l-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome

Human Molecular Genetics -leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome Baoshan Xu 2 Nenja Sowa 0 2 Maria E. Cardenas 3 Jennifer L. Gerton 1 2 0 Medical Faculty, University of Gottingen , Robert-Koch-Str. 40, 37075 Gottingen , Germany 1 Department of Biochemistry and Molecular Biology, University of Kansas School of Medicine , Kansas City, KS 66160 , USA 2 Stowers Institute for Medical Research , Kansas City, MO 64110 , USA 3 Department of Molecular Genetics and Microbiology, Duke University , Durham, NC 27708 , USA 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. -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. -leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, -leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (-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 -leucine or its metabolite -KIC can partially rescue development in zebrafish models for CdLS. Introduction Cohesin is a protein ring structure that holds sister chromatids together from DNA replication until cell division. The cohesin complex is composed of four subunits: Scc1 (Rad21/Mcd1), Scc3, Smc1 and Smc3. Smc1 and Smc3 are family members of Structural Maintenance of Chromosomes proteins. Scc1 and Scc3 interact with the ATPase-containing head domains of Smc1 and Smc3 to stabilize the cohesin ring structure. Cohesin facilitates chromosome segregation, DNA damage repair and gene expression. Reduced cohesin function can cause the differential expression of many genes. The molecular mechanisms behind cohesins regulation of gene expression are still elusive but are critical for animal development. Cohesin is physically associated with several transcriptionally active regions within the metazoan genome, such as the H19 imprinting control region (1) and the -globin locus control region (2), suggesting cohesin plays a critical role in gene expression. Mutations of cohesin subunits as well as its regulators lead to a spectrum of disorders known as cohesinopathies which include both Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS). Characteristics of cohesinopathies can include poor growth, limb, gut and heart defects, craniofacial abnormalities and mental retardation ranging from mild to severe. Mutation of both copies of Establishment of Sister chromatid Cohesion acetyltransferase 2 (ESCO2) causes RBS (3). CdLS is a clinically distinct syndrome caused by mutation in one of the two copies of a number of different genes, suggesting the syndrome is caused by haploinsufficiency for cohesin function. Fly Nipped-B-like protein (NIPBL) is a homolog of fungal Sister Chromatid Cohesin protein (SCC2) that loads the cohesin complex onto the chromosome. The cohesin loading factor was first reported to be mutated in CdLS in 2004 (4,5). Around 60% of CdLS cases are caused by an NIPBL mutation. Smc1 and Smc3 were subsequently found to be mutated in a small number of CdLS patients with mild symptoms (6,7). Recently, HDAC8 mutations were documented in several CdLS patients (8). HDAC8 deacetylates the Smc3 subunit of cohesin (8). A related cohesinopathy, which has overlapping features with CdLS, is caused by mutations in Rad21 (9). Therefore, mutations in many different cohesin-related genes can cause developmental disorders with both overlapping and unique features. Cells derived from CdLS patients do not show high levels of aneuploidy (10), consistent with the hypothesis that changes in gene expression cause this syndrome. Whole animal models have been developed to study pathogenesis in the cohesinopathies. A CdLS mouse model (Nipbl+/) mimics many features of CdLS including small size, craniofacial anomalies, heart defects and perinatal mortality, along with dysregulated expression of many genes across various tissues (11). Multiple studies performed in zebrafish found that rad21-morphant and rad21 transgenic mutant embryos displayed central nervous system necrosis, decreased head and eye size and decreased trunk thickness (9,12,13). There are two copies of nipbl in the zebrafish genome; the nipbla/b-knockdown embryos exhibited disrupted blood circulation, defective cardiac precursor migration and decreased gut thickness (14). The smc3-morphant embryos have brain and eye necrosis (15). The esco2 and rad21 morphant embryos demonstrated mitotic delay and elevated apoptosis (12,16), both of which may contribute to the developmental abnormalities. Two major regulatory proteins are affected in zebrafish cohesin morphant embryosp53 and c-Myc. p53, a tumor suppressor protein and cell cycle regulator, is upregulated in rad21, smc3 and esco2 morphant zebrafish embryos (1417). There are many different triggers for p53, including defects in ribosome biogenesis. It is unclear which triggers cause the upregulation observed in cohesin morphant zebrafish. The cohesin complex physically binds to the promoter of c-Myc, which plays an important role in cell growth and apoptosis and facilitates transcription of many genes involved in translation. In contrast to p53, c-Myc is downregulated in rad21, nipbla/b and smc3-morphant zebrafish embryos as well as in D. melanogaster mutants (13), but upregulated in esco2 morphants. As c-Myc is a positive regulator of ribosome biogenesis and protein synthesis (18) and p53 serves as a cellular sensor for ribosome impairment, then the upregulation of p53 in combination with the downregulation of c-Myc suggests that ribosome function and protein translation could be compromised (...truncated)