Comparison of skin barrier abnormalities and epidermal ceramide profiles among three ω-O-acylceramide synthesis-deficient mouse strains.

BACKGROUND: The epidermis contains many structurally diverse ceramides, which form the skin permeability barrier (skin barrier). Mutations in genes involved in the synthesis of ω-O-acylceramides (acylceramides) and protein-bound ceramides cause ichthyosis. OBJECTIVE: We aimed to elucidate the relationship between the degree of skin barrier impairment and changes in epidermal ceramide profiles caused by mutations in acylceramide synthesis genes. METHODS: Knockout (KO) mice of three genes-fatty acid (FA) ω-hydroxylase Cyp4f39 (human CYP4F22 ortholog), FA elongase Elovl1, and acyl-CoA synthetase Fatp4-were subjected to transepidermal water loss measurement, toluidine blue staining, and epidermal ceramide profiling via liquid chromatography coupled with tandem mass spectrometry. RESULTS: Transepidermal water loss was highest in Cyp4f39 KO mice, followed by Elovl1 KO and Fatp4 KO mice, and Cyp4f39 KO mice also showed the strongest degree of toluidine blue staining. In Cyp4f39 KO, Elovl1 KO, and Fatp4 KO mice, acylceramide levels were 0.6%, 1.6%, and 12%, respectively, of those in wild-type mice. Protein-bound ceramide levels were 0.2%, 30%, and 33%, respectively, of those in wild-type mice. We also observed a near-complete absence of ω-hydroxy ceramides in Cyp4f39 KO mice, reduced total ceramide levels and shortened FA moieties in Elovl1 KO mice, and increased hydroxylated ceramide levels and slightly shortened FA moieties in Fatp4 KO mice. CONCLUSIONS: The degree of reduction in protein-bound ceramide levels is probably related to the severity of skin barrier defects in these three strains. However, reduced acylceramide levels and other changes in ceramide composition unique to each KO strain are also involved.

Involvement of ω-O-acylceramides and protein-bound ceramides in oral permeability barrier formation.

The permeability barrier present in the oral cavity is critical for protection from infection. Although lipids have properties suitable for permeability barrier formation, little is known about their role in oral barrier formation. Here, we show the presence of ω-O-acylceramides (acylceramides) and protein-bound ceramides, which are essential for the formation of permeability barriers in the epidermis, in the oral mucosae (buccal and tongue mucosae), esophagus, and stomach in mice. Conditional knockout of the fatty acid elongase Elovl1, which is involved in the synthesis of ≥C24 ceramides including acylceramides and protein-bound ceramides, in the oral mucosae and esophagus causes increased pigment penetration into the mucosal epithelium of the tongue and enhanced aversive responses to capsaicin-containing water. We find acylceramides in the buccal and gingival mucosae and protein-bound ceramides in the gingival mucosa in humans. These results indicate that acylceramides and protein-bound ceramides are important for oral permeability barrier formation.

Incomplete Elongation of Ultra-long-chain Polyunsaturated Acyl-CoAs by the Fatty Acid Elongase ELOVL4 in Spinocerebellar Ataxia Type 34.

Spinocerebellar ataxias (SCAs) are autosomal dominant diseases characterized by cerebellar atrophy and ataxia. The SCA subtype SCA34 is caused by specific mutations in the gene ELOVL4, which encodes a fatty acid (FA) elongase that synthesizes ultra-long-chain (ULC; ≥C26) FAs. However, the pathogenesis and molecular mechanism that confers dominant inheritance remains unknown. Here, a cell-based assay demonstrated that each of the five known SCA34 mutants produced shorter ULC polyunsaturated FA-containing phosphatidylcholines (ULC-PCs) than wild-type protein, in the following order of severity: Q180P and T233M > W246G > I171T and L168F. Next, we generated knock-in mouse embryonic stem cells that contained heterozygous Q180P, heterozygous W246G, or homozygous W246G mutations. Neuronal differentiation-dependent production of ULC-PCs was reduced in heterozygous Q180P and homozygous W246G cells relative to control cells, and we observed shortening of the FA moiety in all mutant cells. This FA shortening was consistent with our prediction that amino acid residues substituted by SCA34 mutations are located in the transmembrane helices that interact with the ω-end region of the FA moiety of the substrate acyl-CoA. Hence, reduced levels and shortening of ULC-PCs in neurons may cause SCA34, and incomplete elongation of ULC polyunsaturated acyl-CoAs by mutated ELOVL4 may induce dominant inheritance.

Ceramide profiling of stratum corneum in Sjögren-Larsson syndrome.

BACKGROUND: 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. OBJECTIVES: To determine the ceramide classes/species responsible for SLS pathogenesis and the enzymes that are impaired in SLS. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Hypomyelinating spastic dyskinesia and ichthyosis caused by a homozygous splice site mutation leading to exon skipping in ELOVL1.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. DISCUSSION: 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.

Erratum: Diverse meibum lipids produced by Awat1 and Awat2 are important for stabilizing tear film and protecting the ocular surface.

[This corrects the article DOI: 10.1016/j.isci.2021.102478.].

Impaired Skin Barrier Function Due to Reduced ω-O-Acylceramide Levels in a Mouse Model of Sjögren-Larsson Syndrome.

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.

Diverse meibum lipids produced by Awat1 and Awat2 are important for stabilizing tear film and protecting the ocular surface.

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.

Improvement of Evaporative Dry Eye With Meibomian Gland Dysfunction in Model Mice by Treatment With Ophthalmic Solution Containing Mineral Oil.

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.

Comprehensive stratum corneum ceramide profiling reveals reduced acylceramides in ichthyosis patient with CERS3 mutations.

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 ω-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 α-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.

Production of branched-chain very-long-chain fatty acids by fatty acid elongases and their tissue distribution in mammals.

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.

Lipid polarity gradient formed by ω-hydroxy lipids in tear film prevents dry eye disease.

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)-ω-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 ω-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 1ω and 2ω) 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.

Severe Skin Permeability Barrier Dysfunction in Knockout Mice Deficient in a Fatty Acid ω-Hydroxylase Crucial to Acylceramide Production.

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.

Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration.

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.

Reduced chain length in myelin sphingolipids and poorer motor coordination in mice deficient in the fatty acid elongase Elovl1.

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.

Neural symptoms in a gene knockout mouse model of Sjögren-Larsson syndrome are associated with a decrease in 2-hydroxygalactosylceramide.

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.

De novo mutation in ELOVL1 causes ichthyosis, acanthosis nigricans, hypomyelination, spastic paraplegia, high frequency deafness and optic atrophy.

BACKGROUND: 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 dominant ELOVL1 mutation. Independently, Kutkowska-Kazmierczak et al. had investigated the same patients and found the same mutation. We extended our study towards additional biochemical, functional, and therapeutic aspects. METHODS: We 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 situ hybridisation and RT-qPCR. As treatment option, we investigated VLCFA loading of fibroblasts. RESULTS: Both patients carried an identical heterozygous de novo ELOVL1 mutation (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. CONCLUSION: A dominant ELOVL1 mutation causes a neuro-ichthyotic disorder possibly amenable to treatment with PPAR-modulating drugs.

Very long-chain tear film lipids produced by fatty acid elongase ELOVL1 prevent dry eye disease in mice.

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.

Decreased Skin Barrier Lipid Acylceramide and Differentiation-Dependent Gene Expression in Ichthyosis Gene Nipal4-Knockout 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.