Proteomic assessment of a cell model of spinal muscular atrophy

Abstract

BackgroundDeletion or mutation(s) of the survival motor neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), a neuromuscular disease characterized by spinal motor neuron death and muscle paralysis. Complete loss of the SMN protein is embryonically lethal, yet reduced levels of this protein result in selective death of motor neurons. Why motor neurons are specifically targeted by SMN deficiency remains to be determined. In this study, embryonic stem (ES) cells derived from a severe SMA mouse model were differentiated into motor neurons in vitro by addition of retinoic acid and sonic hedgehog agonist. Proteomic and western blot analyses were used to probe protein expression alterations in this cell-culture model of SMA that could be relevant to the disease.ResultsWhen ES cells were primed with Noggin/fibroblast growth factors (bFGF and FGF-8) in a more robust neural differentiation medium for 2 days before differentiation induction, the efficiency of in vitro motor neuron differentiation was improved from ~25% to ~50%. The differentiated ES cells expressed a pan-neuronal marker (neurofilament) and motor neuron markers (Hb9, Islet-1, and ChAT). Even though SMN-deficient ES cells had marked reduced levels of SMN (~20% of that in control ES cells), the morphology and differentiation efficiency for these cells are comparable to those for control samples. However, proteomics in conjunction with western blot analyses revealed 6 down-regulated and 14 up-regulated proteins with most of them involved in energy metabolism, cell stress-response, protein degradation, and cytoskeleton stability. Some of these activated cellular pathways showed specificity for either undifferentiated or differentiated cells. Increased p21 protein expression indicated that SMA ES cells were responding to cellular stress. Up-regulation of p21 was confirmed in spinal cord tissues from the same SMA mouse model from which the ES cells were derived.ConclusionSMN-deficient ES cells provide a cell-culture model for SMA. SMN deficiency activates cellular stress pathways, causing a dysregulation of energy metabolism, protein degradation, and cytoskeleton stability.

Developmental arrest of Drosophila survival motor neuron (Smn) mutants accounts for differences in expression of minor intron-containing genes

“…With this caveat in mind, the RNA-seq data provide evidence for the activation of stress signaling pathways that could represent an important systemic response to dSMN depletion. The activation of cellular stress pathways was previously observed in a mouse embryonic stem (ES) cell model of SMA and in spinal cord tissues from which these ES cells were derived (Wu et al 2011). Thus, activation of stressresponse pathways may be a conserved feature of SMA.…”

Transcriptomic comparison of Drosophila snRNP biogenesis mutants reveals mutant-specific changes in pre-mRNA processing: implications for spinal muscular atrophy

“…Our analysis of severe and intermediate SMA models suggests that snRNP-independent, Smn gene-specific RNA changes may play a bigger role in SMA pathology than previously envisioned. The SMN-specific activation of stress RNA-seq of snRNP biogenesis mutants www.rnajournal.org 1223 signaling pathways is conserved in SMA models (Wu et al 2011;Genabai et al 2015) and correlates well with disease severity in Smn hypomorphs (Fig. 8C).…”

SMN - A chaperone for nuclear RNP social occasions?

“…Interestingly, SMN depletion is associated with innate immune and stress signaling. [144][145][146] Moreover, hypomorphic SMN mutations that cause milder forms of SMA in humans and significant viability defects in SMA model flies are neither associated with defects in premRNA splicing nor spliceosomal snRNP biogenesis. 147 Although potential defects in snRNP biogenesis at specific developmental time points are possible, these findings indicate that snRNP-dependent RNA processing changes are unlikely to be primary drivers of SMA pathology.…”