Dysregulation of Mdm2 and Mdm4 alternative splicing underlies motor neuron death in spinal muscular atrophy

Perturbation of RNA homeostasis is a common theme of many neurodegenerative disorders in which genetic mutations are associated with the direct or indirect impairment of RNA-binding proteins or factors controlling RNA processing at multiple levels (Cooper et al. 2009; Li et al. 2014; Conlon and Manley 2017). Remarkably, while disease-linked proteins are ubiquitously expressed and carry out essential biological processes, pathology is characterized by selective degeneration of specific neuronal populations. Furthermore, these proteins are often multifunctional and involved in the regulation of diverse RNA pathways, making it difficult to discern specific disease-relevant events among many transcriptome abnormalities. These complexities have made it remarkably challenging in most instances to establish causal links between RNA dysregulation and disease etiology. Nonetheless, dissecting the RNA-mediated mechanisms underlying the dysfunction and death of select neurons is necessary to uncover the molecular basis of human disease and identify drivers of neurodegeneration, which may also represent key targets for therapeutic intervention.

One prominent example of a neurodegenerative disease featuring RNA dysfunction is spinal muscular atrophy (SMA), a leading genetic cause of death in infancy that results from ubiquitous deficiency in the survival motor neuron (SMN) protein (Burghes and Beattie 2009; Tisdale and Pellizzoni 2015). SMN is part of a macromolecular complex that mediates assembly of Sm-class small nuclear ribonucleoproteins (snRNPs) of the pre-mRNA splicing machinery (Meister et al. 2001; Pellizzoni et al. 2002) as well as U7 snRNPs that carry out histone mRNA 3′ end processing (Pillai et al. 2003; Tisdale et al. 2013). SMN has also been implicated in the assembly of other RNA-binding proteins with a variety of coding and noncoding RNAs (Li et al. 2014), the most characterized of which is the formation of specific mRNPs for axonal transport and localized translation in neurons (Donlin-Asp et al. 2017). Reflecting the emerging multifaceted role of SMN in RNP assembly and post-transcriptional gene regulation, disruption of several distinct SMN-dependent RNA pathways has been proposed to contribute to SMA pathogenesis (Li et al. 2014; Donlin-Asp et al. 2016). However, proving clear mechanistic links between select SMN-dependent RNA pathways and specific disease-relevant features of SMA remains an outstanding challenge.

Here, we sought to address this issue by focusing on the molecular mechanisms of motor neuron death in SMA. The classical hallmark of SMA in both patients and mouse models is the progressive loss of spinal motor neurons, leading to skeletal muscle atrophy (Burghes and Beattie 2009; Tisdale and Pellizzoni 2015). Studies in SMA mice have shown that motor neuron death occurs cell-autonomously (Gogliotti et al. 2012; Martinez et al. 2012; McGovern et al. 2015; Fletcher et al. 2017), and distinct motor neuron pools display differential vulnerability to SMN deficiency (Mentis et al. 2011; Fletcher et al. 2017; Simon et al. 2017)—a feature reflected in the varying degree of denervation of the respective target muscles (Ling et al. 2012). This faithfully recapitulates clinical characteristics of the disease in which axial and proximal muscles are more affected than distal ones (Oskoui et al. 2017). Importantly, degeneration of vulnerable motor neurons in a mouse model of severe SMA is driven by a p53-dependent death pathway (Simon et al. 2017), and p53 induction has also been documented in motor neurons from milder mouse models of the disease (Murray et al. 2015; Jangi et al. 2017) as well as post-mortem spinal cords of SMA patients (Simic et al. 2000). However, the molecular mechanisms linking SMN deficiency to p53 activation and motor neuron death in SMA are unknown.

Under normal conditions, p53 expression is maintained at low levels by Mdm2 and Mdm4—two nonredundant negative regulators that act in concert to prevent unwarranted induction of the p53 pathway, which can lead to cell cycle arrest or cell death in a context-dependent manner (Vousden and Prives 2009). Mdm2 mainly serves as an E3 ubiquitin ligase that targets p53 for degradation, while Mdm4 inhibits p53 transcriptional activity in addition to enhancing Mdm2 function (Marine et al. 2006; Shadfan et al. 2012). We hypothesized that p53 activation and motor neuron degeneration in SMA could involve dysregulation of the repressive activity of Mdm2 and Mdm4 (i.e., p53 anti-repression) through splicing dysfunction induced by SMN deficiency. Consistent with this possibility, previous studies implicated alternative splicing of critical exons of Mdm2 and Mdm4 in the regulation of p53. Mdm2 transcripts that exclude exon 3 produce a truncated form of the protein that induces p53 stabilization through multiple mechanisms (Saucedo et al. 1999; Perry et al. 2000; Giglio et al. 2010). Skipping of exon 7 in Mdm4 introduces an early stop codon that induces nonsense-mediated decay or production of an inactive protein, leading to p53 activation through loss of function (Bezzi et al. 2013; Bardot et al. 2015; Dewaele et al. 2016).

In this study, we identify Mdm2 exon 3 and Mdm4 exon 7 as critical downstream targets whose coordinated alternative splicing is regulated by SMN through its function in snRNP biogenesis. Importantly, we show that defective splicing of these key regulatory exons induced by SMN deficiency, which occurs early and is most pronounced in vulnerable motor neurons, acts as a biological switch governing initiation of the p53 response in SMA mice. Through selective induction of exon skipping in wild-type mice, we demonstrate that perturbation of Mdm2 and Mdm4 alternative splicing synergistically induces p53 anti-repression such that both splicing events, but neither alone, are necessary and sufficient to elicit robust activation of p53 in vivo. Conversely, correction of either deficit by restoration of full-length Mdm2 or Mdm4 is sufficient to suppress p53 induction and prevent motor neuron degeneration in SMA. Thus, while SMN deficiency induces widespread transcriptome perturbations with the potential to contribute to SMA pathology (Zhang et al. 2008; Li et al. 2014), we show that it is the selective dysregulation of Mdm2 and Mdm4 alternative splicing that underlies p53 anti-repression and motor neuron death in a mouse model of the disease. These findings provide a direct mechanistic link between the disruption of SMN's function in the assembly of the snRNP constituents of the splicing machinery and the molecular basis of neurodegeneration in SMA.


SMN deficiency disrupts Mdm2 and Mdm4 alternative splicing in SMA motor neurons

To determine the mechanisms by which SMN deficiency induces p53 in SMA, we investigated whether SMN regulates alternative splicing of Mdm2 exon 3 and Mdm4 exon 7, changes in which have been linked to p53 activation (Perry et al. 2000; Giglio et al. 2010; Bardot et al. 2015; Dewaele et al. 2016). In previously established NIH3T3-SmnRNAi fibroblasts with doxycycline (Dox)-inducible RNAi knockdown of Smn to ∼10% of normal levels (Lotti et al. 2012; Ruggiu et al. 2012; Tisdale et al. 2013), RT–PCR analysis revealed that the inclusion of both Mdm2 exon 3 and Mdm4 exon 7 was significantly reduced upon Smn deficiency (Fig. 1A–D), while splicing of other Mdm2 and Mdm4 exons was not affected (Supplemental Fig. S1). Moreover, these splicing changes were reversed by expression of RNAi-resistant human SMN in NIH3T3-SMN/SmnRNAi cells (Fig. 1A–D; Lotti et al. 2012; Ruggiu et al. 2012; Tisdale et al. 2013), highlighting the specificity of the effects for SMN depletion. These results demonstrate that SMN regulates the alternative splicing of specific exons of Mdm2 and Mdm4 mRNAs in vitro.