A system-level, molecular evolutionary analysis of mammalian phototransduction

Visual perception is initiated in the photoreceptor cells of the retina via the phototransduction system.This system has shown marked evolution during mammalian divergence in such complex attributes as activation time and recovery time. We have performed a molecular evolutionary analysis of proteins involved in mammalianphototransduction in order to unravel how the action of natural selection has been distributed throughout thesystem to evolve such traits. We found selective pressures to be non-randomly distributed according to both a simple protein classification scheme and a protein-interaction network representation of the signaling pathway. Proteins which are topologically central in the signaling pathway, such as the G proteins, as well as retinoid cycle chaperones and proteins involved in photoreceptor cell-type determination, were found to be more constrained in their evolution. Proteins peripheral to the pathway, such as ion channels and exchangers, as well as the retinoid cycle enzymes, have experienced a relaxation of selective pressures. Furthermore, signals of positive selection were detected in two genes: the short-wave (blue) opsin (OPN1SW) in hominids and the rod-specific Na+/Ca2+,K+ ion exchanger (SLC24A1) in rodents. The functions of the proteins involved in phototransduction and the topology of the interactions between them have imposed non-random constraints on their evolution. Thus, in shaping or conserving system-level phototransduction traits, natural selection has targeted the underlying proteins in a concerted manner.

Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon.

A three-dimensional cell culture system was used as a model to study the influence of low levels of mercury in the developing brain. Aggregating cell cultures of fetal rat telencephalon were treated for 10 days either during an early developmental period (i.e., between days 5 and 15 in vitro) or during a phase of advanced maturation (i.e., between days 25 and 35) with mercury. An inorganic (HgCl2) and an organic mercury compound (monomethylmercury chloride, MeHgCl) were examined. By monitoring changes in cell type-specific enzymes activities, the concentration-dependent toxicity of the compounds was determined. In immature cultures, a general cytotoxicity was observed at 10(-6) M for both mercury compounds. In these cultures, HgCl2 appeared somewhat more toxic than MeHgCl. However, no appreciable demethylation of MeHgCl could be detected, indicating similar toxic potencies for both mercury compounds. In highly differentiated cultures, by contrast, MeHgCl exhibited a higher toxic potency than HgCl2. In addition, at 10(-6) M, MeHgCl showed pronounced neuron-specific toxicity. Below the cytotoxic concentrations, distinct glia-specific reactions could be observed with both mercury compounds. An increase in the immunoreactivity for glial fibrillary acidic protein, typical for gliosis, could be observed at concentrations between 10(-9) M and 10(-7) M in immature cultures, and between 10(-8) M and 3 x 10(-5) M in highly differentiated cultures. A conspicuous increase in the number and clustering of GSI-B4 lectin-binding cells, indicating a microglial response, was found at concentrations between 10(-10) M and 10(-7) M. These development-dependent and cell type-specific effects may reflect the pathogenic potential of long-term exposure to subclinical doses of mercury.

Systèmes de délivrance des médicaments pour le segment antérieur: bases fondamentales et applications cliniques [Drug delivery systems to target the anterior segment of the eye: fundamental bases and clinical applications].

The development of new drug delivery systems to target the anterior segment of the eye may offer many advantages: to increase the biodisponibility of the drug, to allow the penetration of drug that cannot be formulated as solutions, to obtain constant and sustained drug release, to achieve higher local concentrations without systemic effects, to target more specifically one tissue or cell type, to reduce the frequency of instillation and therefore increase the observance and comfort of the patient while reducing side effects of frequent instillation. Several approaches are developed, aiming to increase the corneal contact time by modified formulation or reservoir systems, or by increasing the tissue permeability using iontophoresis. To date, no ocular drug delivery system is ideal for all purposes. To maximize treatment efficacy, careful evaluation of the specific pathological condition, the targeted Intraocular tissue and the location of the most severe pathology must be made before selecting the method of delivery most suitable for each individual patient.

Identification and staining of distinct populations of secretory organelles in astrocytes.

Increasing evidence indicates that astrocytes, the most abundant glial cell type in the brain, respond to an elevation in cytoplasmic calcium concentration ([Ca(2+)]i) by releasing chemical transmitters (also called gliotransmitters) via regulated exocytosis of heterogeneous classes of organelles. By this process, astrocytes exert modulatory influences on neighboring cells and are thought to participate in the control of synaptic circuits and cerebral blood flow. Studying the properties of exocytosis in astrocytes is a challenge, because the cell biological basis of this process is incompletely defined. Astrocytic exocytosis involves multiple populations of secretory vesicles, including synaptic-like microvesicles (SLMVs), dense-core granules (DCGs), and lysosomes. Here we summarize the available information for identifying individual populations of secretory organelles in astrocytes, including DCGs, SLMVs, and lysosomes, and present experimental procedures for specifically staining such populations.

The shaping of invasive glioma phenotype by the ubiquitin-proteasome system.

Abstract Protein degradation is an indispensable process for cells which is often deregulated in various diseases, including malignant conditions. Depending on the specific cell type and functions of expressed proteins, this aberration may have different effects on the determination of malignant phenotypes. A discrete, inherent feature of malignant glioma is its profound invasive and migratory potential, regulated by the expression of signaling and effector proteins, many of which are also subjected to post-translational regulation by the ubiquitin-proteasome system (UPS). Here we provide an overview of this connection, focusing on important pro-invasive protein signals targeted by the UPS.

Effects of trimethyltin (TMT) on glial and neuronal cells in aggregate cultures: dependence on the developmental stage.

Long-term effects of trimethyltin (TMT) applied at concentrations below the cytotoxic level were examined in three-dimensional cell cultures of fetal rat telencephalon using biochemical, immunochemical and morphological criteria. It was found that in immature cultures low concentrations of TMT (10(-8) M) specifically induced a gliotic response in astrocytes, with increased immunoreactivity for glial fibrillary acidic protein, and a greater number of astrocytic processes. Significant changes in oligodendrocytic and neuronal parameters were found only at 10(-6) M of TMT. In differentiated cultures, distinct changes in cell type-specific parameters occurred at 10(-6) M of TMT (the lowest effective concentration). In addition, different patterns of responses were found for astrocytes and oligodendrocytes, as compared to immature cultures. These results suggest that among neural cells, astroblasts are most sensitive to TMT, and that the glial responses to this neurotoxicant are development-dependent.

A qualitative continuous model of cellular auxin and brassinosteroid signaling and their crosstalk.

Motivation: Hormone pathway interactions are crucial in shaping plant development, such as synergism between the auxin and brassinosteroid pathways in cell elongation. Both hormone pathways have been characterized in detail, revealing several feedback loops. The complexity of this network, combined with a shortage of kinetic data, renders its quantitative analysis virtually impossible at present.Results: As a first step towards overcoming these obstacles, we analyzed the network using a Boolean logic approach to build models of auxin and brassinosteroid signaling, and their interaction. To compare these discrete dynamic models across conditions, we transformed them into qualitative continuous systems, which predict network component states more accurately and can accommodate kinetic data as they become available. To this end, we developed an extension for the SQUAD software, allowing semi-quantitative analysis of network states. Contrasting the developmental output depending on cell type-specific modulators enabled us to identify a most parsimonious model, which explains initially paradoxical mutant phenotypes and revealed a novel physiological feature.

Dissection of hormone-responsive plasma membrane to nucleus signaling of Bravis Radix : its role in vascular differentiation and root meristem growth

AbstractPlants continuously grow during their complete life span and understanding the mechanisms that qualitatively regulate their traits remains a challenging topic in biology. The hormone auxin has been identified as a crucial molecule for shaping plant growth, as it has a role in most developmental processes. In the root, the directional, so-called polar transport of auxin generates a peak of concentration that specifies and maintains the stem cell niche and a subsequent gradient of decreasing concentration that also regulates cell proliferation and differentiation. For these reasons, auxin is considered the main morphogen of the root, as it is fundamental for its organization and maintenance. Recently, in Arabidopsis thaliana, a natural variation screen allowed the discovery of BREVIS RADIX (BRX) gene as a limiting factor for auxin responsive gene expression and thus for root growth.In this study, we discovered that BRX is a direct target of auxin that positively feeds back on auxin signaling, as a transcriptional co-regulator, through interaction with the Auxin Response Factor (ARF) MONOPTEROS (MP), modulating the auxin gene response magnitude during the transition between division and differentiation in the root meristem. Moreover, we provide evidence that BRX is activated at the plasma membrane level as an associated protein before moving into the nucleus to modulate cellular growth.To investigate the discrepancy between the auxin concentration and the expression pattern of its downstream targets, we combined experimental and computational approaches. Expression profiles deviating from the auxin gradient could only be modeled after intersection of auxin activity with the observed differential endocytosis pattern and with positive auto- regulatory feedback through plasma- membrane-to-nucleus transfer of BRX. Because BRX is required for expression of certain auxin response factor targets, our data suggest a cell-type-specific endocytosis-dependent input into transcriptional auxin perception. This input sustains expression of a subset of auxin-responsive genes across the root meristem's division and transition zones and is essential for meristem growth. Thus, the endocytosis pattern provides specific positional information to modulate auxin response. RésuméLes plantes croissent continuellement tout au long de leur cycle de vie. Comprendre et expliquer les mécanismes impliqués dans ce phénomène reste à l'heure actuelle, un défi. L'hormone auxine a été identifiée comme une molécule essentielle à la régulation de la croissance des plantes, car impliquée dans la plupart des processus développementaux. Dans la racine, le transport polaire de l'auxine, par la génération d'un pic de concentration, spécifie et maintient la niche de cellules souches, et par la génération d'un gradient de concentration, contrôle la prolifération et la différentiation cellulaire. Puisque l'auxine est essentielle pour l'organisation et la maintenance du système racinaire, il est considéré comme son principal morphogène. Récemment, dans la plante modèle, Arabidopsis thalinana, un criblage des variations génétique a permis d'identifier le gène Brevis radix (BRX) comme facteur limitant l'expression des gènes de réponse à l'auxine et par là même, la croissance de la racine.Dans ce travail, nous avons découvert que BRX est une cible direct de l'auxine qui rétroactive positivement le signalement de l'hormone, agissant ainsi comme un régulateur transcriptionnel à travers l'interaction avec la protéine Monopteros (MP) de la famille des facteurs de réponse à l'auxine (Auxin Responsive Factor, ARF), et modulant ainsi la magnitude de la réponse des gènes reliés à l'auxine durant la division et la différentiation cellulaire dans le méristème de la racine. De plus, nous fournissons des preuves que BRX est activées au niveau de la membrane plasmique, tel une protéine associée se déplaçant à l'intérieur du noyau et modulant la croissance cellulaire.Pour mener à bien l'investigation des divergences entre la concentration de l'auxine et les schémas d'expression de ses propres gènes cibles, nous avons combiné les approches expérimentales et computationnelles. Les profiles d'expressions déviant du gradient d'auxine pourraient seulement être modéliser après intersection de l'activité de l'auxine avec les schémas différentiels d'endocytose observés et les boucles de rétroaction positives et autorégulatrices par le transfert de BRX de la membrane plasmique au noyau. Puisque BRX est requis pour l'expression de certains gènes cibles des facteurs de réponse à l'auxine, nos données suggèrent une contribution dépendante d'une endocytose spécifique au type de cellule dans la perception transcriptionnelle à l'auxine Cette contribution soutient l'expression d'un sous-set de gène de réponse à l'auxine dans la division du méristème racinaire et la zone de transition, et par conséquent, est essentielle pour la croissance méristematique. Ainsi, le schéma d'endocytose fournit des informations positionnelles spécifiques à la modulation de la réponse à l'auxine.

Natural gene-expression variation in Down syndrome modulates the outcome of gene-dosage imbalance.

Down syndrome (DS) is characterized by extensive phenotypic variability, with most traits occurring in only a fraction of affected individuals. Substantial gene-expression variation is present among unaffected individuals, and this variation has a strong genetic component. Since DS is caused by genomic-dosage imbalance, we hypothesize that gene-expression variation of human chromosome 21 (HSA21) genes in individuals with DS has an impact on the phenotypic variability among affected individuals. We studied gene-expression variation in 14 lymphoblastoid and 17 fibroblast cell lines from individuals with DS and an equal number of controls. Gene expression was assayed using quantitative real-time polymerase chain reaction on 100 and 106 HSA21 genes and 23 and 26 non-HSA21 genes in lymphoblastoid and fibroblast cell lines, respectively. Surprisingly, only 39% and 62% of HSA21 genes in lymphoblastoid and fibroblast cells, respectively, showed a statistically significant difference between DS and normal samples, although the average up-regulation of HSA21 genes was close to the expected 1.5-fold in both cell types. Gene-expression variation in DS and normal samples was evaluated using the Kolmogorov-Smirnov test. According to the degree of overlap in expression levels, we classified all genes into 3 groups: (A) nonoverlapping, (B) partially overlapping, and (C) extensively overlapping expression distributions between normal and DS samples. We hypothesize that, in each cell type, group A genes are the most dosage sensitive and are most likely involved in the constant DS traits, group B genes might be involved in variable DS traits, and group C genes are not dosage sensitive and are least likely to participate in DS pathological phenotypes. This study provides the first extensive data set on HSA21 gene-expression variation in DS and underscores its role in modulating the outcome of gene-dosage imbalance.

Multi-scale systems analysis of plant development : beyond cellular signaling and tissue morphodynamics

Les plantes sont essentielles pour les sociétés humaines. Notre alimentation quotidienne, les matériaux de constructions et les sources énergétiques dérivent de la biomasse végétale. En revanche, la compréhension des multiples aspects développementaux des plantes est encore peu exploitée et représente un sujet de recherche majeur pour la science. L'émergence des technologies à haut débit pour le séquençage de génome à grande échelle ou l'imagerie de haute résolution permet à présent de produire des quantités énormes d'information. L'analyse informatique est une façon d'intégrer ces données et de réduire la complexité apparente vers une échelle d'abstraction appropriée, dont la finalité est de fournir des perspectives de recherches ciblées. Ceci représente la raison première de cette thèse. En d'autres termes, nous appliquons des méthodes descriptives et prédictives combinées à des simulations numériques afin d'apporter des solutions originales à des problèmes relatifs à la morphogénèse à l'échelle de la cellule et de l'organe. Nous nous sommes fixés parmi les objectifs principaux de cette thèse d'élucider de quelle manière l'interaction croisée des phytohormones auxine et brassinosteroïdes (BRs) détermine la croissance de la cellule dans la racine du méristème apical d'Arabidopsis thaliana, l'organisme modèle de référence pour les études moléculaires en plantes. Pour reconstruire le réseau de signalement cellulaire, nous avons extrait de la littérature les informations pertinentes concernant les relations entre les protéines impliquées dans la transduction des signaux hormonaux. Le réseau a ensuite été modélisé en utilisant un formalisme logique et qualitatif pour pallier l'absence de données quantitatives. Tout d'abord, Les résultats ont permis de confirmer que l'auxine et les BRs agissent en synergie pour contrôler la croissance de la cellule, puis, d'expliquer des observations phénotypiques paradoxales et au final, de mettre à jour une interaction clef entre deux protéines dans la maintenance du méristème de la racine. Une étude ultérieure chez la plante modèle Brachypodium dystachion (Brachypo- dium) a révélé l'ajustement du réseau d'interaction croisée entre auxine et éthylène par rapport à Arabidopsis. Chez ce dernier, interférer avec la biosynthèse de l'auxine mène à la formation d'une racine courte. Néanmoins, nous avons isolé chez Brachypodium un mutant hypomorphique dans la biosynthèse de l'auxine qui affiche une racine plus longue. Nous avons alors conduit une analyse morphométrique qui a confirmé que des cellules plus anisotropique (plus fines et longues) sont à l'origine de ce phénotype racinaire. Des analyses plus approfondies ont démontré que la différence phénotypique entre Brachypodium et Arabidopsis s'explique par une inversion de la fonction régulatrice dans la relation entre le réseau de signalisation par l'éthylène et la biosynthèse de l'auxine. L'analyse morphométrique utilisée dans l'étude précédente exploite le pipeline de traitement d'image de notre méthode d'histologie quantitative. Pendant la croissance secondaire, la symétrie bilatérale de l'hypocotyle est remplacée par une symétrie radiale et une organisation concentrique des tissus constitutifs. Ces tissus sont initialement composés d'une douzaine de cellules mais peuvent aisément atteindre des dizaines de milliers dans les derniers stades du développement. Cette échelle dépasse largement le seuil d'investigation par les moyens dits 'traditionnels' comme l'imagerie directe de tissus en profondeur. L'étude de ce système pendant cette phase de développement ne peut se faire qu'en réalisant des coupes fines de l'organe, ce qui empêche une compréhension des phénomènes cellulaires dynamiques sous-jacents. Nous y avons remédié en proposant une stratégie originale nommée, histologie quantitative. De fait, nous avons extrait l'information contenue dans des images de très haute résolution de sections transverses d'hypocotyles en utilisant un pipeline d'analyse et de segmentation d'image à grande échelle. Nous l'avons ensuite combiné avec un algorithme de reconnaissance automatique des cellules. Cet outil nous a permis de réaliser une description quantitative de la progression de la croissance secondaire révélant des schémas développementales non-apparents avec une inspection visuelle classique. La formation de pôle de phloèmes en structure répétée et espacée entre eux d'une longueur constante illustre les bénéfices de notre approche. Par ailleurs, l'exploitation approfondie de ces résultats a montré un changement de croissance anisotropique des cellules du cambium et du phloème qui semble en phase avec l'expansion du xylème. Combinant des outils génétiques et de la modélisation biomécanique, nous avons démontré que seule la croissance plus rapide des tissus internes peut produire une réorientation de l'axe de croissance anisotropique des tissus périphériques. Cette prédiction a été confirmée par le calcul du ratio des taux de croissance du xylème et du phloème au cours de développement secondaire ; des ratios élevés sont effectivement observés et concomitant à l'établissement progressif et tangentiel du cambium. Ces résultats suggèrent un mécanisme d'auto-organisation établi par un gradient de division méristématique qui génèrent une distribution de contraintes mécaniques. Ceci réoriente la croissance anisotropique des tissus périphériques pour supporter la croissance secondaire. - Plants are essential for human society, because our daily food, construction materials and sustainable energy are derived from plant biomass. Yet, despite this importance, the multiple developmental aspects of plants are still poorly understood and represent a major challenge for science. With the emergence of high throughput devices for genome sequencing and high-resolution imaging, data has never been so easy to collect, generating huge amounts of information. Computational analysis is one way to integrate those data and to decrease the apparent complexity towards an appropriate scale of abstraction with the aim to eventually provide new answers and direct further research perspectives. This is the motivation behind this thesis work, i.e. the application of descriptive and predictive analytics combined with computational modeling to answer problems that revolve around morphogenesis at the subcellular and organ scale. One of the goals of this thesis is to elucidate how the auxin-brassinosteroid phytohormone interaction determines the cell growth in the root apical meristem of Arabidopsis thaliana (Arabidopsis), the plant model of reference for molecular studies. The pertinent information about signaling protein relationships was obtained through the literature to reconstruct the entire hormonal crosstalk. Due to a lack of quantitative information, we employed a qualitative modeling formalism. This work permitted to confirm the synergistic effect of the hormonal crosstalk on cell elongation, to explain some of our paradoxical mutant phenotypes and to predict a novel interaction between the BREVIS RADIX (BRX) protein and the transcription factor MONOPTEROS (MP),which turned out to be critical for the maintenance of the root meristem. On the same subcellular scale, another study in the monocot model Brachypodium dystachion (Brachypodium) revealed an alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis. In the latter, increasing interference with auxin biosynthesis results in progressively shorter roots. By contrast, a hypomorphic Brachypodium mutant isolated in this study in an enzyme of the auxin biosynthesis pathway displayed a dramatically longer seminal root. Our morphometric analysis confirmed that more anisotropic cells (thinner and longer) are principally responsible for the mutant root phenotype. Further characterization pointed towards an inverted regulatory logic in the relation between ethylene signaling and auxin biosynthesis in Brachypodium as compared to Arabidopsis, which explains the phenotypic discrepancy. Finally, the morphometric analysis of hypocotyl secondary growth that we applied in this study was performed with the image-processing pipeline of our quantitative histology method. During its secondary growth, the hypocotyl reorganizes its primary bilateral symmetry to a radial symmetry of highly specialized tissues comprising several thousand cells, starting with a few dozens. However, such a scale only permits observations in thin cross-sections, severely hampering a comprehensive analysis of the morphodynamics involved. Our quantitative histology strategy overcomes this limitation. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with an automated cell type recognition algorithm, it allows precise quantitative characterization of vascular development and reveals developmental patterns that were not evident from visual inspection, for example the steady interspace distance of the phloem poles. Further analyses indicated a change in growth anisotropy of cambial and phloem cells, which appeared in phase with the expansion of xylem. Combining genetic tools and computational modeling, we showed that the reorientation of growth anisotropy axis of peripheral tissue layers only occurs when the growth rate of central tissue is higher than the peripheral one. This was confirmed by the calculation of the ratio of the growth rate xylem to phloem throughout secondary growth. High ratios are indeed observed and concomitant with the homogenization of cambium anisotropy. These results suggest a self-organization mechanism, promoted by a gradient of division in the cambium that generates a pattern of mechanical constraints. This, in turn, reorients the growth anisotropy of peripheral tissues to sustain the secondary growth.

Robustness of Drosophila early embryo and wing imaginal disc development

Within a developing organism, cells require information on where they are in order to differentiate into the correct cell-type. Pattern formation is the process by which cells acquire and process positional cues and thus determine their fate. This can be achieved by the production and release of a diffusible signaling molecule, called a morphogen, which forms a concentration gradient: exposure to different morphogen levels leads to the activation of specific signaling pathways. Thus, in response to the morphogen gradient, cells start to express different sets of genes, forming domains characterized by a unique combination of differentially expressed genes. As a result, a pattern of cell fates and specification emerges.Though morphogens have been known for decades, it is not yet clear how these gradients form and are interpreted in order to yield highly robust patterns of gene expression. During my PhD thesis, I investigated the properties of Bicoid (Bcd) and Decapentaplegic (Dpp), two morphogens involved in the patterning of the anterior-posterior axis of Drosophila embryo and wing primordium, respectively. In particular, I have been interested in understanding how the pattern proportions are maintained across embryos of different sizes or within a growing tissue. This property is commonly referred to as scaling and is essential for yielding functional organs or organisms. In order to tackle these questions, I analysed fluorescence images showing the pattern of gene expression domains in the early embryo and wing imaginal disc. After characterizing the extent of these domains in a quantitative and systematic manner, I introduced and applied a new scaling measure in order to assess how well proportions are maintained. I found that scaling emerged as a universal property both in early embryos (at least far away from the Bcd source) and in wing imaginal discs (across different developmental stages). Since we were also interested in understanding the mechanisms underlying scaling and how it is transmitted from the morphogen to the target genes down in the signaling cascade, I also quantified scaling in mutant flies where this property could be disrupted. While scaling is largely conserved in embryos with altered bcd dosage, my modeling suggests that Bcd trapping by the nuclei as well as pre-steady state decoding of the morphogen gradient are essential to ensure precise and scaled patterning of the Bcd signaling cascade. In the wing imaginal disc, it appears that as the disc grows, the Dpp response expands and scales with the tissue size. Interestingly, scaling is not perfect at all positions in the field. The scaling of the target gene domains is best where they have a function; Spalt, for example, scales best at the position in the anterior compartment where it helps to form one of the anterior veins of the wing. Analysis of mutants for pentagone, a transcriptional target of Dpp that encodes a secreted feedback regulator of the pathway, indicates that Pentagone plays a key role in scaling the Dpp gradient activity.

Intraperikaryal neurofilamentous accumulations in a subset of retinal ganglion cells in aged mice that express a human neurofilament gene.

Neurofilamentous changes in select groups of neurons are associated with the degenerative changes of many human age-related neurodegenerative diseases. To examine the possible effects of aging on the neuronal cytoskeleton containing human proteins, the retinas of transgenic mice expressing the gene for the human middle-sized neurofilament triplet were investigated at 3 or 12 months of age. Transgenic mice developed tangle-like neurofilamentous accumulations in a subset of retinal ganglion cells at 12 months of age. These neurofilamentous accumulations, which also involved endogenous neurofilament proteins, were present in the perikarya and proximal processes of large ganglion cells and were predominantly located in peripheral retina. The presence of the human protein may thus confer vulnerability of the cytoskeleton to age-related alterations in this specific retinal cell type and may serve as a model for similar cellular changes associated with Alzheimer's disease and glaucoma.

Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons.

The excitatory neurotransmitter glutamate has been reported to have a major impact on brain energy metabolism. Using primary cultures of rat hippocampal neurons, we observed that glutamate reduces glucose utilization in this cell type, suggesting alteration in mitochondrial oxidative metabolism. The aquaglyceroporin AQP9 and the monocarboxylate transporter MCT2, two transporters for oxidative energy substrates, appear to be present in mitochondria of these neurons. Moreover, they not only co-localize but they interact with each other as they were found to co-immunoprecipitate from hippocampal neuron homogenates. Exposure of cultured hippocampal neurons to glutamate 100 μM for 1 h led to enhanced expression of both AQP9 and MCT2 at the protein level without any significant change at the mRNA level. In parallel, a similar increase in the protein expression of LDHA was evidenced without an effect on the mRNA level. These data suggest that glutamate exerts an influence on neuronal energy metabolism likely through a regulation of the expression of some key mitochondrial proteins.

In vivo nuclear translocation of mineralocorticoid and glucocorticoid receptors in rat kidney: differential effect of corticosteroids along the distal tubule.

Aldosterone and corticosterone bind to mineralocorticoid (MR) and glucocorticoid receptors (GR), which, upon ligand binding, are thought to translocate to the cell nucleus to act as transcription factors. Mineralocorticoid selectivity is achieved by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) that inactivates 11β-hydroxy glucocorticoids. High expression levels of 11β-HSD2 characterize the aldosterone-sensitive distal nephron (ASDN), which comprises the segment-specific cells of late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct (CD). We used MR- and GR-specific antibodies to study localization and regulation of MR and GR in kidneys of rats with altered plasma aldosterone and corticosterone levels. In control rats, MR and GR were found in cell nuclei of thick ascending limb (TAL), DCT, CNT, CD cells, and intercalated cells (IC). GR was also abundant in cell nuclei and the subapical compartment of proximal tubule (PT) cells. Dietary NaCl loading, which lowers plasma aldosterone, caused a selective removal of GR from cell nuclei of 11β-HSD2-positive ASDN. The nuclear localization of MR was unaffected. Adrenalectomy (ADX) resulted in removal of MR and GR from the cell nuclei of all epithelial cells. Aldosterone replacement rapidly relocated the receptors in the cell nuclei. In ASDN cells, low-dose corticosterone replacement caused nuclear localization of MR, but not of GR. The GR was redistributed to the nucleus only in PT, TAL, early DCT, and IC that express no or very little 11β-HSD2. In ASDN cells, nuclear GR localization was only achieved when corticosterone was replaced at high doses. Thus ligand-induced nuclear translocation of MR and GR are part of MR and GR regulation in the kidney and show remarkable segment- and cell type-specific characteristics. Differential regulation of MR and GR may alter the level of heterodimerization of the receptors and hence may contribute to the complexity of corticosteroid effects on ASDN function.

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and cellular IRES [X-linked inhibitor-of-apoptosis (XIAP) and c-myc]. Bicistronic lentiviral constructs expressing two fluorescent reporters were used to transduce laboratory and B lymphoblastoid cell lines [15 CEPH pedigrees (n = 205) and 50 unrelated individuals]. IRES efficiency varied according to cell type and among individuals. Control of IRES activity has a significant genetic component (h(2) of 0.47 and 0.36 for EMCV and XIAP, respectively). Quantitative linkage analysis identified a suggestive locus (LOD 2.35) on chromosome 18q21.2, and genome-wide association analysis revealed of a cluster of SNPs on chromosome 3, intronic to the FHIT gene, marginally associated (P = 5.9E-7) with XIAP IRES function. This study illustrates the in vitro generation of intermediate phenotypes by using cell lines for the evaluation of genetic determinants of control of elements such as IRES.