Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid.

PURPOSE: In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. METHODS: Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. RESULTS: Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [(3)H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. CONCLUSIONS: These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.

Insulin-like growth factor-I is a potential trophic factor for amacrine cells.

In this study we show that insulin-like growth factor (IGF)-I selectively promotes survival and differentiation of amacrine neurons. In cultures lacking this factor, an initial degeneration pathway, selectively affecting amacrine neurons, led to no lamellipodia development and little axon outgrowth. Cell lysis initially affected 50% of amacrine neurons; those remaining underwent apoptosis leading to the death of approximately 95% of them by day 10. Apoptosis was preceded by a marked increase in c-Jun expression. Addition of IGF-I or high concentrations (over 1 microM) of either insulin or IGF-II to the cultures prevented the degeneration of amacrine neurons, stimulated their neurite outgrowth, increased phospho-Akt expression and decreased c-Jun expression. The high insulin and IGF-II concentrations required to protect amacrine cells suggest that these neurons depend on IGF-I for their survival, IGF-II and insulin probably acting through IGF-I receptors to mimic IGF-I effects. Inhibition of phosphatidylinositol-3 kinase (PI 3-kinase) with wortmannin blocked insulin-mediated survival. Wortmannin addition had similar effects to IGF-I deprivation: it prevented neurite outgrowth, increased c-Jun expression and induced apoptosis. These results suggest that IGF-I is essential for the survival and differentiation of amacrine neurons, and activation of PI 3-kinase is involved in the intracellular signaling pathways mediating these effects.

Docosahexaenoic acid promotes differentiation of developing photoreceptors in culture.

PURPOSE: The purpose of this work was to study the effects of diverse fatty acids on the composition, metabolism, differentiation, and characteristics of opsin expression in retina photoreceptors. METHODS: Cultures of rat retinal neurons were incubated with or without 22:6 n-3, 22:5 n-3, 20:4 n-6, 18:1 n-9, and 16:0, labeled and unlabeled. RESULTS: In photoreceptor cells incubated with 22:6 n-3 and 22:5 n-3, the proportions of these fatty acids in phospholipids increased four- to sixfold. The remaining fatty acids did not change lipid acyl chain composition. The labeled fatty acids were all actively esterified in neuronal lipids, particularly in phosphatidylcholine. Addition of unlabeled 22:6 n-3 did not affect the distribution among lipids of the other fatty acids but displaced [3H]20:4 n-6 from phosphatidylcholine and phosphatidylethanolamine. These results suggest that retinal neurons have specific mechanisms for processing fatty acids of different lengths and degrees of unsaturation and that 22:6 n-3 incorporation takes priority. Of all fatty acids, 22:6 n-3 was the most effective in promoting photoreceptor differentiation. In 22:6-sufficient photoreceptors, new apical processes formed, the expression of opsin augmented, and its localization improved, concentrating in the apical processes of the cells. CONCLUSIONS: The advancement in differentiation selectively elicited by 22:6 correlates with the fact that 22:6 n-3, but none of the other fatty acids, delays significantly the onset of apoptosis in photoreceptors in culture. The synthesis of 22:6-containing phospholipid molecules could be required for the proper localization of opsin. This could contribute to furthering the differentiation of photoreceptors, preventing their apoptosis, and extending their survival.

Selective outgrowth and differential tropism of amacrine and photoreceptor axons to cell targets during early development in vitro.

The axonal guidance and outgrowth in retinal neurons were investigated in cultures of pure retinal neurons (control) or in cocultures with heterologous BC3H-1 cells. Under control conditions, only about 10% of retinal neurons developed axons; coculturing with BC3H-1 cells induced early axonal outgrowth and guidance to BC3H-1 cells in most amacrine neurons. Both mechanisms were dependent on laminin and neural cell-adhesion molecules (N-CAMs) released by BC3H-1 cells, because they were prevented by antibodies directed against these molecules. The protein kinase C (PKC) inhibitor, staurosporine, reduced the effect of laminin on amacrine axonal outgrowth, suggesting that this effect was mediated by PKC. The occurrence of structures resembling synaptic boutons and the expression of synaptophysin at the amacrine axon ends of heterologous connections suggested that amacrine axons establish true synaptic contacts rather than simply overlapping with the BC3H-1 cells. In contrast to the heterologous contacts with BC3H-1 cells, the amacrine-amacrine axonal contacts observed in the cocultures were independent of laminin and N-CAM. Axonal outgrowth occurred in about 10% of the photoreceptors and was not affected by BC3H-1 cells or by substratum pretreatment with laminin or N-CAM. These results show that different mechanisms affect axonal outgrowth and guidance in amacrine and photoreceptor neurons in vitro, and they suggest that similar mechanisms could contribute to the development of the scaffold of axon pathways in the retina in vivo.

Apoptosis of retinal photoreceptors during development in vitro: protective effect of docosahexaenoic acid.

When rat retinal cells are cultured in a serum-free medium, the photoreceptor cells start dying after 7 days. The addition of docosahexaenoic acid (DHA) to the cultures prevents the selective death of photoreceptors. Here it is shown that, unlike other retinal neurons, photoreceptors die through an apoptotic pathway. Hallmarks of apoptosis, such as nuclear fragmentation and condensation and DNA cleavage forming a ladder pattern on an agarose gel, were observed. The timing and high selectivity of the triggering of photoreceptor cell apoptosis suggest the existence of a programmed cell death. Compared with other fatty acids, DHA not only was the most effective in promoting photoreceptor survival, but also the only one to decrease the number of apoptotic nuclei. The results suggest that DHA is important among the factors preventing apoptosis of photoreceptors in the developing retina. A limitation in the availability of this fatty acid might trigger apoptosis as a result of the failure to develop functional photoreceptor outer segments.

Docosahexaenoic acid is required for the survival of rat retinal photoreceptors in vitro.

The effect of docosahexaenoic acid (DHA) on neuronal survival was studied in cultured cells isolated from newborn rat retina. In vivo, the content of DHA in the retina increased nearly fourfold from days 2 to 12 after birth, whereas in retinal cells in culture it remained constant. Unlike amacrine cells, the photoreceptor cells in control cultures underwent a selective degeneration, starting at day 7, that led to their massive death by day 11. The addition of DHA at day 7 led to its active incorporation by the cultures, increasing from 6 to 21% of total fatty acids in cell lipids, and completely prevented photoreceptor cell death. When other fatty acids were tested, both neuronal fatty acid composition and photoreceptor death were the same as in control cultures. These results indicate that DHA is specifically required for the survival of retinal photoreceptors.

Heterologous retinal cultured neurons and cell adhesion molecules induce clustering of acetylcholine receptors and polynucleation in mouse muscle BC3H-1 clonal cell line.

Several features of the clonal cell line BC3H-1 resemble those of embryonic muscle cells at their early stage of development (Patrick et al.: J Biol Chem 252:2143-2153, 1977). Under normal culture conditions, fully differentiated BC3H-1 cells possess a spindle-shaped, muscle-like morphology with a single nucleus. Like embryonic muscle cell counterparts, they neither express the epsilon subunit nor exhibit the clustered organization of the nicotinic acetylcholine receptor (AChR) characteristic of mature myocytes. Instead, AChRs are evenly distributed upon the sarcolemma upon differentiation. Here we report that BC3H-1 cells can be induced to express AChR clusters by co-culturing the cell line with heterologous retinal neurons, which establish contacts with former cells. Clustering was also triggered by pretreating the culture substratum with several cell adhesion molecules. Polynucleation, a phenomenon observed in mature myotubes in vivo, was also observed in BC3H-1 cells under the two sets of experimental conditions. AChR clustering and polynucleation, both characteristic of the mature postsynaptic region, can thus be induced in BC3H-1 cells by at least two quite distinct pathways: neuronal cell contact and/or cell adhesion molecules. It is suggested that heterologous retinal neurons can elicit only an incomplete differentiation of BC3H-1 cells, failing to concentrate the clusters beneath the neuronal endings, and failing to induce expression of the epsilon subunit, characteristic of the mature AChR.

Myogenic differentiation of the muscle clonal cell line BC3H-1 is accompanied by changes in its lipid composition.

Phospholipid and neutral lipid composition was studied in the course of myogenic differentiation of the clonal cell line BC3H-1. Total phospholipid content increased during differentiation, predominantly in the major classes of choline and ethanolamine glycerophospholipids. The contents of other lipids, such as triacylglycerols, diminished more than 50% during this period. The content and distribution of fatty acids also underwent marked differentiation-dependent changes. The polyunsaturated (tetrapenta- and hexaenoic) fatty acid species of several phospholipid classes diminished during differentiation, especially those in choline, serine and inositol glycerophospholipids. Most noticeable were the changes in phosphatidylserine; long-chain fatty acids having 20 to 22 carbon atoms and 4 to 6 double bonds decreased from about 30 to about 10 mol%. Although increased levels of saturation in other phospholipid fatty acyl chains appear to accompany the myogenic changes of BC3H-1 cells, some unsaturated fatty acids, such as oleic acid (18:1), increased by as much as 80% during the same period, suggesting the activation of a delta 9 desaturase. Sphingomyelin contained only saturated and monoenoic fatty acids and exhibited a four- to five-fold decrease in its content of monoenoic acyl groups. Diacylglycerols became enriched in arachidonate and docosahexaenoate. The amount of cholesterol and its esters increased slightly during differentiation of BC3H-1 cells. The data show that several metabolic pathways change during myogenic differentiation of the BC3H-1 clonal cell line, particularly de novo biosynthetic pathways, elongation/desaturation reactions, and acyl chain turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) by isolated normal and rd mouse retinal photoreceptor neurons in culture.

Cultures of dissociated retinal neurons and photoreceptors from homozygous wild-type, heterozygous rd/+ and homozygous rd/rd retinas have been used to investigate the capacity of isolated photoreceptor cells to synthesize and secrete the interphotoreceptor retinoid-binding protein (IRBP). Retinal cells were dissociated on postnatal day 2 and grown in chemically defined medium in the absence of glial and pigmented epithelial cells. Expression of IRBP immunoreactive materials in these cultures was cell type-specific and developmentally regulated. Thus increasing numbers of rod photoreceptor cells showed immunoreactivity during the first week in culture, whereas nonphotoreceptor cell types remained consistently negative. Photoreceptor immunoreactivity could be detected in permeated (detergent-treated) as well as in unpermeated preparations, the latter suggesting that some IRBP is associated with the photoreceptor cell surface. These materials appeared to be loosely bound to the photoreceptors, since they disappeared when the cultures were exposed for 6 hr to IRBP-free medium but not when they were exposed to IRBP-containing medium. IRBP synthesis and secretion could be demonstrated by analyzing either cell extracts or conditioned medium by "slot blot" and Western blot techniques using affinity purified antibodies against bovine IRBP as well as by fluorographic analysis after metabolic labeling of the cultures with 35S-methionine. Comparisons of cultures from the different genotypes showed many similarities, including the abundance of IRBP-immunoreactive photoreceptors in 7 day cultures. However, immunochemical analysis showed lower conditioned medium/cell extract IRBP ratios in rd/rd cultures, an observation consistent with previous reports suggesting that IRBP secretion may be deficient in rd/rd photoreceptor cells.

Differential susceptibility of isolated mouse retinal neurons and photoreceptors to kainic acid toxicity. In vitro studies.

Dissociated cultures of mouse retinal neurons and photoreceptors in chemically defined medium were used to investigate the susceptibility of these cells to the neurotoxin kainic acid (KA). Cells isolated from the newborn mouse retina were initially insensitive to this toxin, and the cells that differentiated as rod photoreceptors retained this resistance throughout the culture period. However, amacrine neurons became increasingly sensitive to KA toxicity as they differentiated in culture; after the fifth day in vitro approximately 90% of these cells were killed by KA in a concentration- and time-dependent fashion. The neurons showed pronounced swelling within 10 min of treatment onset, and cell lysis and nuclear fragmentation were evident during the next few hours. KA-induced degeneration of these neurons was corroborated using the amacrine cell-specific monoclonal antibody HPC-1, as well as autoradiographic and biochemical determinations of the high affinity uptake for GABA. This inhibitory neurotransmitter was taken up by amacrine neurons but not by photoreceptor cells, and this uptake was completely abolished in KA-treated cultures. Similar results were obtained with the neuronal enzyme choline acetyltransferase. However, both photoreceptor survival and the expression of photoreceptor markers such as opsin and the retinoid-binding protein interphotoreceptor (IRBP) were similar in KA-treated and control cultures. Similarly, the high affinity uptake of glutamate, an excitatory amino acid which is predominantly taken up by photoreceptors, showed only modest changes in KA-treated cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of neonatal mouse retinal neurons and photoreceptors in low density cell culture.

We describe here a culture method which allows the growth of dissociated mouse retinal neurons and photoreceptors in chemically defined medium. Neural retinas from 2-day-old C57/BL mice were dissected from other ocular tissues, including the pigment epithelium, and dissociated into a cell suspension after brief trypsination. Most cells attached as single, unaggregated units to substrata pretreated with polyornithine and the neurite-promoting factor (PNPF). The cells were cultured in serum-free, high pyruvate Dulbecco's modified Eagle's medium containing chemically defined supplements. Under these conditions, onset of cell process development was rapid, giving rise to extensive neurite networks. Three morphologically distinct cell types were apparent during the first week in vitro. Some cells retained a circular outline and failed to produce processes, while 50-60% of the cells developed as multipolar neurons showing a large cell body and several neurites. Approximately 90% of these cells reacted with an amacrine cell-specific monoclonal antibody. Some 30% of the cultured cells expressed phenotypic properties characteristic of rod photoreceptors, including a small cell body, an apical cilium, a short neurite with a spherule-like terminal body, and immunoreactivity with antibodies against opsin as well as a rod cell-specific monoclonal antibody. No further signs of outer segment differentiation were observed in these cells. Non-neuronal "flat" cells, which represented less than 0.5% of the total cell number, reacted with an antibody against the glial fibrillary acidic protein. The number of neurons and photoreceptors remained relatively stable during the first 4-7 days in vitro. During the second week in culture, however, there was specific degeneration of greater than 90% of the photoreceptor cells, while less than 20% of the multipolar neurons were similarly affected. Consequently, in addition to providing a system for studying the differentiation of retinal neurons and photoreceptors, the specific degeneration of photoreceptors in these mouse retinal cell cultures makes this system ideal for investigating factors influencing photoreceptor survival.

Differential sensitivity of cultured retinal neurons and photoreceptors to herpes simplex infection.

Infection of the retina with herpes simplex virus type 1 (HSV-1) causes devastating lesions usually leading to blindness. However, the interactions between individual retinal cell types and this virus have not been well characterized, probably because of limitations posed by the complexity of the intact retina. We have now approached this problem through the use of separate, purified populations of isolated chick embryo retinal neurons and photoreceptor cells, of glial cells, and of pigmented epithelial cells. This manuscript deals with the initial part of these studies, aimed at determining the susceptibility of different retinal types to HSV-1 infection. The different cultures were exposed to HSV-1 for 3-48 hr, and cell infection was evaluated by immunocytochemical detection of viral antigens or by autoradiographic study of viral DNA replication. Practically 100% of the retinal glial cells and pigmented epithelial cells appeared susceptible to HSV-1 infection. On the other hand, as many as 70% of the neurons present in glia-free, pigment epithelium-free cultures, also appeared infected after a 24-hr exposure to the virus. Neuronal susceptibility to HSV-1 was already present in early (2-day) cultures, was time- and concentration-dependent, and led to neuronal degeneration after 24-48 hr. Neuronal infection was also corroborated by the detection of viral particles by transmission electron microscopy. Photoreceptor cells were consistently and selectively resistant to HSV-1 infection at all the concentrations and time points investigated. Both immunocytochemical and autoradiographic studies showed similar results. Photoreceptor resistance to HSV-1 appears to be selective, since they could be readily infected with RNA viruses such as vesicular stomatitis virus and influenza virus. These cell culture preparations offer an attractive system for the investigation of cellular mechanisms involved in the differential susceptibility of retinal cells to viral infection. Moreover, they could also help in the screening of treatments potentially capable of preventing and (or) curing HSV-induced retinal infection.

Generation of enriched populations of cultured photoreceptor cells.

This laboratory has recently described a monolayer culture system that allows the survival and differentiation of chick embryo retinal neurons and photoreceptors in the absence of contamination by connective tissue, endothelial, glial, or pigmented epithelial cells. The authors report here that the photoreceptor cells can be further purified by exploiting the selective toxicity of kainic acid (KA) and beta-bungarotoxin (BBT), which are known to destroy many retinal neurons without affecting photoreceptors. When added to retinal cultures, both KA and BBT caused developmental stage- and concentration-dependent degeneration without affecting the number or qualitative properties of photoreceptors. Some retinal neurons were already sensitive to KA after only 2 days in vitro, and their number increased as the cultures matured. The maximum effect of KA (a loss of 60% of the neurons) was obtained with an 8-24 hr treatment with 2 mM KA after 6 days in vitro. Neuronal sensitivity to BBT showed a developmental pattern similar to KA. However, maximum neuronal losses produced by BBT were higher (70% of the neurons) and occurred at lower concentrations (1-2 nM). Combined treatments with KA and BBT did not show additive effects or potentiation between toxins. KA- or BBT-induced neuronal degeneration was accompanied by a greater than 90% loss of neuronal markers such as choline acetyltransferase activity or high-affinity GABA uptake. Transmission electron microscopy showed the integrity of photoreceptor cells in KA- and BBT-treated cultures. These results show that both KA and BBT are useful tools for the generation of enriched photoreceptor populations in vitro.

Dexamethasone effect on free fatty acid and diacylglycerol accumulation during experimentally induced vasogenic brain edema.

Free fatty acids (FFA), diacylglycerols (DG), and water content were measured in the right and left cerebral hemispheres of rats with brain edema cryogenically induced to the right cerebral hemisphere. The effect of dexamethasone pretreatment was also studied. Twenty-four hours after lesion, maximal edema was attained concomitant with an accumulation of FFA (8.5-fold) and DG (2.9-fold). Polyunsaturated fatty acids (PUFA), primarily docosahexaenoic acid, increased greatly in both lipid pools. In the contralateral hemisphere, only DG levels increased, especially those containing stearate and arachidonate, which increased by almost 50%. By 48 h, FFA had decreased 60%, whereas DG had attained sham levels. These changes occurred prior to edema resolution. Dexamethasone decreased the degree of edema and the accumulation of PUFA-containing DG by 30% in both hemispheres. There was a complete inhibition of FFA and arachidonic acid accumulation in the lesioned side. These results suggest that dexamethasone: (1) inhibits phospholipases A2 that, in turn, decrease membrane phospholipid breakdown; and (2) has a limited effect on the enzymatic systems involved in PUFA-DG accumulation. Hence, in experimentally induced vasogenic brain edema, PUFA-containing DG, rather than FFA, may be related closely to the spreading of edema fluid.