Untangling Amyloid Plaques With Proteases

描述（由申请人提供）：老年斑是阿尔茨海默病（AD）的病理标志之一，主要由A？肽。一个吗?1-42是淀粉样前体蛋白（APP）的42个氨基酸肽片段，在斑块内具有显著的聚集成淀粉样原纤维的倾向。一个吗?聚集似乎与浓度有关；因此，调节细胞外液（或间质液，ISF）的机制水平可能对淀粉样斑块的形成很重要。在正常生理条件下，A？存在于脑细胞外液（ISF和CSF）中。A的稳态水平？是由生产和清理之间的平衡来维持的。而导致家族性阿尔茨海默病的基因突变指向A？作为疾病的原因，已经提出更普遍的散发形式的AD可能是由受损的a ？间隙。越来越多的蛋白酶可以降解可溶性A？（sA?），以及体内抑制或删除其中一些蛋白酶导致大脑A?的稳态水平增加。小鼠，包括NEP，胰岛素降解酶（IDE），内皮素转换酶（ECE）。我们最近发表了细胞外蛋白酶基质金属蛋白酶-9 （MMP-9）能够降解sA？除了纤原纤维A？（fA?）和原位致密淀粉样斑块。降解fA的能力？是MMP-9的一个重要特征，而大多数其他A？有辱人格的蛋白酶。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mmp9基因缺失导致淀粉样斑块负荷增加。在本次拨款申请中，我们将验证MMP-9直接降解细胞外A？，从而减弱APP/PS1小鼠斑块的发病机制。此外，我们将验证抑制内源性MMP-9抑制剂TIMP-1（即去抑制MMP-9）将进一步减弱斑块发病机制的假设。如果这是真的，TIMP-1可能被确定为治疗AD的潜在治疗靶点。在Aim 1中，我们将确定MMP-9活性是否通过直接降解A？APP/PS1小鼠。在Aim 2中，我们将确定MMP-9是否直接分解脑细胞外A？APP/PS1采用体内微透析。在Aim 3中，我们将确定MMP-9是否调节APP/PS1小鼠中预先存在的致密斑块的生长/消退，使用活体双光子显微镜成像。在Aim 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。如果这是真的，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R01拨款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。该基金的中心假设是，淀粉样斑块周围星形胶质细胞分泌的MMP-9，通过降解可溶性和纤维性A？以及构成斑块的淀粉样原纤维。如果这一假设是正确的，那么就可以确定干预以降解斑块的潜在目标。

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.