Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS.

Angiotensin-converting enzyme inhibitors (ACEi) delay progression of the inherited renal disease Alport syndrome. However, the effect of ACEis weakens gradually due to an “aldosterone escape”. Here, we investigate if an aldosterone antagonist can counteract loss of ACEi-efficacy. COL4A3-/- mice were treated with ramipril (ACEi), starting at 4.5 weeks of age, and spironolactone was added at 7 weeks of age. Lifespan until renal failure, as well as kidney function parameters, were investigated. Dual therapy decreased proteinuria levels compared to ACEi monotherapy. Matrix accumulation, as well as tubulointerstitial and glomerular scar-tissue formation, were significantly reduced compared to untreated mice and ACEi-monotherapy at 75 and 100 days. Lifespan in dual treated mice was extended compared to untreated mice. However, lifespan was not superior to ACEi monotherapy–despite improved urea-nitrogen levels in the dual therapy group. In conclusion, adding the aldosterone-antagonist spironolactone to ACEi therapy further improved kidney function and reduced proteinuria and fibrosis. However, survival was not improved further, possibly due to premature death from side effects of dual therapy such as hyperkalemia. Thus, dual therapy could offer an effective therapy option for Alport syndrome patients with progressive proteinuria. However, the risks of adverse events require close monitoring.

Cohesinopathies are genetic disorders characterized by defects in fetal growth retardation such as Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS). These diseases are caused by mutations in genes that contribute to the function of the cohesin complex such as NIPBL, Smc1A, Smc3, HDAC8, Rad21 and ESCO2. Although the essential role of the cohesin complex in chromosome segregation has been well documented, it also plays roles in DNA damage repair, chromosome conformation, and gene expression. We recently reported L‐leucine can partially rescue several defects in zebrafish models for RBS by stimulating the TOR (target of rapamycin) pathway. In this study, we investigate the TOR and p53 pathways in zebrafish models for CdLS through morpholino injection and Western blot analysis, and the effect of L‐leucine on the morphants. Our results show partial inhibition of the TOR pathway in NIPBL, Rad21, Smc3‐defective embryos, and p53 signaling was upregulated in Rad21 and Smc3 morphants, but not NIPBL morphants. L‐leucine treatment partially improved TOR activity in CdLS embryos, promoting many aspects of development, and survival. L‐leucine administration partly prevented increased apoptosis in the cohesinopathy embryos. The Rad21 morphant exhibited a significant increase in mitotic cells, which was strongly suppressed by L‐leucine. In summary, our results suggest that translational impairment may contribute to the etiology of CdLS and L‐leucine treatment may provide some benefit. Grant Funding Source: Supported by Stowers Institute and Cornelia de Lange Syndrome Foundation Research Grants