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 ...

This article is available online at http://www.jlr.org Supplementary key words essential fatty acids • arachidonic acid • highly unsaturated fatty acids • very-long-chain polyunsaturated fatty acids • choelsta-3,5-diene • male reproduction • spermiogenesis Delta-6 desaturase (D6D) is the fi rst and rate-limiting enzyme for highly unsaturated fatty acid (HUFA) synthesis that consists of a series of elongation and desaturation reactions ( 1 ). The dietary essential fatty acids 18:2n-6 (linoleic acid) and 18:3n-3 ( ␣ -linolenic acid) are substrates for D6D and precursors of physiologically important HUFAs, such as 20:4n-6 [arachidonic acid (AA)], 22:5n6 [docosapentaenoic acid (DPAn6)], and 22:6n3 [docosahexaenoic acid (DHA)]. D6D is also required for the fi nal desaturation step for the synthesis of DPAn6 and DHA.These HUFAs are present in high concentration in testes and sperm of mammals. DPAn6, a HUFA derived from AA, dramatically increases in rat testes during the sexual maturation stage ( 2 ). In mice, AA, DPAn6, and DHA are abundant in membrane phospholipids of round spermatids ( 3 ) and mature mouse spermatozoa ( 4 ), suggesting an important role for these fatty acids for proper spermatogenesis. In humans, DHA is the main HUFA in sperm ( 5 ). DHA is specifi cally high in the sperm tail when compared with the sperm head in monkeys ( 6 ), implying a role of DHA in sperm tail function. AA may also have a role in male fertility as a precursor to eicosanoids. Prostaglandin E2, for example, has been shown to increase sperm motility ( 7 ), while inhibition of cycloxygenase-2 in mouse vas deferens results in a decrease of sperm motility and fertilAbstract 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 ( Abbreviations: AA, arachidonic acid; CD, cholesta-3,5-diene; D6D, ⌬ 6 desaturase; DHA, docosahexaenoic acid; DPAn6, docosapentaenoic acid; FAME, fatty acid methyl esters; HUFA, highly unsaturated fatty acid; VLPUFA, very-long-chain polyunsaturated fatty acid.

Lipids are essential components of the nervous system. However, the functions of very long-chain fatty acids (VLC-FA; ≥ 28 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 + Elovl4 mut/mut mice) develop seizures by P19 and die by P21. Electrophysiological analyses of hippocampal slices showed aberrant epileptogenic activity in S + Elovl4 mut/mut mice. FM1-43 dye release studies showed that synapses made by cultured hippocampal neurons from S + Elovl4 mut/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.Electronic supplementary materialThe online version of this article (10.1007/s12035-017-0824-8) contains supplementary material, which is available to authorized users.

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.

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that is expressed in cancer cells. Its role in tumor metabolism is not definitively established, but investigators have suggested that regulation of PKM2 activity can cause accumulation of glycolytic intermediates and increase flux through the pentose phosphate pathway. Recent evidence suggests that PKM2 also may have non-metabolic functions, including as a transcriptional co-activator in gene regulation. We reported previously that PKM2 is abundant in photoreceptor cells in mouse retinas. In the present study, we conditionally deleted PKM2 (rod-cre PKM2-KO) in rod photoreceptors and found that the absence of PKM2 causes increased expression of PKM1 in rods. Analysis of metabolic flux from U-13C glucose shows that rod-cre PKM2-KO retinas accumulate glycolytic intermediates, consistent with an overall reduction in the amount of pyruvate kinase activity. Rod-cre PKM2-KO mice also have an increased NADPH availability could favor lipid synthesis, but we found no difference in phospholipid synthesis between rod-cre PKM2 KO and PKM2-positive controls. As rod-cre PKM2-KO mice aged, we observed a significant loss of rod function, reduced thickness of the photoreceptor outer segment layer, and reduced expression of photoreceptor proteins, including PDE6β. The rod-cre PKM2-KO retinas showed greater TUNEL staining than wild-type retinas, indicating a slow retinal degeneration. In vitro analysis showed that PKM2 can regulate transcriptional activity from the PDE6β promoter in vitro. Our findings indicate that both the metabolic and transcriptional regulatory functions of PKM2 may contribute to photoreceptor structure, function, and viability.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. While the histopathology of the different disease stages is well characterized, the cause underlying the progression, from the early drusen stage to the advanced macular degeneration stage that leads to blindness, remains unknown. Here, we show that photoreceptors (PRs) of diseased individuals display increased expression of two key glycolytic genes, suggestive of a glucose shortage during disease. Mimicking aspects of this metabolic profile in PRs of wild-type mice by activation of the mammalian target of rapamycin complex 1 (mTORC1) caused early drusen-like pathologies, as well as advanced AMD-like pathologies. Mice with activated mTORC1 in PRs also displayed other early disease features, such as a delay in photoreceptor outer segment (POS) clearance and accumulation of lipofuscin in the retinal-pigmented epithelium (RPE) and of lipoproteins at the Bruch’s membrane (BrM), as well as changes in complement accumulation. Interestingly, formation of drusen-like deposits was dependent on activation of mTORC1 in cones. Both major types of advanced AMD pathologies, including geographic atrophy (GA) and neovascular pathologies, were also seen. Finally, activated mTORC1 in PRs resulted in a threefold reduction in di-docosahexaenoic acid (DHA)–containing phospholipid species. Feeding mice a DHA-enriched diet alleviated most pathologies. The data recapitulate many aspects of the human disease, suggesting that metabolic adaptations in photoreceptors could contribute to disease progression in AMD. Identifying the changes downstream of mTORC1 that lead to advanced pathologies in mouse might present new opportunities to study the role of PRs in AMD pathogenesis.

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.Stargardt disease | condensing enzyme | photoreceptor degeneration

Rhodopsins are densely packed in rod outer-segment membranes to maximize photon absorption, but this arrangement interferes with transducin activation by restricting the mobility of both proteins. We attempted to explore this phenomenon in transgenic mice that overexpressed rhodopsin in their rods. Photon capture was improved, and, for a given number of photoisomerizations, bright-flash responses rose more gradually with a reduction in amplification--but not because rhodopsins were more tightly packed in the membrane. Instead, rods increased their outer-segment diameters, accommodating the extra rhodopsins without changing the rhodopsin packing density. Because the expression of other phototransduction proteins did not increase, transducin and its effector phosphodiesterase were distributed over a larger surface area. That feature, as well as an increase in cytosolic volume, was responsible for delaying the onset of the photoresponse and for attenuating its amplification.