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

A major challenge in neurodegenerative diseases is to unravel the mechanisms by which mutations in ubiquitously expressed genes result in selective death of specific neuronal types, leading to the distinctive clinical manifestations of each disease (Brichta and Greengard, 2014; Kanning et al., 2010; Yaron and Schuldiner, 2016). Uncovering these mechanisms would provide insights into the molecular basis of neurodegeneration and offer clues for the development of neuroprotective therapies.

In contrast to neurodegenerative diseases such as Parkinson’s, Huntington’s, and amyotrophic lateral sclerosis (ALS), in which both cell-autonomous and non-cell-autonomous pathways contribute to degeneration of vulnerable neurons (Boillée et al., 2006; Michel et al., 2016; Sambataro and Pennuto, 2012), the cell-autonomous origin of motor neuron death in spinal muscular atrophy (SMA) is well established (Fletcher et al., 2017; Gogliotti et al., 2012; Martinez et al., 2012; McGovern et al., 2015; Simon et al., 2016). Thus, SMA provides an ideal context to identify cellular pathways and key drivers of selective neurodegeneration.

SMA is the most frequent genetic cause of infant mortality. All cases result from homozygous mutation or deletion of the survival motor neuron 1 (SMN1) gene with retention of the hypomorphic SMN2 gene (Lefebvre et al., 1995), leading to ubiquitous deficiency in the SMN protein. Yet despite the reduction of SMN in all tissues, it is selective loss of spinal motor neurons that is the hallmark of SMA in both humans and mouse models of the disease (Burghes and Beattie, 2009; Tisdale and Pellizzoni, 2015). Moreover, there is striking differential vulnerability even among individual motor neuron pools. We previously showed that, consistent with the clinical features of the disease in SMA patients, spinal motor neurons innervating proximal muscles are preferentially affected and degenerate early in the disease process compared with those that innervate distal muscles and are relatively spared (Mentis et al., 2011). To date, several effector pathways have been implicated in the process of motor neuron death in SMA, which include tau phosphorylation (Miller et al., 2015), endoplasmic reticulum (ER) stress (Ng et al., 2015), and c-Jun N-terminal kinase 3 (JNK3) activation (Genabai et al., 2015), among others. However, the events that trigger degeneration of motor neurons, but not of other spinal neurons in SMA, remain poorly defined. Even more puzzling is the reason ubiquitous SMN deficiency would induce the selective death of specific pools of vulnerable motor neurons while sparing resistant motor neurons that share similar developmental programs and functional properties.

Here, we used comparative gene expression profiling of vulnerable and resistant SMA mouse motor neurons prior to the onset of neuronal death in order to identify cellular pathways causally involved in the degenerative process. We found specific upregulation of p53 transcriptional targets in vulnerable, but not resistant, SMA motor neurons, which correlated well with early-onset nuclear accumulation of p53. We subsequently demonstrated that pharmacological or genetic inhibition of the p53 pathway rescues vulnerable motor neurons from degeneration in a well-established mouse model of SMA (Le et al., 2005), identifying p53 as a key mediator of motor neuron death induced by SMN deficiency in vivo. Intriguingly, at late stages of disease, widespread p53 activation was observed in resistant motor neurons and other spinal neurons of SMA mice, which do not degenerate in the disease. To address these seemingly conflicting observations, we identified phosphorylation of p53 serine 18 as a death-specific marker that is selectively present in vulnerable SMA motor neurons. Importantly, this specific modification was not observed in resistant motor neurons or in spinal interneurons despite nuclear accumulation of p53 at late disease stages in SMA mice. Moreover, using a newly devised in vivo replacement strategy, we provide evidence that phosphorylation of specific serines in the amino-terminal transactivation domain (TAD) of p53 is necessary to trigger death of SMA motor neurons. These findings indicate that distinct events induced by SMN deficiency converge on p53 to activate a neuronal death pathway selectively in vulnerable motor neurons.

Results

SMN Deficiency Induces p53 Activation in SMA Motor NeuronsTo gain insight into the death pathway of SMA motor neurons, we performed comparative profiling of gene expression changes induced by SMN deficiency in vulnerable and resistant motor neurons from SMA mice. We specifically used the well-characterized SMNΔ7 mouse model of SMA that harbors homozygous knockout of the endogenous Smn gene, two copies of the human SMN2 gene, and multiple copies of the SMNΔ7 cDNA transgene (Le et al., 2005). Using laser capture microdissection, we isolated retrogradely labeled vulnerable medial motor column (MMC) motor neurons innervating the multifidus muscle and resistant lateral motor column (LMC) motor neurons innervating the gastrocnemius and tibialis anterior muscles from wild-type (WT) and SMA mice (Figure 1A), as we described previously (Lotti et al., 2012). We focused on an early symptomatic stage (P4), preceding motor neuron death in the L5 spinal segment (Mentis et al., 2011). Microarray analysis revealed expression changes in 75 genes in vulnerable MMC SMA motor neurons, but only a single change, the Smn gene, in resistant LMC motor neurons, compared with MMC and LMC motor neurons from WT mice, respectively (Table S1). KEGG pathway analysis using the DAVID gene ontology platform identified p53 signaling as the top pathway altered in vulnerable SMA motor neurons (p = 1.19E-04) and the specific upregulation of ten p53 transcriptional target genes (Table S1), which we validated by RT-qPCR and fluorescence in situ hybridization (Figures 1B and 1C).