Lipoic Acid Therapy for Experimental Autoimmune Encephalomyelitis

6.项目总结/摘要 多发性硬化（MS）是一种常见的中枢神经系统（CNS）致残性疾病。而 MS以前被认为是一种脱髓鞘性疾病，现在已经清楚， 在MS中突出，并且是MS中永久残疾的主要原因。 对MS中的变性的理解并不准确，但认为激活的小胶质细胞参与了MS中的轴突变性。 在MS的各个阶段都有破坏。 硫辛酸是一种有效的治疗MS小鼠模型，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 酸似乎涉及其刺激小的第二分子信使cAMP的产生的能力 通过前列腺素G偶联蛋白受体EP 2和EP 4。初步证据表明， 酸可以抑制小胶质细胞活化，而不依赖于其对T细胞的作用，并且可以通过刺激 cAMP产生。这项研究项目将测试假设，硫辛酸刺激cAMP的生产， 通过EP 2和EP 4受体激活小胶质细胞，从而防止小胶质细胞活化和可溶性神经胶质细胞的释放。 破坏轴突的炎症介质。这将在三个部分所述的工作中完成。 具体目标。对于特定目标1，将使用以下任一种方法激活从小鼠脑中培养的小胶质细胞： 脂多糖（LPS）或细胞因子肿瘤坏死因子-α（TNF-α）和干扰素-γ（IFN-γ） 在存在或不存在不同浓度的硫辛酸的情况下。小胶质细胞活化将通过以下方式测量： 评估一氧化氮（NO）、炎性细胞因子的产生和细胞表面活化的表达 标记。这将确定硫辛酸是否可以在体外抑制小胶质细胞活化。硫辛酸的能力 将通过活细胞荧光成像来确定酸刺激培养的小胶质细胞中cAMP产生 使用基于FRET的技术和cAMP报告基因。为了确定是否抑制小胶质细胞活化 通过硫辛酸依赖于cAMP的产生，将测试cAMP的抑制剂逆转 通过硫辛酸抑制小胶质细胞活化。最后，确定硫辛酸的作用依赖性 硫辛酸对EP 2和EP 4受体的作用，硫辛酸抑制小胶质细胞EP 2和EP 4活化的能力， 将敲除小鼠与野生型小胶质细胞的小鼠进行比较。 对于特异性目标2，通过脑内注射TNF-α/IFN-γ在体内诱导小胶质细胞活化。 或LPS，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CD 11b+小胶质细胞的数量、细胞的上调 通过定量免疫荧光测定MHC II类、半乳糖凝集素-3和iNOS的表面表达。 将使用EP 2敲除和EP 4flox/flox/CD 11bCre条件性敲除小鼠重复实验， 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受体的影响。 对于具体目标3，研究了硫辛酸对野生型和EP 2敲除的EAE的治疗作用， 将比较EP 4flox/flox/CD 11bCre条件性敲除小鼠，以确定 硫辛酸的作用依赖于EP 2和EP 4受体。 这项研究的结果将提供进一步的见解，硫辛酸的作用机制， EAE，并提供指导开发硫辛酸治疗所需的额外临床前数据 女士

6. Project Summary/Abstract 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-¿) and interferon-gamma (IFN-¿) 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-¿/IFN-¿ 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.