β-Sheet breaker peptides inhibit fibrogenesis in a myocyte model of Aβ production: implication for Alzheimer's disease therapy

淀粉样蛋白β蛋白(Aβ)是阿尔茨海默病老年斑的主要成分，当聚集成纤维时具有神经毒性。Aβ的长度从39到43个氨基酸不等，特别是较长的Aβ42，被认为在阿尔茨海默病(AD)的发病机制中发挥着重要作用。最近，与Aβ17-21中心区同源的短合成肽(LPFFD)作为β-Sheet Breaker(IAβ35)在体外被证明能够与Aβ高亲和力结合，部分抑制Aβ的纤维形成，并重新溶解预先形成的纤维。我们建立了氯喹下培养的心肌细胞系统作为实验模型，以研究APP生产Aβ的过程。将IAβ5基因(BRS73)导入人横纹肌肉瘤细胞系。为了了解IAβ5在Aβ的隔离机制中的作用，我们用免疫学方法研究了IAβ5与Aβ1-40和Aβ1-42分子的相互作用。培养的…核周可见微空泡。CQN治疗12h后，CCL-136和BRS73表达增加。24 h后，空泡数量增多，体积增大，边缘增厚。含有CCL136细胞染色颗粒体的空泡周围和异质物质与BC42和BC40(与Aβ42和Aβ40反应的Aβ末端特异性多克隆抗体)反应。BC42和抗IAβ5抗体的双重免疫染色表明，在CQN存在的情况下，Aβ42和IAβ5共定位于BRS73的微泡中。4CQN处理的CCL-136和BRS73细胞线粒体经BC42和BC40免疫印迹分析均可检测到KDA条带。CQN处理的BRS73.4细胞线粒体部分的15 kDa蛋白条带被所有抗体标记。当Aβ1-42与IAβ5孵育时，BC42和抗IAβ5在体外结合实验中观察到4~15 kDa的涂片条带。这一发现表明，iAp5优先与Aβ1-42结合，通过与Aβ42结合形成β-折叠破碎肽淀粉样蛋白，从而阻断β-折叠构象的形成，β-折叠破碎肽可能在体外被证明是一种有效的淀粉样蛋白生成抑制剂，从而为AD的治疗提供了一种重要的治疗方法。较少

Amyloid βprotein (Aβ), the major component of Alzheimer's senile plaques, is neurotoxic when aggregated into fibrils. Varying in length from 39 to 43 amino acids, Aβ, particularly the longer Aβ42, is thought to play a significant role in Alzheimer's disease (AD) pathogenesis. Recently, short synthetic peptides (LPFFD) homologous to the central region of Aβ17-21 (LVFFA) as β-sheet breaker peptide (iAβ35) have been shown in vitro to bind to Aβ with high affinity, partially inhibit Aβ fibrillogenesis, and redissolve preformed fibrils. We developed cultured myocyte systems under chloroquine (CQN) as an experimental model to study APP processing for Aβ production. Human rhabdomyosarcoma (CCL-136) was transfected with iAβ5 cDNA (BRS73). To understand the role iAβ5 plays in the sequestration mechanism of Aβ, we have investigated interactions of iAβ5, with Aβ1-40 and Aβ1-42 molecules by immunological methods in this model.1. Microvacuoles were recognizable in perinuclear region of the cultured … More CCL-136 and BRS73 after 12 h CQN treatment. After 24 h, the vacuoles increased in number and large together with thick rim.2. Perinuclear and heterogeneous materials surrounding vacuoles containing stained granular bodies of CCL136 cells reacted with BC42 and BC40 (C-terminal end-specific polyclonal antibodies of Aβ that react with Aβ42 and Aβ40). Double immunostaining with BC42 and anti-iAβ5 antibodies demonstrated that Aβ42 and iAβ5 were co-localized in microvacuoles of BRS73 in the presence of CQN.3. 4 Kda band was detectable by Western blot analysis using BC42 and BC40 in mitochondrial fraction of CQN-treated CCL-136 and BRS73. 15 kDa protein bands were labeled with all antibodies in mitochondorial fraction of CQN-treated BRS73.4. When Aβ1-42 was incubated with iAβ5, 4〜15 kDa smear bands were observed with BC42 and anti-iAβ5 in in vitro binding assay. This finding showed that iAp5 bound Aβ1-42 preferentially.β-sheet breaker peptide amyloid formation by binding to Aβ42, thereby blocked the formation of β-sheet conformation, β-sheet breaker peptide may prove to be an effective inhibitor of amyloidogenesis in vitro and, hence, would provide an important therapy for AD. Less