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 ...

OBJECTIVEVascular endothelial growth factor (VEGF-A or VEGF) is a major pathogenic factor and therapeutic target for diabetic retinopathy (DR). Since VEGF has been proposed as a survival factor for retinal neurons, defining the cellular origin of pathogenic VEGF is necessary for the effectiveness and safety of long-term anti-VEGF therapies for DR. To determine the significance of Müller cell-derived VEGF in DR, we disrupted VEGF in Müller cells with an inducible Cre/lox system and examined diabetes-induced retinal inflammation and vascular leakage in these conditional VEGF knockout (KO) mice.RESEARCH DESIGN AND METHODSLeukostasis was determined by counting the number of fluorescently labeled leukocytes inside retinal vasculature. Expression of biomarkers for retinal inflammation was assessed by immunoblotting of TNF-α, ICAM-1, and NF-κB. Vascular leakage was measured by immunoblotting of retinal albumin and fluorescent microscopic analysis of extravascular albumin. Diabetes-induced vascular alterations were examined by immunoblotting and immunohistochemistry for tight junctions, and by trypsin digestion assays for acellular capillaries. Retinal integrity was analyzed with morphologic and morphometric analyses.RESULTSDiabetic conditional VEGF KO mice exhibited significantly reduced leukostasis, expression of inflammatory biomarkers, depletion of tight junction proteins, numbers of acellular capillaries, and vascular leakage compared to diabetic control mice.CONCLUSIONSMüller cell-derived VEGF plays an essential and causative role in retinal inflammation, vascular lesions, and vascular leakage in DR. Therefore, Müller cells are a primary cellular target for proinflammatory signals that mediates retinal inflammation and vascular leakage in DR.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

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.

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The relatively saturated lipid environment observed in DRMs is likely to promote the localization of signaling proteins modified with saturated fatty acyl chains. Based on the lipid composition of DRMs, the authors conclude that they would not efficiently support phototransduction.

Exposure to intense light increases 4-HNE and 4-HHE protein modifications in the retina, suggesting that free radical initiated, nonenzymatic reactions are involved in this process. These modifications may be early events that precede photoreceptor cell apoptosis.

Obesity has deleterious effects on cognitive function in the elderly adults. In mice, aging exacerbates obesity-induced oxidative stress, microvascular dysfunction, blood-brain barrier (BBB) disruption, and neuroinflammation, which compromise cognitive health. However, the specific mechanisms through which aging and obesity interact to remain elusive. Previously, we have shown that Nrf2 signaling plays a critical role in microvascular resilience to obesity and that aging is associated with progressive Nrf2 dysfunction, promoting microvascular impairment. To test the hypothesis that Nrf2 deficiency exacerbates cerebromicrovascular dysfunction induced by obesity Nrf2+/+ and Nrf2-/-, mice were fed an adipogenic high-fat diet (HFD). Nrf2 deficiency significantly exacerbated HFD-induced oxidative stress and cellular senescence, impairment of neurovascular coupling responses, BBB disruption, and microglia activation, mimicking the aging phenotype. Obesity in Nrf2-/- mice elicited complex alterations in the amyloidogenic gene expression profile, including upregulation of amyloid precursor protein. Nrf2 deficiency and obesity additively reduced long-term potentiation in the CA1 area of the hippocampus. Collectively, Nrf2 dysfunction exacerbates the deleterious effects of obesity, compromising cerebromicrovascular and brain health by impairing neurovascular coupling mechanisms, BBB integrity and synaptic function and promoting neuroinflammation. These results support a possible role for age-related Nrf2 dysfunction in the pathogenesis of vascular cognitive impairment and Alzheimer's disease.