Cytoskeleton modifications are required for neuronal stem cells to acquire neuronal polarization. Little is known, however, about mechanisms that orchestrate cytoskeleton remodeling along neuritogenesis. Here, we show that the silencing of the cellular prion protein (PrP(C)) impairs the initial sprouting of neurites upon induction of differentiation of the 1C11 neuroectodermal cell line, indicating that PrP(C) is necessary to neuritogenesis. Such PrP(C) function relies on its capacity to negatively regulate the clustering, activation, and signaling activity of β1 integrins at the plasma membrane. β1 Integrin aggregation caused by PrP(C) depletion triggers overactivation of the RhoA-Rho kinase-LIMK-cofilin pathway, which, in turn, alters the turnover of focal adhesions, increases the stability of actin microfilaments, and in fine impairs neurite formation. Inhibition of Rho kinases is sufficient to compensate for the lack of PrP(C) and to restore neurite sprouting. We also observe an increased secretion of fibronectin in the surrounding milieu of PrP(C)-depleted 1C11 cells, which likely self-sustains β1 integrin signaling overactivation and contributes to neuritogenesis defect. Our overall data reveal that PrP(C) contributes to the acquisition of neuronal polarization by modulating β1 integrin activity, cell interaction with fibronectin, and cytoskeleton dynamics.

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BackgroundThe cellular prion protein, PrPC, is GPI anchored and abundant in lipid rafts. The absolute requirement of PrPC in neurodegeneration associated to prion diseases is well established. However, the function of this ubiquitous protein is still puzzling. Our previous work using the 1C11 neuronal model, provided evidence that PrPC acts as a cell surface receptor. Besides a ubiquitous signaling function of PrPC, we have described a neuronal specificity pointing to a role of PrPC in neuronal homeostasis. 1C11 cells, upon appropriate induction, engage into neuronal differentiation programs, giving rise either to serotonergic (1C115-HT) or noradrenergic (1C11NE) derivatives.Methodology/Principal FindingsThe neuronal specificity of PrPC signaling prompted us to search for PrPC partners in 1C11-derived bioaminergic neuronal cells. We show here by immunoprecipitation an association of PrPC with an 80 kDa protein identified by mass spectrometry as the tissue non-specific alkaline phosphatase (TNAP). This interaction occurs in lipid rafts and is restricted to 1C11-derived neuronal progenies. Our data indicate that TNAP is implemented during the differentiation programs of 1C115-HT and 1C11NE cells and is active at their cell surface. Noteworthy, TNAP may contribute to the regulation of serotonin or catecholamine synthesis in 1C115-HT and 1C11NE bioaminergic cells by controlling pyridoxal phosphate levels. Finally, TNAP activity is shown to modulate the phosphorylation status of laminin and thereby its interaction with PrP.Conclusion/SignificanceThe identification of a novel PrPC partner in lipid rafts of neuronal cells favors the idea of a role of PrP in multiple functions. Because PrPC and laminin functionally interact to support neuronal differentiation and memory consolidation, our findings introduce TNAP as a functional protagonist in the PrPC-laminin interplay. The partnership between TNAP and PrPC in neuronal cells may provide new clues as to the neurospecificity of PrPC function.

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol-anchored protein, abundant in lipid rafts and highly expressed in the brain. While PrPC is much studied for its involvement under its abnormal PrPSc isoform in Transmissible Spongiform Encephalopathies, its physiological role remains unclear. Here, we report that GSK3β, a multifunctional kinase whose inhibition is neuroprotective, is a downstream target of PrPC signalling in serotonergic neuronal cells. We show that the PrPC-dependent inactivation of GSK3β is relayed by a caveolin-Lyn platform located on neuronal cell bodies. Furthermore, the coupling of PrPC to GSK3β potentiates serotonergic signalling by altering the distribution and activity of the serotonin 1B receptor (5-HT1BR), a receptor that limits neurotransmitter release. In vivo, our data reveal an increased GSK3β kinase activity in PrP-deficient mouse brain, as well as sustained 5-HT1BR activity, whose inhibition promotes an anxiogenic behavioural response. Collectively, our data unveil a new facet of PrPC signalling that strengthens neurotransmission.

The subversion of the normal function exerted by the cellular prion protein (PrP C ) in neurons by pathogenic prions is assumed to have a central role in the pathogenesis of transmissible spongiform encephalopathies. Using two murine models of prion infection, the 1C11 neuronal cell line and neurospheres, we document that prion infection is associated with the constitutive activation of signaling targets normally coupled with PrP C , including the Fyn kinase, the mitogen-associated protein kinases ERK1/2 and the CREB transcription factor. PrP C -dependent signaling overactivation in infected cells is associated with the recruitment of p38 and JNK stress-associated kinases. Downstream from CREB, prion-infected cells exhibit reduced activity of the matrix metalloprotease (MMP)-9. As MMP-9 catalyzes the degradation of the amyloid A-beta peptide, the decrease in MMP-9 activity in prion-infected cells causes a significant impairment of the clearance of A-beta, leading to its accumulation. By exploiting two 1C11-infected clones accumulating high or moderate levels of prions, we show that the prion-induced changes are correlated with the level of infectivity. Of note, a dose-dependent increase in A-beta levels was also found in the cerebrospinal fluid of mice inoculated with these infected clones. By demonstrating that pathogenic prions trigger increases in A-beta levels through the deviation of PrP C signaling, our data argue that A-beta may exacerbate prion-induced toxicity.

The cellular prion protein PrP(C) is the normal counterpart of the scrapie prion protein PrP(Sc), the main component of the infectious agent of transmissible spongiform encephalopathies (TSEs). It is a ubiquitous cell-surface glycoprotein, abundantly expressed in neurons, which constitute the targets of TSE pathogenesis. The presence of PrP(C) at the surface of neurons is an absolute requirement for the development of prion diseases and corruption of PrP(C) function(s) within an infectious context emerges as a proximal cause for PrP(Sc)-induced neurodegeneration. Experimental evidence gained over the past decade indicates that PrP(C) has the capacity to mobilize promiscuous signal transduction cascades that, notably, contribute to cell homeostasis. Beyond ubiquitous effectors, much data converge onto a neurospecificity of PrP(C) signaling, which may be the clue to neuronal cell demise in prion disorders. In this article, we highlight the requirement of PrP(C) for TSEs-associated neurodegeneration and review the current knowledge of PrP(C)-dependent signal transduction in neuronal cells and its implications for PrP(Sc)-mediated neurotoxicity.

The cellular prion protein PrP(C) is the normal counterpart of the scrapie prion protein PrP(Sc), the main component of the infectious agent of transmissible spongiform encephalopathies (TSEs). It is a ubiquitous cell-surface glycoprotein, abundantly expressed in neurons, which constitute the targets of TSE pathogenesis. Taking advantage of the 1C11 neuroectodermal cell line, endowed with the capacity to convert into 1C11(5-HT) serotonergic or 1C11(NE) noradrenergic neuronal cells, allowed us to ascribe a signaling function to PrP(C). Antibody-mediated ligation of PrP(C) recruits transduction pathways, which involve nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species production and target the extracellular-regulated kinases ERK1/2. In fully differentiated cells only, these effectors are under the control of a PrP(C)-caveolin-Fyn platform, located on neuritic extensions. In addition to its proper signaling activity, PrP(C) modulates the agonist-induced response of the three serotonergic G protein-coupled receptors present on the 1C11(5-HT) differentiated cells. The impact of PrP(C) ligation on the receptor couplings depends on the receptor subtype and the pathway considered. The implementation of the PrP(C)-caveolin complex again is mandatory for PrP(C) to exert its action on 5-HT receptor signaling. Our current data argue that PrP(C) interferes with the intensities and/or dynamics of G protein activation by agonist-bound 5-HT receptors. By mobilizing transduction cascades controlling the cellular redox state and the ERK1/2 kinases and by altering 5-HT receptor-mediated intracellular response, PrP(C) takes part in the homeostasis of serotonergic neuronal cells. These findings may have implications for future research aiming at understanding the fate of serotonergic neurons in prion diseases.