Lipoic Acid Therapy for Experimental Autoimmune Encephalomyelitis

描述(由申请人提供)： 多发性硬化症(MS)是中枢神经系统(CNS)的常见致残性疾病。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，是MS永久性残疾的主要原因。MS轴突变性的发病机制尚不清楚，但激活的小胶质细胞被认为参与了MS各个阶段的轴突破坏。硫辛酸是治疗MS小鼠模型、实验性自身免疫性脑脊髓炎(EAE)的有效药物，并在EAE中抑制T细胞向中枢神经系统的迁移。硫辛酸的作用机制似乎涉及到它通过前列腺素G偶联蛋白受体EP2和EP4刺激小第二分子信使cAMP的产生。初步证据表明，硫辛酸可能会抑制小胶质细胞的激活，而不是依赖于它对T细胞的影响，而且可能是通过刺激cAMP的产生来实现的。这项研究项目将检验硫辛酸通过EP2和EP4受体刺激小胶质细胞产生cAMP，从而阻止小胶质细胞激活和释放破坏轴突的可溶炎性介质的假设。这将在三个具体目标所述的工作中实现。对于特定的目标1，在不同浓度的硫辛酸存在或不存在的情况下，从小鼠脑中培养的小胶质细胞将被脂多糖(LPS)或细胞因子肿瘤坏死因子-α(TNF-1)和干扰素-γ(干扰素-3)激活。小胶质细胞的激活将通过评估一氧化氮(NO)、炎性细胞因子的产生和细胞表面激活标志物的表达来衡量。这将决定硫辛酸是否能在体外抑制小胶质细胞的激活。硫辛酸刺激培养的小胶质细胞产生cAMP的能力将通过使用基于FRET的技术和cAMP报告程序的活细胞荧光成像来确定。为了确定硫辛酸对小胶质细胞激活的抑制是否依赖于cAMP的产生，将测试cAMP的抑制剂是否具有逆转硫辛酸对小胶质细胞激活的抑制的能力。最后，为了确定硫辛酸对EP2和EP4受体作用的依赖性，将硫辛酸抑制EP2和EP4基因敲除小鼠小胶质细胞激活的能力与野生型小胶质细胞进行比较。对于特定的目的2，将在体内通过脑内注射肿瘤坏死因子-1/干扰素-3或脂多糖诱导小胶质细胞激活，并测定全身注射硫辛酸对小胶质细胞激活的阻断能力。体内小胶质细胞活化的指标包括CD11b+小胶质细胞的数量，免疫荧光法测定细胞表面MHC II类分子、Galectin-3和iNOS的表达。使用EP2基因敲除和EP4FLOX/FLOX/CD11bCre条件基因敲除小鼠重复实验，并与野生型小鼠的结果进行比较，以确定硫辛酸对EP2和EP4受体对小胶质细胞的抑制作用的依赖性。针对具体目的3，比较硫辛酸对野生型和EP2基因敲除小鼠以及EP4flx/Flox/CD11bCre条件性基因敲除小鼠的治疗作用，以确定硫辛酸的作用机制是否依赖于EP2和EP4受体。这项研究的结果将为硫辛酸在EAE中的作用机制提供进一步的见解，并为指导硫辛酸作为治疗MS的方法的发展提供额外的临床前数据。 公共卫生相关性： 这项研究项目与退伍军人的健康和保健问题有关，因为它的最终目的是开发硫辛酸作为治疗多发性硬化症(MS)的一种新的、成本效益高的疗法。多发性硬化症是退伍军人中一种重要的神经系统疾病。弗吉尼亚州MS卓越中心编写的一份关于VHA医疗保健利用的报告显示，从2000年到2003年，超过24,000名患有MS的退伍军人通过VHA获得了护理(MS卓越中心，2004年http://www.va.gov/ms).在这些人中，超过6000人与多发性硬化症的服务有关，超过9000人正在服用一种疾病修饰药物。VHA为患有多发性硬化症的退伍军人提供护理的平均年成本超过每名患者每年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抚恤金，这提供了另一个巨大的费用来源。目前FDA批准的多发性硬化症治疗方法每年花费高达3.5万美元，而且只有部分有效。寻找新的、经济有效的治疗方法可以减缓多发性硬化症的进展，这与VHA的卫生保健使命明显相关。

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.