Cyclodextrin induces calcium-dependent lysosomal exocytosis

Abstract

Cyclodextrins (CDs) have long been used to manipulate cellular cholesterol levels both in vitro and in vivo, but their direct effects at a cellular level are not well characterized. Recently, CDs have garnered much interest because of their ability to clear stored cholesterol from Niemann Pick Type C (NPC) cells and markedly prolong the life of NPC1 disease mice. Here, we investigate the hypothesis that treatment with 2-hydroxypropyl- β-cyclodextrin (HPB-CD) stimulates lysosomal exocytosis in a calcium-enhanced manner. We propose that this exocytosis is the mechanism by which HPB-CD ameliorates the endolysosomal cholesterol storage phenotype in NPC cells. These findings have significant implications for the use of HPB-CD in biochemical assays and data interpretation as well as for their use for the treatment for NPC and other disorders.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. The effect of HPB-CD on stress-related protein kinases.

Representative western blot analyses of total (pan) and phosphorylated (p) ERK (A) and JNK (B and C) in lysates from Wt cells treated with HPB-CD. The blots shown are representative of 3 independent experiments.

Figure 2. Filipin staining of free cholesterol in NPC1 cells treated with HPB-CD for 20 hours.

Filipin intensity was quantitated in at least 150 cells for each sample. The bar graph represents average values from 3 independent experiments. *P<0.0001.

Figure 3. HPB-CD toxicity in cell lines derived from BALB/c mice.

Wt (squares) and NPC1 (circles) cells were treated with increasing concentrations of HPB-CD for 2 hours and their percent survival was determined using the MTT assay as described in “Materials and Methods”. * and ** denote statistically significant differences between treated and untreated cells with P<0.01 and P<0.05, respectively.

Figure 4. Enlarged cholesterol-filled vesicles in NPC1 cells treated with HPB-CD.

Cholesterol-filled vesicles fuse to form larger vesicles (arrows), the number of which increases with time. The bar graph represents the percentage of cells with at least one enlarged vesicle (with a minimum size of 10 microns) out of 150 cells and is representative of 2 independent experiments.

Figure 5. Secretion of the lysosomal enzyme β-hexosaminidase by HPB-CD-treated cells.

(A) Wt (circles) and NPC1 (squares) cells were incubated with (closed shapes) or without (open shapes) 2% HPB-CD. At the indicated times, an aliquot of media was assayed for β-hexosaminidase activity. (B) Wt cells were incubated with 2% HPB-CD in the presence (closed shapes) or absence (open shapes) of calcium and an aliquot of media was assayed for β-hexosaminidase activity. (C) Wt cells were incubated with 1 µM ionomycin for 20 min in the presence (closed shapes) or absence (open shapes) of calcium and an aliquot of media was assayed for β-hexosaminidase activity. All enzyme activities are expressed as the percentage of the total enzyme activity found in media and cells. * and ** denote statistically significant differences between treated and untreated cells with P<0.01 and P<0.05, respectively.

Figure 6. Secretion of exosomes in HPB-CD-treated cells.

Wt cells incubated with PBS (−), 2% HPB-CD (CD), or 1 µM ionomycin (Iono) show differential secretion of exosomes in the presence (+) or absence (−) of calcium, as determined by the presence of flotillin in the culture media. Equal loading was determined by normalizing to GAPDH levels in the cell lysate.

Figure 7. Relocalization of endolysosomal membrane proteins in HPB-CD-treated cells.

Wt cells expressing NPC1-GFP exhibit staining in endolysosomal vesicles throughout the cell (A). Following treatment with 2% HPB-CD for 30 minutes, the majority of transfected cells contain protein that is found in vesicles lining the cell periphery (B). This location mimics the position of NPC1-GFP protein found in cells treated with 1 µM ionomycin, which is known to induce endolysosomal exocytosis (C). Greater than 60% of transfected cells in each sample exhibited the phenotypes described.