Beta-Amyloid Protofibrils Cause AD Neuronal Dysfunction

早期阿尔茨海默病（AD）的症状包括轻微的记忆丧失和认知障碍，慢慢发展为严重的痴呆症。临床症状最初可能是由于突触功能障碍，随后是更深刻的形态学改变，可能包括神经性营养不良、突触丧失和直接细胞死亡。这种隐性进展性病理生理的基本机制被认为是与年龄相关的淀粉样蛋白（Abeta）原纤维的积累，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，原纤维，低聚形式的β可以启动神经元功能障碍，并可以直接和/或通过进一步过渡到更高分子量的组装（淀粉样原纤维），引发神经元损失。为了支持这一观点，在阿尔茨海默病患者的脑脊液中发现了稳定的β低聚物，纤维前形式的β引起突触功能障碍和神经元死亡，大脑中可溶性β水平与认知障碍相对相关。此外，产生人类β的转基因小鼠在任何斑块形成之前都表现出电生理变化。我们最近发表的研究表明，原纤维（PFs）是一种亚稳态的Abeta寡聚物形式，可以急剧增加皮质神经元的电活动并可重复性地诱导神经毒性。在这一建议中，我们的4个目标侧重于；1)亚致死（nM）原纤维β诱导电生理活性改变的离子机制；2) pf诱导神经毒性的生化机制；3)内源性原纤维的分离与鉴定；4) 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。原代混合脑培养将用于评估pf诱导的电活动和神经元损伤。利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通过特定的通道拮抗剂进行评估。早期神经元功能障碍将通过细微的形态学、生化和基因表达标记进行评估。我们的假设和数据表明，阿尔茨海默病的临床前和临床进展在一定程度上是由β寡聚化的早期时间变化驱动的，而不仅仅是淀粉样蛋白纤维的形成。破译和抑制PF的生物活性是一种新的方法，有助于阐明早期β中间体在AD中的作用，并有助于设计合理的治疗策略来减缓或阻止神经元损伤的进展。

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.