Notch1 mediates postnatal neurogenesis in hippocampus enhanced by intermittent 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.
Section snippets
Animals
Notch1 (Conlon et al., 1995) and Thy1/YFP transgenic mice (line H) (Feng et al., 2000) were described. Both of the two kinds of mice are on C57BL/6 background. Notch1 heterozygous mice were mated to generate wild type (WT) and Notch1 deficient mice (Notch1 +/−) (no Notch1 homozygous mice were obtained from heterozygous intercrosses of N +/− mice. At E11, all homozygous mice were dead (Conlon et al., 1995). Thy1-YFP mice were generated from intercrosses of Thy1-YFP mice. Notch1 +/− were crossed
Genotype identification, hippocampus and neuron morphology, Notch1 and NICD expression in wild type (WT) and Notch1 heterozygous deficient (N +/−) mice
To address the function of Notch1 in the postnatal neurogenesis after intermittent hypoxia treatment, Notch1 heterozygous deficient (N +/−) mice were adopted in our study. Before all the experiments, genotype, general brain morphology and total Notch1 or NICD expression in WT and N +/− mice were analyzed. Genotypes of WT and N +/− mice were identified by PCR (Fig. 1A). The detection of single wt (200 bp) loci indicated the wild type. Both detection of wt (200 bp) and Neo (280 bp) loci indicated the
Discussion
The major findings provided by the present study are as follows: first, IH, as a novel external stimulus, enhances neurogenesis in postnatal hippocampus at multiple steps, including proliferation of NSCs, survival and migration of newborn neurons, and spine morphogenesis of mature neurons; second, Notch1 signaling is activated by hypoxia in vivo and is required for hypoxia-induced neurogenesis. In wild type (WT) mice, our experiments showed that the number of cells newly-labeled with Brdu in
Funding
This work was supported by a grant from the National Basic Research Program of China (No. 2011CB910800 and 2012CB518200) and by a grant from the National Nature and Sciences Foundation of China (No. 90919025 and 30831160514).
Acknowledgement
The authors would like to thank Yu Zhengping from Department of Occupational Health, Third Military Medical University for some of laboratory apparatus he provided.
References (62)
- et al.
Activity-induced Notch signaling in neurons requires Arc/Arg3.1 and is essential for synaptic plasticity in hippocampal networks
Neuron
(2011) - et al.
Different dendrite and dendritic spine alterations in basal and apical arbors in mutant human amyloid precursor protein transgenic mice
Brain Res.
(2006) - et al.
Learning and memory deficits in Notch mutant mice
Curr. Biol.
(2003) - et al.
Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression
Neuron
(2009) - et al.
Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP
Neuron
(2000) - et al.
Hypoxia requires notch signaling to maintain the undifferentiated cell state
Dev. Cell
(2005) - et al.
Neonatal alcohol exposure disrupts hippocampal neurogenesis and contextual fear conditioning in adult rats
Brain Res.
(2011) - et al.
Interaction between Reelin and Notch signaling regulates neuronal migration in the cerebral cortex
Neuron
(2008) - et al.
Involvement of gamma-secretase in postnatal angiogenesis
Biochem. Biophys. Res. Commun.
(2007) Adult neurogenesis and modulation of neural circuit function
Curr. Opin. Neurobiol.
(2011)
Inhibition of Notch signaling by Dll4-Fc promotes reperfusion of acutely ischemic tissues
Biochem. Biophys. Res. Commun.
Adult neurogenesis in the mammalian brain: significant answers and significant questions
Neuron
Signaling in adult neurogenesis
Curr. Opin. Neurobiol.
Perforant path activation of ectopic granule cells that are born after pilocarpine-induced seizures
Neuroscience
The cerebral effects of ascent to high altitudes
Lancet Neurol.
Neurogenesis in the adult rat brain after intermittent hypoxia
Brain Res.
Not(ch) just development: Notch signalling in the adult brain
Nat. Rev. Neurosci.
Activin in the brain modulates anxiety-related behavior and adult neurogenesis
PLoS ONE
Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods
J. Comp. Neurol.
Stroke and T-cells
Neuromolecular Med.
Prolonged voluntary wheel-running stimulates neural precursors in the hippocampus and forebrain of adult CD1 mice
Hippocampus
Intermittent Hypoxia and Experimental Parkinson’s Disease
Intermittent Hypoxia and Human Diseases.
Notch regulates cell fate and dendrite morphology of newborn neurons in the postnatal dentate gyrus
Proc. Natl. Acad. Sci. U. S. A.
Atorvastatin promotes presenilin-1 expression and Notch1 activity and increases neural progenitor cell proliferation after stroke
Stroke
Notch1 is required for the coordinate segmentation of somites
Development
Neurogenesis in the adult human hippocampus
Nat. Med.
Neuronal differentiation in the adult hippocampus recapitulates embryonic development
J. Neurosci.
Transient spine expansion and learning-induced plasticity in layer 1 primary motor cortex
J. Neurosci.
Dendritic spines: structure, dynamics and regulation
Nat. Rev. Neurosci.
Herp, a new primary target of notch regulated by ligand binding
Mol. Cell. Biol.
More hippocampal neurons in adult mice living in an enriched environment
Nature
Cited by (31)
The relationship between intermittent hypoxemia events and neural outcomes in neonates
2021, Experimental NeurologyCitation Excerpt :In rodents, exposure to short rapid fluctuations in oxygen levels beginning between the 7th and 10th day of life resulted in learning impairment, hyperactivity, and altered dopamine signaling (Decker et al., 2005; Row et al., 2002). In contrast, exposure to a single brief (5 min) cycle of hypoxia during the first 24 h after birth (Martin et al., 2012; Martin et al., 2010), or longer (4 h) and milder cycles of hypoxia during the first 3–4 weeks of life (Zhang et al., 2005) enhanced long-term spatial learning (Zhang et al., 2005), memory (Martin et al., 2010), and structural changes in both the hippocampus (Martin et al., 2010; Zhang et al., 2014) and frontal cortex (Martin et al., 2010). The mechanistic pathway(s) by which these distinct IH patterns induce a pathological cascade is unknown and a key factor in identifying areas of focus for clinical interventions.
Chronic mild hypoxia promotes hippocampal neurogenesis involving Notch1 signaling in epileptic rats
2019, Brain ResearchCitation Excerpt :Taken together, these results suggest that hypoxia promotes NSC proliferation and induces NSCs to differentiate into neurons rather than astrocytes. The Notch1 signaling pathway is an important regulator of neurogenesis (Zhang et al., 2014). Using western blot, we examined expression levels of Notch1 and the target protein Hes1 in vivo and vitro.
HIF-1α is Critical for the Activation of Notch Signaling in Neurogenesis During Acute Epilepsy
2018, NeuroscienceCitation Excerpt :The western blotting and immunoprecipitation analyses also revealed the probable molecular circadian mechanism of the physical interaction between HIF-1α and NICD. Previous studies have shown that there is cross-talk between HIF-1α and Notch signaling in some physiological and pathological conditions, such as in several types of tumors (Pistollato et al., 2010, Landor and Lendahl, 2017), ischemia reperfusion (I/R) injury (Cheng et al., 2014) and postnatal neurogenesis (Zhang et al., 2014), in vivo and in vitro. However, the relationship between HIF-1α and Notch signaling is complex and controversial, and the molecular mechanism is unclear.
Notch signaling and neuronal death in stroke
2018, Progress in NeurobiologyCitation Excerpt :Administration of Notch-activating antibodies enhances ischemic stroke-induced neurogenesis in the SVZ, reduces infarct volume and improves motor deficits in aged ischemic rats (Sun et al., 2013). A recent study showed that intermittent hypoxia enhances neurogenesis and dendritic spine formation in neurons, and these adaptive responses to mild hypoxia of NPCs and neurons are attenuated in Notch heterozygous (Notch+/−) mice (Zhang et al., 2014). As discussed in Section 4, under conditions of severe stress, the collaboration of p53 with other proteins such as NF-κB and HIF-1α may play a major role in detrimental outcomes.
Obstructive sleep apnea affects cognition: dual effects of intermittent hypoxia on neurons
2024, Sleep and Breathing