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A lesion in the nervous system induces cellular and molecular mechanisms leading to irreversible dysfunctions. Post-injuredenvironment is non permissive for axonal regeneration and CNS neurons also exhibit a reduced intrinsic growth capacity.Nevertheless, neuronal plasticity is still possible: injured axons can degenerate or regenerate, but mechanisms governing thisresponse remains elusive. My aim is to understand the relation between microtubules and mitochondria on axon fate after atraumatic injury, in particular by focusing on the corticospinal tract connecting to the spinal cord neurons. Corticospinalneurons are known to show poor regeneration capacities after a spinal cord injury. Axonal degeneration is associated with afast decrease of NAD+ leading to energy drop, cytoskeletal disorganization and finally axonal degradation. Mitochondriarepositioning in the axon can stimulate regeneration and branching by rescuing energy deficits. In addition, microtubules (MT)to which converge extracellular cues for intracellular trafficking are also involved in mitochondrial transport. Thereby,mitochondria and MT are essential in determining the axon fate. I am developping an in vitro set-up with microfluidics orientednetworks of cortico-spinal neurons on which a physical injruy can be apply. This system will allow to study the influence ofpost-translationnal MT modifications on mitochondria dynamic during axonal regeneration or degeneration.

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.

International audience; Background: Mechanisms involved in prion induced neuronal death are still enigmatic. In vivo, both neurons and astrocytes support prion propagation leading to abnormal production of a misfolded protein called scrapie prion protein (PrPSc). Although crucial for prion replication and accumulation of aggregated PrPSc, the role of the normal form of prion protein,the cellular PrP (PrPC), is still controversial in prion-induced neurotoxicity. Objectives : In this study we investigated the role of neuronal PrPC and the contribution of astrocytes in prion-induced neuronal death. Methods: We have previously shown that prions could be efficiently propagated in primarily cultured neurons and astrocytes (Cronier et al. 2004). Here, we have set up a model in which neurons devoid of PrPC or expressing PrPC from different species are cocultured with prion-infected astrocytes that continuously deliver physiological concentrations of PrPSc. Results : In these conditions, we showed that scrapie-infected astrocytes exacerbate prion-induced cell death solely in neurons expressing transconformable PrPC. Although infectious, conditioned medium of prion-infected astrocytes did not display any acute neuronal toxicity. However PrPSc accumulation in neurons led to neuritic damage and cell death associated with an increased sensitivity to glutamate and reactive oxygen species. Discussion : Our results indicate that interaction between neuronal PrPC and PrPSc is not sufficient to induce neuronal death but that PrPC transconformation is required for prion-associated neurotoxicity. In addition, it is unlikely that a dysfunction of prion-infected astrocytes is involved in the initiation of neuronal death process induced by prions. Altogether, our findings support the view that prion infection and subsequent PrPC transconformation trigger impairment of neuronal homeostasis by sensitizing neurons to environmental stress that are regulated by neighbouring cells including astrocyte.

International audience; Background: Activation of glia in prion diseases contributes to TSE pathogenesis through the release of chemokines and pro-inflammatory cytokines acting as neurotoxic mediators. While it is now established that expression of the cellular prion protein (PrPC) at the surface of neuronal cells is mandatory for the scrapie isoform (PrPSC) to exert its toxicity, whether PrPSC replication in neurons potentiates their sensitivity to inflammatory cytokines is unclear. Objectives: Our goal was 1) to assess the impact of prion infection in neuronal cells on TNFα-mediated toxicity and 2) to relate these effects to the normal function of PrPC. Methods: Our study is based on RT-PCR, western blot and immunofluorescence experiments. We take advantage of the neuroectodermal 1C11 cell line endowed with the capacity to differentiate into bioaminergic neuronal cells. 1C11 cells combine functional expression of endogenous PrPC and the ability to chronically replicate several prion strains. Experiments were also carried out on primary cerebellar granule neurons obtained from FVB WT or PrP-null mice and FVB infected mice with 22L prions. Results: In 1C11 precursors and their neuronal progenies, infection with the Fukuoka or 22L strains renders cells more vulnerable to exogenous TNFα. This higher cell sensitivity to TNFα relates to an increased expression of the death receptor TNFR1 at the surface of infected cells, itself resulting from both an upregulation of TNFR1 transcripts and an inhibition of metalloproteinase-dependent TNFR1 shedding. The raise in TNFR1 was also monitored in brains of 22L infected mice. Finally, we provide evidence that PrPC depletion promotes a deregulation in TNFR1 that compares that observed within an infectious context. Discussion: We substantiate an increased vulnerability of infected neuronal cells to inflammatory cytokines, that reflects a loss of PrPC function on TNF-R1 regulation. We propose that the altered cell response to TNFα-mediated toxicity contributes to exacerbate prion-induced pathogenesis.

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International audience; Transmissible spongiform encephalopathies (TSE) arise as a consequence of the infection by prions of the central nervous system and are incurable. Neuronal loss and gliosis, associated with the accumulation of misfolded PrP protein (PrPSc) are hallmarks of prion diseases, however the mechanisms underlying these disorders remain unclear. We recently described a cellular model consisting of cerebellar granule cells derived from transgenic mice expressing ovine PrP. Upon exposure to low infectious doses of natural sheep scrapie agent, such cultures were found to accumulate de novo PrPSc and infectivity, as assessed by protease digestion and mouse bioassay. Both neurons and astrocytes were found to sustain prion propagation, and a late neuronal death was observed in infected cultures (1). In an attempt to generalize these observations, we found that it is also feasible to propagate mouse, hamster and human prions in cultures derived from transgenic mouse lines expressing the cognate PrPc. Primary cell cultures are widely used to search for molecules with protecting activity in neurodegenerative or neuro-infectious diseases as well as to assess their neurotoxicity. Thus we sought to evaluate the activity of Congo red, chlorpromazine and a polyene antibiotics, three compounds known to cure prion-infected cell lines, in these newly developed cell systems. We observed that the anti-prion activity in such cultures is species- or strain-dependent, and is in good agreement with results reported in vivo in rodent models. Our findings suggest that prion-infected primary neuronal cultures may be relevant tools for anti-prion drug appraisal, including human TSE agents.

International audience