Notch1 mediates postnatal neurogenesis in hippocampus enhanced by intermittent hypoxia

Highlights

• Intermittent hypoxia, as a new stimulus, enhances neurogenesis at multiple stages.

• The multiple stages include NSC proliferation, migration and integration.

• The mechanism relies on Notch1 pathway activated by intermittent hypoxia.

• We have provided links between Notch1, neurogenesis and hypoxia in vivo.

Abstract

Notch1 is a transcription factor on the membrane and regulates various stages of neurogenesis. Recently, studies have shown that in vitro neurogenesis is enhanced by hypoxia, and there is cross-coupling between Notch and hypoxia signaling pathways in vitro. However, to date, no data have reported whether Notch1 can be regulated by hypoxia in vivo and mediates hypoxia-induced neurogenesis. To determine causative links between Notch1, neurogenesis and hypoxia, we examined multiple steps of hippocampal neurogenesis followed intermittent hypoxia (IH) in wild type (WT) and Notch1 heterozygous deficient (N +/−) mice. We found that IH increased NSC proliferation, newborn neuron survival and migration, and spine morphogenesis in dentate gyrus of hippocampus, as well as neurogenesis in olfactory bulb in WT mice. However, IH-enhanced neurogenesis was inhibited in N +/− mice. It was shown that Notch1 signaling was activated following IH in WT mice, but not in N +/− mice. Our data indicated that IH, as a novel external stimulus, enhances neurogenesis at multiple stages and that Notch1 is activated by hypoxia in vivo and required for hypoxia-induced neurogenesis. These results suggest IH as a novel therapeutic strategy for degenerative neurological disorders and provide evidence for causative links between Notch1, neurogenesis and hypoxia.

Introduction

Neurogenesis continuously exists throughout adulthood in the subgranular zone (SGZ) of the mammalian dentate gyrus (Eriksson et al., 1998, Kornack and Rakic, 1999, Kuhn et al., 1996, Mu et al., 2010) and is implicated in the mediation of anxiety (Ageta et al., 2008), depression (David et al., 2009, Sahay and Hen, 2007), drug addiction (Noonan et al., 2010), learning and memory (Inokuchi, 2011, Shors et al., 2001). Adult neurogenesis is a dynamic and multistep process involving proliferation of precursor cells, their differentiation into lineage-restricted immature neurons, and the progressive maturation of these newborn cells into fully functional and integrated neurons (Vukovic et al., 2001).

One characteristic of postnatal neurogenesis is its sensitivity to various external stimuli, including physiological ones, e.g. physical exercises and enrich environment (Bednarczyk et al., 2009, Kempermann et al., 1997), and pathological ones, e.g. seizures and stroke (Ohab et al., 2006, Scharfman et al., 2003). These stimuli can exert their effects during specific stages of neurogenesis, from regulating precursor proliferation to promoting the survival of newly generated neurons (Ma et al., 2009). It is clear that every single phase of neurogenesis can be regulated by different stimuli and each stimulus may have multiple targets (Ming and Song, 2011).

Intermittent hypoxia (IH), generally defined as repeated episodes of hypoxia interspersed with normoxic periods, has always been used as a method for training pilots, mountaineers and athletes, and even been applied as treatment and prevention of hypertension (Serebrovskaya et al., 2008), ischemic coronary artery diseases (Zhu et al., 2006), and acute myeloid leukemia (Liu et al., 1998). In the recent years, IH has been found to exert many positive effects on CNS in animal and human studies, such as enhancement of spatial learning and memory (Zhang et al., 2005), production of antidepressant-like effect (Zhu et al., 2010), and treatment of Parkinson's disease (Belikova et al., 2012).

Our previous studies have addressed the enhancement of NSC proliferation (Zhu et al., 2005) in vivo and increase of newborn neurons (Zhu et al., 2010) after hypoxic treatment. The in vitro studies also demonstrated that the reduced oxygen levels can promote the survival, proliferation and catecholaminergic differentiation of CNS stem cells (Morrison et al., 2000, Studer et al., 2000, Zhang et al., 2006, Zhao et al., 2008). Interestingly, our in vivo studies have shown that external hypoxia environment could change the intrinsic oxygen niche of brain, and reduce the oxygen levels in the DG of hippocampus (Zhang et al., 2010). So here we hypothesized that postnatal neurogenesis may be promoted by the reduced oxygen levels in the DG induced by the external hypoxia environment. None have investigated and identified whether IH is a stimulus for postnatal neurogenesis at multiple stages, from proliferation of NSCs to maturation, survival, migration and integration of newborn neurons. In addition, the majority of the hippocampal development takes place in the early postnatal stages (from P1 to P30) (Altman and Bayer, 1990). Stimuli at neonatal stage can also last long and affect the adult neurogenesis (Hamilton et al., 2011). However, little is known whether IH can promote the neonatal neurogenesis.

Notch1 is a transcription factor on the membrane ideally situated to integrate cues from the niche to regulate various stages of neurogenesis (Ables et al., 2011, Yoon and Gaiano, 2005). In response to signals of neighboring cells, Notch1 not only controls the self-renewal and fate in embryonic NSCs (Yoon and Gaiano, 2005), but also regulates dendrite morphology of newborn neurons and maintains the undifferentiated state of NSCs in the postnatal brain (Breunig et al., 2007, Redmond et al., 2000). Researchers have also found precocious neuronal differentiation and defects in the synaptic plasticity and spatial learning and memory in Notch1 mutant mice (Alberi et al., 2011, Costa et al., 2003, Lütolf et al., 2002). Recent in vitro experiments suggested that Notch1 is required for the undifferentiated cell state promoted by hypoxia (Gustafsson et al., 2005) and mediates the hypoxia-induced tumor cell migration and invasion (Sahlgren et al., 2008). In addition, it has been shown that there is cross-coupling between the Notch and hypoxia signaling pathways in vitro (Zheng et al., 2008). However, there is no study reported whether Notch1 is regulated by hypoxia in vivo and mediates hypoxia-induced neurogenesis.

Therefore, our first objective in the present study was to determine whether IH is another external stimulus for postnatal neurogenesis in DG at multiple stages from the proliferation of NSCs to the differentiation, survival, migration and morphogenesis of newborn neurons. The second one was to examine whether Notch1 is regulated by hypoxia in vivo. The third one was to identify causative links between Notch1, neurogenesis and hypoxia. To answer these questions, we assessed the proliferation, differentiation, survival, migration and spine morphogenesis in DG of wild-type (WT) and Notch1 deficient (N +/−) mice after normoxia and IH treatment. We found that IH is a novel stimulus for neurogenesis in DG at multiple stages including NSC proliferation, newborn neuron survival and migration, and spine morphogenesis. We further showed that Notch1 is activated by hypoxia in vivo and required for hypoxia-induced neurogenesis.