An "exacerbate-reverse" strategy in yeast identifies histone deacetylase inhibition as a correction for cholesterol and sphingolipid transport defects in human Niemann-Pick type C disease

Abstract

Niemann-Pick type C (NP-C) disease is a fatal lysosomal lipid storage disorder for which no effective therapy exists. A genome-wide, conditional synthetic lethality screen was performed using the yeast model of NP-C disease during anaerobiosis, an auxotrophic condition that requires yeast to utilize exogenous sterol. We identified 12 pathways and 13 genes as modifiers of the absence of the yeast NPC1 ortholog (NCR1) and quantified the impact of loss of these genes on sterol metabolism in ncr1Δ strains grown under viable aerobic conditions. Deletion of components of the yeast NuA4 histone acetyltransferase complex in ncr1Δ strains conferred anaerobic inviability and accumulation of multiple sterol intermediates. Thus, we hypothesize an imbalance in histone acetylation in human NP-C disease. Accordingly, we show that the majority of the 11 histone deacetylase (HDAC) genes are transcriptionally up-regulated in three genetically distinct fibroblast lines derived from patients with NP-C disease. A clinically approved HDAC inhibitor (suberoylanilide hydroxamic acid) reverses the dysregulation of the majority of the HDAC genes. Consequently, three key cellular diagnostic criteria of NP-C disease are dramatically ameliorated as follows: lysosomal accumulation of both cholesterol and sphingolipids and defective esterification of LDL-derived cholesterol. These data suggest HDAC inhibition as a candidate therapy for NP-C disease. We conclude that pathways that exacerbate lethality in a model organism can be reversed in human cells as a novel therapeutic strategy. This "exacerbate-reverse" approach can potentially be utilized in any model organism for any disease.

FIGURE 1.

Deletion of 13 genes confers significant growth defects to the ncr1Δ yeast model of NP-C disease as a consequence of sterol auxotrophy. Haploid double deletions were derived from a cross of ncr1Δ and the indicated haploid single gene deletions. 5-Fold dilutions (left to right) of saturated, aerobically grown cultures of the single mutants and their derived double mutant were grown at 30 °C aerobically on SCD and anaerobically on SCD + 20 μg/ml ergosterol + 0.5% Tween 80 for 3 days.

FIGURE 2.

Sterol metabolism is disturbed in yeast ncr1Δ eaf1Δ cells. A, analysis of ncr1Δ eaf1Δ strains identifies a bottleneck in aerobic sterol synthesis with increased intracellular accumulation of ergosterol precursors and decreased ergosterol. Cells were grown in triplicate under aerobic conditions in YPD at 30 °C to 100 OD units. Sterol biosynthetic intermediates were measured by GC-MS and are expressed as a percentage of total sterols. *, p < 0.05, two-tailed Student's t test comparison of ncr1Δ eaf1Δ cells with control, ncr1Δ, or eaf1Δ strains. B, sensitivity of ncr1Δ eaf1Δ to fluconazole and nystatin. 5-Fold dilutions (left to right) of saturated, aerobically grown cultures were grown aerobically at 30 °C for 2 days in the presence of the indicated drug.

FIGURE 3.

Histone deacetylase genes are up-regulated in human NP-C fibroblasts. RNA from the indicated mutants and control fibroblast lines were assessed for HDAC gene expression by quantitative real time PCR using the primers described in supplemental Table S1. The majority of the 11 HDAC genes are up-regulated in fibroblasts derived from three patients with NP-C disease (NPC-26, NPC-2, and NPC-29). *, p < 0.05, two-tailed Student's t test for each NP-C fibroblast relative to the control fibroblast. Mis-regulation of HDAC11 has been reported elsewhere (42).

FIGURE 4.

Targets of HDAC genes are dysregulated in NP-C fibroblasts and regulated by SAHA. NPC1 mutant and control fibroblast lines were grown in the presence and absence of SAHA and assessed for expression of genes known to be responsive to HDAC activity. A statistical comparison was conducted separately for untreated control versus untreated NP-C, untreated control versus treated control, and untreated NP-C versus treated NP-C cells using a two-tailed Student's t test (* indicates a significant difference based on p < 0.05).

FIGURE 5.

Treatment of NPC-26 fibroblasts with SAHA restores expression of HDAC genes. NPC1 mutant and control fibroblast lines were grown in the presence and absence of SAHA and assessed for expression of the HDAC genes. *, p < 0.05, two-tailed Student's t test compared treated and untreated cells separately for control and NP-C fibroblasts.

FIGURE 6.

Global histone deacetylase inhibition corrects cholesterol homeostasis in NP-C fibroblasts. Mutant fibroblasts (NPC-26 and NPC-J4) were incubated for 18 h in the presence of 5 μm SAHA, stained with filipin, and assessed for esterification of LDL-cholesterol. A, reduction in lysosomal accumulation of unesterified cholesterol as measured by filipin fluorescence. Quantification of filipin fluorescence was obtained and is expressed as arbitrary units. B, restoration of deficient esterification of LDL-derived cholesterol as measured by percent cholesteryl [³H]oleate formation relative to total lipids. Cells were grown for 4 days in lipoprotein-deficient serum, followed by a 24-h treatment with or without 5 μm SAHA, the last 4 h of which included LDL plus [³H]oleate. Incorporation was quantified by scintillation and normalized to total lipid. *, p < 0.05, treated versus untreated cells by two-tailed Student's t test.

FIGURE 7.

Global histone deacetylase inhibition corrects lysosomal accumulation of sphingolipids in NP-C fibroblasts. Mutant fibroblasts (NPC-26 and NPC-J4) were incubated for 18 h in the presence of 5 μm SAHA and assessed for sphingolipid accumulation. A, reduction in lysosomal accumulation of globotriosylceramide (GL-3) as measured with verotoxin. NPC-26 fibroblasts were incubated for 18 h in the presence of 5 μm SAHA and stained with verotoxin. B, NPC-J4 fibroblasts in the presence and absence of SAHA were stained with Alexa Fluor 555-cholera toxin subunit B to visualize GM1. C, transport of BODIPY-LacCer in NPC-J4 cells was assessed by fluorescent microscopy following a 30-min loading at 4 °C and a 30-min chase. Quantitation of fluorescence was obtained and is expressed as arbitrary units. Statistically significant differences are based on a two-tailed Student's t test (* indicates a significant difference based on p < 0.05).

FIGURE 8.

Class-specific histone deacetylase inhibition ameliorates cholesterol accumulation. Mutant fibroblasts (NPC-26) were incubated for 18 h in the presence of the HDAC class-specific inhibitors MC1568 and MGCD0103 (5 μm) and assessed for cholesterol accumulation by filipin fluorescence. Quantification of filipin fluorescence is expressed as arbitrary units. *, p < 0.05, treated versus untreated cells by two-tailed Student's t test.