糖皮質固醇對寡突膠細胞保護作用之研究

Contents ….…………………………………………………………………… I List of Tables and Figures ……………………………………………… Ⅵ Abstract in Chinese …………………………………………………… Ⅸ Abstract in English …………………….……………………………. ⅩⅠ Chapter 1. Introduction White matter injury ……………………………………………………….… 2 Excitotoxicity in OLGs during white matter injury ……………………….… 4 Glucocorticoid therapy for white matter injury ………………………….… 5 Glucocorticoid receptor (GR) ………….….…….… 5 Effects of glucocorticoids on the survival of OLGs ………….…….… 7 Erythropoietin- a neuroprotective cytokine ……….…….… 9 Transcriptional regulation of Epo and downstream signaling .…….… 10 Activation of HIF-1?? : oxygen-dependent and independent mechanisms …. 11 Rationale ……………….…….… 13 Aims and strategies ……………………….…………………………. 14 Chapter 2. Materials and Methods 1. Materials …………………………………………………………….……. 16 Reagents ….………………………………………….…………………….… 16 Drugs .…………………………………………………………….…………… 18 Antibodies .……………………………….…………………….………….… 19 Commercial kits …………………………………………………………….… 20 Culture medium .………………………………………………….…………… 21 Plasmids .……………………………………………….…………….… 22 Animals .……………………………………………….…………….… 22 2. Methods ……………………………………….………….…….…….…. 23 Primary cultured rat OLGs ….…………………….…………………….… 23 Primary cultured rat cortical neurons .…………………….……….…………… 23 Immunofluorescent staining and confocal microscopy .……………….…….… 24 Semi-quantitative and quantitative real-time RT-PCR ……………….……….… 24 Enzyme-linked immunosorbent assay (ELISA) .………………….…………… 25 Spinal cord injury animal model .…………………….…….…………….… 26 Viability and cell death assay of OLGs .…………………….…………….… 26 Western blot analysis .……………………………………….…………….… 27 RNA knockdown .………………………………….……….…………….… 28 Plasmid construction .…………………………….………….…………….… 28 Luciferase activity assay .…………………………….…….…………….… 29 Nuclear protein extraction .………………………….……….…………….… 29 Co-immunoprecipitation .……………………………………….………….… 30 Yeast two-hybrid assay .……………………………….…….…………….… 30 Electrophoretic Mobility Shift Assay (EMSA) .……………….………….… 30 Chromatin immunoprecipitation (ChIP) assay .…………….…….……….… 31 Computer modeling of protein-protein interaction .……….…….……….… 32 Statistic analysis .…………………………………………….…………….… 32 Chapter 3. Results 1. MP upregulation of Epo expression in OLGs but not in cortical neurons……………………………………………………………….….…… 35 2. MP upregulation of Epo expression in the injured spinal cord in vivo…………………………………………………………………….….…. 35 3. Exogenous administration of EPO protects OLGs against excitotoxicity………………………………………………………….….…. 36 4. Causal role of Epo expression in MP protection of OLGs against excitotoxicity………………………………………………………….….…. 37 5. Glucocorticoid receptor-dependent activation of HIF-1α?nby MP……. 38 6. Reversal of MP activation of pHREEPO-Luc and upregulation of Bcl-xL expression by GR or HIF-1α knockdown………………….…….….….…. 40 7. MP treatment increased both GR and HIF-1α in the nucleus of OLGs…. 40 8. MP enhancement of nuclear GR-HIF-1α interaction in OLGs……….…. 41 9. Co-recruitment of HIF-1α and GR onto the promoter and 3’ enhancer of the Epo gene in OLGs but not in neurons…………….………………….….…. 43 10. HIF-1?? association with GR-HIF-1α complex is required for the Epo induction by MP treatment…………….…………………………….….…. 45 11. Structural analysis of interactions among GR, HIF-1α, ARNT, and DNA………………………………………………………………….………. 45 12. Overexpression of GR-DBD abolished MP-induced HIF-1α activity in OLGs…………………………….………………………………….………. 47 Chapter 4. Discussion . 48 The causal role of Epo expression in MP protection of OLGs against excitotoxicity……………………………………………………………….……. 49 Activation of dual transcription factors (HIF-1α and GR) in MP upregulation of Epo expression…………………………….…………………….……………… 50 MP induction of GR and HIF-1α interaction and recruitment onto the Epo promoter and enhancer…………….………………………….……….….…. 51 OLG-specific MP action on the HIF-1α-Epo cascade…………….…………………………….…………….……….….…. 53 Therapeutic application of MP-mediated neuroprotection in demyelinating disorders…………….…………………………………………….…….….…. 56 Chapter 5. Conclusion and Perspectives . 59 Chapter 6. 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Neurology 57:1239-1247. ; 糖皮質固醇已廣泛應用於中樞神經系統白質損傷之治療，如多發性硬化症 (multiple sclerosis) 、急性瀰漫性腦脊髓腦炎 (acute disseminated encephalomyelitis) 等髓鞘病變相關疾病以及因外傷造成之脊髓損傷 (spinal cord injury)。在先前脊髓損傷研究中已證明糖皮質固醇的保護作用在寡突膠細胞(Oligodendrocytes; OLGs) 而不是神經元細胞（neurons），而寡突膠細胞為中樞神經系統中形成髓鞘之細胞。本論文旨在進一步探討糖皮質固醇專一性保護寡突膠細胞的分子機轉。 本研究使用目前臨床最常使用治療多發性硬化症及脊髓損傷之合成糖皮質固醇 (Methylprednisolone; MP)，研究結果證實MP可以保護初代培養的大白鼠寡突膠細胞免於遭受興奮性胺基酸AMPA所產生之模擬白質損傷後的興奮性毒性，且此一保護機轉為透過增加紅血球生成素 (erythropoietin; Epo) 的基因表現，紅血球生成素已知為一種具有神經保護作用的細胞間素，並可改善脊髓損傷動物的功能性復原。我們進一步發現，MP所調控增加的Epo的機轉必須先透過糖皮質固醇接受體 (glucocorticoid receptor, GR) 活化Epo的上游轉錄因子：缺氧誘導因子-1α (hypoxia inducible factor-1α?z簡稱HIF-1α)。透過免疫沈澱 (Co-immunoprecipitation)、染色質免疫沉澱 (chromatin immunoprecipitation)、酵母菌雙雜交篩選系統 (yeast two hybrid) 、電腦模擬立體蛋白結合結構及選擇性過度表現GR domain等分析，我們證實了MP保護寡突膠細胞的作用為促進HIF-1?? 的累積，進而造成GR DNA binding domain (DBD) 與HIF-1α PAS (PER/ARNT/SIM) domain的結合，使得HIF-1α與HIF-1β結合，而啟動Epo 基因的轉錄與表達。然而，上述這些MP在寡突膠細胞活化HIF-1?? 和增加Epo表達的作用，卻未在初代培養大腦皮質神經細胞中發生。因此，本研究不但闡明了糖皮質固醇選擇性的保護寡突膠細胞而非神經細胞的分子機制，也首度證實了糖皮質固醇可在正常氧分壓下，透過GR活化HIF-1α。此研究成果對於發展神經白質損傷的治療策略提供了重要的學理依據。 Glucocorticoids are commonly used in treating diseases with white matter lesions such as multiple sclerosis, acute disseminated encephalomyelitis, other demyelinating diseases and spinal cord injury. However, glucocorticoids are ineffective in gray matter injuries, such as head injury and stroke. The glucocorticoid effects on the survival of injured oligodendrocytes (OLGs), which wrap around central axons to form myelin sheath, but not central neurons, have been demonstrated in spinal cord injury animal model. In this study, we elucidated the molecular mechanism of this OLG-specific protection. We unraveled a novel mechanism of methylprednisolone (MP), a synthetic glucocorticoid widely used for treating multiple sclerosis and spinal cord injury, in protecting OLGs against death induced by AMPA, an excitotoxin, which has been implicated in white matter damage after spinal cord injury. This cytoprotective action of MP in OLGs is causally related to its upregulation of erythropoietin (Epo), a neuroprotective cytokine that enhances functional recovery in animal models of spinal cord injury and multiple sclerosis. MP transactivation of Epo expression entails complex transcriptional events involving a glucocorticoid receptor (GR) mechanism in the activation of hypoxia-inducible factor-1α (HIF-1α), a cellular stress-adapting transcription factor. Co-immunoprecipitation, chromatin immunoprecipitation analysis, yeast two-hybrid analysis, structure modeling of three-dimensional protein-protein interactions and selective overexpression of GR domains confirm MP induces interaction between GR DNA binding domain and HIF-1α PAS (PER/ARNT/SIM) domain, with subsequent recruitment of HIF-1α to transactivate Epo expression in OLGs. In contrast, MP does not induce GR- HIF-1α interaction, HIF-1α activation, and Epo expression in cultured cortical neurons. This novel mechanism of MP action is specific for OLGs, but not neurons, not only supports the clinical notion regarding the beneficial effects of glucocorticoid therapy on white, but not gray, matter diseases, but also demonstrates for the first time that GR activates HIF-1α under the normoxia condition in a cell-type specific manner. The information obtained from this study provides novel insights into the development of more effective therapies for diseases affecting the white matter.