Lipoic Acid Therapy for Experimental Autoimmune Encephalomyelitis

描述（由申请人提供）： 多发性硬化（MS）是一种常见的中枢神经系统（CNS）致残性疾病。虽然MS被正式认为是一种脱髓鞘疾病，但现在清楚的是，轴突变性在MS中显著发生，并且是MS中永久性残疾的主要原因。MS中轴突变性的发病机制不精确地理解，但活化的小胶质细胞被认为参与MS所有阶段的轴突破坏。实验性自身免疫性脑脊髓炎（EAE），并抑制EAE中T细胞迁移到CNS中。硫辛酸的作用机制似乎涉及其通过前列腺素G偶联蛋白受体EP 2和EP 4刺激小的第二分子信使cAMP产生的能力。初步证据表明，硫辛酸可以抑制小胶质细胞的激活独立于其对T细胞的影响，并可能通过刺激cAMP的产生。该研究项目将测试以下假设：硫辛酸通过EP 2和EP 4受体刺激小胶质细胞中cAMP的产生，从而防止小胶质细胞活化和释放损害轴突的可溶性炎症介质。这将在三个具体目标所述的工作中完成。对于特定目标1，在存在或不存在不同浓度的硫辛酸的情况下，使用脂多糖（LPS）或细胞因子肿瘤坏死因子-α（TNF-1）和干扰素-γ（IFN-3）活化从小鼠脑培养的小胶质细胞。将通过评估一氧化氮（NO）、炎性细胞因子的产生和细胞表面活化标志物的表达来测量小胶质细胞活化。这将确定硫辛酸是否可以在体外抑制小胶质细胞活化。将通过使用基于FRET的技术和cAMP报告基因的活细胞荧光成像来确定硫辛酸刺激培养的小胶质细胞中cAMP产生的能力。为了确定硫辛酸对小胶质细胞活化的抑制是否依赖于cAMP产生，将测试cAMP抑制剂逆转硫辛酸对小胶质细胞活化的抑制的能力。最后，为了确定硫辛酸对EP 2和EP 4受体的作用的依赖性，将硫辛酸抑制小胶质细胞EP 2和EP 4敲除小鼠的活化的能力与野生型小胶质细胞的能力进行比较。 对于特定目标2，将通过脑内注射TNF-1/IFN-3或LPS在体内诱导小胶质细胞活化，并测定全身施用的硫辛酸阻断小胶质细胞活化的能力。体内小胶质细胞活化的测量将包括通过定量免疫荧光测定的CD 11b+小胶质细胞的数量、MHC II类、半乳糖凝集素-3和iNOS的细胞表面表达的上调。使用EP 2敲除和EP 4flox/flox/CD 11bCre条件性敲除小鼠重复实验，并将结果与野生型小鼠中获得的结果进行比较，以确定硫辛酸对小胶质细胞上的EP 2和EP 4受体的抑制作用的依赖性。 对于特定目标3，将比较硫辛酸对野生型和EP 2敲除和EP 4flox/flox/CD 11bCre条件性敲除小鼠中的EAE的治疗作用，以确定硫辛酸的作用机制是否依赖于EP 2和EP 4受体。 这项研究的结果将进一步深入了解硫辛酸在EAE中的作用机制，并提供指导开发硫辛酸作为MS治疗所需的额外临床前数据。 公共卫生相关性： 该研究项目与退伍军人的健康和医疗保健问题有关，因为其目的是最终开发硫辛酸作为多发性硬化症（MS）的新的和具有成本效益的治疗方法。MS是退伍军人中的一种重要神经系统疾病。退伍军人事务部MS卓越中心编写的一份关于VHA保健利用情况的报告显示，2000-2003年，24 000多名患有MS的退伍军人通过VHA接受了保健（MS卓越中心，2004年http：//www.va.gov/ms）。其中超过6，000人与MS服务连接，超过9，000人正在服用一种疾病修饰药物。VHA为患有MS的退伍军人提供护理的平均年成本超过每位患者每年35，000美元。此外，许多患有多发性硬化症的退伍军人成为残疾人，有资格领取退伍军人事务部养老金，这是另一个巨大的开支来源。目前FDA批准的MS治疗每年花费高达35，000美元，并且仅部分有效。寻找新的，具有成本效益的治疗方法，可以减缓MS的进展是明显相关的卫生保健使命的VHA。

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a common and disabling disease of the central nervous system (CNS). While MS was formally believed to be a demyelinating disease, it is now clear that axonal degeneration occurs prominently in MS and is the primary cause of permanent disability in MS. The pathogenesis of axonal degeneration in MS is imprecisely understood but activated microglia are believed to be involved in axonal destructions at all stages of MS. Lipoic acid is an effective therapy for the murine model of MS, experimental autoimmune encephalomyelitis (EAE), and inhibits T cell migration into the CNS in EAE. The mechanism of action of lipoic acid appears to involve its ability to stimulate the production of the small second molecule messenger cAMP via the prostaglandin G-coupled protein receptors EP2 and EP4. Preliminary evidence suggests that lipoic acid may inhibit microglial activation independent of its effects on T cells and may do so via stimulation of cAMP production. This research project will test the hypothesis that lipoic acid stimulates cAMP production in microglia cells via the EP2 and EP4 receptors and thereby prevents microglial activation and release of soluble inflammatory mediators that damage axons. This will be accomplished in the work described in the three Specific Aims. For Specific Aim 1, microglia cultured from mouse brains will be activated using either lipopolysaccharide (LPS) or the cytokines tumor necrosis factor-alpha (TNF-1) and interferon-gamma (IFN-3) in the presence or absence of varying concentrations of lipoic acid. Microglial activation will be measured by assessing production of nitric oxide (NO), inflammatory cytokines, and expression of cell surface activation markers. This will determine whether lipoic acid can inhibit microglial activation in vitro. The ability of lipoic acid to stimulate cAMP production in cultured microglial cells will be determined by live cell fluorescent imaging using a FRET-based technology and a cAMP reporter. To determine whether inhibition of microglial activation by lipoic acid is dependent on cAMP production, an inhibitor of cAMP will be tested for its ability to reverse inhibition of microglial activation by lipoic acid. Finally, to determine the dependence of the effects of lipoic acid on the EP2 and EP4 receptors, the ability of lipoic acid to inhibit activation of microglia EP2 and EP4 knock-out mice will be compared with that of wild type microglia. For Specific Aim 2, microglial activation will be induced in vivo by intracerebral injection of TNF-1/IFN-3 or LPS and the ability of systemically administered lipoic acid to block microglial activation will be determined. Measures of in vivo microglial activation will include number of CD11b+ microglial, upregulation of the cell surface expression of MHC Class II, galectin-3 and iNOS determined by quantitative immunofluorescence. Experiments will be repeated using EP2 knock-out and EP4flox/flox/CD11bCre conditional knock-out mice and results compared with those obtained in wild type mice to determine the dependence of the inhibitory effects of lipoic acid on the EP2 and EP4 receptors on microglia. For Specific Aim 3, the therapeutic effects of lipoic acid on EAE in wild type and EP2 knock-out and EP4flox/flox/CD11bCre conditional knock-out mice will be compared to determine whether the mechanism of action of lipoic acid depends on the EP2 and EP4 receptors. The results of this research will provide further insights into the mechanism of action of lipoic acid in EAE and provide additional pre-clinical data needed for guiding the development of lipoic acid as a treatment for MS. PUBLIC HEALTH RELEVANCE: This research project is relevant to veterans' health and healthcare issue because its purpose is ultimately to develop lipoic acid as a new and cost effective treatment for multiple sclerosis (MS). MS is an important neurologic disease among veterans. A report on VHA health care utilization prepared by the VA MS Centers Excellence revealed that over 24,000 veterans with MS received care through VHA from 2000-2003 (MS Center of Excellence, 2004 http://www.va.gov/ms). Over 6,000 of these individuals were service connected for MS and over 9,000 were taking one of the disease modifying drugs. The average annual cost to VHA for providing care to veterans with MS exceeded $35,000 per patient per year. In addition, many veterans with MS become disabled and are eligible for VA pensions, providing another source of tremendous expense. Current FDA approved therapies for MS cost up to $35,000 a year and are only partially effective. Finding new, cost effective therapies that can slow the progression of MS is of obvious relevance to the health care mission of VHA.