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Sjögren-Larsson syndrome (SLS) is a neurocutaneous disorder whose causative gene is the fatty aldehyde dehydrogenase ALDH3A2 and of which ichthyosis is the major skin symptom. The stratum corneum contains a variety of ceramides, among which ω-O-acylceramides (acylceramides) and protein-bound ceramides are essential for skin permeability barrier formation.To determine the ceramide classes/species responsible for SLS pathogenesis and the enzymes that are impaired in SLS.Genomic DNA was collected from peripheral blood samples from an SLS patient and her parents, and whole-genome sequencing and Sanger sequencing were performed. Lipids were extracted from stratum corneum samples from the SLS patient and healthy volunteers and subjected to ceramide profiling via liquid chromatography coupled with tandem mass spectrometry.A duplication (c.55_130dup) and a missense mutation (p.Lys447Glu) were found in the patient's ALDH3A2 gene. The patient had reduced levels of all acylceramide classes, with total acylceramide levels at 25 % of healthy controls. Reductions were also observed for several nonacylated ceramides: ceramides with phytosphingosine or 6-hydroxysphingosine in the long-chain base moiety were reduced to 24 % and 41 % of control levels, respectively, and ceramides with an α-hydroxy fatty acid as the fatty acid moiety were reduced to 29 %. The fatty acid moiety was shortened in many nonacylated ceramide classes.These results suggest that reduced acylceramide levels are a primary cause of the ichthyosis symptoms of SLS, but reductions in other ceramide classes may also be involved.

The stratum corneum (SC) of the epidermis acts as a skin permeability barrier, and abnormalities in SC formation lead to several skin disorders. Lipids, especially the epidermis-specific ceramide classes omega-O-acylceramides (acylceramides) and protein-bound ceramides, are essential for skin barrier formation. Ceramide synthase 3 (CERS3) is involved in the synthesis of acylceramides and protein-bound ceramides, and CERS3 mutations cause autosomal recessive congenital ichthyosis. In the present study, we measured ceramide synthase activity and performed comprehensive SC ceramide profiling in an ichthyosis patient with compound heterozygous CERS3 mutations: nonsense mutation p.Arg75* and missense mutation p.Arg229His. The activity of p.Arg75* and p.Arg229His mutant CERS3 proteins was reduced to 4% and 56%, respectively, of the wild-type protein. In the patient's SC, acylceramide levels were greatly reduced, but the levels of protein-bound ceramides remained almost unchanged. Non-acylated ceramide levels were also affected in the patient; in particular, the levels of ceramides composed of sphingosine and non-hydroxy or alpha-hydroxy fatty acid were substantially higher than in healthy controls. These results suggest that a reduction in acylceramide levels alone leads to ichthyosis. Although protein-bound ceramides are synthesized from acylceramides, levels of acylceramides and protein-bound ceramides are not necessarily correlated.

FA2H encodes fatty acid 2-hydroxylase, which plays a significant role in maintaining the neuronal myelin sheath. Previous reports have revealed that a FA2H mutation leads to spastic paraplegia, leukodystrophy, and neurodegeneration with brain iron accumulation, collectively referred to as fatty acid hydroxylase-associated neurodegeneration (FAHN). The disease severity of FAHN varies among individual patients and may be explained by the enzyme activity of FA2H mutant proteins. Here we report a 10-year-old Japanese boy with FAHN having novel heterozygous mutations in FA2H. The patient presented with a spastic gait since the age of 5 years and was unable to walk without a cane by the time he was 8 years old. Brain MRI demonstrated a partial thinning of the corpus callosum, slight reduction of cerebellar volume, and posterior dominant periventricular leukodystrophy. Whole exome sequencing revealed two novel missense mutations in FA2H with compound heterozygous inheritance (NM_024306, p.Val149Leu, and p.His260Gln mutations). The enzyme activities of the p.Val149Leu and p.His260Gln variants were 60%–80% and almost 0%, respectively. Our cell-based enzyme assay demonstrated partial functionality for one of the variants, indicating a milder phenotype. However, considered along with previous reports, there was no definite relationship between the disease severity and residual enzyme activity measured using a similar method. Further research is needed to precisely predict the phenotypic severity of this disorder.

Next generation sequencing technologies allow detection of very rare pathogenic gene variants and uncover cerebral palsy. Herein, we describe two siblings with cerebral palsy due to ELOVL1 splice site mutation in autosomal recessive manner. ELOVL1 catalyzes fatty acid elongation to produce very long-chain fatty acids (VLCFAs; ≥C21), most of which are components of sphingolipids such as ceramides and sphingomyelins. Ichthyotic keratoderma, spasticity, hypomyelination, and dysmorphic facies (MIM: 618527) stem from ELOVL1 gene deficiency in human.We have studied a consanguineous family with whole exome sequencing (WES) and performed in depth analysis of cryptic splicing on the molecular level using RNA. Comprehensive analysis of ceramides in the skin stratum corneum of patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS). ELOVL1 protein structure was computationally modelled.The novel c.376-2A G (ENST00000372458.8) homozygous variant in the affected siblings causes exon skipping. Comprehensive analysis of ceramides in the skin stratum corneum of patients using LC-MS/MS demonstrated significant shortening of fatty acid moieties and severe reduction in the levels of acylceramides.It has recently been shown that disease associated variants of ELOVL1 segregate in an autosomal dominant manner. However, our study for the first time demonstrates an alternative autosomal recessive inheritance model for ELOVL1. In conclusion, we suggest that in ultra-rare diseases, being able to identify the inheritance patterns of the disease-associated gene or genes can be an important guide to identifying the molecular mechanism of genetic cerebral palsy.

Very‐long‐chain fatty acids, with a chain length of >C20, are abundant in myelin sphingolipids. Recently, a de novo mutation in the ELOVL1 gene, which encodes fatty acid elongase, was identified in patients with neurocutaneous disorders involving skin ichthyosis and multiple neurological abnormalities, including hypomyelination, spastic paraplegia, and high‐frequency deafness. However, the consequences of ELOVL1 deficiency for lipid composition and detailed pathological changes in the brain remain unclear. Here, we analyzed Elovl1 mutant mice as a model of human ELOVL1 deficiency. The mice exhibited a decreased postnatal survival rate, and some died of startle epilepsy. The acyl chain length of sphingolipids such as galactosylceramides, sulfatides, sphingomyelins, and ceramides in the brains of these mice was markedly shortened. Moreover, the mice exhibited reduced levels of galactosylceramides, which are important for myelin formation and stability. Electron microscope analysis of the corpus callosum in Elovl1 mutant mice revealed modest hypomyelination, especially in large‐diameter axons. Furthermore, behavioral testing of the mice revealed deficits such as poorer motor coordination and reduced acoustic startle response to high‐intensity stimulus. These findings provide clues to the molecular mechanism of the neurological symptoms of patients with the ELOVL1 mutation.

Summary A lipid layer consisting of meibum lipids exists in the tear film and functions in preventing dry eye disease. Although the meibum lipids include diverse lipid classes, the synthesis pathway and role of each class remain largely unknown. Here, we created single and double knockout (KO and DKO, respectively) mice for the two acyl-CoA wax alcohol acyltransferases (Awat1 and Awat2) and investigated their dry eye phenotypes and meibum lipid composition. Awat2 KO and DKO mice exhibited severe dry eye with meibomian gland dysfunction, whereas Awat1 KO mice had mild dry eye. In these mice, specific meibum lipid classes were reduced: (O-acyl)-ω-hydroxy fatty acids and type 1ω wax diesters in Awat1 KO mice, wax monoesters and types 1ω and 2ω wax diesters in Awat2 KO mice, and most of these in DKO mice. Our findings reveal that Awat1 and Awat2 show characteristic substrate specificity and together produce diverse meibum lipids., Graphical abstract, Highlights • Awat2 knockout mice exhibit severe dry eye phenotypes • Awat1 knockout mice exhibit mild dry eye phenotypes • Awat2 is responsible for the production of wax monoesters and type 2ω wax diesters • Awat1 is involved in (O-acyl)-ω-hydroxy fatty acid production

Purpose Meibomian gland dysfunction (MGD) is a major cause of evaporative dry eye. The purpose of this study is to assess the efficacy of a mineral oil-containing ophthalmic solution (MO) in mitigating the evaporative dry eye phenotypes in a mouse model in which fatty acid elongase Elovl1 is disrupted. Methods Elovl1-deficient mice were assessed in terms of number of plugged meibomian gland orifices, tear film breakup time (BUT), corneal fluorescein staining (CFS) score, tear quantity, and histology. The effects of the MO on the dry eye phenotypes were compared with those in groups not treated or treated with blank ophthalmic solution (BL). Results Untreated Elovl1-deficient mice exhibited dry eye phenotypes with MGD symptoms such as plugging of meibomian gland orifices (P = 0.002 compared with control mice), high CFS scores (P = 0.002), and shortened BUT (P < 0.001). Among three groups of Elovl1-deficient mice (MO treated, BL treated, and untreated), the MO-treated group exhibited fewer plugged orifices (MO treated, 7.6; BL treated, 10.5 [P = 0.033]; untreated, 13.0 [P < 0.001]), lower CFS scores (MO treated, 1.1; BL treated, 2.7 [P = 0.013]; untreated, 2.5 [P = 0.050]), and improved BUT (MO treated, 19.4 seconds; BL treated, 8.3 seconds [P = 0.098]; untreated, 1.5 seconds [P = 0.008]). Conclusions Elovl1-deficient mice exhibited multiple MGD symptoms, which were improved by MO. Translational Relevance Our findings reveal the usefulness of Elovl1-deficient mice as a model for dry eye with MGD and suggest the potential of mineral oil eye drops as a treatment for this condition.

Although most mammalian fatty acids (FAs) are straight-chain, there also exist branched-chain FAs such as iso- and anteiso-FAs, especially in the meibomian glands. Meibum lipids, which are secreted from the meibomian glands and are important for dry eye prevention, contain abundant branched-chain lipids, such as cholesteryl esters and wax esters with chain-lengths of ≥C21 (very-long-chain; VLC). However, the exact tissue distribution of branched-chain lipids or the enzymes involved in the production of branched-chain VLCFAs has remained poorly understood. Here, we revealed that FA elongases ELOVL1, ELOVL3, and ELOVL7, of the seven mammalian ELOVL isozymes, elongated saturated branched-chain acyl-CoAs. ELOVL3 was highly active toward iso-C17:0 and anteiso-C17:0 acyl-CoAs and elongated them up to iso-C23:0 and anteiso-C25:0 acyl-CoAs, respectively. ELOVL1 elongated both iso- and anteiso-C23:0 acyl-CoAs to C25:0 acyl-CoAs. By establishing a liquid chromatography-tandem mass spectrometry method capable of separating branched- and straight-chain lipids, we showed that essentially all of the cholesteryl esters and 88% of the wax esters in the mouse meibomian glands are branched. In Elovl1 mutant mice, the levels of ≥C24:0 branched-chain cholesteryl esters and ≥C25:0 branched-chain wax esters were decreased, indicating that ELOVL1 indeed elongates branched-chain VLC acyl-CoAs in vivo. In addition, substantial amounts of ceramides containing branched-chain FAs were present in the skin, meibomian glands, and liver. Our findings provide new insights into the molecular mechanisms that create FA and lipid diversity.

BackgroundVery long-chain fatty acids (VLCFAs) are essential for functioning of biological membranes. ELOVL fatty acid elongase 1 catalyses elongation of saturated and monounsaturated C22-C26-VLCFAs. We studied two patients with a dominantELOVL1mutation. Independently, Kutkowska-Kaźmierczaket al.had investigated the same patients and found the same mutation. We extended our study towards additional biochemical, functional, and therapeutic aspects.MethodsWe did mutation screening by whole exome sequencing. RNA-sequencing was performed in patient and control fibroblasts. Ceramide and sphingomyelin levels were measured by LC-MS/MS. ELOVL1 activity was determined by a stable isotope-labelled [13C]malonyl-CoA elongation assay. ELOVL1 expression patterns were investigated by immunofluorescence,in situhybridisation and RT-qPCR. As treatment option, we investigated VLCFA loading of fibroblasts.ResultsBoth patients carried an identical heterozygousde novo ELOVL1mutation (c.494C>T, NM_001256399; p.S165F) not deriving from a founder allele. Patients suffered from epidermal hyperproliferation and increased keratinisation (ichthyosis). Hypomyelination of the central white matter explained spastic paraplegia and central nystagmus, while optic atrophy was causative for reduction of peripheral vision and visual acuity. The mutation abrogated ELOVL1 enzymatic activity and reduced ≥C24 ceramides and sphingomyelins in patient cells. Fibroblast loading with C22:0-VLCFAs increased C24:0-ceramides and sphingomyelins. We found competitive inhibition for ceramide and sphingomyelin synthesis between saturated and monounsaturated VLCFAs. Transcriptome analysis revealed upregulation of modules involved in epidermal development and keratinisation, and downregulation of genes for neurodevelopment, myelination, and synaptogenesis. Many regulated genes carried consensus proliferator-activated receptor (PPAR)α and PPARγ binding motifs in their 5’-regions.ConclusionA dominantELOVL1mutation causes a neuro-ichthyotic disorder possibly amenable to treatment with PPAR-modulating drugs.

Insulation by myelin lipids is essential to fast action potential conductivity: changes in their quality or amount can cause several neurologic disorders. Sjögren-Larsson syndrome (SLS) is one such disorder, which is caused by mutations in the fatty aldehyde dehydrogenase ALDH3A2. To date, the molecular mechanism underlying SLS pathology has remained unknown. In this study, we found that Aldh3a2 is expressed in oligodendrocytes and neurons in the mouse brain, and neurons of Aldh3a2 knockout (KO) mice exhibited impaired metabolism of the long-chain base, a component of sphingolipids. Aldh3a2 KO mice showed several abnormalities corresponding to SLS symptoms in behavioral tests, including increased paw slips on a balance beam and light-induced anxiety. In their brain tissue, 2-hydroxygalactosylceramide, an important lipid for myelin function and maintenance, was reduced by the inactivation of fatty acid 2-hydroxylase. Our findings provide important new insights into the molecular mechanisms responsible for neural pathogenesis caused by lipid metabolism abnormalities.-Kanetake, T., Sassa, T., Nojiri, K., Sawai, M., Hattori, S., Miyakawa, T., Kitamura, T., Kihara, A. Neural symptoms in a gene knockout mouse model of Sjögren-Larsson syndrome are associated with a decrease in 2-hydroxygalactosylceramide.

Sjögren-Larsson syndrome (SLS) is an inherited neurocutaneous disorder whose causative gene encodes the fatty aldehyde dehydrogenase ALDH3A2. To date, the detailed molecular mechanism of the skin pathology of SLS has remained largely unclear. We generated double-knockout (DKO) mice for Aldh3a2 and its homolog Aldh3b2 (a pseudogene in humans). These mice showed hyperkeratosis and reduced fatty aldehyde dehydrogenase activity and skin barrier function. The levels of ω-O-acylceramides (acylceramides), which are specialized ceramides essential for skin barrier function, in the epidermis of DKO mice were about 60% of those in wild-type mice. In the DKO mice, levels of acylceramide precursors (ω-hydroxy ceramides and triglycerides) were increased, suggesting that the final step of acylceramide production was inhibited. A decrease in acylceramide levels was also observed in human immortalized keratinocytes lacking ALDH3A2. Differentiated keratinocytes prepared from the DKO mice exhibited impaired long-chain base metabolism. Based on these results, we propose that the long-chain-base-derived fatty aldehydes that accumulate in DKO mice and SLS patients attack and inhibit the enzyme involved in the final step of acylceramide production. Our findings provide insight into the pathogenesis of the skin symptoms of SLS, i.e., decreased acylceramide production, and its molecular mechanism.

Differences among fatty acids (FAs) in chain length and number of double bonds create lipid diversity. FA elongation proceeds via a four-step reaction cycle, in which the 3-hydroxyacyl-CoA dehydratases (HACDs) HACD1-4 catalyze the third step. However, the contribution of each HACD to 3-hydroxyacyl-CoA dehydratase activity in certain tissues or in different FA elongation pathways remains unclear. HACD1 is specifically expressed in muscles and is a myopathy-causative gene. Here, we generated Hacd1 KO mice and observed that these mice had reduced body and skeletal muscle weights. In skeletal muscle, HACD1 mRNA expression was by far the highest among the HACDs. However, we observed only an similar to 40% reduction in HACD activity and no changes in membrane lipid composition in Hacd1-KO skeletal muscle, suggesting that some HACD activities are redundant. Moreover, when expressed in yeast, both HACD1 and HACD2 participated in saturated and monounsaturated FA elongation pathways. Disruption of HACD2 in the haploid human cell line HAP1 significantly reduced FA elongation activities toward both saturated and unsaturated FAs, and HACD1 HACD2 double disruption resulted in a further reduction. Overexpressed HACD3 exhibited weak activity in saturated and monounsaturated FA elongation pathways, and no activity was detected for HACD4. We therefore conclude that HACD1 and HACD2 exhibit redundant activities in a wide range of FA elongation pathways, including those for saturated to polyunsaturated FAs, with HACD2 being the major 3-hydroxyacyl-CoA dehydratase. Our findings are important for furthering the understanding of the molecular mechanisms in FA elongation and diversity.

Meibum lipids form a lipid layer on the outermost side of the tear film and function to prevent water evaporation and reduce surface tension. (O-Acyl)-omega-hydroxy fatty acids (OAHFAs), a subclass of these lipids, are thought to be involved in connecting the lipid and aqueous layers in tears, although their actual function and synthesis pathway have to date remained unclear. Here, we reveal that the fatty acid omega-hydroxylase Cyp4f39 is involved in OAHFA production. Cyp4f39-deficient mice exhibited damaged corneal epithelium and shortening of tear film break-up time, both indicative of dry eye disease. In addition, tears accumulated on the lower eyelid side, indicating increased tear surface tension. In Cyp4f39-deficient mice, the production of wax diesters (type 1 omega and 2 omega) and cholesteryl OAHFAs was also impaired. These OAHFA derivatives show intermediate polarity among meibum lipids, suggesting that OAHFAs and their derivatives contribute to lipid polarity gradient formation for tear film stabilization.

The skin permeability barrier is indispensable for maintaining water inside the body and preventing the invasion of pathogens and allergens; abnormalities lead to skin disorders such as atopic dermatitis and ichthyosis. Acylceramide is an essential lipid for skin barrier formation, and CYP4F22 is a fatty acid ω-hydroxylase involved in its synthesis. Mutations in CYP4F22 cause autosomal recessive congenital ichthyosis, although the symptoms vary among mutation sites and types. Here, we generated knockout mice deficient in Cyp4f39, the mouse ortholog of human CYP4F22, to investigate the effects of completely abrogating the function of the fatty acid ω-hydroxylase involved in acylceramide production on skin barrier formation. Cyp4f39 knockout mice died within 8 hours of birth. Large increases in transepidermal water loss and penetration of a dye from outside the body were observed, indicating severe skin barrier dysfunction. Histologic analyses of the epidermis revealed impairment of lipid lamella formation, accumulation of corneodesmosomes in the stratum corneum, and persistence of periderm. In addition, lipid analyses by mass spectrometry showed almost complete loss of acylceramide and its precursor ω-hydroxy ceramide. In conclusion, our findings provide clues to the molecular mechanisms of skin barrier abnormalities and the pathogenesis of ichthyosis caused by Cyp4f39 and CYP4F22 by association.

The fatty aldehyde dehydrogenase (FALDH) ALDH3A2 is the causative gene of Sjogren Larsson syndrome (SLS). To date, the molecular mechanism underlying the symptoms characterizing SLS has been poorly understood. Using Aldh3a2(-/-) mice, we found here that Aldh3a2 was the major FALDH active in undifferentiated keratinocytes. Long-chain base metabolism was greatly impaired in Aldh3a2(-/-) keratinocytes. Phenotypically, the intercellular spaces were widened in the basal layer of the Aldh3a2(-/-) epidermis due to hyperproliferation of keratinocytes. Furthermore, oxidative stress-induced genes were upregulated in Aldh3a2(-/-) keratinocytes. Upon keratinocyte differentiation, the activity of another FALDH, Aldh3b2, surpassed that of Aldh3a2. As a result, Aldh3a2(-/-) mice were indistinguishable from wild-type mice in terms of their whole epidermis FALDH activity, and their skin barrier function was uncompromised under normal conditions. However, perturbation of the stratum corneum caused increased transepidermal water loss and delayed barrier recovery in Aldh3a2(-/-) mice. In conclusion, Aldh3a2(-/-) mice replicated some aspects of SLS symptoms, especially at the basal layer of the epidermis. Our results suggest that hyperproliferation of keratinocytes via oxidative stress responses may partly contribute to the ichthyosis symptoms of SLS.

Ceramide and complex sphingolipids regulate important cellular functions including cell growth, apoptosis, and signaling. Dysregulation of sphingolipid metabolism leads to pathological consequences such as sphingolipidoses and insulin resistance. Ceramides in mammals vary greatly in their acyl-chain composition: six different ceramide synthase isozymes (CERS1-6) that exhibit distinct substrate specificity and tissue distribution account for this diversity. In the present study, we demonstrated that CERS2-6 were phosphorylated at the cytoplasmic C-terminal regions. Most of the phosphorylated residues conformed to a consensus motif for phosphorylation by casein kinase 2 (CK2), and treatment of cells with the CK2-specific inhibitor CX-4945 lowered the phosphorylation levels of CERS2, -4, -5, and -6. Phosphorylation of CERS2 was especially important for its catalytic activity, acting mainly by increasing its V-max value. Phosphorylation modestly increased the catalytic activities of CERS4 and -5 and mildly increased those of CERS3 and -6. Dephosphorylation of endogenous ceramide synthases in the mouse brain led to severely reduced activity toward the Cers2 substrates C22:0/C24:0-CoAs and modestly reduced activity toward the Cers5/6 substrate C16:0-CoA. These results suggest that the phosphorylation of ceramide synthases may be a key regulatory point in the control of the distribution and levels of sphingolipids of various acyl-chain lengths.

Very long-chain fatty acids (VLCFAs) are essential for functioning of biological membranes. ELOVL fatty acid elongase 1 catalyses elongation of saturated and monounsaturated C22-C26-VLCFAs. We studied two patients with a dominantWe did mutation screening by whole exome sequencing. RNA-sequencing was performed in patient and control fibroblasts. Ceramide and sphingomyelin levels were measured by LC-MS/MS. ELOVL1 activity was determined by a stable isotope-labelled [Both patients carried an identical heterozygousA dominant

Lipids secreted from the meibomian gland (meibum) form the superficial layer of the tear film and prevent water evaporation from the ocular surface and infection. Here, we identified the fatty acid (FA) elongases responsible for the synthesis of very long-chain FAs (VLCFAs) that constitute the meibum lipids. Elongation of VLCFAs (ELOVL)1 is primarily responsible for the production of saturated VLCFAs, whereas ELOVL1, ELOVL3, and ELOVL4 redundantly participate in the synthesis of monounsaturated VLCFAs. Gene disruption of Elovl1 in mice shortened acyl moieties in the 2 major meibum lipids: cholesteryl esters and wax esters. These changes were associated with dry eye phenotypes, including increases in eye-blink frequency and water evaporation from the ocular surface at younger ages. Aged Elovl1 mutant mice developed corneal opacity with vascular invasion, accompanied by epidermalization of the cornea. Thus, in addition to the well-known VLC ceramides (acylceramides) in the epidermis, VLC meibum lipids are barrier-forming lipids.-Sassa, T., Tadaki, M., Kiyonari, H., Kihara, A. Very long-chain tear film lipids produced by fatty acid elongase ELOVL1 prevent dry eye disease in mice.

NIPAL4 is one of the causative genes for autosomal recessive congenital ichthyosis. However, the role of NIPAL4 in skin barrier formation and the molecular mechanism of ichthyosis pathology caused by NIPAL4 mutations, have not yet been determined. Here, we found that Nipal4-knockout (KO) mice exhibited neonatal lethality due to skin barrier defects. Histological analyses showed several morphological abnormalities in the Nipal4-KO epidermis, including impairment of lipid multilayer structure formation, hyperkeratosis, immature keratohyalin granules, and developed heterochromatin structures. The levels of the skin barrier lipid acylceramide were decreased in Nipal4-KO mice. Expression of genes involved in skin barrier formation normally increases during keratinocyte differentiation, in which chromatin remodeling is involved. However, the induction of Krt1, Lor, Flg, Elovl1, and Dgat2 was impaired in Nipal4-KO mice. NIPAL4 is a putative Mg2+ transporter, and Mg2+ concentration in differentiated keratinocytes of Nipal4-KO mice was indeed lower than that of wild-type mice. Our results suggest that low Mg2+ concentration causes aberration in the proper chromatin remodeling process, which in turn leads to failure of differentiation-dependent gene induction in keratinocytes. Our findings provide insights into Mg2+-dependent regulation of gene expression and skin barrier formation during keratinocyte differentiation.

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by impaired degradation of very long-chain fatty acids (VLCFAs) due to mutations in the ABCD1 gene responsible for VLCFA transport into peroxisomes. Lorenzo's oil, a 4: 1 mixture of glyceryl trioleate and glyceryl trierucate, has been used to reduce the saturated VLCFA level in the plasma of X-ALD patients; however, the mechanism by which this occurs remains elusive. We report the biochemical characterization of Lorenzo's oil activity toward elongation of very long-chain fatty acid (ELOVL) 1, the primary enzyme responsible for the synthesis of saturated and monounsaturated VLCFAs. Oleic and erucic acids inhibited ELOVL1, and, moreover, their 4:1 mixture (the FA composition of Lorenzo's oil) exhibited the most potent inhibitory activity. The kinetics analysis revealed that this was a mixed (not a competitive) inhibition. At the cellular level, treatment with the 4:1 mixture reduced the level of SM with a saturated VLCFA accompanied by an increased level of SM with a monounsaturated VLCFA, probably due to the incorporation of erucic acid into the FA elongation cycle.(jlr) These results suggest that inhibition of ELOVL1 may be an underlying mechanism by which Lorenzo's oil exerts its action.

Background: The P182L mutation in the trans-2-enoyl-CoA reductase (TER) gene required for very long-chain fatty acid (VLCFA) synthesis causes nonsyndromic mental retardation. Results: This mutation reduces the activity and stability of the TER enzyme. Conclusion: The impaired TER function affects VLCFA synthesis and thereby alters the cellular sphingolipid profile. Significance: Maintenance of a proper VLCFA level may be important for neural function. Very long-chain fatty acids (VLCFAs, chain length >C20) exist in tissues throughout the body and are synthesized by repetition of the fatty acid (FA) elongation cycle composed of four successive enzymatic reactions. In mammals, the TER gene is the only gene encoding trans-2-enoyl-CoA reductase, which catalyzes the fourth reaction in the FA elongation cycle. The TER P182L mutation is the pathogenic mutation for nonsyndromic mental retardation. This mutation substitutes a leucine for a proline residue at amino acid 182 in the TER enzyme. Currently, the mechanism by which the TER P182L mutation causes nonsyndromic mental retardation is unknown. To understand the effect of this mutation on the TER enzyme and VLCFA synthesis, we have biochemically characterized the TER P182L mutant enzyme using yeast and mammalian cells transfected with the TER P182L mutant gene and analyzed the FA elongation cycle in the B-lymphoblastoid cell line with the homozygous TER P182L mutation (TERP182L/P182L B-lymphoblastoid cell line). We have found that TER P182L mutant enzyme exhibits reduced trans-2-enoyl-CoA reductase activity and protein stability, thereby impairing VLCFA synthesis and, in turn, altering the sphingolipid profile (i.e. decreased level of C24 sphingomyelin and C24 ceramide) in the TERP182L/P182L B-lymphoblastoid cell line. We have also found that in addition to the TER enzyme-catalyzed fourth reaction, the third reaction in the FA elongation cycle is affected by the TER P182L mutation. These findings provide new insight into the biochemical defects associated with this genetic mutation.

Intracellular Ca2+ imaging has widely been used to visualize intracellular signals, but the application in an intact animal is still limited due to difficulty of the indicator loading. In addition, the motion of the living animal produces artifacts. To investigate Ca2+ signaling at early embryonic stage, we established transgenic zebrafish line expressing a genetically encoded Ca2+ indicator, cameleon YC2.60, driven by a constitutively active promoter, hspa8. Although the embryo dynamically changes its morphology, the motion artifact could be canceled out by taking the advantage of YC2.60 as a ratiometric indicator. The transgenic zebrafish was used to visualize the propagation of cytosolic Ca2+ during the early embryonic stage upon fertilization and along cleavage furrow, and the rise in Ca2+ in the myocytes contracting spontaneously in the embryo. We also established a transgenic zebrafish line expressing YC2.60 targeted to the mitochondria. The rise in mitochondrial Ca2+ was rather sustained (≈2 min), which is consistent with the requirement of ATP refilling since the mitochondrial Ca2+ upregulates rate-limiting enzymes of Krebs cycle. This is in contrast with the transient rise in the cytosol Ca2+ that directly evokes the muscle contraction. These transgenic zebrafish lines are expected to serve as useful tools further Ca2+ imaging in vivo.

One of the most fascinating questions in evolutionary biology is how traits unique to humans, such as their high cognitive abilities, erect bipedalism, and hairless skin, are encoded in the genome. Recent advances in genomics have begun to reveal differences between the genomes of the great apes. It has become evident that one of the many mutation types, segmental duplication, has drastically increased in the primate genomes, and most remarkably in the human genome. Genes contained in these segmental duplications have a tremendous potential to cause genetic innovation, probably accounting for the acquisition of human-specific traits. In this review, I begin with an overview of the genes, which have increased their copy number specifically in the human lineage, following its separation from the common ancestor with our closest living relative, the chimpanzee. Then, I introduce the recent experimental approaches, focusing on SRGAP2, which has been partially duplicated, to elucidate the role of SRGAP2 protein and its human-specific paralogs in human brain development and evolution.

Sphingolipids, major lipid components of the eukaryotic plasma membrane, have a variety of physiological functions and have been associated with many diseases. They have also been implicated in apoptosis. Sphingolipids are heterogeneous in their acyl chain length, with long-chain (C16) and very long-chain (C24) sphingolipids being predominant in most mammalian tissues. We demonstrate that knockdown of ELOVL1 or CERS2, which catalyze synthesis of C24 acyl-CoAs and C24 ceramide, respectively, drastically reduced C24 sphingolipid levels with a complementary increase in C16 sphingolipids. Under ELOVL1 or CERS2 knockdown conditions, cisplatin-induced apoptosis significantly increased. Enhanced sensitivity to cisplatin-induced apoptosis exhibited close correlation with increases in caspase-3/7 activity. No significant alterations in sphingolipid metabolism such as ceramide generation were apparent with the cisplatin-induced apoptosis, and inhibitors of ceramide generation had no effect on the apoptosis. Apoptosis induced by UV radiation or C6 ceramides also increased in ELOVL1 or CERS2 knockdown cells. Changes in the composition of sphingolipid chain length may affect susceptibility to stimuli-induced apoptosis by affecting the properties of cell membranes, such as lipid microdomain/raft formation.

Very long-chain fatty acids (VLCFAs) have a variety of physiological functions and are related to numerous disorders. The key step of VLCFA elongation is catalyzed by members of the elongase family, ELOVLs. Mammals have seven ELOVLs (ELOVL1-7), yet none of them has been purified and analyzed. In the presented study we purified ELOVL7 and measured its activity by reconstituting it into proteoliposomes. Purified ELOVL7 exhibited high activity toward acyl-CoAs with C18 carbon chain length. The calculated Km values toward C18:3(n-3)-CoA and malonyl-CoA were both in the μM range. We also found that progression of the VLCFA cycle enhances ELOVL7 activity.

Very long-chain fatty acids (VLCFAs) exert a variety of cellular functions and are associated with numerous diseases. However, the precise pathway behind their elongation has remained elusive. Moreover, few regulatory mechanisms for VLCFAs synthesis have been identified. Elongases catalyze the first of four steps in the VLCFA elongation cycle; mammals have seven elongases (ELOVL1–7). In the present study, we determined the precise substrate specificities of all the ELOVLs by in vitro analyses. Particularly notable was the high activity exhibited by ELOVL1 toward saturated and monounsaturated C20- and C22-CoAs, and that it was essential for the production of C24 sphingolipids, which are unique in their capacity to interdigitate within the membrane as a result of their long chain length. We further established that ELOVL1 activity is regulated with the ceramide synthase CERS2, an enzyme essential for C24 sphingolipid synthesis. This regulation may ensure that the production of C24-CoA by elongation is coordinated with its utilization. Finally, knockdown of ELOVL1 caused a reduction in the activity of the Src kinase LYN, confirming that C24-sphingolipids are particularly important in membrane microdomain function.

Fatty acids (FAs) are highly diverse in terms of carbon (C) chain-length and number of double bonds. FAs with C>20 are called very long-chain fatty acids (VLCFAs). VLCFAs are found not only as constituents of cellular lipids such as sphingolipids and glycerophospholipids but also as precursors of lipid mediators. Our understanding on the function of VLCFAs is growing in parallel with the identification of enzymes involved in VLCFA synthesis or degradation. A variety of inherited diseases, such as ichthyosis, macular degeneration, myopathy, mental retardation, and demyelination, are caused by mutations in the genes encoding VLCFA metabolizing enzymes. In this review, we describe mammalian VLCFAs by highlighting their tissue distribution and metabolic pathways, and we discuss responsible genes and enzymes with reference to their roles in pathophysiology.

The sphingolipid backbone ceramide (Cer) is a major component of lipid lamellae in the stratum corneum of epidermis and has a pivotal role in epidermal barrier formation. Unlike Cers in other tissues, Cers in epidermis contain extremely long fatty acids (FAs). Decreases in epidermal Cer levels, as well as changes in their FA chain lengths, cause several cutaneous disorders. However, the molecular mechanisms that produce such extremely long Cers and determine their chain lengths are poorly understood. We generated mice deficient in the Elovl1 gene, which encodes the FA elongase responsible for producing C20 to C28 FAs. Elovl1 knockout mice died shortly after birth due to epidermal barrier defects. The lipid lamellae in the stratum corneum were largely diminished in these mice. In the epidermis of the Elovl1-null mice, the levels of Cers with ≥C26 FAs were decreased, while those of Cers with ≤C24 FAs were increased. In contrast, the levels of C24 sphingomyelin were reduced, accompanied by an increase in C20 sphingomyelin levels. Two ceramide synthases, CerS2 and CerS3, expressed in an epidermal layer-specific manner, regulate Elovl1 to produce acyl coenzyme As with different chain lengths. Elovl1 is a key determinant of epidermal Cer chain length and is essential for permeability barrier formation.

The lipid lamellae in the stratum corneum is important for the epidermal permeability barrier. The lipid lamellae component ceramide (CER), comprising an ultra long-chain (ULC) fatty acid (FA) of >= 26 carbons (ULC CER), plays an essential role in barrier formation. ULC acyl-CoAs, produced by the FA elongase ELOVL4, are converted to ULC CERs by the CER synthase CERS3. In the presented study, we observed that ELOVL4 and CERS3 mRNAs increased during keratinocyte differentiation in vivo and in vitro. We also determined that peroxisome proliferator-activated receptor beta/delta is involved in the up-regulation of the mRNAs. Knockdown of CERS3 caused a reduction in the elongase activities toward ULC acyl-CoAs, suggesting that CERS3 positively regulates ULCFA. Thus, we reveal that the two key players in ULC CER production in epidermis, CERS3 and ELOVL4, are coordinately regulated at both the transcriptional and enzymatic levels.

SUMMARY Structural genomic variations represent a major driving force of evolution, and a burst of large segmental gene duplications occurred in the human lineage during its separation from nonhuman primates. SRGAP2, a gene recently implicated in neocortical development, has undergone two human-specific duplications. Here, we find that both duplications (SRGAP2B and SRGAP2C) are partial and encode a truncated F-BAR domain. SRGAP2C is expressed in the developing and adult human brain and dimerizes with ancestral SRGAP2 to inhibit its function. In the mouse neocortex, SRGAP2 promotes spine maturation and limits spine density. Expression of SRGAP2C phenocopies SRGAP2 deficiency. It underlies sustained radial migration and leads to the emergence of human-specific features, including neoteny during spine maturation and increased density of longer spines. These results suggest that inhibition of SRGAP2 function by its human-specific paralogs has contributed to the evolution of the human neocortex and plays an important role during human brain development.

The zebrafish dorsal habenula (dHb) shows conspicuous asymmetry in its connection with the interpeduncular nucleus (IPN) and is equivalent to the mammalian medial habenula. Genetic inactivation of the lateral subnucleus of dHb (dHbL) biased fish towards freezing rather than the normal flight response to a conditioned fear stimulus, suggesting that the dHbL-IPN pathway is important for controlling experience-dependent modification of fear responses.

During brain development, proper neuronal migration and morphogenesis is critical for the establishment of functional neural circuits. Here we report that srGAP2 negatively regulates neuronal migration and induces neurite outgrowth and branching through the ability of its F-BAR domain to induce filopodia-like membrane protrusions resembling those induced by I-BAR domains in vivo and in vitro. Previous work has suggested that in non-neuronal cells, filopodia dynamics decreases the rate of cell migration and the persistence of leading edge protrusions. srGAP2 knockdown reduces leading process branching and increases the rate of neuronal migration in vivo. Overexpression of srGAP2 or its F-BAR domain has the opposite effects, increasing leading process branching and decreasing migration. These results (1) suggest that F-BAR domains are functionally diverse and (2) highlight the functional importance of proteins directly regulating membrane deformation for proper neuronal migration and morphogenesis.

Recent studies have demonstrated that the topography of thalamocortical (TC) axon projections is initiated before they reach the cortex, in the ventral telencephalon (VTel). However, at this point, the molecular mechanisms patterning the topography of TC projections in the VTel remains poorly understood. Here, we show that a long-range, high-rostral to low-caudal gradient of Netrin-1 in the VTel is required in vivo for the topographic sorting of TC axons to distinct cortical domains. We demonstrate that Netrin-1 is a chemoattractant for rostral thalamic axons but functions as a chemorepulsive cue for caudal thalamic axons. In accordance with this model, DCC is expressed in a high-rostromedial to low-caudolateral gradient in the dorsal thalamus (DTh), whereas three Unc5 receptors (Unc5A–C) show graded expression in the reverse orientation. Finally, we show that DCC is required for the attraction of rostromedial thalamic axons to the Netrin-1–rich, anterior part of the VTel, whereas DCC and Unc5A/C receptors are required for the repulsion of caudolateral TC axons from the same Netrin-1–rich region of the VTel. Our results demonstrate that a long-range gradient of Netrin-1 acts as a counteracting force from ephrin-A5 to control the topography of TC projections before they enter the cortex., Author Summary The functional properties of each structure in the central nervous system are critically dependent on the precision of neuronal connectivity. The cerebral cortex in particular is a highly organized structure divided into many distinct cortical areas underlying important sensory, motor, and cognitive functions in the brain. Each primary cortical area receives its synaptic inputs from the periphery via the dorsal thalamus. The main relay station for sensory information to the cortex, the thalamus, can be divided into specific nuclei projecting topographically to individual cortical areas. How is the complex topography of thalamic axon projection to individual cortical areas specified during development? Recent evidence demonstrated that thalamic axons are routed to different cortical domains before they enter the cortex, by putative axon guidance cues present in the ventral forebrain. In the present study, we provide evidence that a secreted axon guidance cue, Netrin-1, expressed in a long-range gradient in the ventral forebrain, plays a critical role in the establishment of the topography of thalamic projections by directing different subsets of axons to specific cortical domains. These results provide important insights into the molecular mechanisms responsible for shaping the topographical patterns of thalamocortical axon projections in mammals., A long-range gradient of Netrin-1 plays a critical role in the specification of the topography of thalamocortical projections in the ventral telencephalon. The function of Netrin-1 requires both its attractive and repulsive functions to guide different subsets of thalamic axons to specific cortical domains.

The dorsal hindbrain includes distinct classes of neurons for processing various sensory stimuli, but the developmental aspects of these neurons remain largely unknown. We identify here two distinct classes of neurons in the dorsal hindbrain of developing zebrafish: (1) neurons that express the inhibitory neuronal marker Gad1/2, and (2) neurons that express the zn-5 antigen and Lhx2/9 and require the basic helix–loop–helix transcription factor Atoh1a for development. Neurons were traced to their axon terminals by expressing green fluorescent protein using the Gal4VP16-UAS (UAS, upstream activating sequences) system in combination with the promoter/enhancer regions of gad2 for the Gad1/2(+) neurons and zic1 for the zn-5(+)Lhx2/9(+) neurons. The Gad1/2(+) neurons projected to the contralateral hindbrain, while the zn-5(+)Lhx2/9(+) neurons projected to the contralateral midbrain torus semicircularis, suggesting a role in auditory and lateral line sensory processing. Comparison of these projections with those from the cochlear nuclei to the inferior colliculus in mammals suggests similarities across vertebrate species. Developmental Dynamics 236:706–718, 2007. © 2007 Wiley-Liss, Inc.