Department of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University, Biomedical Center A13, SE-221 84 Lund, Sweden.
Glycobiology. 2021 Aug 7;31(7):800-811. doi: 10.1093/glycob/cwaa120.
The Apolipoprotein E4 (ApoE4) genotype is the most influential risk factor for sporadic Alzheimer's disease. It appears to be associated with retarded endosome-to-autophagosome trafficking. The amyloid precursor protein (APP) and the heparan sulfate (HS)-containing proteoglycan glypican-1 (Gpc-1) are both processed in endosomes, and mutually regulated by the APP degradation products and the released HS. We have investigated APP and Gpc-1 processing in ApoE3 and ApoE4 expressing human fibroblasts, in human neural stem cells (NSC) exposed to the cholesterol transport inhibitor U18666A and in induced neurons obtained by reprogramming of ApoE fibroblasts (ApoE-iN). We have used immunofluorescence microscopy, flow cytometry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis western blotting with antibodies recognizing the released HS, APP, amyloid β(Aβ), late endosomes (Rab7), autophagosomes (LC3) and neurons (Tuj1). We found that the capacity to release HS was not fully utilized in ApoE4 expressing fibroblasts and that HS-Aβ complexes accumulated in the nuclei. In ApoE3 fibroblasts, the β-cleaved APP C-terminal fragment (β-CTF) and Aβ were primarily present in late endosomes and autophagosomes. When HS release from Gpc-1 was enhanced by ascorbate in ApoE4/4 fibroblasts, there was efficient transfer of Aβ and HS from the nuclei to autophagosomes. In U18666A-treated NSC as well as in ApoE4/4-iN we repeatedly found accumulation of APP degradation products (β-CTF/Aβ). This was reversed by subsequent exposure to ascorbate or dehydroascorbic acid.
载脂蛋白 E4（ApoE4）基因型是散发性阿尔茨海默病的最主要影响风险因素。它似乎与内体-自噬体运输延迟有关。淀粉样前体蛋白（APP）和含有硫酸乙酰肝素（HS）的蛋白聚糖聚糖蛋白 1（Gpc-1）都在内体中进行加工，并且通过 APP 降解产物和释放的 HS 相互调节。我们已经研究了 ApoE3 和 ApoE4 表达的人成纤维细胞、暴露于胆固醇转运抑制剂 U18666A 的人神经干细胞（NSC）以及通过 ApoE 成纤维细胞重编程获得的诱导神经元（ApoE-iN）中的 APP 和 Gpc-1 加工。我们使用免疫荧光显微镜、流式细胞术和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳western 印迹，用识别释放的 HS、APP、淀粉样β（Aβ）、晚期内体（Rab7）、自噬体（LC3）和神经元（Tuj1）的抗体。我们发现，ApoE4 表达的成纤维细胞中 HS 的释放能力没有得到充分利用，并且 HS-Aβ 复合物在核内积累。在 ApoE3 成纤维细胞中，β-切割 APP C 端片段（β-CTF）和 Aβ主要存在于晚期内体和自噬体中。当 ApoE4/4 成纤维细胞中的 Gpc-1 通过抗坏血酸释放 HS 时，Aβ 和 HS 从核内有效地转移到自噬体中。在 U18666A 处理的 NSC 以及 ApoE4/4-iN 中，我们反复发现 APP 降解产物（β-CTF/Aβ）的积累。这可以通过随后暴露于抗坏血酸或脱氢抗坏血酸来逆转。
