Plasmalogens Regulate Retinal Connexin 43 Expression and Müller Glial Cells Gap Junction Intercellular Communication and Migration.

Plasmalogens are a specific glycerophospholipid subtype characterized by a vinyl-ether bound at their sn-1 moiety. Their biosynthesis is initiated in the peroxisome by dihydroxyacetone phosphate-acyltransferase (DHAPAT), which is encoded by the DAPAT gene. Previous studies have shown that plasmalogen-deficient mice exhibit major physiological dysfunctions including several eye defects, among which abnormal vascular development of the retina and a reactive activation of macroglial Müller cells. Interestingly, plasmalogen deficiency in mice is also associated with a reduced expression of brain connexin 43 (Cx43). Cx43 is the main connexin subtype of retinal glial cells and is involved in several cellular mechanisms such as calcium-based gap junction intercellular communication (GJIC) or cell migration. Thus, the aim of our work was 1) to confirm the alteration of Cx43 expression in the retina of plasmalogen-deficient DAPAT-/- mice and 2) to investigate whether plasmalogens are involved in crucial functions of Müller cells such as GJIC and cell migration. First, we found that plasmalogen deficiency was associated with a significant reduction of Cx43 expression in the retina of DAPAT-/- mice in vivo. Secondly, using a siRNA targeting DHAPAT in vitro, we found that a 50%-reduction of Müller cells content in plasmalogens was sufficient to significantly downregulate Cx43 expression, while increasing its phosphorylation. Furthermore, plasmalogen-depleted Müller cells exhibited several alterations in ATP-induced GJIC, such as calcium waves of higher amplitude that propagated slower to neighboring cells, including astrocytes. Finally, in vitro plasmalogen depletion was also associated with a significant downregulation of Müller cells migration. Taken together, these data confirm that plasmalogens are critical for the regulation of Cx43 expression and for characteristics of retinal Müller glial cells such as GJIC and cell migration.

Is 24(S)-hydroxycholesterol a potent modulator of cholesterol metabolism in Müller cells? An in vitro study about neuron to glia communication in the retina.

Communication between neurons and glia plays a major role in nervous tissue homeostasis. It is thought to participate in tuning cholesterol metabolism to cellular demand, which is a critical issue for neuronal health. Cholesterol is a membrane lipid crucial for nervous tissue functioning, and perturbed regulation of its metabolism has been linked to several neurodegenerative disorders. In the brain, 24(S)-hydroxycholesterol (24S-OHC) is an oxysterol synthesized by neurons to eliminate cholesterol, and 24S-OHC has been shown to regulate cholesterol metabolism in astrocytes, glial cells which provide cholesterol to neurons. In the retina, 24S-OHC is also an elimination product of cholesterol produced by neurons, especially the retinal ganglion cells. However, it is not known whether Müller cells, the major macroglial cells of the retina, play the role of cholesterol provider for retinal neurons and whether they respond to 24S-OHC signaling, similarly to brain glial cells. In the present study, primary cultures of rat Müller cells were treated with 0, 0.5 or 1.5 µM 24S-OHC for 48 hours. The levels of cholesterol, precursors and oxysterols were quantified using gas chromatography coupled to flame-ionization detection or mass spectrometry. In addition, the expression of key genes related to cholesterol metabolism was analyzed using RTq-PCR. Müller cells were shown to express many genes linked to cholesterol metabolism, including genes coding for proteins implicated in cholesterol biosynthesis (HMGCR), cholesterol uptake and export via lipoproteins (LDL-R, SR-BI, ApoE and ABACA1) and regulation of cholesterol metabolism (SREBP2 and LXRß). Cholesterol and several of its precursors and oxidative products were present. CYP27A1, the main retinal enzyme implicated in cholesterol elimination via oxysterol production, was quantified at low transcript levels but neither of its two typical products were detected in Müller cells. Furthermore, our results demonstrate that 24S-OHC has a strong hypocholesterolemic effect in Müller cells, leading to cholesterol depletion (-37 % at 1.5 µM). This was mediated by a decrease in cholesterol synthesis, as illustrated by reduced levels of cholesterol precursors: desmosterol (-38 % at 1.5 µM) and lathosterol (-84 % at 1.5 µM), and strong downregulation of HMGCR gene expression (2.4 fold decrease at 1.5µM). In addition, LDL-R and SR-BI gene expression were reduced in response to 24S-OHC treatment (2 fold and 1.6 fold at 1.5 µM, respectively), suggesting diminished lipoprotein uptake by the cells. On the contrary, there was a dramatic overexpression of ABCA1 transporter (10 fold increase at 1.5 µM), probably mediating an increase in cholesterol efflux. Finally, 24S-OHC induced a small but significant upregulation of the CYP27A1 gene. These data indicate that Müller cells possess the necessary cholesterol metabolism machinery and that they are able to sharply adjust their cholesterol metabolism in response to 24S-OHC, a signal molecule of neuronal cholesterol status. This suggests that Müller cells could be major players of cholesterol homeostasis in the retina via neuron-glia crosstalk.

Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol.

Glaucoma is a progressive and irreversible blinding neuropathy that is characterized by the loss of retinal ganglion cells (RGCs). Muller Glial Cell (MGC) activation is induced in retinal gliosis. MGCs are the most numerous glial cells in the retina and one of their roles is to sustain cholesterol homeostasis. 24S-hydroxycholesterol (24S-OHC) is one of the form of cholesterol elimination from the retina and is overexpressed during glaucoma. The objective of this study was to determine whether 24S-OHC triggers MGC membrane dynamics involving lipid rafts and contributes to gliosis at early and late time points. A proteomic analysis was carried out by nanoLC-MS/MS in raft and non-raft fractions from MGCs after treatment with 24S-OHC (10µM). The expression of structural and functional proteins was further analyzed by Western-blotting, as well as the levels of GM3 ganglioside by LC-MS. Cholesterol, sphingomyelin, saturated fatty acids and ganglioside GM3 are enriched in the rafts fractions in MGCs. Caveolin-1, flotillin-1, connexin-30 and -43 are localized in the MGCs rafts. Proteins implicated in adhesion or oxidative stress pathways in raft fractions were up and down-regulated by the treatment. Our data showed that 24S-OHC induced early changes in protein distribution in raft microdomains; however, further studies are needed to better characterize the surrounded mechanisms.

Early adaptive response of the retina to a pro-diabetogenic diet: Impairment of cone response and gene expression changes in high-fructose fed rats.

The lack of plasticity of neurons to respond to dietary changes, such as high fat and high fructose diets, by modulating gene and protein expression has been associated with functional and behavioral impairments that can have detrimental consequences. The inhibition of high fat-induced rewiring of hypothalamic neurons induced obesity. Feeding rodents with high fructose is a recognized and widely used model to trigger obesity and metabolic syndrome. However the adaptive response of the retina to short term feeding with high fructose is poorly documented. We therefore aimed to characterize both the functional and gene expression changes in the neurosensory retina of Brown Norway rats fed during 3 and 8 days with a 60%-rich fructose diet (n = 16 per diet and per time point). Glucose, insulin, leptin, triacylglycerols, total cholesterol, HDL-cholesterol, LDL-cholesterol and fructosamine were quantified in plasma (n = 8 in each group). Functionality of the inner retina was studied using scotopic single flash electroretinography (n = 8 in each group) and the individual response of rod and cone photoreceptors was determined using 8.02 Hz Flicker electroretinography (n = 8 in each group). Analysis of gene expression in the neurosensory retina was performed by Affymetrix genechips, and confirmed by RT-qPCR (n = 6 in each group). Elevated glycemia (+13%), insulinemia (+83%), and leptinemia (+172%) was observed after 8 days of fructose feeding. The cone photoreceptor response was altered at day 8 in high fructose fed rats (Δ = 0.5 log unit of light stimulus intensity). Affymetrix analysis of gene expression highlighted significant modulation of the pathways of eIF2 signaling and endoplasmic reticulum stress, regulation of eIF4 and p70S6K signaling, as well as mTOR signaling and mitochondrial dysfunction. RT-qPCR analysis confirmed the down regulation of Crystallins, Npy, Nid1 and Optc genes after 3 days of fructose feeding, and up regulation of End2. Meanwhile, a trend towards an increased expression of αA- and αB-crystallin proteins was observed at day 8. Our results are consistent with early alterations of the functioning and gene expression in the retina in a pro diabetogenic environment.

Involvement of plasmalogens in post-natal retinal vascular development.

OBJECTIVE: Proper development of retinal blood vessels is essential to ensure sufficient oxygen and nutrient supplies to the retina. It was shown that polyunsaturated fatty acids (PUFAs) could modulate factors involved in tissue vascularization. A congenital deficiency in ether-phospholipids, also termed "plasmalogens", was shown to lead to abnormal ocular vascularization. Because plasmalogens are considered to be reservoirs of PUFAs, we wished to improve our understanding of the mechanisms by which plasmalogens regulate retinal vascular development and whether the release of PUFAs by calcium-independent phospholipase A2 (iPLA2) could be involved. METHODS AND RESULTS: By characterizing the cellular and molecular steps of retinal vascular development in a mouse model of plasmalogen deficiency, we demonstrated that plasmalogens modulate angiogenic processes during the early phases of retinal vascularization. They influence glial activity and primary astrocyte template formation, endothelial cell proliferation and retinal vessel outgrowth, and impact the expression of the genes involved in angiogenesis in the retina. These early defects led to a disorganized and dysfunctional retinal vascular network at adult age. By comparing these data to those obtained on a mouse model of retinal iPLA2 inhibition, we suggest that these processes may be mediated by PUFAs released from plasmalogens and further signalling through the angiopoietin/tie pathways. CONCLUSIONS: These data suggest that plasmalogens play a crucial role in retinal vascularization processes.

Polyunsaturated fatty acids induce modification in the lipid composition and the prostaglandin production of the conjunctival epithelium cells.

BACKGROUND: This study was conducted to evaluate whether polyunsaturated fatty acids (PUFA) such as γ-linolenic acid (GLA) and eicosapentaenoic acid (EPA), as found in the diet, may affect the lipid composition of conjunctival epithelium and whether these modifications affect prostaglandin (PG) production after inflammatory stimulation. METHODS: Chang and IOBA-NHC conjunctival human cells were treated with GLA and/or EPA at 5, 10, 20, 30, 40, or 50 µg/ml for 72 h and then were stimulated with interferon-gamma (IFN-γ) for 48 h. Changes in the composition of neutral lipids and phospholipids were monitored by gas chromatography. PGE1 and PGE2 levels were measured by enzyme immunoassay. RESULTS: PUFA supplementations in the culture medium induced incorporation of these fatty acids and of their metabolites in neutral lipids and phospholipids of the conjunctival cells. The fatty acid composition of neutral lipids and phospholipids was not affected by stimulation with IFN-γ. The production of PGE1 and PGE2 was affected by GLA supplementation whereas it was not modified by EPA supplementation. A combined supplementation of EPA and GLA did not change the production of PGE1 but decreased the production of PGE2. CONCLUSIONS: These results suggest that modulation of fatty acid composition and PG production by PUFA supplementation is possible in the conjunctival epithelium, which is an important site of inflammation in dry eye syndrome.

Oxysterols induced inflammation and oxidation in primary porcine retinal pigment epithelial cells.

PURPOSE: Aging is associated with an accumulation of cholesterol esters in the Bruch membrane. Cholesterol esters are prone to undergo oxidation and generate oxysterols that have cytotoxic and proinflammatory properties. We investigated the effects of three oxysterols on mitochondrial dysfunctions, inflammation, and oxidative stress in primary cultures of porcine retinal pigment epithelial (RPE) cells. METHODS: RPE cells were incubated with oxysterols (50 micro M of 24-hydroxycholesterol, 25-hydroxycholesterol, or 7-ketocholesterol) for 24 hr and 48 hr. Oxysterol content was determined in cells and in corresponding media by gas chromatography. Mitochondrial activity was measured by mitochondrial dehydrogenase activity. The intracellular formation of reactive oxygen species in RPE cells was detected by using the fluorescent probe DCFH-DA. IL-8 was assayed in the supernatants by ELISA, and the corresponding cellular transcripts were semiquantified by RT-PCR. RESULTS: Analyses of the oxysterols content in the RPE cells and corresponding media suggested a high rate of cellular uptake, although some differences were observed between 7-ketocholesterol on the one hand and 24-hydroxycholesterol and 25-hydroxycholesterol on the other hand. All oxysterols induced slight mitochondrial dysfunctions but a significant 2- to 4-fold increase in reactive oxygen species (ROS) production compared with the control. They also enhanced IL-8 gene expression and IL-8 protein secretion in the following decreasing order: 25-hydroxycholesterol > 24-hydroxycholesterol > 7-ketocholesterol. CONCLUSIONS: We conclude that in confluent primary porcine RPE cells, 24-hydroxycholesterol, 25-hydroxycholesterol, and 7-ketocholesterol are potent inducers of oxidation and inflammation.