Dietary supplement enriched in antioxidants and omega-3 promotes retinal glutamine synthesis.

To prevent ocular pathologies, new generation of dietary supplements have been commercially available. They consist of nutritional supplement mixing components known to provide antioxidative properties, such as unsaturated fatty acid, resveratrol or flavonoids. However, to date, few data evaluating the impact of a mixture mainly composed of those components (Nutrof Total®) on the retina are available. Only one in-vivo preclinical study demonstrated that dietary supplementation (DS) prevents the retina from light-induced retinal degeneration; and only one in-vitro study on Müller cells culture showed that glutamate metabolism cycle was key in oxidative stress response. Therefore, we raised the question about the in-vivo effect of DS on glutamate metabolism in the retina. Herein, we showed that the dietary supplementation promotes in-vivo increase of retinal glutamine amount through a higher glutamine synthesis as observed in-vitro on Muller cells. Therefore, we can suggest that the promotion of glutamine synthesis is part of the protective effect of DS against retinal degeneration, acting as a preconditioning mechanism against retinal degeneration.

FMRP-related retinal phenotypes: Evidence of glutamate-glutamine metabolic cycle impairment.

FMRP, the fragile X mental retardation protein coded by the FMR1 gene, is an RNA-binding protein that assists transport, stabilization and translational regulation of specific synaptic mRNAs. Its expression has been found in multiple cell types of central nervous system (CNS) including glial cells where its involvement in glutamate neurotransmitter homeostasis have been shown. Indeed, glutamate homeostasis deficit has been observed in absence of FMRP in-vivo in cortex and hippocampus structures as well as in vitro on astroglial cell culture. Interestingly, the retina which is an extension of the CNS is presenting electrophysiological alterations in absence of FMRP in both human and murine models suggesting neurotransmitter impairments. Therefore, we investigate the consequences of Fmrp absence on Glutamate-Glutamine cycle in whole retinas and primary retinal Müller cells culture which are the main glial cells of the retina. Using the Fmr1-/y mice, we have shown in vivo and in vitro that the absence of Fmrp in Müller cells is characterized by loss of Glutamate-Glutamine cycle homeostasis due to a lower Glutamine Synthetase protein expression and activity. The lack of Fmrp in the retina induces a reduced flow of glutamine synthesis. Our data established for the first time in literature a direct link between the lack of Fmrp and neurotransmitter homeostasis in the retina.

Dietary Supplement Enriched in Antioxidants and Omega-3 Promotes Glutamine Synthesis in Müller Cells: A Key Process against Oxidative Stress in Retina.

To prevent ocular pathologies, new generation of dietary supplements have been commercially available. They consist of nutritional supplement mixing components known to provide antioxidative properties, such as unsaturated fatty acid, resveratrol or flavonoids. However, to date, only one preclinical study has evaluated the impact of a mixture mainly composed of those components (Nutrof Total®) on the retina and demonstrated that in vivo supplementation prevents the retina from structural and functional injuries induced by light. Considering the crucial role played by the glial Müller cells in the retina, particularly to regulate the glutamate cycle to prevent damage in oxidative stress conditions, we questioned the impact of this ocular supplement on the glutamate metabolic cycle. To this end, various molecular aspects associated with the glutamate/glutamine metabolism cycle in Müller cells were investigated on primary Müller cells cultures incubated, or not, with the commercially mix supplement before being subjected, or not, to oxidative conditions. Our results demonstrated that in vitro supplementation provides guidance of the glutamate/glutamine cycle in favor of glutamine synthesis. These results suggest that glutamine synthesis is a crucial cellular process of retinal protection against oxidative damages and could be a key step in the previous in vivo beneficial results provided by the dietary supplementation.

Visual Behavior Impairments as an Aberrant Sensory Processing in the Mouse Model of Fragile X Syndrome.

Fragile X Syndrome (FXS), the most common inherited form of human intellectual disability (ID) associated with autistic-like behaviors, is characterized by dys-sensitivity to sensory stimuli, especially vision. In the absence of Fragile Mental Retardation Protein (FMRP), both retinal and cerebral structures of the visual pathway are impaired, suggesting that perception and integration of visual stimuli are altered. However, behavioral consequences of these defects remain unknown. In this study, we used male Fmr1 -/y mice to further define visual disturbances from a behavioral perspective by focusing on three traits characterizing visual modality: perception of depth, contrasts and movements. We performed specific tests (Optomotor Drum, Visual Cliff) to evaluate these visual modalities, their evolution from youth to adulthood, and to assess their involvement in a cognitive task. We show that Fmr1 -/y mice exhibit alteration in their visual skills, displaying impaired perspective perception, a drop in their ability to understand a moving contrasted pattern, and a defect in contrasts discrimination. Interestingly, Fmr1 -/y phenotypes remain stable over time from adolescence to late adulthood. Besides, we report that color and shape are meaningful for the achievement of a cognitive test involving object recognition. Altogether, these results underline the significance of visual behavior alterations in FXS conditions and relevance of assessing visual skills in neuropsychiatric models before performing behavioral tasks, such as cognitive assessments, that involve visual discrimination.

Early Retinal Defects in Fmr1-/y Mice: Toward a Critical Role of Visual Dys-Sensitivity in the Fragile X Syndrome Phenotype?

Fragile X Syndrome (FXS) is caused by a deficiency in Fragile X Mental Retardation Protein (FMRP) leading to global sensorial abnormalities, among which visual defects represent a critical part. These visual defects are associated with cerebral neuron immaturity especially in the primary visual cortex. However, we recently demonstrated that retinas of adult Fmr1-/y mice, the FXS murine model, present molecular, cellular and functional alterations. However, no data are currently available on the evolution pattern of such defects. As retinal stimulation through Eye Opening (EO) is a crucial signal for the cerebral visual system maturation, we questioned the precocity of molecular and functional retinal phenotype. To answer this question, we studied the retinal molecular phenotype of Fmr1-/y mice before EO until adult age and the consequences of the retinal loss of Fmrp on retinal function in young and adult mice. We showed that retinal molecular defects are present before EO and remain stable at adult age, leading to electrophysiological impairments without any underlying structural changes. We underlined that loss of Fmrp leads to a wide range of defects in the retina, settled even before EO. Our work demonstrates a critical role of the sensorial dysfunction in the Fmr1-/y mice overall phenotype, and provides evidence that altered peripheral perception is a component of the sensory processing defect in FXS conditions.

A fraction of neurofibromin interacts with PML bodies in the nucleus of the CCF astrocytoma cell line.

Neurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line: CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using "in situ" proximity ligation assay (PLA).

A new putative target for antisense gene therapy of glioma: glycogen synthase.

The treatment of malignant brain gliomas remains a challenge, despite the availability of the classical triad of surgery, radiotherapy, and chemotherapy. There is thus the need for investigations into other forms of treatment strategies, such as gene therapy. Using antisense technology we have targeted glycogen metabolism, since malignant astrocytes present a high content of glycogen. In vitro rat C6­glioma cells, transfected with antisense glycogen synthase (C6­AS cells) exhibited a decreased expression of glycogen synthase and reduced activity of glycogen synthesis, along with attenuated invasiveness. In vivo tumors induced by C6­AS cells in nude mice exhibited a significant reduction in tumor growth compared with controls. This reduction could be mediated by the induction of MCH­I expression. The inhibition of glycogen synthesis by antisense glycogen synthase validates a putative target and a new approach for further study to advance the much­needed efficacy of intervention strategies for malignant gliomas.

Stable transfection of cDNAs targeting specific steps of glycogen metabolism supports the existence of active gluconeogenesis in mouse cultured astrocytes.

In order to assess the participation of astrocytic gluconeogenesis in the synthesis of glycogen, mouse astrocytes were stably transfected with antisense cDNA of fructose-1,6-bisphosphatase (FBPase) and with sense and antisense cDNAs of glycogen synthase (GS). The antisenses of FBPase and GS have similar significant effect in decreasing astrocyte glycogen content by 60%, while sense GS significantly increased glycogen content by 100%. The FBPase activity was decreased by all three cDNAs used, while glycogen phosphorylase was not altered. The activity of GS was decreased by the antisense GS and increased by the sense GS. These data demonstrate that the gluconeogenesis in astrocytes is involved in the glycogenesis modulation.