Alzheimer's therapy: Blocking ApoE/ABeta binding

描述（由申请方提供）：载脂蛋白（apo）E4亚型的遗传是散发性阿尔茨海默病（AD）的主要风险因素。ApoE，特别是其E4同种型，可以作为β-淀粉样蛋白（Abeta）的病理性伴侣，促进其从可溶性Abeta构象转化为毒性Abeta聚集体，其以斑块的形式或在血管壁中积累。已知ApoE在Abeta上的结合位点对应于残基12-28。为了阻断这种结合，我们合成了一种肽，Abeta 12 - 28 P，其与Abeta的残基12-28同源，但残基18处的缬氨酸被脯氨酸取代。这使得该肽无毒且非原纤维形成。Abeta 12 - 28 P是血脑屏障可渗透的（清除率=65+/-20 μ l血清/gr.脑），其末端保护形式的血清半衰期为62.2+/-18 min。我们收集了大量的初步数据，证明Abeta 12 - 28 P在体外和体内均可降低Abeta 12毒性和原纤维形成。与接受安慰剂的年龄匹配的Tg小鼠相比，用Abeta 12 - 28 P治疗一个月的转基因（Tg）APP/PS1 AD小鼠显示出在皮质中Abeta负荷减少63.3%，在海马（p = 0.0047）中减少61.9%（p = 0.0048）。在处理小鼠的血清中未检测到抗Abeta的抗体;因此，观察到的该肽的治疗作用不能归因于抗体清除反应。在给药动物中未观察到毒性。在这项研究计划中，我们计划扩展我们的初步工作，确定我们的先导化合物阻断apoE/Abeta相互作用的治疗是否是一种可行的治疗方法，用于减少AD中的Abeta沉积，使用不同的动物模型，包括双Tg小鼠共表达APP和各种亚型的人apoE在鼠apoE敲除背景。行为测试、Abeta负荷的组织学和生化分析将是本研究的终点。双光子显微镜将用于跟踪Tg小鼠体内的治疗效果。我们还计划确定apoE上Abeta结合位点的位置和结构，这对于开发可能阻断apoE/Abeta相互作用的其他化合物至关重要。

DESCRIPTION (provided by applicant): Inheritance of the apolipoprotein (apo) E4 isoform is a major risk factor for sporadic Alzheimer's disease (AD). ApoE, especially its E4 isoform, can act as a pathological chaperone of beta-amyloid (Abeta) promoting its conformational transformation from soluble Abeta into toxic Abeta aggregates which accumulate in the form of plaques or in vascular walls. It is known that the binding site of apoE on Abeta corresponds to residues 12-28. To block this binding, we synthesized a peptide, Abeta12-28P, which is homologous to residues 12-28 of Abeta but with the valine at residue 18 was substituted by a proline. This renders this peptide non-toxic and non-fibrillogenic. Abeta12-28P is blood-brain-barrier permeable (clearance=65+/-20 mu l serum/gr.brain) and its end-protected version has a serum half-life of 62.2+/- 18 min. We have gathered extensive preliminary data demonstrating that Abeta12-28P reduces Abeta toxicity and fibril formation both in vitro and in vivo. Transgenic (Tg) APP/PS1 AD mice treated for one month with Abeta12-28P demonstrated a reduction in Abeta load by 63.3% in the cortex and by 61.9% (p=0.0048) in the hippocampus (p=0.0047) comparing to age matched Tg mice which received placebo. No antibodies against Abeta were detected in the sera of treated mice; therefore, the observed therapeutic effect of this peptide cannot be attributed to an antibody clearance response. No toxicity was observed in treated animals. In this research proposal we plan to extend our preliminary work, determining if treatment with our lead compound blocking apoE/Abeta interaction is a viable therapeutic approach for reducing Abeta deposition in AD using different animal models including double Tg mice co-expressing APP and various isoforms of human apoE on a murine apoE knock-out background. Behavioral testing, histological and biochemical analysis of Abeta load will be end points of this study. Two-photon microscopy will be used to follow the treatment effect in Tg mice in vivo. We are also planning to identify the location and structure of the Abeta binding site on apoE which is crucial for the development additional compounds which may block the apoE/Abeta interaction.