Search

International audience; No abstract available

Background Epidemiological emerging evidence shows that human exposure to some nanosized materials present in the environment would contribute to the onset and/or progression of Alzheimer’s disease (AD). The cellular and molecular mechanisms whereby nanoparticles would exert some adverse effects towards neurons and take part in AD pathology are nevertheless unknown. Results Here, we provide the prime evidence that titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) bind the cellular form of the prion protein (PrPC), a plasma membrane protein well known for its implication in prion diseases and prion-like diseases, such as AD. The interaction between TiO2- or CB-NPs and PrPC at the surface of neuronal cells grown in culture corrupts PrPC signaling function. This triggers PrPC-dependent activation of NADPH oxidase and subsequent production of reactive oxygen species (ROS) that alters redox equilibrium. Through PrPC interaction, NPs also promote the activation of 3-phosphoinositide-dependent kinase 1 (PDK1), which in turn provokes the internalization of the neuroprotective TACE α-secretase. This diverts TACE cleavage activity away from (i) TNFα receptors (TNFR), whose accumulation at the plasma membrane augments the vulnerability of NP-exposed neuronal cells to TNFα -associated inflammation, and (ii) the amyloid precursor protein APP, leading to overproduction of neurotoxic amyloid Aβ40/42 peptides. The silencing of PrPC or the pharmacological inhibition of PDK1 protects neuronal cells from TiO2- and CB-NPs effects regarding ROS production, TNFα hypersensitivity, and Aβ rise. Finally, we show that dysregulation of the PrPC-PDK1-TACE pathway likely occurs in the brain of mice injected with TiO2-NPs by the intra-cerebro-ventricular route as we monitor a rise of TNFR at the cell surface of several groups of neurons located in distinct brain areas. Conclusion Our in vitro and in vivo study thus posits for the first time normal cellular prion protein PrPC as being a neuronal receptor of TiO2- and CB-NPs and identifies PrPC-coupled signaling pathways by which those nanoparticles alter redox equilibrium, augment the intrinsic sensitivity of neurons to neuroinflammation, and provoke a rise of Aβ peptides. By identifying signaling cascades dysregulated by TiO2- and CB-NPs in neurons, our data shed light on how human exposure to some NPs might be related to AD.

The serotonergic 2B receptor (5-HT2BR), expressed in the central nervous system and at the periphery, contributes to the regulation of several physiological processes such as cardio-vascular, pulmonary, bone and neuronal functions. Due to its multifaceted roles, signaling pathways and cell targets controlled by the 5-HT2BR are still the subject of intensive research. Here, we review how exploiting the dynamics of differentiation of the 1C11 neuronal stem cell line allows to identify several 5-HT2BR-coupled signaling pathways and targets, among which the serotonin transporter, involved in the homeostasis of serotonergic neurons and their adaptive response to selective serotonin-reuptake inhibitors (SSRIs) like fluoxetine (Prozac®). In addition, mesoblastic stem cell lines permit to show that along the osteogenic differentiation, the 5-HT2BR controls the activity of the tissue-non-specific alkaline phosphatase, a major enzyme for bone formation. Finally, the 5-HT2BR expressed by pulpal stem cells takes part to natural tooth repair upon 5-HT release by injury-activated platelets.

The presence of amyloid beta (Aβ) plaques in the brain of some individuals with Creutzfeldt-Jakob or Gertsmann-Straussler-Scheinker diseases suggests that pathogenic prions (PrPSc) would have stimulated the production and deposition of Aβ peptides. We here show in prion-infected neurons and mice that deregulation of the PDK1-TACE α-secretase pathway reduces the Amyloid Precursor Protein (APP) α-cleavage in favor of APP β-processing, leading to Aβ40/42 accumulation. Aβ predominates as monomers, but is also found as trimers and tetramers. Prion-induced Aβ peptides do not affect prion replication and infectivity, but display seedable properties as they can deposit in the mouse brain only when seeds of Aβ trimers are co-transmitted with PrPSc. Importantly, brain Aβ deposition accelerates death of prion-infected mice. Our data stress that PrPSc, through deregulation of the PDK1-TACE-APP pathway, provokes the accumulation of Aβ, a prerequisite for the onset of an Aβ seeds-induced Aβ pathology within a prion-infectious context.

In prion diseases, the brain lesion profile is influenced by the prion "strain" properties, the invasion route to the brain, and still unknown host cell-specific parameters. To gain insight into those endogenous factors, we analyzed the histopathological alterations induced by distinct prion strains in the mouse cerebellum. We show that 22L and ME7 scrapie prion proteins (PrP22L , PrPME7 ), but not bovine spongiform encephalopathy PrP6PB1 , accumulate in a reproducible parasagittal banding pattern in the cerebellar cortex of infected mice. Such banding pattern of PrP22L aggregation did not depend on the neuroinvasion route, but coincided with the parasagittal compartmentation of the cerebellum mostly defined by the expression of zebrins, such as aldolase C and the excitatory amino acid transporter 4, in Purkinje cells. We provide evidence that Purkinje cells display a differential, subtype-specific vulnerability to 22L prions with zebrin-expressing Purkinje cells being more resistant to prion toxicity, while in stripes where PrP22L accumulated most zebrin-deficient Purkinje cells are lost and spongiosis accentuated. In addition, in PrP22L stripes, enhanced reactive astrocyte processes associated with microglia activation support interdependent events between the topographic pattern of Purkinje cell death, reactive gliosis and PrP22L accumulation. Finally, we find that in preclinically-ill mice prion infection promotes at the membrane of astrocytes enveloping Purkinje cell excitatory synapses, upregulation of tumor necrosis factor-α receptor type 1 (TNFR1), a key mediator of the neuroinflammation process. These overall data show that Purkinje cell sensitivity to prion insult is locally restricted by the parasagittal compartmentation of the cerebellum, and that perisynaptic astrocytes may contribute to prion pathogenesis through prion-induced TNFR1 upregulation.

Genetic and pharmacological studies provided evidence that serotonin (5-HT) is an important signaling molecule for the development and the maintenance of mineralized tissues. However, how 5-HT takes part to the homeostasis of teeth and bone remains elusive. In the dental field, a major breakthrough comes from the identification of 5-HT but also dopamine (DA) as "damage" signals necessary for stem cell-based tooth repair. Pulpal stem cells express the overall functions of 5-HT and DA neurons including a definite set of functional 5-HT/DA receptors that render cells responsive for circulating bioamines. Upon tooth injury, activated platelets release bulks of 5-HT/DA that mobilize pulpal stem cells for natural dental repair. The contribution of 5-HT to bone metabolism is more documented with description of both anabolic and resorptive effects. By controlling the tissue-non specific alkaline phosphatase (TNAP), 5-HT2B receptors exert an anabolic function and a pivotal role in mineralization processes. Increasing our understanding of the role of 5-HT receptors in bone metabolism may pave the road for the development of therapeutic strategies towards skeletal-associated pathologies and ectopic calcification.

The presence of amyloid beta (Aβ) plaques in the brain of some individuals with Creutzfeldt-Jakob or Gertsmann-Straussler-Scheinker diseases suggests that pathogenic prions (PrPSc) would have stimulated the production and deposition of Aβ peptides. We here show in prion-infected neurons and mice that deregulation of the PDK1-TACE α-secretase pathway reduces the Amyloid Precursor Protein (APP) α-cleavage in favor of APP β-processing, leading to Aβ40/42 accumulation. Aβ predominates as monomers, but is also found as trimers and tetramers. Prion-induced Aβ peptides do not affect prion replication and infectivity, but display seedable properties as they can deposit in the mouse brain only when seeds of Aβ trimers are co-transmitted with PrPSc. Importantly, brain Aβ deposition accelerates death of prion-infected mice. Our data stress that PrPSc, through deregulation of the PDK1-TACE-APP pathway, provokes the accumulation of Aβ, a prerequisite for the onset of an Aβ seeds-induced Aβ pathology within a prion-infectious context., Aβ plaques have been detected in brains of patients with prion diseases. Here, using mice, the authors show that prion infection enhances Aβ production via a PDK1-TACE mechanism and that brain deposition of Aβ induced by Aβ seeds co-transmitted with PrPSc contributes to mortality in prion disease.

In current dental practices, traditional restorative approaches may have relatively limited long-term survival and may be associated to diverse complications, such as allergy, pulpitis, or periodontal pathologies. To overcome these shortcomings, novel innovative strategies have been envisioned for tooth repair. During the two last decades, the extensive advances in our understanding of tooth development as well as stem cell research provide the foundation for exciting opportunities in dental tissue engineering. The replacement of lost teeth by engineered dental tissue appears as a fascinating goal. However, the feasibility remains an intriguing question. Is the challenge to create a new tooth acting as a substitute for lost tooth or to regenerate only part of this organ that is enamel, dentin, or dental pulp? Is it possible to exploit stem cells for transplantation purposes to promote matrix formation and mineralization in the framework of endodontic treatment? Finally, investigating the functional properties of pulpal stem cells is however mandatory to envision novel therapeutic dental strategies. In this review, we summarize the current knowledge of stem cells used for dental tissue engineering and discuss the ensuing challenges for regenerative dentistry.

Mineralization is a process of deposition of calcium phosphate crystals within a fibrous extracellular matrix (ECM). In mineralizing tissues, such as dentin, bone and hypertrophic cartilage, this process is initiated by a specific population of extracellular vesicles (EV), called matrix vesicles (MV). Although it has been proposed that MV are formed by shedding of the plasma membrane, the cellular and molecular mechanisms regulating formation of mineralization-competent MV are not fully elucidated. In these studies, 17IIA11, ST2, and MC3T3-E1 osteogenic cell lines were used to determine how formation of MV is regulated during initiation of the mineralization process. In addition, the molecular composition of MV secreted by 17IIA11 cells and exosomes from blood and B16-F10 melanoma cell line was compared to identify the molecular characteristics distinguishing MV from other EV. Western blot analyses demonstrated that MV released from 17IIA11 cells are characterized by high levels of proteins engaged in calcium and phosphate regulation, but do not express the exosomal markers CD81 and HSP70. Furthermore, we uncovered that the molecular composition of MV released by 17IIA11 cells changes upon exposure to the classical inducers of osteogenic differentiation, namely ascorbic acid and phosphate. Specifically, lysosomal proteins Lamp1 and Lamp2a were only detected in MV secreted by cells stimulated with osteogenic factors. Quantitative nanoparticle tracking analyses of MV secreted by osteogenic cells determined that standard osteogenic factors stimulate MV secretion and that phosphate is the main driver of their secretion. On the molecular level, phosphate-induced MV secretion is mediated through activation of extracellular signal-regulated kinases Erk1/2 and is accompanied by re-organization of filamentous actin. In summary, we determined that mineralization-competent MV are distinct from exosomes, and we identified a new role of phosphate in the process of ECM mineralization. These data provide novel insights into the mechanisms of MV formation during initiation of the mineralization process.

International audience; Serotonin transporter, SERT (SLC64A for solute carrier family 6, member A4), is a twelve transmembrane domain (TMDs) protein that assumes the uptake of serotonin (5-HT) through dissipation of the Na+ gradient established by the electrogenic pump Na/K ATPase. Abnormalities in 5-HT level and signaling have been associated with various disorders of the central nervous system (CNS) such as depression, obsessive-compulsive disorder, anxiety disorders, and autism spectrum disorder. Since the 50s, SERT has raised a lot of interest as being the target of a class of antidepressants, the Serotonin Selective Reuptake Inhibitors (SSRIs), used in clinics to combat depressive states. Because of the refractoriness of two-third of patients to SSRI treatment, a better understanding of the mechanisms regulating SERT functions is of priority. Here, we review how genetic and epigenetic regulations, post-translational modifications of SERT, and specific interactions between SERT and a set of diverse partners influence SERT expression, trafficking to and away from the plasma membrane and activity, in connection with the neuronal adaptive cell response to SSRI antidepressants.

Although cellular prion protein PrPC is well known for its implication in Transmissible Spongiform Encephalopathies, its functions remain elusive. Combining in vitro and in vivo approaches, we here show that PrPC displays the intrinsic capacity to protect neuronal cells from a pro-inflammatory TNFα noxious insult. Mechanistically, PrPC coupling to the NADPH oxidase-TACE α-secretase signaling pathway promotes TACE-mediated cleavage of transmembrane TNFα receptors (TNFRs) and the release of soluble TNFR, which limits the sensitivity of recipient cells to TNFα. We further show that PrPC expression is necessary for TACE α-secretase to stay at the plasma membrane in an active state for TNFR shedding. Such PrPC control of TACE localization depends on PrPC modulation of β1 integrin signaling and downstream activation of ROCK-I and PDK1 kinases. Loss of PrPC provokes TACE internalization, which in turn cancels TACE-mediated cleavage of TNFR and renders PrPC-depleted neuronal cells as well as PrPC knockout mice highly vulnerable to pro-inflammatory TNFα insult. Our work provides the prime evidence that in an inflammatory context PrPC adjusts the response of neuronal cells targeted by TNFα through TACE α-secretase. Our data also support the view that abnormal TACE trafficking and activity in prion diseases originate from a-loss-of-PrPC cytoprotective function.

International audience; With an estimated life-time prevalence of 15 to 17% and an incapacitating illness in 50% of cases, depressive spectrum disorders represent a heavy public health burden. Despite considerable efforts to underpin the molecular and cellular changes associated with depressive states, a global understanding of the pathophysiology of major depressive disorders (MDD) is still lacking. It is now acknowledged that deficits in synaptic plasticity, such as those resulting from chronic stress, can set the stage for the onset of depression. As a corollary, antidepressants balance neurotransmitter systems and help restore neuronal activity. In recent years, microRNAs have emerged as key protagonists in numerous physiopathological conditions including CNS function and disease. This review summarizes the current evidence for an involvement of microRNAs in the pathophysiology of depression and their contribution to the action of antidepressants.

The role of the serotonin 2B receptor (5-HT(2B) R) in enamel formation and mineralization was explored in adult 5HT(2B) R knockout (KO) mice compared with wild-type (WT) mice. In the molar, quantitative data obtained by micro-computed tomography imaging showed that the overall volume of the enamel layer was firmly reduced in KO mice. Defective mineralization was ascertained by energy-dispersive X-ray microanalysis. We also observed, using scanning electron microscopy, that parazones in the KO mice included two or three helicoidally twisted rods within Hunter-Schreger bands, instead of a single rod, as found in the WT mice. Minor disturbances were also detected in the incisors of KO mice. Structural modifications, thinner enamel crystallites, and porosities observed in KO mice indicate that the 5-HT(2B) R-mediated signaling pathways as part of the enamel formation process. These data provide a basis for evaluating the role of 5-HT(2B) R in ameloblast functions. Defects observed in the mineralization and structure of enamel in KO mice highlight that the 5-HT(2B) R interferes with the mechanisms directing amelogenesis.

Chantier qualité GA; A striking feature of the cellular prion protein (PrPC) is the heterogeneity of its glycoforms, whose contribution to PrPC function has yet to be defined. Using the 1C11 neuronal bioaminergic differentiation model and a glycomics approach, we show here a correlation between differential PrPC N-glycosylations in 1C115-HT serotonergic and 1C11NE noradrenergic cells compared to their 1C11 precursor cells and a variation of the glycogenome expression status in these cells. In particular, expression of genes involved in N-glycan synthesis or in the modeling of chondroitin and heparan sulfate proteoglycans appeared to be modulated. Our results highlight that, the expression of glycosylation-related genes is regulated during bioaminergic neuronal differentiation, consistent with a participation of glycoconjugates in neuronal development and plasticity. A neuronal regulation of glycosylation processes may have direct implications on some neurospecific functions of PrPC and may participate in specific brain targeting of prion strains.

The continuously growing rodent incisor is a widely used model to investigate odontogenesis and mineralized tissue formation. This study focused on evaluating the mouse mandibular incisor as an experimental biological tool for analyzing in vivo the capacity of odontoblast-like progenitors or bioactive molecules to contribute to reparative dentinogenesis. We describe here a surgical procedure allowing direct access to the forming part of the incisor dental pulp Amelogenin peptide A+4 adsorbed on agarose beads, or dental pulp progenitor cells were implanted in the pulp following this procedure. After 10 days A+4 induced the formation of an osteodentin occluding almost the totality of the pulp compartment. Implantation of progenitor cells leads to formation of islets of osteodentin-like structures located centrally in the pulp. These pilot studies validate the incisor as an experimental model to test the capacity of progenitor cells or bioactive molecules to induce the formation of reparative dentin.

To evaluate the biocompatibility and osteoinductive properties of Bioroot™ RCS (BR, Septodont, France) compared to Kerr's Pulp Canal Sealer™ (PCS, Kerr, Italy) using the mouse pulp-derived stem cell line A4, which have an osteo/odontogenic potential in vitro and contribute to efficient bone repair in vivo.A4 cells were cultured at the stem cell stage in the presence of solid disks of BR or PCS, whereas untreated A4 cells were used as control. After 3, 7, 10 days of direct contact with the sealers, cell viability was quantified using Trypan Blue exclusion assay. Immunolabelings were performed to assess the expression of odontoblast markers i.e. type 1 collagen, DMP1 or BSP. Finally, sealer-treated cells were induced toward osteo/odontogenic differentiation to assess the impact of the sealers on mineralization by Von Kossa staining. Statistical significance was evaluated by one-way analysis of variance and t-test (p0.05).BR did not alter the viability and morphology of A4 pulpal cells compared to control group (p0.05); however, living cell percentage of PCS was significantly lower compared to control and BR groups (p0.05). BR preserved the intrinsic ability of A4 cells to express type 1 collagen, DMP1 or BSP at the stem cell stage. It did not alter the integrity of collagen fibers surrounding the cells and promoted overexpression of BSP and DMP1 at the cell surface. In contrast to PCS, BR did not compromise the mineralization potential of pulpal A4 stem cells.Bioroot™ RCS was not as cytotoxic as PCS. It did not recruit the pulpal stem cells toward differentiation but preserve their osteo-odontogenic intrinsic properties. Bioroot™ RCS might provide more suitable environment to induce stem cells for hard tissue deposition.

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-HT(2B) receptors as key players in controlling the overall 5-HT transport system. In the absence of external 5-HT, 5-HT(2B) receptor coupling to NO production ensures SERT phosphorylation to basal level and maximal 5-HT uptake. In the presence of 5-HT, the 5-HT(2B) receptor-PKC coupling promotes additional phosphorylations of both SERT and Na(+),K(+)-ATPase alpha-subunit, impairing the electrochemical gradient necessary to 5-HT uptake. SERT hyperphosphorylation also affects antidepressant recognition. Finally, such 5-HT(2B) 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.

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 1D 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- Converting Enzyme), a member of a disintegrin and metalloproteinase (ADAM) family, as a downstream target of the 5-HT2B and 1D receptor-NADPH oxidase signaling pathways. Upon 5-HT2B or 1D receptor stimulation, ROS fully govern TNF- shedding in the surrounding milieu of 1C115-HT or 1C11NE cells. Third, 5-HT2B and 1Dreceptor 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 1D 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.

The inducible serotonergic 1C115-HT cell line expresses a defined set of serotonergic receptors of the 5-HT2B, 5-HT1B/D, and 5-HT2A subtypes, which sustain a regulation of serotonergic associated functions through G-protein-dependent signaling. 1C115-HT cells have been instrumental to assign a signaling function to the cellular prion protein PrPC. Here, we establish that antibody-mediated ligation of PrPC concomitant to agonist stimulation of 5-HT receptors modulates the couplings of all three serotonergic receptors present on 1C115-HT cells. Specific impacts of PrP antibodies were monitored depending on the receptor and pathway considered. PrPC ligation selectively cancels the 5-HT2A-PLC response, decreases the 5-HT1B/D negative coupling to adenylate cyclase, and potentiates the 5-HT2B-PLA2 coupling. As a result, PrPC ligation disturbs the functional interactions occurring between the signaling pathways of the three receptor subtypes. In 1C115-HT cells, antagonizing cross-talks arising from 5-HT2B and 5-HT2A receptors control the 5-HT1B/D function. PrPC ligation reinforces the negative regulation exerted by 5-HT2B on 5-HT1B/D receptors. On the other hand it abrogates the blocking action of 5-HT2A on the regulatory loop linking 5-HT1B/D receptors. We propose that the ligation of PrPC affects the potency or dynamics of G-protein activation by agonist-bound serotonergic receptors. Finally, the PrPC-dependent modulation of 5-HT receptor couplings is restricted to 1C115-HT cells expressing a complete serotonergic phenotype. It critically involves a PrPC-caveolin platform implemented on the neurites of 1C115-HT cells during differentiation. Our findings define PrPC as a modulator of 5-HT receptor coupling to G-proteins and thereby as a protagonist contributing to the homeostasis of serotonergic neurons. They provide a foundation for uncovering the impact of prion infection on serotonergic functions.

Osteoblasts and chondroblasts are derived from common mesenchymal progenitors. Although bone morphogenetic protein induces mesenchymal differentiation into both osteogenic and chodrogenic lineage cells in vitro, its inhibitor, Noggin, is expressed exclusively during chondrogenic but not osteogenic differentiation in an embryonal carcinoma-derived mesodermal cell line, C1. We hypothesized that Noggin may regulate cell differentiation in a lineage-specific manner. To test this hypothesis, Noggin was overexpressed using recombinant adenovirus (Ad/Noggin) in mesodermal C1 cells to examine whether Noggin specifically inhibits chondrogenic differentiation. Noggin overexpression by recombinant adenovirus infection reduced Sox9, patched, Ihh, and type II, X, and XI collagen mRNA expression levels in C1 cell aggregates that were induced to differentiate into chondrocyte lineage by culturing in differentiation medium. In contrast, Noggin overexpression did not affect osteogenic differentiation in C1 cells because osteoblast phenotypic markers such as osteocalcin and alkaline phosphatase mRNA levels were not altered. We further examined whether Noggin also differentially affects chondrogenesis and osteogenesis in limb development by using organ cultures of long bone. Ad/Noggin infection into 15.5 d post conception limb skeletal rudiments that were cultured on filter membrane in vitro or on the chorioallantoic membranes in ovo inhibited the levels of chondrogenesis, which were evaluated based on alcian blue staining. These results suggest that Noggin specifically blocks chondrogenic differentiation, rather than osteogenic differentiation, in mesodermal stem cell line C1 and skeletal cells.

Studying the apical Na/H exchanger NHE2 is difficult in the intact thick ascending limb (TAL) because of its weak expression and transport activity compared with the co-expressed NHE3. From a mouse transgenic for a recombinant plasmid adeno-SV(40) (PK4), we developed an immortalized TAL cell line, referred to as MKTAL, which selectively expresses NHE2 protein and activity. The immortalized cells retain the main properties of TAL cells. They have a stable homogeneous epithelial-like phenotype, express SV(40) T antigen and exhibit polarity with an apical domain bearing few microvilli and separated from lateral domains by typical epithelial-type junctional complexes expressing ZO1 protein. Tamm-Horsfall protein is present on the apical membrane. MKTAL cells express NHE2 and NHE1 proteins but not NHE3 and NHE4, whereby NHE2 protein is expressed selectively in the apical domain of the plasma membrane. NHE2 contributed about half of the total Na/H exchange activity. mRNAs for the Na-K-2Cl cotransporter-2 (NKCC2) and the anion exchangers AE2 and AE3 were also present. While acute exposure to NO donors did not alter NHE2 activity, chronic exposure inhibited NHE2 activity selectively and down-regulated NHE2 mRNA abundance. In conclusion, MKTAL cells retain structural and functional properties of their in vivo TAL counterparts and express functional NHE2 protein in the apical membrane, which may be inhibited by NO. Thus, MKTAL cells may be an appropriate model for studying the cellular mechanisms of NHE2 regulation.

Bacteriorhodopsin and rhodopsin crystal structures were used as templates to build structural models of the mouse and human serotonin (5-HT)-2B receptors (5-HT(2B)Rs). Serotonin was docked to the receptors, and the amino acids predicted to participate to its binding were subjected to mutagenesis. 5-HT binding affinity and 5-HT-induced inositol triphosphate production were measured in LMTK(-) cells transfected with either wild-type or mutated receptor genes. According to these measurements, the bacteriorhodopsin-based models of the 5-HT(2B)Rs appear more confident than the rhodopsin-based ones. Residues belonging to the transmembrane domains 3 and 6, i.e. Asp(3.32), Ser(3.36), Phe(6.52), and Asn(6.55), make direct contacts with 5-HT. In addition, Trp(3.28), Phe(3.35), Phe(6.52), and Phe(7.38) form an aromatic box surrounding 5-HT. The specificity of human and mouse 5-HT(2B)Rs may be reflected by different rearrangements of the aromatic network upon 5-HT binding. Two amino acids close to Pro(5.50) in the human transmembrane domain 5 sequence were permuted to introduce a "mouse-like" sequence. This change was enough to confer the human 5-HT(2B)R properties similar to those of the mouse. Taken together, the computed models and the site-directed mutagenesis experiments give a structural explanation to (i) the different 5-HT pK(D) values measured with the human and mouse 5-HT(2B)Rs (7.9 and 5.8, respectively) and (ii) the specificity of 5-HT binding to 5-HT(2B)Rs as compared with other serotonergic G-protein coupled receptors.

Cell cultures represent versatile and useful experimental models of transmissible spongiform encephalopathies. These models include chronically prion infected cell lines, as well as cultures expressing variable amounts of wild-type, mutated or chimeric prion proteins. These cultures have been widely used to investigate the biology of both the normal and the pathological isoform of the prion protein. They have also contributed to the comprehension of the pathogenic processes occurring in transmissible spongiform encephalopathies and in the development of new therapeutic approaches of these diseases. To cite this article: S. Lehmann et al., C. R. Biologies, 325 (2002) 59–65.

A strategy based upon the introduction of an adenovirus-SV40 plasmid into multipotential cells was designed to immortalize clones displaying properties of lineage stem cells. The murine 1C11 cell line behaves as a neuroepithelial progenitor. Upon appropriate induction, almost 100% of 1C11 precursor cells develop neurite extensions and convert into either serotonergic or noradrenergic neurons. The two mutually exclusive neuronal programs are autoregulated by serotonergic or adrenergic receptors. PrPc is constitutively expressed by 1C11 cells. Antibody-mediated cross-linking of PrPc promotes the dephosphorylation of the tyrosine kinase Fyn associated to a Fyn kinase activation. The coupling of PrPc to Fyn is dependent on caveolin-1. It is restricted to the fully differentiated serotonergic or noradrenergic cells and occurs mainly at neurites. Thus, PrPc may represent a signal transduction protein which may fine-tune neuronal functions. Since the 1C11 stem cell supports prion replication, it may provide a tool to investigate whether PrPSc accumulation interferes with PrPc signalling activity.

In the field of dentistry, the murine incisor has long been considered as an outstanding model to study amelogenesis. However, it clearly appears that enamel from wild type mouse incisors and molars presents several structural differences. In incisor, exclusively radial enamel is observed. In molars, enamel displays a high level of complexity since the inner part is lamellar whereas the outer enamel shows radial and tangential structures. Recently, the serotonin 2B receptor (5-HT2BR) was shown to be involved in ameloblast function and enamel mineralization. The incisors from 5HT2BR knockout (KO) mice exhibit mineralization defects mostly in the outer maturation zone and porous matrix network in the inner zone. In the molars, the mutation affects both secretory and maturation stages of amelogenesis since pronounced alterations concern overall enamel structures. Molars from 5HT2BR KO mice display reduction in enamel thickness, alterations of inner enamel architecture including defects in Hunter-Schreger Bands arrangements, and altered maturation of the outer radial enamel. Differences of enamel structure were also observed between incisor and molar from other KO mice depleted for genes encoding enamel extracellular matrix proteins. Thus, upon mutation, enamel analysis based exclusively on incisor defects would be biased. In view of the functional relationship between enamel structure and tooth morphogenesis, identification of molecular actors involved in amelogenesis requires comparative studies between mice molars and incisors.

Using the proliferating cell nuclear antigen (PCNA) immunostaining, we previously identified, after pulp exposure, three zones of proliferating cells in the rat molar pulp. Zones I and II were in the crown near the pulp. Zone III was near the apex revealing a recruitment of mitotic cells at distance from the lesion. To gain further insight into the spatio-temporal evolution of proliferating pulp cells of zone III, we performed a longitudinal study of PCNA staining in rat molar mesial root at 3, 8, and 15 d after pulp exposure associated to implantation of unloaded or amelogenin loaded agarose beads. At day 3 after implantation, PCNA-positive cells were located in the central part of the radicular pulp. At day 8, PCNA-labeled cells were aligned in the lateral part of the pulp beneath the odontoblast/sub-odontoblast layer. At day 15, PCNA labeling became undetectable in the root and was located in the coronal pulp. These results suggest that after pulp exposure, PCNA-positive cells may migrate from the central part of the radicular pulp to the sub-odontoblast cell layer and then from the apical root to the crown. Electron microscopy and immunostaining analysis showed that pulpal cells were linked by desmosome-like and gap-junctions. Extracellular matrix was composed of thin collagen fibrils associated with glycosaminoglycans favoring cell mobility. These data suggest that the syncytium-like structure formed by pulp radicular cells may be a pre-request for plithotaxis, a collective cell migration process. This emergent mechanism may govern pulp healing and regeneration after dental lesion.

Many dental pulp stem cells are neural crest derivatives essential for lifelong maintenance of tooth functions and homeostasis as well as tooth repair. These cells may be directly implicated in the healing process or indirectly involved in cell-to-cell diffusion of paracrine messages to resident (pulpoblasts) or nonresident cells (migrating mesenchymal cells). The identity of the pulp progenitors and the mechanisms sustaining their regenerative capacity remain largely unknown. Taking advantage of the A4 cell line, a multipotent stem cell derived from the molar pulp of mouse embryo, we investigated the capacity of these pulp-derived precursors to induce in vivo the formation of a reparative dentin-like structure upon implantation within the pulp of a rodent incisor or a first maxillary molar after surgical exposure. One month after the pulp injury alone, a nonmineralized fibrous matrix filled the mesial part of the coronal pulp chamber. Upon A4 cell implantation, a mineralized osteodentin was formed in the implantation site without affecting the structure and vitality of the residual pulp in the central and distal parts of the pulp chamber. These results show that dental pulp stem cells can induce the formation of reparative dentin and therefore constitute a useful tool for pulp therapies. Finally, reparative dentin was also built up when A4 progenitors were performed by alginate beads, suggesting that alginate is a suitable carrier for cell implantation in teeth.

TRPS1 (tricho-rhino-phalangeal syndrome) is a unique GATA-type transcription factor that acts as a transcriptional repressor. TRPS1 deficiency and dysregulated TRPS1 expression result in skeletal and dental abnormalities implicating TRPS1 in endochondral bone formation and tooth development. Moreover, patients with tricho-rhino-phalangeal syndrome frequently present with low bone mass indicating TRPS1 involvement in bone homeostasis. In addition, our previous data demonstrated accelerated mineralization of the perichondrium in Trps1 mutant mice and impaired dentin mineralization in Col1a1-Trps1 transgenic mice, implicating Trps1 in the mineralization process. To understand the role of Trps1 in the differentiation and function of cells producing mineralized matrix, we used a preodontoblastic cell line as a model of dentin mineralization. We generated both Trps1-deficient and Trps1-overexpressing stable cell lines and analyzed the progression of mineralization by alkaline phosphatase and alizarin red staining. As predicted, based on our previous in vivo data, delayed and decreased mineralization of Trps1-overexpressing odontoblastic cells was observed when compared with control cells. This was associated with down-regulation of genes regulating phosphate homeostasis. Interestingly, Trps1-deficient cells lost the ability to mineralize and demonstrated decreased expression of several genes critical for initiating the mineralization process, including Alpl and Phospho1. Based on these data, we have concluded that Trps1 serves two critical and context-dependent functions in odontoblast-regulated mineralization as follows: 1) Trps1 is required for odontoblast maturation by supporting expression of genes crucial for initiating the mineralization process, and 2) Trps1 represses the function of mature cells and, consequently, restricts the extent of extracellular matrix mineralization.

The cellular prion protein PrP c is a glycosylphosphatidylinositol-anchored cell-surface protein whose biological function is unclear. We used the murine 1C11 neuronal differentiation model to search for PrP c -dependent signal transduction through antibody-mediated cross-linking. A caveolin-1–dependent coupling of PrP c to the tyrosine kinase Fyn was observed. Clathrin might also contribute to this coupling. The ability of the 1C11 cell line to trigger PrP c -dependent Fyn activation was restricted to its fully differentiated serotonergic or noradrenergic progenies. Moreover, the signaling activity of PrP c occurred mainly at neurites. Thus, PrP c may be a signal transduction protein.

The murine F9-derived 1C11 clone exhibits a stable epithelial morphology, expresses nestin, an early neuroectodermal marker, and expresses genes involved in neuroectodermal cell fate. Upon appropriate induction, 100% of 1C11 precursor cells develop neurite extensions and acquire neuronal markers (N-CAM, synaptophysin, gammagamma-enolase, and neurofilament) as well as the general functions of either serotonergic (1C11*(/5HT)) (5HT, 5-hydroxytryptamine) or noradrenergic (1C11**(/NE)) (NE, norepinephrine) neurons. The two programs are shown to be mutually exclusive. 1C11 thus behaves as a neuroepithelial cell line with a dual bioaminergic fate. 1C11*(/5HT) cells implement a functional 5-HT transporter and thereby a complete serotonergic phenotype within 4 days, whereas 5-HT(1B/D), 5-HT(2B), and 5-HT(2A) receptors are sequentially induced. The accurate time schedule of catecholaminergic differentiation was defined. Catecholamine synthesis, storage, and catabolism are acquired within 4 days; the noradrenergic phenotype is complete at day 12 and includes a functional norepinephrine transporter and an alpha(1D)-adrenoreceptor (day 8). The time-dependent onset of neurotransmitter-associated functions proper to either program is similar to in vivo observations. Along each pathway, the selective induction of serotonergic or adrenergic receptors is shown to be an essential part of the differentiation program, since they promote an autoregulation of the corresponding phenotype.

The biological function of the cellular prion protein, PrPc, is currently unknown. The presence of PrPc transcripts in the developing neural tube from embryonic day 13.5 and the predominant expression of PrPc in the adult brain is suggestive of a role in the onset and/or modulation of neuronal functions. We took advantage of the bipotential neuroectodermal 1C11 cell line to monitor PrPc expression during its bioaminergic differentiations. The F9-derived 1C11 precursor cell line displays a stable and immature phenotype in the absence of extracellular signal and, upon induction, has the capacity to acquire a complete serotonergic or noradrenergic phenotype, the two pathways being mutually exclusive. A real-time quantitative PCR assay was developed to assess PrPc gene expression at definite times of the two programs that correspond to sequential acquisition of neurotransmitter-specific functions. 1C11 cells and their differentiated progenies express significant amounts of PrP transcripts and of the corresponding protein. A unique decrease in prnp gene expression is observed upon entry into the serotonergic pathway, correlating with a downregulation at the protein level. Moreover, nerve growth factor (NGF) is shown to induce a decrease in the level of prnp gene expression along the serotonergic – but not the noradrenergic – pathway. Our study accurately establishes that prnp gene expression (i) is strongly upregulated concomitantly with cell fate restriction of multipotential cells towards the neural lineage; (ii) is differentially regulated along the serotonergic versus noradrenergic differentiation program of a unique neuroectodermal progenitor. The 1C11 cell line may provide a new tool for studying prion infectivity in a well-defined neuronal context.

The study of signaling cascades and of functional interactions between 5-hydroxytryptamine (5-HT) receptor pathways with heterogenous brain cell populations remains an arduous task. We took advantage of a serotonergic cell line to elucidate cross-talks between 5-HT receptors and to demonstrate the involvement of two 5-HT2 receptor subtypes in the regulation of 5-HT1B/1D function. The inducible 1C11 cell line has the unique property of acquiring within 4 days a complete serotonergic phenotype (1C11* cells), including three 5-HT receptors. 5-HT1B/1D and 5-HT2B receptors are expressed since day 2 of the serotonergic differentiation while 5-HT2A receptors are induced at day 4. We first established that 5-HT2B receptors are coupled with the phospholipase A2 (PLA2)-mediated release of arachidonic acid (AA) and that the activation of 5-HT2B receptors in 1C11*d2 cells inhibits the 5-HT1B/1D receptor function via a cyclooxygenase-dependent AA metabolite. At day 4, this 5-HT2B-mediated inhibition of the 5-HT1B/1D function can be blocked upon concomitant 5-HT2A activation although a 5-HT2A/PLA2 positive coupling was evidenced. This suggests the existence in 1C11*d4 cells of pathway(s) for 5-HT2A receptors, distinct from PLC and PLA2. Finally, this study reveals the antagonistic roles of 5-HT2A and 5-HT2B receptors in regulating the function of 5-HT1B/1D, a receptor involved in neuropsychiatric disorders and migraine pathogenesis.

Axial skeletons such as vertebrae, ribs, and scapulae develop from the embryonic somitic mesoderm through interactions with neural tube/notochord and skin ectoderm. Bone morphogenetic proteins (BMPs) seem to play important roles in these tissue interactions; however, the relationship between BMP signaling and the early development of axial skeletons is poorly understood. In this report, we investigated possible roles of BMP signaling in axial skeletal formation. First, we describe the expression patterns of BMP4 and type I receptors for BMP during somitogenesis in chick embryos based on whole mount in situ hybridization. Next, the effects of BMP on axial skeletal morphogenesis were investigated by implantation of BMP proteins into the dorsal mesoderm at the time of somitogenesis. Transcripts for both BMP4 ligand and its receptors are expressed in the dorsal ectoderm and mesoderm. Implantation of BMP4 and BMP2 into the dorsal regions of embryos result in subsequent anomalies of vertebrae, ribs, and scapulae. The effects of BMP implantation on the skeleton are shown to be dependent upon the somitic stage. Vertebral anomalies are restricted to the dorsolateral elements of the vertebrae and specifically observed after BMP implantation into embryonic day 2 (E2) embryos, but not E3 embryos. These results indicate that implantation of BMP into the dorsal part of embryos where endogenous BMP ligand and BMP receptors are expressed perturbs BMP signaling and causes axial skeletal malformations. The findings presented here suggest that BMP signaling may be involved in the early developmental process of the axial skeleton.

International audience; alpha-secretase-mediated cleavage of amyloid precursor protein (APP) precludes formation of neurotoxic amyloid-beta (A beta) peptides, and alpha-cleavage of cellular prion protein (Pr-Pc) prevents its conversion into misfolded, pathogenic prions (PrPSc). The mechanisms leading to decreased alpha-secretase activity in Alzheimer's and prion disease remain unclear. Here, we find that tumor necrosis factor-alpha-converting enzyme (TACE)-mediated alpha-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 A beta deposition and in the brains of individuals with Alzheimer's disease. PDK1 induces phosphorylation and caveolin-l-mediated internalization of TACE. This dysregulation of TACE increases PrPSc and A beta accumulation and reduces shedding of TNF-alpha receptor type 1 (TNFR1). Inhibition of PDK1 promotes localization of TACE to the plasma membrane, restores TACE-dependent alpha-secretase activity and cleavage of APP, PrPC and TNFR1, and attenuates PrPSc- and A beta-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 Alzheimer's disease-like pathology and memory impairment.

The teratocarcinoma-derived C1 clone behaves as a mesodermal tripotential progenitor cell whose choice of fate, either osteoblast, chondroblast, or adipoblast, is strictly dependent on the spatial organization of the cells and the nature of the induction. In the absence of cell contact before the addition of inducers, the C1 cells maintain a stable undifferentiated phenotype while expressing potential regulators of embryonic mesodermal stem cell fate such a M-twist and Id1. Upon establishment of cell contacts before the induction of differentiation, the early genes characteristic of the three fates become expressed. In the presence of beta glycerophosphate and ascorbate, provided the cells have formed aggregates, 95% of the C1 cells mineralize with a kinetics of gene expression close to that of osteoblasts (Poliard, A., D. Lamblin, P. J. Marie, M. H. Buc, and O. Kellerman. 1993. J. Cell Sci. 106:503-512). With 10(-6)M dexamethasone, 80% of the same aggregates differentiate into foci of chondroblast-like cells. The kinetics of expression of the genes encoding type II, IX, X, and XI collagens, aggrecan and link protein during the conversion toward cartilage hypertrophy resembles that accompanying in vivo chondrogenesis. The synergistic action of dexamethasone and insulin convert most confluent C1 cells into functional adipocytes and induce a pattern of gene expression close to that reported for adipoblast cell lines. The C1 clone with its capacity to differentiate along three alternative pathways with high frequency, therefore appears as a valid in vitro model for deciphering the molecular basis of mesoblast ontogeny.

Serotonin, first described as a neurotransmitter in invertebrates, has been investigated mostly for its functions in the mature central nervous system of higher vertebrates. Serotonin receptor diversity has been described in the mammalian brain and in insects. We report the isolation of a cDNA coding for a Drosophila melanogaster serotonin receptor that displays a sequence, a gene organization, and pharmacological properties typical of the mammalian 5-HT2 serotonin receptor subtype. Its mRNA can be detected in the adult fly; moreover, a high level of expression occurs at 3 hr of Drosophila embryogenesis. This early embryonic expression is surprisingly organized in a seven-stripe pattern that appears at the cellular blastoderm stage. In addition, this pattern is in phase with that of the even-parasegment-expressed pair-rule gene fushi-tarazu and is similarly modified by mutations affecting segmentation genes. Simultaneously with this pair-rule expression, the complete machinery of serotonin synthesis is present and leads to a peak of ligand concomitant with a peak of 5-HT2-specific receptor sites in blastoderm embryos.

The role of the serotonin 2B receptor (5-HT(2B) R) in enamel formation and mineralization was explored in adult 5HT(2B) R knockout (KO) mice compared with wild-type (WT) mice. In the molar, quantitative data obtained by micro-computed tomography imaging showed that the overall volume of the enamel layer was firmly reduced in KO mice. Defective mineralization was ascertained by energy-dispersive X-ray microanalysis. We also observed, using scanning electron microscopy, that parazones in the KO mice included two or three helicoidally twisted rods within Hunter-Schreger bands, instead of a single rod, as found in the WT mice. Minor disturbances were also detected in the incisors of KO mice. Structural modifications, thinner enamel crystallites, and porosities observed in KO mice indicate that the 5-HT(2B) R-mediated signaling pathways as part of the enamel formation process. These data provide a basis for evaluating the role of 5-HT(2B) R in ameloblast functions. Defects observed in the mineralization and structure of enamel in KO mice highlight that the 5-HT(2B) R interferes with the mechanisms directing amelogenesis.