Characterization of amyloid-β:α7β2-nicotinic acetylcholine receptor interactions relevant to Alzheimer's disease

阿尔茨海默病（AD）是一种神经退行性疾病，其特征是持续进行性认知能力下降。目前还没有治愈或有效的治疗方法，特别是考虑到通过干预去除淀粉样蛋白-β (Aβ)斑块（以前被认为是疾病的病理标志）来保持AD患者的认知功能是徒劳的。然而，人们的注意力已经转向可溶性低聚形式的Aβ (oAβ)的潜在致病作用。此外，仍然相关的是基底前脑胆碱能神经元（BFCN）的损失，该神经元为正常认知表现至关重要的其他脑中心提供广泛的神经支配。我们与AD相关的工作的长期目标是确定导致BFCN功能不稳定和变性的重要和早期疾病过程，并且可以足够早地靶向减缓或阻止神经元死亡和记忆损害。我们的初步研究结果支持并融合了AD的oAβ和胆碱能假说。他们表明，在小鼠器官型培养物中持续暴露于oAβ会导致BFCN过度兴奋和最终死亡。这些作用被含有α7亚基（α7*-nAChR）的烟碱乙酰胆碱受体拮抗剂阻断。在nAChR β2亚基敲除的小鼠中，BFCN基因也缺失。nAChR α7和β2亚基在BFCN中富集，它们结合形成独特的α7β2-nAChR亚型，不同于单独由α7亚基组成的同质受体（α7-nAChR）。此外，在AD小鼠模型中，如果这些小鼠也缺乏β2亚基，则空间参考记忆的缺陷是归一化的。这个探索性R21项目最关键的是我们发现oAβ在激活α7-和α7β2- nachr单通道功能方面模仿乙酰胆碱（ACh）。oAβ，而不是ACh，只改变α7β2-nAChR的单通道动力学。这些研究支持了该项目的总体假设，即oAβ作用于α7β2-nAChR导致观察到的BFCN不稳定和死亡以及最终的认知损害。本开发项目的目标是确定oAβ:α7β2-nAChR相互作用的位点，以及在不干扰ACh自然激活α7β2-nAChR的情况下阻止它们的方法。具体目的1是验证人类oAβ刺激人类α7β2-nAChR功能的位点与ACh作为激动剂作用的位点不同的假设。特异性目的2是验证Aβ片段可以阻断oAβ在α7β2-nAChR上的作用，同时保持ACh激动剂的作用不变的假设。我们成熟的技术将用于nAChR表达、定点诱变、药理学和单通道电生理学。结果将提供oAβ:α7β2-nAChR相互作用导致BFCN死亡的分子描述，并可以解释痴呆的出现。这些事件发生在疾病进展的早期，从而允许新的治疗干预措施，以防止或减轻神经元丧失和有效的，及时的治疗AD。

Alzheimer's disease (AD) is a neurodegenerative condition characterized by relentlessly progressive cognitive decline. There is no cure or effective treatment, especially given futility in preserving cognitive function in AD patients using interventions to remove amyloid-β (Aβ) plaques previously considered to be pathological hallmarks of disease. However, attention has turned to potential etiopathogenic roles of soluble, oligomeric forms of Aβ (oAβ). Moreover, remaining relevant is the loss of basal forebrain cholinergic neurons (BFCN) that provide widespread innervation to other brain centers critical for normal cognitive performance. A long-term goal of our work related to AD is to identify seminal and early disease processes that lead to BFCN functional instability and degeneration and can be targeted early enough to slow or stop neuronal death and memory compromise. Our preliminary findings support and merge aspects of both the oAβ and cholinergic hypotheses of AD. They show that sustained exposure in murine organotypic culture to oAβ causes hyperexcitation and eventual death of BFCN. These effects are blocked by antagonists of nicotinic acetylcholine receptors containing α7 subunits (α7*-nAChR). They also are absent for BFCN from nAChR β2 subunit knock-out mice. nAChR α7 and β2 subunits are enriched in BFCN where they combine to form a unique α7β2-nAChR subtype distinct from homomeric receptors composed of α7 subunits alone (α7-nAChR). Furthermore, deficits in spatial reference memory in an AD mouse model is normalized if those mice also lack β2 subunits. Most critical to this exploratory R21 project is our finding that oAβ mimics acetylcholine (ACh) in the ability to activate α7- and α7β2-nAChR single channel function. oAβ, but not ACh, uniquely alters single channel kinetics only for α7β2-nAChR. These studies support the project's overarching hypothesis that oAβ action at α7β2-nAChR leads to observed instability and demise of BFCN and ultimate cognitive compromise. This developmental project's goals are to identify sites for oAβ:α7β2-nAChR interactions and ways to prevent them without perturbing natural activation of α7β2-nAChR by ACh. Specific Aim 1 is to test the hypothesis that the site for human oAβ stimulation of human α7β2-nAChR function is different than that for ACh action as an agonist. Specific Aim 2 is to test the hypothesis that fragments of Aβ can block effects of oAβ at α7β2-nAChR while leaving ACh agonist action intact. Our proven skills will be used in nAChR expression, site-directed mutagenesis, pharmacology, and single channel electrophysiology. Results will provide a molecular description of oAβ:α7β2-nAChR interactions that lead to BFCN demise and could explain the emergence of dementia. These events occur early enough in disease progression to allow for novel therapeutic interventions to prevent or abate neuronal loss and useful, timely treatment of AD.