Extracellular Zinc Buffering and A-beta Oligomerization

描述（申请人提供）：阿尔茨海默病（Alzheimer's disease， AD）是最常见的痴呆类型，是影响老年人痴呆的主要原因，占所有痴呆病例的60-80%。虽然AD的机制尚不清楚，但已知AD是一种蛋白质病变，导致细胞内tau蛋白和细胞外淀粉样蛋白2 （Ab）聚集体（老年斑）积聚。当突触释放的Ab寡聚并最终聚集时，它通过直接毒性和/或产生活性氧导致神经细胞损伤和炎症反应。目前尚不清楚是什么触发了AD中Ab寡聚和聚集。其中一种理论是“AD的金属假说”，该假说认为细胞外金属离子，特别是突触分泌的Zn2+，刺激Ab寡聚和聚集，导致老年斑的形成。尽管有许多进展支持AD的金属假说，但这一观点仍然存在争议，部分原因是在突触释放Zn2+的时间尺度上没有足够的结合和聚集事件信息。显然，需要更好地理解Zn2+信号的时间模式，并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，这将变得尤其重要，这将允许形成可溶性低聚物，这被认为是最具毒性的，但不是它们聚集成斑块，可能防止毒性。尽管在研究上付出了巨大的努力，但目前还没有治疗阿尔茨海默病的方法。研究表明，Zn2+螯合可以改善AD动物模型中的斑块形成，因此，Zn2+螯合剂被认为是一种通过防止Ab寡聚而治疗AD的潜在药物。Zn2+缓冲途径的调节剂也有望用于阿尔茨海默病的药物开发。了解Ab寡聚化背后的生物物理过程，以及了解驱动寡聚化的Zn2+信号的时间模式，对于开发此类药物至关重要。我们设计了一种超快速的体外动力学测量技术，测量Ca2+与钙结合蛋白的结合，并创建了一个室室动力学模型，首次解决了Ca2+结合的协同性质。该技术将适用于测量Zn2+的快速结合动力学，通过在Ab存在下释放Zn2+，同时光学监测Zn2+的结合动力学。同样的方法将用于测量富集在Zn2+释放突触上的Zn2+结合蛋白金属硫蛋白-3 （MT-3）的Zn2+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的Zn2+信号，因为它们被认为发生在突触。研究Zn2+在类似突触条件下与Ab和其他蛋白质结合的动力学，将首次直接区分Zn2+在Ab寡聚过程中的生理和病理作用，从而为开发新的高效阿尔茨海默病药物提供可能。

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.