Pathological impact of SMN 2 mis‐splicing in adult SMA mice

Abstract

Loss-of-function mutations in SMN1 cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. The related SMN2 gene expresses suboptimal levels of functional SMN protein, due to a splicing defect. Many SMA patients reach adulthood, and there is also adult-onset (type IV) SMA. There is currently no animal model for adult-onset SMA, and the tissue-specific pathogenesis of post-developmental SMN deficiency remains elusive. Here, we use an antisense oligonucleotide (ASO) to exacerbate SMN2 mis-splicing. Intracerebroventricular ASO injection in adult SMN2-transgenic mice phenocopies key aspects of adult-onset SMA, including delayed-onset motor dysfunction and relevant histopathological features. SMN2 mis-splicing increases during late-stage disease, likely accelerating disease progression. Systemic ASO injection in adult mice causes peripheral SMN2 mis-splicing and affects prognosis, eliciting marked liver and heart pathologies, with decreased IGF1 levels. ASO dose–response and time-course studies suggest that only moderate SMN levels are required in the adult central nervous system, and treatment with a splicing-correcting ASO shows a broad therapeutic time window. We describe distinctive pathological features of adult-onset and early-onset SMA.

Motor neuron cell-nonautonomous rescue of spinal muscular atrophy phenotypes in mild and severe transgenic mouse models

“…On the other hand, in several studies, including our own, selective depletion of SMN in motor neurons induces NMJ defects, and restoration of SMN rescues them, indicating a cell-autonomous mechanism (Park et al 2010;Gogliotti et al 2012;Lee et al 2012;Martinez et al 2012;Sahashi et al 2012Sahashi et al , 2013. This apparent discrepancy may be due to the extremely low SMN levels in motor neurons in these studies.…”

Rescue of gene-expression changes in an induced mouse model of spinal muscular atrophy by an antisense oligonucleotide that promotes inclusion of SMN2 exon 7

“…Given that (1) few genotype-driven changes were present in a model of severe SMA and (2) significant early post-natal expression changes in the mouse spinal cord would likely confound any changes mediated by ASO therapy, we decided to analyze gene-expression changes in a more recently characterized induced model of SMA [12,13]. To evaluate the time course of transcriptional changes induced by skipping SMN2 exon 7 beyond background levels of exon 7 exclusion, 15-week-old mice with mild SMA (Smn -/-; SMN2 +/+ ) [7] were given a single ICV bolus injection of 50 μg exon 7 skip ASO 20-37 or a control ASO, and central nervous system tissue (spinal cord and brain) was collected 10, 20 or 30 days after treatment (Table 1).…”

“…Therefore, an induced model of SMA was developed by administering adult mice with mild SMA an ASO, designated ASO 20-37, that targets a purine-rich exon splicing enhancer region in the middle of exon 7 and promotes skipping of SMN2 exon 7 beyond background levels of exon 7 exclusion [12,13]. We recently described the phenotypic and histopathologic effects of induced SMN depletion and repletion with and , respectively, delivered by intracerebroventricular (ICV) injection in neonatal [12] and adult [13] mice with SMA. The consequences of SMN2 mis-splicing were progressive and dose dependent.…”

SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage

“…In an effort to understand the link between RNA processing changes and gene-level changes in SMA and to further investigate the Cdkn1a activation, we characterized spinal cord transcriptome changes by RNAseq at high depth in an ASO-mediated inducible mouse model of type I SMA, which bypasses confounding developmental changes (6,7). Expanding on the aforementioned studies showing snRNP assembly defects and splicing changes, we found that thousands of introns in functionally diverse genes were retained in a manner correlating with splice site strength, such that introns with weaker splicing signals were more sensitive to SMN depletion.…”

“…Importantly, SMN2 copy number varies across individuals and is a modifier of disease severity, with a higher copy number leading to reduced severity. We previously characterized the function of an antisense oligonucleotide (ASO) that binds and blocks an exonic splicing silencer in exon 7 of SMN2 pre-mRNA, enhancing the inclusion of exon 7 and the production of full-length protein when delivered to the cerebrospinal fluid in an inducible mouse model of type I SMA (6,7). We found that early treatment with an ASO that promoted SMN2 exon 7 inclusion prevented and reversed the gene expression changes that occurred on disease induction (6).…”