Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking

Abstract

Niemann Pick type C (NPC) disease is an autosomal recessive disorder characterized by the lysosomal/ late endosomal (LE) accumulation of unesterified cholesterol and other lipids due to a defect in the intracellular lipid trafficking. About 95% of patients present mutations in the NPC1 gene. Among the 290 mutations reported in the NPC1 gene, about 70% are missense. However, little information is available regarding the impact of missense mutations on NPC1 protein stability and function. In this study, we in vitro characterized the pathogenic effect of 7 NPC1 missense mutations. In all cases, the basal levels of mutant NPC1 expression were reduced with respect to wild type. Treatment of fibroblasts carrying NPC1 missense mutations in homo or hemizygosity, with the proteasome inhibitor MG132 or glycerol 10%, a chemical chaperone known to stabilize misfolded proteins, resulted in a significant increase of NPC1 protein levels in all cell lines, indicating that these mutants are subjected to proteasomal degradation due to protein misfolding The increment of NPC1 mutant protein induced by the proteasome inhibitor was associated with a localization of NPC1 protein within lysosomal/LE compartment. In cell lines carrying mutations p.N1156S, p.L1191F, p.V1165M, and p.I1061T, the increment of NPC1 mutant protein resulted in an improvement of the intracellular trafficking of cholesterol and GM1. These findings showed that it is possible to correct the NPC cellular phenotype by increasing the amount of endogenous NPC1 mutated protein, suggesting that at least some NPC1 mutations might be potentially rescued by small molecules-based chaperone therapy.

Endoplasmic Reticulum-associated Degradation of Niemann-Pick C1

“…Similar responses were obtained with a loss-of-function mutant P691S, in accordance with the notion that NPC1 underwent ERAD (21). In the current study, we attempted to confirm NPC1 ERAD and tested whether we could reproduce accelerated ERAD of the I1061T mutant, as suggested by other groups (9,(12)(13)(14).…”

“…They concluded that the I1061T mutant retains NPC1 function but is selected for endoplasmic reticulum-associated degradation (ERAD) due to protein misfolding. This conclusion was supported by subsequent studies that demonstrated the ability of ryanodine receptor antagonists (12) or MG132 (13) to increase the protein level of the I1061T mutant and alleviate cholesterol accumulation in patient-derived cells. Recently, Ohgane et al (14) described oxysterol derivatives that could bind to and suppress degradation of this mutant protein.…”

“…The I1061T mutant protein migrated on electrophoresis faster than the wild-type protein (Fig. 2B), most likely because of differential glycosylation (9,13). MG132 was effective on all of these mutants to induce accumulation of the ubiquitylated form and increase the steady-state levels (Fig.…”

Lysosomal storage diseases and the heat shock response: convergences and therapeutic opportunities

“…MG-132 treatment further increased the association of NPC1 with the lysosomal/late endosomal compartment and reduced the storage of ganglioside GM1 and un-esterifi ed cholesterol, examples of some of the key metabolites that are accumulated due to the loss of function characterizing the disease ( 43 ). MG-132 has furthermore been shown to be effective in primary patient cells from sialidosis (neuraminidase defi ciency).…”

“…As mentioned above, the potential benefi t of modulation of the HSR is now emerging in the LSD fi eld with the reporting of a number of studies of this approach in genetically distinct diseases such as Gaucher's disease, Tay-Sach's disease, Niemann-Pick disease types A and B, Niemann-Pick disease type C, ␣ -mannosidoses, sialidosis, and mucopolysaccharidosis type 3A ( 7,(43)(44)(45)(46)(47)(48)(49). In cellular studies of Gaucher's and Tay-Sach's disease for example, the induction of the HSR or UPR with celastrol and MG-132 results in increases of both cytosolic and ER-resident chaperones, of the HSP70 family in particular, in a process dependent on UPRresponsive transcription factors Ire1, ATF6, and PERK.…”