Stimulation of mTORC1 with L-leucine Rescues Defects Associated with Roberts Syndrome

Citation: Xu B, Lee KK, Zhang L, Gerton JL ( Stimulation of mTORC1 with L-leucine Rescues Defects Associated with Roberts Syndrome Baoshan Xu 0 Kenneth K. Lee 0 Lily Zhang 0 Jennifer L. Gerton 0 Nancy B. Spinner, University of Pennsylvania, United States of America 0 1 Stowers Institute for Medical Research, University of Kansas School of Medicine, Kansas City, Kansas, United States of America, 2 Department of Biochemistry and Molecular Biology, University of Kansas School of Medicine , Kansas City, Kansas , United States of America Roberts syndrome (RBS) is a human disease characterized by defects in limb and craniofacial development and growth and mental retardation. RBS is caused by mutations in ESCO2, a gene which encodes an acetyltransferase for the cohesin complex. While the essential role of the cohesin complex in chromosome segregation has been well characterized, it plays additional roles in DNA damage repair, chromosome condensation, and gene expression. The developmental phenotypes of Roberts syndrome and other cohesinopathies suggest that gene expression is impaired during embryogenesis. It was previously reported that ribosomal RNA production and protein translation were impaired in immortalized RBS cells. It was speculated that cohesin binding at the rDNA was important for nucleolar form and function. We have explored the hypothesis that reduced ribosome function contributes to RBS in zebrafish models and human cells. Two key pathways that sense cellular stress are the p53 and mTOR pathways. We report that mTOR signaling is inhibited in human RBS cells based on the reduced phosphorylation of the downstream effectors S6K1, S6 and 4EBP1, and this correlates with p53 activation. Nucleoli, the sites of ribosome production, are highly fragmented in RBS cells. We tested the effect of inhibiting p53 or stimulating mTOR in RBS cells. The rescue provided by mTOR activation was more significant, with activation rescuing both cell division and cell death. To study this cohesinopathy in a whole animal model we used ESCO2-mutant and morphant zebrafish embryos, which have developmental defects mimicking RBS. Consistent with RBS patient cells, the ESCO2 mutant embryos show p53 activation and inhibition of the TOR pathway. Stimulation of the TOR pathway with L-leucine rescued many developmental defects of ESCO2-mutant embryos. Our data support the idea that RBS can be attributed in part to defects in ribosome biogenesis, and stimulation of the TOR pathway has therapeutic potential. - Funding: This work was funded by the Stowers Institute for Medical Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Cohesin is a protein complex that adheres sister chromatids from the time of their replication until their division [1,2]. Cohesion between sister chromatids is facilitated by acetylation of the Smc3 subunit of the complex by the ECO1 acetyltransferase during S phase [3,4,5]. Human developmental syndromes such as Roberts syndrome (RBS) and Cornelia de Lange syndrome, termed cohesinopathies, arise from mutations in cohesin genes [6]. ESCO2, which is a human ortholog of ECO1 in the yeast Saccharomyces cerevisiae, is inactivated in RBS [7]. RBS is an autosomal recessive, multi-system disorder characterized by prenatal growth retardation (ranging from mild to severe), limb malformations (including bilateral symmetric tetraphocomelia or hypomelia caused by mesomelic shortening), craniofacial abnormalities and mental retardation [8,9,10,11,12]. Previous studies have reported loss of ESCO2 acetyltransferase activity in RBS [13]. Chromosomes show a characteristic pattern of heterochromatin repulsion with the regions affected including centromeres and NORs (nucleolar organizing centers or rDNA). A previous report revealed that mutations in yeast ECO1 and human ESCO2 impaired ribosomal RNA (rRNA) production and protein synthesis in budding yeast and human immortalized RBS cells [14]. Also, mutations in cohesin are associated with aberrant nucleolar morphology in yeast [15]. Cohesin binds to the rDNA in every organism studied, giving cohesin the potential to affect the structure and function of the nucleolus. We hypothesized that defective ribosome biogenesis contributes to the etiology of the RBS disorder. Perturbation of ribosome biogenesis is thought to lead to nucleolar stress and p53 activation. The mechanism appears to be the specific binding of ribosome proteins to Mdm2, which inhibits its E3 ubiquitin ligase function toward p53, leading to p53 stabilization and activation [16,17,18]. This binding happens when there is an imbalance of ribosomal proteins. Once p53 is stabilized, it will act to promote the transcription of Mdm2 in a feedback loop, as well as several other genes such as p21 and p27, cyclin-dependent kinase inhibitors [19]. Depletion of ribosomal proteins such as Rpl5, Rpl11, or Rps7 induces p53 upregulation in various cell lines [17,20,21,22]. Loss of Rpl11 impaired zebrafish embryonic development via a p53-dependent apoptotic response [22]. Furthermore, defects in ribosomal proteins such as Rps6 (S6), Rps19 and Rpl24 have been implicated in congenital malformations and aberrant growth during fetal development [17,18,22,23,24,25]. Taken together, these studies indicate a strong link between p53 activation and the process of ribosome biogenesis. Roberts syndrome is a human developmental disorder caused by mutations in the ESCO2 gene. This gene encodes an acetyltransferase that acetylates the cohesin ring complex to promote a locked configuration. The cohesin complex binds to many locations on chromosomes and mutations that affect its function result in changes in gene expression. In fact, Roberts syndrome and other diseases caused by mutations in cohesin are associated with differential gene expression. We wanted to understand how mutations in ESCO2 affect two important molecular pathways that detect cellular stress, the p53 and mTOR (mammalian target of rapamycin) pathways. We report that mutations in ESCO2 are associated with p53 activation and inhibition of mTOR in human cells and zebrafish. We tested the rescue effect of p53 inhibition and mTOR activation on human Roberts syndrome cells and zebrafish models for Roberts syndrome. While both treatments displayed rescue effects, the activation of mTOR provided more significant rescue. Our work suggests that stimulation of the mTOR pathway with the amino acid L-leucine has therapeutic potential for Roberts syndrome. In addition, our work suggests that some of the differential gene expression in Roberts syndrome may be explained by translational inhibition connected with the inhibition of the mTOR pathway. The TOR (target of rapamycin) pathway is a major node of control for protein translation and ribosome biogenesis. mTOR allows eukaryotic c (...truncated)