Permeability Barrier Disorder in Niemann–Pick Disease: Sphingomyelin–Ceramide Processing Required for Normal Barrier Homeostasis  Matthias Schmuth, Mao-Qiang Man, Florian Weber, WenNi Gao, Kenneth R. Feingold, Peter Fritsch, Peter M. Elias  Journal of Investigative Dermatology  Volume 115, Issue 3, Pages 459-466 (September 2000) DOI: 10.1046/j.1523-1747.2000.00081.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Diminished barrier recovery in NP epidermis. The recovery of normal barrier function is altered in the epidermis of patients with NP disease. The volar forearm(s) of individuals were tape-stripped, and TEWL measured with time following barrier disruption (time 0). The delay in barrier recovery, although evident early after disruption (i.e., 0–24 h), was most pronounced at 24 h (*p<0.01 versus normal control values). By 2–4 d (insert) barrier recovery had normalized in NP patients relative to the normal controls (n=4 patients for each value ± SD, except for normal control value at 9 d only, as three individuals were lost to follow-up at this time point). Journal of Investigative Dermatology 2000 115, 459-466DOI: (10.1046/j.1523-1747.2000.00081.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Characterization of epidermal SMase activity. The dependence of epidermal SMase activity on pH and Ca2+. Activity of SMase was determined with increasing pH (from pH 4.1 to 7.6) using a sodium-acetate buffer (60 m M; see Materials and Methods), with or without added Ca2+ (open and closed circles, respectively). Optimal activity (mean ± SD; n=3 determinations) was observed at pH 5.1–5.6, with minimal activity at neutral pH (i.e., pH 7.4–7.6). Addition of Ca2+ (20 m M) significantly increased aSMase activity (*p<0.05 at pH 4.6, 5.1, and 5.6); note that pH values used for ± Ca2+ experiment were distinct. Journal of Investigative Dermatology 2000 115, 459-466DOI: (10.1046/j.1523-1747.2000.00081.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Activity of epidermal aSMase following barrier disruption ± PDHS. aSMase activity was measured in epidermal homogenates (10 000 × g; pH 5.6) 2 h following acetone disruption of the permeability barrier in hairless mice. Results are presented as nmol SM hydrolyzed per h per mg protein (± SD; n=12, 5, 8 animals for untreated control, acetone-treated, and acetone-treated plus PDHS groups, respectively). Barrier disruption with acetone resulted in a 1.44-fold increase in aSMase activity (p<0.008 versus untreated control). A single topical application of PDHS (0.15 mg per 5 cm2) significantly inhibited aSMase activity (57% inhibition; p<0.0001 versus acetone treated). Journal of Investigative Dermatology 2000 115, 459-466DOI: (10.1046/j.1523-1747.2000.00081.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Inhibition of aSMase delays barrier recovery acetone-treated murine skin. (A) Effect of DMI on barrier recovery. The permeability barrier was abrogated with topical acetone treatment (to TEWL ≈ 8.0 mg per 0.5 cm2 per h), immediately followed by topical vehicle (open bars; n=25 animals) or DMI (0.60 mg per 5 cm2) (solid bars; n=10 animals). Results are presented as the mean TEWL (% of time 0 ± SEM). DMI significantly delayed barrier recovery versus vehicle-treated controls at both 2 and 4 h (experiment 1; i.e., 16 and 30%, respectively; ap<0.005; bp<0.001; note incomplete axis). The co-application of Cer (0.375 mg per 5 cm2) (experiment 2; gray bar) normalized barrier recovery at 4 h in the presence of DMI inhibition (cp<0.005 for DMI versus either control or DMI + Cer values; n≥9 animals for each value). (B) Effect of PDHS on barrier recovery. The permeability barrier of hairless mouse skin was abrogated, as above, and topical vehicle (open bars; n=19 animals); PDHS (0.15 per mg 5 cm2) (solid bars; n=30 animals); or PDHS plus Cer (Cer; (gray bars; n=20 animals) were applied. PDHS treatment significantly delayed barrier recovery at both 2 and 4 h (i.e., delayed 24% and 18%, respectively versus vehicle control; p<0.05). Co-application of Cer again eliminated the inhibitor-induced delay in barrier recovery at 4 h (p<0.05; PDHS-treated versus PDHS + Cer-treated animals; difference at 2 h was not significant). Journal of Investigative Dermatology 2000 115, 459-466DOI: (10.1046/j.1523-1747.2000.00081.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Inhibitor treatment results in persistence of immature extracellular membrane structures. (A) Typical mature lamellar membrane unit structures (solid arrows) in vehicle-treated SC (3 h). (B, C) Whereas acetone + PDHS treatment does not alter lamellar body content or secretion (B, solid arrows), 3 h after acetone followed by inhibitor treatment, the intercellular spaces above the stratum granulosum (SG)-SC interface contain widely dispersed lamellar fragments (open arrows), and areas devoid of mature lamellar membrane structures. (D) Co-application of Cer with PDHS normalizes both the amount and substructure of extracellular lamellar membranes (arrows). (A) ×85 000; (B) ×55 000; (C) ×75 000; (D) ×77 000. (A–D) RuO4 postfixation. Journal of Investigative Dermatology 2000 115, 459-466DOI: (10.1046/j.1523-1747.2000.00081.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions