Extracellular Zinc Buffering and A-beta Oligomerization

描述(申请人提供)：阿尔茨海默病(AD)是最常见的痴呆症类型，也是影响老年人痴呆症的主要原因，占所有痴呆症病例的60%至80%。虽然AD的发病机制尚不清楚，但已知AD是一种蛋白质病，导致tau蛋白和细胞外淀粉样蛋白2(Ab)聚集体(老年斑)积聚。当突触释放的抗体寡聚并最终聚集时，它通过直接毒性和/或产生活性氧物种导致神经元损伤和胶质细胞的炎症反应。在AD中，是什么触发了抗体的寡聚和聚集，目前还不是很清楚。其中一种学说是“AD的金属假说”，该假说认为细胞外金属离子，特别是突触排出的锌离子，引起抗体寡聚和聚集，导致老年斑的形成。尽管AD的金属假说得到了很大的支持，但这一观点仍然是一个有争议的问题，部分原因是关于突触释放的锌离子的时间尺度上的结合和聚集事件没有足够的信息。显然，需要更好地了解锌离子信号的时间模式，并彻底了解这些信号如何触发抗体的病理性寡聚化。如果存在特定的锌离子浓度窗口或锌离子信号的特定时间模式，允许形成被认为毒性最大的可溶低聚物，但不能聚集成可能保护其免受毒性的斑块，这一点就变得特别重要。尽管在研究上付出了巨大的努力，但目前尚无治疗AD的方法。已有研究表明，在AD动物模型中，锌离子的螯合作用可以改善斑块的形成，因此，锌离子螯合剂被认为是一种通过阻止抗体齐聚来治疗AD的潜在药物。锌离子缓冲通路的调节器也为AD的药物开发提供了希望。了解抗体寡聚的生物物理过程以及对驱动寡聚的锌离子信号的时间模式的洞察对于开发此类药物至关重要。我们设计了一种笼状Ca~(2+)闪光光解后与钙结合蛋白结合的超快速体外动力学测量技术，并建立了一个隔室动力学模型，首次解决了Ca~(2+)结合的协同性质。该技术在光学监测锌离子结合动力学的同时，通过在抗体存在下去除锌离子来测量快速的锌离子结合动力学。同样的工作也将被用来测量锌离子结合蛋白金属硫蛋白-3(MT-3)的锌离子结合动力学，金属硫蛋白-3是富含在锌离子释放突触上的。我们的方法允许前所未有的可能性来模拟细胞外锌离子信号，因为它们被认为发生在突触上。在与突触相似的条件下，研究锌离子与抗体和其他蛋白质结合的动力学，将首次直接区分锌离子在抗体齐聚中的生理和病理作用，从而可能开发出治疗AD的新型高效药物。 与公共健康相关：已有研究表明，减少锌在大脑中的释放可以改善阿尔茨海默病(AD)动物模型的斑块形成。因此，细胞外锌离子水平的调节剂被认为是一种潜在的有效治疗阿尔茨海默病的方法，它可以防止老年斑的形成。该项目将确定自然产生的锌离子信号何时驱动抗体寡聚化，而抗体寡聚化是老年斑和许多其他AD症状形成的基础。凭借所获得的洞察力，应该可以设计出专门针对可能导致阿尔茨海默病的病理性锌离子信号的药物，同时最大限度地干扰正常大脑功能所需的锌离子信号。

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.