Benoit Schneider

miR-16 targets the serotonin transporter: a new facet for adaptive responses to antidepressants.

MicroRNA-16 and Depression Signal transmission between neurons is effected by neurotransmitters such as serotonin. Membrane-bound transporters remove excess neurotransmitters. Disruption of the delicate balance between neurotransmitter release and removal can lead to larger disruptions in neuronal circuitry. Depression and anxiety may be linked to dysfunction of some of these circuits. Uptake inhibitors can be used to treat depression, but the molecular pathways affected have been unclear. Baudry et al. (p. 1537) now show that microRNA-16 controls synthesis of the serotonin transporter and that the amount of microRNA-16 can be controlled by the same uptake inhibitors used to treat depression. The uptake transporter for a key neurotransmitter is regulated by a microRNA, yielding new insight into how Prozac functions. The serotonin transporter (SERT) ensures the recapture of serotonin and is the pharmacological target of selective serotonin reuptake inhibitor (SSRI) antidepressants. We show that SERT is a target of microRNA-16 (miR-16). miR-16 is expressed at higher levels in noradrenergic than in serotonergic cells; its reduction in noradrenergic neurons causes de novo SERT expression. In mice, chronic treatment with the SSRI fluoxetine (Prozac) increases miR-16 levels in serotonergic raphe nuclei, which reduces SERT expression. Further, raphe exposed to fluoxetine release the neurotrophic factor S100β, which acts on noradrenergic cells of the locus coeruleus. By decreasing miR-16, S100β turns on the expression of serotonergic functions in noradrenergic neurons. Based on pharmacological and behavioral data, we propose that miR-16 contributes to the therapeutic action of SSRI antidepressants in monoaminergic neurons.

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.

Serotonin transport and serotonin transporter-mediated antidepressant recognition are controlled by 5-HT2B receptor signaling in serotonergic neuronal cells

The plasma membrane 5‐HT transporter (SERT) is the major protagonist in regulating extracellular 5‐HT concentration and constitutes the target of drugs used to treat a host of metabolic and psychiatric disorders. The exact mechanisms sustaining SERT function still remain elusive. The present work exploits the properties of the 1C11 neuroectodermal progenitor, which acquires, upon 4 days of differentiation, a functional SERT within an integrated serotonergic phenotype to investigate regulatory mechanisms involved in SERT onset and functions. We show that poly(A) addition precedes SERT mRNA translation on day 2 of the serotonergic program. The newly translated transporter molecules immediately bind cocaine. Day 4 must be awaited to monitor antidepressant recognition and 5‐HT uptake. Because external 5‐HT reduces both 5‐HT transport and SERT antidepressant binding, we identify 5‐HT2B receptors as key players in controlling the overall 5‐HT transport system. In the absence of external 5‐HT, 5‐HT2B receptor coupling to NO production ensures SERT phosphorylation to basal level and maximal 5‐HT uptake. In the presence of 5‐HT, the 5‐HT2B receptor‐PKC coupling promotes additional phosphorylations of both SERT and Na+,K+‐ATPase α‐subunit, impairing the electrochemical gradient necessary to 5‐HT uptake. SERT hyperphosphorylation also affects antidepressant recognition. Finally, such 5‐HT2B receptor‐mediated control of SERT activity operates in primary neurons from raphe nuclei. Altogether, our data shed new light on the 5‐HT‐driven post‐translational modifications involved in the control of SERT activity.—Launay, J‐M., Schneider, B., Loric, S., Da Prada, M., Kellermann, O. Serotonin transport and serotonin transporter‐mediated antidepressant recognition are controlled by 5‐HT2B receptor signaling in serotonergic neuronal cells. FASEB J. 20, 1843–1854 (2006)

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.

Structural basis for activation of α-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase

AIDS chemotherapy is limited by inadequate intracellular concentrations of the active triphosphate form of nucleoside analogues, leading to incomplete inhibition of viral replication and the appearance of drug‐resistant virus. Drug activation by nucleoside diphosphate kinase and inhibition of HIV‐1 reverse transcriptase were studied comparatively. We synthesized analogues with a borano (BH3−) group on the α‐phosphate, and found that they are substrates for both enzymes. X‐ray structures of complexes with nucleotide diphosphate kinase provided a structural basis for their activation. The complex with d4T triphosphate displayed an intramolecular CH…O bond contributing to catalysis, and the Rp diastereoisomer of thymidine α‐boranotriphosphate bound like a normal substrate. Using α‐(Rp)‐boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the α‐borano group improved both phosphorylation by nucleotide diphosphate kinase and inhibition of reverse transcription. Moreover, repair of blocked DNA chains by pyrophosphorolysis was reduced significantly in variant reverse transcriptases bearing substitutions found in drug‐resistant viruses. Thus, the α‐borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance.

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.

3′-Phosphorylated Nucleotides Are Tight Binding Inhibitors of Nucleoside Diphosphate Kinase Activity

Nucleoside diphosphate (NDP) kinase catalyzes the phosphorylation of ribo- and deoxyribonucleosides diphosphates into triphosphates. NDP kinase is also involved in malignant tumors and was shown to activate in vitro transcription of the c-myc oncogene by binding to its NHE sequence. The structure of the complex of NDP kinase with bound ADP shows that the nucleotide adopts a different conformation from that observed in other phosphokinases with an internal H bond between the 3′-OH and the β-O made free by the phosphate transfer. We use intrinsic protein fluorescence to investigate the inhibitory and binding potential of nucleotide analogues phosphorylated in 3′-OH position of the ribose to both wild type and F64W mutant NDP kinase from Dictyostelium discoideum. Due to their 3′-phosphate, 5′-phosphoadenosine 3′-phosphate (PAP) and adenosine 3′-phosphate 5′-phosphosulfate (PAPS) can be regarded as structural analogues of enzyme-bound ADP. TheK D of PAPS (10 μm) is three times lower than the K D of ADP. PAPS also acts as a competitive inhibitor toward natural substrates during catalysis, with a K I in agreement with binding data. The crystal structure of the binary complex between Dictyostelium NDP kinase and PAPS was solved at 2.8-Å resolution. It shows a new mode of nucleotide binding at the active site with the 3′-phosphate of PAPS located near the catalytic histidine, at the same position as the γ-phosphate in the transition state. The sulfate group is directed toward the protein surface. PAPS will be useful for the design of high affinity drugs targeted to NDP kinases.

Production of reactive oxygen species is turned on and rapidly shut down in epithelial cells infected with Chlamydia trachomatis

ABSTRACT Reactive oxygen species (ROS) are many-faceted compounds involved in cell defense against pathogens, as well as in cell signaling. Their involvement in the response to infection in epithelial cells remains poorly documented. Here, we investigated the production of ROS during infection with Chlamydia trachomatis, a strict intracellular pathogen, in HeLa cells. C. trachomatis induced a transient increase in the ROS level within a few hours, followed by a return to basal level 9 hours after infection. At this time point, the host enzyme dedicated to ROS production, NADPH oxidase, could no longer be activated by external stimuli, such as interleukin-1β. In addition, Rac, a regulatory subunit of the NADPH oxidase complex, was relocated to the membrane of the compartment in which the bacteria develop, the inclusion, while other subunits were not. Altogether, these results indicate that C. trachomatis infection elicits the production of ROS and that the bacteria rapidly target the activity of NADPH oxidase to shut it down. Prevention of ROS production at the onset of the bacterial developmental cycle might delay the host response to infection.