Filtering genes to improve sensitivity in oligonucleotide microarray data analysis

Many recent microarrays hold an enormous number of probe sets, thus raising many practical and theoretical problems in controlling the false discovery rate (FDR). Biologically, it is likely that most probe sets are associated with un-expressed genes, so the measured values are simply noise due to non-specific binding; also many probe sets are associated with non-differentially-expressed (non-DE) genes. In an analysis to find DE genes, these probe sets contribute to the false discoveries, so it is desirable to filter out these probe sets prior to analysis. In the methodology proposed here, we first fit a robust linear model for probe-level Affymetrix data that accounts for probe and array effects. We then develop a novel procedure called FLUSH (Filtering Likely Uninformative Sets of Hybridizations), which excludes probe sets that have statistically small array-effects or large residual variance. This filtering procedure was evaluated on a publicly available data set from a controlled spiked-in experiment, as well as on a real experimental data set of a mouse model for retinal degeneration. In both cases, FLUSH filtering improves the sensitivity in the detection of DE genes compared to analyses using unfiltered, presence-filtered, intensity-filtered and variance-filtered data. A freely-available package called FLUSH implements the procedures and graphical displays described in the article

℮-conome: an automated tissue counting platform of cone photoreceptors for rodent models of retinitis pigmentosa

<p>Abstract</p> <p>Background</p> <p>Retinitis pigmentosa is characterized by the sequential loss of rod and cone photoreceptors. The preservation of cones would prevent blindness due to their essential role in human vision. Rod-derived Cone Viability Factor is a thioredoxin-like protein that is secreted by rods and is involved in cone survival. To validate the activity of Rod-derived Cone Viability Factors (RdCVFs) as therapeutic agents for treating retinitis Pigmentosa, we have developed e-conome, an automated cell counting platform for retinal flat mounts of rodent models of cone degeneration. This automated quantification method allows for faster data analysis thereby accelerating translational research.</p> <p>Methods</p> <p>An inverted fluorescent microscope, motorized and coupled to a CCD camera records images of cones labeled with fluorescent peanut agglutinin lectin on flat-mounted retinas. In an average of 300 fields per retina, nine Z-planes at magnification X40 are acquired after two-stage autofocus individually for each field. The projection of the stack of 9 images is subject to a threshold, filtered to exclude aberrant images based on preset variables. The cones are identified by treating the resulting image using 13 variables empirically determined. The cone density is calculated over the 300 fields.</p> <p>Results</p> <p>The method was validated by comparison to the conventional stereological counting. The decrease in cone density in <it>rd1 </it>mouse was found to be equivalent to the decrease determined by stereological counting. We also studied the spatiotemporal pattern of the degeneration of cones in the <it>rd1 </it>mouse and show that while the reduction in cone density starts in the central part of the retina, cone degeneration progresses at the same speed over the whole retinal surface. We finally show that for mice with an inactivation of the Nucleoredoxin-like genes <it>Nxnl1 </it>or <it>Nxnl2 </it>encoding RdCVFs, the loss of cones is more pronounced in the ventral retina.</p> <p>Conclusion</p> <p>The automated platform ℮-conome used here for retinal disease is a tool that can broadly accelerate translational research for neurodegenerative diseases.</p

Transcriptomic Analysis of Human Retinal Detachment Reveals Both Inflammatory Response and Photoreceptor Death

Background Retinal detachment often leads to a severe and permanent loss of vision and its therapeutic management remains to this day exclusively surgical. We have used surgical specimens to perform a differential analysis of the transcriptome of human retinal tissues following detachment in order to identify new potential pharmacological targets that could be used in combination with surgery to further improve final outcome. Methodology/Principal Findings Statistical analysis reveals major involvement of the immune response in the disease. Interestingly, using a novel approach relying on coordinated expression, the interindividual variation was monitored to unravel a second crucial aspect of the pathological process: the death of photoreceptor cells. Within the genes identified, the expression of the major histocompatibility complex I gene HLA-C enables diagnosis of the disease, while PKD2L1 and SLCO4A1 -which are both down-regulated- act synergistically to provide an estimate of the duration of the retinal detachment process. Our analysis thus reveals the two complementary cellular and molecular aspects linked to retinal detachment: an immune response and the degeneration of photoreceptor cells. We also reveal that the human specimens have a higher clinical value as compared to artificial models that point to IL6 and oxidative stress, not implicated in the surgical specimens studied here. Conclusions/Significance This systematic analysis confirmed the occurrence of both neurodegeneration and inflammation during retinal detachment, and further identifies precisely the modification of expression of the different genes implicated in these two phenomena. Our data henceforth give a new insight into the disease process and provide a rationale for therapeutic strategies aimed at limiting inflammation and photoreceptor damage associated with retinal detachment and, in turn, improving visual prognosis after retinal surgery

Le polymorphisme des genes de l'inter-alpha-trypsine inhibiteur : recherche d'association genetique avec l'emphyseme pulmonaire

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Étude fonctionnelle de NR2E3, un récepteur nucléaire orphelin impliqué dans la différenciation des photorécepteurs

Les récepteurs nucléaires constituent une famille de facteurs de transcription dont l activité est modulée par la fixation d un ligand liposoluble. Ils participent à de nombreux processus physiologiques dont ceux liés au développement. Le récepteur nucléaire orphelin NR2E3 est impliqué dans la différentiation des photorécepteurs, ses mutations affectant en particulier les photorécepteurs à cônes. Ceux-ci sont à l origine de la vision des couleurs et de l acuité visuelle centrale, et constituent la cible des approches thérapeutiques développées dans le laboratoire.Chez l homme, des mutations récessives de NR2E3 sont responsables de l ESCS (Enhanced S-Cone Syndrome), une pathologie rétinienne caractérisée par une hypersensibilité à la lumière bleue liée à un excès de cônes bleus.Le domaine de fixation au ligand (LBD) de NR2E3 exerce une activité d inhibition transcriptionnelle en trans d une manière comparable à celle observée pour d autres récepteurs nucléaires en absence de leur ligand. Pour tester l hypothèse de l existence d un ligand pour NR2E3, nous avons examiné comment les mutations à l origine de l ESCS et localisées dans le LBD affectaient l activité d inhibition transcriptionnelle. Nos résultats indiquent une absence de corrélation entre ces mutations et l activité répressive, celle-ci étant conservée pour certaines mutations, et suggèrent qu une autre fonction du LBD de NR2E3 est altérée, telle une activité transcriptionnelle modulée par un ligand.L hypothèse de l existence d un ligand pour NR2E3 est renforcée par l analyse du modèle moléculaire de son LBD indiquant la présence d une poche de fixation pour un ligand. Nous avons initié une recherche de ligands endogènes pour NR2E3 en utilisant un essai fonctionnel inspiré de l endocrinologie inverse. Partant de l hypothèse qu un récepteur nucléaire doit être co-localisé avec son ligand, cet essai fonctionnel a servi au criblage des fractions lipidiques issues de rétines de porc.En conclusion, ce travail apporte des éléments de preuve à l existence d un ligand pour NR2E3 et permet d interpréter les conséquences de ses mutations pathogènes.Nuclear receptor superfamily is a large group of ligand modulated transcription factors, which regulate a variety of crucial metabolic and developmental events. The orphan nuclear receptor NR2E3 is involved in photoreceptor differentiation and its mutations are responsible for abnormal cone photoreceptor development. Cone photoreceptors are responsible for colour vision and central visual acuity, and constitute the main target for therapeutic approaches.Recessive mutations in the human NR2E3 gene cause ESCS (Enhanced S-Cone Syndrome), a retinal disease characterized by a hypersensitivity to blue light related to an excess of short-wavelength sensitive cones.Similarly to other unliganded nuclear receptors, the NR2E3 ligand binding domain (NR2E3LBD) exerts transcriptional inhibition. In order to test the hypothesis for the existence of a ligand for NR2E3, we have studied how the LBD localized ESCS mutations influence transcriptional inhibition. Our results show an absence of correlation between ESCS mutations and NR2E3 repressive activity, suggesting that another NR2E3LBD function is disturbed in ESCS, such as a ligand modulated transcriptional activity.The hypothesis for the existence of a ligand for NR2E3 is reinforced by the analysis of a NR2E3LBD molecular model, pointing out the existence of a ligand binding pocket. We have initiated a search for NR2E3 putative endogenous ligand using a functional assay inspired by reverse endocrinology. According to the assumption that a nuclear receptor should be co-localized with its ligands, this functional assay was used to screen lipidic fractions from porcine retina.In conclusion, this work brings elements of proof for the existence of a ligand for NR2E3 and makes it possible to interpret the consequences of its pathogenic changes.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Mécanismes de régulation de l'expression du facteur trophique Rod-derived Cone Viability Factor, un espoir pour le traitement des dégénérescences rétiniennes

Notre étude porte sur la régulation de l expression du gène Nucleoredoxin like-1 (Nxnl1) qui code pour le facteur Rod-derived Cone Viability Factor (RdCVF), un facteur de survie des photorécepteurs à cônes. Du fait de son action protectrice, RdCVF est un espoir pour le traitement des dégénérescences rétiniennes héréditaires tel que la rétinopathie pigmentaire (RP). Les résultats obtenus sur les modèles de RP étudiés, la souris rd1 et le rat transgénique Rhodopsine P23H, valident l objectif thérapeutique. Par l utilisation d une approche bioinformatique couplée à la transcriptomique, dix-huit facteurs de transcription ont été testés sur le promoteur de Nxnl1dans un modèle hétérologue. Ceci, a conduit à démontrer l activité de l homéogène Chx10/Vsx2 sur le promoteur de Nxnl1 chez la souris et chez l homme. De plus, nous pouvons également affirmer, qu en plus des photorécepteurs, RdCVF aussi exprimé par les cellules de la rétine interne. Cependant, bien que ces cellules ne soient pas affectées par la dégénérescence des bâtonnets chez la souris rd1, nous observons une perte d expression de RdCVF qui englobe les bâtonnets et les cellules de la rétine interne. Notre travail sous-tend donc une stratégie thérapeutique alternative qui consisterait à rétablir l expression endogène de RdCVF par les cellules de la rétine interne pour protéger les cônes et préserver la vision centrale chez les patients souffrant de RPPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Etude du potentiel endogène de réparation de la rétine adulte

Il est admis que certaines régions du système nerveux central des mammifères adultes sont permissives à une neurogénèse, potentiel susceptible d'être activé par la dégénérescence. Afin de tester si la rétine est capable de régénération en vue de maintenir l'intégrité tissulaire, nous avons comparé le niveau de prolifération cellulaire dans une rétine saine par rapport à une rétine en dégénérescence, la rétine rd1. L'injection puis la détection d'un analogue de base d'ADN (BrdU) a notamment permis de détecter deux populations cellulaires, des noyaux localisés dans l'extrême périphérie de la rétine, ainsi que, dans la rétine en dégénérescence seulement, des noyaux localisés dans la couche des photorécepteurs dont une partie exprime des pigments visuels de photorécepteurs.La quantification du nombre de cellules ayant incorporé de la BrdU dans la zone ciliaire suggère un retard de développement dans la rétine rd1 par rapport à la rétine normale sans montrer de réelle induction d'un potentiel régénératif. Cependant, d'autres résultats attestent d'une certaine plasticité de la zone ciliaire adulte.La couche des photorécepteurs présentant une apoptose massive, nous avons voulu vérifier la spécificité de certains marqueurs couramment utilisés pour visualiser la prolifération cellulaire; nos résultats sont cohérents avec des mécanismes de réparation d'ADN dans les photorécepteurs rd1 impliquant la protéine suppresseur de tumeur, p53 ainsi que son partenaire mdm2, un facteur accessoire de DNA Polymérases, PCNA (Proliferating Cell Nuclear Antigen), une ligase, la DNA Ligase IV et son partenaire dans la réparation de cassures double brin de l'ADN, XRCC4. Selon une seconde approche, nous développons un modèle d'étude du potentiel neurogénique de la glie rétinienne: nous cherchons à appliquer à la rétine un système ayant permis, dans le cerveau en développement et adulte, d'attribuer aux cellules gliales un rôle de précurseur neuronal susceptible d'être exploité lors d'une dégénérescence neuronale. Cette hypothèse, testée chez une souris transgénique exprimant le récepteur d'un rétrovirus aviaire sous contrôle d'un promoteur glial permettant théoriquement l'infection donc le suivi des cellules gliales, n'a pu être vérifiée.In light of different recent results suggesting that the adult mammalian central nervous system can produce new neurons, possibly as an endogenous repair mechanism, we investigated whether neurogenesis occurs in response to photoreceptor degeneration in the rd1 mouse, a model of human-inherited retinal dystrophy. Bromodeoxy-Uridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression experiments detected cell proliferation in the extreme peripheral retina, in both wt and rd1 retina, independent of degeneration. BrdU incorporation and PCNA expression also occurred in rd1 photoreceptors. Our results strongly suggest that these photoreceptors are undergoing DNA repair rather than coming from a proliferative event: p53, MDM2, PCNA, DNA ligase IV and XRCC4 are expressed before photoreceptor death, consistent with a model where photoreceptors expressing the rd1 mutation activate a process of DNA repair but which is overwhelmed by the disease mutation leading to apoptotic death. The existence of such a balance offers potential new targets for neuroprotective approaches. On the other hand, we hypothesised retinal glial cells could be neuronal progenitors, as shown for radial glia during cerebral development and for astrocytes in the subventricular zone of the forebrain. To test if glial cells are able to transform into photoreceptors in response to their degeneration, we used a transgenic mouse enabling to follow glial cells through their specific infection with an avian retrovirus. In the mouse model of retinal degeneration due to rd1 expression, the absence of glial proliferation does not allow to infect glial cells with a retrovirus.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Metabolic and Redox Signaling of the Nucleoredoxin-Like-1 Gene for the Treatment of Genetic Retinal Diseases

International audienceThe loss of cone photoreceptor function in retinitis pigmentosa (RP) severely impacts the central and daily vision and quality of life of patients affected by this disease. The loss of cones follows the degeneration of rods, in a manner independent of the causing mutations in numerous genes associated with RP. We have explored this phenomenon and proposed that the loss of rods triggers a reduction in the expression of rod-derived cone viability factor (RdCVF) encoded by the nucleoredoxin-like 1 (NXNL1) gene which interrupts the metabolic and redox signaling between rods and cones. After providing scientific evidence supporting this mechanism, we propose a way to restore this lost signaling and prevent the cone vision loss in animal models of RP. We also explain how we could restore this signaling to prevent cone vision loss in animal models of the disease and how we plan to apply this therapeutic strategy by the administration of both products of NXNL1 encoding the trophic factor RdCVF and the thioredoxin enzyme RdCVFL using an adeno-associated viral vector. We describe in detail all the steps of this translational program, from the design of the drug, its production, biological validation, and analytical and preclinical qualification required for a future clinical trial that would, if successful, provide a treatment for this incurable disease

Altered Expression of Metallothionein-I and -II and Their Receptor Megalin in Inherited Photoreceptor Degeneration

PURPOSE. To examine in rodent models of retinitis pigmentosa (RP) the expression of the neuroprotectants metallothionein-I and -II and of megalin, an endocytic receptor that mediates their transport into neurons. METHODS. Gene and protein expression were analyzed in retinas of rd1 and rds mice and in those of RCS (Royal College of Surgeons) rats of various ages. Glial cell markers (cellular retinaldehyde binding protein, CRALBP; glial fibrillary acidic protein, GFAP; CD11b; and isolectin B4) were used to establish the identity of the cells. RESULTS. Metallothionein-I and -II gene expression increased with age in normal and degenerating retinas and was significantly greater in the latter. Protein expression, corresponding to metallothionein-I + II, was first observed in rd1 mice in Muller cells at postnatal day (P) 12 and in rds mice at P16, coinciding with the onset of GFAP expression in these cells. In RCS rats, the same distribution was observed, but not until P32, long after the onset of GFAP expression. Metallothionein-I + II was observed also in a small number of microglial cells. Megalin was expressed in the nerve fiber layer and in the region of the inner and outer segments in normal animals, but expression in the outer retina was lost with age in degenerating retinas. CONCLUSIONS. Induction of metallothionein-I and -II occurs in the RP models studied and correlates with glial activation. The progressive loss of megalin suggests that transport of metallothionein- I + II into the degenerating photoreceptors (from e.g., Muller cells), could be impaired, potentially limiting the actions of these metallothioneins. (Invest Ophthalmol Vis Sci. 2010;51:4809 -4820) DOI:10.1167/iovs.09-507

Identification of gene expression changes associated with the progression of retinal degeneration in the rd1 mouse.

PURPOSE: One approach to gaining insight into the biological pathways contributing to rod and cone photoreceptor death is to identify patterns of gene expression changes. In the present study, a custom retinal microarray was developed to analyze the rd1 mouse, a well-characterized animal model of human retinal degeneration. METHODS: A microarray was constructed containing cDNA fragments corresponding to genes known or postulated to be involved in normal retinal function, development, and disease. Gene expression in rd1 retina was compared with age-matched control retinas at three time points: the peak of rod degeneration (postnatal day [P]14), early in cone degeneration (P35), and during cone degeneration (P50). Selected microarray results were confirmed with real-time PCR. The cellular distribution of one of the differentially expressed genes, dickkopf 3 (Dkk3), was assessed by in situ hybridization. RESULTS: At each stage of degeneration, there was only limited overlap of the genes that showed increased expression, suggesting the involvement of temporally distinct molecular pathways. Genes active in transport mechanisms and in signaling pathways were differentially expressed during rod degeneration, whereas genes with functions in protein modification and cellular metabolism were differentially expressed during cone degeneration. Increased expression of genes involved in cell proliferation pathways and oxidative stress was observed at each time point. CONCLUSIONS: These microarray results provide clues to understanding the molecular pathways underlying photoreceptor degeneration and indicate directions for future studies. In addition, comparisons of normal and degenerated retina identified numerous genes and ESTs that are potentially enriched in rod photoreceptors