Gabriela E. Oprea, Sandra Kröber, Michelle L. McWhorter, Wilfried Rossoll, Stefan Müller, Michael Krawczak, Gary J. Bassell, Christine E. Beattie, Brunhilde Wirth

Homozygous deletion of the survival motor neuron 1 gene (SMN1) causes spinal muscular atrophy (SMA), the most frequent genetic cause of early childhood lethality. In rare instances, however, individuals are asymptomatic despite carrying the same SMN1 mutations as their affected siblings, thereby suggesting the influence of modifier genes. We discovered that unaffected SMN1-deleted females exhibit significantly higher expression of plastin 3 (PLS3) than their SMA-affected counterparts. We demonstrated that PLS3 is important for axonogenesis through increasing the F-actin level. Overexpression of PLS3 rescued the axon length and outgrowth defects associated with SMN down-regulation in motor neurons of SMA mouse embryos and in zebrafish. Our study suggests that defects in axonogenesis are the major cause of SMA, thereby opening new therapeutic options for SMA and similar neuromuscular diseases.
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Leading European researchers and clinicians have joined forces in a newly launched Network of Excellence (NoE) on finding new treatments for rare neuromuscular diseases (NMD), such as muscular dystrophies and spinal muscular atrophy.

Dubbed TREAT-NMD (Translational research in Europe – assessment and treatment of neuromuscular), the five-year network is the first of its kind in Europe, bringing together a total of 21 partner organisations from 11 countries. They include charities and companies that will work alongside doctors and researchers in the field.
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Proximal spinal muscular atrophy (SMA) is a common autosomal recessively inherited neuromuscular disorder causing infant death in half of all patients. Homozygous loss of the survival motor neuron 1 (SMN1) gene causes SMA, whereas the number of the SMN2 copy genes modulates the severity of the disease. Due to a silent mutation within an exonic splicing enhancer, SMN2 mainly produces alternatively spliced transcripts lacking exon 7 and only approximately 10% of a full-length protein identical to SMN1. However, SMN2 represents a promising target for an SMA therapy. The correct splicing of SMN2 can be efficiently restored by over-expression of the splicing factor Htra2-beta1 as well as by exogenous factors like drugs that inhibit histone deacetylases (HDACs). Here we show that the novel benzamide M344, an HDAC inhibitor, up-regulates SMN2 protein expression in fibroblast cells derived from SMA patients up to 7-fold after 64 h of treatment. Moreover, M344 significantly raises the total number of gems/nucleus as well as the number of nuclei that contain gems. This is the strongest in vitro effect of a drug on the SMN protein level reported so far. The reversion of Delta7-SMN2 into FL-SMN2 transcripts as demonstrated by quantitative RT-PCR is most likely facilitated by elevated levels of Htra2-beta1. Investigations of the cytotoxicity of M344 using an MTT assay revealed toxic cell effects only at very high concentrations. In conclusion, M344 can be considered as highly potent HDAC inhibitor which is active at low doses and therefore represents a promising candidate for a causal therapy of SMA.
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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 alpha-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 alpha-motoneurone degeneration by epigenetic SMN2 gene activation.
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by Brunhilde Wirth
Institute of Human Genetics of the University of Cologne, Germany

Here we report on our paper entitled “In vivo activation of SMN in SMA carriers and patients treated with valproate” by Brichta L, Holker I, Haug K, Klockgether T & Wirth B. Annals of Neurology, 2006, April 10, advanced online publication.

In July 2003, we reported that valproic acid (VPA) was able to increase full-length SMN2 transcript and protein levels by 2fold to 4fold in fibroblasts derived from SMA patients (Brichta et al. Hum Mol Genet, 2003). Similar results were shown by the group of K. Fischbeck and published in November 2003 (Sumner et al., Ann Neurol 2003). Furthermore, we have been able to demonstrate that VPA significantly increases SMN RNA/protein levels in cultured brain slices from rat and humans (obtained after surgery of epilepsy patients) as well as in cultured rat embryonic motor neurons (Hahnen et al., J Neurochemistry, 2006, in press).

VPA is a well-explored FDA-approved drug that rarely shows any severe side effects in long-term therapy of epilepsy patients. This makes it available for a straightforward application in humans.

Meanwhile, we studied the effect of VPA in blood from SMA carriers and patients to address the following questions: (1) Is VPA capable of acting on the in vivo FL-SMN transcript/protein level, and (2) how suitable is the use of blood for the development of a biomarker that would allow monitoring of the drug response in VPA-treated SMA patients?
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