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 SMAlike 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.

Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca 2+ homeostasis. CRT also has extracellular functions, e.g. tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release is unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here, we show that under apoptotic stress conditions, the cytosolic concentration of CRT increases and associates with phosphatidylserine (PS) in a Ca 2+ -dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT), which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH groups by reactive nitrogen species. During apoptosis, both CRT expression and the concentration of nitric oxide (NO) increase. By using S-nitroso-L-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol-dependent and caspase-independent manner. Furthermore, the CRT and PS are relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS could explain how CRT acts as a bridging molecule during apoptotic cell clearance.

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rRNA sequencing has shown that leuconostocs comprise three distinct phylogenetic lineages which have been designated separate genera (viz., the genera Leuconostoc sensu stricto, Oenococcus, and WeisseZZa). In addition, the 16s rRNA line formed by Oenococcus oeni (formerly Leuconostoc oenos) is exceptionally long; this fact, together with variations in the compositions of conserved positions in the 16s rRNA, has led to the hypothesis (D. Yang and C. R. Woese, Syst. Appl. Microbiol. 12:145-149, 1989) that this organism is a fast-evolving bacterium. Previous evidence that the leuconostocs should be divided into three genera and that 0. oeni is an example of tachytelic evolution has come solely from rRNA analyses. In this study we sequenced the rpoC gene encoding the p' subunit of DNA-dependent RNA polymerase of leuconostocs and performed a comparative phylogenetic analysis. The subdivision of the leuconostocs into three distinct lineages was confirmed by the rpoC gene data, but no evidence that indicated that 0. oeni is evolving at an extraordinary rate was found. If 0. oeni is truly tachytelic, then fast-evolving phenomena would be expected to occur throughout the whole genome, including this independent molecular chronometer.

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