Different Trafficking Phenotypes of Niemann-Pick C1 Gene Mutations Correlate with Various Alterations in Lipid Storage, Membrane Composition and Miglustat Amenability.

Graham Brogden,Hadeel Shammas,Anibh M Das,Katia Maalouf,Sandra Rizk,Friederike Walters,Hassan Y Naim

doi:10.3390/ijms21062101

Abstract

Niemann-Pick Type C (NPC) is an autosomal recessive lysosomal storage disease leading to progressive neurodegeneration. Mutations in the NPC1 gene, which accounts for 95% of the cases, lead to a defect in intra-lysosomal trafficking of cholesterol and an accumulation of storage material including cholesterol and sphingolipids in the endo-lysosomal system. Symptoms are progressive neurological and visceral deterioration, with variable onset and severity of the disease. This study investigates the influence of two different NPC1 mutations on the biochemical phenotype in fibroblasts isolated from NPC patients in comparison to healthy, wild type (WT) cells. Skin derived fibroblasts were cultured from one patient compound-heterozygous for D874V/D948Y mutations, which presented wild-type like intracellular trafficking of NPC1, and a second patient compound- heterozygous for I1061T/P887L mutations, which exhibited a more severe biochemical phenotype as revealed in the delayed trafficking of NPC1. Biochemical analysis using HPLC and TLC indicated that lipid accumulations were mutation-dependent and correlated with the trafficking pattern of NPC1: higher levels of cholesterol and glycolipids were associated with the mutations that exhibited delayed intracellular trafficking, as compared to their WT-like trafficked or wild type (WT) counterparts. Furthermore, variations in membrane structure was confirmed in these cell lines based on alteration in lipid rafts composition as revealed by the shift in flotillin-2 (FLOT2) distribution, a typical lipid rafts marker, which again showed marked alterations only in the NPC1 mutant showing major trafficking delay. Finally, treatment with N-butyldeoxynojirimycin (NB-DNJ, Miglustat) led to a reduction of stored lipids in cells from both patients to various extents, however, no normalisation in lipid raft structure was achieved. The data presented in this study help in understanding the varying biochemical phenotypes observed in patients harbouring different mutations, which explain why the effectiveness of NB-DNJ treatment is patient specific.

Highlights

Niemann-Pick Type C disease (NPC, OMIM #257220) is a progressive neurodegenerative lysosomal disorder that is inherited in a rare autosomal recessive pattern from mutations in NPC1 or NPC2 genes
endoglycosidase H (Endo H) distinguishes between the mannose-rich immature form of proteins that are located in the ER or in the early secretory pathway and their complex glycosylated mature counterparts that have been processed in the Golgi apparatus
Our study shows that lipid accumulations, cholesterol and glycolipids, are significantly higher in fibroblasts derived from both patients as compared to the control cells

Summary

Introduction

Niemann-Pick Type C disease (NPC, OMIM #257220) is a progressive neurodegenerative lysosomal disorder that is inherited in a rare autosomal recessive pattern from mutations in NPC1 or NPC2 genes. A late onset form of the disease with a milder clinical phenotype has been estimated to have an occurrence of up to 1:19,000 [1]. NPC disease is a progressive neurodegenerative disorder with over 400 reported mutations, resulting in a wide diversity of clinical manifestations. NPC1 is synthesised and co-translationally N-glycosylated in the ER, trafficked after acquisition of correct folding to the Golgi apparatus, where it matures to a complex glycosylated protein that is transported via the endosomal/lysosomal system to the lysosomes [4]. A complex of NPC1-cholesterol-NPC2 is formed facilitating the transport of cholesterol out of the endosomal/lysosomal compartment [5]

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