% Triton X-100 in 1×TBS) for ≥20 min followed by primary antibodies in carrier (3% normal donkey serum, 0.3% Tween-20 in 1×TBS) overnight at RT. Antibody staining for DCX, Ki67, AC3, and chicken anti-GFP was revealed using species-specific fluorophore-conjugated secondary antibodies (1:200 in 1×TBS, Cy2, Cy3, and Cy5, Jackson ImmunoResearch). For quantification and morphological assessment of YFP+ cells, slides were stained with rabbit anti-GFP and detected with biotinylated secondary antibody (1:200 in 1×TBS, Jackson ImmunoResearch), amplified using ABC Elite kit (Vector Laboratories, Cat# PK-6100) and revealed with TSA Renaissance fluorescent amplification kit (1:50, PerkinElmer Life Sciences, Cat# NEL701). Endogenous peroxidase activity was quenched in 0.3% H2O2 for 30 min before ABC and TSA. All sections were counterstained with DAPI (1:5000, Roche Applied Science, Cat# 236276) before they were dehydrated and coverslipped with DPX (Sigma-Aldrich, Cat# 44581). Omission of primary or secondary antibodies resulted in no staining and served as a negative control.
For FF IHC to assess colocalization of YFP/S100β/GFAP or YFP/DCX/NeuN, YFP was identified with chicken anti-GFP first, followed by staining for the other antigens. For staining of chicken anti-GFP followed by goat anti-DCX, sections incubated in modified blocking solution (3% normal donkey serum, 0.3% Triton X-100, 2% ABC Elite kit reagent A in 1×TBS) for ≥20 min, followed by primary antibodies in modified carrier (3% normal donkey serum, 0.3% Tween-20, 2% ABC Elite kit reagent B in 1×TBS) overnight at RT. Sections were incubated in 1% H2O2 for 30 min, washed 4×5 min in 1×TBS, then incubated with biotinylated secondary antibody (1:200 in 1.5% normal donkey serum in 1×TBS) for 4-6 hrs at RT, washed, amplified for 1 hr using ABC Elite kit, washed 4×10 min, and revealed for 12 min with TSA Renaissance fluorescent amplification kit. Antibody staining for NeuN, GFAP, and S100β was revealed using species-specific fluorophore-conjugated secondary antibodies (1:200 in 1.5% normal donkey serum in 1×TBS) for 4-6 hrs at RT. After all staining was complete, sections were counterstained with DAPI for 20 min, washed, mounted onto uncharged slides and coverslipped with DPX.
Analysis of immunoreative cells
Quantification was performed with an Olympus BX-51 microscope (400×) as previously described (Mandyam et al., 2004; Donovan et al., 2006; Lagace et al., 2007; Lagace et al., 2008; Lagace et al., 2010). Briefly, an observer blind to experimental groups counted immunoreactive cells in the SGZ in every 9th coronal section throughout the dentate gyrus (-0.82 mm to -4.24 mm from bregma) via the optical fractionator method. Quantification of YFP+ SGZ cells and YFP+ SGZ cells with Type-1 NSC or neuronal morphology was performed similarly via the optical fractionator method by an observer blind to experimental group. The proportion of YFP+ Type-1 NSC or YFP+ neurons was determined by dividing the number of YFP+ cells with either morphology by the total number of YFP+ cells (Donovan et al., 2008; Imayoshi et al., 2008). For phenotypic and dendritic morphology analyses, colocalization of signals was determined by scanning and optical sectioning in the Z plane of triple-immunofluorescence labeled sections with a Zeiss Axiovert 200/LSM510 confocal microscope (emission wavelengths 488, 543, and 633, 630×). For phenotyping, 100-150 YFP+ cells per animal (n≥3 per group) were analyzed and the total number of YFP+ cells immunoreactive for Ki67 or DCX was calculated by multiplying total YFP+ counts by the proportion that expressed the marker for each animal. For dendritic analysis, Z-stacks of DCX+YFP+ cells with a process extending through the GCL were imported into Neurolucida (Version 8, MicroBrightField) to obtain 2D tracings of neurons (3-15 cells/animal, n=3 per group), followed by Sholl analysis (Breunig et al., 2007; Dahlhaus et al., 2008). Statistical significance was generally determined by One- or Two-Way ANOVA, followed by Bonferroni post-hoc, and detailed statistics are provided in Table 2 and Supplemental Table 1.
Table 2
Table 2
Statistical results, numerical analysis.
Neurosphere isolation
Neurospheres were isolated from the subventricular zone as previously described (Brewer and Torricelli, 2007) from mice 40 days post-TAM. Briefly, dissections from two WT or Notch1 iKO mice were pooled and dissociated enzymatically for 40 min at 37°C and plated at equal density (10 cells/μl). Neurospheres were maintained in serum-free culture media supplemented with N2, B27, bFGF, EGF and heparin and passaged when confluent (every 7-10 days) using trypsin-EDTA. Secondary spheres were counted at confluence (7 days after plating) using a 1mm gridded dish. Genomic DNA was isolated from passage 4 neurospheres and PCR confirmed genomic recombination using the forward primer 5′-ctg act tag tag ggg gaa aac and the reverse primer: 5′-tac tcc gac acc caa tac ct.
Generation of Notch1 iKO mice
To target disruption of Notch1 signaling to nestin-expressing cells in the adult hippocampus, we generated Notch1 iKO mice. TAM administration induced expression of YFP in WT and iKO mice, as well as elimination of the promoter and first exon of notch1 from nestin-expressing progenitors in iKO mice (Figure 1a-b). YFP+ cells presented morphologies and phenotypes consistent with the stages of SGZ neurogenesis. YFP+ astrocytes (S100β+) and oligodendrocytes were extremely rare in the dentate gyrus, consistent with our previous finding that progeny of nestin-expressing cells in the SGZ are primarily neurons (Lagace et al., 2007).
Figure 1
Figure 1
Notch1 iKO mice have fewer YFP+ cells in the SGZ
Adult Notch1 iKO mice have fewer SGZ YFP+ cells
To determine the effect of Notch1 ablation from nestin-expressing Type-1 NSCs and their progeny on adult SGZ neurogenesis, we assessed the number of YFP+ cells in WT and Notch1 iKO mice. Qualitatively, Notch1 iKO mice had fewer YFP+ cells 60 and 90 days post-TAM (Figure 1c). Quantitative assessment confirmed that the number of YFP+ cells increased over time in WT mice, as previously shown (Lagace et al., 2007), but not in Notch1 iKO mice (Figure 1d), suggesting that Notch1 is necessary for continuous generation of adult SGZ cells.
YFP+ SGZ Type-1 NSCs and TAPs are reduced in Notch1 iKO mice
To assess which neurogenic stages were affected by elimination of Notch1 from nestin-expressing cells, YFP+ SGZ cells were assigned to categories based on expression of IHC markers (Figure 2a, Table 1, Kempermann et al., 2004). YFP+ TAPs (Ki67+DCX-), proliferating neuroblasts (Ki67+DCX+), and post-mitotic immature neurons (Ki67-DCX+) were evident in both WT and iKO mice. YFP+ Type-1 NSCs (Figure 2b) and mature neurons (Figure 2d) were identified based on their distinctive morphology as previously reported (Lagace et al., 2007). Similar results were obtained using morphological criteria or phenotypic markers (Supplemental Figure 1a-f). At early times post-TAM, there were no differences between WT and iKO mice in the number of YFP+ cells or their phenotypic distribution. While the number of YFP+ Type-1 NSCs remained constant in WT mice, the number of labeled NSCs decreased significantly ≥60 days post-TAM in iKO littermates (Figure 2e), which was confirmed via quantification of YFP+GFAP+S100β– NSCs (Supplemental Figure 1a-c). Notch1 iKO mice had fewer TAPs than WT mice (Figure 2f), and ≥60 days post-TAM there were significantly fewer YFP+ neuroblasts (Figure 2g), immature neurons (Figure 2h), and mature neurons (Figure 2i) in iKO mice, the latter result confirmed via quantification of YFP+NeuN+ neurons (Supplemental Figure 1d-f). Intriguingly, while we found a significant difference in the number of YFP+ cells generated in Notch iKO mice compared to their WT littermates (Table 2), we found no differences in the proportion of cell types generated (Supplemental Figure 1g-k, Supplemental Table 1), suggesting that fate of YFP+ cells is not affected in Notch1 iKO mice. However, we did find that the proportion of proliferating cells was decreased in Notch1 iKO mice (Supplemental Figure 1j). Consistent with decreased proliferation, we found fewer cells in the cell cycle in Notch1 iKO mice (Figure 2j). Further, there was no expansion of Ki67+ cells in iKO mice, resulting in an earlier peak and decreased neurogenesis in Notch1 iKO mice (Figure 2k). We determined that these differences in cell number were not due to increased apoptosis (Figure 2l). Previous reports suggest that Notch1 also modulates dendritic morphology of adult-generated neurons (Breunig et al., 2007). While we saw no difference in the number of branches between genotypes (Figure 3a), Sholl analysis revealed a smaller dendritic tree in YFP+Ki67-DCX+ immature neurons in Notch1 iKO mice (Figure 3b-c). Together, these data show that elimination of Notch1 from nestin-expressing cells and their progeny decreased the number of YFP+ cells at all stages of neurogenesis without affecting cell fate, decreased the proportion of TAPs without changing cell death, and decreased the size of the dendritic tree of immature neurons in vivo.
Figure 2
Figure 2
Elimination of Notch1 from nestin-expressing cells decreases YFP+ SGZ NSC number and neurogenesis
Table 1
Table 1
Classification criteria for SGZ cells in stages of neurogenesis.
Figure 3
Figure 3
Dendritic morphology is decreased in Notch1 iKO mice
In agreement with the decline in Type-1 NSCs and progenitors after eliminating Notch1 in vivo (Figure 2), NSCs isolated from adult Notch1 iKO mice generated 70% fewer secondary neurospheres in vitro compared to WT littermates (Figure 4a). While neurospheres from WT mice could be continuously passaged, neurospheres from Notch1 iKO mice could not be continuously propagated (Figure 4b). Therefore Notch1 is necessary for the expansion and self-renewal of nestin-expressing cells in the adult SGZ in vivo and in vitro.
Figure 4
Figure 4
Elimination of Notch1 from nestin-expressing Type-1 NSCs in vivo decreases neurosphere formation in vitro
Running rescues YFP+ neurogenesis but not Type-1 NSCs or TAPs in Notch1 iKO mice
Given that physical activity potently stimulates hippocampal neurogenesis (van Praag et al., 1999b; Naylor et al., 2008) and specifically increases proliferation of TAPs and neuroblasts (Fabel and Kempermann, 2008), we explored whether physical activity could rescue the deficits in YFP+ cell number in Notch1 iKO mice. Thirty days post-TAM, WT and Notch1 iKO littermates were given access to a running wheel for 30 days (Figure 5a). WT and Notch1 iKO mice ran similar distances and had similar running patterns (Figure 5a). There were no statistical differences found between mice single-housed with a locked running wheel and naïve, group-housed mice, so data from these non-runner groups were combined and compared to the running group. For example, total YFP+ cells in naïve WT mice were not significantly different from WT mice single-housed with a locked wheel (Supplemental Figure 2a-b). While physical activity increased total YFP+ cell number (Figure 5b-c), it failed to rescue either Type-1 NSCs or TAPs in Notch1 iKO mice (Figure 6a-b). However, physical activity restored proliferation (Figure 6e) and neurogenesis (Figure 6f), and specifically rescued YFP+ neuroblast and immature neuron cell numbers (Figure 6c-d, Supplemental Figure 2). These data show that unlike neuroblasts, Type-1 NSCs and TAPs uniquely require Notch1 to regulate their number in the adult SGZ.
Figure 5
Figure 5
Physical activity rescues total YFP+ cell number in Notch1 iKO runner mice
Figure 6
Figure 6
Physical activity rescues YFP+ neurogenesis but not YFP+ Type-1 NSCs or TAPs in Notch1 iKO mice
We provide several lines of evidence that Notch1 signaling in nestin-expressing cells maintains the size of the undifferentiated pool of stem and progenitor cells in the adult SGZ under basal conditions. We find fewer YFP+ SGZ cell numbers in our inducible transgenic mice in which Notch1 was eliminated from nestin-expressing cells. Although the number of cells at all stages of neurogenesis is reduced in iKO mice, the undifferentiated Type-1 NSC and TAP populations seem particularly vulnerable to ablation of Notch1 signaling. There is a dramatic loss of YFP-labeled Type-1 NSCs over time, paralleling a simultaneous significant loss of TAPs. It is unlikely that decreases in cell number are the result of cell death, as we found no evidence of increased apoptosis. Instead, we interpret the decrease in NSCs and TAPs in Notch1 iKO mice as a failure to self-renew and expand the YFP+ population. Ultimately, precocious cell cycle exit of NSCs and TAPs leads to fewer neuroblasts and immature neurons, culminating in 50% fewer YFP+ neurons without Notch1. In addition to these robust in vivo findings, progenitor cells from adult Notch1 iKO mice are impaired in their ability to self-renew and proliferate in the neurosphere assay in vitro, further emphasizing the importance of Notch1 in regulating and maintaining the reservoir of stem-like cells in the adult.
Our time course studies notably extend data from previous reports that suggest Notch1 regulates cell cycle entry and exit of neural stem and progenitor cells (Guentchev and McKay, 2006; Breunig et al., 2007; Guo et al., 2009; Wang et al., 2009). Based on these studies and the anti-differentiation nature of Notch signaling (Yoon and Gaiano, 2005), we hypothesized that eliminating Notch1 from nestin-expressing cells would result in proportionally more YFP+ neurons at the cost of stem and progenitor cells. Surprisingly, WT and Notch1 iKO mice are indistinguishable at early times post-TAM (≤30 days), and it is not until later times (≥ 60 days) that we find significantly fewer Type-1 NSCs and TAPs and fewer YFP+ cells in the cell cycle (Ki67+) in Notch1 iKO mice. We interpret these findings as decreased stem cell renewal and increased cell cycle exit, respectively, although shorter and longer timepoints might reveal whether the absence of Notch1 signaling forces Type-1 NSCs to enter the cell cycle and become TAPs (Imayoshi et al., 2010). However, in contrast to Bruenig and colleagues and our own hypothesis, we never observe an increased proportion of YFP+ cells that were DCX+ in Notch1 iKO mice. Further, we did not observe an increase in neurons at the expense of NSCs; instead, the total number of YFP+ neurons generated in Notch1 iKO mice was reduced but proportional to YFP+ neurons in WT mice. Together, these data suggest that following inducible removal of Notch1 from nestin-expressing cells, Type-1 NSCs and TAPs exit the cell cycle, thereby precluding progenitor cell expansion and decreasing the number of YFP+ progenitors available to mature into new neurons. Importantly, our data do not demonstrate a role for Notch1 in regulating the fate of the progeny of nestin-expressing cells.
The disparities between Breunig and colleagues and our own observations likely reflect differences in the stem cell population that was recombined (GFAP versus nestin), especially given the context-dependent nature of Notch signaling (Poellinger and Lendahl, 2008; Cau and Blader, 2009). Perhaps in GFAP-expressing neural stem cells Notch1 regulates both proliferation and neuronal fate, while in nestin-expressing stem cells - where neuronal fate may already be determined (Lagace et al., 2007; Steiner et al., 2008) - Notch1 may regulate only proliferation. Ultimately, both our data and the data from Breunig and colleagues support that Notch1 is required for the maintenance of a pool of proliferating undifferentiated cells in the adult SGZ. These results underscore the power of the inducible transgenic approach to dissect the role of a multi-purpose protein, like Notch1, in its contribution to a dynamic, complex, and context-dependent physiological process like neurogenesis.
Based on the observation that proliferation was decreased in Notch1 iKO mice, we hypothesized that the deficits in progenitor cells might be rescued by exercise, which is among the most potent inducers of neurogenesis in the adult SGZ (van Praag et al., 1999a; van Praag, 2008). Running increases the proliferative activity and number of TAPs and neuroblasts, with either modest or no detectable changes in the proliferative activity or number of Type-1 NSCs (Kronenberg et al., 2003; Suh et al., 2007; Lugert et al., 2010). Surprisingly, we find that running increases neuroblast proliferation and rescues neurogenesis in Notch1 iKO mice, despite persistent deficits in both the Type-1 NSC and TAP pools. Therefore, it seems that while Notch1 is critical for maintenance of undifferentiated progenitor cells, Notch1 deficits alone cannot inhibit physical activity-dependent neurogenesis (Figure 7). One interpretation is that Type-1 NSCs are not necessary for running-induced proliferation, consistent with the above-mentioned reports that NSCs proliferate modestly, if at all, in response to running. However, nestin+ TAPs, which ordinarily respond to running, may require Notch1 signaling to remain in the cell cycle, in contrast to recent findings (Lugert et al., 2010). Alternatively, the remaining YFP+ neuroblasts lacking Notch1 may be hyper-responsive to running and proliferate more to overcome deficits in progenitor cells. Perhaps neurotrophic factors and components of the neurogenic niche that are necessary for exercise-induced neurogenesis, such as BDNF, VEGF, β-endorphin and/or endocannabinoids, are sufficient to increase neuroblast proliferation in the absence of Notch1 signaling (Fabel et al., 2003; Kitamura et al., 2003; Bjornebekk et al., 2005; Koehl et al., 2008; Hill et al., 2009; Lafenetre et al., 2009). It is also possible that alternate pathways (e.g. mediated by GSK3, Shh, Wnt, or Sox2) can compensate for the lack of Notch1 in DCX+ differentiating cells but not in undifferentiated Type-1 NSCs or TAPs (Hurlbut et al., 2007; Annenkov, 2009; Favaro et al., 2009; D. Chichung Lie, personal communication). Regardless, the observed normalization of neuroblast proliferation and number of YFP+ neurons highlights the complex interplay between stem and progenitor cells, the neurogenic niche, and environmental stimuli, and encourages mechanistic studies into how Notch1 regulates adult neurogenesis in general and Type-1 NSCs in particular.
Figure 7
Figure 7
Proposed model of Notch1 in regulating adult neurogenesis under basal conditions and after physical activity
The data presented here have several implications. Particularly intriguing is our observation that running can bypass a genetic lesion with key cellular consequences (gradual depletion of neural progenitors) and still normalize adult neurogenesis. These data highlight the possibility of physical activity-based therapy in neurogenesis-related memory and mood disorders, and encourage analysis of synaptic plasticity and behavior in our mutants. Further, our demonstration of a critical role for intact Notch1 signaling in NSC maintenance urges caution in the clinical use of gamma-secretase inhibitors (GSI) for the treatment of Alzheimer's and other diseases. Activation of Notch1 requires gamma-secretase cleavage, therefore GSI also inhibit Notch1 activity (Geling et al., 2002) and SGZ progenitor proliferation (Breunig et al., 2007). Our data suggest that the resulting inhibition of Notch1 activity could result in decreased hippocampal neurogenesis and eventual depletion of hippocampal NSCs. Given that Alzheimer's disease models show decreased hippocampal neurogenesis (e.g. Donovan et al., 2006; Zhao et al., 2008; Lazarov and Marr, 2009), our results suggest long-term treatment with broad-spectrum GSI might exacerbate Alzheimer's disease progression. While more work is needed to evaluate the long-term effects of GSIs on NSC maintenance, the inducible model presented here will be useful in elucidating the underlying mechanisms of Notch signaling in the adult SGZ and in exploring how therapeutic targeting may offset the decrease in neurogenesis relevant to memory, mood disorders, and neurodegenerative diseases.
Supplementary Material
Supp1
Acknowledgments
We are grateful to Dr. Diane Lagace, Dr. Scott Russo, Dr. Josh Breunig, Dr. Euiseok Kim and to members of the Eisch laboratory for valuable discussion of this project and its results; Dr. Jane Johnson for mice; Dr. Eric Nestler for use of the confocal microscope in the Fishburg Department of Neuroscience at Mt. Sinai School of Medicine in NYC; Kerstin Ure for dissections for neurospheres; and Allison Quast, Krishna Suri, Michael Robichaux, Lurit Bepo and Amina Igeh for superb technical assistance. This study was supported by NIH/NIDA grants to AJE (DA023701, DA023555, DA016765), NIH T32 funds (T32 DA007290 and MH076690) that supported the training of NAD, MAJ, and JLA, and NIH individual training grant funds (F31 NS064632) to support the training of NAD.
Abbreviations
iKOinducible knockout
TAMtamoxifen
SGZsubgranular zone
DGdentate gyrus
NSCneural stem cell
TAPtransiently-amplifying progenitor

Footnotes
Author Contributions: J.A. designed the study, carried out breeding and treatment of animals, analyzed and documented the Notch elimination, running, colocalization of YFP with IHC markers, and performed the dendritic analysis and neurosphere experiments and wrote the paper. N.D. designed the study, established the parameters for and performed the immunohistochemistry for TAP, neuroblast and immature neuron analysis and dendritic tracing studies, discussed the interpretations, and wrote the manuscript. M.A.J. analyzed and documented AC3+ cells and wrote the paper. P.R. analyzed and documented colocalization of YFP/Ki67 and wrote the paper. Z.G. carried out and documented the neurosphere experiments and wrote the paper. D.C. provided running wheels and wrote the paper. F.R. generated floxed Notch1 mice. J.H. supervised neurosphere experiments and wrote the paper. A.E. designed and supervised the study, discussed results, formulated interpretations, and wrote the paper.
  • Androutsellis-Theotokis A, Leker RR, Soldner F, Hoeppner DJ, Ravin R, Poser SW, Rueger MA, Bae SK, Kittappa R, McKay RD. Notch signalling regulates stem cell numbers in vitro and in vivo. Nature. 2006;442:823–826. [PubMed]
  • Annenkov A. The insulin-like growth factor (IGF) receptor type 1 (IGF1R) as an essential component of the signalling network regulating neurogenesis. Mol Neurobiol. 2009;40:195–215. [PubMed]
  • Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science. 1999;284:770–776. [PubMed]
  • Basak O, Taylor V. Stem cells of the adult mammalian brain and their niche. Cell Mol Life Sci. 2009;66:1057–1072. [PubMed]
  • Bednarczyk MR, Aumont A, Decary S, Bergeron R, Fernandes KJ. Prolonged voluntary wheel-running stimulates neural precursors in the hippocampus and forebrain of adult CD1 mice. Hippocampus 2009 [PubMed]
  • Bjornebekk A, Mathe AA, Brene S. The antidepressant effect of running is associated with increased hippocampal cell proliferation. Int J Neuropsychopharmacol. 2005;8:357–368. [PubMed]
  • Breunig JJ, Silbereis J, Vaccarino FM, Sestan N, Rakic P. Notch regulates cell fate and dendrite morphology of newborn neurons in the postnatal dentate gyrus. Proc Natl Acad Sci U S A. 2007;104:20558–20563. [PubMed]
  • Brewer GJ, Torricelli JR. Isolation and culture of adult neurons and neurospheres. Nat Protoc. 2007;2:1490–1498. [PubMed]
  • Carlen M, Meletis K, Goritz C, Darsalia V, Evergren E, Tanigaki K, Amendola M, Barnabe-Heider F, Yeung MS, Naldini L, Honjo T, Kokaia Z, Shupliakov O, Cassidy RM, Lindvall O, Frisen J. Forebrain ependymal cells are Notch-dependent and generate neuroblasts and astrocytes after stroke. Nat Neurosci. 2009;12:259–267. [PubMed]
  • Cau E, Blader P. Notch activity in the nervous system: to switch or not switch? Neural Dev. 2009;4:36. [PMC free article] [PubMed]
  • Corbin JG, Gaiano N, Juliano SL, Poluch S, Stancik E, Haydar TF. Regulation of neural progenitor cell development in the nervous system. J Neurochem. 2008;106:2272–2287. [PMC free article] [PubMed]
  • Dahlhaus M, Hermans JM, Van Woerden LH, Saiepour MH, Nakazawa K, Mansvelder HD, Heimel JA, Levelt CN. Notch1 signaling in pyramidal neurons regulates synaptic connectivity and experience-dependent modifications of acuity in the visual cortex. J Neurosci. 2008;28:10794–10802. [PubMed]
  • Doetsch F, Hen R. Young and excitable: the function of new neurons in the adult mammalian brain. Curr Opin Neurobiol. 2005;15:121–128. [PubMed]
  • Donovan MH, Yamaguchi M, Eisch AJ. Dynamic expression of TrkB receptor protein on proliferating and maturing cells in the adult mouse dentate gyrus. Hippocampus. 2008;18:435–439. [PMC free article] [PubMed]
  • Donovan MH, Yazdani U, Norris RD, Games D, German DC, Eisch AJ. Decreased adult hippocampal neurogenesis in the PDAPP mouse model of Alzheimer's disease. J Comp Neurol. 2006;495:70–83. [PubMed]
  • Duan X, Kang E, Liu CY, Ming GL, Song H. Development of neural stem cell in the adult brain. Curr Opin Neurobiol. 2008;18:108–115. [PMC free article] [PubMed]
  • Eisch AJ, Cameron HA, Encinas JM, Meltzer LA, Ming GL, Overstreet-Wadiche LS. Adult neurogenesis, mental health, and mental illness: hope or hype? J Neurosci. 2008;28:11785–11791. [PMC free article] [PubMed]
  • Fabel K, Kempermann G. Physical activity and the regulation of neurogenesis in the adult and aging brain. Neuromolecular Med. 2008;10:59–66. [PubMed]
  • Fabel K, Tam B, Kaufer D, Baiker A, Simmons N, Kuo CJ, Palmer TD. VEGF is necessary for exercise-induced adult hippocampal neurogenesis. Eur J Neurosci. 2003;18:2803–2812. [PubMed]
  • Favaro R, Valotta M, Ferri AL, Latorre E, Mariani J, Giachino C, Lancini C, Tosetti V, Ottolenghi S, Taylor V, Nicolis SK. Hippocampal development and neural stem cell maintenance require Sox2-dependent regulation of Shh. Nat Neurosci 2009 [PubMed]
  • Geling A, Steiner H, Willem M, Bally-Cuif L, Haass C. A gamma-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep. 2002;3:688–694. [PubMed]
  • Guentchev M, McKay RD. Notch controls proliferation and differentiation of stem cells in a dose-dependent manner. Eur J Neurosci. 2006;23:2289–2296. [PubMed]
  • Guo D, Ye J, Dai J, Li L, Chen F, Ma D, Ji C. Notch-1 regulates Akt signaling pathway and the expression of cell cycle regulatory proteins cyclin D1, CDK2 and p21 in T-ALL cell lines. Leuk Res. 2009;33:678–685. [PubMed]
  • Hill MN, Titterness AK, Morrish AC, Carrier EJ, Lee TT, Gil-Mohapel J, Gorzalka BB, Hillard CJ, Christie BR. Endogenous cannabinoid signaling is required for voluntary exercise-induced enhancement of progenitor cell proliferation in the hippocampus. Hippocampus 2009 [PMC free article] [PubMed]
  • Hurlbut GD, Kankel MW, Lake RJ, Artavanis-Tsakonas S. Crossing paths with Notch in the hyper-network. Curr Opin Cell Biol. 2007;19:166–175. [PubMed]
  • Imayoshi I, Ohtsuka T, Metzger D, Chambon P, Kageyama R. Temporal regulation of Cre recombinase activity in neural stem cells. Genesis. 2006;44:233–238. [PubMed]
  • Imayoshi I, Sakamoto M, Yamaguchi M, Mori K, Kageyama R. Essential roles of Notch signaling in maintenance of neural stem cells in developing and adult brains. J Neurosci. 2010;30:3489–3498. [PubMed]
  • Imayoshi I, Sakamoto M, Ohtsuka T, Takao K, Miyakawa T, Yamaguchi M, Mori K, Ikeda T, Itohara S, Kageyama R. Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain. Nat Neurosci. 2008;11:1153–1161. [PubMed]
  • Johnson MA, Ables JL, Eisch AJ. Cell-intrinsic signals that regulate adult neurogenesis in vivo: insights from inducible approaches. BMB Rep. 2009;42:245–259. [PMC free article] [PubMed]
  • Kempermann G, Jessberger S, Steiner B, Kronenberg G. Milestones of neuronal development in the adult hippocampus. Trends Neurosci. 2004;27:447–452. [PubMed]
  • Kempermann G, Chesler EJ, Lu L, Williams RW, Gage FH. Natural variation and genetic covariance in adult hippocampal neurogenesis. Proc Natl Acad Sci U S A. 2006;103:780–785. [PubMed]
  • Kitamura T, Mishina M, Sugiyama H. Enhancement of neurogenesis by running wheel exercises is suppressed in mice lacking NMDA receptor epsilon 1 subunit. Neurosci Res. 2003;47:55–63. [PubMed]
  • Koehl M, Meerlo P, Gonzales D, Rontal A, Turek FW, Abrous DN. Exercise-induced promotion of hippocampal cell proliferation requires beta-endorphin. FASEB J. 2008;22:2253–2262. [PubMed]
  • Kronenberg G, Reuter K, Steiner B, Brandt MD, Jessberger S, Yamaguchi M, Kempermann G. Subpopulations of proliferating cells of the adult hippocampus respond differently to physiologic neurogenic stimuli. J Comp Neurol. 2003;467:455–463. [PubMed]
  • Lafenetre P, Leske O, Ma-Hoegemeier Z, Haghikia A, Bichler Z, Wahle P, Heumann R. Exercise can rescue recognition memory impairment in a model with reduced adult hippocampal neurogenesis. Frontiers In Behavioral Neuroscience 2009 [PMC free article] [PubMed]
  • Lagace DC, Benavides DR, Kansy JW, Mapelli M, Greengard P, Bibb JA, Eisch AJ. Cdk5 is essential for adult hippocampal neurogenesis. Proc Natl Acad Sci U S A. 2008;105:18567–18571. [PubMed]
  • Lagace DC, Donovan MH, Decarolis NA, Farnbauch LA, Malhotra S, Berton O, Nestler EJ, Krishnan V, Eisch AJ. Adult hippocampal neurogenesis is functionally important for stress-induced social avoidance. Proc Natl Acad Sci U S A. 2010;107:4436–4441. [PubMed]
  • Lagace DC, Whitman MC, Noonan MA, Ables JL, DeCarolis NA, Arguello AA, Donovan MH, Fischer SJ, Farnbauch LA, Beech RD, DiLeone RJ, Greer CA, Mandyam CD, Eisch AJ. Dynamic contribution of nestin-expressing stem cells to adult neurogenesis. J Neurosci. 2007;27:12623–12629. [PMC free article] [PubMed]
  • Lazarov O, Marr RA. Neurogenesis and Alzheimer's disease: At the crossroads. Exp Neurol 2009
  • Lugert S, Basak O, Knuckles P, Haussler U, Fabel K, Gotz M, Haas CA, Kempermann G, Taylor V, Giachino C. Quiescent and active hippocampal neural stem cells with distinct morphologies respond selectively to physiological and pathological stimuli and aging. Cell Stem Cell. 2010;6:445–456. [PubMed]
  • Mandyam CD, Norris RD, Eisch AJ. Chronic morphine induces premature mitosis of proliferating cells in the adult mouse subgranular zone. J Neurosci Res. 2004;76:783–794. [PubMed]
  • Ming GL, Song H. Adult neurogenesis in the mammalian central nervous system. Annu Rev Neurosci. 2005;28:223–250. [PubMed]
  • Naylor AS, Bull C, Nilsson MK, Zhu C, Bjork-Eriksson T, Eriksson PS, Blomgren K, Kuhn HG. Voluntary running rescues adult hippocampal neurogenesis after irradiation of the young mouse brain. Proc Natl Acad Sci U S A. 2008;105:14632–14637. [PubMed]
  • Pietropaolo S, Sun Y, Li R, Brana C, Feldon J, Yee BK. The impact of voluntary exercise on mental health in rodents: a neuroplasticity perspective. Behav Brain Res. 2008;192:42–60. [PubMed]
  • Poellinger L, Lendahl U. Modulating Notch signaling by pathway-intrinsic and pathway-extrinsic mechanisms. Curr Opin Genet Dev. 2008;18:449–454. [PubMed]
  • Radtke F, Schweisguth F, Pear W. The Notch ‘gospel’ EMBO Rep. 2005;6:1120–1125. [PubMed]
  • Radtke F, Wilson A, Stark G, Bauer M, van Meerwijk J, MacDonald HR, Aguet M. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity. 1999;10:547–558. [PubMed]
  • Steiner B, Zurborg S, Horster H, Fabel K, Kempermann G. Differential 24 h responsiveness of Prox1-expressing precursor cells in adult hippocampal neurogenesis to physical activity, environmental enrichment, and kainic acid-induced seizures. Neuroscience. 2008;154:521–529. [PubMed]
  • Suh H, Consiglio A, Ray J, Sawai T, D'Amour KA, Gage FH. In vivo fate analysis reveals the multipotent and self-renewal capacities of Sox2+ neural stem cells in the adult hippocampus. Cell Stem Cell. 2007;1:515–528. [PMC free article] [PubMed]
  • van Praag H. Neurogenesis and exercise: past and future directions. Neuromolecular Med. 2008;10:128–140. [PubMed]
  • van Praag H, Kempermann G, Gage FH. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci. 1999a;2:266–270. [PubMed]
  • van Praag H, Christie BR, Sejnowski TJ, Gage FH. Running enhances neurogenesis, learning, and long-term potentiation in mice. Proc Natl Acad Sci U S A. 1999b;96:13427–13431. [PubMed]
  • Wang X, Mao X, Xie L, Greenberg DA, Jin K. Involvement of Notch1 signaling in neurogenesis in the subventricular zone of normal and ischemic rat brain in vivo. J Cereb Blood Flow Metab. 2009;29:1644–1654. [PMC free article] [PubMed]
  • Yoon K, Gaiano N. Notch signaling in the mammalian central nervous system: insights from mouse mutants. Nat Neurosci. 2005;8:709–715. [PubMed]
  • Zhao C, Deng W, Gage FH. Mechanisms and functional implications of adult neurogenesis. Cell. 2008;132:645–660. [PubMed]