Untangling Amyloid Plaques With Proteases

描述（由申请人提供）：阿尔茨海默病（AD）的病理标志之一是老年斑，主要由A？肽。一个？1-42，淀粉样前体蛋白（APP）的42个氨基酸的肽片段，具有在斑块内聚集成淀粉样纤维的显著倾向。一个？聚集似乎是浓度依赖性的，因此，调节细胞外（或间质液，ISF）A？水平可能对淀粉样斑块形成很重要。在正常生理条件下，A？存在于脑细胞外液（ISF和CSF）中。这个稳态水平的A？是通过生产和清除之间的平衡来维持的。而导致家族性AD的基因突变指向A？过量生产作为疾病的原因，它已经提出，更普遍的散发形式的AD可能是由受损的A？间隙越来越多的蛋白酶被称为降解可溶性A？(sA?)在体外，和抑制或删除这些蛋白酶在体内的一些导致增加稳态水平的脑A？在小鼠中，包括脑啡肽酶（NEP）、胰岛素降解酶（IDE）、内皮素转换酶（ECE）。我们最近发表的细胞外蛋白酶，基质金属蛋白酶-9（MMP-9），能够降解sA？除了纤维A？(fA?)体外和原位致密淀粉样斑块。降低fA的能力？是MMP-9的一个重要特征，这是大多数其他A？降解蛋白酶。MMP-9免疫反应性见于老年APP/PS1小鼠淀粉样斑块周围的反应性星形胶质细胞。此外，我们最近发现APP/PS1小鼠中mmp 9基因缺失导致淀粉样斑块负荷增加。在这项拨款申请中，我们将测试MMP-9直接降解细胞外A？的假设，从而减弱APP/PS1小鼠中的斑块发病机制。此外，我们将检验抑制MMP-9的内源性抑制剂TIMP-1（即解除MMP-9的抑制）将进一步减弱斑块发病机制的假设。如果是真的，TIMP-1可能被确定为治疗AD的潜在治疗靶点。在目的1，我们将确定是否MMP-9活性减弱淀粉样斑块的积累直接降解A？APP/PS1小鼠。在目标2中，我们将确定MMP-9是否直接分解代谢脑细胞外A？，在APP/PS1中使用体内微透析。在目标3中，我们将确定MMP-9是否调节使用活体2-光子显微镜成像的APP/PS1小鼠中预先存在的致密斑块的生长/消退。在目标4中，我们将确定TIMP-1是否抑制A2的MMP降解，加速淀粉样斑块的形成。如果是真的，TIMP-1可能被确定为治疗AD的潜在治疗靶点。公共卫生相关性：这项R 01资助申请旨在研究蛋白酶MMP-9及其内源性抑制剂TIMP-1在阿尔茨海默病小鼠模型中调节淀粉样斑块生长的作用。该研究的中心假设是，MMP-9由淀粉样斑块周围的星形胶质细胞分泌，通过降解可溶性和纤维状A？以及构成斑块的淀粉样纤维。如果该假设是正确的，则可以识别用于降解斑块的干预的潜在靶点。

DESCRIPTION (provided by applicant): One of the pathological hallmarks of Alzheimer's Disease (AD) is the senile plaque, principally composed of the A? peptide. A?1-42, the 42 amino-acid peptide fragment of the amyloid precursor protein (APP), has a striking propensity to aggregate into amyloid fibrils within plaques. A? aggregation appears to be concentration-dependent; thus, mechanisms that regulate extracellular (or interstitial fluid, ISF) A? levels may be important for amyloid plaque formation. Under normal physiological conditions, A? exists in brain extracellular fluids (ISF and CSF). This steady-state level of A? is maintained by a balance between production and clearance. While genetic mutations that lead to familial AD point to A? over-production as a cause for disease, it has been suggested that the much more prevalent sporadic form of AD may be caused by impaired A? clearance. A growing list of proteases are known to degrade soluble A? (sA?) in vitro, and inhibition or deletion of some of these proteases in vivo have resulted in increased steady-state levels of brain A? in mice, including neprilysin (NEP), insulin-degrading enzyme (IDE), endothelin-converting enzyme (ECE). We have recently published that the extracellular protease, matrix metalloproteinase-9 (MMP-9), is capable of degrading sA? in addition to fibrillar A? (fA?) in vitro and compact amyloid plaques in situ. The ability to degrade fA? is an important characteristic of MMP-9 which is lacking in most other A?-degrading proteases. MMP-9 immunoreactivity is seen in reactive astrocytes surrounding amyloid plaques of aged APP/PS1 mice. Furthermore, we have recently found that gene deletion of mmp9 in APP/PS1 mice resulted in an increase in amyloid plaque load. In this grant application, we will test the hypothesis that MMP-9 directly degrades extracellular A?, thus attenuating plaque pathogenesis in APP/PS1 mice. Furthermore, we will test the hypothesis that inhibiting TIMP-1, an endogenous inhibitor of MMP-9 (i.e. disinhibiting MMP-9), will further attenuate plaque pathogenesis. If true, TIMP-1 may be identified as a potential therapeutic target for the treatment of AD. In Aim 1, we will determine if MMP-9 activity attenuates amyloid plaque accumulation by directly degrading A? in APP/PS1 mice. In Aim 2, we will determine if MMP-9 directly catabolizes brain extracellular A?, using in vivo microdialysis in APP/PS1. In Aim 3, we will determine if MMP-9 regulates the growth/regression of pre- existing compact plaques in APP/PS1 mice imaged using intravital 2-photon microscopy. In Aim 4, we will determine if TIMP-1 inhibits MMP degradation of A2, accelerating amyloid plaque formation. If true, TIMP-1 may be identified as a potential therapeutic target for the treatment of AD. PUBLIC HEALTH RELEVANCE: This R01 grant application proposes to study the role of the protease MMP-9 and its endogenous inhibitor, TIMP-1 in regulating amyloid plaque growth in a mouse model of Alzheimer's disease. The central hypothesis of the grant is that MMP-9, secreted by astrocytes surrounding amyloid plaques, regulates amyloid plaque growth/regression by degrading soluble and fibrillar A? and amyloid fibrils of which plaques are comprised. If the hypothesis is true, potential targets for intervention to degrade plaques may be identified.