Effect of Rosa canina Methanol Extract on Membrane Trafficking in Different Niemann‐Pick C1 Phenotypes

Abstract

Niemann Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder that is caused by mutations in Niemann‐Pick C1 (NPC1) or Niemann‐Pick C2 (NPC2) genes ultimately leading to progressive neurological deterioration. 95% of NPC cases arise from mutations in the NPC1 gene. NPC1 protein mediates the intracellular cholesterol trafficking and plays a crucial role in maintaining lipid homeostasis. Mutations in NPC1 are associated with defective cholesterol mobilization and an accumulation of unesterified cholesterol and subsequently sphingomyelin and glycosphingolipids in the late endo‐lysosomes. N‐butyldeoxynojirimycin, known as Miglustat, is one of the approved therapeutic options. It is an inhibitor of sphingolipid synthesis and is used in substrate reduction therapy. Nevertheless, the efficiency of Miglustat varies among NPC patients depending on the type of the mutation and moreover its accompanied by several side effects, such as diarrhea. The focus of this study is to find an alternative therapy that presents less side effects. Rosa canina L. methanol extract (RCME) has been shown to improve the protein trafficking and maintain cholesterol homeostasis. Here, we used skin‐derived fibroblasts from patients harbouring homozygote or compound heterozygote mutations to assess the effects of RCME on the trafficking of NPC1 and cellular cholesterol contents as compared to wild type cells. For this purpose, the cells were treated for 24 h with RCME (100 µg/ml) with and without Miglustat (100 µM) and the trafficking of NPC1 between the endoplasmic reticulum (ER) and the Golgi was examined by endoglycosidase H treatment. This treatment, which discriminates between mannose‐rich N‐linked glycosylated proteins and mature complex glycosylated forms, demonstrated a significant enhancement of the trafficking of a NPC1 mutants between the ER and the Golgi upon RCME treatment as compared to Miglustat. This trafficking behaviour was observed with those NPC1 mutants that are not entirely blocked in the ER and exit the ER at a slow rate. Markedly, the high cholesterol levels measured in the patients’ fibroblasts are substantially reduced upon RCME treatment, while almost similar cholesterol levels were maintained in the wild type cells. Impaired cellular cholesterol trafficking is associated with partial distortion of the lipid membrane composition, particularly of the cholesterol‐ and sphingolipids‐enriched membrane microdomains or lipid rafts (LRs). Our data show that RCME and also Miglustat maintained a normal LRs distribution as assessed by the LR marker flotillin 2.We conclude that RCME possess the capacity to reverse adverse effects caused by mutations in the NPC1gene including impaired NPC1 trafficking, elevated cholesterol levels and lipid raft alterations. All this together render the constituents of RCME, including quercetin, a promising agents in NPC therapy.