The role of pericytes in white matter disease

作为对RFA-NS-16-021的响应，以解决我们在生物机制方面的一些知识空白 常见的脑血管疾病和与年龄相关的白质病 在分子、细胞和脑电路水平，我们建议用新的方法研究周细胞在西医疾病中的作用。 特定于周细胞的动物模型和先进的分子、细胞和神经成像方法以及电路水平 分析。据估计，全世界高达45%的痴呆症完全或部分是由于与年龄相关的小痴呆症 脑血管疾病(SVD)。周细胞是嵌入在小血液壁中的血管壁细胞。 毛细血管、毛细血管前小动脉和毛细血管后小静脉。在大脑中，它们控制着钥匙 神经血管功能，如血脑屏障(BBB)完整性和脑血流量(CBF)。周细胞 变性导致血脑屏障破坏和血流动力学反应受损，见于神经科。 表现为SVD、WM疾病和认知障碍的疾病，如阿尔茨海默氏症、中风和CADASIL- 缺血性SVD和VCID最常见的遗传原因；然而，周细胞在病理生理学中的作用 SVD和WM病的发病机制在很大程度上还不清楚。根据我们在周细胞缺陷性血小板中获得的试验数据- 衍生生长因子受体-β(PDGFRβF7/F7)小鼠及其在TgNotch3R169C小鼠中表达的研究 NOTCH3 CADASIL在血管平滑肌细胞和周细胞中的突变，我们假设WM周细胞丢失 导致血脑屏障破坏和脑血流量减少，导致少突胶质细胞丢失，脱髓鞘，轴突损伤， 破坏中枢神经系统回路的连接和解体，从而导致功能缺陷和神经元丢失。 由于目前可用的周细胞缺陷的pdgfb/pdgfrβ系和TgNotch3R169C小鼠不是周细胞特异性的， 为了验证我们的假设，我们已经产生了新的周细胞特异性株系，如PDGFRβ-FLP、Cspg4-FSF-Creer、 IDTR小鼠可诱导周细胞消融，并将开发一种新的小鼠品系，可诱导表达 Notch3R169C突变仅见于周细胞。我们将使用i)尖端纵向动态对比度增强 局部血脑屏障完整性的磁共振成像(MRI)，脑血流的动态磁化率对比MRI， 弥散张量成像(DTI)和基于DTI的纤维束成像，用于结构/连通性变化和轨迹追踪 用于CNS电路级分析的基于连接学；ii)行为测试；以及iii)免疫组织学， 神经病理学、流式细胞术和电子显微镜组织分析。我们将确定 整体可诱导周细胞消融(20%-70%)(AIM 1)，局灶性可诱导周细胞丢失在前扣带回 皮质边缘电路(AIM 2)和周细胞特异性诱导Notch3R169C表达(AIM 3)对血脑屏障完整性的影响， CBF减少、WM完整性、中枢神经系统回路中断和功能缺陷(行为)。我们预计， 建议中的研究将有助于更好地理解WM病的机制基础 血管认知障碍和痴呆，并将建立周细胞作为西医疾病的新的关键靶点。

In response to the RFA-NS-16-021 to address some of the gaps in our knowledge of the biologic mechanisms of the commonly occurring cerebrovascular disease and age-related white matter (WM) disease at the molecular, cellular and brain circuit level, we propose to study the role of pericytes in WM disease using new pericyte-specific animal models and advanced molecular, cellular and neuroimaging methods, and circuit level analysis. Up to 45% of all dementias worldwide are estimated to be wholly or partly due to age-related small vessel disease (SVD) of the brain. Pericytes are vascular mural cells embedded in the wall of small blood vessels such as capillaries, pre-capillary arterioles, and post-capillary venules. In the brain, they control key neurovascular functions such as blood-brain barrier (BBB) integrity and cerebral blood flow (CBF). Pericyte degeneration leads to BBB breakdown and impaired hemodynamic responses, and is found in neurologic disorders exhibiting SVD, WM disease and cognitive impairment such as Alzheimer's, stroke and CADASIL - the most common genetic cause of ischemic SVD and VCID; yet, the role of pericytes in the pathophysiology of SVD and WM disease is largely not known. Based on our pilot data obtained in pericyte-deficient platelet- derived growth factor receptor-β (PdgfrβF7/F7) mice and published studies in TgNotch3R169C mice expressing Notch3 CADASIL mutant in vascular smooth muscle cells and pericytes, we hypothesize that WM pericyte loss leads to BBB breakdown and CBF reductions causing loss of oligodendrocytes, demyelination, axon damage, disrupted connectivity and disintegration of CNS circuits, which leads to functional deficits and neuron loss. Since currently available pericyte-deficient Pdgfb/Pdgfrβ lines and TgNotch3R169C mice are not pericyte specific, to test our hypothesis we have generated new pericyte-specific lines such as Pdgfrβ-Flp; Cspg4-FSF-CreER; iDTR mice with inducible pericyte ablation, and will develop a new mouse line with inducible expression of Notch3R169C mutation only in pericytes. We will use i) cutting-edge longitudinal dynamic contrast-enhanced magnetic resonance imaging (MRI) of regional BBB integrity, dynamic susceptibility contrast MRI of CBF, diffusion tensor imaging (DTI) and DTI-based tractography for structural/connectivity changes, and tract-tracing based connectomics for CNS circuit level analysis; ii) behavior tests; and iii) immunohistology, neuropathology, flow cytometry, and electron microscopy tissue analyses. We will determine the effects of global inducible pericyte ablation (20-70%) (AIM 1), focal inducible pericyte loss in the anterior cingulum of the corticolimbic ciruit (AIM 2) and pericyte-specific inducible Notch3R169C expression (AIM 3) on BBB integrity, CBF reductions, WM integrity, disruption of CNS circuits and functional deficits (behavior). We expect that the proposed studies will contribute towards better understanding of the mechanistic basis of WM disease in vascular cognitive impairment and dementia, and will establish pericyte as a new key target for WM disease.