Titanium dioxide and carbon black nanoparticles disrupt neuronal homeostasis via excessive activation of cellular prion protein signaling.

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 Aß40/42 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 Aß 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 Aß 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.

Loss of prion protein control of glucose metabolism promotes neurodegeneration in model of prion diseases.

Corruption of cellular prion protein (PrPC) function(s) at the plasma membrane of neurons is at the root of prion diseases, such as Creutzfeldt-Jakob disease and its variant in humans, and Bovine Spongiform Encephalopathies, better known as mad cow disease, in cattle. The roles exerted by PrPC, however, remain poorly elucidated. With the perspective to grasp the molecular pathways of neurodegeneration occurring in prion diseases, and to identify therapeutic targets, achieving a better understanding of PrPC roles is a priority. Based on global approaches that compare the proteome and metabolome of the PrPC expressing 1C11 neuronal stem cell line to those of PrPnull-1C11 cells stably repressed for PrPC expression, we here unravel that PrPC contributes to the regulation of the energetic metabolism by orienting cells towards mitochondrial oxidative degradation of glucose. Through its coupling to cAMP/protein kinase A signaling, PrPC tones down the expression of the pyruvate dehydrogenase kinase 4 (PDK4). Such an event favors the transfer of pyruvate into mitochondria and its conversion into acetyl-CoA by the pyruvate dehydrogenase complex and, thereby, limits fatty acids ß-oxidation and subsequent onset of oxidative stress conditions. The corruption of PrPC metabolic role by pathogenic prions PrPSc causes in the mouse hippocampus an imbalance between glucose oxidative degradation and fatty acids ß-oxidation in a PDK4-dependent manner. The inhibition of PDK4 extends the survival of prion-infected mice, supporting that PrPSc-induced deregulation of PDK4 activity and subsequent metabolic derangements contribute to prion diseases. Our study posits PDK4 as a potential therapeutic target to fight against prion diseases.

The Cellular Prion Protein-ROCK Connection: Contribution to Neuronal Homeostasis and Neurodegenerative Diseases.

Amyloid-based neurodegenerative diseases such as prion, Alzheimer's, and Parkinson's diseases have distinct etiologies and clinical manifestations, but they share common pathological events. These diseases are caused by abnormally folded proteins (pathogenic prions PrPSc in prion diseases, ß-amyloids/Aß and Tau in Alzheimer's disease, α-synuclein in Parkinson's disease) that display ß-sheet-enriched structures, propagate and accumulate in the nervous central system, and trigger neuronal death. In prion diseases, PrPSc-induced corruption of the physiological functions exerted by normal cellular prion proteins (PrPC) present at the cell surface of neurons is at the root of neuronal death. For a decade, PrPC emerges as a common cell surface receptor for other amyloids such as Aß and α-synuclein, which relays, at least in part, their toxicity. In lipid-rafts of the plasma membrane, PrPC exerts a signaling function and controls a set of effectors involved in neuronal homeostasis, among which are the RhoA-associated coiled-coil containing kinases (ROCKs). Here we review (i) how PrPC controls ROCKs, (ii) how PrPC-ROCK coupling contributes to neuronal homeostasis, and (iii) how the deregulation of the PrPC-ROCK connection in amyloid-based neurodegenerative diseases triggers a loss of neuronal polarity, affects neurotransmitter-associated functions, contributes to the endoplasmic reticulum stress cascade, renders diseased neurons highly sensitive to neuroinflammation, and amplifies the production of neurotoxic amyloids.

Production of seedable Amyloid-ß peptides in model of prion diseases upon PrPSc-induced PDK1 overactivation.

The presence of amyloid beta (Aß) 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 Aß 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 ß-processing, leading to Aß40/42 accumulation. Aß predominates as monomers, but is also found as trimers and tetramers. Prion-induced Aß peptides do not affect prion replication and infectivity, but display seedable properties as they can deposit in the mouse brain only when seeds of Aß trimers are co-transmitted with PrPSc. Importantly, brain Aß deposition accelerates death of prion-infected mice. Our data stress that PrPSc, through deregulation of the PDK1-TACE-APP pathway, provokes the accumulation of Aß, a prerequisite for the onset of an Aß seeds-induced Aß pathology within a prion-infectious context.

Multifaceted Regulations of the Serotonin Transporter: Impact on Antidepressant Response.

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.

Serotonin in stem cell based-dental repair and bone formation: A review.

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.

Cerebellar compartmentation of prion pathogenesis.

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.

Protective role of cellular prion protein against TNFα-mediated inflammation through TACE α-secretase.

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 ß1 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.

Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro.

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.

In vitro bioactivity of Bioroot™ RCS, via A4 mouse pulpal stem cells.

OBJECTIVE: 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. METHODS: 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 (p<0.05). RESULTS: BR did not alter the viability and morphology of A4 pulpal cells compared to control group (p>0.05); however, living cell percentage of PCS was significantly lower compared to control and BR groups (p<0.05). 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. SIGNIFICANCE: 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.

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.

Essential Roles of Dopamine and Serotonin in Tooth Repair: Functional Interplay Between Odontogenic Stem Cells and Platelets.

Characterizing stem cell intrinsic functions is an ongoing challenge for cell therapies. Here, we report that two independent A4 and H8 stem cell lines isolated from mouse molar pulp display the overall functions of bioaminergic cells. Both clones produce neurotrophins and synthesize, catabolize, store, and transport serotonin (5-hydroxytryptamine [5-HT]) and dopamine (DA). They express 5-HT1D,2B,7 and D1,3 autoreceptors, which render pulpal stem cells competent to respond to circulating 5-HT and DA. We show that injury-activated platelets are the source of systemic 5-HT and DA necessary for dental repair since natural dentin reparation is impaired in two rat models with monoamine storage-deficient blood platelets. Moreover, selective inhibition of either D1, D3, 5-HT2B, or 5-HT7 receptor within the pulp of wild-type rat molars after lesion alters the reparative process. Altogether our data argue that 5-HT and DA coreleased by pulp injury-activated platelets are critical for stem cell-mediated dental repair through 5-HT and DA receptor signalings.

Plithotaxis, a collective cell migration, regulates the sliding of proliferating pulp cells located in the apical niche.

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.

Dual role of the Trps1 transcription factor in dentin mineralization.

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.

Pulp stem cells: implication in reparative dentin formation.

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.

Deletion of serotonin 2B receptor provokes structural alterations of mouse dental tissues.

Rampant caries and periodontal diseases occur in patients treated with antidepressants such as serotonin reuptake inhibitors (SRIs; e.g., Prozac) which target the serotonin transporter (SERT). As the serotonin 2B receptor (5HT2BR) regulates SERT functionality and capacity to recognize SRIs, we investigated the potential role of 5HT2BR on dental tissues by exploiting 5HT2BR knockout (KO) mice. Compared to wild-type (WT) mice, several structural differences were identified in the teeth of KO mice. In the molar of KO mice, rod curvatures and twisting were altered compared to WT mice, suggesting involvement of 5HT2BR at early stages of enamel formation. The volume of the KO enamel layer was also reduced, and larger porosities were observed in the prismatic enamel, with smaller crystallite thickness. Crystallite pattern disorganization and occlusal abrasion were enhanced in female KO mice, indicating a sexual dimorphism. In the incisor, no difference was detected in the width of the enamel layer between KO and WT mice; however, enamel maturation differed in absence of 5HT2BR. Specifically, the outer aprismatic enamel border was 1.5- to 2-fold larger in KO compared to WT mice, together with a decreased etching pattern. Finally, although no noticeable difference was observed in dentin, the micro-CT three-dimensional pulp reconstruction evidenced a decrease in both length and width of dentin formation in the root canals of the KO versus WT mice. These data provide evidence that 5HT2BR-mediated signaling pathways are involved in enamel formation and dentinogenesis.