B Fluorescence-based Thioflavin T (ThT) binding A Structure of chemically synthesized C Morphological analysis of Aβ fibrils assembled patients with Alzheimer’s disease6). Moreover, HS S-domains were also abundant in amyloid deposits of transthyretin ATTR amyloidosis patient tissues3). We hypothesized that HS S-domains are central to the shield that inhibits cellular clearance, and that remodeling of the HS S-domains with Sulf2 would promote phagocytotic clearance of the amyloid deposits by microglia. Our preliminary data showed that expression of Figure 2. Accumulation of HS S-domains in cerebral amyloid deposits of Alzheimer’s disease model micehuman Sulf2 facilitates microglial clearance of Aß deposits in brain sections of Alzheimer’s model mice ex vivo, supporting our concept. Specific aims of the project were to analyze mechanisms underlying inhibition of microglial amyloid clearance, and to evaluate the way to breakdown HS-S-domain-shields with Sulf2 to facilitate the microglial phagocytotic clearance of amyloid in vivo.Figure 3. Effects of 6-O-phosphorylated heparan sulfate/heparin oligosaccharide derivatives on amyloid β fibril formationHS S-domains revealed by RB4CD12, an anti-S-domain phage display antibody exist in the basement membrane of brain microvessels in wild-type mice (Non-Tg) (arrows). The S-domains abnormally accumulate in amyloid ß (Aß) deposits in the brain of two Alzheimer’s disease mouse models (J20 and Tg2576) (arrowheads)6).6-O-phosphorylated heparan sulfate analogues8).assay of amyloid β (Aβ) in the absence (Control) or presence of heparin, or HS/heparin oligosaccharide derivative. Aβ (10 μM) was incubated without or with heparin (40 μg/mL) or HS/heparin derivative (100 μM) in the mixture of ThT (10 μM) at 37 oC. ThT fluorescence intensity was measured for 24 h. Ordinary two-way ANOVA with post-hoc Dunnett’s test (vs. Control) showed a significant change in Aβ aggregation in heparin-mixed fractions and in compound 38-mixed fractions. Post-hoc Tukey’s range test showed significant change in compound 28-, 30-, and 38-mixed fractions compared with heparin-mixed fractions. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.in the presence of heparin and compound 30 or 38. Atomic force microscopy images in tapping mode high (z range: 100.00 nm, top) and amplitude in deflection (z range: 1.00 V for Heparin and Heparin + 30, 0.25 V for Heparin + 38, bottom) of Aβ assemblies are shown. These assemblies were prepared in the presence of heparin (40 μg/mL) alone, or co-presence of heparin (40 μg/mL) and compound 30 or 38 (100 μM). Scale bar: 1μm.117
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