Mathéa Pietri

NADPH oxidase and extracellular regulated kinases 1/2 are targets of prion protein signaling in neuronal and nonneuronal cells.

Putative functions of the cellular prion protein, PrPC, include resistance to oxidative stress, copper uptake, cell adhesion, and cell signaling. Here, we report NADPH oxidase-dependent reactive oxygen species (ROS) production and extracellular regulated kinases 1/2 (ERK1/2) phosphorylation on PrPC stimulation in the 1C11 neuroectodermal precursor, in its neuronal differentiated progenies, and in GT1-7 neurohypothalamic and BW5147 lymphoid cells. In neuroprogenitor, hypothalamic, and lymphoid cells, ERK1/2 activation is fully controlled by the NADPH oxidase-dependent ROS production. In 1C11-derived bioaminergic cells, ROS signaling and ERK1/2 phosphorylation are both controlled by Fyn kinase activation, introducing some specificity in PrPC transduction associated with this neuronal context. These data argue for an ubiquitous function of PrPC in cell-redox homeostasis through ROS production.

Raphe-mediated signals control the hippocampal response to SRI antidepressants via miR-16

Serotonin reuptake inhibitor (SRI) antidepressants such as fluoxetine (Prozac), promote hippocampal neurogenesis. They also increase the levels of the bcl-2 protein, whose overexpression in transgenic mice enhances adult hippocampal neurogenesis. However, the mechanisms underlying SRI-mediated neurogenesis are unclear. Recently, we identified the microRNA miR-16 as an important effector of SRI antidepressant action in serotonergic raphe and noradrenergic locus coeruleus (LC). We show here that miR-16 mediates adult neurogenesis in the mouse hippocampus. Fluoxetine, acting on serotonergic raphe neurons, decreases the amount of miR-16 in the hippocampus, which in turn increases the levels of the serotonin transporter (SERT), the target of SRI, and that of bcl-2 and the number of cells positive for Doublecortin, a marker of neuronal maturation. Neutralization of miR-16 in the hippocampus further exerts an antidepressant-like effect in behavioral tests. The fluoxetine-induced hippocampal response is relayed, in part, by the neurotrophic factor S100β, secreted by raphe and acting via the LC. Fluoxetine-exposed serotonergic neurons also secrete brain-derived neurotrophic factor, Wnt2 and 15-Deoxy-delta12,14-prostaglandin J2. These molecules are unable to mimic on their own the action of fluoxetine and we show that they act synergistically to regulate miR-16 at the hippocampus. Of note, these signaling molecules are increased in the cerebrospinal fluid of depressed patients upon fluoxetine treatment. Thus, our results demonstrate that miR-16 mediates the action of fluoxetine by acting as a micromanager of hippocampal neurogenesis. They further clarify the signals and the pathways involved in the hippocampal response to fluoxetine, which may help refine therapeutic strategies to alleviate depressive disorders.

Overstimulation of PrPC signaling pathways by prion peptide 106-126 causes oxidative injury of bioaminergic neuronal cells

Transmissible spongiform encephalopathies, also called prion diseases, are characterized by neuronal loss linked to the accumulation of PrPSc, a pathologic variant of the cellular prion protein (PrPC). Although the molecular and cellular bases of PrPSc-induced neuropathogenesis are not yet fully understood, increasing evidence supports the view that PrPSc accumulation interferes with PrPC normal function(s) in neurons. In the present work, we exploit the properties of PrP-(106-126), a synthetic peptide encompassing residues 106-126 of PrP, to investigate into the mechanisms sustaining prion-associated neuronal damage. This peptide shares many physicochemical properties with PrPSc and is neurotoxic in vitro and in vivo. We examined the impact of PrP-(106-126) exposure on 1C11 neuroepithelial cells, their neuronal progenies, and GT1-7 hypothalamic cells. This peptide triggers reactive oxygen species overflow, mitogen-activated protein kinase (ERK1/2), and SAPK (p38 and JNK1/2) sustained activation, and apoptotic signals in 1C11-derived serotonergic and noradrenergic neuronal cells, while having no effect on 1C11 precursor and GT1-7 cells. The neurotoxic action of PrP-(106-126) relies on cell surface expression of PrPC, recruitment of a PrPC-Caveolin-Fyn signaling platform, and overstimulation of NADPH-oxidase activity. Altogether, these findings provide actual evidence that PrP-(106-126)-induced neuronal injury is caused by an amplification of PrPC-associated signaling responses, which notably promotes oxidative stress conditions. Distorsion of PrPC signaling in neuronal cells could hence represent a causal event in transmissible spongiform encephalopathy pathogenesis.

Neuritogenesis: the prion protein controls β1 integrin signaling activity

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 (PrPC) impairs the initial sprouting of neurites upon induction of differentiation of the 1C11 neuroectodermal cell line, indicating that PrPC is necessary to neuritogenesis. Such PrPC 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 PrPC 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 PrPC and to restore neurite sprouting. We also observe an increased secretion of fibronectin in the surrounding milieu of PrPC‐depleted 1C11 cells, which likely self‐sustains β1 integrin signaling overactivation and contributes to neuritogenesis defect. Our overall data reveal that PrPC contributes to the acquisition of neuronal polarization by modulating β1 integrin activity, cell interaction with fibronectin, and cytoskeleton dynamics.—Loubet, D., Dakowski, C., Pietri, M., Pradines, E., Bernard, S., Callebert, J., Ardila‐Osorio, H., Mouillet‐Richard, S., Launay, J. M., Kellermann, O., Schneider, B. Neuritogenesis: the prion protein controls β1 integrin signaling activity. FASEB J. 26, 678–690 (2012). www.fasebj.org

PDK1 decreases TACE-mediated α-secretase activity and promotes disease progression in prion and Alzheimer's diseases

α-secretase–mediated cleavage of amyloid precursor protein (APP) precludes formation of neurotoxic amyloid-β (Aβ) peptides, and α-cleavage of cellular prion protein (PrPC) prevents its conversion into misfolded, pathogenic prions (PrPSc). The mechanisms leading to decreased α-secretase activity in Alzheimers and prion disease remain unclear. Here, we find that tumor necrosis factor-α–converting enzyme (TACE)-mediated α-secretase activity is impaired at the surface of neurons infected with PrPSc or isolated from APP-transgenic mice with amyloid pathology. 3-phosphoinositide–dependent kinase-1 (PDK1) activity is increased in neurons infected with prions or affected by Aβ deposition and in the brains of individuals with Alzheimers disease. PDK1 induces phosphorylation and caveolin-1–mediated internalization of TACE. This dysregulation of TACE increases PrPSc and Aβ accumulation and reduces shedding of TNF-α receptor type 1 (TNFR1). Inhibition of PDK1 promotes localization of TACE to the plasma membrane, restores TACE-dependent α-secretase activity and cleavage of APP, PrPC and TNFR1, and attenuates PrPSc- and Aβ-induced neurotoxicity. In mice, inhibition or siRNA-mediated silencing of PDK1 extends survival and reduces motor impairment following PrPSc infection and in APP-transgenic mice reduces Alzheimers disease-like pathology and memory impairment.

Cellular Prion Protein Signaling in Serotonergic Neuronal Cells

Abstract:  The cellular prion protein PrPC is the normal counterpart of the scrapie prion protein PrPSc, 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 1C115‐HT serotonergic or 1C11NE noradrenergic neuronal cells, allowed us to ascribe a signaling function to PrPC. Antibody‐mediated ligation of PrPC 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 PrPC‐caveolin‐Fyn platform, located on neuritic extensions. In addition to its proper signaling activity, PrPC modulates the agonist‐induced response of the three serotonergic G protein–coupled receptors present on the 1C115‐HT differentiated cells. The impact of PrPC ligation on the receptor couplings depends on the receptor subtype and the pathway considered. The implementation of the PrPC‐caveolin complex again is mandatory for PrPC to exert its action on 5‐HT receptor signaling. Our current data argue that PrPC 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, PrPC 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.

Reactive oxygen species-dependent TNF-α converting enzyme activation through stimulation of 5-HT2B and α1D autoreceptors in neuronal cells

A major determinant of neuronal homeostasis is the proper integration of cell signaling pathways recruited by a variety of neuronal and non‐neuronal factors. By taking advantage of a neuroectodermal cell line (1C11) endowed with the capacity to differentiate into serotonergic (1C115‐HT) or noradrenergic (1C11NE) neurons, we identified serotonin (5‐hydroxytryptamine, 5‐HT)‐ and norepinephrine (NE)‐dependent signaling cascades possibly involved in neuronal functions. First, we establish that 5‐HT2B receptors and α1D adrenoceptors are functionally coupled to reactive oxygen species (ROS) synthesis through NADPH oxidase activation in 1C115‐HT and 1C11NE cells. This observation constitutes the prime evidence that bioaminergic autoreceptors take part in the control of the cellular redox equilibrium in a neuronal context. Second, our data identify TACE (TNF‐α Converting Enzyme), a member of a disintegrin and metalloproteinase (ADAM) family, as a downstream target of the 5‐HT2B and α1D receptor‐NADPH oxidase signaling pathways. Upon 5‐HT2B or α1D receptor stimulation, ROS fully govern TNF‐α shedding in the surrounding milieu of 1C115HT or 1C11NE cells. Third, 5‐HT2B and α1D receptor couplings to the NADPH oxidase‐TACE cascade are strictly restricted to 1C11‐derived progenies that have implemented a complete serotonergic or noradrenergic phenotype. Overall, these observations suggest that 5‐HT2B and α1D autoreceptors may play a role in the maintenance of neuron‐ and neurotransmitter‐associated functions. Eventually, our study may have implications regarding the origin of oxidative stress as well as up‐regulated expression of proinflammatory cytokines in neurodegenerative disorders, which may relate to the deviation of normal signaling pathways. Pietri, M., Schneider, B., Mouillet‐Richard, S., Ermonval, M., Mutel, V., Launay, J.‐M., Kellermann, O. Reactive oxygen species‐dependent TNF‐α converting enzyme activation through stimulation of 5‐HT2B and α1D autoreceptors in neuronal cells. FASEB J. 19, 1078–1087 (2005)

The Cellular Prion Protein Interacts with the Tissue Non-Specific Alkaline Phosphatase in Membrane Microdomains of Bioaminergic Neuronal Cells

Background The 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 Findings The 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/Significance The 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.

Understanding the neurospecificity of Prion protein signaling.

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.

Double-Edge Sword of Sustained ROCK Activation in Prion Diseases through Neuritogenesis Defects and Prion Accumulation

In prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrPSc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrPSc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrPSc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrPC by TACE α-secretase, which physiologically precludes PrPSc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc. In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrPSc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases.