Mutations in PNPLA1 have been identified as causative for autosomal recessive congenital ichthyosis in humans and dogs. So far, the underlying molecular mechanisms are unknown. In this study, we generated and characterized PNPLA1-deficient mice and found that PNPLA1 is crucial for epidermal sphingolipid synthesis. The absence of functional PNPLA1 in mice impaired the formation of omega-O-acylceramides and led to an accumulation of nonesterified omega-hydroxy-ceramides. As a consequence, PNPLA1-deficient mice lacked a functional corneocyte-bound lipid envelope leading to a severe skin barrier defect and premature death of newborn animals. Functional analyses of differentiated keratinocytes from a patient with mutated PNPLA1 demonstrated an identical defect in omega-O-acylceramide synthesis in human cells, indicating that PNPLA1 function is conserved among mammals and indispensable for normal skin physiology. Notably, topical application of epidermal lipids from wild-type onto Pnpla1-mutant mice promoted rebuilding of the corneocyte-bound lipid envelope, indicating that supplementation of ichthyotic skin with omega-O-acylceramides might be a therapeutic approach for the treatment of skin symptoms in individuals affected by omega-O-acylceramide deficiency.

Loss-of-function mutations in arachidonate lipoxygenase 12B (ALOX12B) are an important cause of autosomal recessive congenital ichthyosis (ARCI). 12R-lipoxygenase (12R-LOX), the protein product of ALOX12B, has been proposed to covalently bind the corneocyte lipid envelope (CLE) to the proteinaceous corneocyte envelope, thereby providing a scaffold for the assembly of barrier-providing, mature lipid lamellae. To test this hypothesis, an in-depth ultrastructural examination of CLEs was performed in ALOX12B^-/- human and Alox12b^-/- mouse epidermis, extracting samples with pyridine to distinguish covalently attached CLEs from unbound (ie, noncovalently bound) CLEs. ALOX12B^--/- stratum corneum contained abundant pyridine-extractable (ie, unbound) CLEs, compared with normal stratum corneum. These unbound CLEs were associated with defective post-secretory lipid processing, and were specific to 12R-LOX deficiency, because they were not observed with deficiency of the related ARCI-associated proteins, patatin-like phospholipase 1 (Pnpla1) or abhydrolase domain containing 5 (Abhd5). These results suggest that 12R-LOX contributes specifically to CLE-corneocyte envelope cross-linking, which appears to be a prerequisite for post-secretory lipid processing, and provide insights into the pathogenesis of 12R-LOX deficiency in this subtype of ARCI, as well as other conditions that display a defective CLE.

Adipose triglyceride lipase (ATGL) and its coactivator comparative gene identification-58 (CGI-58) are limiting in cellular triglyceride catabolism. Although ATGL deficiency is compatible with normal skin development, mice globally lacking CGI-58 die postnatally and exhibit a severe epidermal permeability barrier defect, which may originate from epidermal and/or peripheral changes in lipid and energy metabolism. Here, we show that epidermis-specific disruption of CGI-58 is sufficient to provoke a defect in the formation of a functional corneocyte lipid envelope linked to impaired ω-O-acylceramide synthesis. As a result, epidermis-specific CGI-58-deficient mice show severe skin dysfunction, arguing for a tissue autonomous cause of disease development. Defective skin permeability barrier formation in global CGI-58-deficient mice could be reversed via transgenic restoration of CGI-58 expression in differentiated but not basal keratinocytes suggesting that CGI-58 is essential for lipid metabolism in suprabasal epidermal layers. The compatibility of ATGL deficiency with normal epidermal function indicated that CGI-58 may stimulate an epidermal triglyceride lipase beyond ATGL required for the adequate provision of fatty acids as a substrate for ω-O-acylceramide synthesis. Pharmacological inhibition of ATGL enzyme activity similarly reduced triglyceride-hydrolytic activities in wild-type and CGI-58 overexpressing epidermis implicating that CGI-58 participates in ω-O-acylceramide biogenesis independent of its role as a coactivator of epidermal triglyceride catabolism.

The corneocyte lipid envelope (CLE), a monolayer of ω-hydroxyceramides whose function(s) remain(s) uncertain, is absent in patients with autosomal recessive congenital ichthyoses with mutations in enzymes that regulate epidermal lipid synthesis. Secreted lipids fail to transform into lamellar membranes in certain autosomal recessive congenital ichthyosis epidermis, suggesting the CLE provides a scaffold for the extracellular lamellae. However, because cornified envelopes are attenuated in these autosomal recessive congenital ichthyoses, the CLE may also provide a scaffold for subjacent cornified envelope formation, evidenced by restoration of cornified envelopes after CLE rescue. We provide multiple lines of evidence that the CLE originates as lamellar body-limiting membranes fuse with the plasma membrane: (i) ABCA12 patients and Abca12^-/- mice display normal CLEs; (ii) CLEs are normal in Netherton syndrome, despite destruction of secreted LB contents; (iii) CLEs are absent in VSP33B-negative patients; (iv) limiting membranes of lamellar bodies are defective in lipid-synthetic autosomal recessive congenital ichthyoses; and (v) lipoxygenases, lipase activity, and LIPN co-localize within putative lamellar bodies.

The corneocyte lipid envelope (CLE), a monolayer of ω-hydroxyceramides whose function(s) remain(s) uncertain, is absent in patients with autosomal recessive congenital ichthyoses with mutations in enzymes that regulate epidermal lipid synthesis. Secreted lipids fail to transform into lamellar membranes in certain autosomal recessive congenital ichthyosis epidermis, suggesting the CLE provides a scaffold for the extracellular lamellae. However, because cornified envelopes are attenuated in these autosomal recessive congenital ichthyoses, the CLE may also provide a scaffold for subjacent cornified envelope formation, evidenced by restoration of cornified envelopes after CLE rescue. We provide multiple lines of evidence that the CLE originates as lamellar body-limiting membranes fuse with the plasma membrane: (i) ABCA12 patients and Abca12^-/- mice display normal CLEs; (ii) CLEs are normal in Netherton syndrome, despite destruction of secreted LB contents; (iii) CLEs are absent in VSP33B-negative patients; (iv) limiting membranes of lamellar bodies are defective in lipid-synthetic autosomal recessive congenital ichthyoses; and (v) lipoxygenases, lipase activity, and LIPN co-localize within putative lamellar bodies.