A mouse model of harlequin ichthyosis delineates a key role for Abca12 in lipid homeostasis

Abstract

Harlequin Ichthyosis (HI) is a severe and often lethal hyperkeratotic skin disease caused by mutations in the ABCA12 transport protein. In keratinocytes, ABCA12 is thought to regulate the transfer of lipids into small intracellular trafficking vesicles known as lamellar bodies. However, the nature and scope of this regulation remains unclear. As part of an original recessive mouse ENU mutagenesis screen, we have identified and characterised an animal model of HI and showed that it displays many of the hallmarks of the disease including hyperkeratosis, loss of barrier function, and defects in lipid homeostasis. We have used this model to follow disease progression in utero and present evidence that loss of Abca12 function leads to premature differentiation of basal keratinocytes. A comprehensive analysis of lipid levels in mutant epidermis demonstrated profound defects in lipid homeostasis, illustrating for the first time the extent to which Abca12 plays a pivotal role in maintaining lipid balance in the skin. To further investigate the scope of Abca12's activity, we have utilised cells from the mutant mouse to ascribe direct transport functions to the protein and, in doing so, we demonstrate activities independent of its role in lamellar body function. These cells have severely impaired lipid efflux leading to intracellular accumulation of neutral lipids. Furthermore, we identify Abca12 as a mediator of Abca1-regulated cellular cholesterol efflux, a finding that may have significant implications for other diseases of lipid metabolism and homeostasis, including atherosclerosis.

Figure 1. An ENU recessive mutagenesis screen identifies a lethal mutation Abca12.

Mutagenised 129/Sv males were crossed with C57BL/6 females and the resultant G₁ males crossed again to C57BL/6. Pedigrees were established by crossing G₁ males with their G₂ daughters and the G₃ offspring were then subjected to genome wide screening for absence of homozygosity of the mutagenised (129/Sv) strain (A). The recessive embryo lethal 12 (el12) mutation was identified using this approach and mapped by recombination to Chromosome 1 (B). Open rectangles indicate haplotypes homozygous for the 129/Sv mutagenised background and filled rectangles are C57BL6/J homozygotes or heterozygotes. Recombination frequency and markers position are indicated. A missense G1997D mutation was identified in Abca12 (C, D). The el12 mutation alters a residue in the second TM region of the protein which is conserved in all species examined (human, mouse, dog, chicken, platypus, microbat and shrew) (E).

Figure 2. The Abca12^el12/el12 phenotype.

Abca12^el12/el12 mice display an epidermal phenotype visible from E16.5 and by E18.5, the thickening of the cornified envelope produces a constrictive, taut and shiny epidermis resulting in limb contractures (A). Sections of epidermis at E17.5 and birth demonstrate severe hyperkeratosis characterised by the formation of a 20–30 cell layer thick stratum corneum. Cell architecture in other layers of the epidermis is also affected, with a reduction in the size of the spinous cell layer and lack of dense palisaded basal cell nuclear architecture (B).

Figure 3. Barrier defects in Abca12^el12/el12 mice.

Abca12^el12/el12 mice have defects in barrier formation as evidenced by dye exclusion (A) and trans-epidermal water loss (B) assays at E18.5. Cornified envelopes prepared from Abca12^el12/el12 mice are fragile and reduced in size compared with wild type littermate controls (Abca12^el12/el12 CEs concentrated 15 times, C, D: p = 6×10⁻²³). Western blotting indicates defects in filaggrin processing in Abca12^el12/el12 epidermis (E, arrow) while expression of other CE proteins such as loricrin is unaffected. Expression of “proliferative” keratin VI is present only in the differentiating hair follicle of both mutant and wild type epidermis (F) and cell proliferation at E17.5 is normal as assayed by phospho-histone H3 staining (G; p = 0.27).

Figure 4. Pathology of the Abca12 mutant epidermis.

Abca12 protein is detected in suprabasal keratinocytes in the granular and cornified cell layers of mutant and wild type epidermis (A). Keratinocytes in Abca12 mutant skin undergo premature differentiation highlighted by strong filaggrin expression in cells juxtaposed to K14⁺ basal keratinocytes (B) and increased co-expression of keratins 10 and 14 (white arrows), especially in basal cells (yellow arrows) (C). Increases in co-expression of K10 and K14 are significant at both E18.5 (p = 0.016) and at P1 (p = 4.44×10⁻⁶) (D). Samples are of E18.5 (A) or P1 (B, C) epidermis counterstained with DAPI. Scale bars = 30 µm.

Figure 5. Ultrastructural defects in Abca12^el12/el12 mice.

Thin sections of Abca12^el12/el12 epidermis illustrated hyperkeratosis and expansion of the stratum granulosum (A). Nile red staining shows reduced intercellular lamellae lipids at E18.5 (B). In wild type epidermis intercellular lipid lamellae (white arrow and inset) were noted as well as LBs fusing with the surface of granular cells (red arrow) (C). Lamellar bodies in wild type and heterozygous embryonic epidermis were normally loaded with lipid (D). In mutant skin, LBs lacked lamellar cargo (E, F arrowheads) but fused with the granular cell membrane (F; arrows). Mutant epidermis had a normal cornified envelope (G,H) with persistent corneodesmosomes in distal layers of the stratum corneum (G, H, red arrowheads) and the cornified layer had multiple lipid inclusions (G, black arrowheads). Unlike the uniform contents of wild type cornified cells mutant cell layers contained vesicular fibrillar structures (I, red arrows) and frequent inclusion bodies (I, black arrows). EM scale bars in C–F, H, I equal 200 nm and 2 µm in G. C–F and I were stained with ruthenium tetroxide, G and H with osmium tetroxide.

Figure 6. Defects in skin lipid composition and cellular lipid efflux mediated by Abca12.

Analysis Abca12^el12/el12 epidermal lipids indicate significant increases in levels of ceramide (Cer), glucoslyceramide (GC), sphingosine (Sph) and free cholesterol in Abca12^el12/el12 skin at E18.5. Total phosophatidylcholine (PC) and cholesterol ester (CE) levels were unchanged (A, ***p<0.0001; **p<0.001; *p<0.01 versus wilt type epidermis) (A). Defects in fibroblast cholesterol efflux were assayed using [³H]cholesterol and incubation in the presence (+) or absence (−)) of the LXR agonist TO-901317. Means plus or minus standard deviation of quadruplicate determinations are shown. (**p<0.001 versus non activated cells; ^*# p<0.001 versus ABCA12^+/+ cells; ^*p<0.01 versus ABCA12^−/+ cells) (B). Expression of Abca1 was determined at the protein level by Western blotting (C, upper panel) and decreases in Abca12 homozygous mutant cells were shown to be in part due to decreases in transcription of Abca1 (C, lower panel, fold change in transcription relative to el12/el12, ***p<0.0005). Lipid accumulation in fibroblasts the presence or absence of the lipid donor acetylated LDL (AcLDL) was assayed by Oil Red O staining (magnification ×100) (D).