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.

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.

Neuritogenesis: the prion protein controls ß1 integrin signaling activity.

Cytoskeleton modifications are required for neuronal stem cells to acquire neuronal polarization. Little is known, however, about mechanisms that orchestrate cytoskeleton remodeling along neuritogenesis. Here, we show that the silencing of the cellular prion protein (PrP(C)) impairs the initial sprouting of neurites upon induction of differentiation of the 1C11 neuroectodermal cell line, indicating that PrP(C) is necessary to neuritogenesis. Such PrP(C) function relies on its capacity to negatively regulate the clustering, activation, and signaling activity of ß1 integrins at the plasma membrane. ß1 Integrin aggregation caused by PrP(C) depletion triggers overactivation of the RhoA-Rho kinase-LIMK-cofilin pathway, which, in turn, alters the turnover of focal adhesions, increases the stability of actin microfilaments, and in fine impairs neurite formation. Inhibition of Rho kinases is sufficient to compensate for the lack of PrP(C) and to restore neurite sprouting. We also observe an increased secretion of fibronectin in the surrounding milieu of PrP(C)-depleted 1C11 cells, which likely self-sustains ß1 integrin signaling overactivation and contributes to neuritogenesis defect. Our overall data reveal that PrP(C) contributes to the acquisition of neuronal polarization by modulating ß1 integrin activity, cell interaction with fibronectin, and cytoskeleton dynamics.

Parkin deficiency delays motor decline and disease manifestation in a mouse model of synucleinopathy.

In synucleinopathies, including Parkinson's disease, partially ubiquitylated alpha-synuclein species phosphorylated on serine 129 (P(S129)-alpha-synuclein) accumulate abnormally. Parkin, an ubiquitin-protein ligase that is dysfunctional in autosomal recessive parkinsonism, protects against alpha-synuclein-mediated toxicity in various models.We analyzed the effects of Parkin deficiency in a mouse model of synucleinopathy to explore the possibility that Parkin and alpha-synuclein act in the same biochemical pathway. Whether or not Parkin was present, these mice developed an age-dependent neurodegenerative disorder preceded by a progressive decline in performance in tasks predictive of sensorimotor dysfunction. The symptoms were accompanied by the deposition of P(S129)-alpha-synuclein but not P(S87)-alpha-synuclein in neuronal cell bodies and neuritic processes throughout the brainstem and the spinal cord; activation of caspase 9 was observed in 5% of the P(S129)-alpha-synuclein-positive neurons. As in Lewy bodies, ubiquitin-immunoreactivity, albeit less abundant, was invariably co-localized with P(S129)-alpha-synuclein. During late disease stages, the disease-specific neuropathological features revealed by ubiquitin- and P(S129)-alpha-synuclein-specific antibodies were similar in mice with or without Parkin. However, the proportion of P(S129)-alpha-synuclein-immunoreactive neuronal cell bodies and neurites co-stained for ubiquitin was lower in the absence than in the presence of Parkin, suggesting less advanced synucleinopathy. Moreover, sensorimotor impairment and manifestation of the neurodegenerative phenotype due to overproduction of human alpha-synuclein were significantly delayed in Parkin-deficient mice.These findings raise the possibility that effective compensatory mechanisms modulate the phenotypic expression of disease in parkin-related parkinsonism.

Biochemical analysis of Parkinson's disease-causing variants of Parkin, an E3 ubiquitin-protein ligase with monoubiquitylation capacity.

Mutations in the parkin gene, encoding an E3 ubiquitin-protein ligase, are a frequent cause of autosomal recessive parkinsonism and are also involved in sporadic Parkinson's disease. Loss of Parkin function is thought to compromise the polyubiquitylation and proteasomal degradation of specific substrates, leading to their deleterious accumulation. Several studies have analyzed the effects of parkin gene mutations on the biochemical properties of the protein. However, the absence of a cell-free system for studying intrinsic Parkin activity has limited the interpretation of these studies. Here we describe the biochemical characterization of Parkin and 10 pathogenic variants carrying amino-acid substitutions throughout the sequence. Mutations in the RING fingers or the ubiquitin-like domain decreased the solubility of the protein in detergent and increased its tendency to form visible aggregates. None of the mutations studied compromised the binding of Parkin to a series of known protein partners/substrates. Moreover, only two variants with substitutions of conserved cysteine residues of the second RING finger were inactive in a purely in vitro ubiquitylation assay, demonstrating that loss of ligase activity is a minor pathogenic mechanism. Interestingly, in this in vitro assay, Parkin catalyzed the linkage of single ubiquitin molecules only, whereas the ubiquitin-protein ligases CHIP and Mdm2 promoted the formation of polyubiquitin chains. Similarly, in mammalian cells Parkin promoted the multimonoubiquitylation of its substrate p38, rather than its polyubiquitylation. Thus, Parkin may mediate polyubiquitylation or proteasome-independent monoubiquitylation depending on the protein context. The discovery of monoubiquitylated Parkin species in cells hints at a novel post-translational modification potentially involved in the regulation of Parkin function.

TRAF interactions with raft-like buoyant complexes, better than TRAF rates of degradation, differentiate signaling by CD40 and EBV latent membrane protein 1.

The CD40 receptor and the Epstein-Barr virus oncoprotein LMP1 are both members of the TNF-receptor family and share several signaling mediators, including TRAF2 and TRAF3. Depending on the cell lineage and stage of maturation, LMP1 and CD40 can have synergistic, antagonist or unrelated effects. Previous publications have suggested that both TRAF2 and TRAF3 move into lipid rafts upon LMP1 expression or CD40 activation, whereas their proteolysis is only enhanced by CD40. However CD40-induced proteolysis of TRAF2 has only been reported in murine cells, and there are conflicting data regarding translocation of TRAF2 into lipid rafts. We therefore investigated TRAF2 and TRAF3 modifications induced by CD40 and LMP1 signaling in a panel of human cell lines of lymphoid and epithelial origins. Upon CD40 stimulation, a marked redistribution of TRAF2 into the buoyant raft fraction was observed in all cell lines and was often associated with a similar redistribution of TRAF3. In contrast, only TRAF3 was redistributed into the raft fraction upon LMP1 expression. Moreover parallel changes in subcellular distribution of TRAF2 and TRAF3 were recorded by electron microscopy. A significant decrease in TRAF2 and TRAF3 concentrations triggered by CD40 ligation was observed in only 1 cell line and there was no evidence that this decrease was required for the negative feed-back on JNK activation. TRAF2 redistribution into raft-like complexes thus appears as the most significant event distinctive of CD40 and LMP1 signaling. On the other hand, the parallel influence of CD40 and LMP1 on TRAF3 redistribution is consistent with functional similarities between the CD40-TRAF3 and LMP1-TRAF3 axes.

Apoptosis and TRAF-1 cleavage in Epstein-Barr virus-positive nasopharyngeal carcinoma cells treated with doxorubicin combined with a farnesyl-transferase inhibitor.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinomas (NPC) are much more sensitive to chemotherapy than other head and neck carcinomas. Spectacular regressions are frequently observed after induction chemotherapy. However, these favorable responses are difficult to predict and often of short duration. So far there have been only few experiments to investigate the mechanisms which underline the cytotoxic effects of anti-neoplastic drugs against NPC cells. In addition, these studies were performed almost entirely on EBV-negative cell lines therefore not truly representative of NPC cells. For the first time, we have used two EBV-positive NPC tumor lines derived from a North African (C15) and a Chinese (C666-1) patient as in vitro targets for a panel of anti-neoplastic agents. Doxorubicin, taxol and in a lesser extent cis-platinum efficiently inhibited NPC cell proliferation at clinically relevant concentrations, but all three agents failed to induce apoptosis. However, massive apoptosis of C15 cells was achieved when doxorubicin (1 microM) was combined with a farnesyl-transferase inhibitor, BIM 2001 (5 microM). Moreover, this apoptotic process was associated with a caspase-dependent early cleavage of the TNF-receptor associated factor 1 (TRAF-1) molecule, a signaling adaptor which is specifically expressed in latently EBV-infected cells. TRAF-1 cleavage might become a useful indicator of chemo-induced apoptosis in EBV-associated NPCs.