A missense mutation (c.184C>T) in ovine CLN6 causes neuronal ceroid lipofuscinosis in Merino sheep whereas affected South Hampshire sheep have reduced levels of CLN6 mRNA

AbstractThe neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of fatal recessively inherited neurodegenerative diseases of humans and animals characterised by common clinical signs and pathology. These include blindness, ataxia, dementia, behavioural changes, seizures, brain and retinal atrophy and accumulation of fluorescent lysosome derived organelles in most cells. A number of different variants have been suggested and seven different causative genes identified in humans (CLN1, CLN2, CLN3, CLN5, CLN6, CLN8 and CTSD). Animal models have played a central role in the investigation of this group of diseases and are extremely valuable for developing a better understanding of the disease mechanisms and possible therapeutic approaches. Ovine models include flocks of affected New Zealand South Hampshires and Borderdales and Australian Merinos. The ovine CLN6 gene has been sequenced in a representative selection of these sheep. These investigations unveiled the mutation responsible for the disease in Merino sheep (c.184C>T; p.Arg62Cys) and three common ovine allelic variants (c.56A>G, c.822G>A and c.933_934insCT). Linkage analysis established that CLN6 is the gene most likely to cause NCL in affected South Hampshire sheep, which do not have the c.184C>T mutation but show reduced expression of CLN6 mRNA in a range of tissues as determined by real-time PCR. Lack of linkage precludes CLN6 as a candidate for NCL in Borderdale sheep

Case Report Beneficial Effect of Conversion to Belatacept in Kidney-Transplant Patients with a Low Glomerular-Filtration Rate

Belatacept has been found to be efficient at preserving good kidney function in maintenance kidney-transplant patients. Herein, we report on the use of belatacept as a rescue therapy for two kidney-transplant patients presenting with severe adverse events after treatment with calcineurin inhibitors (CNIs) and mammalian target-of-rapamycin (mTOR) inhibitors. Two kidney-transplant patients developed severely impaired kidney function after receiving CNIs. The use of everolimus was associated with severe angioedema. Belatacept was then successfully used to improve kidney function in both cases, even though estimated glomerularfiltration rate before conversion was <20 mL/min. These case reports show that belatacept can be used as a rescue therapy, even if kidney function is very low in kidney-transplant patients who cannot tolerate CNIs and/or mTOR inhibitors

CXCR3 Antagonism of SDF-1(5-67) Restores Trabecular Function and Prevents Retinal Neurodegeneration in a Rat Model of Ocular Hypertension

Glaucoma, the most common cause of irreversible blindness, is a neuropathy commonly initiated by pathological ocular hypertension due to unknown mechanisms of trabecular meshwork degeneration. Current antiglaucoma therapy does not target the causal trabecular pathology, which may explain why treatment failure is often observed. Here we show that the chemokine CXCL12, its truncated form SDF-1(5-67), and the receptors CXCR4 and CXCR3 are expressed in human glaucomatous trabecular tissue and a human trabecular cell line. SDF-1(5-67) is produced under the control of matrix metallo-proteinases, TNF-α, and TGF-β2, factors known to be involved in glaucoma. CXCL12 protects in vitro trabecular cells from apoptotic death via CXCR4 whereas SDF-1(5-67) induces apoptosis through CXCR3 and caspase activation. Ocular administration of SDF-1(5-67) in the rat increases intraocular pressure. In contrast, administration of a selective CXCR3 antagonist in a rat model of ocular hypertension decreases intraocular pressure, prevents retinal neurodegeneration, and preserves visual function. The protective effect of CXCR3 antagonism is related to restoration of the trabecular function. These data demonstrate that proteolytic cleavage of CXCL12 is involved in trabecular pathophysiology, and that local administration of a selective CXCR3 antagonist may be a beneficial therapeutic strategy for treating ocular hypertension and subsequent retinal degeneration

Rôle de la signalisation des cytokinines dans le contrôle de l'architecture racinaire chez Medicago truncatula

L objectif de cette thèse était de déterminer quels éléments de la signalisation des cytokinines étaient requis pour contrôler la mise en place du système racinaire des légumineuses, et comment cette voie s intégrait avec d autres signaux développementaux. Dans une première partie, le mutant de M.truncatula affectant le récepteur aux cytokinines MtCRE1 s est révélé jouer un rôle fondamental dans le contrôle de l interaction symbiotique avec S.meliloti à la fois à des stades précoces et tardifs. L activation de la voie de signalisation des cytokinines médiée par CRE1,visualisée par le gène de réponse primaire aux cytokinines MtRR4, régule aussi positivement plusieurs gènes de nodulation tels que NIN, NSP2 et ERN1. Cette même voie est en outre impliquée dans la modulation du transport polarisé d auxine, potentiellement en régulant l accumulation de protéines PINs. Enfin l étude du double mutant cre1/sk1, affecté dans le gène de réponse à l éthylène EIN2, a révélé que les voies de signalisation de ces deux phytohormones seraient indépendantes lors de la mise en place des nodules. La deuxième partie de ce travail de thèse a permis d établir une connexion entre le facteur de transcription EFD et les cytokinines. En effet, une des cibles principales d EFD, MtRR4, régulerait négativement le signal cytokinine au niveau des primordia et de la zone d infection des nodules, favorisant ainsi leur différentiation. Enfin, la troisième partie de ce travail visait à identifier de nouvelles cibles directement régulées par la signalisation cytokinine et agissant dans le système racinaire des légumineuses. Une combinaison d approches de biochimie, de transcriptomique et de bioinformatique a permis d identifier des éléments cis présents dans les promoteurs des gènes régulés par les cytokinines. Parmi eux, le gène de nodulation NSP2 a été caractérisé comme étant régulé directement via la signalisation cytokinine dépendant des RRs de type B. Au final, l ensemble de ces résultats met en évidence le rôle crucial de la signalisation cytokinine médiée par MtCRE1, ainsi que son interaction avec d autres signaux dans le contrôle de l architecture racinaire, et a permis l identification de nouveaux gènes de réponse aux cytokinines.The objective of this thesis was to determine which components of the cytokinin signaling pathway were required to control legume root architecture, and how, this process was integrated with other developmental cues. In a first part, mutants in the M. truncatula CRE1 cytokinin receptor were found to play a crucial role in controlling the symbiotic interaction with S. meliloti. Cytokinin signaling mediated by CRE1, followed through the activation of the primary response gene to cytokinins MtRR4, also positively regulates several nodulation genes such as NIN, NSP2 and ERN1. The same pathway is also involved in modulating polar auxin transport, potentially by regulating the accumulation of PIN proteins. Finally, study of cre1/skl double mutants, also affected in the ethylene response gene EIN2, revealed that ethylene control nodule initiation independently of CRE1 cytokinin signaling. The second part of this thesis has established a connection between the transcription factor EFD and cytokinins. Indeed, a major target of EFD, MtRR4, may down regulate cytokinin signaling in nodule primordia and infection zone, thereby promoting their differentiation. Finally, the third part of this work was to indentify new targets directly regulated by cytokinin signalling and acting in the legume root system. A combination of biochemical, transcriptomic and bioinformatic approaches allowed the identification of cis-elements present in promoters of genes regulated by cytokinins. Among them, the nodulation gene NSP2 has been characterized as regulated depending on B type RRs involved in cytokinin signaling. Ultimately, these results highlight the crucial role of MtCRE1 cytokinin signaling and its interactions with other cues in the control of root architecture and enabled the identification of new genes responsive to cytokinins in legumes.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Rôle des chimiokines CX3CL1/FKN et CXCL12/SDF-1 dans la physiopathologie du segment antérieur de l'oeil

Dans le glaucome (GPAO), la dégénérescence du nerf optique est associée à une augmentation de la pression intraoculaire (PIO) causée par la dégénérescence du trabéculum. Nous avons montré que cette chimiokine et son récepteur CX3CR1 sont exprimés au niveau de trabéculums de patients ainsi que dans une lignée humaine trabéculaire. Nous avons montré in vitro que cette chimiokine est impliquée dans la prolifération et la migration des cellules trabéculaires. Les propriétés chimio-attractantes de CX3CL1 nous ont conduits à étudier son implication dans l infiltration leucocytaire de la conjonctive. Nous avons montré que les cellules conjonctivales de patients exposés au chlorure de benzalkonium (BAK), un conservateur toxique des collyres, surexpriment CX3CL1. Des expériences in vitro sur des lignées humaines conjonctivales et in vivo sur des souris témoins et déficientes pour le récepteur CX3CR1 ont montré que CX3CL1, en présence de BAK, induit une infiltration de certains phénotypes leucocytaires. De plus, nous avons montré que des trabéculums humains ainsi qu une lignée cellulaire trabéculaire in vitro expriment CXCL12 et sa forme tronquée, le SDF-1(5-67). Des études in vitro ont mis en évidence le rôle protecteur de CXCL12 via le récepteur CXCR4, ainsi que l action délétère de SDF-1(5-67) via le récepteur CXCR3. Nous avons montré in vivo sur des rats témoins et des rats rendus hypertones par chirurgie que SDF-1(5-67) est responsable d une augmentation de la PIO et que le blocage de CXCR3 permet une restauration de la filtration trabéculaire. Ces trois études mettent en évidence l existence d un système chimiokinergique qui participe à la physiopathologie du GPAOIn primary open-angle glaucoma (POAG), retinal nerve degeneration is associated with pathological ocular hypertension (OHT) due to the degeneration of the trabeculum. We showed that human trabeculum obtained by surgery from glaucomatous patients and a human trabecular cell line express CX3CL1 and its receptor CX3CR1. We showed that CX3CL1 enhanced both proliferation and migration of trabecular cells. The chemoattracting abilities of CX3CL1 led us to investigate its implication in the leukocytic infiltration of conjunctival tissue during inflammation. This tissue was exposed to a toxic preservative present in ophthalmic medications: benxalkonium chloride (BAK). We showed that conjunctival cells from patients exposed to BAK overexpress CX3CL1. In vitro experiments on human conjunctival epithelial cell lines demonstrated that CX3CL1, in the presence of BAK, induces a selective attraction of leukocytes subsets. This observation was verified in vivo using CX3CR1-deficient mice as compared to CX3CR1+/+ controls. We identified the expression of both forms CXCL12 and SDF-1(5-67) in human glaucomatous trabecular tissue as well as in HTM cells. We performed in vitro experiments and demonstrated that CXCL12 acts as a protective chemokine via its CXCR4 receptor whereas the processing of CXCL12 into SDF-1(5-67) has a deleterious effect via CXCR3 receptor. We showed that SDF-1(5-67) induces apoptosis via CXCR3. We also demonstrated that in vivo blockade of CXCR3 by a specific antagonist can restore trabecular filtrating function. These three studies present evidence of a chemokinergic system that plays a role in the cellular physiopathology of POAGPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Blockade of the chemokine receptor CXCR3 lowers intraocular pressure and prevents retinal degeneration in an animal model of glaucoma

International audienc

CX3CL1 is involved in the ocular surface inflammation induced by benzalkonium chloride

International audienc

MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula

Phytohormonal interactions are essential to regulate plant organogenesis. In response to the presence of signals from symbiotic bacteria, the Nod factors, legume roots generate a new organ: the nitrogen-fixing nodule. Analysis of mutants in the Medicago truncatula CRE1 cytokinin receptor and of the MtRR4 cytokinin primary response gene expression pattern revealed that cytokinin acts in initial cortical cell divisions and later in the transition between meristematic and differentiation zones of the mature nodule. MtCRE1 signaling is required for activation of the downstream nodulation-related transcription factors MtERN1, MtNSP2 and MtNIN, as well as to regulate expression and accumulation of PIN auxin efflux carriers. Whereas the MtCRE1 pathway is required to allow the inhibition of polar auxin transport in response to rhizobia, nodulation is still negatively regulated by the MtEIN2/SICKLE- dependent ethylene pathway in cre1 mutants. Hence, MtCRE1 signaling acts as a regulatory knob, integrating positive plant and bacterial cues to control legume nodule organogenesis

Mt ZR1, a PRAF protein, is involved in the development of roots and symbiotic root nodules in Medicago truncatula

International audiencePRAF proteins are present in all plants, but their functions remain unclear. We investigated the role of one member of the PRAF family, MtZR1, on the development of roots and nitrogen-fixing nodules in Medicago truncatula. We found that MtZR1 was expressed in all M.truncatula organs. Spatiotemporal analysis showed that MtZR1 expression in M.truncatula roots was mostly limited to the root meristem and the vascular bundles of mature nodules. MtZR1 expression in root nodules was down-regulated in response to various abiotic stresses known to affect nitrogen fixation efficiency. The down-regulation of MtZR1 expression by RNA interference in transgenic roots decreased root growth and impaired nodule development and function. MtZR1 overexpression resulted in longer roots and significant changes to nodule development. Our data thus indicate that MtZR1 is involved in the development of roots and nodules. To our knowledge, this work provides the first in vivo experimental evidence of a biological role for a typical PRAF protein in plants