ULTRA-STRUCTURAL STUDIES OF THE ACCUMULATION OF CHOLESTEROLS IN LYSOSOME OF FIBROBLAST CELL LINES DERIVED FROM NIEMANN-PICK TYPE C PATIENTS

Abstract

Niemann-Pick disease, type C (NPC), is a fatal autosomal recessive lysosomal storage disorder resulting in fatal neurodegeneration. It is caused by mutations in either of two genes Npc1 or Npc2 that results in accumulation of unesterified cholesterol and glycosphingolipids in lysosomes. Delta-Tocopherol and Methyl-Beta-Cyclodextrin have shown to reduce cholesterol accumulation in NPC cells. In this thesis, we used room temperature TEM and high pressure freeze/freeze substitution TEM methods for ultrastructural analysis of healthy control, NPC, and NPC cells treated with delta-tocopherol and methyl-betacyclodextrin. HPF method significantly improved ultrastructure of these cells. We showed vast and enlarged late endosome/lysosome with multi-lamellar, multi-vesicular and osmiophilic dense bodies as the pathological phenotype of NPC cells. We showed delta-tocopherol and methyl-beta-cyclodextrin significantly reduced cholesterol accumulation by reducing the number and size of enlarged late endosome/lysosome compartments in NPC cells. Our results were based on combined data from fluorescence microscopy, RT-TEM and HPF-TEM ultrastructural analysis.