Effects And Mechanisms Of β-catenin On The Proliferation Of Neonatal Mice Hippocampal Neural Stem Cells Under Hypoxia Condition

Hypoxia in adult mammalian central nerve system (CNS) is often induced by many kinds of disease, such as brain trauma, stroke, epilepsy, et al, which can cause irreversible death of neurons and serious disability. So it is necessary to take effective measures to supplement or replace the lost neurons.The generation of new neurons from neural stem cells (NSCs) is restricted to two regions of the adult mammalian CNS: the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. NSCs in both regions are capable of proliferation, migration and differentiation under hypoxia condition. Endogeneous NSCs offer several advantages for brain repair, such as regardless of wound, immunological incompatibility with the host and ethics controversy, et al, which provides an ideal approach for repair after CNS injury.Hypoxia only induces insufficient NSCs proliferation spontaneously and little is understood about how hypoxia induces quiescent stem cells to proliferate, which is the major barrier for utilizing the endogenous NSCs to repair nervous system injury. Previous researches indicate that Wnt/β-catenin signaling plays an important role in tumorigenesis, embryonic neurogenesis. Low oxygen level existes both in tumorigenesis and embryonic development. Presently it has been reported that Wnt/β-catenin signaling involved in adult mammalian neurogensis. Therefore it is indicated that Wnt/β-catenin might participate in NSCs proliferation under hypoxia condition. To illuminate the answers, the low oxygen models of NSCs and astrocytes derived from hippocampus of newborn green fluorescence protein (GFP) transgenic mice were developed in this study, and then the effcets and mechanisms ofβ-catenin in the proliferation of hippocampal NSCs under hypoxia condition were illustrated.The study consisted of three parts: Part I The influence of hypoxia on the proliferation and differentiation of NSCs derived from newborn GFP transgenic mice hippocampusNSCs were mechanically dissected from hippocampus of newborn GFP transgenic mice at day 0 and plated in defined medium without serum. Nonadherent NSCs in vitro could generate neurosphere again after passaged. Neurosphere was Nestin and Musashi1 positive stained by immunology fluorescence staining and bromodeoxyuridine (BrdU) positive stained by immunology histochemical staining. Neurosphere could differentiate into neurons (detected by NSE), astrocytes (detected by GFAP) and oligodendrocytes (detected by MBP) induced by 10% fetal bovine serum (FBS). GFP was expressed both in proliferation and differentiation conditions. The results showed that NSCs were isolated, cultivated and identified from hippocampus of neonatal GFP transgenic mice postnatal day 0 in vitro successfully.Hippocampal NSCs were cultured at 5% O2 for different time and then collected for analysis. The effect of hypoxia on the proliferation and differentiation of hippocampal NSCs was analyzed by cell counts analysis, BrdU incorporation, MTT and immunohistochemistry, and the results showed that hypoxia promoted the proliferation and differentiation of hippocampal NSCs.Part II The effects ofβ-catenin signaling on the proliferation of hippocampal NSCs under hypoxia conditionThe major components of Wnt/β-catenin signaling in hippocampal NSCs including Wnt receptor (Frz1),β-catenin, Axin1, GSK-3βand LEF1 were detected by RT-PCR, which indicated that hippocampal NSCs had the ability to respond to Wnt signal. It was found that the relative luciferase activity in hippocampal NSCs was promoted by hypoxia, which indirectly suggested that hypoxia could enhance the expression ofβ-catenin. Furthermore, we demonstrated that theβ-catenin protein levels in NSCs, especially in nuleus, and the cyclin D1 protein levels were increased after hypoxia treatment for 12 h and 24 h compared with tranditional O2 condition(p<0.05).Therefore it was considered thatβ-catenin might involve in the proliferation of hippocampal NSCs under hypoxia condition. Beta-catenin in hippocampal NSCs in vitro was overexpressed or inhibited by electroporation and then it was detected the influence ofβ-catenin on the proliferation of hippocampal NSCs. The results showed that overexpressed β-catenin in NSCs could promote the proliferation of hippocampal NSCs and expression of cyclinD1 under hypoxia condition, while decreasedβ-catenin could reduce the proliferation of hippocampal NSCs and expression of cyclinD1 under hypoxia condition. Our observations suggested thatβ-catenin involved in the hippocampal NSCs proliferation under hypoxia condition through regulation of cyclinD1.Part III The mechanisms in the accumulationβ-catenin in hippocampal NSCs promoted by hypoxiaIt was little known that how hypoxia promoted the accumulation ofβ-catenin in hippocampal NSCs. Previous studies showed that Wnt3 and Wnt3a played important roles in embryonic hippocampal development and adut hippocampal neurogenesis. Astrocytes not only acted the major supporting cells, but also interacted with other cells through excreting regulative factors. Therefore the influence of hypoxia on the expression of Wnt3 and Wnt3αin hippocampal astrocytes was analyzed by RT-PCR, and the results showed that Wnt3 was expressed in hippocampal astrocytes and hypoxia reduced its expression compared with traditional culture condition (p<0.05), while Wnt3a was not detected neither in tradition or hypoxia condition. Phosphorylated Akt could inactivate GKS-3βthrough phosphorylation of GSK-3β, which resulted the reduction of the degradation ofβ-catenin. Therefore the influence of hypoxia on the expression of phosphorylated Akt and GKS-3βprotein was analyzed by Western Blotting, and it was found that hypoxia promoted the level of phosphorylated Akt and GKS-3βprotein compared with traditional culture condition (p<0.05). The observations indicated that the amount ofβ-catenin promoted by hypoxia was relative with the phosphorylated Akt and GKS-3βprotein rather than Wnt3 and Wnt3a.In conclusions, Hypoxia could promote the proliferation and differentiation of NSCs deried from hippocampus of neonatal GFP transgenic mice in vitro andβ-catenin involved in the proliferation of hippocampal NSCs under hypoxia condition through regulating the expression of cyclinD1. The promotion ofβ-catenin exprssion was related with the phosphorylated Akt and GKS-3βprotein induced by hypoxia rather than Wnt3 and Wnt3a. This study had important implication for repairment of CNS injury by utilization of endogenous NSCs.