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

Abstract

Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease, is the leading monogenic cause of infant mortality. Homozygous loss of the gene survival of motor neuron 1 (SMN1) causes the selective degeneration of lower motor neurons and subsequent atrophy of proximal skeletal muscles. The SMN1 protein product, survival of motor neuron (SMN), is ubiquitously expressed and is a key factor in the assembly of the core splicing machinery. The molecular mechanisms by which disruption of the broad functions of SMN leads to neurodegeneration remain unclear. We used an antisense oligonucleotide (ASO)-based inducible mouse model of SMA to investigate the SMN-specific transcriptome changes associated with neurodegeneration. We found evidence of widespread intron retention, particularly of minor U12 introns, in the spinal cord of mice 30 d after SMA induction, which was then rescued by a therapeutic ASO. Intron retention was concomitant with a strong induction of the p53 pathway and DNA damage response, manifesting as γ-H2A.X positivity in neurons of the spinal cord and brain. Widespread intron retention and markers of the DNA damage response were also observed with SMN depletion in human SH-SY5Y neuroblastoma cells and human induced pluripotent stem cell-derived motor neurons. We also found that retained introns, high in GC content, served as substrates for the formation of transcriptional R-loops. We propose that defects in intron removal in SMA promote DNA damage in part through the formation of RNA:DNA hybrid structures, leading to motor neuron death.

Converging Mechanisms of p53 Activation Drive Motor Neuron Degeneration in Spinal Muscular Atrophy

“…Consistent with our findings, increased expression of known p53-regulated transcripts was recently reported in vulnerable SMA motor neurons from a milder mouse model of SMA (Murray et al, 2015) and in mice in which SMN deficiency was induced postnatally using antisense oligonucleotides (Jangi et al, 2017). However, no causal link between p53 activation and motor neuron death was demonstrated in SMA mice.…”

“…We focused specifically on candidate targets that are related to p53 and have previously been implicated the neurodegenerative process in SMA, including DNA damage (Jangi et al, 2017) and JNK activation (Genabai et al, 2015) among others. However, immunostaining with antibodies against total H2AX and phosphorylated γH2AX, which is a well-established marker of DNA damage, provided no evidence for γH2AX accumulation and induction of the DNA damage response in SMA motor neurons (Figure S3A–B).…”

“…In principle, disruption of one or more of the multiple functions of SMN in RNA regulation (Donlin-Asp et al, 2016; Li et al, 2014) may induce downstream pathogenic events that converge on p53 and its regulatory network. Interestingly, DNA damage and activation of upstream kinases such as JNK are known to induce p53 and have been implicated in the degeneration of SMA motor neurons in mouse models (Genabai et al, 2015; Jangi et al, 2017). However, we found no evidence for either DNA damage or activation of JNK that might account for p53 induction in vulnerable motor neurons of SMA mice.…”

RNA-binding proteins in neurodegeneration: mechanisms in aggregate

“…Interestingly, a rare class of genes with more than one U11/U12 intron is the voltage-gated ion channel superfamily of genes (Wu and Krainer 1999), which themselves are mutated in neurodegenerative and neuromuscular disease (Andavan and Lemmens-Gruber 2011), suggesting that selectively compromised splicing of these transcripts may have disease relevance. Recently, it was shown in mice that SMN deficiency leads to intron retention, particularly among U11/ U12 introns, which can be reversed by increasing SMN levels (Jangi et al 2017). While the affected genes were not similar in function, their pre-mRNA structures were, as they tended to have weaker 5 ′ and 3 ′ splice sites, be GC-enriched, and contain more R-loop structures to which SMN has been independently been shown to be recruited (Zhao et al 2016;Jangi et al 2017).…”

“…Recently, it was shown in mice that SMN deficiency leads to intron retention, particularly among U11/ U12 introns, which can be reversed by increasing SMN levels (Jangi et al 2017). While the affected genes were not similar in function, their pre-mRNA structures were, as they tended to have weaker 5 ′ and 3 ′ splice sites, be GC-enriched, and contain more R-loop structures to which SMN has been independently been shown to be recruited (Zhao et al 2016;Jangi et al 2017). Efforts are ongoing to determine whether there is indeed a select repertoire of splicing changes associated with SMN deficiency from which one can rationalize disease features or whether SMN depletion leads to global splicing disruption that manifests differently depending on the splicing factors in a given cell type.…”