Hypoxia is a modifier of SMN2 splicing and disease severity in a severe SMA mouse model

Abstract

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease associated with low levels of the essential survival motor neuron (SMN) protein. Reduced levels of SMN is due to the loss of the SMN1 gene and inefficient splicing of the SMN2 gene caused by a C>T mutation in exon 7. Global analysis of the severe SMNΔ7 SMA mouse model revealed altered splicing and increased levels of the hypoxia-inducible transcript, Hif3alpha, at late stages of disease progression. Severe SMA patients also develop respiratory deficiency during disease progression. We sought to evaluate whether hypoxia was capable of altering SMN2 exon 7 splicing and whether increased oxygenation could modulate disease in a severe SMA mouse model. Hypoxia treatment in cell culture increased SMN2 exon 7 skipping and reduced SMN protein levels. Concordantly, the treatment of SMNΔ7 mice with hyperoxia treatment increased the inclusion of SMN2 exon 7 in skeletal muscles and resulted in improved motor function. Transfection splicing assays of SMN minigenes under hypoxia revealed that hypoxia-induced skipping is dependent on poor exon definition due to the SMN2 C>T mutation and suboptimal 5' splice site. Hypoxia treatment in cell culture led to increased hnRNP A1 and Sam68 levels. Mutation of hnRNP A1-binding sites prevented hypoxia-induced skipping of SMN exon 7 and was found to bind both hnRNP A1 and Sam68. These results implicate hypoxic stress as a modulator of SMN2 exon 7 splicing in disease progression and a coordinated regulation by hnRNP A1 and Sam68 as modifiers of hypoxia-induced skipping of SMN exon 7.

Vascular Defects and Spinal Cord Hypoxia in Spinal Muscular Atrophy

“…Aside from obvious deleterious effects on cell metabolism and survival, hypoxia also has a negative effect upon SMN splicing, by increasing levels of exon 7 skipping in the SMN2 gene, 46 which would be expected to result in further depletion of SMN levels in hypoxic tissue. This suggests that hypoxia could be a major modulator of SMN protein levels, the main determinant of disease severity.…”

SMN regulation in SMA and in response to stress: new paradigms and therapeutic possibilities

“…While several splicing factors are known to promote exon 7 inclusion in SMN1 and exon 7 exclusion in SMN2 splicing, the relative abundance of final spliced mRNA product is also affected by stresses and cell signaling processes, such as hypoxia, starvation, and temperature changes (Bebee et al 2012; Sahashi et al 2012). An example of this effect may be seen under the influence of oxidative stress induced by paraquot treatment, which has been shown to result in multiple aberrant SMN2 splicing products in murine tissues (Seo et al 2016).…”

“…Concordantly, a hallmark of late stage disease in a mouse model of severe SMA is increased levels of the hypoxic markers, Hif1alpha (Bebee et al 2012) and Hif3alpha (Zhang et al 2008). Additionally, hypoxic treatment of cultured SMA patient fibroblasts leads to increased levels of exon 7 skipping and subsequent reduced levels of stable, functional SMN protein.…”

“…Additionally, hypoxic treatment of cultured SMA patient fibroblasts leads to increased levels of exon 7 skipping and subsequent reduced levels of stable, functional SMN protein. Importantly, this splicing alteration was found to be due to upregulation of the negative splicing factor hnRNP A1 (Bebee et al 2012). While the mechanism of hnRNP A1 upregulation was not addressed in that study, hypoxia has also been shown to activate the AKT pathway, which phosphorylates hnRNP A1 (Jo et al 2008).…”

“…Notably, hyperbaric hyperoxia treatment of the SMA mice improved motor function and body weight (Bebee et al 2012). The authors proposed that a feed-forward cycle contributes to the progression of the SMA disease state, in which motor neuron denervation of intercostal and diaphragm muscles leads to breathing impairment and hypoxia.…”