CCN1-induced Senescence in the Cerebral Vasculature

项目摘要 血管对认知障碍和痴呆（VCID）的贡献包括许多不同的疾病 通过继发于脑血管病理学的认知缺陷来识别。脑内皮衰老 已被提出作为一种机制，可以驱动血脑屏障破坏（BBB），这之前的VCID。 然而，驱动内皮细胞衰老的机制尚未完全了解。 鸟嘌呤四链体（G4）是DNA中富含G的序列中形成的非典型二级结构， 核糖核酸重要的是，G4稳定促进细胞衰老。特别地，mRNA中G4 s的稳定化 （rG 4s）使核糖体停滞在延伸阶段并阻断翻译。该机制由rG 4- 结合蛋白脆性X智力低下蛋白（FMRP）与肿瘤抑制因子的rG 4结合， 参与衰老的信号通路的组成部分。FMRP缺陷与以下因素有关： 不同组织的衰老。我们发现，在培养的脑内皮细胞（CEC）中过表达FMRP， 减轻DNA损伤，这是细胞衰老的重要原因。 我们发现，G4稳定促进内皮细胞衰老，并增加富含半胱氨酸的水平。 血管生成诱导物61（Cyr 61/CCN 1），由脑中的内皮细胞分泌的基质细胞蛋白。CCN 1是 被认为是衰老相关分泌表型（SASP）的组成部分。在成纤维细胞中，CCN 1 通过与整合素α6β1结合诱导衰老。 在我们的研究中，我们将使用双侧颈动脉狭窄（BCAS）诱导慢性脑灌注不足， 小鼠VCID模型。我们发现BCAS小鼠的G4 s、CCN 1和衰老水平升高 标记。 我们假设，在小鼠中，与G4稳定相关的内皮衰老可以减轻， 从而防止BBB破坏和认知缺陷。为了验证我们的假设，我们提出了两个目标： 在目标1中，我们将用编码突变形式的载体选择性地靶向年轻小鼠的脑内皮细胞， CCN 1不能与α6β1结合。我们将测量运动和认知功能，以及BBB渗透性， 大脑的衰老 在目标2中，我们将确定上调FMRP是否减轻运动和认知缺陷，BBB损伤， 老年BCAS小鼠脑血管系统中的衰老。 总的来说，这项研究提出了两种独立的策略，以减轻由 脑血管系统的衰老：阻碍脑内皮中CCN 1-α6β 1结合;以及 特异性上调小鼠脑血管中的FMPR。

Project Summary Vascular contributions to cognitive impairment and dementia (VCID) includes a number of different disorders identified by cognitive deficits secondary to cerebrovascular pathology. Senescence in the cerebral endothelium has been proposed as a mechanism that can drive blood brain barrier disruption (BBB), which precedes VCID. However, the mechanisms that drive endothelial cells to senescence are not fully understood. Guanine-quadruplex (G4) is a non-canonical secondary structure formed in G-rich sequences in DNA and RNA. Importantly, G4 stabilization promotes cellular senescence. Particularly, stabilization of G4s in mRNA (rG4s) stalls ribosomes in the elongation stage and blocks translation. This mechanism is promoted by rG4- binding proteins. Fragile X mental retardation protein (FMRP) binds to the rG4 of tumor suppressors and components of signaling pathways involved in senescence. FMRP deficiency has been associated with senescence in different tissues. We found that overexpressing FMRP in cultured cerebral endothelial cells (CEC) mitigated DNA damage, an important cause of cell senescence. We found that G4 stabilization promoted endothelial senescence and enhanced the levels of Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1), a matricellular protein secreted by endothelial cells in the brain. CCN1 is considered a component of the senescence-associated secretory phenotype (SASP). In fibroblasts, CCN1 induces senescence by binding to integrin α6β1. In our study, we will use bilateral carotid artery stenosis (BCAS) that induces chronic cerebral hypoperfusion and models VCID in mice. We found that BCAS mice showed enhanced levels of G4s, CCN1 and senescence markers. We hypothesize that the endothelial senescence associated with G4 stabilization could be mitigated in mice and hence prevent BBB disruption and cognitive deficits. To test our hypothesis, we propose two aims: In Aim 1, we will selectively target the brain endothelium of young mice with a vector encoding a mutant form of CCN1 that cannot bind to α6β1. We will measure motor and cognitive function, and BBB permeability, and senescence in the brain. In Aim 2, we will determine if upregulating FMRP mitigates motor and cognitive deficits, BBB impairment, and senescence in the cerebral vasculature of aged BCAS mice. Overall, this study proposes two independent strategies to mitigate VCID-associated phenotypes induced by senescence in the cerebrovasculature: impeding the CCN1-α6β1binding in the cerebral endothelium; and upregulating FMPR specifically in the brain vasculature of mice.