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

Abstract

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. 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.