Astrocytes Play a Critical Role in the Pathology of EAE

描述(申请人提供)：多发性硬化症(MS)及其动物模型实验性自身免疫性脑脊髓炎(EAE)被认为是由T细胞介导的对髓鞘抗原的免疫反应引发的。然而，近年来，大量证据表明，中枢神经系统(CNS)内的各种细胞群，如小胶质细胞和星形胶质细胞，在疾病的发生和发展中起着重要作用。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功能障碍。在所有这些过程中，在多发性硬化症和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，其功能意义仍鲜为人知。例如，反应性星形胶质细胞释放对神经元生存和修复至关重要的神经营养因子，也负责产生不利于功能恢复的促炎分子(细胞因子、趋化因子、生长因子等)生长抑制分子。发生在反应性星形胶质细胞中的许多过程都受到核因子-kB的调控，核因子-kB是炎症和继发性损伤的关键调节因子。本研究旨在探讨星形胶质细胞核因子-kB在实验性自身免疫性脑脊髓炎(EAE)病理生理学中的作用，利用本实验室建立的转基因小鼠模型(GFAP-IkBA-DN小鼠)，在表达GFAP的细胞(如星形胶质细胞和非髓鞘雪旺细胞)中，核因子-kB功能失活。初步数据显示，阻断星形胶质细胞的核因子-kB可显著降低疾病严重程度，改善EAE后的功能恢复，并减少神经炎症和脱髓鞘。这导致我们假设反应性星形胶质细胞在EAE和MS的疾病进展和慢性神经功能障碍的发展中起着重要作用。这一假设将在下面概述的四个具体目标中得到验证。虽然在我们的转基因小鼠中产生的结果非常有希望，但在Aim 1中的研究将把我们的GFAP-IkBaN小鼠与另外两个小鼠系(如下所述)进行比较，以确认到目前为止在我们的实验模型中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。第一个小鼠品系(73.12xffIKKb)是通过将Sofroniew博士的实验室培育的GFAP-Cre品系与Michael Karin博士的实验室培育的(f/f)IKKB品系杂交而获得的。第二个小鼠品系(GFAPCreERT2xffIKKb)是通过将McCarthy博士实验室培育的三苯氧胺可诱导GFAP-Cre品系(CreERT2)培育到相同的花蕾(f/f)IKKB品系而获得的。在目标2和目标3中，我们将在阻断星形胶质细胞NF-kB提供保护的基础上，使用与相应对照小鼠相比提供最强劲临床改善的LINE(S)来进一步研究其机制。具体地说，AIM 2的研究将确定WT疾病小鼠和突变小鼠在血脑渗透性和中枢神经系统白细胞渗透方面是否存在差异。目标3的研究将确定抑制星形胶质细胞核因子-kB促进抗炎反应的机制。这一目标的研究将集中在抑制星形胶质细胞核因子-kB如何改变脊髓中T和B细胞的反应。最后，由于脱髓鞘是这种疾病的一个标志，并可能受到神经炎症的调节，目标4的研究将探讨抑制星形胶质细胞-核因子-kB对少突胶质细胞存活和脱髓鞘的影响。我们的实验不仅将深入了解核因子-kB的信号机制，而且还将阐明星形胶质细胞在病理条件下的反应。最终，我们的目标是确定干扰这些反应是否可以成为治疗多发性硬化症和其他神经疾病的有益策略。公共卫生相关性：这项应用的目标是更好地了解星形胶质细胞在多发性硬化症及其动物模型EAE的发病机制中所起的作用。星形胶质细胞是一种在大脑和脊髓中发现的非神经细胞。了解这种疾病和其他疾病的病理将有助于开发更有效的治疗方法。

DESCRIPTION (provided by applicant): 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 a therapeutic strategy for MS and other neurological disorders. PUBLIC HEALTH RELEVANCE: The objective of this application is to better understand the role astrocytes, a non- neuronal cell found in the brain and spinal cord, play in the pathogenesis of multiple sclerosis and its animal model EAE. Understanding the pathology of this and other diseases will help in the development of more effective therapies.