FMR protein: Evidence of an emerging role in retinal aging?

Aging is a multifactorial process that affects the entire organism by cumulative alterations. Visual function impairments that go along with aging are commonly observed, causing lower visual acuity, lower contrast sensitivity, and impaired dark adaptation. Electroretinogram analysis revealed that the amplitudes of rod- and cone-mediated responses are reduced in aged mice and humans. Reports suggested that age-related changes observed in both rod and cone photoreceptor functionality were linked to oxidative stress regulation or free radical production homeostasis. Interestingly, several recent reports linked the fragile X mental retardation protein (FMRP) cellular activity with oxidative stress regulation in several tissue including brain tissue where FMRP participates to the response to stress via protein translation in neurite or is involved in free radical production and abnormal glutathione homeostasis. Based on these recent literatures, we raised the question about the effect of FMRP absence in the aging retina of Fmr1-/y compared to their WT littermates. Indeed, up to now, only young or adult mice (<6 months) were investigated and have shown a specific retinal phenotype. Herein, we demonstrated that Fmr1-/y mice do not present the aging effect on retinal function observed in WT littermates since ERG a- and b-waves amplitudes as well as oscillatory potentials amplitudes were not collapsed with age (12/18 months old). Absence of FMRP and its consequences seem to protect the retina against aging effect, rising a pivotal role of FMRP in retinal aging process.

Fragile X Mental Retardation Protein expression in the retina is regulated by light.

Fragile X Mental Retardation Protein (FMRP) is a RNA-binding protein that modulates protein synthesis at the synapse and its function is regulated by glutamate. The retina is the first structure that participates in vision, and uses glutamate to transduce electromagnetic signals from light to electrochemical signals to neurons. FMRP has been previously detected in the retina, but its localization has not been studied yet. In this work, our objectives were to describe the localization of FMRP in the retina, to determine whether different exposure to dark or light stimulus alters FMRP expression in the retina, and to compare the pattern in two different species, the mouse and chick. We found that both FMRP mRNA and protein are expressed in the retina. By immunohistochemistry analysis we found that both mouse and chick present similar FMRP expression localized mainly in both plexiform layers and the inner retina. It was also observed that FMRP is down-regulated by 24 h dark adaptation compared to its expression in the retina of animals that were exposed to light for 1 h after 24 h in the dark. We conclude that FMRP is likely to participate in retinal physiology, since its expression changes with light exposure. In addition, the expression pattern and regulation by light of FMRP seems well conserved since it was similar in both mouse and chick.

Immunonutrition improves functional capacities in head and neck and esophageal cancer patients undergoing radiochemotherapy: a randomized clinical trial.

BACKGROUND & AIMS: Malnutrition is frequent in head and neck (HN) and esophageal cancer patients and aggravated by radiochemotherapy (RCT), increasing morbi-mortality and treatment toxicity. Our goal was to investigate the effect of immunonutrition consisting of an arginine, omega-3 fatty acid, nucleotides-enriched diet on nutritional status, and functional capacity in HN or esophageal cancer patients undergoing RCT. METHODS: 37 patients were randomized in a double-blind clinical trial. 5 days before and until the end of RCT (5-7 weeks), they received either an Immunomodulating Enteral Nutrition (IEN) or an isonitrogenous, isoenergetic Standard Enteral Nutrition (SEN). Anthropometrical parameters, nutritional risk index (NRI), serum albumin, plasma antioxidant capacity, and functional capacity were recorded between the beginning and the end of RCT. RESULTS: A significant gain in total body weight (+2.1 ± 3.1 kg) was observed in IEN patients. Albuminemia and NRI were improved concomitantly in IEN malnourished patients. Plasma antioxidant capacity was improved (+100 ± 13 µM EqTrolox) in IEN patients. Functional capacity measured by WHO Performance Status and Karnofsky index was maintained in IEN patients but significantly reduced in SEN patients. CONCLUSIONS: These preliminary data show that immunonutrition could improve the nutritional status together with functional capacity in HN and esophageal cancer patients undergoing RCT. CLINICAL TRIAL REGISTRATION: This clinical trial promoted by the University Hospital Center of Clermont-Ferrand has been registered at ClinicalTrial.gov website under the following reference: NCT00333099.

[Autism, genetics and synaptic function alterations]. / Autisme, génétique et anomalies de la fonction synaptique.

Autism is a neurodevelopmental disorder characterized by a deficit of language and communication both associated with a restricted repertoire of activities and interests. The current prevalence of autistic disorder stricto sensu is estimated at 1/500 whereas autism spectrum disorders (ASD) increases up to 1/150 to 1/200. Mental deficiency (MD) and epilepsy are present in numerous autistic individuals. Consequently, autism is as a major public health issue. Autism was first considered as a non biological disease; however various rational approaches for analysing epidemiological data suggested the possibility of the influence of genetic factors. In 2003, this hypothesis was clearly illustrated by the characterization of genetic mutations transmitted through a mendelian manner. Subsequently, the glutamate synapse appeared as a preferential causal target in autism because the identified genes encoded proteins present in this structure. Strikingly, the findings that an identical genetic dysfunction of the synapse might also explain some MD suggested the possibility of a genetic comorbidity between these neurodevelopmental conditions. To date, various identified genes are considered indifferently as "autism" or "MD" genes. The characterization of mutations in the NLGN4X gene in patients with Asperger syndrome, autism without MD, or MD without autism, was the first example. It appears that a genetic continuum between ASD on one hand, and between autism and MD on the other hand, is present. Consequently, it is likely that genes already involved in MD will be found mutated in autistic patients and will represent future target for finding new factors in autism.

Calpains are activated by light but their inhibition has no neuroprotective effect against light-damage.

Calpain had been shown to be highly activated at one day after exposure to the damaging light (Perche et al. (2007)Caspase-dependent apoptosis in light-induced retinal degeneration. Invest Ophthalmol Vis Sci 48:2753-2759.), suggesting that they might play a critical role in photoreceptor apoptosis induced by light. Therefore in the present study we investigate the role of calpain in light-induced photoreceptor cell death. In a first set of experiments, untreated albino Wistar rats were sacrificed at 0, 2, 4, 6, 12, 24 h of light exposure and at one day after the light was turned off (D1) to measure retinal calpain activity and to study calpain expression. In a second set of experiments, after control electroretinograms (ERGs), rats were uninjected or injected intravitreally with DMSO or the calpain inhibitor Mu-Phe-hPhe-FMK, before being exposed to the damaging light for 24 h. ERGs were then recorded at one day (D1) and fifteen days (D15) after the end of light exposure. Rats were sacrificed at D1 for apoptotic cell detection or D15 for histological analysis (ONL thickness). Calpain activity and expression significantly increased in Untreated retinas, from 0 h to D1. DMSO has no effect on calpain activity. Mu-Phe-hPhe-FMK significantly inhibited retinal calpain activity by 85% at 2 h of light exposure and still 48% at D1. However, Mu-Phe-hPhe-FMK has no effect on light-induced retinal degeneration as evidence by equivalent loss of function, equivalent loss of photoreceptor cells and an equivalent number of apoptotic cells in Mu-Phe-hPhe-FMK and DMSO retinas. Therefore, calpains are up-regulated by light stress but they do not have a pivotal role in photoreceptor apoptosis.

Transient protective effect of caspase inhibitors in RCS rat.

In most retinal degenerations in humans and in animal models, photoreceptor cells die by apoptosis. Although the biochemical features are similar in all apoptotic cells, different molecular events lead the cell to death. In the present study we used a rat model of inherited retinal degeneration, the RCS rats, to investigate the involvement of the proteases, caspases and/or calpains, in photoreceptor apoptosis. In the first experiments, rats were untreated or injected intravitreally at post natal day 27 (P27) with the large broad spectrum caspase inhibitor, ZVAD, the calpain inhibitor, MuhPhe, or with the vehicle, DMSO. Retinal status was evaluated at P35 and P42 by electroretinography, morphometry and apoptotic nuclei detection. DMSO and MuhPhe had no effect on RCS retinas as evidenced by equivalent loss of function and equivalent number of apoptotic cells than in untreated group. ZVAD transiently reduced apoptotic cells and preserved photoreceptor function at P35 but not at P42. These results suggest that caspases but not calpains are involved in retinal degeneration in the RCS. In the second experiments, RCS rats were injected twice at P27 and P35 with ZVAD or DMSO. Although ZVAD-treated retinas were preserved at P35 compared to the DMSO controls, the second injection of ZVAD did not extend the preserving effect to P42. Moreover, a single injection of ZVAD at P35 had no preserving effect at P42. All these data taken together suggest that caspases do not play a pivotal role after P35. In a fourth set of experiments, we used specific caspase inhibitors to elucidate which caspase was activated. The caspase-1/4 inhibitor (YVAD) or the caspase-3/7 inhibitor (DEVD) were injected intravitreally at P27 and retinal status was evaluated at P35 and P42. Electroretinograms and apoptotic nuclei detection demonstrated that YVAD and DEVD preserved photoreceptors at P35 but not at P42. These results suggest that both caspase-1/4 and caspase-3/7 play a major role in the apoptotic pathway between P27 and P35 in retinal degeneration of RCS rats. In this study, we show that 1/ the photoreceptor apoptotic process in the RCS rat involves caspases but not calpains, and 2/ the retinal degeneration seems to be composed of different phases involving different molecular players. Indeed, we have demonstrated that caspases are playing a major role at P35, but not at P42.

Origin of type I collagen localized within oviduct epithelium of quail hyperstimulated by progesterone.

Bird oviduct development is controlled by sex steroid hormones. Estrogens (E) induce cell proliferation, formation of tubular glands by epithelial cell evagination and cell differentiation. Progesterone (P) strongly increases secretory processes in E-treated quails, but inhibits cell proliferation and cell evagination. The balance between E and P is very critical for the development and morphogenesis of the oviduct. After six daily injections of low doses of E (10 micrograms day-1) and high doses of P (5 mg day-1) into ovariectomized quails, cell proliferation and secretory process are stimulated but cell evagination is totally inhibited and distribution of striated collagen is perturbed. Using antibodies against type I collagen the stroma, which is mainly composed of fibroblasts, is brightly stained, as are some regions within the epithelium. Electron microscopy shows that bundles of striated collagen fibrils appear in extracellular spaces between the lateral membranes of the epithelial cells or between the basal lamina and the epithelial basal membrane. After in situ hybridization using a 35S riboprobe specific for mRNA of the alpha 2 chain of type I collagen, mRNA was detected only in the fibroblasts of the stroma and not in epithelial cells. Furthermore electron microscope studies of collagen bundles in serial sections clearly show collagen fibrils passing through the basal lamina. It is assumed that the type I collagen between epithelial cells originates from mesenchymal cells. In the oviduct of immature birds or after physiological E + P stimulation, striated collagen is localized only in the stroma and never within the epithelium. These results indicate a modulation of extracellular matrix by sex steroid hormones in the quail oviduct.

Modification of cell evagination and cell differentiation in quail oviduct hyperstimulated by progesterone.

Quail oviduct development is controlled by sex steroid hormones. Estrogen (E) induce cell proliferation, formation of tubular glands by epithelial cell evagination and cell differentiation. Progesterone (P) strongly increases the secretory process in E-treated quails, but inhibits cell proliferation, cell evagination and differentiation of ciliated cells. The balance between E and P is critical for harmonious development of the oviduct. After 6 daily injections of two doses of estradiol benzoate (10 or 20 micrograms/d) and high doses of P (4 mg/d), tubular gland formation by epithelial cell evagination was inhibited, while epithelial cell proliferation occurred, as shown by the height of the villi and the increase in DNA. Secretory processes were strongly stimulated. Ovalbumin, a tubular gland cell marker and avidin, a mucous cell marker, were localized by immunofluorescence and immunogold labeling. Ovalbumin was localized only in the rudimentary tubular glands, whereas avidin was dispersed throughout the secretory cells. High doses of progesterone inhibited tubular gland cell proliferation, disturbed the distribution of avidin and inhibited differentiation of ciliated cells. Ovalbumin synthesis occurred only in epithelial cells which were evaginated despite the hyperstimulation. Ovalbumin gene expression appeared highly dependent upon the cell position.

Heterogeneity of progesterone receptor expression in epithelial cells of immature and differentiating quail oviduct.

The localization of progesterone receptor (PR) in the quail oviduct was investigated before and after the onset of sexual maturation using an immunohistochemical technique. PR was revealed exclusively in nuclei of target cells whatever the hormonal state of the tissue (immature or not, pretreated or not with progesterone). In the immature or ovariectomized quail oviduct, PR was principally localized in the undifferentiated epithelial cells; some mesothelial cells and a very few stromal cells expressed the PR. Only 40-45% of the epithelial cells were immunoreactive. These positive cells were mainly localized in the furrows of the villi where further evagination of the epithelium will occur to form the tubular glands. The onset of sexual maturation was accompanied by an increase of the proportion of positive epithelial cells and stromal cells. In estradiol-treated animals, more than 90% of the tubular gland cells were strongly stained while only 40% of the luminal epithelial cells were immunoreactive. Our results show that there are two subpopulations of epithelial cells: those expressing the PR before the onset of sexual maturation even in ovariectomized quails (constitutive expression) and those expressing the PR during sexual maturation or after estrogen injection (inductive expression). These results, associated with previously published studies dealing with the cytodifferentiation of epithelial cells during natural development or after various hormonal treatments in ovariectomized animals, suggest that the first are the progenitors of tubular gland cells, and the second the progenitors of ciliated and goblet cells. In stromal cells, PR expression is also inducuible.

Immunolocalization of laminin during estrogen-induced differentiation of quail oviduct epithelial cells.

During estrogen-induced development of the quail oviduct, tubular glands are formed by evagination of epithelial cells into the stroma. The distribution of laminin was studied during the early stages by means of immunofluorescence and immunoperoxidase techniques. Ultrastructural changes in the basal lamina were studied by electron microscopy. Basement membranes at all stages of development were delineated with 3 polyclonal antilaminin antisera. However, in ovariectomized birds, laminin could not be detected by one of the polyclonal antilaminin antisera. Subsequently, this antibody detected laminin as epithelial cell evaginations were induced by estradiol benzoate. The heavy and light chains of Engelbreth Holm sarcoma (EHS) laminin were revealed in immunoblotting by all antibodies. By electron microscopy after the immunoperoxidase technique with antilaminin antisera laminin appears to be accumulated mainly in the lamina densa. Furthermore, the thickness of the basal lamina increases during oviduct development. These data indicate that basal lamina organization is modified during oviduct cell differentiation and that immunoreactivity of epithelial basement membrane laminin changes during development.