Suberoylanilide hydroxamic acid (vorinostat) up-regulates progranulin transcription: rational therapeutic approach to frontotemporal dementia

Abstract

Progranulin (GRN) haploinsufficiency is a frequent cause of familial frontotemporal dementia, a currently untreatable progressive neurodegenerative disease. By chemical library screening, we identified suberoylanilide hydroxamic acid (SAHA), a Food and Drug Administration-approved histone deacetylase inhibitor, as an enhancer of GRN expression. SAHA dose-dependently increased GRN mRNA and protein levels in cultured cells and restored near-normal GRN expression in haploinsufficient cells from human subjects. Although elevation of secreted progranulin levels through a post-transcriptional mechanism has recently been reported, this is, to the best of our knowledge, the first report of a small molecule enhancer of progranulin transcription. SAHA has demonstrated therapeutic potential in other neurodegenerative diseases and thus holds promise as a first generation drug for the prevention and treatment of frontotemporal dementia.

FIGURE 1.

Luciferase reporter-based HTS for small molecule enhancers of progranulin expression. A, schematic representation of a bacterial artificial chromosome (BAC)-based reporter construct. Luciferase coding sequence was inserted into exon 2 of the human GRN gene, replacing the original start codon with the first codon of luciferase cDNA. chr.17, chromosome 17. B, results from screening 1200 compounds composing the Prestwick Chemical Library®. Neuro-2a cells stably expressing the reporter were treated with the compounds at 2.5 μm for 24 h, and luciferase activity was measured afterward. Arrows indicate the compounds that resulted in the top two highest activities.

FIGURE 2.

HDAC inhibitors and resveratrol dose-dependently increase luciferase reporter activity. The Neuro-2a luciferase reporter cell line used for screening was also utilized in follow-up experiments. A, dose-response relationships for HTS hits SAHA (●), resveratrol (△), and leflunomide (■). EC₅₀ values were 0.51 μm for SAHA and 0.24 μm for resveratrol. B, dose-response relationships for HDAC inhibitors M344 (●), PTACH (△), and trichostatin A (TSA; □). C, dose-response relationships for class I specific HDAC inhibitors MS-275 (●) and sodium valproate (△). D, selective HDAC inhibitors MC1568 (class II selective; ●), tubacin (HDAC6-specific; ■), tubastatin A (HDAC6-specific; △), and droxinostat (HDAC3-, HDAC6-, and HDAC8-selective; ♢) did not increase GRN promoter reporter activity. E, SAHA and resveratrol have additive effects. Resveratrol concentrations tested are shown on the x axis. SAHA concentrations tested are noted next to the dose-response curves. ●, resveratrol alone; △, resveratrol + 0.1 μm SAHA; ■, resveratrol + 0.3 μm SAHA; ♢, resveratrol + 0.5 μm SAHA. F, SAHA and resveratrol have additive effects. SAHA concentrations tested are shown on the x axis. Resveratrol concentrations tested are noted next to the dose-response curves. ●, SAHA alone; △, SAHA + 0.1 μm resveratrol; ■, SAHA + 1 μm resveratrol.

FIGURE 3.

HDAC inhibitors moderately decrease cell viability. Neuro-2a cells were treated with the indicated drugs for 24 h, and cell titers were measured with a luciferase-based assay. Lower luminescence values correspond to lower ATP concentrations in the well. Error bars indicate S.E. *, p < 0.05 versus the vehicle control. TSA, trichostatin A.

FIGURE 4.

SAHA enhances progranulin expression in cultured cells. A and B, qPCR analysis measured the relative abundance of GRN mRNA in Neuro-2a cells treated with SAHA or resveratrol for 24 h. 1 μm SAHA significantly increased GRN expression, whereas 1 μm resveratrol did not have any effect either alone or in combination with 1 μm SAHA. In the inset in A, the same samples were analyzed using qPCR primers designed to detect pre-mRNA. *, p < 0.05; ns, p > 0.05 versus the vehicle control or for the indicated comparisons. C–F, Neuro-2a whole cell lysates (C; quantified in D) and cell culture supernatants (E and F) were analyzed for progranulin (PGRN) protein expression by immunoblotting after a 24-h application of indicated drugs. #, significant toxicity was observed with the indicated treatment.

FIGURE 5.

SAHA enhances progranulin expression in haploinsufficient human cells. A, Epstein-Barr virus-immortalized human lymphoblastoid cells from a subject with a nonsense GRN R493X mutation (gray bars) and from a relative carrying wild-type alleles (black bars) were treated with SAHA for 24 h, and the relative abundance of GRN mRNA was measured by qPCR (n = 2 for 0.3 and 1 μm SAHA; n = 3 for control and 2.5 μm SAHA). *, p < 0.05 versus the GRN^+/+ control; **, p < 0.05 for the indicated comparison. The dashed line indicates the relative abundance of 1. B, human dermal fibroblasts from two subjects heterozygous for GRN nonsense mutations (AA and II) and from two control subjects (HH and V) were treated as indicated, and the relative abundance of GRN mRNA was measured by qPCR. All samples were processed in parallel, and ΔΔC_t values were normalized to one of the control samples (HH). The dashed line indicates the relative abundance of 1. C, cumulative data from the four cell lines shown in B plus an independent human dermal fibroblast line with a heterozygous R493X mutation is shown (n = 6 for 0 and 1 μm SAHA; n = 4 for 0.1 and 0.3 μm SAHA). *, p < 0.05 versus the vehicle control. The inset shows Western blot analysis of total cell lysates from HEK293 cells treated with the indicated concentrations of SAHA for 24 h. PGRN, progranulin.