Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on July 29, 2003

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Human Molecular Genetics, 2003, Vol. 12, No. 19 2481-2489
DOI: 10.1093/hmg/ddg256
© 2003 Oxford University Press

Valproic acid increases the SMN2 protein level: a well-known drug as a potential therapy for spinal muscular atrophy

L. Brichta^1,2, Y. Hofmann¹, E. Hahnen³, F. A. Siebzehnrubl³, H. Raschke¹, I. Blumcke³, I. Y. Eyupoglu⁴ and B. Wirth^1,2,*

¹Institute of Human Genetics, University of Bonn, Wilhelmstrasse 31, 53111 Bonn, Germany, ²Institute of Human Genetics, University of Cologne, Kerpener Str. 34, 50931 Cologne, Germany, ³Institute of Neuropathology, University Erlangen-Nuremberg, Krankenhausstrasse 8-10, 91054 Erlangen, Germany and ⁴Department of Neurosurgery, University Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany

Received May 8, 2003; Accepted July 25, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder causing infant death in half of all patients. Homozygous absence of the survival motor neuron gene (SMN1) is the primary cause of SMA, while SMA severity is mainly determined by the number of SMN2 copies. One SMN2 copy produces only about 10% of full-length protein identical to SMN1, whereas the majority of SMN2 transcripts is aberrantly spliced due to a silent mutation within an exonic splicing enhancer in exon 7. However, correct splicing can be restored by over-expression of the SR-like splicing factor Htra2-ß1. We show that in fibroblast cultures derived from SMA patients treated with therapeutic doses (0.5–500 µM) of valproic acid (VPA), the level of full-length SMN2 mRNA/protein increased 2- to 4-fold. Importantly, this up-regulation of SMN could be most likely attributed to increased levels of Htra2-ß1 which facilitates the correct splicing of SMN2 RNA as well as to an SMN gene transcription activation. Especially at low VPA concentrations, the restored SMN level depended on the number of SMN2 copies. Moreover, VPA was able to increase SMN protein levels through transcription activation in organotypic hippocampal brain slices from rats. Finally, VPA also increased the expression of further SR proteins, which may have important implications for other disorders affected by alternative splicing. Since VPA is a drug highly successfully used in long-term epilepsy therapy, our findings open the exciting perspective for a first causal therapy of an inherited disease by elevating the SMN2 transcription level and restoring its correct splicing.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Proximal spinal muscular atrophy (SMA) is one of the most common recessively inherited disorders in humans with an incidence of 1 : 6000 and a heterozygosity frequency of 1 : 35 (1,2). Some 94% of all SMA patients present homozygous absence of the survival motor neuron gene 1 (SMN1), allowing an easy and fast molecular genetic diagnosis of SMA (3–5). The resulting absence of SMN1 protein leads to degeneration of -motor neurons in the anterior horns of the spinal cord, causing weakness and atrophy of the proximal voluntary muscles (3). Approximately 50% of all SMA patients (type I) are never able to sit or stand unaided and usually die before the age of 2 years. Type II SMA patients are able to sit, but are never able to stand, whereas type III SMA patients are able to sit and stand, but often get wheelchair bound while muscle weakness is progressing (6).

The SMN protein has been shown to possess essential functions in snRNP biogenesis, splicing and presumably in axonal transport of RNA (7–9). Knocking out the smn gene in mice leads to early embryonic lethality proving its pivotal function (10). Consistent with the essential function of SMN, SMA patients carry at least one copy gene, SMN2, when SMN1 is homozygously deleted. SMN2 is almost identical to SMN1, but significantly differs in its splicing pattern (3). SMN2 produces minor amounts of full-length (FL) SMN2 RNA, while the vast majority of transcripts is aberrantly spliced due to a silent mutation in SMN2 exon 7 that disrupts an exonic splicing enhancer (11,12) and creates a new splicing silencer site for hnRNP A1 at the same time (13). The aberrantly spliced transcripts (7SMN2) encode a truncated protein that is biochemically unstable and unable to compensate for the loss of SMN1 (14). The severity of the disease is primarily related to the number of SMN2 copies (2,15). Depending on the SMN2 copy number, FL-SMN2 RNA levels in SMA patients range between 20 and 50%; carriers with one SMN1 copy and usually 1–3 SMN2 copies produce about 60–80% FL-SMN RNA and protein and therefore are asymptomatic (2,16). Thus, doubling of FL-SMN RNA and protein in -motor neurons of SMA patients would be sufficient to impede the onset or progression of the disease.

We have already been able to show that the splicing pattern of SMN2 can be restored to about 80% in vitro by over-expression of the splicing factor Htra2-ß1 (17). Additional splicing factors—hnRNP-G, RBM and SRp30c—stabilize the RNA–protein complex and further promote the inclusion of exon 7 into mRNA (18,19). Recently, Chang et al. (20) identified a first compound, butyrate, that significantly restores the FL-SMN2 transcript and protein levels in cell cultures of SMA patients. Further drugs such as aclarubicin and sodium vanadate have also been shown to restore the splicing pattern of SMN2 transcripts; however, side effects and toxicity do not allow consideration for long-term SMA therapy (21,22). The striking disadvantage of butyrate is its very short half-life of only 6 min in human serum, which also makes the drug inadequate for SMA therapy. Chemically, the structure of butyrate is characterized as a short-chain fatty acid. Hypothesizing a connection between the chemical structure of butyrate and its effect on the level of SMN2 RNA and protein, we assumed that valproic acid (VPA, 2-propylpentanoic acid, an eight-carbon branched-chain fatty acid) could show similar effects. Moreover, both butyrate and VPA are known to act as histone deacetylase (HDAC) inhibitors enhancing transcription of some genes (23). In contrast to butyrate, VPA is an FDA approved drug with a terminal half-life of 8–10 h in human serum. VPA is frequently used in long-term epilepsy treatment and has recently been shown to yield therapeutic effects in mood disorders as well as migraine (24). In numerous in vitro studies, VPA suppresses tumor growth and metastasis and anti-cancer activity was demonstrated in many tumor cell lines (25).

In the present study, we demonstrate that VPA significantly increases the SMN protein level in fibroblast cultures derived from SMA patients as well as in hippocampal brain slices from rats. Interestingly, VPA up-regulated the SMN protein level not only via Htra2-ß1, the splicing factor which is essential for the inclusion of exon 7 into SMN2 mRNA, but also via activation of the SMN gene transcription, most probably due to the AP1- and/or Sp1-dependent pathway.

Since VPA is an excellently examined drug in terms of toxicology, side effects and dose and has already been in clinical use for more than 3 decades, it may open the exciting possibility of a causal pharmacological treatment of SMA by transcription activation of SMN2 and modulation of the SMN2 splicing pattern at the same time.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
VPA increases SMN protein levels in fibroblast cultures from SMA patients
Fibroblast cultures from five SMA patients with homozygous absence of SMN1 were used to determine the influence of VPA on the SMN2 expression level. Three patients suffer from type I SMA and carry either two (ML17 and ML39) or three (ML16) SMN2 copies, respectively. The remaining two patients carry three SMN2 copies, one of which presents a type II SMA (ML5) whereas the other one a type III SMA (ML12).

Fibroblast cultures were treated with 0.5–1000 µM of VPA for 16 h. The optimal time period of treatment was established by a time course experiment covering a range of 12, 16, 24, 36 and 48 h (data not shown). Proteins of untreated (mock) and treated fibroblasts were analyzed by western blotting, whereas, in a first step, all blots were verified for equal amounts of protein by staining with anti-ß-tubulin and in a second step with anti-SMN antibodies. In Figure 1A and B, a representative western blot analysis of the culture ML5 is shown as an example together with a diagram displaying a dose-dependent increase of the SMN protein level with rising VPA concentrations. Mean values±SEM for SMN protein levels relative to ß-tubulin for each of the investigated SMA fibroblast lines are presented in Table 1. We observed a significant up-regulation of the SMN protein after VPA treatment with highest values ranging between the 1.8- and 4.2-fold SMN protein level compared to untreated cells. Importantly, even the lowest concentration of VPA used (0.5 µM) still increased the SMN protein level 1.6–2.3-fold (Fig. 1A and B; Table 1). Furthermore, depending on the respective SMN2 copy number, SMN protein levels varied among the investigated SMA patients. Especially at lower VPA concentrations (0.5 and 5 µM), a stronger increase of SMN protein was observed in patients carrying three SMN2 copies than in those possessing only two SMN2 copies (Table 1).

View larger version (21K): [in this window] [in a new window]   Figure 1. Increase of SMN protein and splicing factors Htra2-ß1, SF2/ASF and SRp20 in fibroblast cultures of SMA patients treated with increasing concentrations of VPA (0.5–1000 µM) for 16 h. (A) Western blots of equal amounts of protein were stained with anti-ß-tubulin antibody and then re-probed with the respective antibodies. A representative western blot is shown for ML5. Two bands for Htra2-ß1 result from the phosphorylated and the unphosphorylated form of the protein, respectively. (B) Diagrammatic presentation of the mean levels (±SEM) of SMN protein relative to ß-tubulin shown for ML5. Corresponding values are given in Table 1. (C) Levels of Htra2-ß1, SF2/ASF and SRp20 under VPA treatment in cell line ML5. Corresponding values are given in Table 3.

 

View this table: [in this window] [in a new window]   Table 1. SMN protein levels (relative to ß-tubulin) in SMA fibroblasts after VPA treatment. Average data (±SEM) from repeated experiments are shown and highest levels are marked in bold

 
VPA increases FL-SMN mRNA by transcription activation and restoring the splicing pattern
In order to identify the mechanism(s) by which VPA causes the SMN2 protein increase, analysis of SMN2 mRNA was performed: (i) by determining the FL-SMN2 versus 7SMN2 transcript ratio as a measure for an effect on exon 7 inclusion and therefore a reversion of the splicing pattern; and (ii) by determining the total amount of SMN2 transcripts (FL-SMN2 plus 7SMN2) relative to an internal control as a measure for the stimulating effect of VPA on the SMN2 transcription rate.

Therefore, after treatment of fibroblast cultures with VPA, a multiplex RT–PCR was carried out under quantitative conditions using primers within exon 6 and 8 of SMN2. Both, FL-SMN2 and 7SMN2 mRNA values, as well as the ratio among them, were calculated relative to the internal control (GAPDH). A summary of the results obtained for each investigated SMA fibroblast culture is shown in Table 2, a representative gel analysis of the multiplex PCR (ML5) together with the corresponding bar graphs showing the average data from repeated experiments is given in Figure 2A–D. Values for the total amount of SMN2 transcripts relative to the internal control were confirmed separately by a second independent quantitative multiplex RT–PCR amplifying parts of exons 1 and 2 of SMN2 (data not shown).

View this table: [in this window] [in a new window]   Table 2. Levels of FL-SMN RNA, 7SMN RNA, total SMN transcripts FL+7 (relative to GAPDH) and the FL/7 ratio in SMA fibroblasts with increasing concentrations of VPA. Average data (±SEM) from repeated experiments are shown and highest levels are marked in bold

 

View larger version (25K): [in this window] [in a new window]   Figure 2. Up-regulation of SMN mRNA in fibroblast cultures of SMA patients treated with increasing concentrations of VPA. (A) Representative multiplex RT–PCR for cell line ML5 carried out under quantitative conditions, showing a steady increase of both FL-SMN and 7SMN. GAPDH served as internal standard. Additionally, a reversion of the splicing pattern is visible. (B) Diagrammatic presentation of the mean values (±SEM) for FL-SMN and 7SMN-RNA relative to GAPDH for cell line ML5. FL-SMN shows a 2.1-fold increase, 7SMN a 1.5-fold, suggesting two mechanisms for FL-RNA generation: reversion of 7-SMN into FL-SMN mRNA shown by the FL-SMN versus 7SMN ratio (C) and transcription activation through the promoter demonstrated by the total transcript level of SMN2 mRNA (D). See also Table 2.

 
Our data clearly demonstrate that VPA treatment of primary fibroblasts derived from SMA patients results in a 1.8–5.2-fold increase of FL-SMN2 mRNA (Table 2; Fig. 2A and B). However, the correction of the SMN2 splicing pattern due to the preferential inclusion of SMN2 exon 7 only partially contributes to the 5.2-fold augmentation of FL-SMN2 transcripts, since the FL-SMN2 versus 7SMN2 ratio increases up to about 1.5-fold only (Fig. 2A and C; Table 2). These data suggest that the supplementary up-regulation is the result of a higher SMN2 gene transcription activity (Fig. 2A, B and D; Table 2). This observation is supported by the increase of both the 7SMN2 transcripts as well as the total amount of SMN2 transcripts (Fig. 2B and D; Table 2). Thus, a synergistic effect of transcription activation and reversion of the SMN2 splicing pattern is responsible for an increase of FL-SMN2 mRNA after treatment of SMA fibroblasts with 0.5–1000 µM VPA.

VPA has been shown to increase the DNA binding capacity of activating protein-1 (AP1) and the Sp family of transcription factors (26,27). The human SMN promoter contains several AP1- and Sp1-binding motifs, which may explain the VPA induced transcriptional activation of SMN2 (28).

VPA increases the level of SR and SR-like splicing factors
An increased amount of SMN2 pre-mRNA would require higher levels of splicing factors in general to process these transcripts and eventually requires higher levels of Htra2-ß1, the most important trans-acting splicing factor shown to restore FL-SMN2 mRNA (17). In order to explain the preferred processing of SMN2 pre-mRNA to FL-SMN2 transcripts, we restained the western blots with an anti-Htra2-ß1 antibody (18). Htra2-ß1 is significantly up-regulated under increasing concentrations of VPA with highest levels varying between 2.7- and 4.1-fold as compared to untreated cells (Fig. 1A and C; Table 3). Furthermore, we analyzed whether this effect is specific for Htra2-ß1 or valid for SR proteins in general, and restained the blots with antibodies against two additional SR splicing factors, SF2/ASF and SRp20. The levels of both splicing proteins were elevated. The amounts of all three splicing factors, however, varied within one particular cell line and among different cell lines (Table 3 and Fig. 1C). Nevertheless, here we demonstrate for the first time that VPA also activates the expression of SR and SR-like splicing factors, including Htra2-ß1 which specifically restores the correct splicing of SMN2 exon 7 (17).

View this table: [in this window] [in a new window]   Table 3. Levels of the splicing factors Htra2-ß1, SF2/ASF and SRp20 (relative to ß-tubulin) in SMA fibroblasts with increasing concentrations of VPA. Average data (±SEM) from repeated experiments are shown and highest levels are marked in bold

 
In addition, we investigated whether butyrate, a short-chain fatty acid which has already been shown to restore the splicing pattern of SMN2 and to increase SMN protein levels (20), might also act via Htra2-ß1. As in the case of VPA, butyrate treatment of fibroblasts leads to increased Htra2-ß1, SF2/ASF and SRp20 protein levels (Table 4), demonstrating that both drugs share similar pathways of activation.

View this table: [in this window] [in a new window]   Table 4. SMN, Htra2-ß1, SF2/ASF and SRp20 protein levels (relative to ß-tubulin) in SMA fibroblasts with increasing concentrations of sodium butyrate. Average data (±SEM) from repeated experiments are shown and highest levels are marked in bold

 
VPA increases rat smn RNA/protein in organotypic hippocampal slice cultures
Since SMA is a neuromuscular disorder, the effect of VPA, especially on neuronal tissue, is of importance in considering the drug as suitable for a potential therapy. Most of the native tissues derived from the CNS, however, hardly survive in culture and are not well established. An extremely potent drug screening and drug validation tool available for CNS disorders is the use of organotypic hippocampal slice cultures (OHSCs) from rat (29,30). Thus, we analyzed the effects of VPA on the expression of rat smn (31). In contrast to humans, rodents carry one smn gene only, which is not subject to alternative splicing. Potential VPA dependent up-regulation of smn in rat OHSCs would therefore solely be triggered by transcriptional activation. Since humans and rats are known to metabolize VPA differently (the terminal half-life of VPA in humans is 8–10 h and in rats 2.5 h, respectively), optimal VPA concentrations and time periods for the stimulation of the OHSCs were determined in pilot experiments. Different concentrations of VPA (50, 500 and 2000 µM) were added to the OHSCs and harvested after 48 h. Expression of smn was analyzed by real time PCR. In all experiments, we observed a prominent increase in the expression of smn mRNA with increasing VPA concentration (Fig. 3A). After determining the most effective VPA concentration (2 mM), OHSCs were stimulated for different time periods (12, 24, 36 and 48 h). With elapsing time, the smn expression is steadily increasing with a maximum of about 1.6-fold at 48 h as compared to untreated OHSCs (Fig. 3B). To validate those experiments, OHSCs were stimulated with the optimal concentration of 2 mM VPA for 48 h in a final experiment and analysis of smn mRNA and protein was performed. VPA treatment induced an up-regulation of smn mRNA as well as an 1.8-fold up-regulation of smn protein levels (Fig. 4A and B). In addition, a moderate increase for Htra2-ß1 and a more significant up-regulation for SF2/ASF were observed (Fig. 4A and C).

View larger version (21K): [in this window] [in a new window]   Figure 3. Up-regulation of smn mRNA in OHSCs treated with VPA. Smn mRNA and ß-actin were determined by real-time quantitative PCR on an ABI Prism 7700 System. (A) Diagrammatic representation of increasing smn mRNA (relative to ß-actin) with increasing concentrations of VPA. (B) Influence of 2 mM VPA on OHSCs as compared to untreated OHSCs in a time course experiment (12–48 h).

 

View larger version (16K): [in this window] [in a new window]   Figure 4. Increase of rat smn protein in OHSCs treated with VPA (2 mM, 48 h). (A) Western blot analysis of proteins derived from untreated and VPA treated brain slices (2 mM, 48 h), respectively. Two additional diagrams demonstrate the corresponding mean values±SEM for smn protein (B) as well as for splicing factors Htra2-ß1 and SF2/ASF (C), each of these proteins relative to ß-actin, from three untreated OHSCs (controls) as compared to three OHSCs treated with VPA.

 
In accordance with the human SMN promoter (accession number AF092925), the rat smn promoter (accession number AC093971) contains several AP1- and Sp1-binding sites, suggesting an AP1- and/or Sp1-dependent pathway for smn transcription activation.

The obtained results clearly demonstrate that VPA increases SMN not only in fibroblast cultures, but also in neuronal tissue. Importantly, these data show that, in humans, VPA achieves high levels of SMN through both, reversion of the splicing pattern of exon 7 via increased levels of Htra2-ß1 and activation of the SMN promoter, whereas in rats a more moderate elevation is obtained exclusively based on transcription activation of the smn promoter probably via AP1- and Sp1-transcription factors.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
In this study, we were able to present a realistic possibility for a causative pharmacological treatment of SMA. The VPA doses used in human fibroblast cultures derived from SMA patients were within the therapeutic range common in epilepsy treatment. Therapy of epilepsy patients with VPA usually requires serum levels of 480–700 µM VPA (corresponding to about 70–100 µg/ml). About 15% of the VPA present in serum cross the blood–brain barrier leading to a concentration of 72–105 µM VPA (10.5–15 µg/ml) in brain and spinal liquor (32). In our experiments, we showed that an 2-fold increase of FL-SMN2 transcript and protein, respectively, could be achieved in the presence of 0.5–50 µM VPA (corresponding to 0.072–7.2 µg VPA/ml). Thus, compared to the VPA level required in epilepsy treatment, our data show that an efficient increase of SMN protein can be obtained at even lower concentrations which might be sufficient to stop the so far inevitable progression of the disease. Through axonal sprouting, even a slight improvement is conceivable.

Among the investigated human fibroblast cell lines, variable maximum values for SMN2 RNA and protein are obtained after treatment with different concentrations of VPA. This is consistent with the interindividual metabolic variability of VPA observed among epilepsy patients during therapy. The discrepancy between the different VPA concentrations required for the maximal FL-SMN2 RNA level and the maximal SMN protein level within one cell line suggests that VPA stimulates transcription and translation of SMN2 differently.

VPA is known to influence the expression of a large variety of genes through different pathways (25), two of which are most likely responsible for the elevated SMN2 transcription rates: (i) the inhibition of histon deacetylases; and (ii) the increase of the DNA binding activity of AP1- and Sp1-transcription factors. On the one hand, inhibition of histon deacetylases and simultaneous hyperacetylation of histones by the respective acetyltransferases releases DNA from histones, which allows access for transcription factors and by these means facilitates gene activation/transcription. Remarkably, a large number of genes (2%) in general is regulated by histon acetylation and deacetylation (33). However, the observed negative side effects of VPA in long-term therapies are rare (34), except for teratogenic effects during the first trimenon of pregnancy (35). On the other hand, DNA binding activity of transcription factors AP1 and Sp1 is stimulated by VPA (26,27). Strikingly, the SMN promotor (28) contains binding motifs for both proteins. In consequence, two mechanisms triggered by VPA have to be considered as causative for the up-regulated SMN2 transcription rate.

In this study, we also show for the first time that various SR and SR-like splicing factors (SF2/ASF, SRp20 and Htra2-ß1) are up-regulated by VPA and similar effects were achieved with butyrate, another inhibitor of HDACs. For Htra2-ß1, it has already been shown that over-expression leads to a reversion of the SMN2 splicing pattern and most importantly also to an increase of endogenous SMN protein (17,18). Since Htra2-ß1 is efficiently up-regulated by VPA in human fibroblasts from SMA patients, it most likely explains the achieved reversion of the SMN2 splicing pattern. It has been demonstrated that Htra2-ß1, but not SF2/ASF or SRp20 are able to restore the SMN2 splicing pattern. While SF2/ASF binds to SMN1 RNA and facilitates the correct splicing, the C-to-T transition in exon 7 abolishes the ability of SF2/ASF to bind SMN2 RNA (12). Additionally, while this paper was in review, data from Manley's group were published, which reveal that depletion of SF2/ASF has no effect on the splicing of SMN exon 7 mRNA (13). For SRp20, only an effect on its own splicing has been shown so far (36); other genes regulated by SRp20 are yet unknown.

In conclusion, among the three splicing factors shown to be up-regulated by VPA, only Htra2-ß1 can be considered directly responsible for the correct splicing of SMN2 mRNA when it is over-expressed.

For the increased levels of splicing factors, several explanations can be given. (i) The expression of the splicing factors is activated through inhibition of HDACs by VPA. (ii) Transcription activator Sp1 binding motifs are also present in the promotor of Htra2-ß1 (37) and may lead to increased expression of this protein. Definitely, Sp1 can up-regulate many transcripts, however, only Htra2-ß1 has been shown to restore FL-SMN2 mRNA. (iii) It has been demonstrated that SMN regulates its own splicing factor Htra2-ß1 such that reduced levels of SMN protein lead to reduced levels of Htra2-ß1 but not of other splicing factors, although no protein–protein interaction between SMN and Htra2-ß1 occurs (16). One plausible explanation would be that the increase of target transcripts, in this case SMN pre-mRNA, triggers the demand for splicing factors in order to guarantee correct pre-mRNA splicing (38). (iv) The increased amount of transcripts observed after VPA treatment in about 2% of genes (33) requires an elevated level of various splicing factors. (v) A common pathway (e.g. activation of a kinase or phosphorylation of SR-domains) may be responsible for an elevation of splicing factors containing an SR-domain. However, the exact mechanism of action remains to be elucidated.

Our findings open the perspective that the use of VPA or other inhibitors of HDACs, which increase the level of SR or SR-like splicing factors and affect the alternative splicing of genes, may also have therapeutic implications for diseases other than SMA.

Moreover, VPA is able to increase SMN protein levels in OHSCs from rats through transcription activation. These data show that the use of rat OHSCs is an extremely potent drug screening and drug validation tool for CNS disorders by testing the efficiency of various compounds in neuronal tissue.

So far, no therapy for SMA is available. Recently, it was demonstrated that small synthetically designed compounds are able to restore the wild type SMN2 splicing pattern in vitro by promoting exon 7 inclusion (39). However, these compounds are not suitable for in vivo delivery and therefore have to be further optimized and characterized. The identification of VPA as a well-known drug that restores FL-SMN protein to significant levels opens the exciting perspective of a causal therapy for SMA in the near future. In case of availability of an SMA therapy, one can envisage an even more successful scenery for SMA treatment: (i) postnatal screening for SMN1 deletions which is easy and fast before motor neurons are degraded; (ii) determination of the SMN2 copy number; and (iii) treatment of children carrying homozygous deletions of SMN1 with an adequate dose based on the number of SMN2 copies.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Treatment of human primary fibroblast cultures with VPA
Patient samples.
Skin biopsies were carried out from five SMA patients (three with type I, one with type II and one with type III) who fulfilled the diagnostic criteria for SMA (6) and carry homozygous deletions of SMN1. Informed consent was obtained from all individuals. From skin biopsies, primary fibroblast cultures were established according to standard protocols.

Cell culture.
Some 2x10⁵ cells of fibroblast cultures derived from SMA patients were transferred into 10 cm dishes using DMEM medium with 0.11 g/l NaPyr (Invitrogen) supplemented with 10% FCS, 1% P/S, 0.3% Amphotericin. VPA (Sigma) was dissolved in aqua dest. and added dropwise for a final concentration of 0.5, 5, 50, 500 and 1000 µM, respectively. For each experiment, to one of the dishes only aqua dest. was added to serve as a control (mock). Cells were incubated for 16 h at 5% CO₂ and 37°C. Prior to lysis in 50 µl RIPA buffer, cells were washed twice in 1x PBS buffer.

Western blot analysis.
SMA fibroblast cultures were harvested in RIPA buffer (150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, 50 mM Tris, pH 8.0) to prepare protein extracts. Denatured protein samples (10 µg protein/sample) were resolved by 12% SDS–PAGE and transferred to nitrocellulose membrane (Schleicher and Schuell) by wet blotting. Immunostaining of the membranes and detection of the signals with Chemiluminescence reagent (Super Signal West Pico, Pierce) was carried out according to standard protocols. Each experiment was repeated at least twice with different passages of the respective cell line. Intensity of the signals was measured using the ONE-Dscan program (Scanalytics). Obtained protein data were normalized to the respective mock value and given as mean±standard error of the mean (SEM).

Antibodies.
The following antibodies were used: mouse monoclonal anti-ß-tubulin (Sigma, 1 : 2000), mouse monoclonal anti-SMN (BD Transduction Laboratories, 1 : 5000), rabbit polyclonal anti-Htra2-ß1 (18, 1 : 1000), mouse monoclonal anti-SRp20 (Santa Cruz Biotechnology, 1 : 100), mouse monoclonal anti-ß-actin (Sigma, 1 : 5000), mouse monoclonal anti-SF2/ASF (kindly provided by A. Krainer, 1 : 200), horseradish peroxidase conjugated goat anti-Mouse IgG (Dianova, 1 : 2500) and horseradish peroxidase conjugated goat anti-Rabbit IgG (Pierce, 1 : 10 000).

Quantitative analysis of SMN2 mRNA from fibroblast cultures treated with VPA.
Fibroblast cultures from SMA patients were treated with VPA for 16 h as described above. Total RNA was isolated by use of the EZNA total RNA Kit (PeqLab) and transcribed into cDNA (17). Subsequently, quantitative PCR was performed using primers within SMN exon 6 (5'-ATA ATT CCC CCA CCA CCT C-3') and exon 8 (5'-GCC TCA CCA CCG TGC TGG-3') or within exon 1 (5'-ATC CGC GGG TTT GCT ATG-3') and exon 2 (5'-GTT GTA AGG AAG CTG CAG TA-3'), respectively. As internal control, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was co-amplified using primers in exon 1 (GAPDH-Fw 5'-TCC GCG CAG CCG AGC CA-3') and exon 4 (GAPDH-Rev 5'-ACG CCA GTG GAC TCC ACG-3'). The PCR reaction was carried out as follows: 95°C for 5 min, followed by 23 cycles to ensure quantitative measurements during the linear phase (95°C for 15 s, 55°C for 30 s, 72°C for 45 s) and final extension at 72°C for 10 min. PCR products were visualized on a 10% polyacrylamide gel by ethidium bromide staining. Gels were 3D-scanned (BIORAD-imaging system) and densitometric measurements were carried out with the ONE-Dscan program (Scanalytics). Each experiment was repeated at least twice with different passages of the respective cell line. Resulting data are given as mean±SEM.

Treatment of organotypic hippocampal slices fromrats with VPA
Organotypic hippocampal slice cultures (OHSCs).
Five-day-old Wistar rats were used for the preparation of OHSCs. After decapitation, brains were rapidly removed and placed in ice-cold preparation medium containing Hank's balanced salt solution (Invitrogen) with 10% normal horse serum (Biochrom). After dissection of the frontal pole of the hemispheres and the cerebellum, brains were cut into 350 µm thick horizontal slices on a vibratome (Leica Microsystems) in preparation medium as described above. For each experiment, three slices were transferred into culture plate insert membrane dishes (BD Biosciences, pore size 0.4 µm) and thereafter into 6-well culture dishes (BD Biosciences) containing 1.2 ml culture medium (MEM/HBSS=2/1, 25% normal horse serum, 2% L-glutamine, 2.64 mg/ml glucose, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10 µg/ml insulin–transferrin–sodium selenite supplement and 0.8 µg/ml vitamin C), according to the interface technique described by Stoppini et al. (29) and modified by Savaskan et al. (30). The slices were cultivated at 35°C and 5% CO₂. The medium was changed 1 day after preparation and every second day thereafter. After 6 days in vitro, the slices were incubated with VPA for different time periods and snap-frozen in liquid nitrogen.

Protein extraction.
Proteins were extracted from rat OHSCs by homogenization of three pooled slices for each experiment in 100 µl lysis-buffer containing 50 mM Tris-buffer (pH 8), 150 mM NaCl, 1% Triton-X 100, 1 mM EDTA (pH 8), 0.1% SDS, 1 mM PMFS and 1 pill Complete-Mini (Roche Applied Science) per 10 ml. Subsequently, the supernatants of homogenized samples were subjected to western blot analysis as described above. Results are normalized to the respective mock value and given as mean±SEM.

RNA extraction and quantitative real-time RT–PCR.
Total RNA was extracted from every three pooled rat OHSCs using the RNeasy Kit (Qiagen) and QIAshredder according to the manufacturer's protocols.

Quantification of mRNA was performed on an ABI Prism 7700 Sequence Detection System (Applied Biosystems). For relative quantification, the target gene was normalized to an internal reference gene (ß-actin). In preliminary experiments, we verified that rat ß-actin (AC: NM031144) and rat smn (AC: AF044910, NM022509) mRNA amplification efficiencies are nearly identical. Reference mRNA was serially diluted and run with ß-actin primers and probe to generate a standard curve. Real-time PCR was conducted three times for each RNA sample. For the one tube/single enzyme RT–PCR reaction, we used the TaqMan EZ RT–PCR Core Reagents Kit (Applied Biosystems). Each reaction used 100 ng of total RNA, 300 nM primers and 100 nM probes. Primers and probes were designed to span exon borders as follows: rat ß-actin (5'-AGG CCC CTC TGA ACC CTA AG-3', 5'-CCA GAG GCA TAC AGG GAC AAC-3', 5'-FAM-TTT GAG ACC TTC AAC ACC CCA GCC A-TAMRA-3') and rat smn (5'-GGA TGC CTC CGT TCC CTT-3', 5'-TCC AGA CAG TCG GGA GAT ATG G-3', 5'-FAM-AGG ACC ACC AAT AAT TCC TCC ACC CCC T-TAMRA-3'). Cycling conditions were 50°C for 2 min, 60°C for 30 min RT step, 95°C for 5 min followed by 40 cycles of 94°C for 20 s and 60°C for 1 min. Analysis of the real-time raw data was performed using the Sequence Detection Software, version 1.7 (Applied Biosystems).

	ACKNOWLEDGEMENTS

 
We are grateful to C.-M. Becker (Erlangen) for helpful discussion on VPA. We thank all SMA patients and clinicians who contributed to this work. We are grateful to S. Raeder (Bonn) and T. Jungbauer (Erlangen) for excellent technical assistance and to A. Krainer for providing the anti-SF2/ASF antibody. This study has been supported by grants to B.W. from the Deutsche Forschungsgemeinschaft (SFB400 A6, Graduiertenkolleg 246 TP6), Families of SMA and BONFOR and to E.H., I.Y.E. and I.B. by the Deutsche Gesellschaft für Muskelkranke e.V.

	FOOTNOTES

 
^* To whom correspondence should be addressed at: Institute of Human Genetics, Wilhelmstrasse 31, 53111 Bonn, Germany. Tel: +49 2282872344; Fax: +49 2282872380; Email: bwirth{at}uni-bonn.de

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