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Phagocytosis of tips of rod outer segments (ROS) by retinal pigment epithelial (RPE) cells is vitally important for maintaining structural and functional integrity of the retina. We previously reported that receptor-mediated specific phagocytosis of ROS induces expression of early response genes coding for transcription factors. Here we study the expression of peroxisome proliferator-activated receptors (PPAR) -alpha, -delta (beta) and -gamma during ROS phagocytosis of rat RPE cells in primary cell culture, using competitive quantitative RT-PCR. During phagocytosis of ROS (but not of latex particles) by RPE cells, RT-PCR revealed a transient increase in PPARgamma mRNA expression, that peaked at 4-6 hr. We sequenced and described two alternatively spliced variants of rat PPARgamma: rPPARgamma1a and rPPARgamma1b. Both of these, along with the recently described rPPARgamma2 were induced by ROS phagocytosis. PPARalpha and PPARdelta mRNA expression was also detected in RPE cells, but the level of expression did not change during ROS phagocytosis. All-trans-retinoic acid and prostaglandin E(2) (PGE(2)) selectively potentiated both basal and ROS-phagocytosis-induced PPARgamma expression. All-trans-retinoic acid had the opposite inhibitory effect on PPARalpha and PPARdelta expression. Cycloheximide had a dual action on PPARgamma expression in RPE cells: it enhanced expression under basal conditions but repressed expression induced by ROS phagocytosis. It also stimulated expression of PPARalpha but had no effect on PPARdelta. Selective activation of PPARgamma may play an important role in regulating the expression of target genes that are involved in lipid and fatty acid metabolism in the photoreceptor renewal process., (Copyright 2000 Wiley-Liss, Inc.)

Interleukin-1 beta (IL-1beta) is an inflammatory cytokine whose expression is elevated in brain during seizures, ischemia, and injury. Expression of IL-1beta and its receptor can also be observed in normal brain. Platelet-activating factor (PAF) is also a dual mediator that promotes neuronal plasticity responses as well as inflammation. We have determined the role of PAF in the regulation of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) genes by IL-1beta in rat primary hippocampal cultures. As assessed by reverse transcriptase/polymerase chain reaction (RT/PCR), recombinant mouse IL-1beta (1 nM) led to an induction of COX-2 mRNA which peaked at 2 hours, declined to baseline levels by 4 hours, began to rise again by 6 hours, and remained elevated at 24 hours post-treatment. iNOS mRNA was also induced, but unlike COX-2, its abundance peaked at 4 hours and decreased by 6 hours to a plateau lasting through 24 hours. Pretreatment with PAF antagonist BN50730 blocked induction of COX-2 mRNA by 2-hour IL-1beta treatment, and 2-hour treatment with the PAF analog mcPAF mimicked the effects of IL-1beta on COX-2 mRNA levels. Following injury, synaptic plasticity changes may be affected by IL-1beta-PAF-COX-2 neuronal signaling., (Copyright 1999 Wiley-Liss, Inc.)

Retinal pigment epithelium (RPE) cells in culture display selective induction of certain early response transcription factors at the onset of photoreceptor rod outer segment (ROS)-specific phagocytosis (Ershov et al., 1996a). Moreover, this response is modulated by prostaglandins. The purpose of this study is to examine the expression of the key enzymes in prostaglandin synthesis: cyclooxygenase-1 (COX-1, constitutive) and cyclooxygenase-2 (COX-2, inducible), during phagocytosis of ROS by RPE cells. Rat RPE cells in primary cell culture were fed with a suspension of freshly isolated rat ROS. COX-1 and COX-2 mRNA expression was studied by quantitative competitive reverse transcriptase-polymerase chain reaction (RT-PCR). During phagocytosis of ROS by RPE cells, RT-PCR revealed an increase in mRNA expression of COX-2, but not of COX-1. COX-2 was also induced by the phospholipid growth factor lyso-phosphatidic acid (LPA) and by the peptide growth factors platelet derived growth factor (PDGF), basic fibroblast growth factor (FGF), and transforming growth factor (TGFbeta), but not nerve growth factor (NGF). Induction of COX-2 by ROS phagocytosis and growth factors through the modulation of prostanoid synthesis may play an important role in the regulation of cell functions associated with photoreceptor cell renewal., (Copyright 1999 Wiley-Liss, Inc.)

Retinal pigment epithelial cells (RPE) actively retrieve and recycle docosahexaenoic acid (DHA, 22:6n-3) from phagosomal phospholipids back to photoreceptor cells. Here we studied the fate of DHA in primary culture rat RPE cells after feeding with a suspension of rod outer segments (ROS) for 4 hr. Phospholipids (PLs), triacylglycerols (TAG), and free fatty acids were isolated from cells and media by thin layer chromatography (TLC), and their acyl groups quantified by gas liquid chromatography (GLC). In RPE cells, DHA-PLs increased 3. 5-fold by 4 hr, decreasing thereafter to 1.6-fold above basal by 24 hr. In contrast, 18:1-PLs were decreased by 13%-18% below RPE basal values by 8-24 hr, respectively. DHA-TAG showed the highest increase (21-fold) by 8 hr. Free DHA displayed a small increase in the cells with a preferential release and accumulation into the media by 24 hr. These results show that in rat RPE cells, photoreceptor cell DHA is transiently incorporated into TAG prior to its release and uptake into 18:1-PLs. These metabolic pathways and remodeling may be critical in the conservation of this essential, photoreceptor cell fatty acid., (Copyright 1999 Wiley-Liss, Inc.)

Pigment epithelial-derived factor (PEDF) has been shown to be a survival factor for cerebellar granule neurons. Here we investigated the ability of PEDF to enhance the survival of hippocampal neurons in culture, and to protect these neurons against acute glutamate toxicity. Hippocampal neurons prepared from 1- to 3-day postnatal rat brain were cultured for either 7 or 14 days in vitro (DIV). At 14 DIV, neurons were only slightly protected (13% +/- 4%) against 50 microM glutamate toxicity when treated with 1 microg/ml of PEDF for 3 successive days before glutamate exposure as measured by lactate dehydrogenase (LDH) release. In comparison, basic fibroblast growth factor (bFGF) at 10 ng/ml for the same treatment period protected 58% +/- 8% of the neurons against glutamate. Using quantitative image analysis of digitized micrographs, we found that the average size of neurons in young, developing hippocampal cultures (7 DIV), was greatly decreased by treatment with 50 microM glutamate. Treatment for up to 5 successive days with 1 microg/ml of PEDF before glutamate addition dramatically increased the average hippocampal neuron soma size, compared to cells treated with glutamate alone. Thus, PEDF may promote the growth of hippocampal neurons, and, if added to developing hippocampal neurons, can also protect these cells from subsequent injury, such as the excitotoxicity of glutamate.

The bioactive lipid platelet-activating factor (PAF) accumulates in brain during injury, seizures and ischemia and may, in addition, be significant in AIDS dementia and in other neurodegenerative diseases. We have used plasma-type recombinant PAF acetylhydrolase (rPAF-AH) to test the hypothesis that PAF accumulation is involved in early events leading to neuronal apoptosis during excitotoxic neuronal injury. Neuronal cultures were labeled with FITC-12-dUTP (TUNEL technique) and propidium iodide, digitized using fluorescence microscopy and a chilled 3CCD color camera, and analyzed with 2D graphics analysis software. N-methyl-D-aspartate (NMDA) (50 microM, 2 hr) induced a 2.5-fold increase in apoptosis of hippocampal neurons compared with controls when analyzed 24 hr after NMDA treatment. Hippocampal neurons receiving rPAF-AH (20 microg/ml) before, during, and after NMDA treatment demonstrated a concentration-dependent neuroprotective effect which resulted in 47% and 30% neuroprotection against 50 and 100 microM NMDA, respectively. The noncompetitive NMDA receptor antagonist MK-801(300 nM) completely inhibited apoptosis caused by NMDA. The neuroprotective effect of rPAF-AH against NMDA-induced apoptosis was confirmed using as additional criteria, histone release, electron microscopy, and DNA laddering. Neuroprotection elicited by rPAF-AH demonstrates that PAF is an injury mediator in NMDA-induced neuronal apoptosis and that the recombinant protein is potentially useful as a therapeutic approach.

Cyclooxygenase-2 (COX-2; EC 1.14.99.1) RNA message abundance in 25 control and Consortium to Establish a Registry for Alzheimer's Disease (CERAD)-confirmed sporadic Alzheimer's disease (AD) brains is remarkably heterogeneous when compared with 55 other AD brain RNA message levels that were previously characterized (Lukiw and Bazan: J Neurosci Res 50:937-945, 1997). Examination of nuclear protein extracts (NPXTs) that were derived from control and AD-affected brain neocortical nuclei (n = 20; age range, 60-82 years; postmortem interval, 0.5-6.5 hours) by using gel shift, gel supershift, and cold oligonucleotide competition assay revealed a highly significant relationship between the extent of inflammatory transcription factor, nuclear factor (NF)-kappaB: DNA binding and the abundance of the COX-2 RNA signal (P < 0.0001; analysis of variance). No strong correlation with AP-1-DNA binding was noted (P > 0.045). These data are the first linking inflammation-related transcription factor NF-KB-DNA binding to up-regulation of transcription from a key inflammatory gene, COX-2, in both normally aging brain and in AD-affected neocortex. Systematic deletion of NF-KB-DNA binding sites in human COX-2 promoter constructs attenuates COX-2 transcriptional induction by mediators of inflammation. Strong NF-kappaB-DNA binding has been reported previously to temporally precede COX-2 gene transcription in human epithelial (A549), hamster B-cell (HIT-T15), human endothelial (HUVEC), human lymphoblast (IM9), human fibroblast (IMR90), rat glioma/mouse neuroblastoma (NG108-15), human keratinocyte (NHEK), mouse fibroblast (NIH 3T3), rat neuroblastoma (SH-SY5Y) cell lines and in mouse and rat brain hippocampus, indicating a highly conserved inflammatory signaling pathway that is common to diverse species and cell types. The mouse, rat, and human COX-2 immediate promoters, despite 7.5 x 10(7) years of DNA sequence divergence, each retain multiple recognition sites specific for NF-kappaB-DNA binding. These data suggest that basic gene induction mechanisms, which have been conserved over long periods of evolution, that increase NF-kappaB-DNA binds ing may be fundamental in driving transcription from inflammation-related genes, such as COX-2, that operate in stressed tissues, in normally aging cell lines, and in neurodegenerative disorders that include AD brain.

Excitatory amino acids transduce physiological and pathological signals to neurons. Similarly, the neuroactive lipid platelet-activating factor (PAF) has been implicated in modulating long-term potentiation and neuronal survival. Excitatory amino acids and PAF have been shown to increase mitogen-activated protein (MAP) kinases in different cell types. Here, we have investigated the similarities and differences between PAF and kainate in activating MAP kinases in primary hippocampal neurons in vitro. Extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 kinases were activated by kainate or PAF in hippocampal neurons. This activation was blocked by the receptor antagonists CNQX and BN 50730 for kainate and PAF, respectively. The PAF receptor antagonist BN 50730 also blocked kainate activation. CNQX had no effect on PAF activation of the kinases, indicating that PAF is downstream of kainate activation. Coapplication of submaximal concentrations of PAF and kainate resulted in a less than additive activation, suggesting similar routes of activation by the two agonists. Both CNQX and BN 50730 blocked kainate-induced neurotoxicity. These results indicate that PAF and kainate activate similar kinase pathways. Therefore, PAF acts downstream of the kainate subtype of glutamate receptors, and when excessive receptor activation takes place, this bioactive lipid may contribute to neuronal cell death.

Long-term treatment by nonsteroidal anti-inflammatory drugs has been shown to decrease the incidence of Alzheimer's disease (AD). Both platelet-activating factor and interleukin-1beta, potent mediators of the inflammatory and immune response, strongly induce transcription of the cyclooxygenase-2 (COX-2) gene in brain cells. Using Northern and RT-PCR analysis, we have determined in 15 control and 10 sporadic AD human neocortical samples (age range, 60-82 yr; postmortem interval [PMI] range, 0.7-16.0 hr) the levels of COX-2 RNA in relation to the constitutively expressed COX-1 and beta-actin RNA message levels. Our results indicate that in short PMI brain, COX-1 and COX-2 transcripts are relatively low abundance RNA messages, ranging from a mean of 6.8% of the beta-actin signal in controls to 8.5% of the beta-actin signal in AD-affected brain. A large variation in the signal intensity for COX-2 RNA was noted in both control and AD; although there was a trend for higher COX-2 RNA message abundance in AD neocortex to +11.5% of that of controls, it did not reach statistical significance (ANOVA = 0.45). Several human tissues, including heart, skeletal muscle, lung, kidney, and spinal cord, displayed 4.6- and 2.8-kb COX-2 RNA message isoforms; however, the 4.6-kb COX-2 RNA predominated in the hippocampus and association neocortex. COX-2 RNA message was found to be degraded at similar rates in both control and AD tissues, and a strong positive correlation between the PMI and the intensity of the COX-2 RNA signal was noted (ANOVA = 0.006). Linear regression analysis indicated that the 4.6-kb COX-2 RNA is an unstable short-lived RNA species with a half-life of not more than 3.5 hr, a feature characteristic of immediate early gene transcripts. Individual hypervariability in COX-2 RNA message abundance may reflect various degrees of expression of AD-related inflammatory processes.

The uptake, metabolism, and cellular distribution of [3H]docosahexaenoic acid (DHA) in human and monkey retinas were studied with biochemical and autoradiographic techniques. In specimens from two human retina biopsies, incubated for 4 hr or 6 hr with [3H]docosahexaenoic acid (110 nM), 80% of the esterified [3H]fatty acid was recovered in phospholipids and the remainder in triacylglycerols and diacylglycerols. The distribution of [3H]DHA in individual phospholipids (PL) was similar in both retinas, with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounting for most of the label. A similar labeling profile was observed in glycerolipids from monkey retina, and after 1 hr of incubation, high labeling of phosphatidic acid (PA, 11%) and phosphatidylinositol (PI, 20%) was observed. In both human and monkey retinas, a preferential uptake of [3H]DHA by photoreceptor cells was revealed by autoradiography. Cone photoreceptors showed a slightly higher density of silver grains in their inner segments than did rod photoreceptors. Photoreceptors accounted for 59% and 79% of the total [3H]DHA taken up by the human and monkey retinas, respectively, the remainder being distributed throughout the neural retina. In conclusion, this study shows for the first time that in human and monkey retinas, DHA is taken up with a high degree of selectivity by photoreceptor cells, and then becomes esterified mainly into phospholipids that will be subsequently utilized for the synthesis of new disc membranes.

Synthesis of arachidonoyl CoA and docosahexaenoyl CoA in homogenates and microsomes from cerebrum, cerebellum, and brain stem and in synaptic plasma membranes from cerebrum of control rats and rats undergoing bicuculline-induced status epilepticus were studied. Arachidonoyl CoA synthesis was 3-5 times higher than docosahexaenoyl CoA in homogenates and microsomes. The synaptic plasma membranes showed only 1.5- to 2.5-fold higher activity. The presence of Triton X-100 (0.1%) in the incubation medium did not alter the activity of arachidonoyl CoA synthesis but did increase the synthesis of docosahexaenoyl CoA in homogenates, microsomes, and especially in synaptic plasma membranes. The synthesis of these polyenoic fatty acyl CoAs were 4-6 times higher in microsomes than in homogenates. Synaptic plasma membranes exhibited about the same amount of activity as homogenates in the synthesis of docosahexaenoyl CoA, but only half the activity of the latter in arachidonoyl CoA synthesis. The synthesis of arachidonyl CoA and docosahexaenoyl CoA in cerebral homogenates and microsomes was higher than that of cerebellum and brain stem. The apparent Km values for labeled arachidonic acid (17 microM) and docosahexaenoic acid (12 microM) in synaptic plasma membranes were lower than the values for microsomes isolated from different brain regions. The Vmax values were also 4-10 times lower. Microsomes from different regions did not differ in their apparent Km values, but did show variations in apparent Vmax values. Cerebellar microsomes showed lower Vmax values than the other two regions. The presence of Triton X-100 caused a significant decrease in the apparent Km values with little change in the Vmax values. Bicuculline-induced seizures did not alter the kinetic properties of arachidonoyl CoA and docosahexaenoyl CoA synthesis, except there was a significant decrease in the apparent Km and Vmax values for cerebellar microsomal docosahexaenoyl CoA synthesis. In conclusion, there were marked differences in the activation of polyenoic fatty acids in different parts of the brain and in subcellular fractions. Although bicuculline-induced convulsions accumulate free polyenoic fatty acids in the brain, no changes were detected when the fatty activation was assayed with exogenous cofactors, except in cerebellum.

The phospholipid and fatty acid contents of developing rod photoreceptor cells were determined in dissociated photoreceptor cells obtained from normal mice and from rd mice exhibiting an inherited retinal degeneration. Photoreceptors were dissociated from retinas by mechanical agitation after mild protease treatment and characterized by light and electron microscopy. Phospholipid classes were isolated by thin-layer chromatography, and fatty acyl groups separated and quantitated by capillary gas-liquid chromatography. Developing photoreceptor cells of normal retinas accumulated all phospholipid classes, but in proportions which shifted with age. The mole % contents of phosphatidylcholine (PC) and phosphatidylinositol (PI) decreased with age, whereas phosphatidylethanolamine (PE) and phosphatidylserine (PS) increased. The content of the polyunsaturated fatty acid docosahexaenoate (22:6), expressed as nmol/microgram lipid phosphorus, increased rapidly during development, whereas arachidonate (20:4) content tended to decline. Mono-unsaturated fatty acid levels (palmitoleate, 16:1; oleate, 18:1) declined with age. Among saturated fatty acids, palmitate (16:0) decreased during normal development, whereas stearate (18:0) increased. The total mass of phospholipid/photoreceptor cell in the normal, adult mouse retina was estimated to be approximately 14 pg. The total phospholipid content and mole % distribution of individual phospholipid classes in immature rd photoreceptors were similar to values for normal cells. In contrast, significant changes in fatty acid composition were detected between immature rd cells and normal cells. Rd cells generally had higher levels of saturated (myristate, 14:0; palmitate, 16:0) and monounsaturated fatty acids (oleate, 18:1) and lower levels of polyunsaturated fatty acids (arachidonate, 20:4; docosahexaenoate, 22:6), suggesting that fatty acid metabolism is altered by expression of the rd gene and/or by the associated impairment of photoreceptor cell differentiation.