Search

Additional file 2: Fig. S1. Hydrodynamic diameter (nm) of TiO2- and CB-NPs measured by DLS in DMEM/F12 at 37 °C. Diameter (nm) of aggregates of TiO2 and CB nanoparticles was measured by DLS following sonication of NPs, dilution in DMEM/F12, and centrifugation for 2 sec at 2000 g to remove large aggregates. At 10, 25 and 50 µg ml−1, TiO2-NPs displayed an average diameter of 130 nm (Polydispersity Index-PDI = 0.21), 206 nm (PDI = 0.53), and 375 nm (PDI = 0.22), respectively, and CB-NPs 171 nm (PDI = 0.13), 188 nm (PDI = 0.15), and 327 nm (PDI = 0.52), respectively. The experiments were performed in triplicates.

Additional file 3: Fig. S2. TiO2 and CB nanoparticles provoke TACE depletion and TNFR1 accumulation at the plasma membrane of serotonergic 1C115-HT neuronal cells in a PDK1-dependent manner. TNFR1 a and TACE b immunostaining at the cell surface of 1C115-HT neuronal cells exposed for 4 h to TiO2- or CB-NPs (1 µg cm−2) in the presence or not of the PDK1 inhibitor BX912 (1 µM) and related quantification histograms. Representative images of three experiments performed in triplicates are shown. Values are means ± SEM. * denotes p < 0.05 and **p < 0.01 versus unexposed cells.

Additional file 1: Table S1. Hydrodynamic diameter (nm) of TiO2- and CB-NPs in PBS at 22 °C measured by DLS. Diameter (nm) of aggregates of TiO2 and CB nanoparticles (5 up to 80 µg ml−1) measured by DLS after NP sonication, dilution in PBS, and centrifugation for 2 sec at 2000 g to remove large aggregates. The hydrodynamic diameter could not be measured for the 5 µg ml−1 NP concentration. The experiments were performed in triplicates.

Additional file 5: Fig. S4. Corruption of PrPC-coupled signaling pathways by TiO2- and CB-NPs in neuronal cells: toward a pro-Alzheimer effect of some TiO2 and CB nanoparticles. Cellular prion protein PrPC is a plasma membrane receptor recognized by TiO2- and CB-NPs in neuronal cells. The interaction between full-length PrPC and NPs mobilizes PrPC-coupled signaling pathways, leading to (i) the activation of NADPH oxidase and the production of ROS, and (ii) the activation of PDK1 that promotes the internalization of TACE α-secretase and thereby down-regulates TACE shedding activity at the root of plasma membrane TNFR1 accumulation and rise in Aβ40/42 production. Such NP interferences with the PrPC signaling network triggers molecular signs of Alzheimer’s disease: modification of cell redox equilibrium, neuronal priming to TNFα inflammatory stress, and accumulation of neurotoxic Aβ40/42 peptides (Image drawn using Servier medical art).

Additional file 4: Fig. S3. TiO2 and CB nanoparticles enhance Aβ40 production in 1C11 precursors and 1C115-HT neuronal cells. a ELISA-based quantification of Aβ40 peptides in 1C11 and 1C115-HT neuronal cells exposed to TiO2- or CB-NPs (1 µg cm−2) up to 24 h. b APP and BACE1 expression level as assessed by RT-qPCR and Western-blotting in 1C11 cells exposed to TiO2- or CB-NPs (1 µg cm−2) for 4 h. c ELISA-based quantification of Aβ40 peptides in 1C115-HT neuronal cells exposed to TiO2- or CB-NPs (1 µg cm−2) for 4 h in the presence or not of a siRNA toward PrPC (siPrP) or the PDK1 inhibitor, BX912 (1 µM). The experiments were performed three times in triplicates. Values are means ± SEM. * denotes p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus unexposed cells.

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., Author Summary Transmissible Spongiform Encephalopathies (TSEs), commonly named prion diseases, are caused by deposition in the brain of pathogenic prions PrPSc that trigger massive neuronal death. Because of our poor understanding of the mechanisms sustaining prion-induced neurodegeneration, there is to date no effective medicine to combat TSEs. The current study demonstrates that ROCK kinases are overactivated in prion-infected cells and contribute to prion pathogenesis at two levels. First, PrPSc-induced ROCK overactivation affects neuronal polarity with synapse disconnection, axon/dendrite degradation, and disturbs neuronal functions. Second, ROCK overactivity amplifies the production of pathogenic prions. The pharmacological inhibition of ROCK protects diseased neurons from PrPSc toxicity by preserving neuronal architecture and functions and lowering PrPSc level. Inhibition of ROCK in prion-infected mice reduces brain PrPSc levels, improves motor activity and extends lifespan. This study opens up new avenues to design ROCK-based therapeutic strategies to fight TSEs.

Expression of the cellular prion protein (PrP(c)) by host cells is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. As a consequence, identification of the cell types expressing PrP(c) is necessary to determine the target cells involved in the cerebral propagation of prion diseases. To identify the cells expressing PrP(c) in the mouse brain, the immunocytochemical localization of PrP(c) was investigated at the cellular and ultrastructural levels in several brain regions. In addition, we analyzed the expression pattern of a green fluorescent protein reporter gene under the control of regulatory sequences of the bovine prion protein gene in the brain of transgenic mice. By using a preembedding immunogold technique, neuronal PrP(c) was observed mainly bound to the cell surface and presynaptic sites. Dictyosomes and recycling organelles in most of the major neuron types also exhibited PrP(c) antigen. In the olfactory bulb, neocortex, putamen, hippocampus, thalamus, and cerebellum, the distribution pattern of both green fluorescent protein and PrP(c) immunoreactivity suggested that the transgenic regulatory sequences of the bovine PrP gene were sufficient to promote expression of the reporter gene in neurons that express immunodetectable endogenous PrP(c). Transgenic mice expressing PrP-GFP may thus provide attractive murine models for analyzing the transcriptional activity of the Prnp gene during prion infections as well as the anatomopathological kinetics of prion diseases.