No description available.

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This study demonstrates the direct delivery of plasmid gene constructs into spinal motor neurons utilizing retrograde axoplasmic transport. The plasmid vectors contained the Lac Z gene under the control of both the Rous sarcoma virus (RSV) and Simian virus (SV)40 promoters. β-Galactosidase expression was observed in α and γ motor neurons by histochemical staining following direct injection into the sciatic nerve or gastrocnemius muscle. The presence of LacZ gene constructs was confirmed by the polymerase chain reaction (PCR). The ability to introduce gene constructs into motor neurons allows for the study of gene regulation and permits the development of gene therapy strategies for motor neuron diseases including the spinal muscular atrophies (SMA) and amyotrophic lateral sclerosis (ALS).

No description available.

Vaccinia virus (VACV), the prototypic poxvirus, undergoes a complex life cycle, with multiple stages that are not yet fully understood. This work studied two cellular proteins which had previously been identified by siRNA screens as playing proviral roles in the replication cycle of VACV: the dual specificity mitogen-activated protein kinase kinase 3 (MKK3) and vacuolar protein sorting 52 (Vps52). MKK3 is an upstream regulator in the p38 pathway which, along with MKK6, phosphorylates and therefore activates p38. In HeLa cell cultures, siRNA depletion experiments confirmed that MKK3 supported VACV replication. MKK3 knockdown reduced production of both early and late-class VACV proteins, suggesting that it facilitates viral gene expression. However, this difference did not translate to an in vivo model, as comparison between wild type and MKK3 knockout mice infected with VACV revealed no significant differences in virus replication or overall disease. The Golgi-associated retrograde protein complex (GARP) is composed of four large heteromeric proteins: Vps51, Vps52, Vps53 and Vps54, and plays a key role in retrograde transport from endosomes to the TGN. The effects of loss of GARP function were investigated using three techniques: mouse embryonic fibroblasts (MEFs) containing the hypomorphic Vps54 “wobbler” mutation, Vps52-targetting siRNA in HeLa cells and pharmacological inhibition of retrograde transport using the drug Retro-2. GARP loss resulted in a marked reduction in VACV spread due to a reduction specifically in “double wrapped” extracellular enveloped virion (EEV) production. Investigation of the mechanism by which GARP facilitates EEV production revealed a disruption of the VACV morphogenesis pathway prior to the double wrapping event, resulting in mislocalisation and aggregation of the viral membrane protein B5 within the cytoplasm. The effects of GARP loss translated to an in vivo model, as mice infected with VACV and treated with Retro-2 exhibited reduced viral replication and overall disease. These results identify GARP as a pro-viral host complex required for EEV production, and suggest that cellular retrograde transport pathways are required for double-wrapping of VACV virions. Overall, the study illustrates both the potential pitfalls of carrying out genetic screens in a transformed cell line and the power of such studies to nevertheless identify novel features of virus biology as well as druggable targets for antiviral intervention.

Chez la levure et les mammifères le complexe rétromère est impliqué dans différentes étapes de trafic intracellulaire qui modulent divers processus cellulaires et développementaux. Chez les mammifères, le rétromère se compose des protéines Vacuolar Protein Sorting (VPS) 35, VPS26, VPS29 et d’un dimere de Sorting Nexins (SNX). Les composants du rétromère sont conservés chez les plantes et sont localisés dans le même endosome de tri. Dans ce travail, nous avons étudié le mécanisme d’assemblage et de recrutement du complexe à la membrane et analysé le rôle du rétromère dans le développement d’Arabidopsis. Nos resultsts montrent que chez les plantes, contrairement aux mammifères, VPS35 recrute le sous-complexe VPS à la membrane, indépendamment des SNXs. Ceci nous a permis de proposer un modèle d’assemblage du rétromère très original. L’analyse de mutants perte de fonction pour le rétromère révèle que VPS26, VPS29 et VPS35 n’ont pas de fonctions indépendantes, mais agissent ensemble dans la régulation de processus développementaux. De plus, par un crible double hybride chez la levure, nous avons isolé un interacteur de VPS35 : Ethylene Insensitive 2 (EIN2). EIN2 est une protéine localisée au Réticulum Endoplasmique et impliquée dans la voie de signalisation d’une hormone végétale : l’éthylène. Nous montrons que des mutants perte de fonction pour le rétromère présentent des défauts dans la réponse à l’éthylène, indiquant un rôle du rétromère dans la perception de cette hormone par les plantes. Ces résultats, combinés avec nos données antérieures montrant que le rétromère est impliqué dans la voie de signalisation de l’auxine, révèlent un lien entre signalisation de l’auxine et de l’éthylènevia le complexe rétromère. / In yeast and mammals, the retromer complex mediates various steps of intracellular trafficking and regulates a variety of cellular and developmental processes. In mammals, it is composed of Vacuolar Protein Sorting (VPS) 35, VPS26, VPS29 and a dimer of Sorting Nexins (SNX). Retromer components are conserved in plants and we showed that they colocalize to the same sorting endosome. In this work, we have investigated the mechanism of assembly and recruitment of the retromer to the endosomal membrane and studied its function in Arabidopsis development. We report that, unlike animals, plant VPS35 recruits the other VPS retromer components to the membrane of endosomes, independently of SNXs. This data allowed us to propose an original model of assembly of the plant retromer complex. By analyzing a series of retromer loss-of-function mutants, we show that VPS26, VPS29 and VPS35 always act together in modulating developmental processes. To identify retromer partners, we carried out a Yeast-2-Hybrid (Y2H) screen using VPS35 as a bait. We found that VPS35 can bind, among other proteins, Ethylene Insensitive 2 (EIN2). EIN2 is an Endoplasmic Reticulum-located protein involved in the signaling pathway of a plant hormone: ethylene. We demonstrate that retromer mutants are affected in ethylene signaling, which indicates that the retromer complex participates in a proper perception of ethylene by plants. Combined with our previous data in which we showed that the retromer is involved in the auxin signalling pathway, our present work reveals a link between auxin and ethylene signaling through the retromer complex.

The vagal postganglionic control of cardiac rate is mediated by two intracardiac ganglia, i.e., the sinoatrial (SA) and posterior atrial (PA) ganglia. Nothing is known about the vagal preganglionic neurons (VPNs) that innervate the PA ganglion or about the neurochemical anatomy of central afferents that innervate these VPNs. These issues were examined using light microscopic retrograde labeling methods and dual-labeling electron microscopic histochemical and immunocytochemical methods. VPNs projecting to the PA ganglion are found in a narrow column exclusively in the ventrolateral nucleus ambiguus (NA-VL). These neurons are relatively large (37.6 ± 2.7 μm by 21.3 ± 3.4 μm) with abundant cytoplasm and intracellular organelles, rare somatic and dendritic spines, round uninvaginated nuclei, and myelinated axons. Previous physiological data indicated that microinjections of neuropeptide Y (NPY) into the NA-VL cause negative chronotropic effects. The present morphological data demonstrate that NPY-immunoreactive nerve terminals formed 18 ± 4% of the axodendritic or axosomatic synapses and close appositions on VPNs projecting to the PA ganglion. Three approximately equal populations of VPNs in the NA-VL were retrogradely labeled from the SA and PA ganglia. One population each projects to the SA ganglion, the PA ganglion, or to both the SA and PA ganglia. Therefore, there are both shared and independent pathways involved in the vagal preganglionic controls of cardiac rate. These data are consistent with the hypothesis that the central and peripheral parasympathetic controls of cardiac rate are coordinated by multiple potentially redundant and/or interacting pathways and mechanisms.

Intracardiac pathways mediating the parasympathetic control of various cardiac functions are incompletely understood. Several intracardiac ganglia have been demonstrated to potently influence cardiac rate [the sinoatrial (SA) ganglion], atrioventricular (AV) conduction (the AV ganglion), or left ventricular contractility (the cranioventricular ganglion). However, there are numerous ganglia found throughout the heart whose functions are poorly characterized. One such ganglion, the posterior atrial (PA) ganglion, is found in a fat pad on the rostral dorsal surface of the right atrium. We have investigated the potential impact of this ganglion on cardiac rate and AV conduction. We report that microinjections of a ganglionic blocker into the PA ganglion significantly attenuates the negative chronotropic effects of vagal stimulation without significantly influencing negative dromotropic effects. Because prior evidence indicates that the PA ganglion does not project to the SA node, we neuroanatomically tested the hypothesis that the PA ganglion mediates its effect on cardiac rate through an interganglionic projection to the SA ganglion. Subsequent to micro-injections of the retrograde tracer fast blue into the SA ganglion, >70% of the retrogradely labeled neurons found within five intracardiac ganglia throughout the heart were observed in the PA ganglion. The neuroanatomic data further indicate that intraganglionic neuronal circuits are found within the SA ganglion. The present data support the hypothesis that two interacting cardiac centers, i.e., the SA and PA ganglia, mediate the peripheral parasympathetic control of cardiac rate. These data further support the emerging concept of an intrinsic cardiac nervous system.

Mouse polyomavirus (MPyV) is a model virus of the Polyomaviridae family. Polyomaviruses are small non-enveloped DNA viruses. They cause severe problems to immunocompromised patients. Their oncogenic potential is known in animals and humans. Trafficking of MPyV within the cell is not clear yet. The virus enters via smooth monopinocytic vesicles and continues to early and late endosomes. From there, the virus is transported to the ER by unknown mechanism. It bypasses Golgi aparatus (GA). One possible pathway is from late endosomes to trans-Golgi network (TGN) facilitated by Rab9 GTPase and then in COPI vesicles to the ER. In this thesis, the effect of inhibitors of retrograde transport (Brefeldin A, Golgicide A) on MPyV infection was evaluated. Brefeldin A is not completely specific; it has effect on whole endosomal system. Golgicide A causes specific disruption of transport via TGN and GA. Both inhibitors suppressed infection of MPyV. Confocal microscopy revealed colocalization of some MPyV virions with markers of TGN and COPI vesicles. MPyV didn't colocalize with cis-Golgi marker. Unfortunately, the effect of overexpression of Rab9 dominant negative mutant couldn't been evaluated due to its high cytotoxicity. However, overexpression of wild type Rab9 slightly increased infectivity. The results...

Les toxines Shiga (Stx) sont produites par Shigella dysenteriae et certaines espèces d’E. coli transmisent aux humains par la consommation d'aliments contaminés et causant des maladies graves. La toxine Stx est libérée par les bactéries dans l'intestin et par la suite, traverse les vaisseaux sanguins en aval pour atteindre leurs principaux organes cibles, notamment les reins. Les dommages causés aux reins peuvent entraîner des complications graves notamment Le syndrome hémolytique urémique (SHU). A ce jour, il n’existe aucun traitement disponible contre le SHU. Les toxines Stx usent du transport rétrograde intracellulaire pour infester les cellules endothéliales rénales et atteindre leur cible cytosolique, l'ARN ribosomal 28S. Via un screening à haut débit, il a été démontré que le composé Rétro-2 bloque le trafic rétrograde de Stx à l'interface Endosome-TGN, sans affecter la morphologie des organites cellulaires et le trafic des protéines endogènes. Au cours de cette thèse, une analyse des relations structure fonction du composé Retro-2 nous a permis d’identifier les régions de l'inhibiteur qui sont critiques pour l'activité de protection. Nous avons identifié un dérivé dihydroquinazolinone nommé Rétro-2.1 qui est à ce jour l'inhibiteur le plus puissant contre les toxines Stx. Afin d’identifier la cible moléculaire de Retro-2.1, nous avons développé des sondes photo-activables bio-actives. En outre, les données de diffraction des rayons X ont révélé que de l'activité antitoxine réside principalement dans l’énantiomère S. (S) -Retro-2.1 est 500 fois plus puissant contre Stx (50 nM) que la molécule initiale. Cette étude peut donner lieu à un nouveau concept thérapeutique ciblant la voie de transport rétrograde de la toxine à l'intérieur de la cellule hôte. Une telle stratégie thérapeutique pourrait donc être étendue à d'autres agents pathogènes qui usent également du trafic rétrograde pour une intoxication des cellules hôtes. Ce nouveau concept thérapeutique qui permet de cibler les cellules hôtes et non l'agent pathogène représente une véritable percée dans la découverte de médicaments à large spectre et réduit le risque de développement d’une résistance chez l’agent pathogène. / Shiga toxins (Stx) are produced by Shigella dysenteriae and certain species of E. coli that can be transmitted to humans primarily through consumption of contaminated foods and may cause severe disease. Stx is released by the bacteria in the intestine and subsequently, could cross the downstream blood vessels to reach their main target organs such as kidney. Damage to the kidney can result in serious life-threatening complication hemolytic uremic syndrome, for which there is no proven safe treatment available other than supportive care. Stx invades renal endothelial cells in a retrograde manner from cell surface to the endoplasmic reticulum in order to gain access to its cytosolic target, 28S rRNA. By using HTS, it was previously demonstrated that the compound Retro-2 blocks retrograde trafficking of Stx at the early endosome-TGN interface, without affecting the morphology of cellular organelles and trafficking of other endogenous proteins. In this work, different regions of the lead inhibitor Retro-2 that are critical for the protective activity have been determined by systematic structure-activity relationship studies. It allowed us to identify a dihydroquinazolinone derivative, named Retro-2.1 that is the most potent inhibitor of Stx to date and also to develop bio-active photo-activatable probes with the aim of identifying the molecular target of Retro-2 derivatives. Further, crystal X-ray diffraction data revealed that the antitoxin activity resides mainly in the S-enantiomer. (S)-Retro-2.1 has displayed 500 fold more potency (50 nM) than parent molecule against Stx cytotoxicity. This study may result in a new therapeutic concept - targeting the retrograde transport route of toxin inside host cell - for the treatment of Stx-producing E. coli infections and could therefore be extended to other pathogens that also traffic via the retrograde transport. Such a new therapeutic concept that target the host cells and not the pathogen itself would represent a real breakthrough in drug discovery leading to broad spectrum drugs.