Martin Paul Agbaga

Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids

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.

Curcumin protects retinal cells from light-and oxidant stress-induced cell death.

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 anti-inflammatory and antioxidative properties, in a 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-kappaB activation and down-regulation of cellular inflammatory genes. When tested on retina-derived cell lines (661W and ARPE-19), pretreatment 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-kappaB, 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.

Retinal very long-chain PUFAs: new insights from studies on ELOVL4 protein.

Compared with other mammalian tissues, retina is highly enriched in PUFA. Long-chain PUFA (LC-PUFA; C18-C24) are essential FAs that are enriched in the retina and are necessary for maintenance of normal retinal development and function. The retina, brain, and sperm also contain very LC-PUFA (VLC-PUFA; >C24). Although VLC-PUFA were discovered more than two decades ago, very little is known about their biosynthesis and functional roles in the retina. This is due mainly to intrinsic difficulties associated with working on these unusually long polyunsaturated hydrocarbon chains and their existence in small amounts. Recent studies on the FA elongase elongation of very long chain fatty acids-4 (ELOVL4) protein, however, suggest that VLC-PUFA probably play some uniquely important roles in the retina as well as the other tissues. Mutations in the ELOVL4 gene are found in patients with autosomal dominant Stargardt disease. Here, we review the recent literature on VLC-PUFA with special emphasis on the elongases responsible for their synthesis. We focus on a novel elongase, ELOVL4, involved in the synthesis of VLC-PUFA, and the importance of these FAs in maintaining the structural and functional integrity of retinal photoreceptors.

Docosahexaenoic acid supplementation fully restores fertility and spermatogenesis in male delta-6 desaturase-null mice

Delta-6 desaturase-null mice (−/−) are unable to synthesize highly unsaturated fatty acids (HUFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), and n6-docosapentaenoic acid (DPAn6). The −/− males exhibit infertility and arrest of spermatogenesis at late spermiogenesis. To determine which HUFA is essential for spermiogenesis, a diet supplemented with either 0.2% (w/w) AA or DHA was fed to wild-type (+/+) and −/− males at weaning until 16 weeks of age (n = 3–5). A breeding success rate of DHA-supplemented −/− was comparable to +/+. DHA-fed −/− showed normal sperm counts and spermiogenesis. Dietary AA was less effective in restoring fertility, sperm count, and spermiogenesis than DHA. Testis fatty acid analysis showed restored DHA in DHA-fed −/−, but DPAn6 remained depleted. In AA-fed −/−, AA was restored at the +/+ level, and 22:4n6, an AA elongated product, accumulated in testis. Cholesta-3,5-diene was present in testis of +/+ and DHA-fed −/−, whereas it diminished in −/− and AA-fed −/−, suggesting impaired sterol metabolism in these groups. Expression of spermiogenesis marker genes was largely normal in all groups. In conclusion, DHA was capable of restoring all observed impairment in male reproduction, whereas 22:4n6 formed from dietary AA may act as an inferior substitute for DHA.

Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy

Autosomal-dominant Stargardt-like macular dystrophy [Stargardt3 (STGD3)] results from single allelic mutations in the elongation of very-long–chain fatty acids-like 4 (ELOVL4), whereas recessive mutations lead to skin and brain dysfunction. ELOVL4 protein localizes to the endoplasmic reticulum, where it mediates the condensation reaction catalyzing the formation of very-long–chain (VLC) (C-28 to C-40) fatty acids, saturated and polyunsaturated (PUFA). The defective gene product is truncated at the C terminus, leading to mislocalization and aggregation in other organelles. We hypothesized that the STGD3 truncated mutant may generate mislocalized, and therefore toxic, keto intermediates of fatty acid elongation, thereby contributing to the disease process. Using cell-based and cell-free microsome assays, we found that the truncated protein lacked innate condensation activity. Coexpression of different forms of wild-type and mutant ELOVL4 revealed a large dominant-negative effect of mutant protein on ELOVL4 localization and enzymatic activity, resulting in reduced VLC-PUFA synthesis. The reduction in VLC-PUFA levels in STGD3 and age-related macular degeneration may be a contributing factor to their retinal pathology.

Role of Elovl4 protein in the biosynthesis of docosahexaenoic acid

The disk membranes of retinal photoreceptor outer segments and other neuronal and reproductive tissues are enriched in docosahexaenoic acid (DHA, 22:6n3), which is essential for their normal function and development. The fatty acid condensing enzyme Elongation of Very Long chain fatty acids-4 (ELOVL4) is highly expressed in retina photoreceptors as well as other tissues with high 22:6n3 content. Mutations in the ELOVL4 gene are associated with autosomal dominant Stargardt-like macular dystrophy (STGD3) and results in synthesis of a truncated protein that cannot be targeted to the endoplasmic reticulum (ER), the site of fatty acid biosynthesis. Considering the abundance and essential roles of 22:6n3 in ELOVL4-expressing tissues (except the skin), it was proposed that the ELOVL4 protein may be involved in 22:6n3 biosynthesis. We tested the hypothesis that the ELOVL4 protein is involved in 22:6n3 biosynthesis by selectively silencing expression of the protein in the cone photoreceptors derived cell line 661 w and showed that the ELOVL4 protein is not involved in DHA biosynthesis from the short chain fatty acid precursors 18:3n3 and 22:5n3.

Regional changes in CNS and retinal glycerophospholipid profiles with age: a molecular blueprint

We present here a quantitative molecular blueprint of the three major glycerophospholipid (GPL) classes, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), in retina and six regions of the brain in C57Bl6 mice at 2, 10, and 26 months of age. We found an age-related increase in molecular species containing saturated and monoenoic FAs and an overall decrease in the longer-chain PUFA molecular species across brain regions, with loss of DHA-containing molecular species as the most consistent and dramatic finding. Although we found very-long-chain PUFAs (VLC-PUFAs) (ue024C28) in PC in the retina, no detectable levels were found in any brain region at any of the ages examined. All brain regions (except hippocampus and retina) showed a significant increase with age in PE plasmalogens. All three retina GPLs had di-PUFA molecular species (predominantly 44:12), which were most abundant in PS (∼30%). In contrast, low levels of di-PUFA GPL (1–2%) were found in all regions of the brain. This study provides a regional and age-related assessment of the brain’s lipidome with a level of detail, inclusion, and quantification that has not heretofore been published.

Cell-Specific Promoters Enable Lipid-Based Nanoparticles to Deliver Genes to Specific Cells of the Retina In Vivo

Non-viral vectors, such as lipid-based nanoparticles (liposome-protamine-DNA complex [LPD]), could be used to deliver a functional gene to the retina to correct visual function and treat blindness. However, one of the limitations of LPD is the lack of cell specificity, as the retina is composed of seven types of cells. If the same gene is expressed in multiple cell types or is absent from one desired cell type, LPD-mediated gene delivery to every cell may have off-target effects. To circumvent this problem, we have tested LPD-mediated gene delivery using various generalized, modified, and retinal cell-specific promoters. We achieved retinal pigment epithelium cell specificity with vitelliform macular dystrophy (VMD2), rod cell specificity with mouse rhodopsin, cone cell specificity with red/green opsin, and ganglion cell specificity with thymocyte antigen promoters. Here we show for the first time that cell-specific promoters enable lipid-based nanoparticles to deliver genes to specific cells of the retina in vivo. This work will inspire investigators in the field of lipid nanotechnology to couple cell-specific promoters to drive expression in a cell- and tissue-specific manner.

In vivo effect of mutant ELOVL4 on the expression and function of wild-type ELOVL4.

PURPOSEnMutations in the elongation of very long chain fatty acids 4 (ELOVL4) gene cause human Stargardts macular dystrophy 3 (STGD3), a juvenile onset dominant form of macular degeneration. To understand the role of the ELOVL4 protein in retinal function, several mouse models have been developed by using transgenic (TG), knock-in (Elovl4(+/mut)), and knockout (Elovl4(+/-)) approaches. Here we analyzed quantitatively the ELOVL4 protein and its enzymatic products (very long chain saturated fatty acid [VLC-FA] and VLC-polyunsaturated fatty acid [VLC-PUFA]) in the retinas of 8 to 10-week-old TG1(+), TG2(+), and Elovl4(+/mut) mice that harbor the mutant ELOVL4 and compared them to their wild-type littermates and Elovl4(+/-) that do not express the mutant protein. We also analyzed skin from these mice to gain insight into the pathogenesis resulting from the ELOVL4 mutation.nnnMETHODSnELOVL4 protein localization in the retina was determined by immunohistochemistry. Levels of wild-type ELOVL4 protein in skin and retinas were determined by Western blotting. Total lipids from skin and retinas were measured by gas chromatography-mass spectrometry (GC-MS). Retinal glycerophosphatidylcholines (PC) were analyzed by tandem mass spectrometry.nnnRESULTSnImmunohistochemical and Western analysis indicated that wild-type ELOVL4 protein was reduced in heterozygous Elovl4(+/mut) and Elovl4(+/-) retinas, but not in TG2(+) retinas. We found that VLC-FA was reduced by 50% in the skin of Elovl4(+/-) and by 60% to 65% in Elovl4(+/mut). We found VLC-PUFA levels at ∼ 50% in both the retinas, and wild-type levels of VLC-PUFA in TG2(+) retinas.nnnCONCLUSIONSnWe conclude that the presence of the mutant ELOVL4 does not affect the function of wild-type ELOVL4 in the fully developed 8- to 10-week-old retinas.

Homozygous Expression of Mutant ELOVL4 Leads to Seizures and Death in a Novel Animal Model of Very Long-Chain Fatty Acid Deficiency

Lipids are essential components of the nervous system. However, the functions of very long-chain fatty acids (VLC-FA; ≥u200928 carbons) in the brain are unknown. The enzyme ELOngation of Very Long-chain fatty acids-4 (ELOVL4) catalyzes the rate-limiting step in the biosynthesis of VLC-FA (Agbaga et al., Proc Natl Acad Sci USA 105(35): 12843–12848, 2008; Logan et al., J Lipid Res 55(4): 698–708, 2014), which we identified in the brain as saturated fatty acids (VLC-SFA). Homozygous mutations in ELOVL4 cause severe neuropathology in humans (Ozaki et al., JAMA Neurol 72(7): 797–805, 2015; Mir et al., BMC Med Genet 15: 25, 2014; Cadieux-Dion et al., JAMA Neurol 71(4): 470–475, 2014; Bourassa et al., JAMA Neurol 72(8): 942–943, 2015; Aldahmesh et al., Am J Hum Genet 89(6): 745–750, 2011) and are post-natal lethal in mice (Cameron et al., Int J Biol Sci 3(2): 111–119, 2007; Li et al., Int J Biol Sci 3(2): 120–128, 2007; McMahon et al., Molecular Vision 13: 258–272, 2007; Vasireddy et al., Hum Mol Genet 16(5): 471–482, 2007) from dehydration due to loss of VLC-SFA that comprise the skin permeability barrier. Double transgenic mice with homozygous knock-in of the Stargardt-like macular dystrophy (STDG3; 797-801_AACTT) mutation of Elovl4 with skin-specific rescue of wild-type Elovl4 expression (S+Elovl4mut/mut mice) develop seizures by P19 and die by P21. Electrophysiological analyses of hippocampal slices showed aberrant epileptogenic activity in S+Elovl4mut/mut mice. FM1-43 dye release studies showed that synapses made by cultured hippocampal neurons from S+Elovl4mut/mut mice exhibited accelerated synaptic release kinetics. Supplementation of VLC-SFA to cultured hippocampal neurons from mutant mice rescued defective synaptic release to wild-type rates. Together, these studies establish a critical, novel role for ELOVL4 and its VLC-SFA products in regulating synaptic release kinetics and epileptogenesis. Future studies aimed at understanding the molecular mechanisms by which VLC-SFA regulate synaptic function may provide new targets for improved seizure therapies.