PrP^C regulates epidermal growth factor receptor function and cell shape dynamics in Neuro2a cells

Abstract

The prion protein (PrP) plays a key role in prion disease pathogenesis. Although the misfolded and pathologic variant of this protein (PrP SC ) has been studied in depth, the physiological role of PrP C remains elusive and controversial.PrP C is a cell-surface glycoprotein involved in multiple cellular functions at the plasma membrane, where it interacts with a myriad of partners and regulates several intracellular signal transduction cascades. However, little is known about the gene expression changes modulated by PrP C in animals and in cellular models. In this article, we present PrP C -dependent gene expression signature in N2a cells and its implication in the most overrepresented functions: cell cycle, cell growth and proliferation, and maintenance of cell shape. PrP C overexpression enhances cell proliferation and cell cycle re-entrance after serum stimulation, while PrP C silencing slows down cell cycle progression. In addition, MAP kinase and protein kinase B (AKT) pathway activation are under the regulation of PrP C in asynchronous cells and following mitogenic stimulation. These effects are due in part to the modulation of epidermal growth factor receptor (EGFR) by PrP C in the plasma membrane, where the two proteins interact in a multimeric complex. We also describe how PrP C overexpression modulates filopodia formation by Rho GTPase regulation mainly in an AKT-Cdc42-N-WASP-dependent pathway.

Domain-Specific Activation of Death-Associated Intracellular Signalling Cascades by the Cellular Prion Protein in Neuroblastoma Cells

“…We chose the N2A cell line as our culture model to dissect the N-terminal part of PrP C because it shows neuronal-like characteristics [41] and has been successfully used in in vitro prion propagation and signalling [42,43,22]. Deletion of domains CD, CC, HR, CR and even all the unstructured N-terminal domain of PrP C (PrP ΔF35 ) does not seem to interfere with maturation or protein trafficking of the PrP C forms to the plasma membrane.…”

“…One day before transfection, cells were cultured in DMEM supplemented with 10 % FBS and without antibiotics, on poly-D-lysine (Sigma)-coated plates (Nunc). Transfection was performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions as indicated [22].…”

Physiological Functions of the Cellular Prion Protein

“…In addition to its reported role in neuroprotection, several studies have shown that PrP C promotes neurite outgrowth, and potential explanations include interactions of PrP C with STI1 (Lopes et al, 2005), neural cell adhesion molecule 1 (NCAM1) (Santuccione et al, 2005), epidermal growth factor receptors (Llorens et al, 2013), integrins (Loubet et al, 2012), laminin (Graner et al, 2000a), or metabotropic glutamate receptors (mGluRs) (Beraldo et al, 2011). The downstream signalling responsible may include inhibition of the ras homolog gene family, member A (RhoA)-Rho-associated protein kinase (ROCK) pathway (Loubet et al, 2012).…”

“…When activated, this signalling pathway stabilises the actin cytoskeleton, thereby inhibiting the development of filopodia—dynamic protrusions from the neurite growth cone that respond to the extracellular environment to guide migration of the developing neurite (O'Connor et al, 1990). Activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2), PI3K-Akt, and protein kinase c (PKC) signalling pathways may also be involved in mediating PrP C -dependent neurite outgrowth (Lopes et al, 2005; Caetano et al, 2008; Beraldo et al, 2011; Llorens et al, 2013). It should be cautioned that signal-regulatory protein α-1 reportedly modulates neurite outgrowth (Wang and Pfenninger, 2006) and, as previously mentioned, polymorphisms between Zurich I PrP C -null mice and their wild type counterparts in the gene encoding signal-regulatory protein α-1 can be present even after extensive backcrossing to an inbred strain (Nuvolone et al, 2016).…”

“…This confounding factor affects the interpretation of several studies that used cells derived from these mice to show apparent regulation of neurite outgrowth by PrP C (Lopes et al, 2005; Santuccione et al, 2005; Beraldo et al, 2011). However, investigations of other cell lines are free from this problem and a connection between PrP C expression and neurite outgrowth has been demonstrated repeatedly in such models (Graner et al, 2000a,b; Loubet et al, 2012; Llorens et al, 2013). …”