Normalization of cholesterol homeostasis by 2-hydroxypropyl-β-cyclodextrin in neurons and glia from Niemann-Pick C1 (NPC1)-deficient mice

Abstract

Niemann-Pick C (NPC) disease is an inherited, progressive neurodegenerative disorder caused by mutations in the NPC1 or NPC2 gene that result in an accumulation of unesterified cholesterol in late endosomes/lysosomes (LE/L) and impaired export of cholesterol from LE/L to the endoplasmic reticulum (ER). Recent studies demonstrate that administration of cyclodextrin (CD) to Npc1(-/-) mice eliminates cholesterol sequestration in LE/L of many tissues, including the brain, delays neurodegeneration, and increases lifespan of the mice. We have now investigated cholesterol homeostasis in NPC1-deficient cells of the brain in response to CD. Primary cultures of neurons and glial cells from Npc1(-/-) mice were incubated for 24 h with 0.1 to 10 mm CD after which survival and cholesterol homeostasis were monitored. Although 10 mm CD was profoundly neurotoxic, and altered astrocyte morphology, 0.1 and 1 mm CD were not toxic but effectively mobilized stored cholesterol from the LE/L as indicated by filipin staining. However, 0.1 and 1 mm CD altered cholesterol homeostasis in opposite directions. The data suggest that 0.1 mm CD releases cholesterol trapped in LE/L of neurons and astrocytes and increases cholesterol availability at the ER, whereas 1 mm CD primarily extracts cholesterol from the plasma membrane and reduces ER cholesterol. These studies in Npc1(-/-) neurons and astrocytes establish a dose of CD (0.1 mm) that would likely be beneficial in NPC disease. The findings are timely because treatment of NPC disease patients with CD is currently being initiated.

FIGURE 1.

Intracellular cholesterol sequestration in Npc1^−/− neurons and glia. Cerebellar granule neurons (A and B), cortical astrocytes (C and D), and cortical microglia (E and F) from Npc1^−/− and Npc1^+/+ mice were cultured for 7 days. The distribution of unesterified cholesterol was qualitatively assessed by filipin staining. Similar results were obtained in at least three additional experiments.

FIGURE 2.

Low doses (0.1 and 1 mm) of CD are not toxic to Npc1^+/+ mouse neurons or astrocytes. Representative phase contrast images of 7-day-old Npc1^+/+ cerebellar granule neurons (A–D) and cortical astrocytes (E–H) incubated for 24 h with vehicle or the indicated CD concentration. Cells were fixed with paraformaldehyde and stained with Hoechst dye for assessment of cell death by apoptosis. The number of apoptotic nuclei was quantified as % of total number of neurons (I) and astrocytes (J). Data are means ± S.E. for >750 cells from three independent cell preparations. For CD versus vehicle (Veh), p > 0.05.

FIGURE 3.

Low doses of CD (0.1 and 1 mm) are not toxic to Npc1^−/− neurons or astrocytes. Representative phase contrast images of 7-day-old Npc1^−/− cerebellar granule neurons (A–D) and cortical astrocytes (E–H) incubated for 24 h with vehicle or the indicated CD concentration. Cells were fixed with paraformaldehyde and stained with Hoechst dye for assessment of cell death by apoptosis. The number of apoptotic nuclei was quantified as % of total number of neurons (I) and astrocytes (J). Data are means ± S.E. for >750 cells from three independent cell preparations. For CD versus vehicle (Veh), p > 0.05.

FIGURE 4.

CD reduces cholesterol sequestration in Npc1^−/− neurons and glia. Filipin staining of unesterified cholesterol in 7-day-old Npc1^−/− cerebellar granule neurons (A–C), astrocytes (D–F), and microglia (G–I) incubated for 24 h with vehicle (Veh) or indicated CD concentration. Shown are representative images from three independent cell preparations.

FIGURE 5.

CD does not alter cholesterol distribution in Npc1^+/+ neurons or astrocytes. Filipin staining of Npc1^+/+ neurons (A–C) and astrocytes (D–F) incubated for 24 h with vehicle (Veh) or indicated CD concentration. Shown are representative images from three independent cell preparations.

FIGURE 6.

Incorporation of [³H]acetate into cholesterol of Npc1^+/+ and Npc1^−/− neurons and astrocytes. Neurons (A) and astrocytes (B) from Npc1^+/+ (black) and Npc1^−/− (white) mice were cultured for 7 days then incubated with [³H]acetate for 4 h. Lipids were extracted, separated by thin-layer chromatography, and radioactivity was quantified in cholesterol and phospholipids. Data for neurons are means ± S.E. of duplicate measurements from three independent experiments, and for astrocytes, data are means ± S.E. from four independent experiments.

FIGURE 7.

CD modulates [³H]acetate incorporation into cholesterol in neurons and astrocytes. Neurons and astrocytes were incubated for 28 h with vehicle (dark gray), 0.1 mm CD (light gray), or 1 mm CD (white); [³H]acetate was included for the final 4 h. Lipids were separated by thin-layer chromatography, and radioactivity was quantified in cholesterol and phospholipids. Data for neurons are means ± S.E. from duplicate determinations of three independent cell preparations. The experiments with Npc1^+/+ neurons were performed independently of those with Npc1^−/− neurons and therefore are not comparable. For astrocytes, the data are means ± S.E. from four independent cell preparations. The experiments with Npc1^+/+ and Npc1^−/− astrocytes were performed concurrently. *, p < 0.05; ***, p < 0.001.

FIGURE 8.

CE formation in astrocytes. [¹⁴C]oleate incorporation into CE and phospholipids was quantified in Npc1^−/− and Npc1^+/+ astrocytes. Cells were incubated for 28 h with vehicle (dark gray), 0.1 mm CD (light gray), or 1 mm CD (white); [¹⁴C]oleate ± Sandoz 58-035 were included for the final 4 h. Lipids were separated, and radioactivity was measured in CE and phospholipids. Data are means ± S.E. from three independent cell preparations. *, p < 0.05; **, p < 0.01.

FIGURE 9.

CE formation in neurons. [¹⁴C]oleate incorporation into CE and phospholipids was quantified in the following: Npc1^−/− neurons incubated with vehicle (dark gray) or 0.1 mm CD (light gray) ± Sandoz 58-035 (A) and Npc1^+/+ neurons incubated with vehicle (B). Data are means ± S.E. from three independent experiments. C, qPCR analysis of ACAT1 mRNA relative to GAPDH mRNA in Npc1^+/+ (black) and Npc1^−/− (white) neurons, and astrocytes were incubated for 24 h with vehicle. Data are means ± S.E. from duplicate analyses of three independent cell preparations. p > 0.05.

FIGURE 10.

qPCR analysis of mRNAs involved in cholesterol metabolism in Npc1^+/+ and Npc1^−/− neurons and astrocytes. mRNA levels were quantified relative to GAPDH mRNA in vehicle-treated (24 h) neurons (A) and astrocytes (B) from Npc1^+/+ (black) and Npc1^−/− (white) mice. LDLR, low density lipoprotein receptor; SREBP, sterol response element-binding protein; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; ABC, ATP-binding cassette. Data are means ± S.E. of triplicate analyses from three independent experiments. *, p < 0.05; **, p < 0.001.

FIGURE 11.

qPCR analysis of mRNAs involved in cholesterol synthesis and efflux in neurons (A and B) and astrocytes (C and D) from Npc1^−/− (A and C) and Npc1^+/+ (B and D) mice. Cells were incubated for 24 h with vehicle (dark gray), 0.1 mm CD (light gray), or 1 mm CD (white), and mRNA levels were quantified relative to GAPDH mRNA. Gene abbreviations are defined in the legend to Fig 10. Data are means ± S.E. from triplicate analyses of three independent experiments. *, p < 0.05; ** p < 0.01; ***, p < 0.001.