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
), 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 (). 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.
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.