Evaluation of Treatment with Cyclodextrin, Allopregnanolone and Miglustat for Niemann-Pick Disease type C1 in a Knock-out Mouse Model

Abstract

Purpose: : Niemann-Pick disease type C1 (NPC1) is a lysosomal storage disease caused by mutations in NPC1 gene which lead to widespread intracellular accumulation of unesterified cholesterol and glycosphingolipids. We have previously shown corneal involvement in NPC1 pathology in form of hyperreflective intracellular inclusions. In this study we examined corneal response to the combined therapy with cyclodextrin, allopregnanolone and miglustat (Cyclo/ALLO/miglustat).

Methods: : Twenty NPC1 homozygous knock-out mice (NPC1-/-) and 20 age-matched controls (NPC1+/+) were involved in this study. NPC1-/- and NPC1+/+ mice were divided in 2 subgroups, undergoing i) combined (Cyclo/ALLO/miglustat) or ii) sham therapy. For the combined therapy, mice were injected with cyclodextrin/allopregnanolone weekly, starting at postnatal day 7 (P7). Additionally, a daily miglustat injection started at P10 until P23. Starting at P23 the mice were fed powdered chow with daily addition of miglustat. For sham treatment, mice were injected with 0.9% NaCl and fed powdered chow. In vivo confocal laser scanning microscopy (CLSM) was performed on both eyes. At P 70 corneas were harvested for histology, electron microscopy, and lipid analysis.

Results: : In vivo CLSM revealed a significant decrease in quantity and intensity of corneal inclusions by 10 weeks of therapy in all treated NPC1-/- mice. These findings could be confirmed by lipid analysis, which also revealed a reduction of glycosphingolipids and free cholesterol concentration. Electron microscopy demonstrated normal corneal ultrastructure, without apparent intracellular inclusions. In contrast, the reflectivity and density of inclusions remained unchanged in sham treated NPC1-/- mice. After both kinds of therapy NPC1-/- and NPC1+/+ mice exhibited a significant accumulation of dendritic cells in the central cornea.

Conclusions: : Understanding the mechanisms by which combined Cyclo/ALLO/miglustat therapy leads to reduced lipid storage may provide new insights on opportunities for NPC1 disease treatment.In vivo CLSM appears to be a useful tool not only to demonstrate the presence, nature and localisation of corneal changes in pathological conditions, but also to reliably evaluate the effectiveness of therapy.

Keywords: cornea: basic science • imaging/image analysis: non-clinical • cornea: epithelium