Myriam Ermonval

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.

Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein

Dengue virus (DENV) causes the major arboviral disease of the tropics, characterized in its severe forms by signs of hemorrhage and plasma leakage. DENV encodes a nonstructural glycoprotein, NS1, that associates with intracellular membranes and the cell surface. NS1 is eventually secreted as a soluble hexamer from DENV-infected cells and circulates in the bloodstream of infected patients. Extracellular NS1 has been shown to modulate the complement system and to enhance DENV infection, yet its structure and function remain essentially unknown. By combining cryoelectron microscopy analysis with a characterization of NS1 amphipathic properties, we show that the secreted NS1 hexamer forms a lipoprotein particle with an open-barrel protein shell and a prominent central channel rich in lipids. Biochemical and NMR analyses of the NS1 lipid cargo reveal the presence of triglycerides, bound at an equimolar ratio to the NS1 protomer, as well as cholesteryl esters and phospholipids, a composition evocative of the plasma lipoproteins involved in vascular homeostasis. This study suggests that DENV NS1, by mimicking or hijacking lipid metabolic pathways, contributes to endothelium dysfunction, a key feature of severe dengue disease.

Identification of a cellular ligand for the natural cytotoxicity receptor NKp44

With an array of activating and inhibitory receptors, natural killer (NK) cells are involved in the eradication of infected, transformed, and tumor cells. NKp44 is a member of the natural cytotoxicity receptor family, which is exclusively expressed on activated NK cells. Here, we identify natural cytotoxicity receptor NKp44 (NKp44L), a novel isoform of the mixed-lineage leukemia-5 protein, as a cellular ligand for NKp44. Unlike the other MLL family members, NKp44L is excluded from the nucleus, but expressed at the cell-surface level; its subcellular localization is being associated with the presence of a specific C-terminal motif. Strikingly, NKp44L has not been detected on circulating cells isolated from healthy individuals, but it is expressed on a large panel of the tumor and transformed cells. The sharply decreased NK lysis activity induced by anti-NKp44L antibodies directly demonstrates the role of NKp44L in cytotoxicity. Taken together, these results show that NKp44L could be critical for NK cell-mediated innate immunity. The identification and cellular distribution of NKp44L highlight the role of this self-molecule as a danger signal to alert the NK cell network.

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.

The Effects of Culture Conditions on the Glycosylation of Secreted Human Placental Alkaline Phosphatase Produced in Chinese Hamster Ovary Cells

The effects of different culture conditions, suspension and microcarrier culture and temperature reduction on the structures of N‐linked glycans attached to secreted human placental alkaline phosphatase (SEAP) were investigated for CHO cells grown in a controlled bioreactor. Both mass spectrometry and anion‐exchange chromatography were used to probe the N‐linked glycan structures and distribution. Complex‐type glycans were the dominant structures with small amounts of high mannose glycans observed in suspension and reduced temperature cultures. Biantennary glycans were the most common structures detected by mass spectrometry, but triantennary and tetraantennary forms were also detected. The amount of sialic acid present was relatively low, approximately 0.4 mol sialic acid/mol SEAP for suspension cultures. Microcarrier cultures exhibited a decrease in productivity compared with suspension culture due to a decrease in both maximum viable cell density (15–20%) and specific productivity (30–50%). In contrast, a biphasic suspension culture in which the temperature was reduced at the beginning of the stationary phase from 37 to 33°C, showed a 7% increase in maximum viable cell density, a 62% increase in integrated viable cell density, and a 133% increase in specific productivity, leading to greater than threefold increase in total productivity. Both microcarrier and reduced temperature cultures showed increased sialylation and decreased fucosylation when compared to suspension culture. Our results highlight the importance of glycoform analysis after process modification as even subtle changes (e.g., changing from one microcarrier to another) may affect glycan distributions. Biotechnol. Bioeng. 2008;100: 1178–1192.

Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I

Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI(332), which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive alpha1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI(332) in the ERAD pathway. In a first step, the Man(8) isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI(332) intermediates. Some other trimmed N-glycan species, in particular Glc(1)Man(7)GlcNAc(2), were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI(332) accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation.

Evolving views in prion glycosylation: functional and pathological implications

Prion diseases form a group of neurodegenerative disorders with the unique feature of being transmissible. These diseases involve a pathogenic protein, called PrP(Sc) for the scrapie isoform of the cellular prion protein (PrP(C)) which is an abnormally-folded counterpart of PrP(C). Many questions remain unresolved concerning the function of PrP(C) and the mechanisms underlying prion replication, transmission and neurodegeneration. PrP(C) is a glycosyl-phosphatidylinositol-anchored glycoprotein expressed at the cell surface of neurons and other cell types. PrP(C) may be present as distinct isoforms depending on proteolytic processing (full length and truncated), topology(GPI-anchored, transmembrane or soluble) and glycosylation (non- mono- and di-glycosylated). The present review focuses on the implications of PrP(C) glycosylation as to the function of the normal protein, the cellular pathways of conversion into PrP(Sc), the diversity of prion strains and the related selective neuronal targeting.

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)

Differential expression of the bone and the liver tissue non-specific alkaline phosphatase isoforms in brain tissues

The enzyme tissue non-specific alkaline phosphatase (TNAP) belongs to the ectophosphatase family. It is present in large amounts in bone in which it plays a role in mineralization but little is known about its function in other tissues. Arguments are accumulating for its involvement in the brain, in particular in view of the neurological symptoms accompanying human TNAP deficiencies. We have previously shown, by histochemistry, alkaline phosphatase (AP) activity in monkey brain vessels and parenchyma in which AP exhibits specific patterns. Here, we clearly attribute this activity to TNAP expression rather than to other APs in primates (human and marmoset) and in rodents (rat and mouse). We have not found any brain-specific transcripts but our data demonstrate that neuronal and endothelial cells exclusively express the bone TNAP transcript in all species tested, except in mouse neurons in which liver TNAP transcripts have also been detected. Moreover, we highlight the developmental regulation of TNAP expression; this also acts during neuronal differentiation. Our study should help to characterize the regulation of the expression of this ectophosphatase in various cell types of the central nervous system.