Extracellular Zinc Buffering and A-beta Oligomerization

描述（由申请人提供）：阿尔茨海默病（AD）是最常见的痴呆类型，是影响老年人的痴呆症的主要原因，占所有痴呆症病例的60 - 80%。虽然AD的机制还不清楚，但已知AD是导致tau蛋白和细胞外淀粉样蛋白2（Ab）聚集体（老年斑）的细胞内积累的蛋白质病。当突触释放的Ab寡聚化并最终聚集时，其通过直接毒性和/或活性氧物质的产生导致神经元损伤和神经胶质细胞中的炎症反应。目前还不清楚是什么触发了AD中的Ab寡聚化和聚集。其中一个理论是"AD的金属假说"，其假定细胞外金属离子，特别是突触分泌的Zn 2+，引发Ab寡聚化和聚集，导致老年斑的形成。尽管有很多进展支持AD的金属假说，这个想法仍然是一个有争议的问题，部分原因是没有足够的信息在突触释放的Zn 2+的时间尺度上的结合和聚集事件。显然，需要更好地理解Zn2+信号的时间模式，同时深入了解这些信号如何触发Ab的病理性寡聚化。如果存在特定的Zn2+浓度窗口或Zn2+信号的特定时间模式，这变得特别重要，所述特定的Zn2+浓度窗口或Zn2+信号的特定时间模式将允许形成可溶性低聚物，所述可溶性低聚物被认为是毒性最大的，但不允许它们聚集成可能防止毒性的斑块。尽管在研究方面做出了巨大努力，但目前还没有可用于AD的治疗方法。已经显示，Zn 2+的螯合可以改善AD动物模型中的斑块形成，并且因此，Zn 2+螯合剂已经被建议作为通过防止Ab寡聚化的AD的潜在治疗。Zn2+缓冲途径的调节剂也有望用于AD的药物开发。了解Ab寡聚化的生物物理过程以及对驱动寡聚化的Zn2+信号的时间模式的了解对于开发此类药物至关重要。我们已经设计了一种超快速的体外动力学测量技术的钙结合蛋白的闪光光解后笼中的钙离子，并创建了一个房室动力学模型，解决了第一次的合作性质的钙离子结合。该技术将适于通过在Ab存在下解开Zn2+同时光学监测Zn2+结合动力学来测量快速Zn2+结合动力学。将进行相同的操作以测量在Zn 2+释放突触处富集的Zn 2+结合蛋白金属硫蛋白-3（MT-3）的Zn 2+结合动力学。我们的方法允许前所未有的可能性来模拟细胞外Zn2+信号，因为它们被认为发生在突触。在类似于突触的条件下，研究Zn 2+与Ab和其他蛋白质结合的动力学将首次直接区分Zn 2+在Ab寡聚化中的生理和病理作用，从而可能开发新的高效AD药物。 公共卫生相关性：在阿尔茨海默病（AD）的动物模型中，减少脑中锌（Zn 2+）的释放可以改善斑块形成。因此，细胞外Zn 2+水平的调节剂已被认为是通过预防老年斑的形成而潜在地有效治疗AD的药物。该项目将确定自然发生的Zn 2+信号何时驱动Ab寡聚化，这是老年斑和许多其他AD症状形成的基础。随着获得的洞察力，应该可以设计专门针对可能导致AD的病理性Zn2+信号的药物，同时最小限度地干扰正常大脑功能所需的Zn2+信号。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common type of dementia and it is the major cause of dementia affecting the elderly, accounting for 60-80% of all dementia cases. Although the mechanisms of AD are not well understood it is known that AD is a proteopathy leading to intracellular accumulation of tau protein and extracellular amyloid 2 (Ab) aggregates (senile plaques). When synaptically released Ab oligomerizes, and eventually aggregates, it leads to neuronal damage and an inflammatory response in glial cells through either direct toxicity and/or production of reactive oxygen species. It is not very well understood what triggers Ab oligomerization and aggregation in AD. One of the theories is the "metal hypothesis of AD" which posits that extracellular metal ions, especially synaptically excreted Zn2+, instigate the Ab oligomerization and aggregation leading to the formation of senile plaques. Despite much progress supporting the metal hypothesis of AD, this idea remains a debated issue, partly because there is no adequate information on the binding and aggregation events at the timescale of synaptically released Zn2+. Clearly, a better understanding of the temporal patterns of Zn2+ signals is required together with a thorough insight into how these signals might trigger pathological oligomerization of Ab. This becomes especially important if there is a specific Zn2+ concentration window or a specific temporal pattern of the Zn2+ signals that would allow the formation of soluble oligomers, which are thought to be the most toxic, but not their aggregation into plaques which possibly protect against toxicity. In spite of enormous efforts in research, presently there is no treatment available for AD. It has been shown that chelation of Zn2+ can ameliorate plaque formation in animal models of AD, and accordingly, Zn2+ chelators have been suggested as a potential treatment for AD by preventing Ab oligomerization. Modulators of Zn2+ buffering pathways also hold promise for drug development in AD. Understanding the biophysical processes underlying the oligomerization of Ab as well as insights into the temporal patterns of Zn2+ signals that drive oligomerization are essential for developing such drugs. We have devised an ultra-fast in vitro kinetic measurement technique of Ca2+- binding to calcium binding proteins following flash photolysis of caged Ca2+ and created a compartmental kinetic model that resolved for the first time the cooperative nature of Ca2+ binding. This technique will be adapted to measure the fast Zn2+ binding kinetics by uncaging Zn2+ in the presence of Ab while optically monitoring Zn2+ binding dynamics. The same will be done to measure the Zn2+ binding dynamics of the Zn2+-binding protein metallothionein-3 (MT-3) which is enriched at Zn2+ releasing synapses. Our approach allows the unprecedented possibility to simulate extracellular Zn2+ signals as they are thought to occur at synapses. Studying the dynamics of Zn2+ binding to Ab and to other proteins under conditions resembling those at synapses will provide a first direct distinction between the physiological and pathological roles of Zn2+ in Ab oligomerization, resulting in the potential development of new and highly effective drugs for AD. PUBLIC HEALTH RELEVANCE: It has been shown that reducing zinc (Zn2+) release in the brain can ameliorate plaque formation in animal models of Alzheimer's disease (AD). Accordingly, modulators of extracellular Zn2+ levels have been suggested as potentially an effective treatment for AD by preventing the formation of senile plaques. This project will determine when naturally occurring Zn2+ signals drive Ab oligomerizatoin which underlies formation of senile plaques and many other the symptoms of AD. With the acquired insight it should be possible to design drugs that specifically target pathological Zn2+ signals that may cause AD while minimally disturbing the Zn2+ signals necessary for normal brain functioning.