PACT increases mammalian embryonic neural stem cell properties by facilitating activation of the notch signaling pathway

Highlights

• PACT enhances mammalian embryonic neural stem cell properties as assessed in vitro and in vivo.

• PACT-increased neural stemness is independent of PKR.

• PACT augments the Notch signaling pathway, a master regulator of neural stem cell fate, at the transcriptional level.

• PACT stabilizes the Notch coactivational complex by enhancing association between NICD and CBF1.

Abstract

The protein activator of protein kinase R (PKR) (PACT) is known to play important roles in PKR regulation and microRNA biogenesis. Based on the observation that PACT is specifically expressed in the ventricular zone (VZ) at the mid-neurogenic period, we examine the role of PACT in this embryonic neural stem cell niche. Here, we provide the first evidence that PACT increases neurosphere formation, as well as expression of Notch target genes and the neural stem cell marker Sox2 in primary neural stem cells in vitro. Consistently, introduction of PACT into the mouse embryonic brain in utero increased the fraction of cells localizing to the VZ. We also show that the PACT-enhanced stemness of neural stem cells is PKR-independent. At the molecular level, PACT was revealed to physically interact with C promoter binding factor 1 (CBF1) and dramatically strengthen the association between CBF1 and Notch intracellular domain (NICD), which indicates stabilization of the Notch transcriptional coactivation complex responsible for Notch target gene expression. Taken together, our study indicates that PACT is a novel transcriptional coactivator of the Notch pathway playing a pivotal role during mammalian brain development.

Introduction

In mammalian brain development, proliferation and differentiation of neural stem cells are strictly regulated to build up proper brain structure. At the early stages, when neuroepithelial cells divide symmetrically to expand the neural stem cell population, multiple signaling pathways coordinate to maintain the balance between stem cell proliferation and differentiation [[1], [2], [3]]. This balance is important not only for maintaining an appropriate neural stem cell pool, but also for forming proper laminar organization and attaining the size of a mature brain [4,5]. However, the mechanisms governing these processes are not fully understood.

The Notch signaling pathway is evolutionally conserved across species and plays key roles in various cellular processes both in the embryonic and adult stages [1]. During brain development, Notch signaling maintains the neural stem cell pool and regulates differentiation of neural progenitors [6]. The Notch pathway is activated by cell-to-cell interaction between Notch receptor- and ligand-expressing cells [7,8]. After binding to the ligands, Notch receptors are cleaved by γ-secretase activity proximal to the membrane. The released Notch intracellular domain (NICD) then moves into the nucleus to associate with coactivational proteins including C promoter-binding factor 1 (CBF1) and Mastermind-like 1 (MAML1) to induce Notch target gene expression [9]. Notch target genes such as Hes1 and Hes5 suppress neurogenesis through inhibiting proneural transcription factors, thereby maintaining neural stem cells [10].

The protein activator of protein kinase R (PKR) (PACT) was originally identified as a PKR regulator [11]. PACT binds to PKR through double-stranded RNA binding domain 1 (dsRBD1) and dsRBD2, and the PKR activation domain was mapped to dsRBD3 which is located in the C-terminus of PACT [12]. Because viral infection-induced PKR prevents viral protein synthesis [13,14], PACT has been regarded as a positive regulator of antiviral defense mechanisms. However, recently, PACT has been shown to have PKR-independent functions such as regulation of the dsRNA endoribonuclease Dicer, which produces miRNA duplexes [15]. These observations raise the possibility that PACT may be a pleiotropic factor regulating diverse cellular events. Interestingly, in situ hybridization data from Eurexpress (www.eurexpress.org) show that PACT is strongly expressed in the ventricular zone (VZ) of the embryonic brain, but its role in this neural stem cell niche has not yet been elucidated.

In this study, we show that PACT increases embryonic neural stem cell properties by enhancing the Notch signaling pathway. Furthermore, we find that PACT greatly strengthens the association between NICD and CBF1, which should inevitably lead to increased Notch target gene expression and subsequent neural stem cell maintenance in the embryonic stages.