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Autophagy is a process leading to the degradation of cellular material, in organelles called lysosomes, to supply energy or generate building blocks for the synthesis of new materials. Over the past decades, its role has been evidenced in several indications, notably in neurodegenerative disorders and orphan diseases called lysosomal storage disorders and its modulation is largely envisioned as a therapeutic avenue to alleviate the symptoms and reverse the clinical courses of these indications. Identifying new chemical classes and drugs is, hence, of huge importance. In this study, we developed automated assays to assess the potential efficacy of chemical compounds on different steps of autophagy, notably its induction through the localization of a largely involved transcription factor, transcription factor EB (TFEB). These assays were then used to screen a collection of 1,520 approved drugs. This study led to the identification of five candidate hits modulating autophagy and TFEB subcellular localization. Our results suggest the repurposing potential of already approved drugs in central nervous system disorders with lysosomal storage impairments.

Background: There is an urgent need to understand and reverse cognitive impairment. The lack of appropriate animal models combined with the limited knowledge of pathophysiological mechanisms makes the development of new cognition-enhancing drugs complex. Scopolamine is a pharmacologic agent which impairs cognition and functional imaging in a wide range of animal species, similarly to what is seen in cognitive impairment in humans. Methods: In this study, using a functional ultrasound (fUS) neuroimaging technique, we monitored the impact of donepezil (DPZ), a potent acetylcholinesterase inhibitor and first-line treatment in patients with mild to moderate Alzheimer's disease, in a scopolamine-induced mouse model. Results: We demonstrated that despite its low impact on the cerebral blood volume (CBV) signal, scopolamine injection produced an overall decrease in functional connectivity between various brain areas. In addition, we revealed that DPZ induced a strong decrease in CBV signal without causing a difference in functional connectivity. Conclusion: Finally, our work highlighted that DPZ counteracted the impact of scopolamine on functional connectivity changes and confirmed the interest of using pharmaco-fUS imaging on cognitive disorders, both in frequent and rare neurological disorders.

International audience; Central nervous system disorders remain the leading causes of mortality and morbidity worldwide, affecting more than 1 billion patients. This therapeutic area suffers from high unmet medical needs and the search for innovative approaches to identify therapeutic strategies is urgent in the field. This review will first cover the challenges and opportunities of drug discovery in neurology, and then as well as the new models, such as human model stem cells and animal models under development in the field. In addition, innovative and translational neuroimaging techniques will be discussed, as the use of big data and artificial intelligence to discovery new drugs in neurological and neuropsychiatric disorders.

International audience; In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca 2+ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFP QL , by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.

Ten years after the launch of the Future Investment Program (Programme d'Investissement d'Avenir, PIA) and the implementation of these tools, one of Giens' roundtable workshop wanted to further explore the impact of PIA on health research and innovation with the aim of preparing action reports (bibliometrics, valuation, reputation) based on 2019 findings and the history of PIA deployment in relation to the healthcare sector; to analyze the development of the industrial sector vis-a-vis the PIA actions and to examine how the specific actions and the healthcare sector in general were able to duly articulate themselves, or, take form, given existing structures or organizations and contribute to site policies through Idex/Isite. Five success keys have been identified, which should serve as a strategic compass for future action plans to develop health innovation: Full trust governance between the project manager and the institution, driven by project objectives; An increased role of universities in the steering of PIA objects, joining together in a federation, in a site policy with the Hospital University Centres and Public Scientific and Technological Establishments; A simplification of public/private partnership schemes, in the nature of the Assessment and Action Plans, and in the responsiveness of the institutions; help with the development of local ecosystems, the fostering and support of young researchers; early cross-fertilization between the academic and industrial worlds.

Supplemental Digital Content is Available in the Text. Mefloquine/amitriptyline THN101 combination reduces cuff-induced neuropathic hypersensitivity by recruiting noradrenergic descending pathways and α2 adrenoceptors and by blocking connexin in mice., Tricyclic antidepressants that inhibit serotonin and noradrenaline reuptake, such as amitriptyline, are among the first-line treatments for neuropathic pain, which is caused by a lesion or disease affecting the somatosensory nervous system. These treatments are, however, partially efficient to alleviate neuropathic pain symptoms, and better treatments are still highly required. Interactions between neurons and glial cells participate in neuropathic pain processes, and importantly, connexins—transmembrane proteins involved in cell–cell communication—contribute to these interactions. In a neuropathic pain model in rats, mefloquine, a connexin inhibitor, has been shown to potentiate the antihyperalgesic effect of amitriptyline, a widely used antidepressant. In this study, we further investigated this improvement of amitriptyline action by mefloquine, using the cuff model of neuropathic pain in mice. We first observed that oral mefloquine co-treatment prolonged the effect of amitriptyline on mechanical hypersensitivity by 12 hours after administration. In addition, we showed that this potentiation was not due to pharmacokinetic interactions between the 2 drugs. Besides, lesional and pharmacological approaches showed that the prolonged effect was induced through noradrenergic descending pathways and the recruitment of α2 adrenoceptors. Another connexin blocker, carbenoxolone, also improved amitriptyline action. Additional in vitro studies suggested that mefloquine may also directly act on serotonin transporters and on adenosine A1 and A2A receptors, but drugs acting on these other targets failed to amplify amitriptyline action. Together, our data indicate that pharmacological blockade of connexins potentiates the therapeutic effect of amitriptyline in neuropathic pain.

Background Recent advances using functional ultrasound (fUS) imaging have opened new avenues to evaluate brain activity through the regional monitoring of cerebral blood volume (CBV) dynamics. In particular, this technology paves the way for understanding physiological or pathological cerebral processes or exploring the pharmacological profiles of new drugs targeting brain disorders. One of the main difficulties of this technology is the lack of standardized and validated tools, in particular relevant brain atlases, to help improving the accuracy, automation and reproducibility of fUS data analysis. New method Here, we demonstrate the possibility to use the MRI-validated SIGMA brain atlas in rat to perform fast and precise analysis of CBV changes in numerous functionally relevant regions of interest using fUS imaging. We applied this atlas to a dataset obtained in anesthetized rats evaluating the cerebral effects of atomoxetine, a norepinephrine reuptake inhibitor currently marketed in attention-deficit/hyperactivity-disorder. Results This approach enabled to show the subregional effects of atomoxetine in the rat with very few inter-individual differences in some areas, such as the dentate gyrus. Conclusions We show the feasibility of inter-individual registration of 2D pharmaco-fUS data and subsequent detailed analysis using the SIGMA atlas.

TFEB is a mammalian transcription factor that binds directly to the CLEAR consensus sequence (5'-GTCACGTGAC-3') present in the regulatory regions of genes inducing autophagosome formation, autophagosome-lysosome fusion, hydrolase enzyme expression, and lysosomal exocytosis. By modulating these activities, TFEB coordinates on-demand control over each cell's degradation pathway. Thus, a nuclear signaling pathway regulates cellular energy metabolism through TFEB. Our growing understanding of the role of TFEB and CLEAR in the promotion of healthy clearance together with in vitro and in vivo preclinical findings in various animal models of disease supports the conclusion that the pharmacological activation of TFEB could clear toxic proteins to treat both rare and common forms of neurodegenerative disease.

International audience; Highlights• A first evaluation of the potential of fUS during a pharmacological challenge.• ATX modulates cerebral blood volume in areas associated with visual processing.• fUS as a new step to move forwards new drugs to development in neurological disorders.Functional ultrasound (fUS) is a new tool enabling the imaging of brain activity through the regional monitoring of cerebral blood volume (CBV) dynamics. This innovative technique has not yet demonstrated its full potential in pharmacological applications and drug development. In the current proof-of-concept study, the impact of atomoxetine (ATX), a potent norepinephrine reuptake inhibitor and non-stimulant treatment marketed in attention-deficit/hyperactivity-disorder, was evaluated in anesthetized rat using pharmacological functional ultrasound (pharmaco-fUS) at increasing doses (0.3, 1 and 3 mg/kg). Using regions of interest (acute changes of CBV and functional connectivity) or pixel-based (general linear modeling and independent component analysis) analysis, we here demonstrated that ATX consistently displayed a hemodynamic effect in the visual cortex, the dentate gyrus and thalamus, especially visual areas such as lateral posterior thalamic nuclei and lateral geniculate nuclei (LGN). The time profile of ATX effects was dose-dependent, with fastest CBV increases at the highest dose, and longer CBV increases at the intermediate dose. Standardizing the use of pharmaco-fUS could improve our understanding of the mechanism of action of drugs active in the brain and might constitute a new step to move forward in drug development for neurological disorders.

In late 2021, the health technology assessment of the French National Authority for Health was seized by the French Ministry of Solidarity and Health to address a specific challenge, the identification of solutions contributing to the development of the methodological expertise in new types of clinical studies for rare diseases. Experts from the rare diseases environment were gathered by OrphanDev, the French network of expertise dedicated to rare diseases. They allowed to identify some of the current issues in France concerning rare diseases, and then present different solutions, in particular related to the evaluation process of orphan drugs and the collection of data on rare diseases.

Astrocytes are glial cells organized in dynamic and structured networks in the brain. These plastic networks, involving key proteins such as connexin 43 (Cx43), are engaged in fine neuronal tuning and have recently been considered as emerging therapeutic targets in central nervous system disorders. We developed and validated a new application of the manganese-enhanced magnetic resonance imaging (MEMRI) technique allowing in vivo investigations of astrocyte-neuron interactions through quantification of brain Cx43 functional activity. The proof of concept has been achieved by quantification of MEMRI signals in brain after either local astrocyte-specific Cx43 knockdown with shRNA or systemic administration of Cx43 blockers. Unilateral hippocampal Cx43 genetical silencing was associated with an ipsilateral local increase of MEMRI signal. Furthermore, Cx43 blockers also enhanced MEMRI signal responses in hippocampus. Altogether, these data reveal the MEMRI technique as a tool for quantitative imaging of in vivo Cx43-dependent function in astrocytes under physiological and pathological conditions.

International audience; Background: Modafinil, a nonamphetaminic wake-promoting compound, is prescribed as first line therapy in narcolepsy, an invalidating disorder characterized by excessive daytime sleepiness and cataplexy. Although its mode of action remains incompletely known, recent studies indicated that modafinil modulates astroglial connexin-based gap junctional communication as administration of a low dose of flecainide, an astroglial connexin inhibitor, enhanced the wake-promoting and procognitive activity of modafinil in rodents and healthy volunteers. The aim of this study is to investigate changes in glucose cerebral metabolism in rodents, induced by the combination of modafinil+flecainide low dose (called THN102).Methods: The impact of THN102 on brain glucose metabolism was noninvasively investigated using 18F-2-fluoro-2-deoxy-D-glucose Positron Emission Tomography imaging in Sprague-Dawley male rats. Animals were injected with vehicle, flecainide, modafinil, or THN102 and further injected with 18F-2-fluoro-2-deoxy-D-glucose followed by 60-minute Positron Emission Tomography acquisition. 18F-2-fluoro-2-deoxy-D-glucose Positron Emission Tomography images were coregistered to a rat brain template and normalized from the total brain Positron Emission Tomography signal. Voxel-to-voxel analysis was performed using SPM8 software. Comparison of brain glucose metabolism between groups was then performed.Results: THN102 significantly increased regional brain glucose metabolism as it resulted in large clusters of 18F-2-fluoro-2-deoxy-D-glucose uptake localized in the cortex, striatum, and amygdala compared with control or drugs administered alone. These regions, highly involved in the regulation of sleep-wake cycle, emotions, and cognitive functions were hence quantitatively modulated by THN102.Conclusion: Data presented here provide the first evidence of a regional brain activation induced by THN102, currently being tested in a phase II clinical trial in narcoleptic patients.

Gap junctions (GJs) are dynamic structures composed of hexamers of connexins (Cxs), a class of transmembrane proteins enabling channel-mediated direct intercellular communication through cell-cell diffusion of ions and small metabolites. In defined conditions, Cxs also work as hemichannels allowing exchanges between the cytoplasm and the extracellular medium. The most common GJ channel is formed by connexin 43 (Cx43) and plays an important role in physiological and pathological processes in excitable tissues, such as heart and brain. Hence, Cx43 has been largely envisioned as a new therapeutic target in cancer, neurological and psychiatric indications, or cardiovascular diseases. Identifying new pharmacological inhibitors of Cx43 GJs with different mechanisms of action and from diverse chemical classes is thus highly challenging. We present here a high-content screening method, based on the evaluation of fluorescent dye transfer rates between adjacent cells to monitor the function of GJs in U251 glioblastoma cells expressing high levels of Cx43. This assay was validated using well-described pharmacological GJ inhibitors such as mefloquine. The method was adapted to screen a library of 1,280 Food and Drug Administration- and European Medicines Agency-approved drugs that led to the selection of both known and new inhibitors of GJ channel function. We further focused on a specific class of microtubule-targeting agents, confirming that a proper tubulin network is required for functional Cx43 GJ channels.

Antidepressants, prescribed as first line treatment of neuropathic pain, have a limited efficacy and poorly tolerated side effects. Because recent studies pointed out the implication of astroglial connexins (Cx) in both neuropathic pain and antidepressive treatment, we investigated whether their blockade by mefloquine could modulate the action of the tricyclic antidepressant amitriptyline. Using primary cultures, we found that both mefloquine and amitriptyline inhibited Cx43-containing gap junctions, and that the drug combination acted synergically. We then investigated whether mefloquine could enhance amitriptyline efficacy in a preclinical model of neuropathic pain. Sprague-Dawley rats that underwent chronic unilateral constriction injury (CCI) to the sciatic nerve (SN) were treated with either amitriptyline, mefloquine or the combination of both drugs. Whereas acute treatments were ineffective, chronic administration of amitriptyline reduced CCI-SN-induced hyperalgesia-like behavior, and this effect was markedly enhanced by co-administration of mefloquine, which was inactive on its own. No pharmacokinetic interactions between both drugs were observed and CCI-SN-induced neuroinflammatory and glial activation markers remained unaffected by these treatments in dorsal root ganglia and spinal cord. Mechanisms downstream of CCI-SN-induced neuroinflammation and glial activation might therefore be targeted. Connexin inhibition in astroglia could represent a promising approach towards improving neuropathic pain therapy by antidepressants.

International audience; Misfolding of the prion protein (PrP) is the central feature of prion diseases. The conversion of the normal α-helical PrP(C) into a pathological β-enriched PrP(Sc) constitutes an early event in the infectious process. Several hypotheses, involving different regions of the protein, endeavor to delineate the structural mechanism underlying this change of conformation. All current working hypotheses, however, are based on biophysical and modeling studies, the biological relevance of which still needs to be assessed. We have studied the effect of positively charged polymers on the conversion, using polylysine as a model system, and have investigated a possible mechanism of structural stabilization. We have shown that poly-D-lysine removes proteinase K-resistant PrP from prion-infected SN56 neuroblastoma cells without affecting PrP(C). The effect is enantiospecific since the levorotary isomer, poly-L-lysine, has a markedly weaker effect, likely because of its higher susceptibility to degradation. In vitro cross-linking and NMR studies confirm a direct interaction between polylysine and PrP, which mainly maps to the PrP region containing helices 2 and 3 (H2H3). Interaction prevents conformational conversion and protein aggregation. Our results establish a central role of H2H3 in PrP(Sc) amyloidogenesis and replication and provide biological relevance for the pathological misfolding of this domain.

International audience; Models for the polymerization process involved in prion self-replication are well-established and studied [H. Engler, J. Pruss, and G.F. Webb, Analysis of a model for the dynamics of prions II, J. Math. Anal. Appl. 324 (2006), pp. 98-117; M.L. Greer, L. Pujo-Menjouet, and G.F. Webb, A mathematical analysis of the dynamics of prion proliferation, J. Theoret. Biol. 242 (2006), pp. 598-606; J. Pruss, L. Pujo-Menjouet, G.F. Webb, and R. Zacher, Analysis of a model for the dynamics of prions, Discrete Cont. Dyn. Sys. Ser. B 6(1) (2006), pp. 215-225] in the case where the dynamics coefficients do not depend on the size of polymers. However, several experimental studies indicate that the structure and size of the prion aggregates are determinant for their pathological effect. This motivated the analysis in Calvez et al. [Size distribution dependence of prion aggregates infectivity, Math Biosci. 217 (2009), pp. 88-99] where the authors take into account size-dependent replicative properties of prion aggregates. We first improve a result concerning the dynamics of prion aggregates when a pathological state exists (high production of the normal protein). Then we study the strain phenomena and more specifically we wonder what specific replicative properties are determinant in strain propagation. We propose to interpret it also as a dynamical property of size repartitions.

STUDY OBJECTIVES: Modafinil is a non-amphetaminic wake-promoting compound used as therapy against sleepiness and narcolepsy. Its mode of action is complex, but modafinil has been recently proposed to act as a cellular-coupling enhancer in glial cells, through modulation of gap junctions constituted by connexins. The present study investigated in mice the impact of connexins on the effects of modafinil using connexin inhibitors. METHODS: Modafinil was administered alone or combined with inhibitors of astrocyte connexin, meclofenamic acid, or flecainide, respectively, acting on Cx30 and Cx43. Sleep-wake states were monitored in wild-type and narcoleptic orexin knockout mice. A spontaneous alternation task was used to evaluate working memory in wild-type mice. The effects of the compounds on astroglial intercellular coupling were determined using dye transfer in acute cortical slices. RESULTS: Meclofenamic acid had little modulation on the effects of modafinil, but flecainide enhanced the wake-promoting and pro-cognitive effects of modafinil. Co-administration of modafinil/flecainide resulted in a marked decrease in the number and duration of direct transitions to rapid eye movement sleep, which are characteristic of narcoleptic episodes in orexin knockout mice. Furthermore, modafinil enhanced the connexin-mediated astroglial cell coupling, whereas flecainide reduced it. Finally, this modafinil-induced effect was reversed by co-administration with flecainide. CONCLUSIONS: Our study indicates that flecainide impacts the pharmacological effects of modafinil, likely through the normalization of Cx30-dependent gap junctional coupling in astroglial networks. The enhancement of the wake-promoting, behavioral, and cognitive outcomes of modafinil demonstrated here with flecainide would open new perspectives in the management of sleep disorders such as narcolepsy. COMMENTARY: A commentary on this article appears in this issue on page 1175.

Prions are in many ways a real biological enigma, given that the exact nature of the pathogen is still unclear. Bovine Spongiform Encephalopathy (BSE) in Great-Britain, and its related disorder the variant form of Creutzfeldt-Jakob Disease (v-CJD) represent major challenges in terms of animal and human health, given the risks of food-borne contamination, and of secondary man-to-man contamination. Improved knowledge of the biology of these agents has led to major progress over the past few years, notably in the field of diagnosis, decontamination and risk analysis, thereby improving the management of these diseases., Les prions constituent par bien des points une énigme biologique. Notamment, leur nature exacte en tant qu’agent infectieux n’est pas définie précisément. L’Encéphalopathie Spongiforme Bovine (ESB) en Grande-Bretagne, et son corollaire chez l’Homme, la variante de la Maladie de Creutzfeldt-Jakob (v-MCJ), représentent des défis importants en termes de santés animale et humaine, compte tenu des risques de contaminations par voie alimentaire d’une part, et de contaminations secondaires interhumaines d’autre part. La meilleure connaissance de la biologie de ces agents a permis des avancées pratiques importantes ces dernières années, notamment en termes de diagnostic, de décontamination et d’évaluation des risques, permettant ainsi une meilleure gestion de ces maladies., Comoy Emmanuel, Mouthon Franck, Deslys Jean-Philippe. Biologie des prions : de l'énigme moléculaire aux risques actuels. In: Bulletin de l'Académie Vétérinaire de France tome 158 n°4, 2005. pp. 407-414.

In multiple sclerosis (MS), the T-cell receptors (TCRS) of autoreactive T lymphocytes recognize various myelin components or derivatives including peptides of the myelin basic protein (MBP). Using the exhaustive immunoscope approach we showed that the T-cell repertoires of MS patients differ from those of healthy controls, with expansion of Vbeta13 cell clones in cerebrospinal fluid (CSF) and in peripheral blood lymphocytes (PBLs). Sequencing of the beta13(+) chains of T cells recovered from the CSF revealed high interindividual diversity, and no particular Vbeta13(+) rearrangements were shown to be myelin-autoreactive. Within the overall Vbeta13 repertoire in the CSF of patient MS3 at the onset of the disease, most of the overrepresented (N)Dbeta(N)Jbeta junctional regions were found to be associated with two or three different Vbeta13 segments. These rearrangements were most common in the PBLs of patient MS3. No such associations were detected in the Vbeta5 multigene family that was used as a control. Thus, Vbeta13 T cells infiltrating the CSF from patient MS3 may have been selected on the basis of both the Vbeta13 segments and the (N)Dbeta(N)Jbeta junctional CDR3 sequence.

An RT-PCR-hybridization was developed that amplified genetic material from the M protein gene of HCoV-229E and HCoV-OC43. The analytic sensitivity of these original primers were compared with primers defined in the N gene and described previously. The results show that 0.05 TCID50 of HCoV-229E and 0.01 TCID50 of HCoV-OC43 can be detected by this molecular method using the original method. Detection of HCoV-229E and HCoV-OC43 in clinical specimens is possible using this method: 348 respiratory specimens (202 sputum and 146 nasal aspirates) were tested with this RT-PCR-hybridization and 12 human coronavirus are detected (3%). The method could provide a useful tool for demonstrating the role of human coronavirus in infections of the respiratory tract.

The study of the prion protein (PrP) physiological functions or its specific role in transmissible spongiform encephalopathies (TSE) requires new tools, particularly those able to induce PrP overexpression in a large range of cells, in vivo as well as in vitro. Here we describe the construction of two recombinant adenoviruses encoding the human PrP either with a valine at position 129 (AdTRVal) or a methionine (AdTRMet). Both genes were put under the control of the tetracycline-responsive promoter, allowing tight regulation of PrP expression. AdTRVal and AdTRMet induced high expression of the human PrP in CHO-KI cells and in organotypic brain slices in culture. The proteins expressed from these viruses exhibited a glycosylphosphatidyl inositol (GPI) anchor, proper glycosylation and sensitivity to proteinase K digestion. AdTRVal and AdTRMet will allow future studies on the human PrP and on the role of the codon 129 polyphormism in human TSE.

Spongiform transmissible encephalopathies are neurodegenerative diseases characterized by the accumulation, in infected brains, of a pathological form of a normal host-encoded protein called PrP. Previous data have shown that PrP could interact with cytosolic factors, including nuclear molecules, emphasizing the possible function of such interactions. Moreover, in infected cells, PrP is observed not only at the plasma membrane but also in the nuclear compartment. The N-terminal extremity of the mature PrP has been thought to harbor a nuclear localization signal reminiscent of the nuclear localization signal of the simian virus 40 large T antigen. By designing a fusion protein between the putative nuclear localization signal of PrP and the green fluorescent protein, we have shown that the N-terminal sequence of PrP is not efficient in targeting the protein in the nuclear compartment. This implies new insights regarding the way by which PrP could, however, reach the nuclear compartment.

Connexins are transmembrane proteins involved in gap junction intercellular communication. They present cell- and tissue-specific expression, with own electric and metabolic coupling specificities. These proteins are involved in numerous physiological processes in the brain and among them neuronal synchronization and trafficking of glucose. Such proteins are also described as being misregulated in various pathologies in the central nervous system. Thus, connexin blockers have been proposed as pharmacological tools to dissect these implications. However, such approaches lack accurate characterization of known inhibitors toward gap junction isoform specificity. In addition, those compounds are limited to few chemical classes and exhibit other activities, for example, an anti-inflammatory effect. The aims of this study were to evaluate the selectivity of described inhibitors and to enrich this pharmacopeia by new chemical classes. In this study, we present the specificity of published inhibitors toward several connexin isoforms expressed in the brain. Furthermore, after a screening of compounds using cellular models, we identified seven new inhibitors, with high functional reversibility and different relative selectivity toward isoforms. They constitute new chemical classes of connexin modulators completing those previously described. These new inhibitors might also provide new insights in understanding numerous pathophysiological processes involving gap junctions.

At present, there is no effective therapy for any of the neurodegenerative amyloidoses, despite renewed efforts to identify compounds active against the various implicated pathogenetic molecules. We have screened a library of 2960 natural and synthetic compounds in two cell lines chronically infected with mouse prions, and have identified eight new inhibitors of prion replication in vitro. They belong to two distinct chemical families that have not previously been recognised as effective in the field of transmissible spongiform encephalopathies: seven are 3-aminosteroids and one is a derivative of erythromycin A with an oxime functionality. Our results suggest that these aminosteroids inhibit prion replication by triggering a common target, possibly implicated in the regulatory pathways of cellular prion protein metabolism. Furthermore, using a quantitative approach for the study of protein stability, it was shown that the erythromycin A derivative altered prion protein stability by direct interaction. Such direct targeting of this amyloid precursor might provide new clues for the understanding of prion diseases and, more importantly, help to define new molecules that are active against prion diseases.

International audience; We consider a model for the polymerization (fragmentation) process involved in infectious prion self-replication and study both its dynamics and non-zero steady state. We address several issues. Firstly, we extend a previous study of the nucleated polymerization model [M.L. Greer, L. Pujo-Menjouet, G.F. Webb, A mathematical analysis of the dynamics of prion proliferation, J. Theoret. Biol. 242 (2006) 598; H. Engler, J. Pruss, G.F. Webb, Analysis of a model for the dynamics of prions II, J. Math. Anal. Appl. 324 (2006) 98] to take into account size dependent replicative properties of prion aggregates. This is achieved by a choice of coefficients in the model that are not constant. Secondly, we show stability results for this steady state for general coefficients where reduction to a system of differential equations is not possible. We use a duality method based on recent ideas developed for population models. These results confirm the potential influence of the amyloid precursor production rate in promoting amyloidogenic diseases. Finally, we investigate how the converting factor may depend upon the aggregate size. Besides the confirmation that size-independent parameters are unlikely to occur, the present study suggests that the PrPsc aggregate size repartition is amongst the most relevant experimental data in order to investigate this dependence. In terms of prion strain, our results indicate that the PrPsc aggregate repartition could be a constraint during the adaptation mechanism of the species barrier overcoming, that opens experimental perspectives for prion amyloid polymerization and prion strain investigation.

Summary The uncertain extent of human exposure to bovine spongiform encephalopathy (BSE)—which can lead to variant Creutzfeldt-Jakob disease (vCJD)—is compounded by incomplete knowledge about the efficiency of oral infection and the magnitude of any bovine-to-human biological barrier to transmission. We therefore investigated oral transmission of BSE to non-human primates. We gave two macaques a 5 g oral dose of brain homogenate from a BSE-infected cow. One macaque developed vCJD-like neurological disease 60 months after exposure, whereas the other remained free of disease at 76 months. On the basis of these findings and data from other studies, we made a preliminary estimate of the food exposure risk for man, which provides additional assurance that existing public health measures can prevent transmission of BSE to man. Published online January 27, 2005 http://image.thelancet.com/extras/05let1056web.pdf

Experimental infection of macaques with pathogenic strains of simian immunodeficiency virus (SIV) represents one of the most relevant animal models for studying HIV pathogenesis. In this study, we demonstrated a significant decrease in the generation of CD4+ T cells from bone marrow (BM) CD34+ progenitors in macaques infected with SIVmac251. This decrease appears to result from changes in the clonogenic potential of BM progenitors of both the myeloid and lymphoid lineages. We also demonstrated a significant decrease in the numbers of the most immature long-term culture-initiating cells (LTC-ICs). Hematopoietic failure occurred as early as primary infection, in the absence of CD34+ BM cell infection and was not related to plasma viral load. No major change was observed in the phenotype of BM CD34+ cells from infected macaques, including apoptosis markers such as annexin V staining and BcL-2 expression, but a significantly higher that normal proportion of cells were in the G0/G1 phase. This is the first demonstration that failure of BM hematopoiesis results in impaired T-cell production, which may contribute to the disruption of T-lymphocyte homeostasis characteristic of pathogenic lentiviral infections in primates.

We report the course of a French cohort of patients treated with growth hormone purified from human hypophyses (hGH) contaminated with the Creutzfeldt-Jakob disease (CJD) agent. 51 of the 54 confirmed probable cases of CJD in this cohort have been studied by methods that include allele-specific oligonucleotide hybridisation and sequencing of the open reading frame of the PRNP gene. The distribution of genotype at codon 129 is shown in the figure. In the Met/Met and Val/Val groups, the number of cases peaked in 1992, 8 years after growth-hormone treatment. In the Met/Val group (six cases) the earliest was identified in 1994, 5 years after the first patients in the RESEARCH LETTERS