Proinflammatory cytokines stimulate expression of β-secretase, which increases processing of amyloid precursor protein (APP), ultimately leading to the deposition of amyloid beta (Aβ). The N-terminal domain of β-cleaved APP supports Cu/NO-dependent release of heparan sulfate (HS) from the glypican-1 (Gpc-1) proteoglycan. HS is an inhibitor of β-secretase, thereby constituting a regulatory, negative feedback loop. Here, we have investigated the effect of the proinflammatory cytokines TNF-α, IL-1β and IL-6 on the interplay between APP processing and release of HS from Gpc-1 in neuronal cells. We have used deconvolution immunofluorescence microscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a panel of monoclonal/polyclonal antibodies recognizing the released HS, the N-terminus of Aβ, Aβ, the C-terminus of APP and the autophagosome marker LC3 as well as the chemical lysosome marker LysoTrackerRed (LTR). We repeatedly found that N2a neuroblastoma cells and human neural stem cells grown in the presence of the cytokines developed large cytoplasmic clusters, which stained positive for HS, the N-terminus of Aβ, Aβ, the C-terminus of APP, LC3 and LTR, indicating accumulation of HS and APP/APP degradation products in enlarged autophagosomes/lysosomes. The SDS-PAGE of immunoisolates obtained from TNF-α-treated N2a cells by using anti-C-terminus of APP revealed the presence of SDS-stable complexes between HS and the C-terminal fragment of β-cleaved APP (βCTF) migrating in the range 10–18 kDa. Clustered accumulation of βCTF disappeared when HS release was prevented and slightly enhanced when HS release was increased. Hence, when proinflammatory cytokines induce increased processing of APP, inhibition of β-secretase by HS is insufficient, which may lead to the impaired autophagosomal degradation.

中文翻译：

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促炎细胞因子诱导 Glypican-1 衍生的硫酸乙酰肝素和 β 裂解 APP 的 C 端片段在分裂神经元细胞的自噬体中积累。

促炎细胞因子刺激 β-分泌酶的表达，这会增加淀粉样前体蛋白 (APP) 的加工，最终导致淀粉样蛋白 β (Aβ) 的沉积。β 切割的 APP 的 N 端结构域支持硫酸乙酰肝素 (HS) 从 glypican-1 (Gpc-1) 蛋白聚糖中的 Cu/NO 依赖性释放。HS 是 β-分泌酶的抑制剂，因此构成了一个调节性的负反馈回路。在这里，我们研究了促炎细胞因子 TNF-α、IL-1β 和 IL-6 对神经元细胞中 APP 加工和 Gpc-1 释放 HS 之间相互作用的影响。我们使用去卷积免疫荧光显微镜和十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS-PAGE) 和一组单克隆/多克隆抗体识别释放的 HS、Aβ、Aβ、APP 的 C 端和自噬体标志物 LC3 以及化学溶酶体标志物 LysoTrackerRed (LTR)。我们反复发现在细胞因子存在下生长的 N2a 神经母细胞瘤细胞和人类神经干细胞形成大的细胞质簇，其染色呈 HS 阳性，Aβ、Aβ 的 N 端、APP、LC3 和 LTR 的 C 端，表明 HS 和 APP/APP 降解产物在扩大的自噬体/溶酶体中积累。通过使用 APP 的抗 C 端从 TNF-α 处理的 N2a 细胞获得的免疫分离物的 SDS-PAGE 显示 HS 和 β-裂解的 APP (βCTF) 的 C 端片段之间存在 SDS 稳定的复合物。范围 10–18 kDa。当 HS 释放被阻止时，βCTF 的聚集积累消失，而当 HS 释放增加时，βCTF 的聚集积聚略有增强。因此，