CCN1-induced Senescence in the Cerebral Vasculature

项目摘要 血管对认知损害和痴呆的贡献(VCID)包括许多不同的疾病 由继发于脑血管病理的认知缺陷来识别。脑内皮细胞的衰老 已被提出作为一种机制，可以推动血脑屏障破坏(BBB)，这是VCID之前的。 然而，驱动内皮细胞衰老的机制尚不完全清楚。 鸟嘌呤四链(G4)是DNA和DNA中富含G的序列中形成的非规范二级结构 核糖核酸。重要的是，G4的稳定促进了细胞衰老。尤其是G4S在mRNA中的稳定性 (RG4s)在伸长阶段阻止核糖体，并阻止翻译。这一机制是由Rg4- 结合蛋白。脆性X智力低下蛋白(FMRP)与肿瘤抑制因子Rg4结合 参与衰老的信号通路的组成部分。FMRP缺乏症与 不同组织中的衰老。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的稳定促进了内皮细胞的衰老，并提高了富含半胱氨酸的水平 血管生成诱导物61(Cyr61/CCN1)是由脑内皮细胞分泌的一种基质细胞蛋白。CCN1是 被认为是衰老相关分泌表型(SASP)的一个组成部分。在成纤维细胞中，CCN1 通过与整合素α6β1结合而诱导衰老。 在我们的研究中，我们将使用双侧颈动脉狭窄(BCAS)，它会导致慢性脑低灌注和 用小鼠建立VCID模型。我们发现BCAS小鼠表现出G4S、CCN1水平升高和衰老 记号笔。 我们假设，与G4稳定相关的内皮衰老可以在小鼠和 从而防止血脑屏障紊乱和认知缺陷。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幼鼠的脑内皮细胞 不能与α6β1结合的CN1。我们将测量运动和认知功能，以及血脑屏障通透性，以及 大脑中的衰老。 在目标2中，我们将确定上调FMRP是否可以缓解运动和认知障碍、血脑屏障损害，以及 老龄BCAS小鼠脑血管系统的衰老。 总体而言，这项研究提出了两种独立的策略来缓解由VCID引起的表型 脑血管衰老：阻碍脑血管内皮细胞CCN1-α6β1结合； 上调FMPR在小鼠脑血管中的特异性。

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.