Astrocytes Play a Critical Role in the Pathology of EAE

项目摘要 多发性硬化(MS)及其动物模型实验性自身免疫性脑脊髓炎(EAE) 据信是由T细胞介导的对髓鞘抗原的免疫反应启动的。然而，近年来，一种 大量的证据已经汇编，表明不同的细胞群体在 中枢神经系统(CNS)，如小胶质细胞和星形胶质细胞，对糖尿病的发生和发展起重要作用 疾病。然而，这些细胞类型的作用还远未被清楚地了解。慢性 神经炎症和脱髓鞘也可能导致疾病进展和慢性神经系统疾病。 赤字。在所有这些过程中，在多发性硬化症以及许多其他神经退行性疾病中，星形胶质细胞 被证明发挥了积极的作用。 星形胶质细胞对损伤的反应是“反应性”或“胶质化”，这是一种复杂的细胞反应，其 人们对其功能意义仍知之甚少。例如，反应性星形胶质细胞释放神经营养因子 对神经元的生存和修复是必不可少的，也是促炎因子的产生 分子(细胞因子、趋化因子、生长因子、一氧化氮等)有害的生长抑制分子 功能恢复。反应性星形胶质细胞中发生的许多过程都受到核因子-kB的调节，核因子-kB是一个关键 炎症和继发性损伤的调节剂。 本研究旨在探讨星形胶质细胞核因子-kB在脑缺血再灌注损伤中的作用。 实验性自身免疫性脑脊髓炎(EAE)的病理生理学研究 本实验室建立的NF-kB功能失活的小鼠模型(GFAP-IkBA型糖尿病小鼠) 表达GFAP，如星形胶质细胞和非髓鞘雪旺细胞。初步数据显示， 阻断星形胶质细胞核因子-kB显著减轻EAE后的疾病严重程度，促进功能恢复 并减少神经炎症和脱髓鞘。这让我们假设反应性星形胶质细胞 显著促进EAE和慢性神经功能障碍的疾病进展和发展 女士，这一假设将在下面概述的四个具体目标中得到检验。虽然在我们的 转基因小鼠非常有希望，Aim 1中的研究将把我们的GFAP-IkBa-dN小鼠与两只 额外的小鼠品系(如下所述)以确认到目前为止在我们的实验模型中所获得的结果 与星形胶质细胞特异性抑制核因子-kB途径有关。第一行鼠标 (73.12xffIKKb)是通过将Sofroniew博士的实验室培育的GFAP-Cre系选育到有花的(f/f)获得的。 在迈克尔·卡林博士的实验室里产生了IKKB基因。第二个鼠标行(GFAPCreERT2xffIKKb)是 通过将麦卡锡博士实验室培育的三苯氧胺可诱导GFAP-Cre系(CreERT2)培育成 相同的浮雕(f/f)IKKB线。在目标2和目标3中，我们将使用提供最强健临床的线(S) 对相应的对照小鼠进行改良，在此基础上进一步研究其作用机制 通过阻断星形胶质细胞的核因子-kB提供保护。具体地说，目标2中的研究将确定是否存在 WT病变者与突变者血脑通透性及中枢神经系统白细胞浸润的差异 老鼠。AIM 3中的研究将确定抑制星形胶质细胞NF-kB促进 抗炎反应。这一目标的研究将集中在抑制星形胶质细胞核因子-kB如何改变T和B细胞 脊髓的反应。最后，由于脱髓鞘是这种疾病的一个标志，可以通过调节 通过神经炎症，目标4的研究将调查星形胶质细胞-核因子-kB的抑制对 少突胶质细胞存活和脱髓鞘。 我们的实验不仅将深入了解核因子-kB的信号机制，而且还将阐明 病理条件下星形胶质细胞的反应。最终，我们的目标是确定是否干扰这些 作为多发性硬化症和其他神经疾病的一种治疗策略，这些反应可能是有益的。

Project Summary Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are believed to be initiated by T cell-mediated immune responses to myelin antigens. In recent years, however, a significant body of evidence has been compiled indicating the contribution of various cell populations within the central nervous system (CNS), such as microglia and astrocytes, to the development and progression of the disease. Nevertheless, the role of these cell types is far from being clearly understood. Chronic neuroinflammation and demyelination may also contribute to disease progression and chronic neurological deficits. In all these processes, in MS as well as in many other neurodegenerative diseases, astrocytes have been demonstrated to play an active role. Astrocytes respond to injury by becoming "reactive" or "gliotic", a complex cellular response whose functional significance is still poorly understood. For instance, reactive astrocytes release neurotrophins essential for neuronal survival and repair, and are also responsible for the production of pro-inflammatory molecules (cytokines, chemokines, growth factors, NO etc) growth-inhibitory molecules detrimental to functional recovery. Many of the processes occurring in reactive astrocytes are regulated by NF-kB, a key modulator of inflammation and secondary injury. The studies outlined in this proposal are designed to investigate the role of astroglial NF-kB in the pathophysiology of experimental autoimmune encephalomyelitis (EAE), taking advantage of a transgenic mouse model generated in our laboratory (GFAP-IkBa-dn mice) where NF-kB is functionally inactivated in cells expressing GFAP, such as astrocytes and non-myelinating Schwann cells. Preliminary data indicate that blocking astroglial NF-kB significantly reduces disease severity, improves functional recovery following EAE and reduces neuroinflammation and demyelination. This leads us to hypothesize that reactive astrocytes significantly contribute to disease progression and development of chronic neurological deficits in EAE and MS. This hypothesis will be tested in the four specific aims outlined below. While the results generated in our transgenic mice are very promising, the studies in Aim 1 will compare our GFAP-IkBa-dn mice to two additional mouse lines (described below) to confirm that the results obtained so far in our experimental model are uniquely associated with the astrocyte-specific inhibition of the NF-kB pathway. The first mouse line (73.12xffIKKb) is obtained by breeding a GFAP-Cre line developed in Dr. Sofroniew's laboratory to a floxed (f/f) IKKb line generated in the laboratory of Dr. Michael Karin. The second mouse line (GFAPCreERT2xffIKKb) is obtained by breeding a tamoxifen inducible GFAP-Cre line (CreERT2) developed in Dr. McCarthy's lab to the same floxed (f/f) IKKb line. In Aims 2 and 3 we will use the line(s) that provides the most robust clinical improvement over the corresponding control mice to further investigate the mechanisms at the basis of the protection provided by blocking astroglial NF-kB. Specifically, studies in Aim 2 will determine if there are differences in blood brain permeability and infiltration of leukocytes in the CNS of diseased WT and mutant mice. Studies in Aim 3 will determine the mechanisms through which inhibiting astroglial NF-kB promotes an anti-inflammatory response. Studies in this aim will focus on how inhibiting astroglial NF-kB alters T and B cell responses in the spinal cord. Finally, since demyelination is a hallmark of this disease and could be modulated by neuroinflammation, studies in Aim 4 will investigate the effect of the inhibition of astroglial-NF-kB on oligodendrocyte survival and demyelination. Our experiments will not only give insights into NF-kB signaling mechanisms, but also elucidate astrocyte responses under pathological conditions. Ultimately, our goal is to determine if interfering with these responses could be beneficial as a therapeutic strategy for MS and other neurological disorders.