Astrocytes Play a Critical Role in the Pathology of EAE

描述（由申请人提供）：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应答引发的。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）对疾病的发展和进展有贡献。然而，这些细胞类型的作用远未被清楚地理解。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缺损。在所有这些过程中，在MS以及许多其他神经退行性疾病中，星形胶质细胞已被证明发挥积极作用。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，其功能意义仍然知之甚少。例如，反应性星形胶质细胞释放神经元存活和修复所必需的神经营养因子，并且还负责产生促炎分子（细胞因子、趋化因子、生长因子、NO等），即对功能恢复有害的生长抑制分子。反应性星形胶质细胞中发生的许多过程受NF-κ B调节，NF-κ B是炎症和继发性损伤的关键调节剂。在这个建议中概述的研究旨在研究星形胶质细胞NF-kB在实验性自身免疫性脑脊髓炎（EAE）的病理生理学中的作用，利用我们实验室产生的转基因小鼠模型（GFAP-IkBa-dn小鼠），其中NF-kB在表达GFAP的细胞中功能失活，如星形胶质细胞和非髓鞘形成的Schwann细胞。初步数据表明，阻断星形胶质细胞NF-κ B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。这使我们假设，反应性星形胶质细胞显着有助于疾病进展和发展的慢性神经功能缺损的EAE和MS。这一假设将在下面列出的四个具体目标进行测试。虽然在我们的转基因小鼠中产生的结果是非常有希望的，但目标1中的研究将比较我们的GFAP-IkBa-dn小鼠与另外两个小鼠系（如下所述），以证实迄今为止在我们的实验模型中获得的结果与NF-κ B通路的星形胶质细胞特异性抑制唯一相关。通过将Sofroniew博士实验室开发的GFAP-Cre系与Michael Karin博士实验室产生的floxed（f/f）IKKb系进行育种，获得第一个小鼠系（73.12xffIKKb）。第二个小鼠品系（GFAPCreERT 2xffIKKb）是通过将McCarthy博士实验室中开发的他莫昔芬诱导型GFAP-Cre品系（CreERT 2）与相同的floxed（f/f）IKKb品系交配而获得的。在目的2和3中，我们将使用相对于相应的对照小鼠提供最稳健的临床改善的品系，以进一步研究基于通过阻断星形胶质细胞NF-κ B提供的保护的机制。具体而言，目标2中的研究将确定患病WT和突变小鼠的CNS中的血脑渗透性和白细胞浸润是否存在差异。目标3中的研究将确定抑制星形胶质细胞NF-κ B促进抗炎反应的机制。在这方面的研究将集中在如何抑制星形胶质细胞NF-κ B改变T和B细胞在脊髓中的反应。最后，由于脱髓鞘是这种疾病的标志，并且可以通过神经炎症来调节，因此目的4中的研究将调查星形胶质细胞-NF-kB的抑制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NF-κ B信号转导机制，而且还将阐明病理条件下星形胶质细胞的反应。最终，我们的目标是确定干扰这些反应是否是MS和其他神经系统疾病的治疗策略。公共卫生相关性：本申请的目的是更好地理解星形胶质细胞（一种在脑和脊髓中发现的非神经元细胞）在多发性硬化症及其动物模型EAE的发病机制中的作用。了解这种疾病和其他疾病的病理学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.