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的认知能力下降。目前还没有治愈或有效的治疗方法，特别是考虑到使用干预措施移除之前被认为是疾病病理特征的淀粉样蛋白-β(Aβ)斑块来保护AD患者的认知功能是徒劳的。然而，人们的注意力已经转向了可溶性、低聚形式的A-β(OA-β)的潜在致病作用。此外，与此相关的是基底前脑胆碱能神经元(BFCN)的丧失，这些神经元为其他大脑中心提供广泛的神经支配，对正常认知能力至关重要。我们与AD相关工作的一个长期目标是识别导致BFCN功能不稳定和退化的种子和早期疾病过程，并可以及早进行靶向，以减缓或停止神经元死亡和记忆损害。我们的初步发现支持并合并了AD的OA、β和胆碱能假说。他们表明，在小鼠器官培养中持续暴露于OAβ会导致BFCN的过度兴奋和最终死亡。这些作用可被含有α7亚单位的烟碱型乙酰胆碱受体拮抗剂(α7*-nAChR)所阻断。在nAChRβ2亚单位敲除小鼠中，它们也不存在于BFCN中。NAChRα7和β2亚基在BFCN中富含，它们结合形成独特的α7β2-nAChR亚型，与仅由α7亚基组成的同源受体(α7-nAChR)不同。此外，如果AD小鼠模型中的小鼠也缺乏β2亚单位，那么这些小鼠的空间参考记忆缺陷就会正常化。对于这个探索性的R21项目来说，最关键的是我们的发现，OAβ在激活α7-和α7β2-nAChR单通道功能方面模仿乙酰胆碱(ACh)。只有α7β2-nAChR的单通道动力学才是唯一改变的，而不是ACh。这些研究支持该项目的总体假设，即OAβ在α7β2-nAChR的作用导致观察到的BFCN的不稳定和死亡以及最终的认知妥协。这个开发项目的目标是确定OAβ:α7β2-nAChR相互作用的位置和防止它们的方法，而不干扰ACh对α7β2-nAChR的自然激活。具体目的1是验证这样一种假设，即人OAβ刺激人α7β2-nAChR功能的部位不同于ACh作为激动剂作用的部位。具体目的2是验证Aβ片段可以在保持ACh激动剂作用不变的情况下阻断OAβ在α7β2-nAChR的作用的假说。我们成熟的技术将用于nAChR表达、定点突变、药理学和单通道电生理学。这些结果将提供导致BFCN死亡的OAβ:α7β2-nAChR相互作用的分子描述，并可能解释痴呆的出现。这些事件发生在疾病进展的足够早的时间，从而允许采取新的治疗干预措施来预防或减轻神经元丢失，并对AD进行有效、及时的治疗。

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.