The circadian glucocorticoid-Krüppel-like factor 15-branched-chain amino acid (GC-KLF15-BCAA) signaling pathway is a key regulatory axis in muscle, whose imbalance has wide-reaching effects on metabolic homeostasis. Spinal muscular atrophy (SMA) is a neuromuscular disorder also characterized by intrinsic muscle pathologies, metabolic abnormalities and disrupted sleep patterns, which can influence or be influenced by circadian regulatory networks that control behavioral and metabolic rhythms. We therefore set out to investigate the contribution of the GC-KLF15-BCAA pathway in SMA pathophysiology of Taiwanese Smn−/−;SMN2 and Smn2B/− mouse models. We thus uncover substantial dysregulation of GC-KLF15-BCAA diurnal rhythmicity in serum, skeletal muscle and metabolic tissues of SMA mice. Importantly, modulating the components of the GC-KLF15-BCAA pathway via pharmacological (prednisolone), genetic (muscle-specific Klf15 overexpression) and dietary (BCAA supplementation) interventions significantly improves disease phenotypes in SMA mice. Our study highlights the GC-KLF15-BCAA pathway as a contributor to SMA pathogenesis and provides several treatment avenues to alleviate peripheral manifestations of the disease. The therapeutic potential of targeting metabolic perturbations by diet and commercially available drugs could have a broader implementation across other neuromuscular and metabolic disorders characterized by altered GC-KLF15-BCAA signaling.
In mammalian cells under oxidative stress, the methionyl-tRNA synthetase (MetRS) misacylates noncognate tRNAs at frequencies as high as 10% distributed among up to 28 tRNA species. Instead of being detrimental for the cell, misincorporation of methionine residues in the proteome reduces the risk of oxidative damage to proteins, which aids the oxidative stress response. tRNA microarrays have been essential for the detection of the full pattern of misacylated tRNAs, but have limited capacity to investigate the misacylation and mistranslation mechanisms in live cells. Here we develop a dual-fluorescence reporter to specifically measure methionine misincorporation at glutamic acid codons GAA and GAG via tRNA Glu mismethionylation in human cells. Our method relies on mutating a specific Met codon in the active site of the fluorescent protein mCherry to a Glu codon that renders mCherry nonfluorescent when translation follows the genetic code. Mistranslation utilizing mismethionylated tRNA Glu restores fluorescence in proportion to the amount of misacylated tRNA Glu . This cellular approach works well for both transient transfection and established stable HEK293 lines. It is rapid, straightforward, and well suited for high-throughput activity analysis under a wide range of physiological conditions. As a proof of concept, we apply this method to characterize the effect of human tRNA Glu isodecoders on mistranslation and discuss the implications of our findings.
A pharmaco-EEG method was used to determine the influence of a new anti-epileptic drug zonisamide on the development and course of alcohol dependence in rabbits. The drug co-administered with ethanol decreased alcohol-induced changes in selected brain structures. Zonisamide also decreases abstinence-induced changes in the EEG recordings.
Calorie restriction is the only intervention proved to prolong both average and maximum lifespan in yeast, worms, fi sh, rodents and possibly primates. Not only does the regimen prolong life, but it also reduces the incident of numerous age-related diseases like diabetes, atherosclerosis or cancer and slows down ageing. Mechanisms by which that is thought to occur have not yet been elucidated, but they probably involve reactive oxygen species signaling, insulin growth factor and transcriptional factors. Here, special emphasis is given to SIRT1 -silent information regulator. There is sound evidence showing that SIRT1 is a key player in mediating physiological response to calorie restriction and that its overexpression is correlated with extended lifespan. The possible mechanism leading to its elevated levels is high NAD/NADH ratio, observed in Sir2 in yeast. SIRT1 increases glucose production, enhances fat mobilization, stimulates angiogenesis, prevents neuronal degeneration and rises insulin sensitivity. Therefore, it seems to be a very benefi cial factor activated by such a simple intervention that is calorie restriction. Mini
Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. The advent of approved treatments for this devastating condition has significantly changed SMA patients' life expectancy and quality of life. Nevertheless, these are not without limitations, and research efforts are underway to develop new approaches to be used alone and in combination, to ensure improved and long-lasting benefits for SMA patients. Protein arginine methyltransferases (PRMT) are emerging as druggable epigenetic targets, with several small molecule PRMT inhibitors already in clinical trial stage. From a screen of highly potent and selective next generation epigenetic small molecules, we have identified MS023, a potent and selective type I PRMT inhibitor, able to promote SMN2 exon 7 inclusion and increase SMN protein levels in preclinical SMA model, by inhibiting the binding of splicing factor hnRNPA1 to SMN2 pre-mRNA. Treatment of SMA mice with MS023 results in amelioration of the disease phenotype, with strong synergistic amplification of the positive effect when delivered in combination with the SMN2-targeting antisense oligonucleotide nusinersen. Moreover, transcriptomic analysis revealed that MS023 treatment has very minimal off-target effects and that the added benefit of the combination therapy is mainly attributable to targeting neuroinflammation. Our study warrants further clinical investigation of PRMT inhibition both as a stand-alone and add-on therapy for SMA patients.
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