Differential uptake of dehydroergosterol as a marker for Niemann-Pick Type C: Assay development and potential for small molecule screening

Rachel E Wright, Purdue University

Abstract

Niemann-Pick Type C (NPC) is an autosomal recessive lysosomal storage disorder caused by mutations in one of two genes npc1 or npc2 which encode important cholesterol trafficking proteins (1). Both NPC1 and NPC2 are necessary for the movement of free cholesterol from the late endosomes/lysosomes to the ER where it can be esterified and further modified for biological use. Disruption of cholesterol trafficking leads to build up of cholesterol and other lipids in the late endosomes and lysosomes in all cells of the body (Fig. 1). Patients experience progressive neurodegeneration and liver dysfunction leading to premature death (2). 2-hydroxypropyl-β-cyclodextrin (HPβCD) and Miglustat are currently the only FDA approved treatments for this disease, and more treatment options are needed. I used the fluorescent cholesterol analog, dehydroergosterol (DHE) packaged in low-density lipoprotein (DHE-LDL) for easy uptake by cells to monitor cholesterol uptake and storage in a cell culture model of NPC. I found that cells containing NPC1 mutation retained more DHE after 12 hour incubation than wild-type (WT) cells. Moreover, treatment with HPβCD reduces DHE labeling of NPC cells to the level of WT cells. I attempted to use this assay to test several polyrotaxane compounds but found that the drug vehicle, dimethyl sulfoxide, eliminated both differential uptake of DHE and HPβCDs ability to decrease DHE labeling in NPC cells.

Degree

M.S.

Advisors

Barker, Purdue University.

Subject Area

Pharmacology|Health sciences

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