Trafficking defects in endogenously synthesized cholesterol in fibroblasts, macrophages, hepatocytes, and glial cells from Niemann-Pick type C1 mice

Abstract

Niemann-Pick type C1 disease (NPC1) is an inherited neurovisceral lipid storage disorder, hallmarked by the intracellular accumulation of unesterified cholesterol and glycolipids in endocytic organelles. Cells acquire cholesterol through exogenous uptake and endogenous biosynthesis. NPC1 participation in the trafficking of LDL-derived cholesterol has been well studied; however, its role in the trafficking of endogenously synthesized cholesterol (endo-CHOL) has received much less attention. Previously, using mutant Chinese hamster ovary cells, we showed that endo-CHOL moves from the endoplasmic reticulum (ER) to the plasma membrane (PM) independent of NPC1. After arriving at the PM, it moves between the PM and internal compartments. The movement of endoCHOL from internal membranes back to the PM and the ER for esterification was shown to be defective in NPC1 cells. To test the generality of these findings, we have examined the trafficking of endoCHOL in four different physiologically relevant cell types isolated from wild-type, heterozygous, and homozygous BALB/c NPC1 NIH mice. The results show that all NPC1 homozygous cell types (embryonic fibroblasts, peritoneal macrophages, hepatocytes, and cerebellar glial cells) exhibit partial trafficking defects, with macrophages and glial cells most prominently affected. Our findings suggest that endoCHOL may contribute significantly to the overall cholesterol accumulation observed in selective tissues affected by Niemann-Pick type C disease.

Secretion of Sterols and the NPC2 Protein from Primary Astrocytes

“…Recently, NPC1 Ϫ/Ϫ astrocytes were shown to have impaired efflux of cholesterol to methyl-␤-cyclodextrin (11). We found no impairment in the secretion of biosynthetic sterols in NPC1 Ϫ/Ϫ astrocytes.…”

“…[4][5][6][7][8][9][10][11][12][13][14] C]Cholesterol (specific activity 58 mCi/mmol), [1␣,2␣(n)- 3 H]cholesterol (specific activity 41.0 Ci/mmol), [ 3 H]acetic acid (specific activity 5.10 -9.00 Ci/mmol), Hybond-C membrane, and the ECL reagents were purchased from Amersham Biosciences. The polyclonal anti-glial fibrillary acidic protein antibody was from Dako, and anti-lysobisphosphatidic acid antibody (13) was a gift from Jean Gruenberg (University of Geneva).…”

“…Interestingly, NPC1 Ϫ/Ϫ astrocytes were shown to have defects in the trafficking of endogenously synthesized cholesterol (11).…”

Transport of LDL-derived cholesterol from the NPC1 compartment to the ER involves the trans-Golgi network and the SNARE protein complex

“…CT43 cells are NPC1-null cells derived from 25RA cells (19). Findings made in this cell system are generally applicable to other cell types (8,(20)(21)(22). To monitor the hydrolysis and transport of 3 H-LDL-CHOL liberated from 3 H-CL, we pulse-labeled cells with 3 H-CL-LDL for 30 min, then chased the cells without 3 H-CL-LDL for up to 6 h [supporting information (SI) Fig.…”

Niemann-Pick Type C Disease and Intracellular Cholesterol Trafficking

“…The recycling of these sterols from the late endosomes to the PM, and the esterification of these molecules within the ER are also partially defective in NPC1 cells (22)(23)(24). The effect of NPC1 on trafficking of endogenously synthe-sized sterols is cell-type dependent: macrophages and glial cells are prominently affected by the NPC1 mutation, whereas embryonic fibroblasts are less affected (25,26).…”