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 β1 integrins at the plasma membrane. β1 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 β1 integrin signaling overactivation and contributes to neuritogenesis defect. Our overall data reveal that PrP ^C contributes to the acquisition of neuronal polarization by modulating β1 integrin activity, cell interaction with fibronectin, and cytoskeleton dynamics.—Loubet, D., Dakowski, C., Pietri, M., Pradines, E., Bernard, S., Callebert, J., Ardila‐Osorio, H., Mouillet‐Richard, S., Launay, J. M., Kellermann, O., Schneider, B. Neuritogenesis: the prion protein controls β1 integrin signaling activity. FASEB J. 26, 678–690 (2012). www.fasebj.org

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 (PrP^Sc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrP^Sc-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 PrP^Sc 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 PrP^C by TACE α-secretase, which physiologically precludes PrP^Sc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrP^Sc. In mice challenged with prions, inhibition of ROCK also lowered brain PrP^Sc 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 PrP^Sc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases. Transmissible Spongiform Encephalopathies (TSEs), commonly named prion diseases, are caused by deposition in the brain of pathogenic prions PrP^Sc 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, PrP^Sc-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 PrP^Sc toxicity by preserving neuronal architecture and functions and lowering PrP^Sc level. Inhibition of ROCK in prion-infected mice reduces brain PrP^Sc levels, improves motor activity and extends lifespan. This study opens up new avenues to design ROCK-based therapeutic strategies to fight TSEs.

Abundant evidence has accumulated showing that fetal alcohol exposure broadly modifies DNA methylation profiles in the brain. DNA methyltransferases (DNMTs), the enzymes responsible for DNA methylation, are likely implicated in this process. However, their regulation by ethanol exposure has been poorly addressed. Here, we show that alcohol exposure modulates DNMT protein levels through multiple mechanisms. Using a neural precursor cell line and primary mouse embryonic fibroblasts (MEFs), we found that ethanol exposure augments the levels of Dnmt3a, Dnmt3b, and Dnmt3l transcripts. We also unveil similar elevation of mRNA levels for other epigenetic actors upon ethanol exposure, among which the induction of lysine demethylase Kdm6a shows heat shock factor dependency. Furthermore, we show that ethanol exposure leads to specific increase in DNMT3A protein levels. This elevation not only relies on the upregulation of Dnmt3a mRNA but also depends on posttranscriptional mechanisms that are mediated by NADPH oxidase-dependent production of reactive oxygen species (ROS). Altogether, our work underlines complex regulation of epigenetic actors in response to alcohol exposure at both transcriptional and posttranscriptional levels. Notably, the upregulation of DNMT3A emerges as a prominent molecular event triggered by ethanol, driven by the generation of ROS.

Although cellular prion protein PrP^Cis well known for its implication in Transmissible Spongiform Encephalopathies, its functions remain elusive. Combiningin vitroandin vivoapproaches, we here show that PrP^Cdisplays the intrinsic capacity to protect neuronal cells from a pro-inflammatory TNFα noxious insult. Mechanistically, PrP^Ccoupling 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 PrP^Cexpression is necessary for TACE α-secretase to stay at the plasma membrane in an active state for TNFR shedding. Such PrP^Ccontrol of TACE localization depends on PrP^Cmodulation of β1 integrin signaling and downstream activation of ROCK-I and PDK1 kinases. Loss of PrP^Cprovokes TACE internalization, which in turn cancels TACE-mediated cleavage of TNFR and renders PrP^C-depleted neuronal cells as well as PrP^Cknockout mice highly vulnerable to pro-inflammatory TNFα insult. Our work provides the prime evidence that in an inflammatory context PrP^Cadjusts 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-PrP^Ccytoprotective function.

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 (PrP^C), 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 PrP^C at the surface of neuronal cells grown in culture corrupts PrP^C signaling function. This triggers PrP^C-dependent activation of NADPH oxidase and subsequent production of reactive oxygen species (ROS) that alters redox equilibrium. Through PrP^C 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 PrP^C 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 PrP^C-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 PrP^C as being a neuronal receptor of TiO₂- and CB-NPs and identifies PrP^C-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.