Targeting SR proteins improves SMN expression in spinal muscular atrophy cells.

Spinal muscular atrophy (SMA) is one of the most common inherited causes of pediatric mortality. SMA is caused by deletions or mutations in the survival of motor neuron 1 (SMN1) gene, which results in SMN protein deficiency. Humans have a centromeric copy of the survival of motor neuron gene, SMN2, which is nearly identical to SMN1. However, SMN2 cannot compensate for the loss of SMN1 because SMN2 has a single-nucleotide difference in exon 7, which negatively affects splicing of the exon. As a result, most mRNA produced from SMN2 lacks exon 7. SMN2 mRNA lacking exon 7 encodes a truncated protein with reduced functionality. Improving SMN2 exon 7 inclusion is a goal of many SMA therapeutic strategies. The identification of regulators of exon 7 inclusion may provide additional therapeutic targets or improve the design of existing strategies. Although a number of regulators of exon 7 inclusion have been identified, the function of most splicing proteins in exon 7 inclusion is unknown. Here, we test the role of SR proteins and hnRNP proteins in SMN2 exon 7 inclusion. Knockdown and overexpression studies reveal that SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF11, hnRNPA1/B1 and hnRNP U can inhibit exon 7 inclusion. Depletion of two of the most potent inhibitors of exon 7 inclusion, SRSF2 or SRSF3, in cell lines derived from SMA patients, increased SMN2 exon 7 inclusion and SMN protein. Our results identify novel regulators of SMN2 exon 7 inclusion, revealing potential targets for SMA therapeutics.

The DcpS inhibitor RG3039 improves motor function in SMA mice.

Spinal muscular atrophy (SMA) is caused by mutations of the survival motor neuron 1 (SMN1) gene, retention of the survival motor neuron 2 (SMN2) gene and insufficient expression of full-length survival motor neuron (SMN) protein. Quinazolines increase SMN2 promoter activity and inhibit the ribonucleic acid scavenger enzyme DcpS. The quinazoline derivative RG3039 has advanced to early phase clinical trials. In preparation for efficacy studies in SMA patients, we investigated the effects of RG3039 in severe SMA mice. Here, we show that RG3039 distributed to central nervous system tissues where it robustly inhibited DcpS enzyme activity, but minimally activated SMN expression or the assembly of small nuclear ribonucleoproteins. Nonetheless, treated SMA mice showed a dose-dependent increase in survival, weight and motor function. This was associated with improved motor neuron somal and neuromuscular junction synaptic innervation and function and increased muscle size. RG3039 also enhanced survival of conditional SMA mice in which SMN had been genetically restored to motor neurons. As this systemically delivered drug may have therapeutic benefits that extend beyond motor neurons, it could act additively with SMN-restoring therapies delivered directly to the central nervous system such as antisense oligonucleotides or gene therapy.

Increased IGF-1 in muscle modulates the phenotype of severe SMA mice.

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by the mutation of the survival motor neuron 1 (SMN1) gene and deficiency of the SMN protein. Severe SMA mice have abnormal motor function and small, immature myofibers early in development suggesting that SMN protein deficiency results in retarded muscle growth. Insulin-like growth factor 1 (IGF-1) stimulates myoblast proliferation, induces myogenic differentiation and generates myocyte hypertrophy in vitro and in vivo. We hypothesized that increased expression of IGF-1 specifically in skeletal muscle would attenuate disease features of SMAΔ7 mice. SMAΔ7 mice overexpressing a local isoform of IGF-1 (mIGF-1) in muscle showed enlarged myofibers and a 40% increase in median survival compared with mIGF-1-negative SMA littermates (median survival = 14 versus 10 days, respectively, log-rank P = 0.025). Surprisingly, this was not associated with a significant improvement in motor behavior. Treatment of both mIGF-1(NEG) and mIGF-1(POS) SMA mice with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in a further extension of survival and improved motor behavior, but the combination of mIGF-1 and TSA treatment was not synergistic. These results show that increased mIGF-1 expression restricted to muscle can modulate the phenotype of SMA mice indicating that therapeutics targeted to muscle alone should not be discounted as potential disease-modifying therapies in SMA. IGF-1 may warrant further investigation in mild SMA animal models and perhaps SMA patients.

The genetics of spinal muscular atrophies.

PURPOSE OF REVIEW: This article reviews clinical, genetic, and therapeutic advances in spinal muscular atrophies (SMAs), inherited disorders characterized by motor neuron loss and muscle weakness. RECENT FINDINGS: There has been progress in defining the clinical and genetic features of at least 16 distinct forms of SMA. The genes associated with 14 of these disorders have been identified in the last decade, including four within the last year: TRPV4, ATP7A, VRK1, and HSPB3. Genetic testing is now available for many SMAs, providing important diagnostic and prognostic information. Cell and animal models of SMAs have been used to further understand how mutations in SMA-associated genes, which code for proteins involved in diverse functions such as transcriptional regulation, RNA processing, and cytoskeletal dynamics, lead to motor neuron dysfunction and loss. In the last year, there has also been remarkable progress in preclinical therapeutics development for proximal SMA using gene therapy, antisense oligonucleotides, and small molecules. SUMMARY: The advances in the clinical and genetic characterization of different forms of SMAs have important implications for clinical evaluation and management of patients. The identification of multiple, novel SMA-causing genes will lead to an improved understanding of motor neuron disease biology and may provide novel targets for therapeutics development.

Inhibition of myostatin does not ameliorate disease features of severe spinal muscular atrophy mice.

There is currently no treatment for the inherited motor neuron disease, spinal muscular atrophy (SMA). Severe SMA causes lower motor neuron loss, impaired myofiber development, profound muscle weakness and early mortality. Myostatin is a transforming growth factor-beta family member that inhibits muscle growth. Loss or blockade of myostatin signaling increases muscle mass and improves muscle strength in mouse models of primary muscle disease and in the motor neuron disease, amyotrophic lateral sclerosis. In this study, we evaluated the effects of blocking myostatin signaling in severe SMA mice (hSMN2/delta7SMN/mSmn(-/-)) by two independent strategies: (i) transgenic overexpression of the myostatin inhibitor follistatin and (ii) post-natal administration of a soluble activin receptor IIB (ActRIIB-Fc). SMA mice overexpressing follistatin showed little increase in muscle mass and no improvement in motor function or survival. SMA mice treated with ActRIIB-Fc showed minimal improvement in motor function, and no extension of survival compared with vehicle-treated mice. Together these results suggest that inhibition of myostatin may not be a promising therapeutic strategy in severe forms of SMA.