Secondary structures that regulate mRNA translation provide insights for ASO-mediated modulation of cardiac hypertrophy.

Translation of upstream open reading frames (uORFs) typically abrogates translation of main (m)ORFs. The molecular mechanism of uORF regulation in cells is not well understood. Here, we data-mined human and mouse heart ribosome profiling analyses and identified a double-stranded RNA (dsRNA) structure within the GATA4 uORF that cooperates with the start codon to augment uORF translation and inhibits mORF translation. A trans-acting RNA helicase DDX3X inhibits the GATA4 uORF-dsRNA activity and modulates the translational balance of uORF and mORF. Antisense oligonucleotides (ASOs) that disrupt this dsRNA structure promote mORF translation, while ASOs that base-pair immediately downstream (i.e., forming a bimolecular double-stranded region) of either the uORF or mORF start codon enhance uORF or mORF translation, respectively. Human cardiomyocytes and mice treated with a uORF-enhancing ASO showed reduced cardiac GATA4 protein levels and increased resistance to cardiomyocyte hypertrophy. We further show the broad utility of uORF-dsRNA- or mORF-targeting ASO to regulate mORF translation for other mRNAs. This work demonstrates that the uORF-dsRNA element regulates the translation of multiple mRNAs as a generalizable translational control mechanism. Moreover, we develop a valuable strategy to alter protein expression and cellular phenotypes by targeting or generating dsRNA downstream of a uORF or mORF start codon.

MiR-107 inhibits the sprouting of intersegmental vessels of zebrafish embryos.

MicroRNAs (miRNAs) play important roles in various biological processes. Our previous study showed that inhibition of MTOR with rapamycin treatment suppressed human endothelial cell tube formation, concomitant with the down-regulation of miR-107. Similarly, inhibition of Ztor by rapamycin also suppressed vascular development in zebrafish embryos. To gain a better understanding of the role of miR-107 and MTOR in vascular development, the present study sought to validate its function by over-expressing miR-107 in zebrafish embryos via microinjection with mimic miR-107 duplexes. Alkaline phosphatase (ALP) staining was used to visualise blood vessels in the zebrafish embryo, and expressions of Pten, Ztor and Rap1 were investigated by immunoblotting. Over-expression of miR-107 in zebrafish embryos inhibited the sprouting of intersegmental vessels (ISVs) with concomitant down-regulation of phosphorylated Rps6 expression, which confirmed the inhibition of Ztor signalling. As expected, pten, which is the target of miR-107, was down-regulated. Interestingly, Rap1, a small GTPase protein that is involved in intersomitic vessels sprouting during angiogenesis, was also down-regulated when miR-107 was over-expressed. Overall, our findings suggest that miR-107 and Ztor-mediated suppression of vascular development in zebrafish embryo involves Rap1.

Fecal Calprotectin in Parkinson's Disease and Multiple System Atrophy.

OBJECTIVE: Converging evidence suggests that intestinal inflammation is involved in the pathogenesis of neurodegenerative diseases. Previous studies on fecal calprotectin in Parkinson's disease (PD) were limited by small sample sizes, and literature regarding intestinal inflammation in multiple system atrophy (MSA) is very scarce. We investigated the levels of fecal calprotectin, a marker of intestinal inflammation, in PD and MSA. METHODS: We recruited 169 subjects (71 PD, 38 MSA, and 60 age-similar nonneurological controls). Clinico-demographic data were collected. PD and MSA were subtyped and the severity assessed using the MDS-UPDRS and UMSARS, respectively. Fecal calprotectin and blood immune markers were analyzed. RESULTS: Compared to controls (median: 35.7 [IQR: 114.2] µg/g), fecal calprotectin was significantly elevated in PD (median: 95.6 [IQR: 162.1] µg/g, p = 0.003) and even higher in MSA (median: 129.5 [IQR: 373.8] µg/g, p = 0.002). A significant interaction effect with age was observed; between-group differences were significant only in older subjects (i.e., ≥ 61 years) and became more apparent with increasing age. A total of 28.9% of MSA and 18.3% of PD patients had highly abnormal fecal calprotectin levels (≥ 250 µg/g); however, this difference was only significant for MSA compared to controls. Fecal calprotectin correlated moderately with selected blood immune markers in PD, but not with clinical features of PD or MSA. CONCLUSION: s Elevated fecal calprotectin suggests a role for intestinal inflammation in PD and MSA. A more complete understanding of gut immune alterations could open up new avenues of research and treatment for these debilitating diseases.

Glutamyl-Prolyl-tRNA Synthetase Regulates Proline-Rich Pro-Fibrotic Protein Synthesis During Cardiac Fibrosis.

RATIONALE: Increased protein synthesis of profibrotic genes is a common feature in cardiac fibrosis and heart failure. Despite this observation, critical factors and molecular mechanisms for translational control of profibrotic genes during cardiac fibrosis remain unclear. OBJECTIVE: To investigate the role of a bifunctional ARS (aminoacyl-tRNA synthetase), EPRS (glutamyl-prolyl-tRNA synthetase) in translational control of cardiac fibrosis. METHODS AND RESULTS: Results from reanalyses of multiple publicly available data sets of human and mouse heart failure, demonstrated that EPRS acted as an integrated node among the ARSs in various cardiac pathogenic processes. We confirmed that EPRS was induced at mRNA and protein levels (≈1.5-2.5-fold increase) in failing hearts compared with nonfailing hearts using our cohort of human and mouse heart samples. Genetic knockout of one allele of Eprs globally (Eprs+/-) using CRISPR-Cas9 technology or in a Postn-Cre-dependent manner (Eprsflox/+; PostnMCM/+) strongly reduces cardiac fibrosis (≈50% reduction) in isoproterenol-, transverse aortic constriction-, and myocardial infarction (MI)-induced heart failure mouse models. Inhibition of EPRS using a PRS (prolyl-tRNA synthetase)-specific inhibitor, halofuginone, significantly decreases translation efficiency (TE) of proline-rich collagens in cardiac fibroblasts as well as TGF-ß (transforming growth factor-ß)-activated myofibroblasts. Overexpression of EPRS increases collagen protein expression in primary cardiac fibroblasts under TGF-ß stimulation. Using transcriptome-wide RNA-Seq and polysome profiling-Seq in halofuginone-treated fibroblasts, we identified multiple novel Pro-rich genes in addition to collagens, such as Ltbp2 (latent TGF-ß-binding protein 2) and Sulf1 (sulfatase 1), which are translationally regulated by EPRS. SULF1 is highly enriched in human and mouse myofibroblasts. In the primary cardiac fibroblast culture system, siRNA-mediated knockdown of SULF1 attenuates cardiac myofibroblast activation and collagen deposition. Overexpression of SULF1 promotes TGF-ß-induced myofibroblast activation and partially antagonizes anti-fibrotic effects of halofuginone treatment. CONCLUSIONS: Our results indicate that EPRS preferentially controls translational activation of proline codon rich profibrotic genes in cardiac fibroblasts and augments pathological cardiac remodeling. Graphical Abstract: A graphical abstract is available for this article.

The roles of MTOR and miRNAs in endothelial cell senescence.

Accumulation of senescent cells in vascular endothelium is known to contribute to vascular aging and increases the risk of developing cardiovascular diseases. The involvement of classical pathways such as p53/p21 and p16/pRB in cellular senescence are well described but there are emerging evidence supporting the increasingly important role of mammalian target of rapamycin (MTOR) as driver of cellular senescence via these pathways or other effector molecules. MicroRNAs (miRNAs) are a highly conserved group of small non-coding RNAs (18-25 nucleotides), instrumental in modulating the expression of target genes associated with various biological and cellular processes including cellular senescence. The inhibition of MTOR activity is predominantly linked to cellular senescence blunting and prolonged lifespan in model organisms. To date, known miRNAs regulating MTOR in endothelial cell senescence remain limited. Herein, this review discusses the roles of MTOR and MTOR-associated miRNAs in regulating endothelial cell senescence, including the crosstalk between MTOR Complex 1 (MTORC1) and cell cycle pathways and the emerging role of MTORC2 in cellular senescence. New insights on how MTOR and miRNAs coordinate underlying molecular mechanisms of endothelial senescence will provide deeper understanding and clarity to the complexity of the regulation of cellular senescence.

Understanding the Role of ztor in Aging-related Diseases Using the Zebrafish Model.

The mammalian target of rapamycin (mTOR), a 289 kDa serine/threonine protein kinase of the phosphoinositide 3-kinase (PI3K)-related family is known for its role in regulating lifespan and the aging process in humans and rodents. Aging in zebrafish very much resembles aging in humans. Aged zebrafish often manifest with spinal curvature, cataracts and cognitive frailty, akin to human age-related phenotypical effects such as osteoarthritis, dwindling vision and cognitive dysfunction. However, the role of the zebrafish orthologue of mTOR, ztor, is less defined in these areas. This review paper discusses the tale of growing old in the zebrafish, the physiological roles of ztor in normal developmental processes and its involvement in the pathogenesis of aging-related diseases such as metabolic disorders and cancers.

Senescent HUVECs-secreted exosomes trigger endothelial barrier dysfunction in young endothelial cells.

Accumulation of senescent endothelial cells can cause endothelium dysfunction which eventually leads to age-related vascular disorders. The senescent-associated secretory phenotype (SASP) cells secrete a plethora of soluble factors that negatively influence the surrounding tissue microenvironment. The present study sought to investigate the effects of exosomes, which are nano-sized extracellular vesicles known for intercellular communications secreted by SASP cells on young endothelial cells. Exosomes were isolated from the condition media of senescent human umbilical vein endothelial cells (HUVECs) and then confirmed by the detection of exosome specific CD63 and CD9 expressions, electron microscopy and acetylcholinesterase assay. The purified exosomes were used to treat young HUVECs. Exposure to exosomes repressed the expression of adherens junction proteins including vascular endothelial (VE)-cadherin and beta-catenin, decreased cell growth kinetics and impaired endothelial migration potential of young endothelial cells. These findings suggest that senescent HUVECs-secreted exosomes could disrupt barrier integrity that underpins endothelial barrier dysfunction in healthy young endothelial cells.

Endothelial replicative senescence delayed by the inhibition of MTORC1 signaling involves MicroRNA-107.

Accumulation of senescent endothelial cells can contribute to endothelium dysfunction. Suppression of MTOR signaling has been shown to delay senescence but the mechanism that underpins this effect, particularly one that involves miRNAs, remains to be further defined. This study sought to identify miRNAs involved in MTORC1-mediated inhibition of replicative senescence in endothelial cells. Pre-senescent HUVECs were prolonged treated with low dose rapamycin (1 nM), an MTOR inhibitor. Rapamycin treatment down-regulated the phosphorylated MTOR, RPS6 and 4EBP1 expressions, which confirmed MTORC1 suppression. Prolonged low dose rapamycin treatment has significantly reduced the percentage of senescence-associated beta galactosidase (SA-ß gal) positively stained senescent cells and P16INK4A expression in these cells. On the contrary, the percentage of BrdU-labelled proliferating cells has significantly increased. RPTOR, a positive regulator of MTORC1 was knockdown using RPTOR siRNA to inhibit MTORC1 activation. RPTOR knockdown was evidenced by significant suppressions of RPTOR mRNA and protein expression levels. In these cells, the expression of miR-107 was down-regulated whereas miR-145-5p and miR-217 were up-regulated. Target gene prediction revealed PTEN as the target of miR-107 and this was confirmed by biotin pull-down assay. Over-expression of miR-107 has decreased PTEN expression, increased MTORC1 activity, induced cell cycle arrest at G0/G1 phase and up-regulated P16INK4A expression but mitigated tube formation. Collectively, our findings revealed that delayed endothelial replicative senescence caused by the inhibition of MTORC1 activation could be modulated by miR-107 via its influence on PTEN.

Deregulation of hsa-miR-20b expression in TNF-α-induced premature senescence of human pulmonary microvascular endothelial cells.

miRNAs are important regulators of cellular senescence yet the extent of their involvement remains to be investigated. We sought to identify miRNAs that are involved in cytokine-induced premature senescence (CIPS) in endothelial cells. CIPS was established in young human pulmonary microvascular endothelial cells (HMVEC-Ls) following treatment with a sublethal dose (20ng/ml) of tumor necrosis factor alpha (TNF-α) for 15days. In parallel, HMVEC-Ls were grown and routinely passaged until the onset of replicative senescence (RS). Differential expression analysis following miRNA microarray profiling revealed an overlapped of eight deregulated miRNAs in both the miRNA profiles of RS and TNF-α-induced premature senescence cells. Amongst the deregulated miRNAs were members of the miR 17-92 cluster which are known regulators of angiogenesis. The role of hsa-miR-20b in TNF-α-induced premature senescence, a paralog member of the miR 17-92 cluster, was further investigated. Biotin-labeled hsa-miR-20b captured the enriched transcripts of retinoblastoma-like 1 (RBL1), indicating that RBL1 is a target of hsa-miR-20b. Knockdown of hsa-miR-20b attenuated premature senescence in the TNF-α-treated HMVEC-Ls as evidenced by increased cell proliferation, increased RBL1 mRNA expression level but decreased protein expression of p16INK4a, a cellular senescence marker. These findings provide an early insight into the role of hsa-miR-20b in endothelial senescence.

Expression of zTOR-associated microRNAs in zebrafish embryo treated with rapamycin.

AIMS: MicroRNAs (miRNAs) are vital in modulating lifespan and various biological processes including vascular function. The pivotal roles of mammalian target of rapamycin (mTOR) in regulating senescence and angiogenesis have been extensively described. However, the roles of its orthologue, zebrafish target of rapamycin (zTOR) in senescence and angiogenesis remain to be unravelled. In the present study, we aimed to investigate the role of zTOR and identify miRNAs associated with senescence and angiogenesis. MAIN METHODS: Zebrafish embryos were treated with rapamycin and the inhibition of zTOR and its downstream proteins were validated by immunoblotting. Following the treatment, melanocyte density was quantitated, and senescence and angiogenic responses were determined by senescence-associated beta-galactosidase (SA-ß-gal) and endogenous alkaline phosphatase (ALP) staining, respectively. Relative expression of microRNAs were determined by quantitative RT-PCR. KEY FINDINGS: Rapamycin (400 nM) suppressed zTOR pathway by down-regulating the phosphorylation of zTOR-associated proteins such as P70S6K and S6K at both 4h post-fertilisation (hpf) and 8hpf while 4E-BP1 was only down-regulated at 8hpf when compared to their respective vehicle controls. Treatment with rapamycin also resulted in significant suppression of melanocyte development and senescence-associated beta-galactosidase (SA-ß-gal) activity, and perturbed the development of intersegmental vessels (ISVs) of zebrafish embryos. In addition, the expressions of dre-miR-9-5p and -3p, dre-miR-25-3p and dre-miR-124-3p were significantly up-regulated in embryos treated with rapamycin from 4hpf. SIGNIFICANCE: Our findings suggest the involvement of zTOR in embryonic senescence and angiogenesis which could be potentially mediated by selected miRNAs.