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Thematic Review Series: Seeing 2020: Lipids and Lipid-Soluble Molecules in the Eye Thematic Review Series Thematic Review Series: Seeing 2020: Lipids and Lipid-Soluble Molecules in the Eye| Volume 62, 100030, January 01, 2021

Very long chain fatty acid-containing lipids: a decade of novel insights from the study of ELOVL4

Open AccessPublished:February 05, 2021DOI:https://doi.org/10.1016/j.jlr.2021.100030

      Abstract

      Lipids play essential roles in maintaining cell structure and function by modulating membrane fluidity and cell signaling. The fatty acid elongase-4 (ELOVL4) protein, expressed in retina, brain, Meibomian glands, skin, testes and sperm, is an essential enzyme that mediates tissue-specific biosynthesis of both VLC-PUFA and VLC-saturated fatty acids (VLC-SFA). These fatty acids play critical roles in maintaining retina and brain function, neuroprotection, skin permeability barrier maintenance, and sperm function, among other important cellular processes. Mutations in ELOVL4 that affect biosynthesis of these fatty acids cause several distinct tissue-specific human disorders that include blindness, age-related cerebellar atrophy and ataxia, skin disorders, early-childhood seizures, mental retardation, and mortality, which underscores the essential roles of ELOVL4 products for life. However, the mechanisms by which one tissue makes VLC-PUFA and another makes VLC-SFA, and how these fatty acids exert their important functional roles in each tissue, remain unknown. This review summarizes research over that last decade that has contributed to our current understanding of the role of ELOVL4 and its products in cellular function. In the retina, VLC-PUFA and their bioactive “Elovanoids” are essential for retinal function. In the brain, VLC-SFA are enriched in synaptic vesicles and mediate neuronal signaling by determining the rate of neurotransmitter release essential for normal neuronal function. These findings point to ELOVL4 and its products as being essential for life. Therefore, mutations and/or age-related epigenetic modifications of fatty acid biosynthetic gene activity that affect VLC-SFA and VLC-PUFA biosynthesis contribute to age-related dysfunction of ELOVL4-expressing tissues.

      Graphical abstract

      Supplementary key words

      Abbreviations:

      AA (arachidonic acid (20:4n6)), AMD (age-related macular degeneration), BBS (Bardet-Biedl syndrome), ELOVL (elongation of very long-chain fatty acid), ERG (electroretinography), IS (inner segment), OS (outer segment), PC (phosphatidylcholine), POS (photoreceptor outer segment), RPE (retinal pigment epithelium), SCA34 (spinocerebellar ataxia-34), STGD3 (Stargardt-like macular dystrophy), VLC-SFA (very long chain saturated fatty acid), VLC-PUFA (very long chain polyunsaturated fatty acid), VLC-FA (very long chain fatty acid)
      Evolving discovery of novel mutations in the ELOngation of Very Long chain fatty acids-4 (ELOVL4) gene as the cause of different tissue-specific disorders in humans is providing novel insights into the role of lipids in health and disease in a number of important organs. In 2001, Zhang et al. (
      • Zhang K.
      • Kniazeva M.
      • Han M.
      • Li W.
      • Yu Z.
      • Yang Z.
      • Li Y.
      • Metzker M.L.
      • Allikmets R.
      • Zack D.J.
      • Kakuk L.E.
      • Lagali P.S.
      • Wong P.W.
      • MacDonald I.M.
      • et al.
      A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.
      ) and Edwards et al. (
      • Edwards A.O.
      • Donoso L.A.
      • Ritter 3rd., R.
      A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family.
      ) independently reported a five base pair deletion (797–801delAACT) in exon 6 of the ELOVL4 gene located on human chromosome 6q14 as the cause of early-onset macular degeneration in patients with autosomal dominant Stargardt-like macular dystrophy (STGD3). The deletion results in truncation of the last 51 amino acids, including an endoplasmic reticulum retention/retrieval signal located at the C-terminal end of the ELOVL4 protein (
      • Zhang K.
      • Kniazeva M.
      • Han M.
      • Li W.
      • Yu Z.
      • Yang Z.
      • Li Y.
      • Metzker M.L.
      • Allikmets R.
      • Zack D.J.
      • Kakuk L.E.
      • Lagali P.S.
      • Wong P.W.
      • MacDonald I.M.
      • et al.
      A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.
      ,
      • Edwards A.O.
      • Donoso L.A.
      • Ritter 3rd., R.
      A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family.
      ). The same year, Bernstein et al. (
      • Bernstein P.S.
      • Tammur J.
      • Singh N.
      • Hutchinson A.
      • Dixon M.
      • Pappas C.M.
      • Zabriskie N.A.
      • Zhang K.
      • Petrukhin K.
      • Leppert M.
      • Allikmets R.
      Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene.
      ) reported two different one base pair deletions separated by four nucleotides (790delT+794delT) within the same region of exon 6 of ELOVL4. These mutations also result in truncation of the ELOVL4 protein in a manner similar to the reported 5 bp deletion (
      • Zhang K.
      • Kniazeva M.
      • Han M.
      • Li W.
      • Yu Z.
      • Yang Z.
      • Li Y.
      • Metzker M.L.
      • Allikmets R.
      • Zack D.J.
      • Kakuk L.E.
      • Lagali P.S.
      • Wong P.W.
      • MacDonald I.M.
      • et al.
      A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.
      ,
      • Edwards A.O.
      • Donoso L.A.
      • Ritter 3rd., R.
      A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family.
      ,
      • Bernstein P.S.
      • Tammur J.
      • Singh N.
      • Hutchinson A.
      • Dixon M.
      • Pappas C.M.
      • Zabriskie N.A.
      • Zhang K.
      • Petrukhin K.
      • Leppert M.
      • Allikmets R.
      Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene.
      ). Three years later, Maugeri et al. (
      • Maugeri A.
      • Meire F.
      • Hoyng C.B.
      • Vink C.
      • Van Regemorter N.
      • Karan G.
      • Yang Z.
      • Cremers F.P.
      • Zhang K.
      A novel mutation in the ELOVL4 gene causes autosomal dominant Stargardt-like macular dystrophy.
      ) reported a fourth mutation also in exon 6 of ELOVL4 (c. 810C > G; p Tyr270X) that leads to truncation of the ELOVL4 protein in a European family with STGD3 pathology. A major characteristic of these STGD3-causing mutations is early-onset loss of central vision and macular degeneration akin to age-related macular degeneration (AMD), characterized by accumulation of high levels of lipofuscin in the retinal pigment epithelium (RPE), and macular degeneration (
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      Age-Related Eye Disease Study Group
      The Age-Related Eye Disease Study severity scale for age-related macular degeneration: AREDS Report No. 17.
      ,
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      • Stone E.M.
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      Autosomal dominant Stargardt-like macular dystrophy.
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      • Donoso L.A.
      Autosomal dominant Stargardt-like macular dystrophy: I. Clinical characterization, longitudinal follow-up, and evidence for a common ancestry in families linked to chromosome 6q14.
      ).
      These discoveries generated significant interest in understanding the biological function of the ELOVL4 protein, which shares sequence homology with a group of yeast fatty acid elongase (ELO) proteins involved in fatty acid elongation (
      • Zhang X.M.
      • Yang Z.
      • Karan G.
      • Hashimoto T.
      • Baehr W.
      • Yang X.J.
      • Zhang K.
      Elovl4 mRNA distribution in the developing mouse retina and phylogenetic conservation of Elovl4 genes.
      ,
      • Oh C.S.
      • Toke D.A.
      • Mandala S.
      • Martin C.E.
      ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation.
      ,
      • Paul S.
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      • Dunn T.M.
      Members of the Arabidopsis FAE1-like 3-ketoacyl-CoA synthase gene family substitute for the Elop proteins of Saccharomyces cerevisiae.
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      • Westerberg R.
      • Silve S.
      • Asadi A.
      • Jakobsson A.
      • Cannon B.
      • Loison G.
      • Jacobsson A.
      Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids.
      ). A number of laboratories, including ours, carried out a series of in vitro (
      • Ambasudhan R.
      • Wang X.
      • Jablonski M.M.
      • Thompson D.A.
      • Lagali P.S.
      • Wong P.W.
      • Sieving P.A.
      • Ayyagari R.
      Atrophic macular degeneration mutations in ELOVL4 result in the intracellular misrouting of the protein.
      ,
      • Karan G.
      • Yang Z.
      • Howes K.
      • Zhao Y.
      • Chen Y.
      • Cameron D.J.
      • Lin Y.
      • Pearson E.
      • Zhang K.
      Loss of ER retention and sequestration of the wild-type ELOVL4 by Stargardt disease dominant negative mutants.
      ,
      • Karan G.
      • Yang Z.
      • Zhang K.
      Expression of wild type and mutant ELOVL4 in cell culture: Subcellular localization and cell viability.
      ,
      • Vasireddy V.
      • Jablonski M.M.
      • Khan N.W.
      • Wang X.F.
      • Sahu P.
      • Sparrow J.R.
      • Ayyagari R.
      Elovl4 5-bp deletion knock-in mouse model for Stargardt-like macular degeneration demonstrates accumulation of ELOVL4 and lipofuscin.
      ,
      • Vasireddy V.
      • Vijayasarathy C.
      • Huang J.
      • Wang X.F.
      • Jablonski M.M.
      • Petty H.R.
      • Sieving P.A.
      • Ayyagari R.
      Stargardt-like macular dystrophy protein ELOVL4 exerts a dominant negative effect by recruiting wild-type protein into aggresomes.
      ,
      • Grayson C.
      • Molday R.S.
      Dominant negative mechanism underlies autosomal dominant Stargardt-like macular dystrophy linked to mutations in ELOVL4.
      ) and in vivo (
      • Vasireddy V.
      • Vijayasarathy C.
      • Huang J.
      • Wang X.F.
      • Jablonski M.M.
      • Petty H.R.
      • Sieving P.A.
      • Ayyagari R.
      Stargardt-like macular dystrophy protein ELOVL4 exerts a dominant negative effect by recruiting wild-type protein into aggresomes.
      ,
      • Karan G.
      • Lillo C.
      • Yang Z.
      • Cameron D.J.
      • Locke K.G.
      • Zhao Y.
      • Thirumalaichary S.
      • Li C.
      • Birch D.G.
      • Vollmer-Snarr H.R.
      • Williams D.S.
      • Zhang K.
      Lipofuscin accumulation, abnormal electrophysiology, and photoreceptor degeneration in mutant ELOVL4 transgenic mice: A model for macular degeneration.
      ,
      • McMahon A.
      • Butovich I.A.
      • Mata N.L.
      • Klein M.
      • Ritter 3rd, R.
      • Richardson J.
      • Birch D.G.
      • Edwards A.O.
      • Kedzierski W.
      Retinal pathology and skin barrier defect in mice carrying a Stargardt disease-3 mutation in elongase of very long chain fatty acids-4.
      ,
      • McMahon A.
      • Jackson S.N.
      • Woods A.S.
      • Kedzierski W.
      A Stargardt disease-3 mutation in the mouse Elovl4 gene causes retinal deficiency of C32-C36 acyl phosphatidylcholines.
      ,
      • Raz-Prag D.
      • Ayyagari R.
      • Fariss R.N.
      • Mandal M.N.
      • Vasireddy V.
      • Majchrzak S.
      • Webber A.L.
      • Bush R.A.
      • Salem Jr., N.
      • Petrukhin K.
      • Sieving P.A.
      Haploinsufficiency is not the key mechanism of pathogenesis in a heterozygous Elovl4 knockout mouse model of STGD3 disease.
      ,
      • Vasireddy V.
      • Jablonski M.M.
      • Mandal M.N.
      • Raz-Prag D.
      • Wang X.F.
      • Nizol L.
      • Iannaccone A.
      • Musch D.C.
      • Bush R.A.
      • Salem Jr., N.
      • Sieving P.A.
      • Ayyagari R.
      Elovl4 5-bp-deletion knock-in mice develop progressive photoreceptor degeneration.
      ) experiments aimed at elucidating the biological function of the ELOVL4 protein and how the mutations cause blindness in patients with STGD3. Since the retina contains high levels of PUFA, especially DHA, which represents approximately 45–50% of retinal outer segment phospholipids, initial hypotheses suggested that ELOVL4 may be involved in retinal DHA biosynthesis (
      • Zhang K.
      • Kniazeva M.
      • Han M.
      • Li W.
      • Yu Z.
      • Yang Z.
      • Li Y.
      • Metzker M.L.
      • Allikmets R.
      • Zack D.J.
      • Kakuk L.E.
      • Lagali P.S.
      • Wong P.W.
      • MacDonald I.M.
      • et al.
      A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.
      ). However, our exhaustive in vitro studies showed that ELOVL4 is not involved in the elongation steps required for DHA biosynthesis (
      • Agbaga M.P.
      • Brush R.S.
      • Mandal M.N.
      • Elliott M.H.
      • Al-Ubaidi M.R.
      • Anderson R.E.
      Role of elovl4 protein in the biosynthesis of docosahexaenoic Acid.
      ). In 2008, our laboratory discovered and unequivocally reported that the wild-type (WT) ELOVL4 protein is an essential enzyme that mediates the initial rate-limiting step of the fatty acyl chain condensation reaction in very long fatty acid elongation (
      • Agbaga M.P.
      • Brush R.S.
      • Mandal M.N.
      • Henry K.
      • Elliott M.H.
      • Anderson R.E.
      Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.
      ). We showed that WT ELOVL4 is involved in synthesis of both very long chain polyunsaturated fatty acids (VLC-PUFA) and very long chain saturated fatty acids (VLC-SFA), which we collectively refer to as very long chain fatty acids (VLC-FA), in ELOVL4-expressing tissues (
      • Agbaga M.P.
      • Brush R.S.
      • Mandal M.N.
      • Henry K.
      • Elliott M.H.
      • Anderson R.E.
      Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.
      ). We subsequently showed that STGD3 mutant ELOVL4 lacks both VLC-PUFA and VLC-SFA biosynthesis and exerts a dominant negative effect on WT ELOVL4 biosynthetic activity in vitro (
      • Logan S.
      • Agbaga M.P.
      • Chan M.D.
      • Brush R.S.
      • Anderson R.E.
      Endoplasmic reticulum microenvironment and conserved histidines govern ELOVL4 fatty acid elongase activity.
      ,
      • Logan S.
      • Agbaga M.P.
      • Chan M.D.
      • Kabir N.
      • Mandal N.A.
      • Brush R.S.
      • Anderson R.E.
      Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy.
      ). The results from these in vitro studies are supported by in vivo studies in which homozygous expression of mutant ELOVL4 or global deletion of ELOVL4 causes neonatal lethality in mice (
      • McMahon A.
      • Butovich I.A.
      • Mata N.L.
      • Klein M.
      • Ritter 3rd, R.
      • Richardson J.
      • Birch D.G.
      • Edwards A.O.
      • Kedzierski W.
      Retinal pathology and skin barrier defect in mice carrying a Stargardt disease-3 mutation in elongase of very long chain fatty acids-4.
      ,
      • Vasireddy V.
      • Uchida Y.
      • Salem Jr., N.
      • Kim S.Y.
      • Mandal M.N.
      • Reddy G.B.
      • Bodepudi R.
      • Alderson N.L.
      • Brown J.C.
      • Hama H.
      • Dlugosz A.
      • Elias P.M.
      • Holleran W.M.
      • Ayyagari R.
      Loss of functional ELOVL4 depletes very long-chain fatty acids (> or =C28) and the unique omega-O-acylceramides in skin leading to neonatal death.
      ,
      • Cameron D.J.
      • Tong Z.
      • Yang Z.
      • Kaminoh J.
      • Kamiyah S.
      • Chen H.
      • Zeng J.
      • Chen Y.
      • Luo L.
      • Zhang K.
      Essential role of Elovl4 in very long chain fatty acid synthesis, skin permeability barrier function, and neonatal survival.
      ,
      • Li W.
      • Sandhoff R.
      • Kono M.
      • Zerfas P.
      • Hoffmann V.
      • Ding B.C.
      • Proia R.L.
      • Deng C.X.
      Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice.
      ).
      Apart from the initial three ELOVL4 mutations that cause STGD3 (
      • Zhang K.
      • Kniazeva M.
      • Han M.
      • Li W.
      • Yu Z.
      • Yang Z.
      • Li Y.
      • Metzker M.L.
      • Allikmets R.
      • Zack D.J.
      • Kakuk L.E.
      • Lagali P.S.
      • Wong P.W.
      • MacDonald I.M.
      • et al.
      A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.
      ,
      • Edwards A.O.
      • Donoso L.A.
      • Ritter 3rd., R.
      A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family.
      ,
      • Bernstein P.S.
      • Tammur J.
      • Singh N.
      • Hutchinson A.
      • Dixon M.
      • Pappas C.M.
      • Zabriskie N.A.
      • Zhang K.
      • Petrukhin K.
      • Leppert M.
      • Allikmets R.
      Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene.
      ,
      • Maugeri A.
      • Meire F.
      • Hoyng C.B.
      • Vink C.
      • Van Regemorter N.
      • Karan G.
      • Yang Z.
      • Cremers F.P.
      • Zhang K.
      A novel mutation in the ELOVL4 gene causes autosomal dominant Stargardt-like macular dystrophy.
      ), in the last decade a number of different ELOVL4 mutations that cause different tissue-specific disorders in humans have been reported (Table 1). These novel ELOVL4 mutations comprise both the heterozygous (
      • Cadieux-Dion M.
      • Turcotte-Gauthier M.
      • Noreau A.
      • Martin C.
      • Meloche C.
      • Gravel M.
      • Drouin C.A.
      • Rouleau G.A.
      • Nguyen D.K.
      • Cossette P.
      Expanding the clinical Phenotype associated with ELOVL4 mutation: Study of a Large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia.
      ,
      • Ozaki K.
      • Doi H.
      • Mitsui J.
      • Sato N.
      • Iikuni Y.
      • Majima T.
      • Yamane K.
      • Irioka T.
      • Ishiura H.
      • Doi K.
      • Morishita S.
      • Higashi M.
      • Sekiguchi T.
      • Koyama K.
      • Ueda N.
      • et al.
      A novel mutation in ELOVL4 leading to spinocerebellar ataxia (SCA) with the hot cross bun sign but lacking erythrokeratodermia: A broadened spectrum of SCA34.
      ,
      • Bourassa C.V.
      • Raskin S.
      • Serafini S.
      • Teive H.A.
      • Dion P.A.
      • Rouleau G.A.
      A New ELOVL4 mutation in a case of spinocerebellar ataxia with erythrokeratodermia.
      ,
      • Bourque P.R.
      • Warman-Chardon J.
      • Lelli D.A.
      • LaBerge L.
      • Kirshen C.
      • Bradshaw S.H.
      • Hartley T.
      • Boycott K.M.
      Novel ELOVL4 mutation associated with erythrokeratodermia and spinocerebellar ataxia (SCA 34).
      ,
      • Agbaga M.P.
      Different mutations in ELOVL4 affect very long chain fatty acid biosynthesis to cause variable neurological disorders in humans.
      ,
      • Beaudin M.
      • Sellami L.
      • Martel C.
      • Touzel-Deschenes L.
      • Houle G.
      • Martineau L.
      • Lacroix K.
      • Lavallee A.
      • Chrestian N.
      • Rouleau G.A.
      • Gros-Louis F.
      • Laforce Jr., R.
      • Dupre N.
      Characterization of the phenotype with cognitive impairment and protein mislocalization in SCA34.
      ) and homozygous forms (
      • Aldahmesh M.A.
      • Mohamed J.Y.
      • Alkuraya H.S.
      • Verma I.C.
      • Puri R.D.
      • Alaiya A.A.
      • Rizzo W.B.
      • Alkuraya F.S.
      Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.
      ,
      • Mir H.
      • Raza S.I.
      • Touseef M.
      • Memon M.M.
      • Khan M.N.
      • Jaffar S.
      • Ahmad W.
      A novel recessive mutation in the gene ELOVL4 causes a neuro-ichthyotic disorder with variable expressivity.
      ), with the latter leading to even more severe human disorders characterized by seizures, intellectual disability, and childhood mortality. Taken together, these results have clearly established that functional ELOVL4 and its VLC-FA products are essential for life.
      Table 1Different tissue-specific disorders caused by different ELOVL4 mutations in humans
      Genetic MutationsExonGenetic Consequence/Resultant ProteinInheritanceRetinal PathologyBrain PathologySkin PathologyReference
      797–801 del AACTT “5 bp deletion”6Premature stop, truncated proteinAutosomal dominantSTGD3None reportedNone reported(
      • Zhang K.
      • Kniazeva M.
      • Han M.
      • Li W.
      • Yu Z.
      • Yang Z.
      • Li Y.
      • Metzker M.L.
      • Allikmets R.
      • Zack D.J.
      • Kakuk L.E.
      • Lagali P.S.
      • Wong P.W.
      • MacDonald I.M.
      • et al.
      A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.
      ,
      • Edwards A.O.
      • Donoso L.A.
      • Ritter 3rd., R.
      A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family.
      )
      789 del T, 794 del T “2 bp deletion”6Premature stop, truncated proteinAutosomal dominantSTGD3None reportedNone reported(
      • Bernstein P.S.
      • Tammur J.
      • Singh N.
      • Hutchinson A.
      • Dixon M.
      • Pappas C.M.
      • Zabriskie N.A.
      • Zhang K.
      • Petrukhin K.
      • Leppert M.
      • Allikmets R.
      Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene.
      )
      c.810C > G6p.Y270X, truncationAutosomal dominantSTGD3None reportedNone reported(
      • Maugeri A.
      • Meire F.
      • Hoyng C.B.
      • Vink C.
      • Van Regemorter N.
      • Karan G.
      • Yang Z.
      • Cremers F.P.
      • Zhang K.
      A novel mutation in the ELOVL4 gene causes autosomal dominant Stargardt-like macular dystrophy.
      )
      c.-90G > CPromoter, rs62407622Downregulation of ELOVL4 expressionAutosomal dominantSTGD3None reportedNone reported(
      • Donato L.
      • Scimone C.
      • Rinaldi C.
      • Aragona P.
      • Briuglia S.
      • D'Ascola A.
      • D'Angelo R.
      • Sidoti A.
      Stargardt phenotype associated with two ELOVL4 promoter variants and ELOVL4 downregulation: New possible perspective to etiopathogenesis?.
      )
      c.-236C > TPromoter, rs240307Downregulation of ELOVL4 expressionAutosomal dominantSTGD3None reportedNone reported(
      • Donato L.
      • Scimone C.
      • Rinaldi C.
      • Aragona P.
      • Briuglia S.
      • D'Ascola A.
      • D'Angelo R.
      • Sidoti A.
      Stargardt phenotype associated with two ELOVL4 promoter variants and ELOVL4 downregulation: New possible perspective to etiopathogenesis?.
      )
      c.504G > C4p.L168FAutosomal dominantNone reportedAge-related cerebellar atrophy causing ataxia in patients with SCA34Erythrodermia variabilis, a skin lesion disorder(
      • Cadieux-Dion M.
      • Turcotte-Gauthier M.
      • Noreau A.
      • Martin C.
      • Meloche C.
      • Gravel M.
      • Drouin C.A.
      • Rouleau G.A.
      • Nguyen D.K.
      • Cossette P.
      Expanding the clinical Phenotype associated with ELOVL4 mutation: Study of a Large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia.
      )
      c.736T > G6p.W246GAutosomal dominantNone reportedAge-related cerebellar atrophy causing ataxia in patients with SCA34None reported in human, but present in homozygous W246G knockin rats(
      • Ozaki K.
      • Doi H.
      • Mitsui J.
      • Sato N.
      • Iikuni Y.
      • Majima T.
      • Yamane K.
      • Irioka T.
      • Ishiura H.
      • Doi K.
      • Morishita S.
      • Higashi M.
      • Sekiguchi T.
      • Koyama K.
      • Ueda N.
      • et al.
      A novel mutation in ELOVL4 leading to spinocerebellar ataxia (SCA) with the hot cross bun sign but lacking erythrokeratodermia: A broadened spectrum of SCA34.
      )
      c.539A > C4p.G180PAutosomal dominantNone reportedAge-related cerebellar atrophy causing ataxia in patients with SCA34Erythrodermia variabilis, a skin lesion disorder(
      • Bourassa C.V.
      • Raskin S.
      • Serafini S.
      • Teive H.A.
      • Dion P.A.
      • Rouleau G.A.
      A New ELOVL4 mutation in a case of spinocerebellar ataxia with erythrokeratodermia.
      )
      c698C > T4p.T233MAutosomal dominantNone reportedAge-related cerebellar atrophy causing ataxia in patients with SCA34Erythrodermia variabilis, a skin lesion disorder(
      • Bourque P.R.
      • Warman-Chardon J.
      • Lelli D.A.
      • LaBerge L.
      • Kirshen C.
      • Bradshaw S.H.
      • Hartley T.
      • Boycott K.M.
      Novel ELOVL4 mutation associated with erythrokeratodermia and spinocerebellar ataxia (SCA 34).
      )
      c.512T > C4p.I171TAutosomal dominantRetinitis pigmentosaAge-related cerebellar atrophy causing ataxia in patients with SCA34None reported(
      • Xiao C.
      • M. B. E
      • Rexach J.
      • Knight-Johnson A.
      • Khemani P.
      • Fogel B.L.
      • Das S.
      • Stone E.M.
      • Gomez C.M.
      A family with spinocerebellar ataxia and retinitis pigmentosa attributed to an ELOVL4 mutation.
      )
      c.689delT6p.Ile230Metfs∗22, truncationHomozygous recessiveLimited retinal examination but no functional retinal data reportedSeizures, intellectual disability, and early childhood mortalityIchthyosis(
      • Aldahmesh M.A.
      • Mohamed J.Y.
      • Alkuraya H.S.
      • Verma I.C.
      • Puri R.D.
      • Alaiya A.A.
      • Rizzo W.B.
      • Alkuraya F.S.
      Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.
      )
      c.646C > T5p.Arg216X, truncationHomozygous recessiveLimited retinal examination but no functional retinal data reportedSeizures, intellectual disability, and early childhood mortalityIchthyosis(
      • Aldahmesh M.A.
      • Mohamed J.Y.
      • Alkuraya H.S.
      • Verma I.C.
      • Puri R.D.
      • Alaiya A.A.
      • Rizzo W.B.
      • Alkuraya F.S.
      Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.
      )
      c.78C > G1p.Tyr26∗, truncationHomozygous recessiveTortuos vessel in macular area with subtle macular changesNone reportedIchthyosis(
      • Mir H.
      • Raza S.I.
      • Touseef M.
      • Memon M.M.
      • Khan M.N.
      • Jaffar S.
      • Ahmad W.
      A novel recessive mutation in the gene ELOVL4 causes a neuro-ichthyotic disorder with variable expressivity.
      )
      Although this review focuses on STGD3 and ELOVL4 mutation-associated disorders, recessive STGD1 (also known as fundus flavimaculatus) represents the more prevalent cases of juvenile-onset Stargardt macular dystrophy diseases (
      • Allikmets R.
      • Shroyer N.F.
      • Singh N.
      • Seddon J.M.
      • Lewis R.A.
      • Bernstein P.S.
      • Peiffer A.
      • Zabriskie N.A.
      • Li Y.
      • Hutchinson A.
      • Dean M.
      • Lupski J.R.
      • Leppert M.
      Mutation of the Stargardt disease gene (ABCR) in age-related macular degeneration.
      ,
      • Molday L.L.
      • Rabin A.R.
      • Molday R.S.
      ABCR expression in foveal cone photoreceptors and its role in Stargardt macular dystrophy.
      ,
      • Maugeri A.
      • Klevering B.J.
      • Rohrschneider K.
      • Blankenagel A.
      • Brunner H.G.
      • Deutman A.F.
      • Hoyng C.B.
      • Cremers F.P.
      Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy.
      ,
      • Zhang K.
      • Garibaldi D.C.
      • Kniazeva M.
      • Albini T.
      • Chiang M.F.
      • Kerrigan M.
      • Sunness J.S.
      • Han M.
      • Allikmets R.
      A novel mutation in the ABCR gene in four patients with autosomal recessive Stargardt disease.
      ,
      • Allikmets R.
      • Singh N.
      • Sun H.
      • Shroyer N.F.
      • Hutchinson A.
      • Chidambaram A.
      • Gerrard B.
      • Baird L.
      • Stauffer D.
      • Peiffer A.
      • Rattner A.
      • Smallwood P.
      • Li Y.
      • Anderson K.L.
      • Lewis R.A.
      • et al.
      A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy.
      ,
      • Arnell H.
      • Mantyjarvi M.
      • Tuppurainen K.
      • Andreasson S.
      • Dahl N.
      Stargardt disease: Linkage to the ABCR gene region on 1p21-p22 in Scandinavian families.
      ,
      • Sun H.
      • Nathans J.
      Stargardt's ABCR is localized to the disc membrane of retinal rod outer segments.
      ,
      • Eksandh L.
      • Ekstrom U.
      • Abrahamson M.
      • Bauer B.
      • Andreasson S.
      Different clinical expressions in two families with Stargardt's macular dystrophy (STGD1).
      ). Unlike STGD3 caused by autosomal dominant mutations in ELOVL4, STGD1 is caused by inheritance of recessive mutations in ATP-binding cassette, sub-family A, member 4 (ABCA4 or ABCR) (
      • Maugeri A.
      • Klevering B.J.
      • Rohrschneider K.
      • Blankenagel A.
      • Brunner H.G.
      • Deutman A.F.
      • Hoyng C.B.
      • Cremers F.P.
      Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy.
      ,
      • Zhang K.
      • Kniazeva M.
      • Hutchinson A.
      • Han M.
      • Dean M.
      • Allikmets R.
      The ABCR gene in recessive and dominant Stargardt diseases: A genetic pathway in macular degeneration.
      ,
      • Kniazeva M.
      • Chiang M.F.
      • Morgan B.
      • Anduze A.L.
      • Zack D.J.
      • Han M.
      • Zhang K.
      A new locus for autosomal dominant stargardt-like disease maps to chromosome 4.
      ). ABCA4 is exclusively expressed in retina photoreceptor cells and is essential for the visual cycle by catalyzing the translocation of specific phosphatidylethanolamines from the extracellular/lumenal to the cytoplasmic leaflet of membranes through the hydrolysis of ATP to generate retinoid substrates (
      • Quazi F.
      • Molday R.S.
      Differential phospholipid substrates and directional transport by ATP-binding cassette proteins ABCA1, ABCA7, and ABCA4 and disease-causing mutants.
      ,
      • Radu R.A.
      • Mata N.L.
      • Bagla A.
      • Travis G.H.
      Light exposure stimulates formation of A2E oxiranes in a mouse model of Stargardt's macular degeneration.
      ). The retinoid substrates imported by ABCA4 from the extracellular or intradiscal (rod) membrane surfaces to the cytoplasmic membrane surface are reduced to vitamin A by trans-retinol dehydrogenase and then transferred to the RPE where it is converted to 11-cis-retinal in both rod and cone photoreceptor cells (
      • Kim H.J.
      • Zhao J.
      • Sparrow J.R.
      Vitamin A aldehyde-taurine adduct and the visual cycle.
      ). Owing to its essential function in the visual cycle, mutations in ABCA4 are also associated with retinitis pigmentosa-19, cone-rod dystrophy type 3, early-onset severe retinal dystrophy, fundus flavimaculatus, and other macular degenerative diseases (
      • Maugeri A.
      • Klevering B.J.
      • Rohrschneider K.
      • Blankenagel A.
      • Brunner H.G.
      • Deutman A.F.
      • Hoyng C.B.
      • Cremers F.P.
      Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy.
      ,
      • Eksandh L.
      • Ekstrom U.
      • Abrahamson M.
      • Bauer B.
      • Andreasson S.
      Different clinical expressions in two families with Stargardt's macular dystrophy (STGD1).
      ,
      • Green J.S.
      • O'Rielly D.D.
      • Pater J.A.
      • Houston J.
      • Rajabi H.
      • Galutira D.
      • Benteau T.
      • Sheaves A.
      • Abdelfatah N.
      • Bautista D.
      • Whelan J.
      • Young T.L.
      The genetic architecture of Stargardt macular dystrophy (STGD1): A longitudinal 40-year study in a genetic isolate.
      ,
      • Michaelides M.
      • Hunt D.M.
      • Moore A.T.
      The genetics of inherited macular dystrophies.
      ,
      • Conley S.M.
      • Cai X.
      • Makkia R.
      • Wu Y.
      • Sparrow J.R.
      • Naash M.I.
      Increased cone sensitivity to ABCA4 deficiency provides insight into macular vision loss in Stargardt's dystrophy.
      ,
      • Rudolph G.
      • Kalpadakis P.
      • Haritoglou C.
      • Rivera A.
      • Weber B.H.
      ,
      • Hu F.
      • Gao F.
      • Li J.
      • Xu P.
      • Wang D.
      • Chen F.
      • Zhang S.
      • Wu J.
      Novel variants associated with Stargardt disease in Chinese patients.
      ,
      • Zhong M.
      • Molday L.L.
      • Molday R.S.
      Role of the C terminus of the photoreceptor ABCA4 transporter in protein folding, function, and retinal degenerative diseases.
      ,
      • Molday R.S.
      • Zhong M.
      • Quazi F.
      The role of the photoreceptor ABC transporter ABCA4 in lipid transport and Stargardt macular degeneration.
      ).
      In this review, however, we will focus on ELOVL4 mutations of the last decade and their related phenotypes and impacts on human health and disease in ELOVL4-expressing tissues.

      Different mutations in Elovl4 cause different tissue-specific disorders in humans

      Heterozygous ELOVL4 mutations affecting retinal structure and function

      The initial ELOVL4 mutations (Fig. 1) that cause blindness in patients with STGD3 were reported in exon 6 of ELOVL4 (
      • Zhang K.
      • Kniazeva M.
      • Han M.
      • Li W.
      • Yu Z.
      • Yang Z.
      • Li Y.
      • Metzker M.L.
      • Allikmets R.
      • Zack D.J.
      • Kakuk L.E.
      • Lagali P.S.
      • Wong P.W.
      • MacDonald I.M.
      • et al.
      A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.
      ,
      • Edwards A.O.
      • Donoso L.A.
      • Ritter 3rd., R.
      A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family.
      ,
      • Bernstein P.S.
      • Tammur J.
      • Singh N.
      • Hutchinson A.
      • Dixon M.
      • Pappas C.M.
      • Zabriskie N.A.
      • Zhang K.
      • Petrukhin K.
      • Leppert M.
      • Allikmets R.
      Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene.
      ,
      • Maugeri A.
      • Meire F.
      • Hoyng C.B.
      • Vink C.
      • Van Regemorter N.
      • Karan G.
      • Yang Z.
      • Cremers F.P.
      • Zhang K.
      A novel mutation in the ELOVL4 gene causes autosomal dominant Stargardt-like macular dystrophy.
      ). Although these mutations lead to a truncated ELOVL4 protein that lacks VLC-FA biosynthesis, in the last decade Bardak et al. (
      • Bardak H.
      • Gunay M.
      • Ercalik Y.
      • Bardak Y.
      • Ozbas H.
      • Bagci O.
      • Ayata A.
      • Sonmez M.
      • Alagoz C.
      Analysis of ELOVL4 and PRPH2 genes in Turkish Stargardt disease patients.
      ) reported two novel genetic base pair substitution mutations (c.814G > C and c.895A > G) in exon 6 as the cause of STGD3 (
      • Bardak H.
      • Gunay M.
      • Ercalik Y.
      • Bardak Y.
      • Ozbas H.
      • Bagci O.
      • Ayata A.
      • Sonmez M.
      • Alagoz C.
      Analysis of ELOVL4 and PRPH2 genes in Turkish Stargardt disease patients.
      ). Donato et al. (
      • Donato L.
      • Scimone C.
      • Rinaldi C.
      • Aragona P.
      • Briuglia S.
      • D'Ascola A.
      • D'Angelo R.
      • Sidoti A.
      Stargardt phenotype associated with two ELOVL4 promoter variants and ELOVL4 downregulation: New possible perspective to etiopathogenesis?.
      ) then reported two point mutations (c. −236C > and c. −90G > C) (Fig. 1) in the promoter region of ELOVL4 in a patient with the retinal phenotypic characteristics of patients with STGD3. In 2020, a sporadic novel missense in ELOVL4, c.59A > G (p.N20S) variant, was reported in a Chinese patient with clinical manifestations similar to those of STGD1 (
      • Hu F.
      • Gao F.
      • Li J.
      • Xu P.
      • Wang D.
      • Chen F.
      • Zhang S.
      • Wu J.
      Novel variants associated with Stargardt disease in Chinese patients.
      ). Of interest, apart from retinal degeneration, there are no reported brain and skin pathologies in patients with STGD3, as we previously reviewed (
      • Agbaga M.P.
      Different mutations in ELOVL4 affect very long chain fatty acid biosynthesis to cause variable neurological disorders in humans.
      ).
      Figure thumbnail gr1
      Fig. 1ELOVL4 (A) and its translated protein (B) showing the location of the various mutations (A-B). All STGD3 causing mutations (orange) in exon 6 lead to a truncated protein, resulting in the loss of the ER retention motif. The two mutations in the promoter region of ELOVL4 downregulate ELOVL4 expression based on luciferase activity and cause STGD3. Most of the SCA34 ELOVL4 (black) mutations cluster in the exon 4 region, except for an exon 6 mutation that produces a full-length protein with a single amino acid substitution. Homozygous mutations (red) produce an even more severe truncation of the protein, with an exon 1 mutation lacking the catalytic motif and ER retention motif. None of the mutations occur in the active region of ELOVL4, yet the STGD3 5 bp deletion mutant is enzymatically inactive due to loss of the ER targeting motif.
      The discovery by Donato et al. (
      • Donato L.
      • Scimone C.
      • Rinaldi C.
      • Aragona P.
      • Briuglia S.
      • D'Ascola A.
      • D'Angelo R.
      • Sidoti A.
      Stargardt phenotype associated with two ELOVL4 promoter variants and ELOVL4 downregulation: New possible perspective to etiopathogenesis?.
      ) that mutations in the ELOVL4 promoter cause STGD3 suggests that transcriptional regulation of ELOVL4 and its products may contribute to the quantity and quality of VLC-FA products synthesized by ELOVL4-expressing tissues, especially the retina, brain, and skin. Compared with the heterozygous mutations within the coding domain of ELOVL4 that cause a truncation or potentially altered protein structure and function, STGD3 pathology arising from single nucleotide polymorphisms (SNPs) in the promoter region of ELOVL4 further suggests that depletion of retinal VLC-FA due to mutant ELOVL4 activity could be the cause of retinal and other tissue-specific pathologies.

      Heterozygous ELOVL4 mutations affecting the brain cause age-related spinocerebellar ataxia with or without skin disorders

      In the last decade, a number of different heterozygous mutations in different exons of the ELOVL4 gene (Fig. 1) have been reported to cause age-related autosomal dominant spinocerebellar ataxia-34 (SCA34) with or without the skin condition erythrokeratodermia variabilis (
      • Cadieux-Dion M.
      • Turcotte-Gauthier M.
      • Noreau A.
      • Martin C.
      • Meloche C.
      • Gravel M.
      • Drouin C.A.
      • Rouleau G.A.
      • Nguyen D.K.
      • Cossette P.
      Expanding the clinical Phenotype associated with ELOVL4 mutation: Study of a Large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia.
      ,
      • Ozaki K.
      • Doi H.
      • Mitsui J.
      • Sato N.
      • Iikuni Y.
      • Majima T.
      • Yamane K.
      • Irioka T.
      • Ishiura H.
      • Doi K.
      • Morishita S.
      • Higashi M.
      • Sekiguchi T.
      • Koyama K.
      • Ueda N.
      • et al.
      A novel mutation in ELOVL4 leading to spinocerebellar ataxia (SCA) with the hot cross bun sign but lacking erythrokeratodermia: A broadened spectrum of SCA34.
      ,
      • Bourassa C.V.
      • Raskin S.
      • Serafini S.
      • Teive H.A.
      • Dion P.A.
      • Rouleau G.A.
      A New ELOVL4 mutation in a case of spinocerebellar ataxia with erythrokeratodermia.
      ,
      • Bourque P.R.
      • Warman-Chardon J.
      • Lelli D.A.
      • LaBerge L.
      • Kirshen C.
      • Bradshaw S.H.
      • Hartley T.
      • Boycott K.M.
      Novel ELOVL4 mutation associated with erythrokeratodermia and spinocerebellar ataxia (SCA 34).
      ,
      • Xiao C.
      • M. B. E
      • Rexach J.
      • Knight-Johnson A.
      • Khemani P.
      • Fogel B.L.
      • Das S.
      • Stone E.M.
      • Gomez C.M.
      A family with spinocerebellar ataxia and retinitis pigmentosa attributed to an ELOVL4 mutation.
      ,
      • Beaudin M.
      • Sellami L.
      • Martel C.
      • Touzel-Deschenes L.
      • Houle G.
      • Martineau L.
      • Lacroix K.
      • Lavallee A.
      • Chrestian N.
      • Rouleau G.A.
      • Gros-Louis F.
      • Laforce Jr., R.
      • Dupre N.
      Characterization of the phenotype with cognitive impairment and protein mislocalization in SCA34.
      ). The first known heterozygous ELOVL4 mutation to cause SCA34 and EKV was reported in a large French-Canadian family by Cadieux-Dion et al. in 2014 (
      • Cadieux-Dion M.
      • Turcotte-Gauthier M.
      • Noreau A.
      • Martin C.
      • Meloche C.
      • Gravel M.
      • Drouin C.A.
      • Rouleau G.A.
      • Nguyen D.K.
      • Cossette P.
      Expanding the clinical Phenotype associated with ELOVL4 mutation: Study of a Large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia.
      ). Affected family members carry a transversion mutation, c.540G > C (p.L168F), in exon 4 of ELOVL4, which segregates with a skin phenotype consisting of early-onset patches of erythema and hyperkeratosis with the ataxia pathology manifesting in the fourth or fifth decade of life (
      • Cadieux-Dion M.
      • Turcotte-Gauthier M.
      • Noreau A.
      • Martin C.
      • Meloche C.
      • Gravel M.
      • Drouin C.A.
      • Rouleau G.A.
      • Nguyen D.K.
      • Cossette P.
      Expanding the clinical Phenotype associated with ELOVL4 mutation: Study of a Large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia.
      ). Magnetic resonance imaging (MRI) of the brain of these patients revealed severe atrophy of the cerebellum and the pons (
      • Cadieux-Dion M.
      • Turcotte-Gauthier M.
      • Noreau A.
      • Martin C.
      • Meloche C.
      • Gravel M.
      • Drouin C.A.
      • Rouleau G.A.
      • Nguyen D.K.
      • Cossette P.
      Expanding the clinical Phenotype associated with ELOVL4 mutation: Study of a Large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia.
      ). Some affected individuals showed cerebellar hypometabolism, as determined by brain fluorodeoxyglucose positron emission tomography (
      • Cadieux-Dion M.
      • Turcotte-Gauthier M.
      • Noreau A.
      • Martin C.
      • Meloche C.
      • Gravel M.
      • Drouin C.A.
      • Rouleau G.A.
      • Nguyen D.K.
      • Cossette P.
      Expanding the clinical Phenotype associated with ELOVL4 mutation: Study of a Large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia.
      ).
      The following year, another SCA34-causing mutation, c.736T > G (p.W246G) in exon 6 of ELOVL4 (Fig. 1), was discovered in a Japanese family who also have selective degeneration of pontocerebellar tracts (hot cross bun sign) but did not have EKV like the French-Canadian patients (
      • Ozaki K.
      • Doi H.
      • Mitsui J.
      • Sato N.
      • Iikuni Y.
      • Majima T.
      • Yamane K.
      • Irioka T.
      • Ishiura H.
      • Doi K.
      • Morishita S.
      • Higashi M.
      • Sekiguchi T.
      • Koyama K.
      • Ueda N.
      • et al.
      A novel mutation in ELOVL4 leading to spinocerebellar ataxia (SCA) with the hot cross bun sign but lacking erythrokeratodermia: A broadened spectrum of SCA34.
      ). However, as in the French-Canadian family, the affected Japanese family members also showed age-related progressive ataxia, ocular movement disturbances, dysarthria, pyramidal tract signs, and pontocerebellar atrophy (
      • Ozaki K.
      • Doi H.
      • Mitsui J.
      • Sato N.
      • Iikuni Y.
      • Majima T.
      • Yamane K.
      • Irioka T.
      • Ishiura H.
      • Doi K.
      • Morishita S.
      • Higashi M.
      • Sekiguchi T.
      • Koyama K.
      • Ueda N.
      • et al.
      A novel mutation in ELOVL4 leading to spinocerebellar ataxia (SCA) with the hot cross bun sign but lacking erythrokeratodermia: A broadened spectrum of SCA34.
      ). The same year, Bourassa et al. (
      • Bourassa C.V.
      • Raskin S.
      • Serafini S.
      • Teive H.A.
      • Dion P.A.
      • Rouleau G.A.
      A New ELOVL4 mutation in a case of spinocerebellar ataxia with erythrokeratodermia.
      ) reported another heterozygous mutation, c.539A > C; p.G180P in exon 4 of ELOVL4, in a man in his thirties who developed progressive ataxia disorder starting in his mid-twenties and whose MRI showed cerebellar and pontine atrophy (
      • Bourassa C.V.
      • Raskin S.
      • Serafini S.
      • Teive H.A.
      • Dion P.A.
      • Rouleau G.A.
      A New ELOVL4 mutation in a case of spinocerebellar ataxia with erythrokeratodermia.
      ). Like the French-Canadian patients, this patient also had erythematous skin lesions on his forearms and legs during adolescence (
      • Bourassa C.V.
      • Raskin S.
      • Serafini S.
      • Teive H.A.
      • Dion P.A.
      • Rouleau G.A.
      A New ELOVL4 mutation in a case of spinocerebellar ataxia with erythrokeratodermia.
      ). These reports were followed by that of Bourque et al. of another novel heterozygous ELOVL4 mutation, c. 698C > T, p T233M in exon 6 of ELOVL4, that caused both EKV and SCA34 in an English-Canadian woman with clinical features similar to the L168F mutation reported in the 32 affected members of the French-Canadian family (
      • Cadieux-Dion M.
      • Turcotte-Gauthier M.
      • Noreau A.
      • Martin C.
      • Meloche C.
      • Gravel M.
      • Drouin C.A.
      • Rouleau G.A.
      • Nguyen D.K.
      • Cossette P.
      Expanding the clinical Phenotype associated with ELOVL4 mutation: Study of a Large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia.
      ,
      • Bourque P.R.
      • Warman-Chardon J.
      • Lelli D.A.
      • LaBerge L.
      • Kirshen C.
      • Bradshaw S.H.
      • Hartley T.
      • Boycott K.M.
      Novel ELOVL4 mutation associated with erythrokeratodermia and spinocerebellar ataxia (SCA 34).
      ). The patient reported noticing ataxia during her teenage years but had early-onset skin pathologies starting around the age of four that progressed to localized skin thickening (
      • Bourque P.R.
      • Warman-Chardon J.
      • Lelli D.A.
      • LaBerge L.
      • Kirshen C.
      • Bradshaw S.H.
      • Hartley T.
      • Boycott K.M.
      Novel ELOVL4 mutation associated with erythrokeratodermia and spinocerebellar ataxia (SCA 34).
      ). Although oculomotor symptoms were reported in the patient, detailed ophthalmological fundus examination of the macula was not reported (
      • Bourque P.R.
      • Warman-Chardon J.
      • Lelli D.A.
      • LaBerge L.
      • Kirshen C.
      • Bradshaw S.H.
      • Hartley T.
      • Boycott K.M.
      Novel ELOVL4 mutation associated with erythrokeratodermia and spinocerebellar ataxia (SCA 34).
      ). In 2019, another heterozygous ELOVL4 mutation, c.698C > T (p.T233M), was discovered in a patient with multisystem neurodegeneration, including ataxia and EKV skin lesions (
      • Ozaki K.
      • Ansai A.
      • Nobuhara K.
      • Araki T.
      • Kubodera T.
      • Ishii T.
      • Higashi M.
      • Sato N.
      • Soga K.
      • Mizusawa H.
      • Ishikawa K.
      • Yokota T.
      Prevalence and clinicoradiological features of spinocerebellar ataxia type 34 in a Japanese ataxia cohort.
      ). MRI of the brain of the patient and his father, who also presented with ataxia but no skin pathology, showed pontine and cerebellar atrophy as well as the hot cross bun sign as previously reported in patients with SCA34 with p. L198F and p. W246G ELOVL4 mutations (
      • Cadieux-Dion M.
      • Turcotte-Gauthier M.
      • Noreau A.
      • Martin C.
      • Meloche C.
      • Gravel M.
      • Drouin C.A.
      • Rouleau G.A.
      • Nguyen D.K.
      • Cossette P.
      Expanding the clinical Phenotype associated with ELOVL4 mutation: Study of a Large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia.
      ,
      • Ozaki K.
      • Doi H.
      • Mitsui J.
      • Sato N.
      • Iikuni Y.
      • Majima T.
      • Yamane K.
      • Irioka T.
      • Ishiura H.
      • Doi K.
      • Morishita S.
      • Higashi M.
      • Sekiguchi T.
      • Koyama K.
      • Ueda N.
      • et al.
      A novel mutation in ELOVL4 leading to spinocerebellar ataxia (SCA) with the hot cross bun sign but lacking erythrokeratodermia: A broadened spectrum of SCA34.
      ,
      • Ozaki K.
      • Ansai A.
      • Nobuhara K.
      • Araki T.
      • Kubodera T.
      • Ishii T.
      • Higashi M.
      • Sato N.
      • Soga K.
      • Mizusawa H.
      • Ishikawa K.
      • Yokota T.
      Prevalence and clinicoradiological features of spinocerebellar ataxia type 34 in a Japanese ataxia cohort.
      ). In 2020, Beaudin et al. (
      • Beaudin M.
      • Sellami L.
      • Martel C.
      • Touzel-Deschenes L.
      • Houle G.
      • Martineau L.
      • Lacroix K.
      • Lavallee A.
      • Chrestian N.
      • Rouleau G.A.
      • Gros-Louis F.
      • Laforce Jr., R.
      • Dupre N.
      Characterization of the phenotype with cognitive impairment and protein mislocalization in SCA34.
      ) reported further characterization of the C.504G > C p.L168F mutation previously reported by Cadiuex et al. (
      • Cadieux-Dion M.
      • Turcotte-Gauthier M.
      • Noreau A.
      • Martin C.
      • Meloche C.
      • Gravel M.
      • Drouin C.A.
      • Rouleau G.A.
      • Nguyen D.K.
      • Cossette P.
      Expanding the clinical Phenotype associated with ELOVL4 mutation: Study of a Large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia.
      ) in another French-Canadian family in which the mean age of ataxia onset was 47 years. Unlike the family reported by Cadiuex et al. (
      • Cadieux-Dion M.
      • Turcotte-Gauthier M.
      • Noreau A.
      • Martin C.
      • Meloche C.
      • Gravel M.
      • Drouin C.A.
      • Rouleau G.A.
      • Nguyen D.K.
      • Cossette P.
      Expanding the clinical Phenotype associated with ELOVL4 mutation: Study of a Large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia.
      ), none of the nine clinically affected family members reported by Beaudin et al. (
      • Beaudin M.
      • Sellami L.
      • Martel C.
      • Touzel-Deschenes L.
      • Houle G.
      • Martineau L.
      • Lacroix K.
      • Lavallee A.
      • Chrestian N.
      • Rouleau G.A.
      • Gros-Louis F.
      • Laforce Jr., R.
      • Dupre N.
      Characterization of the phenotype with cognitive impairment and protein mislocalization in SCA34.
      ) had EKV. Most of the affected members, however, exhibited horizontal nystagmus and saccadic pursuits, and one of the nine affected individuals had pisiform perimacular lesions (
      • Beaudin M.
      • Sellami L.
      • Martel C.
      • Touzel-Deschenes L.
      • Houle G.
      • Martineau L.
      • Lacroix K.
      • Lavallee A.
      • Chrestian N.
      • Rouleau G.A.
      • Gros-Louis F.
      • Laforce Jr., R.
      • Dupre N.
      Characterization of the phenotype with cognitive impairment and protein mislocalization in SCA34.
      ). Further clinical characterization of the patients revealed cerebellar and pontine atrophy (four of six patients) and cruciform hypersignal in the pons (two of six patients). Fluorodeoxyglucose-positron emission tomography imaging revealed diffuse cerebellar hypometabolism in all five tested patients, with subtle parietal hypometabolism in three of them. The patients also exhibited cognitive deficits in executive functioning that were statistically significantly different from age- and education-matched controls (
      • Beaudin M.
      • Sellami L.
      • Martel C.
      • Touzel-Deschenes L.
      • Houle G.
      • Martineau L.
      • Lacroix K.
      • Lavallee A.
      • Chrestian N.
      • Rouleau G.A.
      • Gros-Louis F.
      • Laforce Jr., R.
      • Dupre N.
      Characterization of the phenotype with cognitive impairment and protein mislocalization in SCA34.
      ).
      As the list of heterozygous SCA34 ELOVL4 mutations grew (Table 1), in 2019 another ELOVL4 mutation, c.512T > C, p.I171T in exon 4 of ELOVL4, (Fig. 1) was reported in a family that presented with both SCA34 and retinal dystrophy characteristic of ELOVL4 mutations that cause STGD3 (
      • Xiao C.
      • M. B. E
      • Rexach J.
      • Knight-Johnson A.
      • Khemani P.
      • Fogel B.L.
      • Das S.
      • Stone E.M.
      • Gomez C.M.
      A family with spinocerebellar ataxia and retinitis pigmentosa attributed to an ELOVL4 mutation.
      ). Ophthalmologic evaluation showed that four of the eight individuals in the family had retinal abnormalities consistent with retinitis pigmentosa (
      • Xiao C.
      • M. B. E
      • Rexach J.
      • Knight-Johnson A.
      • Khemani P.
      • Fogel B.L.
      • Das S.
      • Stone E.M.
      • Gomez C.M.
      A family with spinocerebellar ataxia and retinitis pigmentosa attributed to an ELOVL4 mutation.
      ). This was the first reported case where an individual with SCA34 pathology also presented with a retinal deficit from the ELOVL4 mutation (
      • Xiao C.
      • M. B. E
      • Rexach J.
      • Knight-Johnson A.
      • Khemani P.
      • Fogel B.L.
      • Das S.
      • Stone E.M.
      • Gomez C.M.
      A family with spinocerebellar ataxia and retinitis pigmentosa attributed to an ELOVL4 mutation.
      ). It is also the first reported case where an ELOVL4 mutation was linked to retinitis pigmentosa. Thus, even with the SCA34-causing ELOVL4 mutations, there are tissue-specific variabilities in the phenotypes reported. Of more importance, there are differences in the time of disease onset and rate of progression of the different tissue-specific disorders caused by different ELOVL4 mutations. Some patients with STGD3 seem to have early-onset vison loss (
      • Zhang K.
      • Kniazeva M.
      • Han M.
      • Li W.
      • Yu Z.
      • Yang Z.
      • Li Y.
      • Metzker M.L.
      • Allikmets R.
      • Zack D.J.
      • Kakuk L.E.
      • Lagali P.S.
      • Wong P.W.
      • MacDonald I.M.
      • et al.
      A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.
      ,
      • Edwards A.O.
      • Donoso L.A.
      • Ritter 3rd., R.
      A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family.
      ,
      • Bernstein P.S.
      • Tammur J.
      • Singh N.
      • Hutchinson A.
      • Dixon M.
      • Pappas C.M.
      • Zabriskie N.A.
      • Zhang K.
      • Petrukhin K.
      • Leppert M.
      • Allikmets R.
      Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene.
      ,
      • Maugeri A.
      • Meire F.
      • Hoyng C.B.
      • Vink C.
      • Van Regemorter N.
      • Karan G.
      • Yang Z.
      • Cremers F.P.
      • Zhang K.
      A novel mutation in the ELOVL4 gene causes autosomal dominant Stargardt-like macular dystrophy.
      ,
      • Donoso L.A.
      • Edwards A.O.
      • Frost A.
      • Vrabec T.
      • Stone E.M.
      • Hageman G.S.
      • Perski T.
      Autosomal dominant Stargardt-like macular dystrophy.
      ,
      • Donato L.
      • Scimone C.
      • Rinaldi C.
      • Aragona P.
      • Briuglia S.
      • D'Ascola A.
      • D'Angelo R.
      • Sidoti A.
      Stargardt phenotype associated with two ELOVL4 promoter variants and ELOVL4 downregulation: New possible perspective to etiopathogenesis?.
      ,
      • Donoso L.A.
      • Frost A.T.
      • Stone E.M.
      • Weleber R.G.
      • MacDonald I.M.
      • Hageman G.S.
      • Cibis G.W.
      • Ritter 3rd, R.
      • Edwards A.O.
      Autosomal dominant Stargardt-like macular dystrophy: Founder effect and reassessment of genetic heterogeneity.
      ), whereas patients with SCA34 with or without EKV develop relatively slow progression of ataxia, with average onset in adulthood (
      • Cadieux-Dion M.
      • Turcotte-Gauthier M.
      • Noreau A.
      • Martin C.
      • Meloche C.
      • Gravel M.
      • Drouin C.A.
      • Rouleau G.A.
      • Nguyen D.K.
      • Cossette P.
      Expanding the clinical Phenotype associated with ELOVL4 mutation: Study of a Large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia.
      ,
      • Ozaki K.
      • Doi H.
      • Mitsui J.
      • Sato N.
      • Iikuni Y.
      • Majima T.
      • Yamane K.
      • Irioka T.
      • Ishiura H.
      • Doi K.
      • Morishita S.
      • Higashi M.
      • Sekiguchi T.
      • Koyama K.
      • Ueda N.
      • et al.
      A novel mutation in ELOVL4 leading to spinocerebellar ataxia (SCA) with the hot cross bun sign but lacking erythrokeratodermia: A broadened spectrum of SCA34.
      ,
      • Bourassa C.V.
      • Raskin S.
      • Serafini S.
      • Teive H.A.
      • Dion P.A.
      • Rouleau G.A.
      A New ELOVL4 mutation in a case of spinocerebellar ataxia with erythrokeratodermia.
      ,
      • Bourque P.R.
      • Warman-Chardon J.
      • Lelli D.A.
      • LaBerge L.
      • Kirshen C.
      • Bradshaw S.H.
      • Hartley T.
      • Boycott K.M.
      Novel ELOVL4 mutation associated with erythrokeratodermia and spinocerebellar ataxia (SCA 34).
      ,
      • Xiao C.
      • M. B. E
      • Rexach J.
      • Knight-Johnson A.
      • Khemani P.
      • Fogel B.L.
      • Das S.
      • Stone E.M.
      • Gomez C.M.
      A family with spinocerebellar ataxia and retinitis pigmentosa attributed to an ELOVL4 mutation.
      ,
      • Beaudin M.
      • Sellami L.
      • Martel C.
      • Touzel-Deschenes L.
      • Houle G.
      • Martineau L.
      • Lacroix K.
      • Lavallee A.
      • Chrestian N.
      • Rouleau G.A.
      • Gros-Louis F.
      • Laforce Jr., R.
      • Dupre N.
      Characterization of the phenotype with cognitive impairment and protein mislocalization in SCA34.
      ,
      • Ozaki K.
      • Ansai A.
      • Nobuhara K.
      • Araki T.
      • Kubodera T.
      • Ishii T.
      • Higashi M.
      • Sato N.
      • Soga K.
      • Mizusawa H.
      • Ishikawa K.
      • Yokota T.
      Prevalence and clinicoradiological features of spinocerebellar ataxia type 34 in a Japanese ataxia cohort.
      ).

      Homozygous ELOVL4 mutations cause severe intellectual disability, seizures, and early childhood death

      To date, three different homozygous mutations in ELOVL4 have been reported to cause very severe pathologies that affect the skin and brain, resulting in early childhood mortality (
      • Aldahmesh M.A.
      • Mohamed J.Y.
      • Alkuraya H.S.
      • Verma I.C.
      • Puri R.D.
      • Alaiya A.A.
      • Rizzo W.B.
      • Alkuraya F.S.
      Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.
      ,
      • Mir H.
      • Raza S.I.
      • Touseef M.
      • Memon M.M.
      • Khan M.N.
      • Jaffar S.
      • Ahmad W.
      A novel recessive mutation in the gene ELOVL4 causes a neuro-ichthyotic disorder with variable expressivity.
      ). Overall, inheritance of the homozygous ELOVL4 mutations, c.78C > G; p.Tyr26∗, and c.646C > T, p.Arg216X (
      • Mir H.
      • Raza S.I.
      • Touseef M.
      • Memon M.M.
      • Khan M.N.
      • Jaffar S.
      • Ahmad W.
      A novel recessive mutation in the gene ELOVL4 causes a neuro-ichthyotic disorder with variable expressivity.
      ) in exon 5, and c.690del p.Ile230Metfs∗22 in exon 6 of ELOVL4 (
      • Aldahmesh M.A.
      • Mohamed J.Y.
      • Alkuraya H.S.
      • Verma I.C.
      • Puri R.D.
      • Alaiya A.A.
      • Rizzo W.B.
      • Alkuraya F.S.
      Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.
      ), causes dry, scaly, and thickened skin disorders (ichthyosis), intellectual disability, seizures, hypertonia, and premature death (
      • Aldahmesh M.A.
      • Mohamed J.Y.
      • Alkuraya H.S.
      • Verma I.C.
      • Puri R.D.
      • Alaiya A.A.
      • Rizzo W.B.
      • Alkuraya F.S.
      Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.
      ,
      • Mir H.
      • Raza S.I.
      • Touseef M.
      • Memon M.M.
      • Khan M.N.
      • Jaffar S.
      • Ahmad W.
      A novel recessive mutation in the gene ELOVL4 causes a neuro-ichthyotic disorder with variable expressivity.
      ). An important characteristic of the neuroichthyotic disorders in these patients is that it shares clinical features resembling Sjogren-Larsson syndrome (MIM 270200) (
      • Aldahmesh M.A.
      • Mohamed J.Y.
      • Alkuraya H.S.
      • Verma I.C.
      • Puri R.D.
      • Alaiya A.A.
      • Rizzo W.B.
      • Alkuraya F.S.
      Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.
      ). These findings suggest that there are potentially other novel ELOVL4 mutations that have not yet been reported because the patients may have been assigned to other clinical categories based on their phenotype. We therefore speculate that new ELOVL4 mutation variants will be discovered and reported.
      Consistent with the human pathologies, mice that are homozygous for the STDG3 alleles or homozygous knockout of mouse Elovl4 die at birth (
      • McMahon A.
      • Butovich I.A.
      • Mata N.L.
      • Klein M.
      • Ritter 3rd, R.
      • Richardson J.
      • Birch D.G.
      • Edwards A.O.
      • Kedzierski W.
      Retinal pathology and skin barrier defect in mice carrying a Stargardt disease-3 mutation in elongase of very long chain fatty acids-4.
      ,
      • Vasireddy V.
      • Uchida Y.
      • Salem Jr., N.
      • Kim S.Y.
      • Mandal M.N.
      • Reddy G.B.
      • Bodepudi R.
      • Alderson N.L.
      • Brown J.C.
      • Hama H.
      • Dlugosz A.
      • Elias P.M.
      • Holleran W.M.
      • Ayyagari R.
      Loss of functional ELOVL4 depletes very long-chain fatty acids (> or =C28) and the unique omega-O-acylceramides in skin leading to neonatal death.
      ,
      • Cameron D.J.
      • Tong Z.
      • Yang Z.
      • Kaminoh J.
      • Kamiyah S.
      • Chen H.
      • Zeng J.
      • Chen Y.
      • Luo L.
      • Zhang K.
      Essential role of Elovl4 in very long chain fatty acid synthesis, skin permeability barrier function, and neonatal survival.
      ,
      • Li W.
      • Sandhoff R.
      • Kono M.
      • Zerfas P.
      • Hoffmann V.
      • Ding B.C.
      • Proia R.L.
      • Deng C.X.
      Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice.
      ). Transgenic expression of wild-type ELOVL4 in the skin using skin-specific promoters rescued these mice from neonatal lethality (
      • McMahon A.
      • Butovich I.A.
      • Kedzierski W.
      Epidermal expression of an Elovl4 transgene rescues neonatal lethality of homozygous Stargardt disease-3 mice.
      ,
      • Hopiavuori B.R.
      • Deak F.
      • Wilkerson J.L.
      • Brush R.S.
      • Rocha-Hopiavuori N.A.
      • Hopiavuori A.R.
      • Ozan K.G.
      • Sullivan M.T.
      • Wren J.D.
      • Georgescu C.
      • Szweda L.
      • Awasthi V.
      • Towner R.
      • Sherry D.M.
      • Anderson R.E.
      • et al.
      Homozygous expression of mutant ELOVL4 leads to seizures and death in a novel animal model of very long-chain fatty acid deficiency.
      ). However, the skin-rescued mice develop seizure-like phenotypes similar to the symptoms seen in humans homozygous for ELOVL4 and die by postnatal day 21 owing to the lack of brain ELOVL4 VLC-FA products (
      • Hopiavuori B.R.
      • Deak F.
      • Wilkerson J.L.
      • Brush R.S.
      • Rocha-Hopiavuori N.A.
      • Hopiavuori A.R.
      • Ozan K.G.
      • Sullivan M.T.
      • Wren J.D.
      • Georgescu C.
      • Szweda L.
      • Awasthi V.
      • Towner R.
      • Sherry D.M.
      • Anderson R.E.
      • et al.
      Homozygous expression of mutant ELOVL4 leads to seizures and death in a novel animal model of very long-chain fatty acid deficiency.
      ), which supports the essential role of ELOVL4-synthesized lipids in health and disease. How the different mutations in ELOVL4 cause such different tissue-specific phenotypes and rates of progression of disease is a puzzling question that remains to be answered.

      Deciphering the mutant ELOVL4 disease pathology by understanding the structure and tissue distribution of ELOVL4

      There are seven members of the fatty acid elongase (ELOVL1-7) family that work in collaboration with other fatty acid biosynthetic enzymes to elongate specific chain lengths of fatty acids in a tissue-specific manner (Fig. 2) (
      • Hopiavuori B.R.
      • Anderson R.E.
      • Agbaga M.P.
      ELOVL4: Very long-chain fatty acids serve an eclectic role in mammalian health and function.
      ,
      • Jakobsson A.
      • Westerberg R.
      • Jacobsson A.
      Fatty acid elongases in mammals: Their regulation and roles in metabolism.
      ,
      • Guillou H.
      • Zadravec D.
      • Martin P.G.
      • Jacobsson A.
      The key roles of elongases and desaturases in mammalian fatty acid metabolism: Insights from transgenic mice.
      ,
      • Ohno Y.
      • Suto S.
      • Yamanaka M.
      • Mizutani Y.
      • Mitsutake S.
      • Igarashi Y.
      • Sassa T.
      • Kihara A.
      ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis.
      ,
      • Bedell M.
      • Harkewicz R.
      • Wang X.
      • Zhang K.
      Focus on molecules: ELOVL4.
      ,
      • Jump D.B.
      Mammalian fatty acid elongases.
      ,
      • Okuda A.
      • Naganuma T.
      • Ohno Y.
      • Abe K.
      • Yamagata M.
      • Igarashi Y.
      • Kihara A.
      Hetero-oligomeric interactions of an ELOVL4 mutant protein: Implications in the molecular mechanism of Stargardt-3 macular dystrophy.
      ). These enzymes are transmembrane resident proteins of the endoplasmic reticulum (ER) within which they elongate both saturated and unsaturated fatty acids with specificity toward a particular fatty acid chain length (
      • Denic V.
      • Weissman J.S.
      A molecular caliper mechanism for determining very long-chain fatty acid length.
      ,
      • Riezman H.
      The long and short of fatty acid synthesis.
      ). ELOVL4 specifically synthesizes both VLC-SFA and VLC-PUFA from C26 fatty acids (Fig. 2A, B) (
      • Agbaga M.P.
      • Brush R.S.
      • Mandal M.N.
      • Henry K.
      • Elliott M.H.
      • Anderson R.E.
      Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.
      ,
      • Yu M.
      • Benham A.
      • Logan S.
      • Brush R.S.
      • Mandal M.N.
      • Anderson R.E.
      • Agbaga M.P.
      ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3.
      ). These enzymes contain three characteristic motifs, namely, (1) an N-glycosylation consensus motif at the N terminus, (2) a catalytic histidine core (HXXHH), and (3) an ER retention/retrieval motif (KXKXX) located at the C terminus. Site-specific mutations within these motifs in cell culture experiments established that the activity of ELOVL enzymes is dependent on an intact catalytic histidine core and the ER retention/retrieval motif (
      • Logan S.
      • Agbaga M.P.
      • Chan M.D.
      • Brush R.S.
      • Anderson R.E.
      Endoplasmic reticulum microenvironment and conserved histidines govern ELOVL4 fatty acid elongase activity.
      ,
      • Logan S.
      • Agbaga M.P.
      • Chan M.D.
      • Kabir N.
      • Mandal N.A.
      • Brush R.S.
      • Anderson R.E.
      Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy.
      ,
      • Logan S.
      • Anderson R.E.
      Dominant Stargardt Macular Dystrophy (STGD3) and ELOVL4.
      ). Despite the ability of ELOVL4 to elongate both PUFA and SFA, the quantitative and qualitative distribution of VLC-PUFA and VLC-SFA varies in the different ELOVL4-expressing tissues. In terms of its tissue distribution, our current knowledge is that ELOVL4 is expressed in the retina, Meibomian glands, brain, skin, and testes (
      • Zhang K.
      • Kniazeva M.
      • Han M.
      • Li W.
      • Yu Z.
      • Yang Z.
      • Li Y.
      • Metzker M.L.
      • Allikmets R.
      • Zack D.J.
      • Kakuk L.E.
      • Lagali P.S.
      • Wong P.W.
      • MacDonald I.M.
      • et al.
      A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.
      ,
      • Agbaga M.P.
      • Brush R.S.
      • Mandal M.N.
      • Henry K.
      • Elliott M.H.
      • Anderson R.E.
      Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.
      ,
      • Mandal M.N.
      • Ambasudhan R.
      • Wong P.W.
      • Gage P.J.
      • Sieving P.A.
      • Ayyagari R.
      Characterization of mouse orthologue of ELOVL4: Genomic organization and spatial and temporal expression.
      ,
      • McMahon A.
      • Lu H.
      • Butovich I.A.
      A role for ELOVL4 in the mouse Meibomian gland and sebocyte cell biology.
      ). However, there is the probability that, as we continue to study the ELOVL4 protein, with time, we may discover other tissues that express it under specific conditions. In the eye, the highest ELOVL4 expression is seen in the rod and cone photoreceptor inner segments and the outer nuclear layer, where VLC-PUFA are the main ELOVL4 products that are selectively incorporated into the sn-1 position in phosphatidylcholine (PC) with DHA esterified at the sn-2 position (
      • Grayson C.
      • Molday R.S.
      Dominant negative mechanism underlies autosomal dominant Stargardt-like macular dystrophy linked to mutations in ELOVL4.
      ,
      • Agbaga M.P.
      • Brush R.S.
      • Mandal M.N.
      • Henry K.
      • Elliott M.H.
      • Anderson R.E.
      Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.
      ,
      • Mandal M.N.
      • Ambasudhan R.
      • Wong P.W.
      • Gage P.J.
      • Sieving P.A.
      • Ayyagari R.
      Characterization of mouse orthologue of ELOVL4: Genomic organization and spatial and temporal expression.
      ,
      • Aveldano M.I.
      A novel group of very long chain polyenoic fatty acids in dipolyunsaturated phosphatidylcholines from vertebrate retina.
      ,
      • Aveldano M.I.
      • Sprecher H.
      Very long chain (C24 to C36) polyenoic fatty acids of the n-3 and n-6 series in dipolyunsaturated phosphatidylcholines from bovine retina.
      ,
      • Aveldano M.I.
      Phospholipid species containing long and very long polyenoic fatty acids remain with rhodopsin after hexane extraction of photoreceptor membranes.
      ,
      • Lagali P.S.
      • Liu J.
      • Ambasudhan R.
      • Kakuk L.E.
      • Bernstein S.L.
      • Seigel G.M.
      • Wong P.W.
      • Ayyagari R.
      Evolutionarily conserved ELOVL4 gene expression in the vertebrate retina.
      ,
      • Umeda S.
      • Ayyagari R.
      • Suzuki M.T.
      • Ono F.
      • Iwata F.
      • Fujiki K.
      • Kanai A.
      • Takada Y.
      • Yoshikawa Y.
      • Tanaka Y.
      • Iwata T.
      Molecular cloning of ELOVL4 gene from cynomolgus monkey (Macaca fascicularis).
      ). Although VLC-PUFA are the main products of ELOVL4 in the retina and testes in which they are incorporated into PC and sphingolipids, respectively, VLC-SFA are predominantly synthesized in the skin, brain, and Meibomian glands and incorporated into sphingolipids (Fig. 2D, E). Even within the retina, VLC-PUFA are incorporated mainly into PC lipid species (Fig. 2C), whereas in the testes and sperm, VLC-PUFA are incorporated into sphingolipids suggesting that different tissues have different specific needs for ELOVL4 products. Also, in the eye, ELOVL4 is expressed within the Meibomian glands, which produce a lipid mixture of meibum that mixes with aqueous tears produced by the lachrymal glands to form the tear film that covers the cornea (
      • McMahon A.
      • Lu H.
      • Butovich I.A.
      A role for ELOVL4 in the mouse Meibomian gland and sebocyte cell biology.
      ). This lipid mixture contains VLC-SFA produced by ELOVL4 that are incorporated into (O-acyl)-ω-hydroxy FA (
      • Butovich I.A.
      On the lipid composition of human meibum and tears: Comparative analysis of nonpolar lipids.
      ,
      • Butovich I.A.
      Lipidomic analysis of human meibum using HPLC-MSn.
      ,
      • Butovich I.A.
      The Meibomian puzzle: Combining pieces together.
      ,
      • Butovich I.A.
      Cholesteryl esters as a depot for very long chain fatty acids in human meibum.
      ,
      • Butovich I.A.
      • Uchiyama E.
      • McCulley J.P.
      Lipids of human meibum: Mass-spectrometric analysis and structural elucidation.
      ,
      • Butovich I.A.
      • Wojtowicz J.C.
      • Molai M.
      Human tear film and meibum. Very long chain wax esters and (O-acyl)-omega-hydroxy fatty acids of meibum.
      ). Reduced VLC-SFA relative to other lipids may alter the rate of evaporation of the tear film. Of significance, changes in the quality and quantity of the lipids secreted by the Meibomian glands are believed to be an underlying cause of the evaporative form of dry eye disease (
      • McMahon A.
      • Lu H.
      • Butovich I.A.
      A role for ELOVL4 in the mouse Meibomian gland and sebocyte cell biology.
      ,
      • Joffre C.
      • Souchier M.
      • Gregoire S.
      • Viau S.
      • Bretillon L.
      • Acar N.
      • Bron A.M.
      • Creuzot-Garcher C.
      Differences in meibomian fatty acid composition in patients with meibomian gland dysfunction and aqueous-deficient dry eye.
      ). Meibum is believed to be excreted spontaneously or when blinking. It is interesting that STGD3 5 bp deletion heterozygous mice, likely having reduced VLC-SFA in their tear film, exhibited increased blinking of the eyelids and an aversion to keeping their eyes fully open and showed an evaporative type of dry eye disease phenotype (
      • McMahon A.
      • Lu H.
      • Butovich I.A.
      A role for ELOVL4 in the mouse Meibomian gland and sebocyte cell biology.
      ). This supports the important role of ELOVL4-synthesized VLC-SFA in ocular health and the prevention of rapid evaporation of the eye's tear film, which needs further studies to unravel the role of ELOVL4 in age-related dry eye diseases.
      Figure thumbnail gr2
      Fig. 2Biosynthesis of VLC-PUFA and VLC-SFA. A: Schematic in vivo biosynthetic pathway from 18:3n3 and 18:2n6 mediated by ELOVL4 and other ELOVL family proteins. Desaturase and elongation steps are consecutively performed by fatty acid desaturase-1 (FADS1 or Δ5 desaturase), fatty acid desaturase-2 (FADS2 or Δ6 desaturase), and ELOVL1-5. Although some elongases are specific for a single step, others are nonspecific or multifunctional and act at several steps (e.g., human ELOVL5 and murine ELOVL2). Panel A is an adapted reproduction from Man Yu et al. (
      • Yu M.
      • Benham A.
      • Logan S.
      • Brush R.S.
      • Mandal M.N.
      • Anderson R.E.
      • Agbaga M.P.
      ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3.
      ). ©2012 by the American Society for Biochemistry and Molecular Biology, Inc. B: VLC-SFA biosynthesis pathway. Elongation steps from 18:0 to VLC-SFA by the different ELOVLs. C: Example of VLC-PUFA esterification in the retina: phosphatidylcholine containing the VLC-PUFA, 34:5n3 and the LC-PUFA, 22:6n3 (DHA). D: Example of VLC-SFA amidification in the skin: ω-O-acylceramide containing the VLC-SFA, 28:0 ω-O-linked with 18:2n6. E: Example of VLC-SFA amidification in the brain: sphingomyelin containing the VLC-SFA, 30:0. Panels C–E, adapted from Hopiavuori et al. (
      • Hopiavuori B.R.
      • Anderson R.E.
      • Agbaga M.P.
      ELOVL4: Very long-chain fatty acids serve an eclectic role in mammalian health and function.
      ) used with permission.
      In the brain, the ELOVL4 products are essential for normal brain function such that depletion of brain ELOVL4 leads to seizures and premature death in rodents and humans (
      • Aldahmesh M.A.
      • Mohamed J.Y.
      • Alkuraya H.S.
      • Verma I.C.
      • Puri R.D.
      • Alaiya A.A.
      • Rizzo W.B.
      • Alkuraya F.S.
      Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.
      ,
      • Mir H.
      • Raza S.I.
      • Touseef M.
      • Memon M.M.
      • Khan M.N.
      • Jaffar S.
      • Ahmad W.
      A novel recessive mutation in the gene ELOVL4 causes a neuro-ichthyotic disorder with variable expressivity.
      ,
      • Hopiavuori B.R.
      • Deak F.
      • Wilkerson J.L.
      • Brush R.S.
      • Rocha-Hopiavuori N.A.
      • Hopiavuori A.R.
      • Ozan K.G.
      • Sullivan M.T.
      • Wren J.D.
      • Georgescu C.
      • Szweda L.
      • Awasthi V.
      • Towner R.
      • Sherry D.M.
      • Anderson R.E.
      • et al.
      Homozygous expression of mutant ELOVL4 leads to seizures and death in a novel animal model of very long-chain fatty acid deficiency.
      ). We have shown that ELOVL4 is expressed in neurons in the brain, with the most abundant expression in most neurons by embryonic day 18 in mice (
      • Sherry D.M.
      • Hopiavuori B.R.
      • Stiles M.A.
      • Rahman N.S.
      • Ozan K.G.
      • Deak F.
      • Agbaga M.P.
      • Anderson R.E.
      Distribution of ELOVL4 in the developing and adult mouse brain.
      ). We detected 28:0 and 30:0 VLC-SFA, which are incorporated into complex sphingolipids and enriched in synaptic vesicle preparations from baboon brains (
      • Hopiavuori B.R.
      • Deak F.
      • Wilkerson J.L.
      • Brush R.S.
      • Rocha-Hopiavuori N.A.
      • Hopiavuori A.R.
      • Ozan K.G.
      • Sullivan M.T.
      • Wren J.D.
      • Georgescu C.
      • Szweda L.
      • Awasthi V.
      • Towner R.
      • Sherry D.M.
      • Anderson R.E.
      • et al.
      Homozygous expression of mutant ELOVL4 leads to seizures and death in a novel animal model of very long-chain fatty acid deficiency.
      ). We showed increased synaptic release kinetics in the hippocampal neurons from homozygous Elovl4Stgd3/Stgd3 mice compared with neurons from wild-type littermates (
      • Hopiavuori B.R.
      • Deak F.
      • Wilkerson J.L.
      • Brush R.S.
      • Rocha-Hopiavuori N.A.
      • Hopiavuori A.R.
      • Ozan K.G.
      • Sullivan M.T.
      • Wren J.D.
      • Georgescu C.
      • Szweda L.
      • Awasthi V.
      • Towner R.
      • Sherry D.M.
      • Anderson R.E.
      • et al.
      Homozygous expression of mutant ELOVL4 leads to seizures and death in a novel animal model of very long-chain fatty acid deficiency.
      ). Supplementing the primary neurons from the Elovl4 homozygous mice with 28:0 and 30:0 rescued the synaptic release rates to wild-type levels (
      • Hopiavuori B.R.
      • Deak F.
      • Wilkerson J.L.
      • Brush R.S.
      • Rocha-Hopiavuori N.A.
      • Hopiavuori A.R.
      • Ozan K.G.
      • Sullivan M.T.
      • Wren J.D.
      • Georgescu C.
      • Szweda L.
      • Awasthi V.
      • Towner R.
      • Sherry D.M.
      • Anderson R.E.
      • et al.
      Homozygous expression of mutant ELOVL4 leads to seizures and death in a novel animal model of very long-chain fatty acid deficiency.
      ). Thus, brain VLC-SFA seem to modulate presynaptic transmitter release kinetics that requires further scientific investigation.
      The expression of ELOVL4 in the skin is important for biosynthesis of VLC-SFA that are essential for skin barrier function and overall survival (
      • Vasireddy V.
      • Uchida Y.
      • Salem Jr., N.
      • Kim S.Y.
      • Mandal M.N.
      • Reddy G.B.
      • Bodepudi R.
      • Alderson N.L.
      • Brown J.C.
      • Hama H.
      • Dlugosz A.
      • Elias P.M.
      • Holleran W.M.
      • Ayyagari R.
      Loss of functional ELOVL4 depletes very long-chain fatty acids (> or =C28) and the unique omega-O-acylceramides in skin leading to neonatal death.
      ,
      • Uchida Y.
      • Holleran W.M.
      Omega-O-acylceramide, a lipid essential for mammalian survival.
      ). The essential role of VLC-SFA for skin function and survival is unequivocally demonstrated in homozygous STGD3 knockin mice and homozygous global Elovl4 knockout mice that die at birth (
      • McMahon A.
      • Butovich I.A.
      • Mata N.L.
      • Klein M.
      • Ritter 3rd, R.
      • Richardson J.
      • Birch D.G.
      • Edwards A.O.
      • Kedzierski W.
      Retinal pathology and skin barrier defect in mice carrying a Stargardt disease-3 mutation in elongase of very long chain fatty acids-4.
      ,
      • Vasireddy V.
      • Uchida Y.
      • Salem Jr., N.
      • Kim S.Y.
      • Mandal M.N.
      • Reddy G.B.
      • Bodepudi R.
      • Alderson N.L.
      • Brown J.C.
      • Hama H.
      • Dlugosz A.
      • Elias P.M.
      • Holleran W.M.
      • Ayyagari R.
      Loss of functional ELOVL4 depletes very long-chain fatty acids (> or =C28) and the unique omega-O-acylceramides in skin leading to neonatal death.
      ,
      • Cameron D.J.
      • Tong Z.
      • Yang Z.
      • Kaminoh J.
      • Kamiyah S.
      • Chen H.
      • Zeng J.
      • Chen Y.
      • Luo L.
      • Zhang K.
      Essential role of Elovl4 in very long chain fatty acid synthesis, skin permeability barrier function, and neonatal survival.
      ,
      • Li W.
      • Sandhoff R.
      • Kono M.
      • Zerfas P.
      • Hoffmann V.
      • Ding B.C.
      • Proia R.L.
      • Deng C.X.
      Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice.
      ) and in humans with homozygous ELOVL4 mutations who die within the first decade of life from a number of neurological and skin disorders (
      • Aldahmesh M.A.
      • Mohamed J.Y.
      • Alkuraya H.S.
      • Verma I.C.
      • Puri R.D.
      • Alaiya A.A.
      • Rizzo W.B.
      • Alkuraya F.S.
      Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.
      ,
      • Mir H.
      • Raza S.I.
      • Touseef M.
      • Memon M.M.
      • Khan M.N.
      • Jaffar S.
      • Ahmad W.
      A novel recessive mutation in the gene ELOVL4 causes a neuro-ichthyotic disorder with variable expressivity.
      ). We and others have established the essential role of ELOVL4 and its VLC-FA products for life by demonstrating that the expression of ELOVL4 in the skin rescues the skin defects that cause neonatal lethality in homozygous STGD3 knockin mice (
      • McMahon A.
      • Butovich I.A.
      • Kedzierski W.
      Epidermal expression of an Elovl4 transgene rescues neonatal lethality of homozygous Stargardt disease-3 mice.
      ,
      • Hopiavuori B.R.
      • Deak F.
      • Wilkerson J.L.
      • Brush R.S.
      • Rocha-Hopiavuori N.A.
      • Hopiavuori A.R.
      • Ozan K.G.
      • Sullivan M.T.
      • Wren J.D.
      • Georgescu C.
      • Szweda L.
      • Awasthi V.
      • Towner R.
      • Sherry D.M.
      • Anderson R.E.
      • et al.
      Homozygous expression of mutant ELOVL4 leads to seizures and death in a novel animal model of very long-chain fatty acid deficiency.
      ).

      What factors determine the different tissue-specific disorders caused by different ELOVL4 mutations?

      The tissue-specific biosynthesis of VLC-PUFA relative to VLC-SFA in different ELOVL4-expressing tissues is likely determined by tissue-specific factors, such as availability of substrates and/or proteins that interact with ELOVL4. In the retina, the biosynthesis of VLC-PUFA is likely due to the higher presence of PUFA substrates, whereas in the skin and Meibomian glands, the preference toward VLC-SFA biosynthesis is probably due to the lack of PUFA precursors and presence of VLC-SFA precursors (Fig. 2A, B). DHA and arachidonic acid (20:4n6) (AA) are the predominant PUFA in the retina and brain (
      • Kitajka K.
      • Sinclair A.J.
      • Weisinger R.S.
      • Weisinger H.S.
      • Mathai M.
      • Jayasooriya A.P.
      • Halver J.E.
      • Puskas L.G.
      Effects of dietary omega-3 polyunsaturated fatty acids on brain gene expression.
      ,
      • Alessandri J.M.
      • Guesnet P.
      • Vancassel S.
      • Astorg P.
      • Denis I.
      • Langelier B.
      • Aid S.
      • Poumes-Ballihaut C.
      • Champeil-Potokar G.
      • Lavialle M.
      Polyunsaturated fatty acids in the central nervous system: Evolution of concepts and nutritional implications throughout life.
      ,
      • Alessandri J.M.
      • Poumes-Ballihaut C.
      • Langelier B.
      • Perruchot M.H.
      • Raguenez G.
      • Lavialle M.
      • Guesnet P.
      Incorporation of docosahexaenoic acid into nerve membrane phospholipids: Bridging the gap between animals and cultured cells.
      ,
      • Anderson G.J.
      • Neuringer M.
      • Lin D.S.
      • Connor W.E.
      Can prenatal N-3 fatty acid deficiency be completely reversed after birth? Effects on retinal and brain biochemistry and visual function in rhesus monkeys.
      ,
      • Bazan N.G.
      Omega-3 fatty acids, pro-inflammatory signaling and neuroprotection.
      ,
      • Carlson S.E.
      Docosahexaenoic acid and arachidonic acid in infant development.
      ,
      • Freemantle E.
      • Vandal M.
      • Tremblay-Mercier J.
      • Tremblay S.
      • Blachere J.C.
      • Begin M.E.
      • Brenna J.T.
      • Windust A.
      • Cunnane S.C.
      Omega-3 fatty acids, energy substrates, and brain function during aging.
      ,
      • Greiner R.C.
      • Winter J.
      • Nathanielsz P.W.
      • Brenna J.T.
      Brain docosahexaenoate accretion in fetal baboons: Bioequivalence of dietary alpha-linolenic and docosahexaenoic acids.
      ,
      • Kaduce T.L.
      • Chen Y.
      • Hell J.W.
      • Spector A.A.
      Docosahexaenoic acid synthesis from n-3 fatty acid precursors in rat hippocampal neurons.
      ,
      • Levant B.
      • Ozias M.K.
      • Carlson S.E.
      Specific brain regions of female rats are differentially depleted of docosahexaenoic acid by reproductive activity and an (n-3) fatty acid-deficient diet.
      ). However, to date, despite our exhaustive search, we have not been able to detect VLC-PUFA in the normal brain, but we have readily detected VLC-PUFA in retinal PC and sperm sphingolipids and VLC-SFA in skin (
      • Agbaga M.P.
      • Brush R.S.
      • Mandal M.N.
      • Henry K.
      • Elliott M.H.
      • Anderson R.E.
      Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.
      ,
      • Aveldano M.I.
      A novel group of very long chain polyenoic fatty acids in dipolyunsaturated phosphatidylcholines from vertebrate retina.
      ,
      • Aveldano M.I.
      • Sprecher H.
      Very long chain (C24 to C36) polyenoic fatty acids of the n-3 and n-6 series in dipolyunsaturated phosphatidylcholines from bovine retina.
      ,
      • Aveldano M.I.
      Phospholipid species containing long and very long polyenoic fatty acids remain with rhodopsin after hexane extraction of photoreceptor membranes.
      ,
      • Brush R.S.
      • Tran J.T.
      • Henry K.R.
      • McClellan M.E.
      • Elliott M.H.
      • Mandal M.N.
      Retinal sphingolipids and their very-long-chain fatty acid-containing species.
      ,
      • Aveldano M.I.
      Long and very long polyunsaturated fatty acids of retina and spermatozoa: The whole complement of polyenoic fatty acid series.
      ,
      • Craig L.B.
      • Brush R.S.
      • Sullivan M.T.
      • Zavy M.T.
      • Agbaga M.P.
      • Anderson R.E.
      Decreased very long chain polyunsaturated fatty acids in sperm correlates with sperm quantity and quality.
      ,
      • Roqueta-Rivera M.
      • Stroud C.K.
      • Haschek W.M.
      • Akare S.J.
      • Segre M.
      • Brush R.S.
      • Agbaga M.P.
      • Anderson R.E.
      • Hess R.A.
      • Nakamura M.T.
      Docosahexaenoic acid supplementation fully restores fertility and spermatogenesis in male delta-6 desaturase-null mice.
      ). This suggests VLC-PUFA and VLC-SFA may be differentially metabolized in the brain and in the retina. Since fatty acid oxidation occurs in peroxisomes when fatty acid chains are too long to be oxidized by the mitochondria, the presence of VLC-PUFA in the brain and other tissues of Zellweger spectrum disorders, but not in normal brain, is attributed to impaired peroxisome dysfunction (
      • Poulos A.
      • Christodoulou J.
      • Chow C.W.
      • Goldblatt J.
      • Paton B.C.
      • Orii T.
      • Suzuki Y.
      • Shimozawa N.
      Peroxisomal assembly defects: Clinical, pathologic, and biochemical findings in two patients in a newly identified complementation group.
      ,
      • Poulos A.
      • Sharp P.
      • Johnson D.
      Plasma polyenoic very-long-chain fatty acids in peroxisomal disease: Biochemical discrimination of Zellweger's syndrome from other phenotypes.
      ,
      • Poulos A.
      • Sharp P.
      • Johnson D.
      • Easton C.
      The occurrence of polyenoic very long chain fatty acids with greater than 32 carbon atoms in molecular species of phosphatidylcholine in normal and peroxisome-deficient (Zellweger's syndrome) brain.
      ). On the other hand, it may be argued that the presence of VLC-PUFA in the brains of Zellweger spectrum disorders suggests that, in the normal brain, VLC-PUFA are efficiently utilized for brain function, hence their lack of accumulation. We need genetic animal models and in vitro studies to test these proposals.
      Studies have shown that some fatty acids may be utilized more for energy purposes. DHA is demonstrated to be a relatively poor substrate for beta-oxidation in the mitochondria and peroxisomes (
      • Madsen L.
      • Rustan A.C.
      • Vaagenes H.
      • Berge K.
      • Dyroy E.
      • Berge R.K.
      Eicosapentaenoic and docosahexaenoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference.
      ,
      • Madsen L.
      • Froyland L.
      • Dyroy E.
      • Helland K.
      • Berge R.K.
      Docosahexaenoic and eicosapentaenoic acids are differently metabolized in rat liver during mitochondria and peroxisome proliferation.
      ,
      • Lazarow P.B.
      Rat liver peroxisomes catalyze the beta oxidation of fatty acids.
      ). EPA is present in the retina and brain at low levels probably because they are more readily metabolized or oxidized than DHA; as a result, there is limited retroconversion of DHA back to EPA in peroxisomes (
      • Madsen L.
      • Rustan A.C.
      • Vaagenes H.
      • Berge K.
      • Dyroy E.
      • Berge R.K.
      Eicosapentaenoic and docosahexaenoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference.
      ,
      • Madsen L.
      • Froyland L.
      • Dyroy E.
      • Helland K.
      • Berge R.K.
      Docosahexaenoic and eicosapentaenoic acids are differently metabolized in rat liver during mitochondria and peroxisome proliferation.
      ,
      • Chen C.T.
      • Liu Z.
      • Ouellet M.
      • Calon F.
      • Bazinet R.P.
      Rapid beta-oxidation of eicosapentaenoic acid in mouse brain: An in situ study.
      ). To sustain the high energy needs of the brain, substantial EPA may therefore not be sufficient for significant FA elongation to VLC-PUFA, since FA elongation and FA catabolism cannot occur simultaneously (
      • Lazarow P.B.
      Rat liver peroxisomes catalyze the beta oxidation of fatty acids.
      ,
      • Leyton J.
      • Drury P.J.
      • Crawford M.A.
      Differential oxidation of saturated and unsaturated fatty acids in vivo in the rat.
      ). Coincidentally, based on our in vitro studies, we showed that EPA is a preferred substrate for VLC-PUFA biosynthesis by ELOVL4-expressing cells treated with equimolar concentrations of DHA and EPA or DHA and AA (
      • Yu M.
      • Benham A.
      • Logan S.
      • Brush R.S.
      • Mandal M.N.
      • Anderson R.E.
      • Agbaga M.P.
      ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3.
      ). These findings are consistent with the discovery that in Atlantic salmon (Salmo salar), Elovl4 synthesized greater amounts of VLC-PUFA from EPA than from AA and DHA (
      • Carmona-Antonanzas G.
      • Monroig O.
      • Dick J.R.
      • Davie A.
      • Tocher D.R.
      Biosynthesis of very long-chain fatty acids (C>24) in Atlantic salmon: Cloning, functional characterisation, and tissue distribution of an Elovl4 elongase.
      ). Also, in the retina, DHA molecules are principal components of retinal PC-containing VLC-PUFA and hence may not undergo extensive metabolism while incorporated into phospholipids (
      • Zemski Berry K.A.
      • Gordon W.C.
      • Murphy R.C.
      • Bazan N.G.
      Spatial organization of lipids in the human retina and optic nerve by MALDI imaging mass spectrometry.
      ). For instance, when vitreal fluid of rat eyes was injected with tritium-labeled [3H]-DHA, 90% of the [3H]-DHA remained as 22:6n-3 in the phospholipids after 48 h in vivo metabolism (
      • Suh M.
      • Clandinin M.T.
      20:5n-3 but not 22:6n-3 is a preferred substrate for synthesis of n-3 very-long- chain fatty acids (C24-C36) in retina.
      ). These observations indicate that DHA is likely utilized more for structural purposes, influencing fluidity of the membrane without further metabolism (
      • Stillwell W.
      • Wassall S.R.
      Docosahexaenoic acid: Membrane properties of a unique fatty acid.
      ). Therefore, despite the presence of relatively high DHA in the brain, it may be serving more as structural and signaling elements and increasingly not available as substrates for ELOVL4 elongation into VLC-PUFA (
      • Lauritzen L.
      • Hansen H.S.
      • Jorgensen M.H.
      • Michaelsen K.F.
      The essentiality of long chain n-3 fatty acids in relation to development and function of the brain and retina.
      ). As we previously proposed (
      • Hopiavuori B.R.
      • Deak F.
      • Wilkerson J.L.
      • Brush R.S.
      • Rocha-Hopiavuori N.A.
      • Hopiavuori A.R.
      • Ozan K.G.
      • Sullivan M.T.
      • Wren J.D.
      • Georgescu C.
      • Szweda L.
      • Awasthi V.
      • Towner R.
      • Sherry D.M.
      • Anderson R.E.
      • et al.
      Homozygous expression of mutant ELOVL4 leads to seizures and death in a novel animal model of very long-chain fatty acid deficiency.
      ), absence of VLC-PUFA in the normal brain could be due to the fact that any VLC-PUFA produced are efficiently biotransformed into Elovanoids that serve a neuroprotective signaling function (
      • Bhattacharjee S.
      • Jun B.
      • Belayev L.
      • Heap J.
      • Kautzmann M.A.
      • Obenaus A.
      • Menghani H.
      • Marcell S.J.
      • Khoutorova L.
      • Yang R.
      • Petasis N.A.
      • Bazan N.G.
      Elovanoids are a novel class of homeostatic lipid mediators that protect neural cell integrity upon injury.
      ). This thought is further supported by the fact that the absence of Adiponectin receptor 1, a receptor necessary for DHA uptake into the retina, leads to depletion of not only DHA but also Elovanoids and their VLC-PUFA precursors 32:6n3 or 34:6n3 (
      • Rice D.S.
      • Calandria J.M.
      • Gordon W.C.
      • Jun B.
      • Zhou Y.
      • Gelfman C.M.
      • Li S.
      • Jin M.
      • Knott E.J.
      • Chang B.
      • Abuin A.
      • Issa T.
      • Potter D.
      • Platt K.A.
      • Bazan N.G.
      Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival.
      ). Also, sufficient availability of a particular fatty acid may interfere with the elongation of another type of fatty acid in a given tissue. For example, using liver microsomal assays, it was reported that the chain elongation of linoleic acid (18:2n6) was more than 60% inhibited by saturated fatty acids myristic acid (14:0) and pentadecanoic acid (15:0) (
      • Mohrhauer H.
      • Christiansen K.
      • Gan M.V.
      • Deubig M.
      • Holman R.T.
      Chain elongation of linoleic acid and its inhibition by other fatty acids in vitro.
      ). This form of competitive inhibition may play a role in the abundance of VLC-SFA or VLC-PUFA in a tissue-specific manner.
      Another factor that could determine the predominant product of ELOVL4 in a tissue-specific manner may be the ELOVL4 enzyme elongation complex substrate discrimination as determined by specific interacting proteins within a given tissue. Saturated and polyunsaturated FA have different physiochemical properties, with straight-chained saturated fatty acids being able to pack closely, whereas PUFA, owing to the presence of double bonds, occupy more space. The double bonds of PUFA cause them to have bent conformations along the acyl chain, so they do not pack tightly. ELOVL1-7 exhibits characteristic substrate specificities toward acyl-CoAs. Some elongases, such as ELOVL6 and ELOVL7, have preferential elongation activity toward saturated fatty acids rather than PUFA. Thus, it is possible that differential expression of the type of ELOVL proteins in a given tissue could determine the type of fatty acid synthesized within that tissue (
      • Guillou H.
      • Zadravec D.
      • Martin P.G.
      • Jacobsson A.
      The key roles of elongases and desaturases in mammalian fatty acid metabolism: Insights from transgenic mice.
      ). In vitro studies showed that elongation by ELOVL6 was limited to long-chain saturated and monounsaturated fatty acyl-CoAs with chain lengths of 12–16 carbons. This finding shows that an increase in carbon length and degree of unsaturation in the FA limit the ability of ELOVL6 to elongate it (
      • Burns T.A.
      • Duckett S.K.
      • Pratt S.L.
      • Jenkins T.C.
      Supplemental palmitoleic (C16:1 cis-9) acid reduces lipogenesis and desaturation in bovine adipocyte cultures.
      ). Although VLC-SFA biosynthesis requires expression of combinations of ELOVL1, 3, 4, 6, and 7 in cells, ELOVL2 and ELOVL5 have substrate preference for PUFA by elongating C18–C20 PUFA (
      • Gregory M.K.
      • Cleland L.G.
      • James M.J.
      Molecular basis for differential elongation of omega-3 docosapentaenoic acid by the rat Elovl5 and Elovl2.
      ,
      • Gregory M.K.
      • James M.J.
      Functional characterization of the duck and turkey fatty acyl elongase enzymes ELOVL5 and ELOVL2.
      ). However, only ELOVL2 further converts 22 carbon PUFA n3/n6 to 24 carbon PUFA n3/n6 (
      • Gregory M.K.
      • Cleland L.G.
      • James M.J.
      Molecular basis for differential elongation of omega-3 docosapentaenoic acid by the rat Elovl5 and Elovl2.
      ). Subsequently, the 24 carbon PUFA can be further elongated into 26 carbon PUFA (which are substrates of ELOVL4) by ELOVL1 and 3. In vitro, both ELOVL2 and 5 enzymes elongated EPA (20:5n3, a substrate common to both ELOVL2 and 5) to DPA (22:5n3). However, only ELOVL2 further elongated the DPA product to 24:5n3 (
      • Gregory M.K.
      • Cleland L.G.
      • James M.J.
      Molecular basis for differential elongation of omega-3 docosapentaenoic acid by the rat Elovl5 and Elovl2.
      ).
      Using a yeast expression system, the cysteine at position 217 in ELOVL2 and a tryptophan at the corresponding position in ELOVL5 have been identified as the molecular reason for the differences in activity toward elongation of DPA to 24:5n3. As a result, C217W-ELOVL2 mutant loses the ability to convert DPA to 24:5n3 while retaining enzymatic activity to elongate EPA to DPA (
      • Gregory M.K.
      • Cleland L.G.
      • James M.J.
      Molecular basis for differential elongation of omega-3 docosapentaenoic acid by the rat Elovl5 and Elovl2.
      ).
      We recently generated a knock-in Long Evans rat model of SCA34 (SCA34-KI) that expresses the 736T > G (p.W246G) form of ELOVL4 that causes human SCA34. Analyses of retina and skin lipids of the SCA34-KI rats showed that the W246G ELOVL4 mutation selectively impaired synthesis of VLC-SFA in the skin, but not retinal VLC-PUFA (Fig. 3) (
      • Agbaga M.P.
      • Stiles M.A.
      • Brush R.S.
      • Sullivan M.T.
      • Machalinski A.
      • Jones K.L.
      • Anderson R.E.
      • Sherry D.M.
      The Elovl4 spinocerebellar Ataxia-34 mutation 736T>G (p.W246G) impairs retinal function in the absence of photoreceptor degeneration.
      ). Consistent with the human SCA34 pathology, the homozygous SCA34 rats that developed progressive motor learning defects with age were significantly smaller than their heterozygous and wild-type control littermates and developed reddish pink scaly skin similar to the human EKV pathology (
      • Agbaga M.P.
      • Stiles M.A.
      • Brush R.S.
      • Sullivan M.T.
      • Machalinski A.
      • Jones K.L.
      • Anderson R.E.
      • Sherry D.M.
      The Elovl4 spinocerebellar Ataxia-34 mutation 736T>G (p.W246G) impairs retinal function in the absence of photoreceptor degeneration.
      ). Analyses of VLC-SFA in skin showed significantly reduced levels of 28:0 and 30:0 in homozygous mutant rats (MUT) compared with wild-type (WT) and heterozygous (HET) ones (Fig. 3A, B). We also showed the levels of 24:0 were significantly elevated in the skin of HET and MUT rats compared with WT rats (Fig. 3A). Of interest, total retinal VLC-PUFA-PC levels showed no differences among the WT, HET, and MUT rat retinas (Fig. 3C), which suggests that the W246G mutation affected skin VLC-SFA biosynthesis but had no effect on retinal VLC-PUFA biosynthesis. Recent studies by Parisi et al. (
      • Parisi L.R.
      • Sowlati-Hashjin S.
      • Berhane I.A.
      • Galster S.L.
      • Carter K.A.
      • Lovell J.F.
      • Chemler S.R.
      • Karttunen M.
      • Atilla-Gokcumen G.E.
      Membrane disruption by very long chain fatty acids during necroptosis.
      ,
      • Parisi L.R.
      • Li N.
      • Atilla-Gokcumen G.E.
      Very long chain fatty acids are functionally involved in necroptosis.
      ) demonstrated that increased levels of C24 fatty acids can disturb membrane integrity more readily than C16 fatty acids. Using molecular dynamics simulations, they showed that 24:0 can interdigitate between leaflets of the lipid bilayer, whereas C16 FA does not, and that increases in the levels of saturated VLC-FA result in cellular membrane permeabilization during necroptosis (
      • Parisi L.R.
      • Sowlati-Hashjin S.
      • Berhane I.A.
      • Galster S.L.
      • Carter K.A.
      • Lovell J.F.
      • Chemler S.R.
      • Karttunen M.
      • Atilla-Gokcumen G.E.
      Membrane disruption by very long chain fatty acids during necroptosis.
      ,
      • Parisi L.R.
      • Li N.
      • Atilla-Gokcumen G.E.
      Very long chain fatty acids are functionally involved in necroptosis.
      ). They proposed that dysregulation of VLC-SFA can cause membrane disruption either by directly disrupting membrane packing or facilitating permeabilization by targeting proteins to the plasma membrane. This observation could explain the EKV-like skin disorders seen in humans and in our SCA34 rat model (
      • Agbaga M.P.
      • Stiles M.A.
      • Brush R.S.
      • Sullivan M.T.
      • Machalinski A.
      • Jones K.L.
      • Anderson R.E.
      • Sherry D.M.
      The Elovl4 spinocerebellar Ataxia-34 mutation 736T>G (p.W246G) impairs retinal function in the absence of photoreceptor degeneration.
      ,
      • Parisi L.R.
      • Sowlati-Hashjin S.
      • Berhane I.A.
      • Galster S.L.
      • Carter K.A.
      • Lovell J.F.
      • Chemler S.R.
      • Karttunen M.
      • Atilla-Gokcumen G.E.
      Membrane disruption by very long chain fatty acids during necroptosis.
      ,
      • Parisi L.R.
      • Li N.
      • Atilla-Gokcumen G.E.
      Very long chain fatty acids are functionally involved in necroptosis.
      ). Taken together, these results suggest that the W246G ELOVL4 protein may have substrate preference for PUFA but not SFA, which would support the hypothesis that ELOVL4 mutations that affect the skin and brain but not the retina impair the synthesis of VLC-SFA, which are the ELOVL4 products of the skin, and not VLC-PUFA, the ELOVL4 products in the retina. It is also possible that mutations in ELOVL4 that significantly change the conformation of the protein structure and its active site may steer ELOVL4 substrate preference for either SFA or PUFA, hence the tissue-specific disorders caused by the different ELOVL4 mutations.
      Figure thumbnail gr3
      Fig. 3The W246G mutation in ELOVL4 impairs VLC-SFA synthesis but retains the ability to synthesize VLC-PUFA. A: Analysis of VLC-SFA in skin. Levels of VLC-SFA (28:0 and 30:0) and total VLC-SFA (28:0 + 30:0) were significantly reduced in the skin of MUT rats compared with WT and HET rats. B: Levels of 26:0, the direct precursor for VLC-SFA synthesis, did not differ significantly across genotypes. However, levels of 24:0 were significantly elevated in the skin of HET and MUT rats compared with WT rats. (Data shown as mean ± SD. Analysis by one-way ANOVA with Tukey's post hoc test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001). C: VLC-PUFA were detected specifically in the phosphatidylcholine fraction (PC), but total VLC-PUFA levels showed no differences among WT, HET, and MUT rat retinas. However, significant differences were detected in non-VLC-FA species (PC 34:01 and PC 40:06) among genotypes.

      Roles of VLC-FA-containing lipids in retinal function

      Genetic and aging components that could affect retinal fatty acid biosynthesis and cause retinal pathologies

      A pathologic hallmark of STGD3 is the presence of lipid-containing residue lipofuscin in the RPE, RPE atrophy, and macular degeneration (
      • Donoso L.A.
      • Edwards A.O.
      • Frost A.
      • Vrabec T.
      • Stone E.M.
      • Hageman G.S.
      • Perski T.
      Autosomal dominant Stargardt-like macular dystrophy.
      ,
      • Edwards A.O.
      • Miedziak A.
      • Vrabec T.
      • Verhoeven J.
      • Acott T.S.
      • Weleber R.G.
      • Donoso L.A.
      Autosomal dominant Stargardt-like macular dystrophy: I. Clinical characterization, longitudinal follow-up, and evidence for a common ancestry in families linked to chromosome 6q14.
      ,
      • Donoso L.A.
      • Frost A.T.
      • Stone E.M.
      • Weleber R.G.
      • MacDonald I.M.
      • Hageman G.S.
      • Cibis G.W.
      • Ritter 3rd, R.
      • Edwards A.O.
      Autosomal dominant Stargardt-like macular dystrophy: Founder effect and reassessment of genetic heterogeneity.
      ). VLC-PUFA are the main products of ELOVL4 in the retina and testes, whereas VLC-SFA are predominantly synthesized in the skin, brain, and Meibomian glands. Although the role of VLC-PUFA in the retina is not completely understood, the current data suggest that retinal VLC-PUFA confer fluidity on retinal outer segment membranes and are essential for synaptic function and overall health and function of the retina and RPE (
      • Bennett L.D.
      • Brush R.S.
      • Chan M.
      • Lydic T.A.
      • Reese K.
      • Reid G.E.
      • Busik J.V.
      • Elliott M.H.
      • Anderson R.E.
      Effect of reduced retinal VLC-PUFA on rod and cone photoreceptors.
      ,
      • Bennett L.D.
      • Hopiavuori B.R.
      • Brush R.S.
      • Chan M.
      • Van Hook M.J.
      • Thoreson W.B.
      • Anderson R.E.
      Examination of VLC-PUFA-deficient photoreceptor terminals.
      ,
      • Harkewicz R.
      • Du H.
      • Tong Z.
      • Alkuraya H.
      • Bedell M.
      • Sun W.
      • Wang X.
      • Hsu Y.H.
      • Esteve-Rudd J.
      • Hughes G.
      • Su Z.
      • Zhang M.
      • Lopes V.S.
      • Molday R.S.
      • Williams D.S.
      • et al.
      Essential role of ELOVL4 protein in very long chain fatty acid synthesis and retinal function.
      ). Indeed, Liu et al. (
      • Liu A.
      • Chang J.
      • Lin Y.
      • Shen Z.
      • Bernstein P.S.
      Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
      ) reported the presence of 21 different VLC-PUFA belonging to six different groups in human retina and RPE/choroid. These groups include n-3 and n-6 VLC-PUFA from C24 to C34 with variable double bonds (
      • Liu A.
      • Chang J.
      • Lin Y.
      • Shen Z.
      • Bernstein P.S.
      Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
      ). They showed that, in the whole retina, the levels of 28:4n-6, 30:6n-3, 30:5n-3, 32:4n-6, and 32:5n-3 peaked in middle age, with the levels of 24:6n-3, 26:4n-6, 28:4n-6, 28:5n-3, 30:4n-6, 32:5n-6, 32:6n-3, 32:5n-3, and 34:4n-6 being significantly higher in normal old age retinas compared with the age-matched AMD group (
      • Liu A.
      • Chang J.
      • Lin Y.
      • Shen Z.
      • Bernstein P.S.
      Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
      ). Compared with the retina, one-tenth of C30–C34 VLC-PUFA were detected in RPE/choroid, which also showed a trend in age-related changes in VLC-PUFA, with levels peaking in middle age donor eyes and severely decreasing in AMD donors (
      • Liu A.
      • Chang J.
      • Lin Y.
      • Shen Z.
      • Bernstein P.S.
      Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
      ). These findings suggest that, even in the normal eye, normal aging affects the levels of retinal VLC-PUFA.
      An important finding from the Liu et al. study (
      • Liu A.
      • Chang J.
      • Lin Y.
      • Shen Z.
      • Bernstein P.S.
      Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
      ) is the decrease in C24–C26 PUFA in AMD retinas compared with age-matched normal retinas. This decrease was proposed to be likely due to lower levels of precursors such as 22:4n-6 and 22:5n-3 in AMD retinas or impaired enzymatic processing (
      • Liu A.
      • Chang J.
      • Lin Y.
      • Shen Z.
      • Bernstein P.S.
      Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration.
      ). In a follow-up study (
      • Gorusupudi A.
      • Liu A.
      • Hageman G.S.
      • Bernstein P.S.
      Associations of human retinal very long-chain polyunsaturated fatty acids with dietary lipid biomarkers.
      ) to the Liu et al. study from 2010, the Bernstein lab collected and analyzed donor eyes and serum from patients with AMD (n = 15; aged 73–91 years) and non-AMD control patients (n = 21; aged 74–88 years). They showed that dietary intake of LC-PUFA, which are precursors for VLC-PUFA, influenced the retinal lipid profile and confirmed that AMD eyes have decreased VLC-PUFA by showing that retinal VLC-PUFA levels were significantly lower in AMD eyes compared with levels in age-matched control eyes (
      • Gorusupudi A.
      • Liu A.
      • Hageman G.S.
      • Bernstein P.S.
      Associations of human retinal very long-chain polyunsaturated fatty acids with dietary lipid biomarkers.
      ). They proposed that serum LC-PUFA levels are good predictors of retinal LC-PUFA and VLC-PUFA levels (
      • Gorusupudi A.
      • Liu A.
      • Hageman G.S.
      • Bernstein P.S.
      Associations of human retinal very long-chain polyunsaturated fatty acids with dietary lipid biomarkers.
      ). Furthermore, they explored the potential contribution of TT and CT variants of ELOVL4 (rs3812153) to AMD pathogenesis. They reported that AMD donors with the CT ELOVL4 variant allele had one form or the other of pigmentary irregularities, soft drusen deposits (>63 and ≤125 μm), geographic atrophy, and choroidal neovascular membranes compared with carriers with the lower-risk TT allele variant (
      • Gorusupudi A.
      • Liu A.
      • Hageman G.S.
      • Bernstein P.S.
      Associations of human retinal very long-chain polyunsaturated fatty acids with dietary lipid biomarkers.
      ). However, consistent with previous studies, they did not find a statistically significant correlation between the lower-risk TT allele compared with the CT ELOVL4 variant and macular degeneration (
      • Gorusupudi A.
      • Liu A.
      • Hageman G.S.
      • Bernstein P.S.
      Associations of human retinal very long-chain polyunsaturated fatty acids with dietary lipid biomarkers.
      ,
      • Ayyagari R.
      • Zhang K.
      • Hutchinson A.
      • Yu Z.
      • Swaroop A.
      • Kakuk L.E.
      • Seddon J.M.
      • Bernstein P.S.
      • Lewis R.A.
      • Tammur J.
      • Yang Z.
      • Li Y.
      • Zhang H.
      • Yashar B.M.
      • Liu J.
      • et al.
      Evaluation of the ELOVL4 gene in patients with age-related macular degeneration.
      ,
      • DeAngelis M.M.
      • Ji F.
      • Kim I.K.
      • Adams S.
      • Capone Jr., A.
      • Ott J.
      • Miller J.W.
      • Dryja T.P.
      Cigarette smoking, CFH, APOE, ELOVL4, and risk of neovascular age-related macular degeneration.
      ). In light of the new discoveries of different ELOVL4 mutations causing different tissue-specific disorders in humans, researchers should consider including analyses of the CT and TT variants in ELOVL4 in patients with AMD in future studies. In line with the essential role of VLC-PUFA in retinal function, the report by Donato et al. (
      • Donato L.
      • Scimone C.
      • Rinaldi C.
      • Aragona P.
      • Briuglia S.
      • D'Ascola A.
      • D'Angelo R.
      • Sidoti A.
      Stargardt phenotype associated with two ELOVL4 promoter variants and ELOVL4 downregulation: New possible perspective to etiopathogenesis?.
      ) that SNP in the human ELOVL4 promoter causes STGD3 suggests that loss of ELOVL4 enzymatic activity, and therefore a decrease in VLC-FA products, could cause an age-related decline in visual function, RPE atrophy, and photoreceptor death.
      Indeed, it is now emerging that age-related hypermethylation of genes involved in retinal fatty acid metabolism that suppresses transcription of these genes potentially contributes to some of the age-related decrease in retinal LC-PUFA and VLC-PUFA (
      • Li X.
      • Wang J.
      • Wang L.
      • Feng G.
      • Li G.
      • Yu M.
      • Li Y.
      • Liu C.
      • Yuan X.
      • Zang G.
      • Li Z.
      • Zhao L.
      • Ouyang H.
      • Quan Q.
      • Wang G.
      • et al.
      Impaired lipid metabolism by age-dependent DNA methylation alterations accelerates aging.
      ). ELOVL2 is expressed in the retina and plays critical roles in the biosynthesis of LC-PUFA that are essential precursors for VLC-PUFA biosynthesis (
      • Chen D.
      • Chao D.L.
      • Rocha L.
      • Kolar M.
      • Nguyen Huu V.A.
      • Krawczyk M.
      • Dasyani M.
      • Wang T.
      • Jafari M.
      • Jabari M.
      • Ross K.D.
      • Saghatelian A.
      • Hamilton B.A.
      • Zhang K.
      • Skowronska-Krawczyk D.
      The lipid elongation enzyme ELOVL2 is a molecular regulator of aging in the retina.
      ). ELOVL2 has emerged as a prominent aging biomarker (
      • Li X.
      • Wang J.
      • Wang L.
      • Feng G.
      • Li G.
      • Yu M.
      • Li Y.
      • Liu C.
      • Yuan X.
      • Zang G.
      • Li Z.
      • Zhao L.
      • Ouyang H.
      • Quan Q.
      • Wang G.
      • et al.
      Impaired lipid metabolism by age-dependent DNA methylation alterations accelerates aging.
      ,
      • Garagnani P.
      • Bacalini M.G.
      • Pirazzini C.
      • Gori D.
      • Giuliani C.
      • Mari D.
      • Di Blasio A.M.
      • Gentilini D.
      • Vitale G.
      • Collino S.
      • Rezzi S.
      • Castellani G.
      • Capri M.
      • Salvioli S.
      • Franceschi C.
      Methylation of ELOVL2 gene as a new epigenetic marker of age.
      ,
      • Kananen L.
      • Marttila S.
      • Nevalainen T.
      • Jylhava J.
      • Mononen N.
      • Kahonen M.
      • Raitakari O.T.
      • Lehtimaki T.
      • Hurme M.
      Aging-associated DNA methylation changes in middle-aged individuals: The Young Finns study.
      ). Age-related increased methylation in ELOVL2 occurs not only in humans but also in mouse retina and liver, where it is highly expressed (
      • Chen D.
      • Chao D.L.
      • Rocha L.
      • Kolar M.
      • Nguyen Huu V.A.
      • Krawczyk M.
      • Dasyani M.
      • Wang T.
      • Jafari M.
      • Jabari M.
      • Ross K.D.
      • Saghatelian A.
      • Hamilton B.A.
      • Zhang K.
      • Skowronska-Krawczyk D.
      The lipid elongation enzyme ELOVL2 is a molecular regulator of aging in the retina.
      ). Recent studies have also reported that genetic variations in ELOVL2 affect the level of blood EPA and DHA, thereby providing an inconsistent degree of protection from cardiovascular disease (
      • Alsaleh A.
      • Maniou Z.
      • Lewis F.J.
      • Hall W.L.
      • Sanders T.A.
      • O'Dell S.D.
      ELOVL2 gene polymorphisms are associated with increases in plasma eicosapentaenoic and docosahexaenoic acid proportions after fish oil supplement.
      ). Carriers of the SNPs rs3734398, rs2236212, and rs953413 in ELOVL2 have lower proportions of plasma DHA than do noncarriers at baseline, which could be improved by dietary EPA and DHA supplementation (
      • Alsaleh A.
      • Maniou Z.
      • Lewis F.J.
      • Hall W.L.
      • Sanders T.A.
      • O'Dell S.D.
      ELOVL2 gene polymorphisms are associated with increases in plasma eicosapentaenoic and docosahexaenoic acid proportions after fish oil supplement.
      ). These findings suggest that, in the general population, age-related methylation of the regulatory regions of ELOVL2 coupled with SNPs in ELOVL2, and potentially in other ELOVLs and fatty acid desaturases that are essential for fatty acid metabolism, could be one of the possible molecular explanations for the age-related decrease in retinal LC-PUFA and VLC-PUFA levels.
      Recently, the Skowronska-Krawczyk lab showed that the Elovl2 promoter region is increasingly methylated with age in the retina and caused an age-dependent decrease in ELOVL2 expression in the liver and retina (
      • Chen D.
      • Chao D.L.
      • Rocha L.
      • Kolar M.
      • Nguyen Huu V.A.
      • Krawczyk M.
      • Dasyani M.
      • Wang T.
      • Jafari M.
      • Jabari M.
      • Ross K.D.
      • Saghatelian A.
      • Hamilton B.A.
      • Zhang K.
      • Skowronska-Krawczyk D.
      The lipid elongation enzyme ELOVL2 is a molecular regulator of aging in the retina.
      ). Their analyses of the fundus of wild-type C57BL/6J mice showed age-dependent accumulation of autofluorescent aggregates and a decrease in visual function at 24 months, which could be reversed in vivo by intravitreal injection of 5-Aza-2′-deoxycytidine (
      • Chen D.
      • Chao D.L.
      • Rocha L.
      • Kolar M.
      • Nguyen Huu V.A.
      • Krawczyk M.
      • Dasyani M.
      • Wang T.
      • Jafari M.
      • Jabari M.
      • Ross K.D.
      • Saghatelian A.
      • Hamilton B.A.
      • Zhang K.
      • Skowronska-Krawczyk D.
      The lipid elongation enzyme ELOVL2 is a molecular regulator of aging in the retina.
      ). To further understand how downregulation of ELOVL2 could cause age-related changes in visual function in the mouse retina, they used the CRISPR-Cas9 technology to generate mutant Elovl2 knockin mice that encode a cysteine-to-tryptophan substitution (C234W) in ELOVL2 (
      • Chen D.
      • Chao D.L.
      • Rocha L.
      • Kolar M.
      • Nguyen Huu V.A.
      • Krawczyk M.
      • Dasyani M.
      • Wang T.
      • Jafari M.
      • Jabari M.
      • Ross K.D.
      • Saghatelian A.
      • Hamilton B.A.
      • Zhang K.
      • Skowronska-Krawczyk D.
      The lipid elongation enzyme ELOVL2 is a molecular regulator of aging in the retina.
      ). The C234W ELOVL2 mutation caused selective inhibition of elongation of C22-PUFA to C24-PUFA, while its activity toward other ELOVL2 substrates was retained (
      • Chen D.
      • Chao D.L.
      • Rocha L.
      • Kolar M.
      • Nguyen Huu V.A.
      • Krawczyk M.
      • Dasyani M.
      • Wang T.
      • Jafari M.
      • Jabari M.
      • Ross K.D.
      • Saghatelian A.
      • Hamilton B.A.
      • Zhang K.
      • Skowronska-Krawczyk D.
      The lipid elongation enzyme ELOVL2 is a molecular regulator of aging in the retina.
      ). The heterozygous Elovl2C234W mice were fertile and bred to produce homozygous Elovl2C234W mice that developed normally. However, fatty acid analyses of the retina and liver of the Elovl2C234W mice revealed accumulation of 22:5 fatty acids and decreased levels of 24:5 and 22:6 fatty acids, which supports loss of ELOVL2 enzymatic activity toward C22 elongation (
      • Chen D.
      • Chao D.L.
      • Rocha L.
      • Kolar M.
      • Nguyen Huu V.A.
      • Krawczyk M.
      • Dasyani M.
      • Wang T.
      • Jafari M.
      • Jabari M.
      • Ross K.D.
      • Saghatelian A.
      • Hamilton B.A.
      • Zhang K.
      • Skowronska-Krawczyk D.
      The lipid elongation enzyme ELOVL2 is a molecular regulator of aging in the retina.
      ). What is remarkable about the loss of ELOVL2 activity and decrease in the C24 fatty acids in the retina of these mice is that the homozygous Elovl2C234W mice exhibited an accelerated aging phenotype as determined by a decline in retinal electrophysiological responses consistent with premature visual dysfunction and showed well-established aging markers related to autofluorescent deposits by 6 months of age (
      • Chen D.
      • Chao D.L.
      • Rocha L.
      • Kolar M.
      • Nguyen Huu V.A.
      • Krawczyk M.
      • Dasyani M.
      • Wang T.
      • Jafari M.
      • Jabari M.
      • Ross K.D.
      • Saghatelian A.
      • Hamilton B.A.
      • Zhang K.
      • Skowronska-Krawczyk D.
      The lipid elongation enzyme ELOVL2 is a molecular regulator of aging in the retina.
      ). The results of age-related pathological changes in the wild-type C57BL/6J mouse retina induced by age-related hypermethylation of the Elovl2 promoter were similar to the changes observed in the Elovl2C234W mouse model (
      • Chen D.
      • Chao D.L.
      • Rocha L.
      • Kolar M.
      • Nguyen Huu V.A.
      • Krawczyk M.
      • Dasyani M.
      • Wang T.
      • Jafari M.
      • Jabari M.
      • Ross K.D.
      • Saghatelian A.
      • Hamilton B.A.
      • Zhang K.
      • Skowronska-Krawczyk D.
      The lipid elongation enzyme ELOVL2 is a molecular regulator of aging in the retina.
      ). These results suggest that age-related dysregulation of retinal fatty acid biosynthetic genes contributes to some forms of age-related retinal degenerative diseases. One factor that was not studied in the Elovl2C234W mouse was the potential contribution of the mutant C234W ELOVL2 protein to the pathology observed. We expect that, in future experiments, if the homozygous Elovl2C234W mice are fed the missing fatty acids and are shown to be rescued from the accelerated aging phenotype due to increased retinal LC-PUFA and VLC-PUFA levels, this will validate the essential role of LC-PUFA and VLC-PUFA in retinal and brain function.
      It must, however, be emphasized that the essential role of LC-PUFA and especially VLC-PUFA in the retina is still not completely understood. It is known that visual function and the accumulation of autofluorescent aggregates in the retina are highly correlated with aging (
      • Birch D.G.
      • Anderson J.L.
      Standardized full-field electroretinography. Normal values and their variation with age.
      ,
      • Birch D.G.
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      • Hoffman D.R.
      • Uauy R.D.
      Retinal development in very-low-birth-weight infants fed diets differing in omega-3 fatty acids.
      ,
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      • Bernardino Jr., V.B.
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      Retinal pigment epithelial abnormalities in fundus flavimaculatus: A light and electron microscopic study.
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      • Finnemann S.C.
      • Leung L.W.
      • Rodriguez-Boulan E.
      The lipofuscin component A2E selectively inhibits phagolysosomal degradation of photoreceptor phospholipid by the retinal pigment epithelium.
      ,
      • Marmorstein A.D.
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      • Sakaguchi H.
      • Hollyfield J.G.
      Spectral profiling of autofluorescence associated with lipofuscin, Bruch's Membrane, and sub-RPE deposits in normal and AMD eyes.
      ,
      • Zanzottera E.C.
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      • Freund K.B.
      • Curcio C.A.
      Visualizing retinal pigment epithelium phenotypes in the transition to atrophy in neovascular age-related macular degeneration.
      ). In heterozygous Elovl4 knockout (Elovl4+/−) animal models that lack expression of the STGD3 mutant ELOVL4 (
      • Raz-Prag D.
      • Ayyagari R.
      • Fariss R.N.
      • Mandal M.N.
      • Vasireddy V.
      • Majchrzak S.
      • Webber A.L.
      • Bush R.A.
      • Salem Jr., N.
      • Petrukhin K.
      • Sieving P.A.
      Haploinsufficiency is not the key mechanism of pathogenesis in a heterozygous Elovl4 knockout mouse model of STGD3 disease.
      ,
      • Li W.
      • Chen Y.
      • Cameron D.J.
      • Wang C.
      • Karan G.
      • Yang Z.
      • Zhao Y.
      • Pearson E.
      • Chen H.
      • Deng C.
      • Howes K.
      • Zhang K.
      Elovl4 haploinsufficiency does not induce early onset retinal degeneration in mice.
      ) protein, there are no abnormal changes in rod and cone morphology or decline in photoreceptor function compared with transgenic mouse models expressing the STGD3 ELOVL4 protein (
      • Vasireddy V.
      • Jablonski M.M.
      • Khan N.W.
      • Wang X.F.
      • Sahu P.
      • Sparrow J.R.
      • Ayyagari R.
      Elovl4 5-bp deletion knock-in mouse model for Stargardt-like macular degeneration demonstrates accumulation of ELOVL4 and lipofuscin.
      ,
      • Karan G.
      • Lillo C.
      • Yang Z.
      • Cameron D.J.
      • Locke K.G.
      • Zhao Y.
      • Thirumalaichary S.
      • Li C.
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      • Williams D.S.
      • Zhang K.
      Lipofuscin accumulation, abnormal electrophysiology, and photoreceptor degeneration in mutant ELOVL4 transgenic mice: A model for macular degeneration.
      ,
      • Vasireddy V.
      • Jablonski M.M.
      • Mandal M.N.
      • Raz-Prag D.
      • Wang X.F.
      • Nizol L.
      • Iannaccone A.
      • Musch D.C.
      • Bush R.A.
      • Salem Jr., N.
      • Sieving P.A.
      • Ayyagari R.
      Elovl4 5-bp-deletion knock-in mice develop progressive photoreceptor degeneration.