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

β-片层破坏肽抑制 Aβ 产生的肌细胞模型中的纤维形成：对阿尔茨海默氏病治疗的意义

• 批准号: 11670955
• 负责人: TSUZUKI Kayo
• 金额: $ 2.11万
• 依托单位: Sapporo Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670955/
• 关键词: Alzheimer's disease; amyloid β protein; β-sheet breaker peptide; Embryonal rhabdomyosarcoma; amyloid precursor protein; Liposome method; Chloroquine; in vitro model

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

β淀粉样蛋白（Aβ）是阿尔茨海默病老年斑的主要成分，聚集成纤维后具有神经毒性。Aβ，特别是较长的Aβ42，长度从39至43个氨基酸不等，被认为在阿尔茨海默病（AD）发病机制中起重要作用。近年来，与Aβ17-21中心区（LVFFA）同源的短合成肽（LPFFD），如β折叠破坏肽（iAβ35），在体外已显示出与Aβ高亲和力结合，部分抑制Aβ纤维形成，并重新溶解预先形成的纤维。我们开发了氯喹（CQN）下培养的心肌细胞系统作为实验模型，以研究APP加工产生Aβ。用iAβ5 cDNA（BRS 73）转染人横纹肌肉瘤（CCL-136）细胞。为了了解iAβ5在Aβ隔离机制中的作用，我们在该模型中通过免疫学方法研究了iAβ5与Aβ1-40和Aβ1-42分子的相互作用.培养的细胞核周区可见微空泡 ...更多信息 CCL-136和BRS 73在CQN处理12小时后的变化。24 h后，空泡数量增多，体积增大，边缘增厚. CCL 136细胞的核周和含有染色颗粒体的空泡周围的异质材料与BC 42和BC 40（与Aβ42和Aβ40反应的Aβ C末端特异性多克隆抗体）反应。BC 42和抗iA β5抗体的双重免疫染色表明，在存在CQN的情况下，Aβ42和iAβ5共定位于BRS 73的微空泡中。4.BC42和BC 40在CQN处理的CCL-136和BRS 73的线粒体组分中通过Western印迹分析检测到Kda条带。用CQN处理的BRS 73.4的线粒体级分中的所有抗体标记15 kDa蛋白条带。将Aβ1-42与iAβ5共同孵育，在体外结合试验中，BC 42和抗iA β5抗体可观察到4 ~ 15 kDa的条带。iAp 5通过与Aβ1-42结合形成β折叠破坏肽（β-sheet breaker peptide，β-sheet breaker peptide，β-sheet breaker peptide），从而阻断β-sheet构象的形成，在体外实验中β-sheet breaker peptide可能是一种有效的淀粉样蛋白生成抑制剂，从而为AD的治疗提供重要的药物。少

项目成果

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Fukatsu R, Tsuzuki K, et al.: "Cultured myocyte systems under chloroquine as an experimental model to study APP, PS-1 processing for Aβ production"Brain Research. (2002)

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