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Reactive astrocytes control leukocyte and humoral trafficking into the CNS

反应性星形胶质细胞控制白细胞和体液转运至中枢神经系统

基本信息

批准号：
9334935
负责人：
GARETH R JOHN
金额：
$ 37.08万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-30 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9334935
关键词：
Acute Disease Address Antibodies Antigens Area Astrocytes Award Bladder Blood - brain barrier anatomy Cells Clinical Data Defect Dimerization Disease Drug Delivery Systems Epithelium Experimental Autoimmune Encephalomyelitis Funding Gatekeeping Genetic Goals Inflammation Mediators Inflammatory Knockout Mice Label Lesion Leukocytes Lymphocyte Mediating Modeling Molecular Multiple Sclerosis Multiple Sclerosis Lesions Mus Neuroglia Neurologic Deficit Pathogenesis Pathogenicity Pathology Pathway interactions Patients Peptide Hydrolases Permeability Pilot Projects Plasma Population Sizes Proteins Role Serum Proteins Severities Severity of illness Signal Pathway Signal Transduction Skin System Testing Therapeutic Tight Junctions Tissues Vascular Endothelial Growth Factors Work design dimer disability disorder control in vitro Model junctional adhesion molecule monocyte multiple sclerosis patient mutant novel novel strategies novel therapeutic intervention novel therapeutics occludin prevent public health relevance scaffold seal targeted treatment trafficking translational study

项目摘要

DESCRIPTION (provided by applicant): In multiple sclerosis (MS), entry of inflammatory leukocytes and humoral factors into the CNS drives the onset of clinical disease. Permanent damage can be reduced by limiting ingress of pathogenic lymphocytes and monocytes, and soluble factors including antibodies and serum proteins. This project seeks to identify CNS mechanisms controlling cell and humoral entry in MS lesions, to define new therapeutic strategies to limit damage and disability. To enter the CNS from the vasculature, cells and soluble factors must traverse two distinct barriers. Initially, they must cross the endothelial blood-brain barrier (BBB) into the perivascular space (PVS). Then, to enter the parenchyma they must penetrate the astrocytic glia limitans (GL). The first funding period of this award focused on the BBB. We identified reactive astrocytes as key drivers of BBB opening in lesions, acting via the permeability factors Vegf-a and Ecgf1. The BBB is sealed by endothelial tight junctions (TJ) of claudin (Cldn)-5 and occludin (Ocln) subunits. Vegf-a and Ecgf1 repress both, opening the BBB. Their blockade prevents BBB disruption, limiting pathology and disability. Here, we now focus on the second key barrier, the astrocytic GL, which controls CNS access upon BBB disruption. Critically, our data reveal that following astrocyte-driven opening of the BBB, these cells then control entry at the GL by forming TJ of their own. In inflammatory lesions, reactive astrocytes at the GL express Cldn-1/4, junction adhesion molecule-a (Jam-a), and Ocln-like tricellulin (Tcl) - the same TJ proteins which tightly seal skin and bladder epithelia. These results suggest astrocytes first drive BBB disruption in lesions, then act as gatekeepers to the CNS, at the GL. Importantly, pilot data further propose differential roles for astrocyte TJ proteins in control of cell vs. humoral entry. Claudins drive TJ strand formation, while Tcl may regulate strand complexity. Jam-a regulates function via intracellular signaling. Our results suggest that in astrocytic Cldn-4 knockout mice, leukocytes may not accumulate in the PVS in lesions, but directly transit the GL into the CNS. Conversely, humoral entry may be more severe in Jam-a conditional mice. Moreover, our data also propose leukocytes target astrocytic Cldn-4 for degradation, to facilitate transmigration. Here, we will test the hypothesis that reactive astrocytes control inflammatory lesion expansion via formation of a TJ barrier at the glia limitans. We propose three Aims. In Aim 1, we will test the extent to which astrocytic Cldn4 and Jam-a differentially regulate cellular vs. humoral trafficking. This work uses focal lesions in conditional knockout mice. In Aim 2, we will use culture models to identify the critical molecular mechanisms controlling leukocyte vs. humoral entry. In Aim 3, we will identify key leukocyte proteases targeting astrocytic Cldn-4 at the GL. We will then test the extent to which astrocytic TJ control disease severity in an MS model, via genetic targeting and therapeutic blockade. This proposal directly addresses control of leukocyte and humoral entry, and severity of neurologic deficit in MS. It also has broader implications for CNS access, drug delivery, and inflammatory disease more widely. The goal of this work is to identify novel therapeutic avenues to restrict lesion expansion and clinical exacerbation in patients.

描述（由申请人提供）：在多发性硬化（MS）中，炎性白细胞和体液因子进入CNS驱动临床疾病的发作。通过限制致病性淋巴细胞和单核细胞以及包括抗体和血清蛋白在内的可溶性因子的进入，可以减少永久性损伤。该项目旨在确定控制MS病变中细胞和体液进入的CNS机制，以确定新的治疗策略来限制损伤和残疾。为了从脉管系统进入CNS，细胞和可溶性因子必须穿过两个不同的屏障。最初，它们必须穿过内皮血脑屏障（BBB）进入血管周围空间（PVS）。然后，为了进入实质，它们必须穿透星形胶质细胞界膜（GL）。该奖项的第一个资助期专注于BBB。我们确定了反应性星形胶质细胞作为病变中BBB开放的关键驱动因素，通过渗透性因子Vegf-a和Ecgf 1起作用。血脑屏障由封闭蛋白（Cldn）-5和闭合蛋白（Ocln）亚基的内皮紧密连接（TJ）密封。Vegf-a和Ecgf 1抑制两者，打开BBB。它们的阻断防止BBB破坏，限制病理和残疾。在这里，我们现在关注第二个关键屏障，星形胶质细胞GL，它控制BBB破坏后的CNS通路。重要的是，我们的数据显示，在星形胶质细胞驱动的BBB开放后，这些细胞通过形成自己的TJ来控制GL的进入。在炎性病变中，GL处的反应性星形胶质细胞表达Cldn-1/4、连接粘附分子-a（Jam-a）和Ocln样三纤维素（Tcl）-紧密密封皮肤和膀胱上皮的相同TJ蛋白。这些结果表明星形胶质细胞首先驱动病变中的BBB破坏，然后在GL处充当CNS的守门人。重要的是，试验数据进一步提出了星形胶质细胞TJ蛋白在控制细胞与体液进入中的不同作用。紧密连接蛋白驱动TJ链的形成，而Tcl可以调节链的复杂性。Jam-a通过细胞内信号传导调节功能。我们的研究结果表明，在星形胶质细胞Cldn-4基因敲除小鼠，白细胞可能不会积累在PVS病变，但直接过境GL进入中枢神经系统。相反，体液进入可能在Jam-a条件小鼠中更严重。此外，我们的数据还提出白细胞靶向星形胶质细胞Cldn-4降解，以促进迁移。在这里，我们将测试的假设，反应性星形胶质细胞控制炎症病变的扩大，通过形成一个TJ屏障在胶质细胞界。我们提出三个目标。在目标1中，我们将测试星形胶质细胞Cldn 4和Jam-a差异调节细胞与体液运输的程度。这项工作使用条件性基因敲除小鼠的局灶性病变。在目标2中，我们将使用培养模型来确定控制白细胞与体液进入的关键分子机制。在目标3中，我们将鉴定在GL处靶向星形胶质细胞Cldn-4的关键白细胞蛋白酶。然后，我们将测试星形胶质细胞TJ通过遗传靶向和治疗阻断在MS模型中控制疾病严重程度的程度。该提案直接解决了白细胞和体液进入的控制，以及MS中神经功能缺损的严重程度。它还对CNS通路、药物递送和更广泛的炎症性疾病具有更广泛的影响。这项工作的目标是确定新的治疗途径，以限制病变扩大和临床恶化的患者。