Reduced secreted clusterin as a mechanism for Alzheimer-associated CLU mutations.
Reduced secreted clusterin as a mechanism for Alzheimer-associated CLU mutations.
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DOI：
10.1186/s13024-015-0024-9
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发表时间：
2015-07-16
影响因子：
15.1
通讯作者：
Sleegers K
中科院分区：
文献类型：
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作者：
Bettens K;Vermeulen S;Van Cauwenberghe C;Heeman B;Asselbergh B;Robberecht C;Engelborghs S;Vandenbulcke M;Vandenberghe R;De Deyn PP;Cruts M;Van Broeckhoven C;Sleegers K
clusterin（CLU）基因已被确定为阿尔茨海默病（AD）的一个重要危险位点。虽然该位点的实际风险增加多态性仍有待确定，但我们先前观察到AD患者中CLU的罕见非同义突变和小插入-缺失频率增加，这些突变和小插入-缺失特异性聚集在CLU的β链结构域中。尽管如此，这些变体的致病性仍不清楚。在这里，我们报告了一种新的非同义CLU突变（p.I360N）在比利时阿尔茨海默病患者，并探讨了致病性的这和10个额外的CLU突变蛋白定位和分泌在体外使用免疫细胞化学，免疫检测和ELISA。β链中的三个患者特异性CLU突变（p.I303NfsX13、p.R338W和p.I360N）导致亚细胞CLU定位改变，并减少了CLU通过分泌途径的转运，表明可能的降解机制。对于这些突变，显着降低CLU强度，观察到在高尔基体，而几乎所有的CLU蛋白是专门存在于内质网。这通过HEK 293 T和HEK 293 FLp-In细胞系中CLU分泌减少进一步证实。我们的数据进一步支持了罕见的编码CLU突变在神经退行性疾病发病机制中的作用。功能分析表明，减少分泌的CLU蛋白作为三个检查CLU突变的作用模式。其中一个是导致分泌蛋白丢失的移码突变，另外两个突变是二硫桥区的氨基酸取代，可能干扰CLU α和β链的异源二聚化。本文的在线版本（doi：10.1186/s13024-015-0024-9）包含补充材料，可供授权用户使用。
The clusterin (CLU) gene has been identified as an important risk locus for Alzheimer’s disease (AD). Although the actual risk–increasing polymorphisms at this locus remain to be identified, we previously observed an increased frequency of rare non-synonymous mutations and small insertion-deletions of CLU in AD patients, which specifically clustered in the β-chain domain of CLU. Nonetheless the pathogenic nature of these variants remained unclear. Here we report a novel non-synonymous CLU mutation (p.I360N) in a Belgian Alzheimer patient and have explored the pathogenic nature of this and 10 additional CLU mutations on protein localization and secretion in vitro using immunocytochemistry, immunodetection and ELISAs. Three patient-specific CLU mutations in the β-chain (p.I303NfsX13, p.R338W and p.I360N) caused an alteration of the subcellular CLU localization and diminished CLU transport through the secretory pathway, indicative of possible degradation mechanisms. For these mutations, significantly reduced CLU intensity was observed in the Golgi while almost all CLU protein was exclusively present in the endoplasmic reticulum. This was further confirmed by diminished CLU secretion in HEK293T and HEK293 FLp-In cell lines. Our data lend further support to the contribution of rare coding CLU mutations in the pathogenesis of neurodegenerative diseases. Functional analyses suggest reduced secretion of the CLU protein as the mode of action for three of the examined CLU mutations. One of those is a frameshift mutation leading to a loss of secreted protein, and the other two mutations are amino acid substitutions in the disulfide bridge region, possibly interfering with heterodimerization of the α- and β-chain of CLU. The online version of this article (doi:10.1186/s13024-015-0024-9) contains supplementary material, which is available to authorized users.