In vitro and ex vivo evaluation of second-generation histone deacetylase inhibitors for the treatment of spinal muscular atrophy

Authors


Address correspondence and reprint requests to Eric Hahnen, PhD, Institute of Human Genetics, University of Cologne, Kerpenerstrasse 34, 50931 Cologne, Germany. E-mail: eric.hahnen@uk-koeln.de

Abstract

Among a panel of histone deacetylase (HDAC) inhibitors investigated, suberoylanilide hydroxamic acid (SAHA) evolved as a potent and non-toxic candidate drug for the treatment of spinal muscular atrophy (SMA), an α-motoneurone disorder caused by insufficient survival motor neuron (SMN) protein levels. SAHA increased SMN levels at low micromolar concentrations in several neuroectodermal tissues, including rat hippocampal brain slices and motoneurone-rich cell fractions, and its therapeutic capacity was confirmed using a novel human brain slice culture assay. SAHA activated survival motor neuron gene 2 (SMN2), the target gene for SMA therapy, and inhibited HDACs at submicromolar doses, providing evidence that SAHA is more efficient than the HDAC inhibitor valproic acid, which is under clinical investigation for SMA treatment. In contrast to SAHA, the compounds m-Carboxycinnamic acid bis-Hydroxamide, suberoyl bishydroxamic acid and M344 displayed unfavourable toxicity profiles, whereas MS-275 failed to increase SMN levels. Clinical trials have revealed that SAHA, which is under investigation for cancer treatment, has a good oral bioavailability and is well tolerated, allowing in vivo concentrations shown to increase SMN levels to be achieved. Because SAHA crosses the blood–brain barrier, oral administration may allow deceleration of progressive α-motoneurone degeneration by epigenetic SMN2 gene activation.

Abbreviations used
CBHA

m-Carboxycinnamic acid bis-Hydroxamide

DIV

days in vitro

DMSO

dimethylsulphoxide

FL

full length

HDAC

histone deacetylase

M344

4-Dimethylamino-N-(6-hydroxycarbamoylhexyl)benzamide N-Hydroxy-7-(4-dimethylaminobenzoyl)aminoheptanamide

MN/SC

motoneurones seeded on Schwann cell layer

MS-275

N-(2-Aminophenyl)-4-[N-(pyridine-3-ylmethoxycarbonyl)aminomethyl]benzamide

OHSC

organotypic hippocampal brain slice culture

PI

propidium iodide

SAHA

suberoylanilide hydroxamic acid

SBHA

suberoyl bishydroxamic acid

SC

Schwann cell layer

SMA

spinal muscular atrophy

SMN

survival motor neuron

VPA

valproic acid

Introduction

Spinal muscular atrophy (SMA) is a common autosomal recessively inherited α-motoneurone disorder that causes weakness and atrophy of voluntary muscles. The disease-determining survival motor neurone gene 1 (SMN1) is homozygously absent in 96% of all patients with SMA and intragenic SMN1 mutations are correspondingly rare (Wirth 2000). Within the SMA region on chromosome 5q, the human SMN gene exists in two copies, SMN1 and SMN2, which are ubiquitously expressed and encode identical proteins (Lefebvre et al. 1995). Even though all patients with SMA lacking SMN1 carry at least one SMN2 gene copy, the amount of functional SMN protein produced by SMN2 is not sufficient to prevent progressive α-motoneurone degeneration. This finding has been assigned to a single translationally silent C to T transition within exon 7, affecting the splicing of primary SMN transcripts (Lorson et al. 1999). As a consequence, the disease-determining SMN1 gene produces full-length transcripts only (FL-SMN), whereas the majority of SMN2 transcripts lack exon 7 owing to alternative splicing (Δ7-SMN). Truncated Δ7-SMN proteins are reduced in their ability to self-oligomerize, which is essential for proper SMN function (Lorson et al. 1998; Wolstencroft et al. 2005) and recently have been shown to ameliorate, but not to prevent, the SMA phenotype in vivo (Le et al. 2005). The disease-modifying property of the SMN2 gene has been verified in transgenic mouse models, confirming SMN2 as a therapeutic target (Monani et al. 2000). Consequently, transcriptional SMN2 activation and/or modulation of the SMN2 splicing pattern to increase FL-SMN levels may be an effective strategy for SMA treatment.

The fatty acids butyrate, phenylbutyrate and valproic acid (VPA) have shown to transcriptionally activate human SMN2 and to modulate its splicing pattern to increase FL-SMN levels (Chang et al. 2001; Brichta et al. 2003; Sumner et al. 2003; Andreassi et al. 2004). Similar to (phenyl)butyrate, the anticonvulsant VPA possesses histone deacetylase (HDAC) inhibitor properties (Gottlicher et al. 2001; Phiel et al. 2001), indicating that the effects observed occur by a common HDAC inhibitory pathway. HDACs operate in concert with histone acetyltransferases to determine the acetylation status of core histones, thus modifying the expression of a subset of genes by chromatin remodelling. Three classes of HDACs have been identified, but only class I (HDAC 1–3, 8) and class II (HDAC 4–7, 9–11) enzymes are susceptible to HDAC inhibitors (Marks et al. 2004).

Although the finding that (phenyl)butyrate and VPA increase SMN protein levels is promising, the clinical potential of the weak fatty acid class of HDAC inhibitors awaits confirmation. The propensity of HDAC inhibitors to initiate growth arrest, differentiation and/or apoptosis of neoplastic cells fostered the development of novel and highly potent HDAC inhibitors, some of which are under clinical investigation for cancer treatment (Marks et al. 2004). The purpose of this study was to evaluate experimentally the second-generation HDAC inhibitors, including the hydroxamic acids suberoylanilide hydroxamic acid (SAHA), m-Carboxycinnamic acid bis-Hydroxamide (CBHA), suberoyl bishydroxamic acid (SBHA) (Richon et al. 1996), as well as the benzamides N-(2-Aminophenyl)-4-[N-(pyridine-3-ylmethoxycarbonyl)aminomethyl]benzamide (MS-275) and 4-Dimethylamino-N-(6-hydroxycarbamoylhexyl)benzamide N-Hydroxy-7-(4-dimethylaminobenzoyl)aminoheptanamide (M344) (Jung et al. 1999; Saito et al. 1999) for SMA treatment. Potential SMN2 gene activation and modulation of its splicing pattern was analysed using SMN1-deleted fibroblasts derived from patients with SMA, which represent a commonly used tool for SMA drug screening. However, it is not known whether HDAC inhibitors increase SMN transcript and protein levels in native, CNS-derived cells and tissues. To address this issue, the test compounds were further analysed using organotypic hippocampal brain slice cultures (OHSCs) and motoneurone-enriched cell fractions derived from rat. The rodent orthologues of the human SMN genes, Smn, are not subject to alternative splicing and exist in one copy only (Battaglia et al. 1997). However, it has been shown that the SMN2/Smn genes share a reproducible pattern of histone acetylation that is largely conserved between tissues and species (Kernochan et al. 2005). Moreover, the SMN2/Smn promoters are highly conserved (Germain-Desprez et al. 2001) and both have shown to be associated with HDAC1 and 2, whereas no specific association was observed for other HDAC isoenzymes (Kernochan et al. 2005). Because the SMN2/Smn promoters share similar chromatin architecture, the analysis of native CNS-derived cells/tissues derived from rat provides crucial data for the identification and characterization of novel candidate drugs for SMA therapy.

Materials and methods

Inhibitors of HDACs

Sources of HDAC inhibitors were as follows: CBHA (Calbiochem, San Diego, CA, USA), M344 (Calbiochem), MS-275 (Calbiochem), SAHA (Alexis Biochemicals, San Diego, CA, USA), SBHA (Alexis Biochemicals), VPA (Sigma-Aldrich, St Louis, MO, USA). The test compounds were dissolved in H20 (VPA) or 100% dimethylsulphoxide (DMSO).

Cell culture

F98 rat glioma cells were cultured as described previously (Eyupoglu et al. 2005). The primary SMN1-deleted fibroblast lines ML5 and ML16 were established from skin biopsies derived from patients with SMA showing three SMN2 copies, and grown as described by Brichta et al. (2003).

Cell viability assay

Viable cell numbers were estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Mosmann 1983). In brief, 8000 cells/mL were seeded in a 96-well plate, treated either with the test compound or solvent only, and analysed at 96 h after incubation as described previously (Eyupoglu et al. 2005).

Hippocampal brain slice cultures

OHSCs derived from 5-day-old Wistar rats were prepared according to the interface technique (Stoppini et al. 1991). After dissection of the frontal pole of the hemispheres and the cerebellum, the brains were cut in 350-µm thick horizontal slices on a vibratome (Leica Microsystems, Wetzlar, Germany). For each experiment, three slices were transferred into culture plate insert membranes (BD Biosciences, San Jose, CA, USA) and thereafter into six-well culture dishes (BD Biosciences) containing 1.2 mL culture medium as described in detail by Eyupoglu et al. (2005). One day after preparation, the culture medium was changed, and OHSCs were exposed to the test compound for 48 h and snap-frozen in liquid nitrogen. Propidium iodide (PI) staining of OHSCs was performed as described previously (Eyupoglu et al. 2005). Human hippocampi were removed from samples obtained during epilepsy surgery. For scientific use of tissue specimens, informed consent was obtained from each patient with the approval of the local ethics committee of the University of Erlangen. Surgical specimens were prepared and processed as described for rat OHSCs.

Motoneurone-enriched co-cultures

Motoneurones were enriched and cultured on a Schwann cell feeder layer as described previously (Haastert et al. 2005). In brief, motoneurone-rich cell fractions were prepared by density gradient centrifugation of enzymatically dissociated ventral horns of the lumbar spinal cords of Sprague–Dawley rat embryos (gestational age 14–15 days) and seeded at 2.7 × 104 cells/cm2 on a layer of neonatal Schwann cells. Motoneurone–Schwann cell co-cultures were exposed to the test compounds, whereas non-treated cultures served as controls. Tests were done in triplicate (three sister cultures) for each compound and concentration.

RT–PCR quantification of Smn/SMN2 transcript levels

Real-time RT–PCR quantification of rat Smn transcript levels relative to those of β-actin (F98 cells, OHSCs), including PCR cycling conditions, as well as primer and probe sequences, have been described in detail previously (Brichta et al. 2003). For analysis of SMN2-derived FL-SMN and Δ7-SMN transcript levels in SMN1-deleted fibroblasts, quantitative RT–PCR was performed using primers within exon 6 and exon 8 respectively. As internal control, the glyceraldehyde-3-phosphate dehydrogenase gene was co-amplified using primers in exon 1 and exon 4. The PCR reaction included 23 cycles only to ensure quantitative measurements during the linear phase. PCR conditions, primer sequences, visualization of PCR products and densitometric quantification were performed as described in detail elsewhere (Brichta et al. 2003).

Immunoblot analyses

Protein lysates of motoneurone-enriched Schwann cell co-cultures were subjected to immunoblot analyses using monoclonal anti-SMN antibody (BD Transduction Laboratories, Lexington, KY, USA) and neurone specific anti-tubulin β III (Upstate, Lake Placid, NY, USA). Western blots were analysed using Scion Image Beta 4.02. (Scion, Frederick, MA, USA) Immunoblot analyses of rat Smn and human SMN protein levels were performed using monoclonal anti-SMN antibody (BD Transduction Laboratories), mouse monoclonal anti β-tubulin (Sigma-Aldrich) and mouse monoclonal β-actin (Sigma-Aldrich) as described in detail previously (Brichta et al. 2003).

Assessment of HDAC inhibition in vitro

HDAC activity was determined as described by Heltweg and Jung (2002) by applying a BioVisionTM fluorimetric assay (BioCat, Heidelberg, Germany). In brief, a HDAC fluorometric substrate, which comprises an acetylated lysine side chain, was incubated with a sample containing HDAC activity. Deacetylation sensitized the substrate so that, in the second step, treatment with the lysine developer produced a fluorophore. Fluorescence was measured with a NOVOstar plate reader (BMG LABTECH, Jena, Germany) at excitation(max) 380 nm and emission(max) 460 nm. Class I and class II HDACs have been shown to be generally expressed in all tissues examined (de Ruijter et al. 2003). For in vitro analyses, HDAC isolated from rat liver was purchased from Alexis Biochemicals. The extraction procedure has been described previously in detail (Heltweg and Jung 2002). Some 30 µL undiluted preparation from rat liver was applied per well. Its specific HDAC activity amounted to ∼ 100 pmol of substrate per min at 37°C.

Data analysis

Concentration–effect curves for the inhibition of cell viability and HDAC activity by the test compounds were analysed by non-linear regression analysis using GraphPad Prism version 4.03 (GraphPad Software, San Diego, CA, USA). Data were fitted to a four-parameter logistic equation comprising the top plateau, bottom plateau, inflection point IC50 and curve slope nH. The parameters IC50 and nH were treated as variables; ‘top’ as the control value of cell viability or HDAC activity was set at 100%. Inhibitor concentrations inducing a 50% (IC50) and 90% (IC90) reduction in cell viability or HDAC activity were read from the best fit curves to give the individually effective concentration range for each compound. Multiple comparisons among the data sets were performed by applying one-way anova with Dunnett’s post test (GraphPad Prism version 4.03). Three levels of statistical significance were discriminated, p < 0.05, p < 0.01 and p < 0.001.

Results

Assessment of effective HDAC inhibitor concentrations for the analysis of rat cells and tissues

It was not known whether the test compounds would increase Smn expression levels in rat cells and tissues. A lack of change in Smn expression following drug treatment might indicate an inability of the test compound to modulate Smn expression or the wrong choice of drug concentration. Preliminary experiments were performed to identify biologically active doses of each test compound. HDAC inhibitors have been shown to reduce tumour cell growth, whereas the viability of normal cells is unaffected (Ungerstedt et al. 2005). In the first set of experiments, neoplastic cells (F98, rat glioma) were exposed to HDAC inhibitors at varying concentrations. All compounds reduced tumour cell viability in a dose-dependent manner, although with different efficacies. MS-275, M344 and SAHA were effective at low micromolar doses (IC90 < 10 µm). CBHA and SBHA appeared to be far less potent than the structurally related drug SAHA, whereas VPA reduced cell growth at millimolar concentrations. IC50 and IC90 values are given in the legend to Fig. 1. F98 glioma cells exposed to HDAC inhibitors displayed a concentration-dependent process outgrowth, which is a common finding after HDAC inhibitor treatment of transformed cells (Marks et al. 2004). MS-275, M344 and SAHA induced phenotypic alterations at doses of 4 µm (MS-275, M344) or 8 µm (SAHA). CBHA and SBHA required concentrations of 160 µm, wheras VPA induced process outgrowth at 2 mm. No morphological changes were observed using half of these doses. A representative experiment is shown inFig. 1a.

Figure 1.

 Assessment of effective HDAC inhibitor concentrations and Smn induction in neoplastic cells derived from rat. All compounds investigated reduced the viability of F98 rat glioma cells in a dose-dependent manner. IC50 and IC90 concentrations respectively were: MS-275, 0.8 and 3.7 µm; M344, 1.2 and 3.3 µm; SAHA, 2.6 and 7.6 µm; SBHA, 53.7 and 239.9 µm; CBHA, 61.7 and 247.5 µm; VPA, 2.6 and 6.8 mm. (a) HDAC inhibitor-induced process outgrowth of F98 cells at 48 h after drug administration (8 µm SAHA). Arrowheads mark process outgrowth not observed in time- and solvent-matched controls. (b, c) Treatment of F98 cells with M344, SAHA, SBHA, CBHA or VPA for 48 h at concentrations shown to induce phenotypic alterations increased Smn protein levels relative to those of β-tubulin. Smn protein levels were expressed as mean ± SD percentage of those in vehicle-treated, time-matched controls: M344, 172 ± 19%; SAHA, 179 ± 42%; SBHA, 187 ± 19%; CBHA, 159 ± 30%; VPA, 143 ± 23%. For quantification, western blot analyses were repeated four times. *p < 0.05. (d) Smn transcript levels (real-time PCR, relative to β-actin) in F98 cells after treatment with 4 µm MS-275, 4 µm M344, 8 µm SAHA or 2 mm VPA for 12 h. Relative Smn transcript levels were expressed as a mean ± SD percentage of those in time-matched controls: MS-275, 108 ± 13%; M344, 230 ± 28%; SAHA, 255 ± 44%; VPA, 182 ± 10%. Real-time analyses were repeated three times. *p < 0.05. Intervention data were statistically compared to control values by applying one-way anova.

In the case of VPA, M344 and SAHA, as well as the SAHA derivatives CBHA and SBHA, concentrations shown to promote phenotypic alterations in F98 cells were associated with increased Smn protein levels (Figs 1b and c), but this was not observed after treatment with 4 µm MS-275 (data not shown).

Quantitative real-time PCR analysis revealed increased Smn transcript levels by 12 h after treatment with 4 µm M344, 8 µm SAHA or 2 mm VPA, but no induction of Smn was detected after treatment with 4 µm MS-275 (Fig. 1d).

Neurotoxicity profiles of HDAC inhibitors in OHSCs

To evaluate whether HDAC inhibitors increase Smn transcript and protein levels in native, CNS-derived tissue, OHSCs obtained from early postnatal rats were used. In a first set of experiments, we investigated the cytotoxicity risk by measuring cellular PI uptake after drug administration, as described previously (Noraberg 2004). OHSCs incubated with HDAC inhibitors at concentrations shown to promote phenotypic alternations in rat glioma cells did not show increased PI incorporation levels after treatment with MS-275, M344, SAHA or VPA, whereas SBHA and CBHA induced increased PI uptake, indicative of cell damage (Figs 2a and b). In an extended analysis of SBHA and CBHA using different drug concentrations (1–80 µm), both compounds caused significantly increased levels of PI incorporation at doses of 10 µm and above (see legend to Fig. 2), indicating that both SAHA derivatives had a considerable risk of neurotoxicity. They were therefore excluded from further analyses. MS-275 failed to increase Smn levels in rat OHSCs (see below), whereas M344 and SAHA appeared to be potent candidate drugs for SMA treatment and were further evaluated in dose escalation experiments. Significantly increased PI incorporation levels were identified following administration of M344 at doses of 20 µm and above, whereas SAHA treatment did not compromise the viability of OHSCs at doses up to 80 µm, indicating a superior neurotoxicity profile of SAHA compared with M344 (Fig. 2c).

Figure 2.

 Neurotoxicity profiles of HDAC inhibitors and Smn induction using OHSCs. (a, b) PI uptake of rat OHSCs after HDAC inhibitor treatment. No significantly altered PI incorporation levels were observed following treatment with 4 µm MS-275, 4 µm M344, 8 µm SAHA or 2 mm VPA for 48 h, whereas CBHA and SBHA caused significantly increased PI uptake at 160 µm. Vehicle (DMSO) concentrations did not exceed 0.16% of the total cell/tissue culture volume in any experiment. PI incorporation levels were not changed after administration of 0.1 or 1% DMSO compared with those in non-treated controls. For quantification relative to the non-treated control, analyses were repeated six times. ***p < 0.001. (c) PI uptake of rat OHSCs 48 h after administration of increasing M344 or SAHA concentrations. For quantification, analyses were repeated three times. **p < 0.01. CBHA and SBHA caused significantly increased PI incorporation levels at doses of 10 µm and above. PI incorporation levels were expressed as a mean ± SD percentage compared with levels in time-matched controls: CBHA: 1 µm 110 ± 8%, 10 µm 127 ± 1% (p < 0.01), 20 µm 145 ± 21% (p < 0.01), 40 µm 157 ± 14% (p < 0.01), 80 µm 193 ± 23% (p < 0.01); SBHA: 1 µm 118 ± 16%, 10 µm 142 ± 9% (p < 0.01), 20 µm 131 ± 9% (p < 0.01), 40 µm 133 ± 5% (p < 0.01), 80 µm 144 ± 5 (p < 0.01). (d) Real-time PCR analyses of rat OHSCs treated with 4 µm M344, 8 µm SAHA or 2 mm VPA for 48 h revealed increased Smn transcript levels. Values were expressed as mean ± SD percentage relative to β-actin: M344, 149% (+ 39%, − 30%); SAHA, 191% (+ 12%, − 12%); VPA, 142% (+ 24%, − 30%); control, 100% (+ 11%, − 10%). No up-regulation was observed following administration of 4 µm MS-275. For quantification, real-time analyses were repeated six times. *p < 0.05, **p < 0.01, ***p < 0.001. (e) Western blot analysis (Smn, β-actin) of rat OHSCs after treatment with 4 µm M344, 8 µm SAHA or 2 mm VPA for 48 h. M344 moderately increased Smn protein levels (128 ± 14%; p < 0.05), whereas SAHA and VPA substantially increased Smn protein levels to 167 ± 26% (p < 0.01) and 200 ± 30% (p < 0.01) respectively. For quantification, western blot analyses were repeated three times.

SAHA, M344 and VPA, but not MS-275, increase Smn expression levels in OHSCs

We showed previously that administration of 2 mm VPA increased Smn transcript levels in OHSCs by 12 h, with a maximum reached at 48 h after treatment (Brichta et al. 2003). Quantitative real-time PCR analysis of OHSCs exposed to 4 µm M344, 8 µm SAHA or 2 mm VPA for 48 h revealed increased Smn transcript levels (Fig. 2d). In contrast, no Smn activation was detectable after administration of 4 µm MS-275 (Fig. 2d), compatible with a lack of Smn promoter susceptibility for MS-275, already noted in neoplastic cells (see above). Smn gene activation induced by M344, SAHA and VPA was confirmed by western blot analyses (Fig. 2e).

SAHA, M344 and VPA increase Smn protein levels in motoneurone-enriched cell cultures

To further validate the impact of HDAC inhibitors on Smn protein expression in the CNS target tissue, motoneurone-enriched cell cultures isolated from rat embryos with a gestational age of 14–15 days (E14/15) were used, as described previously in detail (Haastert et al. 2005). After 3 days in vitro (DIV), more than 70% of neurones were motoneurones (Haastert et al. 2005), indicating a high level of enrichment by the chosen preparation and purification method. Motoneurone-enriched cell fractions were cultured on a non-confluent Schwann cell feeder layer for 3 DIV, and subsequently exposed to M344, SAHA or VPA. In each experiment, drugs were used at concentrations that were shown to reduce the viability of rat glioma cells by 50 or 90% (see above). At 3 DIV, motoneurone-enriched cell cultures exposed to IC50 concentrations demonstrated increased Smn protein levels relative to levels of neurone-specific β III tubulin (VPA, 305%; M344, 264%; SAHA, 176%), whereas Smn induction was not further increased when IC90 doses were used (VPA, 207%; M344, 156%; SAHA, 166%) (Fig. 3a). At 6 DIV, co-cultures treated with IC50 or IC90 doses of SAHA showed an approximately two-fold increase in Smn protein levels (184% and 199% respectively) relative to levels of neurone-specific β III tubulin (Fig. 3b).

Figure 3.

 SAHA, M344 and VPA increase Smn protein levels in motoneurone-enriched cell cultures. (a) Western blot analysis (Smn, neurone-specific βIII tubulin) of motoneurone-rich cell fractions seeded on a Schwann cell layer and cultivated for 3 DIV followed by treatment with VPA (2.6 mm, 6.8 mm), M344 (1.2 µm, 3.3 µm) or SAHA (2.6 µm, 7.6 µm) for 20 h. (b) Western blot analysis (Smn, neurone-specific β III tubulin) of motoneurone-rich cell fractions seeded on a Schwann cell layer (MN/SC) and cultivated for 6 DIV, followed by treatment with 2.6 µm or 7.6 µm SAHA for 20 h. Both proteins (Smn, β III tubulin) were barely detectable in non-confluent Schwann cell feeder layers (SC) dissolved in equal amounts of lysis buffer as used for co-cultures, indicating that increased Smn expression levels were due to Smn gene activation in motoneurone-enriched cell fractions.

SAHA and M344, but not MS-275, activate the human SMN2 gene in fibroblasts derived from patients with SMA

In contrast to the human SMN2 gene, the rat Smn gene is not subject to alternative splicing. Thus, the Smn induction described so far was based solely on transcriptional activation. To evaluate the capacity of HDAC inhibitors to affect human SMN2 expression and/or splicing pattern, SMN1-deleted fibroblasts derived from two unrelated patients with SMA were investigated (cell lines ML16 and ML5). At 24 h after treatment, low micromolar doses of SAHA (0.05–10 µm) increased SMN2-derived SMN protein levels with a peak value of 296 ± 61% at 5 µm (cell line ML16) (Figs 4a and b). SMN2 gene activation following SAHA treatment was confirmed using a second fibroblast cell line (ML5). Again, a significant (p < 0.05), more than two-fold induction in SMN protein was observed after treatment with 0.5, 1 and 5 µm SAHA (233 ± 17, 241 ± 67 and 202 ± 53% respectively). Analysis of SMN2 transcript levels revealed increased amounts of both SMN2-derived isoforms (FL-SMN2, Δ7-SMN2) at 24 h after treatment, again with a maximum observed at a dose of 5 µm SAHA (Fig. 4c). Similar to VPA and butyrate, which have been shown to alter the SMN2 splicing pattern (Chang et al. 2001; Brichta et al. 2003; Sumner et al. 2003; Andreassi et al. 2004), the FL-SMN2/Δ7-SMN2 ratio was moderately changed towards increased FL-SMN transcript levels, which reached significance at 10 µm SAHA (Fig. 4d). Exposure of ML16 cells to non-toxic concentrations of M344 (≤ 20 µm, see above) for 24 h increased SMN protein levels to 148 ± 11% (0.5 µm), 163 ± 15% (5 µm) and 168 ± 6% (10 µm). As with SAHA, increased FL-SMN2 and Δ7-SMN2 transcript levels were detectable using doses of 5 and 10 µm, with a shift towards FL-SMN2 transcripts (data not shown). In contrast to SAHA and M344, treatment of patient-derived fibroblasts with 0.05–5.0-µm doses of MS-275 for 24 h did not significantly affect SMN2-derived protein or transcript levels. Representative western blot and RT–PCR analyses are shown in Figs 4(e) and (f).

Figure 4.

 SAHA and M344, but not MS-275, activate the human SMN2 gene in fibroblasts derived from patients with SMA. (a) Western blot analysis of SMN protein levels in fibroblast cultures (ML16) treated with increasing SAHA concentrations (0.05–10 µm) for 24 h. (b) Bar graph showing mean ± SEM SMN protein levels relative to levels of β-tubulin. For quantification, western blot analyses were repeated three times. *p < 0.05. (c, d) Quantitative RT–PCR analysis of SMN2-derived FL-SMN and Δ7-SMN transcripts in fibroblast cultures (ML16) treated with increasing SAHA concentrations for 24 h. SMN2 transcript levels (FL-SMN and Δ7SMN respectively) were expressed as a mean ± SEM percentage of values in time-matched controls: 0.05 µm, 100 ± 1 and 101 ± 2%; 0.5 µm, 108 ± 10 and 117 ± 6%; 1 µm, 117 ± 16 and 120 ± 5%; 5 µm, 148 ± 12 and 135 ± 11%; 10 µm, 136 ± 16 and 113 ± 6%. For quantification, analyses were repeated three times. *p < 0.05. (e) Western blot analysis (SMN, β-tubulin) of SMN protein levels in fibroblast cultures (ML16) treated with increasing concentrations of MS-275 for 24 h. (f) Quantitative RT–PCR analysis of SMN2-derived transcripts in fibroblast cultures (ML16) treated with increasing concentrations of MS-275 for 24 h.

SAHA increases SMN protein levels in human brain tissue

A novel ex vivo assay using human OHSCs obtained from epilepsy surgery was established to further evaluate candidate drugs for SMA therapy. Multiple OHSCs were dissected from a surgical specimen of the human hippocampus, cultured for 24 h in a humified atmosphere, and exposed to 8 µm SAHA or 2 mm VPA for 48 h. Significantly increased SMN protein levels were observed after VPA administration (190 ± 12%), indicating the viability of neuroepithelial cell populations within human OHSCs. However, administration of 8 µm SAHA did not alter the SMN protein level in this particular sample (data not shown). Using a second surgical specimen, human OHSCs were exposed to increasing, non-toxic doses of SAHA, i.e. 16, 32 and 64 µm. Moderately increased SMN protein levels were observed after administration of 16 µm SAHA (123 ± 12%), whereas SMN induction was pronounced following treatment with 32 µm (212 ± 25%) and 64 µm (162 ± 28%) doses (Fig. 5a). To confirm this observation, OHSCs derived from a third patient were exposed to SAHA for 48 h. Repeated western blot analyses revealed significantly increased SMN protein levels after treatment with 16 µm (178 ± 23%), 32 µm (184 ± 31%) and 64 µm (187 ± 30%) doses (Fig. 5b), indicating that SAHA is capable of increasing SMN levels in human CNS.

Figure 5.

 SAHA increases SMN protein levels in human brain tissue. (a, b) Western blot analysis of SMN protein levels in human OHSCs (derived from two independent patients with epilepsy) treated with increasing SAHA concentrations (16, 32 and 64 µm) for 48 h. For quantification, analyses were repeated three times.

Isoenzyme-specific HDAC inhibition by VPA and MS-275, but not by M344 and SAHA

To confirm the potencies of MS-275, M344, SAHA and VPA in reducing HDAC activity, and to gain insight into the inability of MS-275 to increase Smn/SMN2 protein levels, HDAC inhibition by the test compounds was investigated in vitro as described previously (Heltweg and Jung 2002). Detailed quantitative analysis confirmed a concentration-dependent inhibition of total HDAC activity in all experiments, although with considerable variation in potency and efficacy. M344 and SAHA inhibited HDAC activity in a concentration-dependent manner and completely at comparatively low concentrations (Fig. 6); half-maximum inhibition by M344 and SAHA was observed at submicromolar doses (M344, 0.3 µm; SAHA, 0.5 µm). In contrast, inhibition of total HDAC activity by MS-275 and VPA required higher concentrations and plateaued at 54 ± 5% (MS-275) and 40 ± 3% (VPA) of control HDAC activity, suggesting that not all HDAC isoenzymes may be susceptible to MS-275 and VPA even at excessive concentrations (Fig. 6). With an inflection point of 7.9 mm, VPA appeared to be at least 10 000 fold less potent than the second-generation HDAC inhibitors M344 and SAHA. Interestingly, the curve for the inhibition of HDAC activity by MS-275 (inflection point 9.3 µm) was shallow with a slope nH = 0.66 ± 0.12 (F-test, p = 0.014), indicating a deviation from simple mass action behaviour.

Figure 6.

 Isoenzyme-specific HDAC inhibition by VPA and MS-275, but not by M344 and SAHA. Concentration–effect curves for the inhibition of HDAC activity isolated from rat liver by MS-275, M344, SAHA and VPA as determined by non-linear regression analysis. Half-maximum inhibition of total HDAC activity by each test compound, defined as inflection point (IC0.5), were as follows: IC0.5, M344 = 0.3 µM, IC0.5, SAHA = 0.5 µM, IC0.5, MS-275 = 9.3 µM, and IC0.5, VPA =7.9 mM). Inhibition of HDAC activity by MS-275 and VPA plateaued at 54 ± 5% and 40 ± 3% of control HDAC activity respectively. Values are mean ± SEM of three to nine independent experiments performed as duplicate determinations.

Discussion

Our results indicate that the second-generation HDAC inhibitor SAHA seems a promising candidate drug for the pharmacological treatment of SMA. Its ability to substantially increase Smn/SMN protein levels at very low micromolar concentrations has been confirmed in various experimental paradigms, including rat OHSCs (Fig. 2), rat motoneurone-enriched cell cultures (Fig. 3), fibroblasts derived from patients with SMA (Fig. 4) as well as in a novel human OHSC assay (Fig. 5). SAHA and M344 effectively reduced HDAC activity at submicromolar concentrations with comparable potencies (Fig. 6). Complete inhibition of HDAC activity by SAHA is compatible with recent data indicating that this hydroxamic acid is not selective for class I and II HDAC isoenzymes (Richon et al. 1998). The isoenzyme selectivity of the benzamide M344 for class II compared with class I HDACs is comparatively weak (Heltweg et al. 2004), suggesting that both compounds essentially do not discriminate between HDAC isoenzymes. Compared with M344, which showed definite toxicity to rat brain parenchyma at 20 µm, the hydroxamic acid SAHA has a more favourable cytotoxicity profile. SAHA caused no detectable toxicity to highly vulnerable rat brain parenchyma even at a concentration of 80 µm (Fig. 2). This feature is particularly promising, considering recent clinical phase I trials with patients suffering from advanced cancer (Kelly et al. 2003). In vivo drug concentrations can be obtained for SAHA in the range 3–54 µm (Kelly et al. 2003), doses that were shown to increase Smn/SMN protein levels in vitro and ex vivo in the present study. In addition, daily intravenous administration of SAHA is well tolerated with no dose-limiting toxicity.

Ongoing clinical trials are currently evaluating the efficacy of the fatty acid class of HDAC inhibitors for SMA treatment. VPA induces accumulation of acetylated histones starting at 0.25 mm (Gottlicher et al. 2001), with a maximum effect observed at millimolar concentrations (Gottlicher et al. 2001; Phiel et al. 2001). We have shown that millimolar doses of VPA are capable of increasing Smn/SMN protein levels with no toxicity to rat brain parenchyma. VPA inhibits HDAC activity in a concentration-dependent manner but does not completely abolish it even at excessive doses. This finding suggests that not all HDAC isoenzymes may be susceptible to inhibition by VPA. However, the HDAC inhibitory action of VPA requires high doses, which might be difficult to achieve in CNS tissue. Recently, alternative pathways of VPA action have been described, which may contribute to the induction of SMN2 observed in SMN1-deleted fibroblasts after treatment with submillimolar doses of VPA (Brichta et al. 2003). Induction of proteosomal HDAC2 isoenzyme degradation was observed with VPA but not with other HDAC inhibitors (Kramer et al. 2003). Furthermore, VPA triggers replication-independent DNA demethylation by enhancing DNA demethylase activity (Detich et al. 2003). However, it remains to be elucidated whether the SMN2 promoter is subject to DNA methylation.

MS-275, which is also under advanced clinical investigation for cancer treatment (Marks et al. 2004), had no apparent impact on Smn/SMN2 expression (Figs 1d, 2d, 4e and 4f). In contrast to M344 and SAHA, MS-275 was not able to completely diminish HDAC activity in a mixture of HDAC isoenzymes (Fig. 6). This finding suggests a pronounced HDAC isoenzyme specificity of MS-275, as already described. MS-275 has been shown to have a considerably higher inhibitory activity against HDAC1 (IC50 0.3 µm) than HDAC3 (IC50 8 µm) (Hu et al. 2003) and an approximately 40 000-fold higher inhibitory activity against HDAC1 than HDAC6 (Miller et al. 2003). Based on the findings that (i) MS-275 is a potent inhibitor of HDAC1 at submicromolar doses (Hu et al. 2003), (ii) MS-275 is not able to increase Smn/SMN2 expression levels (Figs 1d, 2d, 4e and 4f), and (ii) a clear association between Smn/SMN2 gene promoters and HDAC1 has been shown (Kernochan et al. 2005), we hypothesize that HDAC1 does not serve as major target for epigenetic SMA therapy. A definite association between HDAC2 and Smn/SMN promoters has been described, which appears to increase during development (Kernochan et al. 2005). However, the impact of HDAC2 as a specific therapeutic target remains to be further elucidated.

Our present study highlights SAHA as a promising compound for the treatment of an autosomal recessively inherited disease. Ongoing clinical phase II evaluation of SAHA in patients with cancer has demonstrated its good oral bioavailability and its biological activity (Marks et al. 2004). However, it has not been established whether SAHA crosses the blood–brain barrier in humans, although earlier studies in rodents showed an accumulation of acetylated histones in brain tissue following SAHA administration, suggesting diffusion across the blood–brain barrier (Hockly et al. 2003). Therefore, oral administration of SAHA may be a promising means of decelerating or arresting progressive α-motoneurone degeneration caused by insufficient amounts of SMN protein by epigenetic SMN2 gene activation.

Acknowledgements

We thank Tajana Jungbauer (Erlangen), Dorit Müller (Erlangen), Iris Jusen (Bonn) and Hella Brinkmann (Hannover) for excellent technical assistance. This study was supported by the Initiative Forschung und Therapie für SMA, Deutsche Gesellschaft für Muskelkranke, Deutsche Forschungsgemeinschaft (SFB400 A6, Wi945/12–1, Bl421/1–2), Families of SMA (WIR0507), the Center for Molecular Medicine Cologne (TV-98) and the Fritz Thyssen Stiftung.

Ancillary