Beta-Amyloid Protofibrils Cause AD Neuronal Dysfunction

β-淀粉样原纤维导致 AD 神经元功能障碍

• 批准号: 7030239
• 负责人: DEAN M HARTLEY
• 金额: $ 4.38万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7030239
• 关键词: Alzheimer' s disease; amyloid proteins; biomarker; brain; calcium channel; cell death; cellular pathology; electrophysiology; embryo /fetus cell /tissue; gene expression; genetically modified animals; human fetus tissue; laboratory mouse; laboratory rat; membrane activity; mixed tissue /cell culture; neuritic plaques; neurons; neurotoxicology; pathologic process; potassium channel; protein structure; protein structure function; voltage /patch clamp

Early Alzheimer's disease (AD) symptoms include mild memory loss and cognitive impairment that slowly progresses to severe dementia. Clinical symptomology may initially be due to synaptic dysfunction, followed by more profound morphological changes that may include neuritic dystrophy, synaptic loss, and frank cell death. The fundamental mechanism underlying this insidiously progressive pathophysiology is thought to be an age- related accumulation of amyloid beta-protein (Abeta) fibrils, ultimately observed as mature amyloid plaques at autopsy. However, the focus on end-stage tissue has led to the assumption that fibrils per se underlie the progression of AD. Our working hypothesis is that prefibrillar, oligomeric forms of Abeta can initiate neuronal dysfunction and can directly and/or via further transition to higher molecular weight assemblies (amyloid fibrils), trigger neuronal loss. In support of this, stable Abeta oligomers have been identified in the cerebrospinal fluid of AD patients, prefibrillar forms of Abeta cause synaptic dysfunction and neuronal death, and soluble Abeta levels in brain correlate relatively well with cognitive impairment. Furthermore, transgenic mice producing human Abeta show electrophysiological changes prior to any plaque formation. Our recently published studies have shown that protofibrils (PFs), a metastable oligomeric form of Abeta, can acutely increase the electrical activity of cortical neurons and reproducibly induce neurotoxicity. In this proposal, our 4 aims focus on; 1) ionic mechanisms of altered electrophysiological activity induced by sub-lethal (nM) protofibrillar Abeta; 2) biochemical mechanisms mediating PF-induced neurotoxicity; 3) isolation and characterization of endogenous protofibrils; and 4) long-term changes in neuronal function caused by PF-induced alterations in specific gene expression. Primary mixed brain cultures will be used to assess the PF-induced electrical activity and neuronal injury. Using whole-cell patch-clamp electrophysiology, the role of calcium and potassium channels in mediating PF-induced activity will be assessed with specific channel antagonists. Early neuronal dysfunction will be assessed using subtle morphological, biochemical, and gene-expression markers. Our hypothesis and data suggest that the preclinical and clinical progression of AD is driven, in part, by early temporal changes occurring in Abeta oligomerization, not just amyloid fibril formation. Deciphering and inhibiting the biological activity of PF is a novel approach that should help in elucidating the role of early Abeta intermediates in AD and in designing rational therapeutic strategies to slow or block the progression of neuronal injury.

早期阿尔茨海默病（AD）症状包括轻度记忆丧失和认知障碍，缓慢进展为严重痴呆。 临床形态学最初可能是由于突触功能障碍，随后是更深刻的形态学变化，可能包括神经炎性营养不良，突触丢失和坦率的细胞死亡。 这种潜在的进行性病理生理学的基本机制被认为是淀粉样β蛋白（Abeta）原纤维的年龄相关性积累，最终在尸检时观察到成熟的淀粉样斑块。然而，对终末期组织的关注导致了原纤维本身是AD进展的基础的假设。 我们的工作假设是，Abeta的前原纤维、寡聚体形式可以引发神经元功能障碍，并且可以直接和/或通过进一步转变为更高分子量的组装体（淀粉样蛋白原纤维）引发神经元损失。 为了支持这一点，已经在AD患者的脑脊液中鉴定出稳定的Abeta寡聚体，Abeta的前原纤维形式引起突触功能障碍和神经元死亡，并且脑中的可溶性Abeta水平与认知障碍相关性相对较好。此外，产生人Abeta的转基因小鼠在任何斑块形成之前显示出电生理学变化。 我们最近发表的研究表明，原纤维（PFs），一种亚稳定的寡聚体形式的Abeta，可以急剧增加皮层神经元的电活动，并可重复地诱导神经毒性。 在这项提案中，我们的4个目标集中在：1）由亚致死（nM）原纤维A β诱导的电生理活性改变的离子机制; 2）介导PF诱导的神经毒性的生化机制; 3）内源性原纤维的分离和表征;以及4）由PF诱导的特定基因表达改变引起的神经元功能的长期变化。 原代混合脑培养物将用于评估PF诱导的电活动和神经元损伤。 使用全细胞膜片钳电生理学，钙和钾通道在介导PF诱导的活性中的作用将用特异性通道拮抗剂进行评估。早期神经元功能障碍将使用微妙的形态学，生物化学和基因表达标记物进行评估。 我们的假设和数据表明，AD的临床前和临床进展部分是由Abeta寡聚化中发生的早期时间变化驱动的，而不仅仅是淀粉样原纤维形成。 破译和抑制PF的生物活性是一种新的方法，应该有助于阐明早期Abeta中间体在AD中的作用，并设计合理的治疗策略，以减缓或阻断神经元损伤的进展。