No abstract

Stargardt-like macular dystrophy (STGD3) is a dominantly inherited juvenile macular degeneration that eventually leads to loss of vision. Three independent mutations causing STGD3 have been identified in exon six of a gene named Elongation of very long chain fatty acids 4 (ELOVL4). The ELOVL4 protein was predicted to be involved in fatty acid elongation, although evidence for this and the specific step(s) it may catalyze have remained elusive. Here, using a gain-of-function approach, we provide direct and compelling evidence that ELOVL4 is required for the synthesis of C28 and C30 saturated fatty acids (VLC-FA) and of C28-C38 very long chain polyunsaturated fatty acids (VLC-PUFA), the latter being uniquely expressed in retina, sperm, and brain. Rat neonatal cardiomyocytes and a human retinal epithelium cell line (ARPE-19) were transduced with recombinant adenovirus type 5 carrying mouse Elovl4 and supplemented with 24:0, 20:5n3, or 22:5n3. The 24:0 was elongated to 28:0 and 30:0; 20:5n3 and 22:5n3 were elongated to a series of C28-C38 PUFA. Because retinal degeneration is the only known phenotype in STGD3 disease, we propose that reduced VLC-PUFA in the retinas of these patients may be the cause of photoreceptor cell death.fatty acid biosynthesis ͉ macular degeneration T hree independent mutations in the last exon (exon-VI) of the ELOVL4 gene are associated with dominant Stargardt-like macular dystrophy (STGD3) in humans (1-4). These mutations cause a frame-shift that introduces a stop codon, resulting in premature termination of the protein and removal of the signal sequence for targeting the protein to its putative cellular location, the endoplasmic reticulum (1, 4). As a result, the mutant protein mis-localizes and aggregates (3,5,6), and, when coexpressed with the wild type protein, the mutant and wild-type proteins associate and mis-localize (3, 7). Based on sequence homology with a group of functional yeast genes and other mammalian ELOVLs, the ELOVL4 protein was predicted to be involved in elongation of very long chain fatty acids (1,5,8). For example, the yeast microsomal Elo1p is responsible for elongation of carbon chains between 14:0 and 16:0 (9). Yeast Elo2p and Elo3p, and mammalian ELOVL1, 2, 3, and 5 have been shown to be involved in elongation of saturated, monounsaturated, or polyunsaturated fatty acids (PUFA) from 18 to 26 carbons (10-12). However, a role for ELOVL4 protein in fatty acid elongation and the specific step(s) it may catalyze have remained elusive (13,14). Based on the abundant expression of ELOVL4 protein in photoreceptor cells of the retina (15-17) and to lesser extents in brain, testis, and skin (17), it was first hypothesized that ELOVL4 may be involved in the biosynthetic pathway of docosahexaenoic acid (22:6n3, DHA), the most abundant PUFA in the retina and the brain (1,16,18). A series of experiments carried out in our laboratory (unpublished data) does not support this hypothesis.Recent reports establish ELOVL4 as an essential protein for growth and development, as neonatal ...

Retinal rods signal the activation of a single receptor molecule by a photon. To ensure efficient photon capture, rods maintain about 109 copies of rhodopsin densely packed into membranous disks. But a high packing density of rhodopsin may impede other steps in phototransduction that take place on the disk membrane, by restricting the lateral movement of, and hence the rate of encounters between, the molecules involved. Although it has been suggested that lateral diffusion of proteins on the membrane sets the rate of onset of the photoresponse, it was later argued that the subsequent processing of the complexes was the main determinant of this rate. The effects of protein density on response shut-off have not been reported. Here we show that a roughly 50% reduction in protein crowding achieved by the hemizygous knockout of rhodopsin in transgenic mice accelerates the rising phases and recoveries of flash responses by about 1.7-fold in vivo. Thus, in rods the rates of both response onset and recovery are set by the diffusional encounter frequency between proteins on the disk membrane.

No abstract

Age-related macular degeneration (AMD) is a complex disease that has potential involvement of inflammatory and oxidative stress-related pathways in its pathogenesis. In search of effective therapeutic agents, we tested curcumin, a naturally-occurring compound with known antiinflammatory and anti-oxidative properties, in rat model of light induced retinal degeneration (LIRD) and in retina derived cell lines. We hypothesized that any compound effective against LIRD, which involves significant oxidative stress and inflammation, would be a candidate for further characterization for its potential application in AMD.We observed significant retinal neuroprotection in rats fed diets supplemented with curcumin (0.2% in diet) for 2 weeks. The mechanism of retinal protection from LIRD by curcumin involves inhibition of NF-κB activation and down-regulation of cellular inflammatory genes. When tested on retinaderived cell lines (661W and ARPE-19), pre-treatment of curcumin protected these cells from H 2 O 2 -induced cell death by up-regulating cellular protective enzymes, such as HO-1, thioredoxin.Since, curcumin with its pleiotropic activities can modulate the expression and activation of many cellular regulatory proteins such as NF-κB, AKT, NRF2 and growth factors, which in turn inhibit cellular inflammatory responses and protect cells; we speculate that curcumin would be an effective nutraceutical compound for preventive and augmentative therapy of AMD.

Rat electroretinograms were measured as a function of dietary supplements of purified ethyl esters of linolenic acid, linoleic acid, and oleic acid. Polyunsaturated fatty acids derived from precursors of linolenic and linoleic acids appear to be important functional components of photoreceptor cell membranes, although in equal dietary concentrations, linolenic acid precursors affect electroretinogram amplitudes to a greater extent than linoleic acid precursors. The electrical response of photoreceptor cell membranes appears to be a function of the position of the double bonds as well as a function of the total number of double bonds in fatty acid supplements.

To determine whether docosahexaenoic acid (DHA) supplementation of breast-feeding mothers increases the DHA contents of breast milk and infant plasma phospholipids (PPs), breast-feeding women were randomly assigned to 3 DHA-supplementation groups (170-260 mg/d) or a control group. Group 1 (n = 6) consumed an algae-produced high-DHA triacylglycerol; group 2 (n = 6) consumed high-DHA eggs; group 3 (n = 6) consumed a high-DHA, low-eicosapentaenoic acid marine oil; and group 4 (n = 6) received no supplementation. From before to after supplementation (2 and 8 wk postpartum), mean (+/-SD) maternal PP DHA increased in groups 1, 2, and 3 by 1.20 +/- 0.53, 0.63 +/- 0.82, and 0.76 +/- 0.35 mol% of fatty acids, respectively (23-41%), but decreased in group 4 by 0.44 +/- 0.34 mol% (15%). Breast-milk DHA of groups 1, 2, and 3 increased by 0.21 +/- 0.16, 0.07 +/- 0.11, and 0. 12 +/- 0.07 mol%, respectively (32-91%) but decreased in group 4 by 0.03 +/- 0.04 mol% (17%). Mean infant PP DHA in groups 1, 2, and 3 increased by 1.63 +/- 0.79, 0.40 +/- 1.0, and 0.98 +/- 0.61 mol%, respectively (11-42%), but only by 0.18 +/- 0.74 mol% (5%) in group 4. Correlations between the DHA contents of maternal plasma and breast milk and of milk and infant PPs were significant. Breast-milk and maternal and infant PP 22:5n-6 concentrations were lowest in group 2. DHA supplementation increases the plasma and breast-milk DHA concentrations of lactating women, resulting in higher PP DHA concentrations in infants.

Recently, we have shown that phosphoinositide 3-kinase (PI3K) in bovine rod outer segment (ROS) is activated in vitro by tyrosine phosphorylation of the C-terminal tail of the insulin receptor (Rajala, R. V. S., and Anderson, R. E. (2001) Invest. Ophthal. Vis. Sci. 42, 3110 -3117). In this study, we have investigated the in vivo mechanism of PI3K activation in the rodent retina and report the novel finding that light stimulates tyrosine phosphorylation of the ␤-subunit of the insulin receptor (IR␤) in ROS membranes, which leads to the association of PI3K enzyme activity with IR␤. Retinas from light-or dark-adapted mice and rats were homogenized and immunoprecipitated with antibodies against phosphotyrosine, IR␤, or the p85 regulatory subunit of PI3K, and PI3K activity was measured using PI-4,5-P 2 as substrate. We observed a light-dependent increase in tyrosine phosphorylation of IR␤ and an increase in PI3K enzyme activity in isolated ROS and in anti-phosphotyrosine and anti-IR␤ immunoprecipitates of retinal homogenates. The light effect was localized to photoreceptor neurons and is independent of insulin secretion. Our results suggest that light induces tyrosine phosphorylation of IR␤ in outer segment membranes, which leads to the binding of p85 through its N-terminal Src homology 2 domain and the generation of PI-3,4,5-P 3 . We suggest that the physiological role of this process may be to provide neuroprotection of the retina against light damage by activating proteins that protect against stressinduced apoptosis.Many cell proliferation and cell survival pathways are initiated upon activation of tyrosine kinase receptors, which transduce their signals by recruiting a variety of cytoplasmic signaling proteins (1, 2). Many of the signaling proteins contain phosphotyrosine binding domains, Src homology 2 (SH2) 1 domains, and Src homology 3 (SH3) domains, which are involved in mediating protein-protein interactions (4, 5).The phosphotyrosine-dependent interaction between different phosphotyrosine binding and SH2 domain-containing proteins with activated receptors initiates cellular changes that take place in response to a wide range of extracellular signals (2).One of the SH2 domain-containing proteins, phosphoinositide 3-kinase (PI3K), consists of an ϳ85-kDa regulatory subunit (p85) and a ϳ110-kDa catalytic subunit (p110), the latter being responsible for the phosphorylation of phosphoinositides at the D3 position and of serine residues in proteins (7, 8). The p85 subunit contains an SH3 domain capable of binding to proline-rich sequences, a p110 binding domain, and two SH2 domains. PI3K was initially found to be associated with middle-T/pp60c-Src (9), pp60v-Src, and platelet-derived growth factor receptors in both normal and transformed NIH3T3 fibroblast cells (10, 11). PI3K activity increases in response to platelet-derived growth factor binding to its receptor, in large part because the p85-p110 complex is translocated from the cytosol to the plasma membrane, by the direct binding of the p85 SH2 domain to tyro...