and that these compounds are unable to serve as scaffolds due to their short saccharide chain lengths.Establishing methodological approaches of extracellular remodeling of brain carbohydrates in vivo, especially HS, would provide a high impact on researchers in the field of neuroscience. These would facilitate an easier access to investigate functions of extracellular molecules that bind to HS S-domains. The transgenic model mice would also aide to investigate potential roles of post-translational modifications of Sulf29) in its biological functions in vivo. The Sulf2 project will be extended to study prion-like behavior of p53 aggregates10). Microglial clearance of Aß facilitated by the Sulf2 action could contribute to development of a new approach in AD therapeutics. The new findings that the small sulfated and phosphorylated HS glycans could interfere with the interaction between HS/heparin and Aβ provide significant information for the understanding of amyloid formation in tissues of the associated diseases. By sharing the Sulf2 conditional transgenic mice and outcomes of this project, we strengthen the international partnerships. As such, we acknowledge and promote the international visibility and attractiveness of the Mizutani Foundation for Glycoscience, and fulfill the prestigious aim of the Foundation: “To contribute to the welfare of mankind through promotion of basic studies on glycoconjugates”.Regulation of amyloid clearance by heparan sulfate remodelingKenji UchimuraWe developed an ex vivo microglial phagocytosis assay for Aß amyloid deposits and showed that lentiviral expression of human Sulf2 in a mouse microglia cell line facilitates clearance of Aß amyloid deposits in Alzheimer’s model brain sections. Moreover, we generated Sulf2 conditional transgenic mouse models that allow cell type-specific expression of the human Sulf2 gene by a Cre-loxP system (unpublished). We bred these mice with an activated microglia/macrophage specific Cre-driver mouse and then crossbred these transgenic mice with J20 Alzheimer’s disease model mice7). We have generated HSulf2-Cre-J20 triple transgenic mice. We are analyzing degree of amyloid deposits and Alzheimer’s disease pathogenesis in the brain of these mice with age.To further analyze possible roles of C-6 sulfate groups of HS S-domains in amyloid formation, we utilized chemically synthesized 6-O-phosphorylated HS oligosaccharide derivatives8) (Figure 3). We tested them for assays of amyloid β fibril formation. Compounds 30 and 38 showed inhibitory effects on Aβ fibril formation in the presence of heparin, a structural analogue of HS S-domains, as measured by the ThT fluorescence intensities and studied by atomic force microscopy. Upon aggregation the transition of an unstructured state to a β-sheet structure can create sulfated GAG-binding sites. It was suggested that compounds 30 and 38 may bind to these conformers and interfere with the subsequent elongation process, References 1) Nishitsuji K & Uchimura K. Sulfated glycosaminoglycans in protein aggregation diseases. Glycoconj J (34):453-466, 2017. 2) Morimoto-Tomita M *, Uchimura K *, Werb Z, Hemmerich S & Rosen SD. Cloning and characterization of two extracellular heparin-degrading 3) Kameyama H, Uchimura K, Yamashita T, Kuwabara K, Mizuguchi M, Hung SC, Okuhira K, Masuda T, Kosugi T, Ohgita T, Saito H, Ando Y & Nishitsuji K. The Accumulation of Heparan Sulfate S-Domains in Kidney Transthyretin Deposits Accelerates Fibril Formation and Promotes Cytotoxicity. Am J Pathol (189):308-319, 2019. 4) Kuwabara K, Nishitsuji K, Uchimura K, Hung SC, Mizuguchi M, Nakajima H, Mikawa S, Kobayashi N, Saito H & Sakashita N. Cellular interaction and cytotoxicity of the iowa mutation of apolipoprotein A-I (ApoA-IIowa) amyloid mediated by sulfate moieties of heparan sulfate. J Biol Chem (290):24210-24221, 2015. 5) Kameyama H, Nakajima H, Nishitsuji K, Mikawa S, Uchimura K, Kobayashi N, Okuhira K, Saito H & Sakashita N. Iowa Mutant Apolipoprotein A-I 6) Hosono-Fukao T, Ohtake-Niimi S, Hoshino H, Britschgi M, Akatsu H, Hossain MM, Nishitsuji K, van Kuppevelt TH, Kimata K, Michikawa M, Wyss-Coray T & Uchimura K. Heparan sulfate subdomains that are degraded by Sulf accumulate in cerebral amyloid ss plaques of Alzheimer's disease: evidence from mouse models and patients. Am J Pathol (180):2056-2067, 2012. 7) Zhang Z, Takeda-Uchimura Y, Foyez T, Ohtake-Niimi S, Narentuya, Akatsu H, Nishitsuji K, Michikawa M, Wyss-Coray T, Kadomatsu K & Uchimura. 8) Uchimura K, Nishitsuji K, Chiu LT, Ohgita T, Saito H, Allain F, Gannedi V, Wong CH & Hung SC. Design and synthesis of 6-O-phosphorylated heparan 9) El Masri R, Seffouh A, Roelants C, Seffouh I, Gout E, Pérard J, Dalonneau F, Nishitsuji K, Noborn F, Nikpour M, Larson G, Crétinon Y, Friedel-Arboleas M, Uchimura K, Daniel R, Lortat-Jacob H, Filhol O & Vivés RR. Extracellular endosulfatase Sulf-2 harbors a chondroitin/dermatan sulfate chain that modulates ist enzymatic activity. Cell Rep (38):110516, 2022.endosulfatases in mice and humans. J Biol Chem (277):49175-49185, 2002. *equal contribution.(ApoA-IIowa) Fibrils Target Lysosomes. Sci Rep (6):30391, 2016.Deficiency of a sulfotransferase for sialic acid-modified glycans mitigates Alzheimer's pathology. Proc Natl Acad Sci USA (114):E2947-E2954, 2017. sulfate oligosaccharides to inhibit amyloid ß aggregation. Chembiochem in press, 2022.10) Iwahashi N, Ikezaki M, Nishikawa T, Namba N, Ohgita T, Saito H, Ihara Y, Shimanouchi T, Ino K, Uchimura K & Nishitsuji K. Sulfated glycosaminoglycans mediate prion-like behavior of p53 aggregates. Proc Natl Acad Sci USA (117):33225-33234, 2020.118
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