The loss of motor neurons (MN) is a hallmark of the neuromuscular disease spinal muscular atrophy (SMA); however it is unclear whether this phenotype autonomously originates within the MN. To address this question, we developed an inducible mouse model of severe SMA that has reduced survival and motor function, motor unit pathology and hyperexcitable MNs. Using an Hb9-Cre allele, we increased Smn levels autonomously within MNs, and demonstrate that MN-rescue significantly improves all phenotypes and pathologies commonly described in SMA mice. MN-rescue also corrects the hyperexcitability in SMA motor neurons, and prevents sensory-motor synaptic stripping. Survival in MN-rescued SMA mice is extended by only 5 days, due in part to failed autonomic innervation of the heart. Collectively, this work demonstrates that the SMA phenotype autonomously originates in MNs and that sensory-motor synapse loss is a consequence, not a cause, of MN dysfunction.

Spinal muscular atrophy (SMA) is caused by loss of the survival motor neuron gene (SMN1) and retention of the SMN2 gene. The copy number of SMN2 affects the amount of SMN protein produced and the severity of the SMA phenotype. While loss of mouse Smn is embryonic lethal, two copies of SMN2 prevents this embryonic lethality resulting in a mouse with severe SMA that dies 5 days after birth. Here we show that expression of full-length SMN under the prion promoter (PrP) rescues severe SMA mice. The PrP results in high levels of SMN in neurons at embryonic day 15. Mice homozygous for PrP-SMN with two copies of SMN2 and lacking mouse Smn survive for an average of 210 days and lumbar motor neuron root counts in these mice were normal. Expression of SMN solely in skeletal muscle using the human skeletal actin (HSA) promoter resulted in no improvement of the SMA phenotype or extension of survival. One HSA line displaying nerve expression of SMN did affect the SMA phenotype with mice living for an average of 160 days. Thus, we conclude that expression of full-length SMN in neurons can correct the severe SMA phenotype in mice. Furthermore, a small increase of SMN in neurons has a substantial impact on survival of SMA mice while high SMN levels in mature skeletal muscle alone has no impact.

Proximal spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. Traditionally, SMA has been described as a motor neuron disease; however, there is a growing body of evidence that arrhythmia and/or cardiomyopathy may present in SMA patients at an increased frequency. Here, we ask whether SMA model mice possess such phenotypes. We find SMA mice suffer from severe bradyarrhythmia characterized by progressive heart block and impaired ventricular depolarization. Echocardiography further confirms functional cardiac deficits in SMA mice. Additional investigations show evidence of both sympathetic innervation defects and dilated cardiomyopathy at late stages of disease. Based upon these data, we propose a model in which decreased sympathetic innervation causes autonomic imbalance. Such imbalance would be characterized by a relative increase in the level of vagal tone controlling heart rate, which is consistent with bradyarrhythmia and progressive heart block. Finally, treatment with the histone deacetylase inhibitor trichostatin A, a drug known to benefit phenotypes of SMA model mice, produces prolonged maturation of the SMA heartbeat and an increase in cardiac size. Treated mice maintain measures of motor function throughout extended survival though they ultimately reach death endpoints in association with a progression of bradyarrhythmia. These data represent the novel identification of cardiac arrhythmia as an early and progressive feature of murine SMA while providing several new, quantitative indices of mouse health. Together with clinical cases that report similar symptoms, this reveals a new area of investigation that will be important to address as we move SMA therapeutics towards clinical success.

Proximal spinal muscular atrophy (SMA) is a neuromuscular disorder for which there is no available therapy. SMA is caused by loss or mutation of the survival motor neuron 1 gene, SMN1, with retention of a nearly identical copy gene, SMN2. In contrast to SMN1, most SMN2 transcripts lack exon 7. This alternatively spliced transcript, Delta7-SMN, encodes a truncated protein that is rapidly degraded. Inhibiting this degradation may be of therapeutic value for the treatment of SMA. Recently aminoglycosides, which decrease translational fidelity to promote readthrough of termination codons, were shown to increase SMN levels in patient cell lines. Amid uncertainty concerning the role of SMN's C-terminus, the potential of translational readthrough as a therapeutic mechanism for SMA is unclear. Here, we used stable cell lines to demonstrate the SMN C-terminus modulates protein stability in a sequence-independent manner that is reproducible by translational readthrough. Geneticin (G418) was then identified as a potent inducer of the Delta7-SMN target sequence in vitro through a novel quantitative assay amenable to high throughput screens. Subsequent treatment of patient cell lines demonstrated that G418 increases SMN levels and is a potential lead compound. Furthermore, treatment of SMA mice with G418 increased both SMN protein and mouse motor function. Chronic administration, however, was associated with toxicity that may have prevented the detection of a survival benefit. Collectively, these results substantiate a sequence independent role of SMN's C-terminus in protein stability and provide the first in vivo evidence supporting translational readthrough as a therapeutic strategy for the treatment of SMA.