Evidence for glutamate-mediated excitotoxic mechanisms during photoreceptor degeneration in the rd1 mouse retina

PURPOSE: Kinetic studies of photoreceptor cell death in the retinal degeneration (rd1) mouse model suggest that photoreceptor degeneration could result from cumulative damage. Since alterations in glutamate metabolism have been described in different models of retinitis pigmentosa, we investigated in the present work whether changes in glutamate turnover occur in the degenerating rd1 retina and whether glutamate-mediated excitotoxic mechanisms may contribute to rod photoreceptor death in this model. METHODS: Free amino acid levels were quantified in rd1 and wild-type retinas using an amino acid analyzer selecting times corresponding to early, intermediate, and terminal phases of rod photoreceptor degeneration. Reverse transcription-polymerase chain reaction (RT-PCR) was used to compare the mRNA expression levels of the glial L-glutamate/L-aspartate transporter GLAST, glutamine synthetase (GS), and vimentin, a marker for retinal glia, between rd1 and wild-type mouse retinas. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an antagonist of both AMPA and kainate subtypes of ionotropic glutamate receptors, was then daily administered from postnatal day 3 (PN3) to PN21 to rd1 mice while control rd1 mice received only physiological saline solution (7 per treatment). At PN22, the respective numbers of surviving rods i

Does GDNF exert its neuroprotective effects on photoreceptors in the rd1 retina through the glial glutamate transporter GLAST?

PURPOSE: We previously demonstrated that exogenous glial cell line-derived neurotrophic factor (GDNF) induces histological and functional protection of photoreceptors in the retinal degeneration (rd1) mouse model. The mechanisms underlying such neuroprotection remain elusive. In parallel to this work, we provided evidence for the occurrence of glutamate-mediated excitotoxic phenomena contributing to rod photoreceptor death in the rd1 retina in the companion paper. In the present study, we investigated whether, as demonstrated in other models, GDNF could exert its neuroprotective effect on photoreceptors through Muller glial cells (MGC) by promoting the expression of the glial L-glutamate/L-aspartate transporter (GLAST), an endogenous neuroprotective mechanism against glutamate-mediated excitotoxicity. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to compare the mRNA expression levels of GDNF receptors between rd1 and wild-type mouse retinas as well as between MGC and mixed retinal cell cultures. Recombinant GDNF was applied to pure MGC cultures, to rd1 retinal organ cultures and injected subretinally into rd1 mouse eyes. GLAST expression following GDNF treatment was measured by RT-PCR, immunoblotting and immunohistochemistry. Free glutamate and glutamine levels were quantified in rd1 retinas after GDNF or control treatment using an amino acid analyzer. RESULTS: mRNA expression studies of GDNF receptors, GFRalpha-1 and Ret, demonstrated that GDNF receptors were not exclusively expressed by the degenerating photoreceptor cells but mainly by MGC. Exogenous GDNF application to MGC cultures, rd1 mouse retinal explants and in vivo rd1 mouse retinas increased the expression of GLAST by 48% in retinal explants (p<0.005) and by 25% in vivo (p<0.0005). GLAST protein expression in MGC was particularly increased around degenerative photoreceptors. Free glutamate and glutamine levels in the rd1 retina were not significantly modified by exogenous GDNF. CONCLUSIONS: Our data suggest that, in the rd1 mouse retina, GDNF neuroprotective effect on photoreceptors can be mediated indirectly through the activation of MGC. We demonstrate that injection of recombinant GDNF enhances the expression of GLAST and more particularly around the degenerating photoreceptors. Since we failed to demonstrate that GDNF decreases free glutamate levels, we could not ascertain whether GDNF promoted photoreceptor-survival via an increase of glutamate uptake and, therefore, a change in glutamate distributio

Minimisation de la pollution générée par un atelier de traitement de surface

International audienceLe traitement de surface est une activité industrielle générant un chiffre d'affaire annuel de 6 milliards d'euros en France, 900 000 tonnes de matériaux d'apport sont appliqués sur 3 milliards de mètres-carrés. Lorsque cette branche industrielle utilise la voie aqueuse, cette activité est grande consommatrice d'eau et de produits chimiques et donc génératrice de rejets aqueux pollués. L'objectif de ce travail est de proposer une méthodologie simple de limitation des flux de pollution ainsi que de la consommation spécifique en eau. Cette méthodologie se base notamment sur le postulat que l'entraînement est le vecteur principal de la pollution au sein d'une chaîne de traitement de surface. Il a été montré (thèse Florine Leveillard, Saint-Etienne, 2010) que la géométrie des pièces traitées, la durée et l'agitation des rinçages, la durée d'égouttage avaient une forte influence sur l'entrainement alors que la tension de surface des bains de traitement avait une influence moyenne et la durée de rinçage une influence nulle. Le modèle développé répond à trois types d'objectifs : qualitatif (qualité des rinçages optimal), conformité réglementaire (limitation à 8 L/m2/fonction de rinçage) et environnemental (quantification des flux polluants émis par l'entreprise). Ce modèle comprend deux étapes : dans la première la chaîne de traitement est décrite, puis dans la seconde la géométrie des pièces traitées conduit, grâce à l'utilisation d'équations théoriques ou empiriques, à une estimation de l'entraînement corrigée par les valeurs de tension superficielle des bains et de la durée d'égouttage des pièces. Le volume entraîné par les pièces dilué dans les bains de rinçage conduit (i) à l'estimation de la pollution induite nécessaire au dimensionnement de la station d'épuration interne à l'entreprise et (ii) à la vérification du fonctionnement optimum des bains de rinçage

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

WDR34, a candidate gene for non-syndromic rod-cone dystrophy

Rod-cone dystrophy (RCD), also called retinitis pigmentosa, is characterized by rod followed by cone photoreceptor degeneration, leading to gradual visual loss. Mutations in over 65 genes have been associated with non-syndromic RCD explaining 60% to 70% of cases, with novel gene defects possibly accounting for the unsolved cases. Homozygosity mapping and whole-exome sequencing applied to a case of autosomal recessive non-syndromic RCD from a consanguineous union identified a homozygous variant in WDR34. Mutations in WDR34 have been previously associated with severe ciliopathy syndromes possibly associated with a retinal dystrophy. This is the first report of a homozygous mutation in WDR34 associated with non-syndromic RCD

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

Cirrhotic livers reveal genetic changes in the MDM2-P14ARF system of cell cycle regulators

The genesis of hepatocellular carcinoma is promoted by changes in the regulatory MDM2-P14ARF system. The incidence of such changes has to date not been analysed in non-tumourous livers showing regenerative proliferation. In the present study, 24 cirrhotic livers of alcohol-, autoimmue disorder- or HCV-caused genesis were screened for MDM2-P14ARF alterations at the level of protein, DNA and mRNA. Using confocal laser scanning microscopy, the absence of MDM2 and P14ARF expression was detected in all samples except three HCV-infected livers (four livers) which contained hepatocytes overexpressing MDM2 (P14ARF) protein. In two of the samples lacking P14ARF expression, laser microdissection and PCR demonstrated deletion of the P14ARF gene. The P14ARF gene amplified from other specimens did not carry mutations. MDM2 splicing variants were present in tissues from alcohol- and autoimmune disorder-induced cirrhoses. Sequencing of full-size mRNA revealed a MDM2 mis-sense mutation in an alcohol-induced cirrhosis. One sample contained regenerative nodules with genetic instability occurring at MDM2 locus D12S83 according to the data of automatic PCR fragment analysis. In summary, this study gives first evidence for different types of MDM2 and P14ARF alterations in cirrhotic livers. We suggest that the changes impair the regulatory MDM2-P14ARF system, thus possibly favouring regenerative proliferation and transformation

Neuroprotective effects of the cannabinoid agonist HU210 on retinal degeneration

Cannabinoids have been demonstrated to exert neuroprotective effects on different types of neuronal insults. Here we have addressed the therapeutic potential of the synthetic cannabinoid HU210 on photoreceptor degeneration, synaptic connectivity and functional activity of the retina in the transgenic P23H rat, an animal model for autosomal dominant retinitis pigmentosa (RP). In P23H rats administered with HU210 (100 μg/kg, i.p.) from P24 to P90, ERG recordings showed an amelioration of vision loss, as compared to vehicle-administered animals. Under scotopic conditions, the maximum a-wave amplitudes recorded at P60 and P90 were higher in HU210-treated animals, as compared to the values obtained in untreated animals. The scotopic b-waves were significantly higher in treated animals than in untreated rats at P30, P60 and P90. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. HU210-treated animals had 40% more photoreceptors than untreated animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were also preserved in HU210-treated P23H rats. These results indicate that HU210 preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in P23H rats. These data suggest that cannabinoids are potentially useful to delay retinal degeneration in RP patients.This research was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2012-36845-FEDER), Instituto de Salud Carlos III (RETICS RD12/0034/0010), Universidad de Alicante (UA2010-48536273), and the Organización Nacional de Ciegos Españoles (ONCE)