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International audience; The regulation of cholesterol, an essential brain lipid, ensures proper neuronal development and function, as demonstrated by links between perturbations of cholesterol metabolism and neurodegenerative diseases, including Alzheimer's disease. The central nervous system (CNS) acquires cholesterol via de novo synthesis, where glial cells provide cholesterol to neurons. Both lipoproteins and lipoprotein receptors are key elements in this intercellular transport, where the latter recognize, bind and endocytose cholesterol containing glia-produced lipoproteins. CNS lipoprotein receptors are like those in the periphery, among which include the ApoB, E binding lipolysis stimulated lipoprotein receptor (LSR). LSR is a multi-meric protein complex that has multiple isoforms including α and α', which are seen as a dou-blet at 68 kDa, and β at 56 kDa. While complete inactivation of murine lsr gene is embryonic lethal, studies on lsr +/-mice revealed altered brain cholesterol distribution and cognitive functions. In the present study, LSR profiling in different CNS regions revealed regiospecific expression of LSR at both RNA and protein levels. At the RNA level, the hippocampus, hypo-thalamus, cerebellum, and olfactory bulb, all showed high levels of total lsr compared to whole brain tissues, whereas at the protein level, only the hypothalamus, olfactory bulb, and retina showed the highest levels of total LSR. Interestingly, major regional changes in LSR expression were observed in aged mice which suggests changes in cholesterol homeostasis in specific structures in the aging brain. Immunocytostaining of primary cultures of mature murine neurons and glial cells isolated from different CNS regions showed that LSR is expressed in both neurons and glial cells. However, lsr RNA expression in the cerebellum was predominantly higher in glial cells, which was confirmed by the immunocytostaining profile of cerebellar neurons and glia. Based on this observation, we would propose that LSR in glial cells may play a key role in glia-neuron cross talk, particularly in the feedback control of cholesterol synthesis to avoid cholesterol overload in neurons and to maintain proper functioning of the brain throughout life.

Chronic low dose exposure and possible cumulative effects of various pollutants could affect consumer health and may contribute to the development of neurodegenerative pathologies. Due to the highly complex and long exposure, a clear link between diet contaminants and neurodegenerative diseases has often been suspected, but rarely proven. An additional drawback is the high diversity of in vitro models (cancer cell lines, stem cells, primary embryonic cells), bringing additional complexity in the deciphering of the observed effects of pollutants. To properly assess the risks and to reevaluate the maximal acceptable dose of specific pollutants in the food chain, there is a need for efficient modeling of pollutant effects on the central nervous system (CNS). To address this need, we are developing a new approach to evaluate the consequences on neuronal health of long time exposure to pollutants. We are actually re-evaluating the neurotoxic effects of chlordecone (CLD) as proof-of-concept of our strategy. Several concentrations of CLD were used to treat a variety of mouse primary neurons isolated from different postnatal CNS areas. We then assessed neuronal functions using specific markers for neuronal death, neurite development and synapse formation. In parallel, we produced cerebrospinal fluid (CSF) from pigs exposed to CLD via contaminated food. This CSF containing CLD and its by-products that are able to cross the blood brain barrier could then be used on the same cultures to compare its effect with that obtained following direct treatment with the molecule. We were able to identify a direct neurotoxic effect (10 µM) on specific purified neuronal cultures together with more subtle damages at lower concentrations including neurite arborization defects (0.1 to 1µM depending on the respective neuronal cultures). Additional effects on pure glia cell cultures at higher concentrations (100 µM) suggest a reactive gliosis in the whole animal. These observations were confirmed by CSF treatment using CLD-contaminated CSF but not with CSF from control pigs. In addition, the dose with no observable effects is at least 10 times lower using primary postnatal neuronal cultures compared to embryonic cultures. Our experimental model is therefore much more sensitive and may reflect more precisely the consequences of chronic CLD exposure. Our strategy could help to re-evaluate the CNS effects of this remnant pollutant present in West Indies soils.

International audience; Oligomeric amyloid-beta (A beta) peptide contributes to impaired synaptic connections and neurodegenerative processes, and as such, represents a primary therapeutic target for Alzheimer's disease (AD)-modifying approaches. However, the lack of efficacy of drugs that inhibit production of A beta demonstrates the need for a better characterization of its toxic effects, both on synaptic and neuronal function. Here, we used conditioned medium obtained from recombinant HEK-A beta PP cells expressing the human amyloid-beta protein precursor (A beta-CM), to investigate A beta-induced neurotoxic and synaptotoxic effects. Characterization of A beta-CM revealed that it contained picomolar amounts of cell-secreted A beta in its soluble form. Incubation of primary cortical neurons with A beta-CM led to significant decreases in synaptic protein levels as compared to controls. This effect was no longer observed in neurons incubated with conditioned medium obtained from HEK-A beta PP cells grown in presence of the B-secretase inhibitor, Semagacestat or LY450139 (LY-CM). However, neurotoxic and pro-apoptotic effects of A beta-CM were only partially prevented using LY-CM, which could be explained by other deleterious compounds related to chronic oxidative stress that were released by HEK-A beta PP cells. Indeed, full neuroprotection was observed in cells exposed to LY-CM by additional treatment with the antioxidant resveratrol, or with the pluripotent n-3 polyunsaturated fatty acid docosahexaenoic acid. Inhibition of A beta production appeared necessary but insufficient to prevent neurodegenerative effects associated with AD due to other neurotoxic compounds that could exert additional deleterious effects on neuronal function and survival. Therefore, association of various types of protective agents needs to be considered when developing strategies for AD treatment.

International audience; Photodynamic therapy (PDT) has shown to be a promising technique to treat various forms of malignant neoplasia. The photodynamic eradication of the tumor cells is achieved by applying a photosensitizer either locally or systemically and following local activation through irradiation of the tumor mass with light of a specific wavelength after a certain time of incubation. Due to preferential accumulation of the photosensitizer in tumor cells, this procedure allows a selective inactivation of the malignant tumor while sparing the surrounding tissue to the greatest extent. These features and requirements make the PDT an attractive therapeutic option for the treatment of retinoblastoma, especially when surgical enucleation is a curative option. This extreme solution is still in use in case of tumours that are resistant to conventional chemotherapy or handled too late due to poor access to medical care in less advanced country. In this study we initially conducted in-vitro investigations of the new cationic water-soluble photo sensitizer tetrahydroporphyrin-tetratosylat (THPTS) regarding its photodynamic effect on human Rb-1 and Y79 retinoblastoma cells. We were able to show, that neither the incubation with THPTS without following illumination, nor the sole illumination showed a considerable effect on the proliferation of the retinoblastoma cells, whereas the incubation with THPTS combined with following illumination led to a maximal cytotoxic effect on the tumor cells. Moreover the phototoxicity was lower in normal primary cells from retinal pigmented epithelium demonstrating a higher phototoxic effect of THPTS in cancer cells than in this normal retinal cell type. The results at hand form an encouraging foundation for further in-vivo studies on the therapeutic potential of this promising photosensitizer for the eyeball and vision preserving as well as potentially curative therapy of retinoblastoma.

The repair of central nerves remains a major challenge in regenerative neurobiology. Regenerative guides possessing critical features such as cell adhesion, physical guiding and topical stimulation are needed. To generate such a guide, silk protein materials are prepared using electrospinning. The silk is selected for this study due to its biocompatibility and ability to be electrospun for the formation of aligned biofunctional nanofibers. The addition of Brain Derived Neurotrophic Factor (BDNF), Ciliary Neurotrophic Factor (CNTF) or both to the electrospun fibers enable enhanced function without impact to the structure or the surface morphology. Only a small fraction of the loaded growth factors is released over time allowing the fibers to continue to provide these factors to the cells for extended periods of time. The entrapped factors remain active and available to the cells as rat retinal ganglion cells (RGCs) exhibit longer axonal growth when in contact with the biofunctionalized fibers. Compare to non-functionalized fibers, the growth of neurites increased 2 fold on fibers containing BDNF, 2.5 fold with fibers containing CNTF and by almost 3-fold on fibers containing both factors. The results demonstrate the potential of aligned and functionalized electrospun silk fibers to promote nerve growth in the central nervous system, underlying the great potential of complex biomaterials in neuroregenerative strategies following axotomy and nerve crush traumas.

There is increasing evidence that synapse function depends on interactions with glial cells, namely astrocytes. Studies on specific neurons of the central nervous system (CNS) indicated that glial signals also control synapse development, but it remained unclear whether this is a general principle that applies to other neuronal cell types. To address this question, we developed new methods to immunoisolate neurons from different brain regions of postnatal mice and to culture them in a chemically defined medium. Electrophysiological recordings and immunocytochemical staining revealed vigorous synaptogenesis in hippocampal and cerebellar neurons, but not in retinal ganglion cells (RGCs) in the absence of glial cells. Co-culture with glia promoted synapse formation in RGCs as indicated by a strong increase in the incidence and frequency of action potential-independent miniature synaptic currents, but showed no such effects in hippocampal or cerebellar neurons. On the other hand, glial signals promoted the efficacy of excitatory synapses in all regions as indicated by an increase in the size of spontaneous synaptic events in cerebellar cultures and of miniature synaptic currents in hippocampal neurons and RGCs. Inhibitory synaptic currents remained largely unaffected by glia. Our results indicate that in the mammalian CNS, the way that glial signals promote the development of excitatory synapses depends on the type of neuron.

Cholesterol is a multifacetted molecule, which serves as essential membrane component, as cofactor for signaling molecules and as precursor for steroid hormones. Despite intense research on the diverse aspects of cholesterol, the role of cholesterol in the nervous system is still little understood. Our recent studies on primary cultures of highly purified neurons from the rodent central nervous system (CNS) suggest that during development, neurons reduce or even abandon cholesterol synthesis and import cholesterol from astrocytes via lipoproteins. Neurons use the glia-derived cholesterol to form numerous and efficient synapses. This provokes new ideas about the role of astrocytes as cholesterol producers and about the function of cholesterol in the CNS and its involvement in neurodegenerative diseases.

International audience

The abnormal retinal electrophysiology observed in patients with Duchenne muscular dystrophy (DMD) has been attributed to an altered expression of C-terminal products of the dystrophin gene. It has been shown that Dp260 is expressed by photoreceptor cells, whereas Dp71 is present in glial cells. The present study was intended to identify all known members of the dystrophin superfamily and their associated proteins expressed in Müller glial cells (MGC).The expression of the proteins and of their messengers was studied in MGC cultures from 2-week-old rats, by polymerase chain reaction amplification, Western blot analysis, and immunocytochemistry. An immunocytochemical localization of the proteins was also performed on enzymatically dissociated Müller cells from adult rat retinas.MGCs expressed a spliced isoform of Dp71 called Dp71f, as well as utrophin, beta-dystroglycan, delta and gamma-sarcoglycans, and alpha1-syntrophin. In morphologically preserved differentiated Müller cells, Dp71f was localized in clusters, utrophin was diffusely distributed in the cytoplasm, and dystrophin-associated proteins (DAPs) were membrane-bound. Most of these proteins were preferentially expressed in the vitread portion of the cells. Dp71f and utrophin expression was restricted to MGCs, whereas all DAPs were also present in other retinal cell types.The exclusive localization of Dp71f and utrophin in MGCs suggests that these proteins, together with DAPs, play a specific role in these cells. Further knowledge of possible interactions of these proteins within a functional complex may provide new insights into the molecular basis of the electroretinogram phenotype in DMD.

International audience; The abnormal retinal neurotransmission observed in Duchenne muscular dystrophy patients has been attributed to altered expression of C-terminal products of the dystrophin gene in this tissue. Müller glial cells from rat retina express dystrophin protein Dp71, utrophin and the members of the dystrophin-associated glycoprotein complex (DGC), namely beta-dystroglycan, delta- and gamma-sarcoglycans and alpha1-syntrophin. The DGC could function in muscle as a link between the cystoskeleton and the extracellular matrix, as well as a signaling complex. However, other than in muscle the composition and intermolecular associations among members of the DGC are still unknown. Here we demonstrate that Dp71 and/or utrophin from rat retinal Müller glial cells form a complex with beta-dystroglycan, delta-sarcoglycan and alpha1-syntrophin. We also show that beta-dystroglycan is associated with alpha-dystrobrevin-1 and PSD-93 and that anti-PSD antibodies coimmunoprecipitated alpha-syntrophin with PSD-93. By overlay experiments we also found that Dp71and/or utrophin and alpha-dystroglycan from Müller cells could bind to actin and laminin, respectively. These results indicate that the DGC could have both structural and signaling functions in retina. On the basis of our accumulated evidence, we propose a hypothetical model for the molecular organization of the dystrophin-associated glycoprotein complex in retinal Müller glial cells, which would be helpful for understanding its function in the central nervous system.

Duchenne muscular dystrophy is frequently associated with a reduced amplitude of b-wave under scotopic conditions in the electroretinogram. This suggests that the dystrophin gene-encoded proteins play a role in retinal neurotransmission. The abnormal neurotransmission has been attributed to altered expressions of C-terminal products of the dystrophin gene in the outer plexiform layer, where photoreceptor cells form synapses with secondary neurons. The present study was undertaken to determine the cellular distribution of each member of the dystrophin superfamily in rat retina.Examined in the study were the developmental pattern of dystrophins in rat retinae that exhibit inherited progressive photoreceptor degeneration; dystrophins messengers expression in the outer and the inner retina of normal rats, prepared by mechanical fractionation through the outer plexiform layer; and immunolocalization of dystrophin proteins and utrophin in normal and degenerated adult rat retinae, with several antibodies prepared against specific regions of the dystrophin superfamily.The results showed that Dp260 is exclusively localized in photoreceptor cells; Dp140 seems to be present in perivascular astrocytes; the exon 78 spliced isoform of Dp71 and the unspliced form are located in Müller glial cells and in perivascular astrocytes, respectively. Müller glial cells also contain utrophin.Although the role of these membrane cytoskeletal proteins remains to be elucidated in retina, the results support the hypothesis that b-wave reduction may be caused by molecular anomalies of C-terminal products of the dystrophin gene expressed in both neuron and glial cells.