SRF/MYOCD: new targets in Alzheimer's neurovasculature

描述(申请人提供)：身体中没有一个器官像大脑那样依赖持续的血液供应。如果大脑血流(CBF)中断，大脑功能在几秒钟内停止，其细胞成分在几分钟内就会受到不可逆转的损害。在老年患者中，越来越多的证据表明血管危险因素和脑血流量降低在阿尔茨海默病(AD)的发展中起着重要作用，甚至可能触发痴呆的发生。我们发现AD患者软脑膜脑动脉的血管平滑肌细胞(VSMC)中存在两种相互作用的转录因子，它们对CBF具有重要的调节作用。这些因子，血清反应因子(SRF)和肌钙蛋白(MYOCD)，是与VSMC功能相关的收缩基因程序的有力诱导者。因此，AD-VSMC表现出几种收缩蛋白的表达增加，这些蛋白影响这些细胞的过度收缩状态。基于这些新的发现，我们假设大脑小动脉VSMC中SRF/MYOCD活性升高导致过度收缩表型，从而导致AD患者CBF减少和神经血管解偶联。在这个第一阶段的应用中，我们努力进一步验证这些发现，并优化依赖于SRF/MYOCD的基因转录的高通量筛选方法。为此，我们描绘了三个里程碑，我们相信我们可以实现这三个里程碑，作为我们在本快速通道提案中同时提交的第二阶段申请的关键基础。里程碑1将评估MYOCD在年龄匹配的对照组VSMC中诱导AD-VSMC过度收缩表型的能力。里程碑2将确定AD-VSMC过度收缩表型是否可以通过RNAi到SRF来正常化。在里程碑3中，我们将优化最近开发的大规模人类细胞荧光素酶分析，为第二阶段描述的研究做准备。这一发现可能是已知的脑低灌流和AD认知功能下降的基础，这一发现代表了治疗干预的新范式。这里要解决的里程碑将为本快速通道应用程序第二阶段中描述的AIMS提供一个重要的平台，其中将筛选一个多样化的小分子化合物库，以寻找专门破坏SRF/MYOCD相互作用的有机化合物，从而使VSMC过度收缩正常化，进而使AD患者的脑灌流不足和调节失调的CBF正常化。

DESCRIPTION (provided by applicant): There is no organ in the body as dependent on a continuous supply of blood as the brain. If cerebral blood flow (CBF) is interrupted, brain function ceases within seconds and irreversible damage to its cellular constituents ensues within minutes. In elderly patients, increasing evidence suggests that vascular risk factors and lowered CBF have a major role in the development of Alzheimer's disease (AD) and possibly even trigger the onset of dementia. We have discovered an over-abundance of two interactive transcription factors in vascular smooth muscle cells (VSMC) from pial cerebral arteries of patients with AD which critically regulate CBF. The factors, serum response factor (SRF) and myocardin (MYOCD), are potent inducers of a contractile gene program linked to VSMC function. Consequently, AD-VSMC display elevated expression of several contractile proteins that effect a hypercontractile state in these cells. Based on these novel findings, we hypothesize that elevated SRF/MYOCD activity in VSMC of small cerebral arteries leads to a hypercontractile phenotype, which contributes to CBF reductions and neurovascular uncoupling in AD. In this Phase I application, we endeavor to further validate these findings and optimize a high-throughput screening assay for SRF/MYOCD-dependent gene transcription. To this end, we have delineated three milestones we are confident we can achieve as a critical foundation for a Phase II application that we have simultaneously submitted in this Fast Track proposal. Milestone 1 will assess the ability of MYOCD to elicit an AD-VSMC hypercontractile phenotype in age-matched control VSMC. Milestone 2 will ascertain whether the AD-VSMC hypercontractile phenotype can be normalized with an RNAi to SRF. In Milestone 3, we will optimize a recently developed large-scale luciferase assay for human cells in preparation for studies described in Phase II. The finding that two interactive transcription factors may underlie the known brain hypoperfusion and cognitive decline in AD represents a new paradigm for therapeutic interventions. The milestones to be addressed here will provide a vital platform for aims delineated in Phase II of this Fast Track Application in which a diverse small molecule library of compounds is to be screened for organic chemicals that specifically disrupt the SRF/MYOCD interaction thereby normalizing VSMC hypercontraction and, by extension, brain hypoperfusion and dysregulated CBF in AD.