Supplement: Stalled capillary flow affects protein clearance by modulating interstitial fluid flow

项目摘要 阿尔茨海默病(AD)患者和小鼠模型的脑血流量(CBF)减少了~20%，但 目前对引起这种低灌注率的机制或潜在的治疗方法仍知之甚少。 拯救CBF赤字的好处。在之前的奖项下，慢性活体双光子激发荧光 显微镜下观察AD模型小鼠的脑血流量。而皮质小动脉未见血流中断 或观察到小静脉，在小鼠模型中发现~2%的皮质毛细血管血流停滞 AD，而野生型对照为~0.4%。这些毛细血管停滞出现在疾病进展的早期， 是由滞留的中性粒细胞引起的，对脑血流量有极大的影响，因为它们降低了血流速度 上游和下游船只。偶然发现抗中性粒细胞表面蛋白Ly6G的抗体 立即减少毛细血管停滞的发生率，从而挽救三分之二的CBF赤字，以及， 值得注意的是，在几个小时内改善了记忆功能。初步数据进一步将这一毛细管停滞与 活性氧(ROS)对细胞的损伤。在这场竞争性更新中，潜在的机制 阿尔茨海默病模型小鼠毛细血管内中性粒细胞滞留及改善脑血流量对AD相关性的影响 对病理学进行了探讨。首先，关于中性粒细胞在毛细血管中停止的机制有三种不同的假说。 节段检测：周细胞对毛细血管的局限性收缩，阻止中性粒细胞通过；结合 中性粒细胞对内皮细胞的炎性黏附分子增加；或中性粒细胞的结合 基底膜和黏附分子暴露在内皮细胞之间扩大的间隙中。第二, 利用细胞类型特异性确定导致中性粒细胞停滞的ROS的分子和细胞来源 ROS产生酶的敲除。第三，长期抢救CBF对淀粉样蛋白沉积的影响- AD病理和神经病理学的驱动因素β(Aβ)将被量化。对这项研究至关重要的是最近- 开发了敲打AD的小鼠模型，可以更好地捕捉CBF减少对表达的反馈 淀粉样前体蛋白(APP)的一部分，它被切割产生Aβ。最后，尖端的三光子激发 使用荧光显微镜对海马区进行成像，以确定毛细血管停滞的作用 大脑中最早表现为AD病理的区域之一的CBF缺陷。大脑的假说 AD的低灌流是由于毛细血管中性粒细胞停滞所致，这一发现既是新的，也得到了有力的支持 在上一次的奖励下。在这次竞争性更新中提出的工作将揭示 在中性粒细胞受阻的基础上，这可能表明改善脑血流的治疗目标将是 补充抗淀粉样蛋白等治疗阿尔茨海默病的方法。

Project Summary Cerebral blood flow (CBF) is reduced in Alzheimer’s disease (AD) patients and mouse models by ~20%, but there remains a limited understanding of the mechanisms causing this hypoperfusion or the potential therapeutic benefit of rescuing CBF deficits. Under the previous award, chronic in vivo two-photon excited fluorescence microscopy was used to study CBF in mouse models of AD. While no blood flow disruption in cortical arterioles or venules was observed, blood flow was found to be stalled in ~2% of cortical capillaries in mouse models of AD, as compared to ~0.4% in wild type controls. These capillary stalls appeared early in disease progression, were caused by arrested neutrophils, and had outsized impacts on CBF because they decreased flow speed in up- and down-stream vessels. Antibodies against the neutrophil surface protein Ly6G were serendipitously found to reduce the incidence of capillary stalls immediately, leading to a rescue of two-thirds of the CBF deficit, and, remarkably, to improved memory function within hours. Preliminary data further link this capillary stalling to cellular damage from reactive oxygen species (ROS). In this competitive renewal, the mechanisms underlying neutrophil arrest in capillaries in mouse models of AD and the consequences of improving CBF on AD-related pathology are explored. First, three different hypotheses about the mechanism of neutrophil arrest in capillary segments are tested: a focal constriction of the capillary by a pericyte that prevents neutrophil passage; binding of the neutrophil to increased inflammatory adhesion molecules on endothelial cells; or binding of the neutrophil to basement membrane and adhesion molecules exposed at widened gaps between endothelial cells. Second, the molecular and cellular origin of the ROS that leads to neutrophil arrest is determined using cell type-specific knockouts of ROS producing enzymes. Third, the impact of long-term CBF rescue on the deposition of amyloid- beta (Aβ), a driver of AD pathology, and on neuropathology will be quantified. Critical for this study are recently- developed knock-in mouse models of AD that may better capture the feedback of CBF reductions on expression of amyloid precursor protein (APP), which is cleaved to produce Aβ. Finally, cutting-edge three-photon excited fluorescence microscopy is used to enable imaging of the hippocampus to determine the role of capillary stalling in CBF deficits in one of the first regions of the brain that exhibits AD pathology. The hypothesis that brain hypoperfusion in AD is due to neutrophil arrest in capillaries is both novel and strongly supported by the findings under the previous award. The work proposed in this competitive renewal would uncover the mechanisms underlying that neutrophil arrest, which could suggest therapeutic targets to improve CBF that would be complementary to anti-amyloid and other treatment approaches for AD.