1Yukishige Ito2Kunio Shiota1Osaka University and RIKEN, 2Emeritus Professor, The University of TokyoThe hypocretin (HCRT) gene encodes prepro-orexin, the cleavage of which produces neuropeptides Orexin-A and Orexin-B. Orexin neurons regulate critical brain activities for controlling sleep, eating, emotions, and metabolism, and impaired orexin neuron function results in several neurologic disorders. Therefore, restoring normal orexin function and understanding the mechanisms of loss or impairment of orexin neurons represent important goals. BackgroundThe biological meaning of the diversity and complexity of glycan chains comprising glycoproteins and glycolipids is the central issue in glycoscience. In particular, the role of hexosamine biosynthetic pathway (HBP) is of paramount importance in producing a variety of glycans. It generates UDP-GlcNAc, a donor substrate of a group of enzymes, N-acetylglucosaminyltransferases (GlcNAc-Ts). UDP-GlcNAc is also an important precursor of sialic acid biosynthesis pathway, a downstream event of HBP. Sialic acid containing molecules are highly important in many recognition events in biological systems. In this case, UDP-GlcNAc is converted to ManNAc by an isomerizing enzyme (GNE) which catalyzes epimerization at the 2-position. Dysfunction of the HBP results in various problems in multicellular living systems, in association with the shortage of sialic acid. Several studies in recent years reported results that point toward the potential of simple monosaccharides, which are either related to or able to perturb HBP as master key glyco-molecules.Aims and resultsIn this project, we planned to conduct the systematic synthesis of N-acetylmannosamine (ManNAc) derivatives in light of their potential as 1) inducers of orexin neurons and 2) modulators of epigenetic control of gene expression.Histone modifications and DNA methylation play a central role in epigenetics by altering the structure and function of chromatin. O-GlcNAc modification is a form of post-translational modification of proteins at the serine (Ser)/threonine (Thr) residues. We have previously reported a novel O-GlcNAc modification at serine 40 (S40) of H2A (H2AS40Gc). S40-type H2A isoforms susceptible to O-GlcNAcylation are evolutionarily new and restricted to viviparous animals1) .H2A isoforms consist of S40 and alanine 40 (A40) types and this residue on H2A is located in the L1 of the globular domain, which is also known as a variable portion that distinguishes between the canonical and non-canonical H2A variants. Importantly, H2AS40Gc level increased owing to chemical-induced DNA damage, similar to phosphorylated H2AX (γH2AX) and acetylated H2AZ (AcH2AZ)2). These data suggest that H2AS40Gc contributes to maintaining genome integrity through the DNA repair mechanism in association with AcH2AZ and γH2AX.We found the induction of orexin neurons (miONs) from mouse embryonic stem cells (mESCs) by ManNAc in a neural culture condition3). ManNAc induced switching of epigenetic factors from Sirt/Ogt to Mgea5 at Hcrt gene locus, suggesting the involvement of HBP in orexin neurogenesis and the link between metabolic disorders and the loss of orexin neurons.Based on the study of miONs, we generated human orexin neurons (hiON) from induced pluripotent stem cells (hiPSCs) by treatment with ManNAc (Figure 1)4). To develop more potent compounds for orexin neurogenesis, we prepared derivatives of ManNAc. The activities of ManNFAc, 5S-ManNAc, 5S-ManNAcF, ManNCOOMe, and ManNCOOEt, evaluated by the expression of HCRT expression normalized to ACBT levels, were markedly higher than of ManNAc (Figure 2)4). The generation of orexin neurons was associated with DNA hypomethylation, histone H3/H4 hyperacetylation, and hypo-O-GlcNAcylation on the HCRT gene locus, and, thereby, the treatment of inhibitors of SIRT1 and OGT was effective at inducing orexin neurons from hiPSCs4). As sialic acid is the end-product in the HBP, the metabolism of ManNAc analogs was analyzed by using the fluorescently-labeled glycolipid Lac-Sph-BODIPY5) using COS-7 cells (Figure 3). Whereas ManNAcF entered the sialic acid 50Creation of carbohydrate derivedepigenetic controlling molecules
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