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.

Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron (SMN) protein due to the functional loss of the SMN1 gene and the inability of its paralog, SMN2, to fully compensate due to reduced exon 7 splicing efficiency. Since SMA patients have at least one copy of SMN2, drug discovery campaigns have sought to identify SMN2 inducers. C5-substituted quinazolines increase SMN2 promoter activity in cell-based assays and a derivative, RG3039, has progressed to clinical testing. It is orally bioavailable, brain-penetrant and has been shown to be an inhibitor of the mRNA decapping enzyme, DcpS. Our pharmacological characterization of RG3039, reported here, demonstrates that RG3039 can extend survival and improve function in two SMA mouse models of varying disease severity (Taiwanese 5058 Hemi and 2B/- SMA mice), and positively impacts neuromuscular pathologies. In 2B/- SMA mice, RG3039 provided a >600% survival benefit (median 18 days to >112 days) when dosing began at P4, highlighting the importance of early intervention. We determined the minimum effective dose and the associated pharmacokinetic (PK) and exposure relationship of RG3039 and DcpS inhibition ex vivo. These data support the long PK half-life with extended pharmacodynamic outcome of RG3039 in 2B/- SMA mice. In motor neurons, RG3039 significantly increased both the average number of cells with gems and average number of gems per cell, which is used as an indirect measure of SMN levels. These studies contribute to dose selection and exposure estimates for the first studies with RG3039 in human subjects.

Spinal muscular atrophy (SMA) is caused by low survival motor neuron (SMN) levels and patients represent a clinical spectrum due primarily to varying copies of the survival motor neuron-2 (SMN2) gene. Patient and animals studies show that disease severity is abrogated as SMN levels increase. Since therapies currently being pursued target the induction of SMN, it will be important to understand the dosage, timing and cellular requirements of SMN for disease etiology and potential therapeutic intervention. This requires new mouse models that can induce SMN temporally and/or spatially. Here we describe the generation of two hypomorphic Smn alleles, SmnC-T-Neo and Smn2B-Neo. These alleles mimic SMN2 exon 7 splicing, titre Smn levels and are inducible. They were specifically designed so that up to three independent lines of mice could be generated, herein we describe two. In a homozygous state each allele results in embryonic lethality. Analysis of these mutants indicates that greater than 5% of Smn protein is required for normal development. The severe hypomorphic nature of these alleles is caused by inclusion of a loxP-flanked neomycin gene selection cassette in Smn intron 7, which can be removed with Cre recombinase. In vitro and in vivo experiments demonstrate these as inducible Smn alleles. When combined with an inducible Cre mouse, embryonic lethality caused by low Smn levels can be rescued early in gestation but not late. This provides direct genetic evidence that a therapeutic window for SMN inductive therapies may exist. Importantly, these lines fill a void for inducible Smn alleles. They also provide a base from which to generate a large repertoire of SMA models of varying disease severities when combined with other Smn alleles or SMN2-containing mice.