Among γ-secretase substrates Notch1 alone is sufficient to block neurogenesis but does not confer self-renewal properties to neural stem cells

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Abstract

Notch signaling pathway enhances neural stem cell characters and regulates cell fate decisions during neural development. Interestingly, besides Notch, other γ-secretase substrates such as APP, LRP2, and ErbB4 have also proven to have biological functions in neural development. We designed a unique experimental setting, combining gain-of- (expression of Notch intracellular domain, NICD) and loss-of-function (γ-secretase inhibition) methods, and were able to examine the function of Notch alone by excluding the activity of other γ-secretase substrates. Here, we show that the frequency and size of neurospheres generated from embryonic neural stem cells (NSCs) significantly decreased by 62.7% and 37.2%, respectively, in the presence of γ-secretase inhibitor even when NICD was expressed. Under the condition of differentiation, however, the γ-secretase inhibitor treatment did not influence the promotion of astrogenesis at the expense of neurogenesis by NICD. These results indicate that other γ-secretase substrate(s) along with Notch are important in the maintenance of the stemness of NSCs, but that Notch alone can sufficiently inhibit neurogenesis without the action of the other γ-secretase substrates during differentiation.

Research highlights

► We were able to examine the function of Notch alone by excluding the activity of other γ-secretase substrates. ► Despite the presence of NICD, the frequency of formation and size of neurospheres were greatly influenced by γ-secretase inhibitors. ► During differentiation, NICD alone can induce astrogliogenesis at the expense of neurogenesis without the action of the other γ-secretase substrates.

Introduction

In the mammalian nervous system, the Notch signaling pathway is thought to regulate cell fate specification during neural development [1]. Notch activation also maintains the neural progenitor state and expands the neural progenitor pool while preventing progenitors from differentiating into neurons [2]. Consistently, deletion of the Notch gene leads to precocious expression of early neuronal markers [3].

Most of the Notch-related gain-of-function studies have used the truncated form of the intracellular region of Notch receptors (Notch intracellular domain, NICD) to induce Notch signaling without binding of ligands [1], [2], [4], [5], [6]. Endogenously, NICD is generated from cleavage of the Notch receptor by γ-secretase activity upon ligand binding. NICD then translocates into the nucleus, associated with CBF1 and activates expression of its target genes [7].

γ-Secretase complex consists of presenilin (PS), nicastrin (NCT), anterior pharynx defective 1 (APH-1), and presenilin enhancer 2 (PEN-2) [8]. The substrates of this enzyme that have so far been identified are amyloid precursor protein (APP), low-density lipoprotein receptor-related protein (LRP)-2, E-cadherin, and ErbB-4 in addition to the Notch family genes. Interestingly, besides the ICD of Notch, the ICDs of APP, LRP2, and ErbB4 are also known to have biological functions in neural development by exerting influence on neurogenesis and astrogenesis [9], [10], [11], [12].

The experimental method that has been commonly used to investigate the effect of Notch on progenitor cells including neural and hematopoietic stem cells, as well as cancer cells in a loss-of-function manner is the use of γ-secretase inhibitors (e.g., N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, DAPT). However, as treatment with the γ-secretase inhibitor hampers the processing not only of Notch but also of other γ-secretase substrates, it cannot be concluded that Notch solely exerts influence on these cells. In this study, NICD in its fully processed form was expressed in the presence of γ-secretase inhibitors to investigate the function only of Notch among the γ-secretase substrates (Fig. 1A). In this unique experimental setting, combination of gain-of- (forced expression of constitutively active form of Notch) and loss-of-function (γ-secretase inhibitor treatment) methods, it was possible to exclude the activity of other γ-secretase substrates. Here, we show that the frequency of generation and size of neurospheres considerably decreased in the presence of γ-secretase inhibitors even when NICD was expressed. During differentiation, however, the promotion of astrogenesis at the expense of neurogenesis by NICD was not influenced by the γ-secretase inhibitor. These results suggest that other γ-secretase substrate(s) along with Notch are important in the maintenance of the stemness of NSCs, but that Notch alone can sufficiently inhibit neurogenesis without the action of the other γ-secretase substrates during differentiation.

Section snippets

Animals and neural progenitor cell preparation

Female mice (CD1; 8 week) were purchased from Orient Bio Inc. (Osan, Korea) and maintained in specific pathogen-free condition before sacrifice. All animal protocols were approved by the Institutional Review Board and conducted in the Laboratory Animal Research Center of Sungkyunkwan University. Neurosphere cultures were prepared from the lateral and medial ganglionic eminences of embryonic day (E) 14.5 embryos (the day of vaginal plug was considered E0.5.). Dissected brain tissue was minced,

Inhibition of γ-secretase does not block NICD transactivation of its target gene

We designed an experimental setting by which the sole effect of NICD, not of the ICDs of the other γ-secretase substrates, could be investigated. First, we tested whether DAPT could alter the level of Notch signaling induced by forced expression of the fully processed form of Notch, NICD. The NICD that was used in the study was derived from Notch1 and about 45% of its C-terminal region including the PEST domain was removed (Fig. 1B). This truncated form of NICD showed constitutive signaling

Discussion

There are two major effects of Notch on NSCs; the promotion of NSC character (e.g., self-renewal capability) and the inhibition of neurogenesis/concurrent promotion of astrogenesis. Various genes, including Notch as well as Shh and Wnt, are known to be involved in the maintenance of NSCs. Among the γ-secretase substrates, however, only Notch has been known to be important in this function and extensive studies are under way. In this study, attempts were made to quantify the contribution of

Acknowledgments

This work was supported by Brain Research Center of the 21st Century Frontier Research Program (#2010K000829) and Basic Science Research Program through the National Research Foundation of Korea (#20100011388) funded by the Ministry of Education, Science and Technology (MEST).

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    These authors contributed equally to this work.

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