Beta-Amyloid Protofibrils Cause AD Neuronal Dysfunction

早期阿尔茨海默病(AD)的症状包括轻度记忆丧失和认知障碍，慢慢发展为严重痴呆症。临床症状最初可能是由于突触功能障碍，然后是更深刻的形态变化，可能包括神经炎性营养不良、突触丢失和坦率的细胞死亡。这种潜伏的进行性病理生理学的基本机制被认为是与年龄相关的淀粉样β蛋白(Abeta)纤维的积累，最终在尸检中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这样的假设，即纤维本身是阿尔茨海默病进展的基础。我们的工作假设是，前纤维状、低聚形式的Abeta可以引发神经元功能障碍，并可以直接和/或通过进一步转变为更高分子量的组件(淀粉样纤维)，触发神经元丢失。为了支持这一点，在AD患者的脑脊液中发现了稳定的Abeta寡聚体，前纤维形式的Abeta导致突触功能障碍和神经元死亡，脑中可溶性Abeta水平与认知功能障碍有较好的相关性。此外，产生人Abeta的转基因小鼠在任何斑块形成之前都会表现出电生理变化。我们最近发表的研究表明，原纤维(PFS)是Abeta的一种亚稳定低聚形式，可以显著增加皮质神经元的电活动，并可重复地诱导神经毒性。在这项建议中，我们的4个目标集中在：1)亚致死(NM)原纤维蛋白Abeta引起的电生理活动改变的离子机制；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鉴定；以及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，将用特定的通道拮抗剂评估钙通道和钾通道在介导PF诱导的活动中的作用。早期神经元功能障碍将使用微妙的形态、生化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寡聚发生的早期时间变化驱动的，而不仅仅是淀粉样原纤维的形成。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Abeta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.