Role of Cortical Microvascular Lesions in Amyloid-Beta Accumulation

描述(申请人提供)：临床证据表明阿尔茨海默病(AD)和脑微血管功能障碍之间有很强的联系，但尚不清楚它们是否独立地导致痴呆症，或者AD的病理是否触发微血管疾病，或者反之亦然。在阿尔茨海默病中，淀粉样多肽聚集形成神经毒性低聚物，最终以淀粉样斑块的形式积累。A？的聚集依赖于浓度，因此增加A？的产量或减少A？的清除可能是AD的触发因素。从大脑中清除A？的一个主要途径是通过血管系统，这表明微血管病变可能会干扰A？的清除。此外，血管损伤会导致活性氧的增加和炎症，这与A值的增加有关。我们最近开发了光学方法来在啮齿动物皮质中个别的、特定靶向的微血管中产生损伤。我们现在建议检验这一假说，即微血管病变启动或加速A积聚和淀粉样斑块的形成。为了进行这些研究，将使用飞秒激光照射来损伤特定目标血管，导致微出血和/或阻止血液流动的凝块的形成。我们将在表达淀粉样前体蛋白(Mo/HuAPP695swe)和突变的早老素1(PS1-dE9)的转基因AD小鼠以及年龄匹配的对照组中，在单个皮质穿透小动脉和毛细血管中产生微血管凝块和出血。体内将通过全身注射甲氧基-X04来标记淀粉样斑块，甲氧基-X04是刚果红的衍生物。病变的位置将在几天内每天用双光子激发荧光显微镜进行成像，以确定闭塞或出血的持续性，以及先前存在的和新的淀粉样斑块的存在。将使用硫代黄素-S和A？抗体进行死后标记，以进一步阐明微血管损伤对A？蓄积和淀粉样斑块形成的影响。在目标1中，我们测试微血管凝块和出血是否会引发血管树中不同位置的病变快速形成淀粉样斑块，以及相对于病变位置形成淀粉样斑块的位置。在目标2中，我们确定微血管病变后淀粉样斑块形成所需的时间，以及由微血管病变引发的淀粉样斑块是否随着时间的推移而稳定。在最终目标中，我们调查了动物的年龄，或先前存在的斑块负荷，如何影响通过微血管病变种植淀粉样斑块。在初步工作中，我们发现微血管凝块在一天内导致新的淀粉样斑块的形成(3只动物中有3/3的凝块，见图7)。在凝结的微血管和附近的实质组织上都形成了新的斑块。附近直径相似但未受损的血管未显示新的淀粉样斑块。这些初步结果表明，单个微血管闭塞导致的血流量严重下降可以触发淀粉样斑块的形成。这些数据表明，微血管凝块可能在阿尔茨海默病的发病机制中发挥重要作用。公共卫生相关性：阿尔茨海默病(AD)是导致老年人痴呆症的主要原因，尽管最近的一些治疗方法略微减缓了疾病的进展，但没有治愈方法。临床研究表明，血管健康是影响AD患者病情严重程度的重要因素，然而血管功能障碍与AD之间的联系机制尚不清楚。这项工作使用独特的光学和生物学工具的组合，直接研究脑内微血管凝块和出血如何影响AD的发展，我们的初步结果表明，微血管功能障碍可能在AD的发病中发挥作用，这表明成功的AD预防将取决于血管疾病的治疗。

DESCRIPTION (provided by applicant): Clinical evidence suggests a strong link between Alzheimer's disease (AD) and cerebral microvascular dysfunction, but it remains unclear whether they contribute independently to dementia, or if AD pathology triggers microvascular disease, or vice versa. In AD, amyloid-¿ (A¿) peptides aggregate to form neurotoxic oligomers and eventually accumulate as amyloid plaques. Aggregation of A¿ depends on concentration, so events that increase production or decrease clearance of A¿ could be triggers for AD. A major pathway for the removal of A¿ from the brain is through the vasculature, suggesting that microvascular lesions could interfere with A¿ clearance. In addition, vascular lesions can lead to increased reactive oxygen species and to inflammation, which are linked to A¿ increase. We recently developed optical methods to create lesions in individual, specifically-targeted microvessels in rodent cortex. We now propose to test the hypothesis that microvascular lesions initiate or accelerate A¿ accumulation and amyloid plaque formation. To perform these studies, femtosecond laser irradiation will be used to injure a specifically targeted blood vessel, causing the formation of a microhemorrhage and/or of a clot that stops blood flow. We will produce microvascular clots and hemorrhages in individual cortical penetrating arterioles and capillaries in transgenic AD mice that express amyloid precursor protein (Mo/HuAPP695swe) and mutant presenilin1 (PS1-dE9), as well as in age-matched controls. Amyloid plaques will be labeled in vivo with systemic injections of methoxy-X04, a Congo-red derivative. The location of the lesions will be imaged daily over several days with two-photon excited fluorescence microscopy to determine the persistence of the occlusion or hemorrhage and the presence of previously existing and new amyloid plaques. Post-mortem labeling with thioflavin-S and A¿ antibodies will be used to further elucidate the impact of the microvascular lesion on A¿ accumulation and amyloid plaque formation. In Aim 1, we test whether microvascular clots and hemorrhages trigger rapid amyloid plaque formation for lesions at different locations in the vascular tree, and where amyloid plaques form relative to the lesion site. In Aim 2, we determine the time required for amyloid plaques to form following microvascular lesions and whether amyloid plaques that are initiated by microvascular lesions are stable over time. In the final Aim, we investigate how the age of the animal, or the pre-existing plaque burden, influences the seeding of amyloid plaques by microvascular lesions. In preliminary work, we found that microvascular clots led to the formation of new amyloid plaques within one day (3/3 clots in 3 animals, see Fig. 7). New plaques were formed both on the clotted microvessel and in the nearby parenchymal tissue. Nearby vessels of similar diameter that were not lesioned showed no new amyloid plaques. These initial results indicate that a severe decrease in blood flow resulting from the occlusion of a single microvessel can trigger amyloid plaque formation. These data suggest that microvascular clots could play an important role in Alzheimer's disease pathogenesis. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is the leading cause of dementia in the elderly and although some recent treatments modestly slow progression of disease, there is no cure. Clinical research has shown that vascular health is an important factor in the severity of AD in patients, yet the mechanisms that link vascular dysfunction and AD remain unclear. This work uses a unique combination of optical and biological tools to directly study how microvascular clots and hemorrhages in the brain affect the development of AD and our preliminary results show that microvascular dysfunction may play a role in initiating AD, suggesting that successful AD prevention will depend on treatment of vascular disease.