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Pathophysiology of Niemann-Pick Type C and Fabry diseases with Emphasis on Membrane Composition and Protein Trafficking

Lysosomal storage diseases (LSDs) are a group of progressive inherited metabolic disorders caused by compromised function of lysosomal proteins/enzymes. This leads to a progressive lysosomal accumulation of metabolites or untrafficked molecules in the late endosomes/lysosomes (LE/L). 
 
 
 
In our study we focus on two LSDs: Niemann-Pick disease type C (NP-C) (cholesterol storage disease) and Fabry disease (FD) (glycolipid storage disease). 
 
 
 
NP-C disease is caused by a mutation in the lysosomal protein NPC1 (95 % of the cases) or NPC2 (5 % of the cases). The clinical spectrum of NP-C ranges from a neonatal fatal disorder to adult-onset cases. Our aim was to understand the implication of particular mutations at the molecular level and its correlation with clinical parameters like the age at onset of symptoms and the severity of the disease. 
 
 
 
To analyse the structural features, maturation pathways and subcellular localization in common mutations of NPC1, a cDNA construct harboring the mutation of interest was generated using site-directed mutagenesis PCR and expressed in mammalian cells. Interestingly, we were able to classify NPC1 mutants into three different clusters according to their trafficking pattern along the secretory pathway and their localization in the cellular compartments. The first cluster included mutations that were blocked in the ER as immature mannose-rich proteins, while the second group comprised mutations that were partially trafficked through the Golgi to the lysosomes. The third cluster included mutations that exhibited a pattern of trafficking and localization similar to the wild type. In addition, the three classes varied also in their association with lipid rafts (LRs) and in their turnover rate. This is the first molecular analysis that relates different mutations in NPC1 with the trafficking behavior of NPC1 protein along the secretory pathway. These results can explain the variable biochemical presentation among the patients, the heterogeneity of symptoms and the response towards the current therapeutic agent for NP-C N-butyldeoxynojirimycin (NB-DNJ, Miglustat). The concept in this study might help to correlate the genotype with the clinical phenotype of NP-C disease and propose a mutation-based personalized therapeutical approach. 
 
 
 
On the other hand, the disruption of lipid homeostasis is a major feature in FD,in which the malfunction of alpha-galactosidase A (GLA) causes accumulation of glycosphingolipids (GSLs) in the lysosomes. Since GSLs are one of the major components in LRs together with cholesterol, we suggested that the LRs composition in FD might be impaired as downstream consequences of cellular lipid alteration. Our results indicate that the N215S mutation causes impairment of GLA trafficking revealed by the treatment of the protein with endoglycosidase H (endo H). The consequence of the defect in trafficking of GLA was investigated by analyzing LR composition. Interestingly, abnormalities in LRs composition in the Fabry fibroblasts were observed based on the altered distribution of flotillin-2 in addition to cholesterol and GLSs analysis. Moreover, treatment of Fabry cells with 50µM or 100µM Miglustat reverted the LRs to a wild type-pattern. In this study we were able to show that LRs play a key role in LSDs. This may help to better understand the pathophysiology in LSDs and possibly identify novel biomarkers.