title: Corneal alterations during combined therapy with cyclodextrin/allopregnanolone and miglustat in a knock-out mouse model of NPC1 disease.
abstract: BACKGROUND
  Niemann Pick disease type C1 is a neurodegenerative disease caused by mutations in the NPC1 gene, which result in accumulation of unesterified cholesterol and glycosphingolipids in the endosomal-lysosomal system as well as limiting membranes. We have previously shown the corneal involvement in NPC1 pathology in form of intracellular inclusions in epithelial cells and keratocytes. The purpose of the present study was to clarify if these inclusions regress during combined substrate reduction- and by-product therapy (SRT and BPT).
  
  
  RESULTS
  Starting at postnatal day 7 (P7) and thereafter, NPC1 knock-out mice (NPC1(-/-)) and wild type controls (NPC1(+/+)) were injected with cyclodextrin/allopregnanolone weekly. Additionally, a daily miglustat injection started at P10 until P23. Starting at P23 the mice were fed powdered chow with daily addition of miglustat. The sham group was injected with 0.9% NaCl at P7, thereafter daily starting at P10 until P23, and fed powdered chow starting at P23. For corneal examination, in vivo confocal laser-scanning microscopy (CLSM) was performed one day before experiment was terminated. Excised corneas were harvested for lipid analysis (HPLC/MS) and electron microscopy. In vivo CLSM demonstrated a regression of hyperreflective inclusions in all treated NPC1(-/-)mice. The findings varied between individual mice, demonstrating a regression, ranging from complete absence to pronounced depositions. The reflectivity of inclusions, however, was significantly lower when compared to untreated and sham-injected NPC1(-/-) mice. These confocal findings were confirmed by lipid analysis and electron microscopy. Another important CLSM finding revealed a distinct increase of mature dendritic cell number in corneas of all treated mice (NPC1(-/-) and NPC1(+/+)), including sham-treated ones.
  
  
  CONCLUSIONS
  The combined substrate reduction- and by-product therapy revealed beneficial effects on the cornea. In vivo CLSM is a non-invasive tool to monitor disease progression and treatment effects in NPC1 disorder.
authors.affiliation: Department of Ophthalmology, University of Rostock, Rostock, Germany. marine.hovakimyan@uni-rostock.de
authors.forename: Marine
authors.initials: M
authors.lastname: Hovakimyan
authors.forename: Jana
authors.initials: J
authors.lastname: Petersen
authors.forename: Fabian
authors.initials: F
authors.lastname: Maass
authors.forename: Maria
authors.initials: M
authors.lastname: Reichard
authors.forename: Martin
authors.initials: M
authors.lastname: Witt
authors.forename: Jan
authors.initials: J
authors.lastname: Lukas
authors.forename: Oliver
authors.initials: O
authors.lastname: Stachs
authors.forename: Rudolf
authors.initials: R
authors.lastname: Guthoff
authors.forename: Arndt
authors.initials: A
authors.lastname: Rolfs
authors.forename: Andreas
authors.initials: A
authors.lastname: Wree
mesh_terms: D017485:1-Deoxynojirimycin / Q000008:administration & dosage / Q000031:analogs & derivatives*; D000777:Anesthetics / Q000008:administration & dosage; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D003505:Cyclodextrins / Q000008:administration & dosage*; D018450:Disease Progression; D004359:Drug Therapy, Combination / Q000379:methods; D004791:Enzyme Inhibitors / Q000008:administration & dosage; D005838:Genotype; D047908:Intracellular Signaling Peptides and Proteins; D008247:Lysosomes / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal / Q000379:methods; D008954:Models, Biological; D009154:Mutation; D000077296:Niemann-Pick C1 Protein; D052556:Niemann-Pick Disease, Type C / Q000378:metabolism*; D011280:Pregnanolone / Q000008:administration & dosage*; D011506:Proteins / Q000235:genetics
chemical_list: D000777:Anesthetics; D003505:Cyclodextrins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D000077296:Niemann-Pick C1 Protein; C106831:Npc1 protein, mouse; D011506:Proteins; D017485:1-Deoxynojirimycin; C059896:miglustat; D011280:Pregnanolone