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

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.

Rosa canina L. Can Restore Endoplasmic Reticulum Alterations, Protein Trafficking and Membrane Integrity in a Dextran Sulfate Sodium-Induced Inflammatory Bowel Disease Phenotype

“…The disruption of LRs distribution induced by DSS is mainly attributed to the decrease of cholesterol level in LRs [ 9 ]. This result is also in line with pervious observations in which we have demonstrated a subtle reduction in the intensity of FLOT2 in the top fraction of LRs upon treatment of fibroblasts with N-butyl-deoxynojirimycin (known as miglustat), which inhibits sphingolipid synthesis and thus alters the composition of LRs in these cells [ 59 ]. The effect of RCME is likely linked to its phenolic composition, specifically luteolin [ 60 ], which has the capacity to inhibit lipid peroxidation that occurs in experimental colitis induced by DSS [ 16 ].…”

Staphylococcus aureus Infection Influences the Function of Intestinal Cells by Altering the Lipid Raft-Dependent Sorting of Sucrase–Isomaltase

“…Taking all this together, it can be postulated that an impaired trafficking of LR-enriched vesicles harboring SI—and other membrane proteins—and their intracellular retention leads to an intracellular accumulation of the two main lipid components comprising LRs, cholesterol, and sphingolipids. This in turn could elicit redistribution and alterations in the membrane lipid composition as has been demonstrated in several lysosomal storage diseases, such as Niemann-Pick Type C and Fabry diseases ( Brogden et al, 2017 , 2020 ; Shammas et al, 2019 ). In addition to altered membrane and protein trafficking in infected Caco-2 cells, S. aureus infection resulted in a reduction of the overall enzymatic activity of SI, most likely due to altered LRs.…”

NPC1 confers metabolic flexibility in Triple Negative Breast Cancer

“…However, recent studies identified “cholesterol auxotrophy”, the inability to survive without exogenous cholesterol, in varying cancer subtypes (27,28) and suggest cholesterol uptake as a targetable pathway. NPC1 specifically transports lysosomal cholesterol to the endoplasmic reticulum (29) where cellular cholesterol levels are sensed and controlled (17,30) . By depleting the endoplasmic reticulum of cholesterol, NPC1 inhibition affects both supply of exogenous cholesterol as well and disrupts cholesterol homeostasis (29,31) .…”

“…However, recent studies identified "cholesterol auxotrophy", the inability to survive without exogenous cholesterol, in varying types of cancer (27,28) ,suggesting cholesterol uptake as a targetable pathway. NPC1 specifically transports lysosomal cholesterol to the endoplasmic reticulum (29) where cellular cholesterol levels are sensed and controlled (17,30) .…”

“…Random fields (25)(26)(27)(28)(29)(30) were analyzed and each cell was classified into 3 categories according to the main mitochondrial morphology (small, medium, elongated). For automated analysis of mitochondrial size, slides were scanned in a Zeiss LSM780 confocal microscope with 63X oil objective.…”