High Dub3 expression in mouse ESC couples the G1/S checkpoint to pluripotency

International audienceThe molecular mechanism underlying G1/S checkpoint bypass in mouse embryonic stem cells (ESCs) remains unknown. DNA damage blocks S phase entry by inhibiting the CDK2 kinase through destruction of its activator, the Cdc25A phosphatase. We observed high Cdc25A levels in G1 that persist even after DNA damage in mouse ESCs. We also found higher expression of Dub3, a deubiquitylase that controls Cdc25A protein abundance. Moreover, we demonstrate that the Dub3 gene is a direct target of Esrrb, a key transcription factor of the self-renewal machinery. We show that Dub3 expression is strongly downregulated during neural conversion and precedes Cdc25A destabilization, while forced Dub3 expression in ESCs becomes lethal upon differentiation, concomitant to cell-cycle remodeling and lineage commitment. Finally, knockdown of either Dub3 or Cdc25A induced spontaneous differentiation of ESCs. Altogether, these findings couple the self-renewal machinery to cell-cycle control through a deubiquitylase in ESCs

Anti-PrP antibodies block PrPSc replication in prion-infected cell cultures by accelerating PrPC degradation.

manuscript received October 15, 2003; revised manuscript received December 15, 2003; accepted December 16, 2003. We thanks P. Rondard, O Bischof, J.-L. Laplanche and J.-P. Pin for their fruitful discussions. we are grateful to S. barrère for her assistance in the statistical analysis of the data and H. McMahon for her assistance in reading the manuscript

Antikinetoplastid SAR study in 3-nitroimidazopyridine series:identification of a novel non-genotoxic and potent anti-T. b. brucei hit-compound with improved pharmacokinetic properties.

International audienceTo study the antikinetoplastid 3-nitroimidazo[1,2-a]pyridine pharmacophore, a structure-activity relationship study was conducted through the synthesis of 26 original derivatives and their in vitro evaluation on both Leishmania spp and Trypanosoma brucei brucei. This SAR study showed that the antitrypanosomal pharmacophore was less restrictive than the antileishmanial one and highlighted positions 2, 6 and 8 of the imidazopyridine ring as key modulation points. None of the synthesized compounds allowed improvement in antileishmanial activity, compared to previous hit molecules in the series. Nevertheless, compound 8, the best antitrypanosomal molecule in this series (EC50 = 17 nM, SI = 2650 & E° = −0.6 V), was not only more active than all reference drugs and previous hit molecules in the series but also displayed improved aqueous solubility and better in vitro pharmacokinetic characteristics: good microsomal stability (T1/2 > 40 min), moderate albumin binding (77%) and moderate permeability across the blood brain barrier according to a PAMPA assay. Moreover, both micronucleus and comet assays showed that nitroaromatic molecule 8 was not genotoxic in vitro. It was evidenced that bioactivation of molecule 8 was operated by T. b. brucei type 1 nitroreductase, in the same manner as fexinidazole. Finally, a mouse pharmacokinetic study showed that 8 displayed good systemic exposure after both single and repeated oral administrations at 100 mg/kg (NOAEL) and satisfying plasmatic half-life (T1/2 = 7.7 h). Thus, molecule 8 appears as a good candidate for initiating a hit to lead drug discovery program

Effective Gene Therapy in a Mouse Model of Prion Diseases

Classical drug therapies against prion diseases have encountered serious difficulties. It has become urgent to develop radically different therapeutic strategies. Previously, we showed that VSV-G pseudotyped FIV derived vectors carrying dominant negative mutants of the PrP gene are efficient to inhibit prion replication in chronically prion-infected cells. Besides, they can transduce neurons and cells of the lymphoreticular system, highlighting their potential use in gene therapy approaches. Here, we used lentiviral gene transfer to deliver PrPQ167R virions possessing anti-prion properties to analyse their efficiency in vivo. Since treatment for prion diseases is initiated belatedly in human patients, we focused on the development of a curative therapeutic protocol targeting the late stage of the disease, either at 35 or 105 days post-infection (d.p.i.) with prions. We observed a prolongation in the lifespan of the treated mice that prompted us to develop a system of cannula implantation into the brain of prion-infected mice. Chronic injections of PrPQ167R virions were done at 80 and 95 d.p.i. After only two injections, survival of the treated mice was extended by 30 days (20%), accompanied by substantial improvement in behaviour. This delay was correlated with: (i) a strong reduction of spongiosis in the ipsilateral side of the brain by comparison with the contralateral side; and (ii) a remarkable decrease in astrocytic gliosis in the whole brain. These results suggest that chronic injections of dominant negative lentiviral vectors into the brain, may be a promising approach for a curative treatment of prion diseases

SOURIS TRANSGENIQUES POUR LA PROTEINE PRION OVINE (TRANSMISSION D'ENCEPHALOPATHIES SUBAIGUES SPONGIFORMES TRANSMISSIBLES NATURELLES ET EXPERIMENTALES ; CONTRIBUTION A LA CARACTERISATION DES MALADIES A PRION (DOCTORAT : DIFFERENCIATION, GENETIQUE ET IMMUNOLOGIE))

LYON1-BU Santé (693882101) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Les prions

En 1986 était décrite pour la première fois la maladie de la vache folle ou encéphalopathie spongiforme bovine (ESB). Dans cet article, nous retraçons 20 ans d’histoire de cette maladie, marquée par sa transmission à l’homme il y a dix ans et par des conséquences socio-économiques majeures. En parallèle, nous faisons le point sur les avancées scientifiques récentes et les questions en suspens concernant les Prions : agents infectieux responsables de ces maladies

Cellular pathogenesis in prion diseases

Prion diseases are characterised by neuronal loss, vacuolation (spongiosis), reactive astrocytosis, microgliosis and in most cases by the accumulation in the central nervous system of the abnormal prion protein, named PrP$^{\rm Sc}$. In this review on the “cellular pathogenesis in prion diseases”, we have chosen to highlight the main mechanisms underlying the impact of PrP$^{\rm C}$/PrP$^{\rm Sc}$ on neurons: the neuronal dysfunction, the neuronal cell death and its relation with PrP$^{\rm Sc}$ accumulation, as well as the role of PrP$^{\rm Sc}$ in the microglial and astrocytic reaction

Prion diseases and adult neurogenesis: How do prions counteract the brain's endogenous repair machinery?

International audienceScientific advances in stem cell biology and adult neurogenesis have raised the hope that neurodegenerative disorders could benefit from stem cell-based therapy. Adult neurogenesis might be part of the physiological regenerative process, however it might become impaired by the disease's mechanism and therefore contribute to neurodegeneration. In prion disorders this endogenous repair system has rarely been studied. Whether adult neurogenesis plays a role or not in brain repair or in the propagation of prion pathology remains unclear. We have recently investigated the status of adult neural stem cells isolated from prion-infected mice. We were able to show that neural stem cells accumulate and replicate prions thus resulting in an alteration of their neuronal destiny. We also reproduced these results in adult neural stem cells, which were infected in vitro. The fact that endogenous adult neurogenesis could be altered by the accumulation of misfolded prion protein represents another great challenge. Inhibiting prion propagation in these cells would thus help the endogenous neurogenesis to compensate for the injured neuronal system. Moreover, understanding the endogenous modulation of the neurogenesis system would help develop effective neural stem cell-based therapies

Efficient transmission of two different sheep scrapie isolates in transgenic mice expressing the ovine PrP gene

International audienc

Florid plaques in ovine PrP transgenic mice infected with an experimental ovine BSE

The occurrence of the variant Creutzfeldt–Jakob disease (vCJD), related to bovine spongiform encephalopathy (BSE), raises the important question of the sources of human contamination. The possibility that sheep may have been fed with BSE-contaminated foodstuff raises the serious concern that BSE may now be present in sheep without being distinguishable from scrapie. Sensitive models are urgently needed given the dramatic consequences of such a possible contamination on animal and human health. We inoculated transgenic mice expressing the ovine PrP gene with a brain homogenate from sheep experimentally infected with BSE. We found numerous typical florid plaques in their brains. Such florid plaques are a feature of vCJD in humans and experimental BSE infection in macaques. Our observation represents the first description, after a primary infection, of this hallmark in a transgenic mouse model. Moreover, these mice appear to be a promising tool in the search for BSE in sheep