Dysregulation of ubiquitin homeostasis and β-catenin signaling promote spinal muscular atrophy
Thomas M. Wishart, … , Brunhilde Wirth, Thomas H. Gillingwater
Thomas M. Wishart, … , Brunhilde Wirth, Thomas H. Gillingwater
Published March 3, 2014
Citation Information: J Clin Invest. 2014;124(4):1821-1834. https://doi.org/10.1172/JCI71318.
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Dysregulation of ubiquitin homeostasis and β-catenin signaling promote spinal muscular atrophy

Abstract

The autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) results from low levels of survival motor neuron (SMN) protein; however, it is unclear how reduced SMN promotes SMA development. Here, we determined that ubiquitin-dependent pathways regulate neuromuscular pathology in SMA. Using mouse models of SMA, we observed widespread perturbations in ubiquitin homeostasis, including reduced levels of ubiquitin-like modifier activating enzyme 1 (UBA1). SMN physically interacted with UBA1 in neurons, and disruption of Uba1 mRNA splicing was observed in the spinal cords of SMA mice exhibiting disease symptoms. Pharmacological or genetic suppression of UBA1 was sufficient to recapitulate an SMA-like neuromuscular pathology in zebrafish, suggesting that UBA1 directly contributes to disease pathogenesis. Dysregulation of UBA1 and subsequent ubiquitination pathways led to β-catenin accumulation, and pharmacological inhibition of β-catenin robustly ameliorated neuromuscular pathology in zebrafish, Drosophila, and mouse models of SMA. UBA1-associated disruption of β-catenin was restricted to the neuromuscular system in SMA mice; therefore, pharmacological inhibition of β-catenin in these animals failed to prevent systemic pathology in peripheral tissues and organs, indicating fundamental molecular differences between neuromuscular and systemic SMA pathology. Our data indicate that SMA-associated reduction of UBA1 contributes to neuromuscular pathogenesis through disruption of ubiquitin homeostasis and subsequent β-catenin signaling, highlighting ubiquitin homeostasis and β-catenin as potential therapeutic targets for SMA.

Authors

Thomas M. Wishart, Chantal A. Mutsaers, Markus Riessland, Michell M. Reimer, Gillian Hunter, Marie L. Hannam, Samantha L. Eaton, Heidi R. Fuller, Sarah L. Roche, Eilidh Somers, Robert Morse, Philip J. Young, Douglas J. Lamont, Matthias Hammerschmidt, Anagha Joshi, Peter Hohenstein, Glenn E. Morris, Simon H. Parson, Paul A. Skehel, Thomas Becker, Iain M. Robinson, Catherina G. Becker, Brunhilde Wirth, Thomas H. Gillingwater

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Figure 1

Perturbations in UBA1 levels and ubiquitin homeostasis in mouse and Drosophila models of SMA.

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Perturbations in UBA1 levels and ubiquitin homeostasis in mouse and Dros...
(A–C) Significant reduction in levels of UBA1 protein in spinal cord and skeletal muscle from severe SMA mice at P5 compared with littermate controls (con), quantified using fluorescent Western blot (n = 3 mice/genotype; unpaired 2-tailed t test). (D–F) Reduced levels of both monomeric and multimeric ubiquitin in the spinal cord of Taiwanese SMA mice at P10 (tubulin: loading control; n = 3 mice/genotype). (G) Representative confocal micrographs of striated muscle from WT and SMA Drosophila larvae immunolabeled with an antibody that recognizes mono- and polyubiquitinated proteins (green). Diffuse staining in muscle and muscle nuclei (stained with Hoechst, blue) of WT flies contrasted with a distinct lack of nuclear staining and increased perinuclear staining in SMA flies. Each panel in G is 75 μm in length. (H and I) UBA1 (green) and NeuN (red) immunolabeling of motor neurons from Taiwanese SMA and littermate control mouse spinal cords at P3 (H) and P7 (I). Note how UBA1 was predominantly cytoplasmic at P3 but nuclear at P7. Scale bars: 10 μm. (J) Significant increase in the ratio of nuclear to cytoplasmic UBA1 in SMA motor neurons at P7 compared with littermate controls (n = 24 motor neurons per genotype). (K) UBA1 levels in whole spinal cord of Taiwanese SMA mice remained unchanged at P3 and P7, but were significantly reduced by P10 (n > 3 mice per time point/genotype; ANOVA with Tukey’s post-hoc test). (L) Levels of UBA1 in skeletal muscle were significantly reduced at an early symptomatic age (P7) in Taiwanese SMA mice (n = 4 mice per genotype). *P < 0.05; **P < 0.01; ***P < 0.001.