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  • 學位論文

發展以IRES機制篩選可治療阿茲海默症與抗病毒之方法

Development of an IRES based drug screening method for Alzheimer’s Disease and Enterovirus Infection

指導教授 : 葉瑞銘 吳宗遠

摘要


摘要 本研究主要分為兩個部分,皆使用IRES元件構築在雙效表現載體中,透過分析在真核細胞中可抑制Cap-independent之轉譯起始,但不影響Cap端之轉譯活性的化合物,用以發展藥物篩選平台。 阿茲海默症 (Alzheimer's disease, AD) 現為已被高度重視之神經退化性疾病,因為此疾病若無給予病人及時的診斷及適當的醫療照護,極可能危及生命。此疾病主要病徵是由於在阿茲海默症病人腦中與記憶相關之神經元漸進性的死亡,並且在病理組織中會發現由-澱粉樣蛋白 (-amyloid) 所構成的斑塊 (Plaques) 以及神經纖維糾結 (Neurofibrillary Tangles, NFTs) 存在於其中。先前研究發現APP及Tau蛋白的表現在阿茲海默症致病機轉中扮演重要角色,而此兩種蛋白質也可藉由IRES調控而表現。因此在本研究中,我們使用雙效表現載體作為藥物篩選平台,以CMV啟動子啟動半乳糖苷酶 (-galatosidase) 之基因表達,再由APP或Tau蛋白之IRES調控分泌型鹼性磷酸酶 (SEAP) 之表現,並且在神經細胞中添加測試之化合物,在不影響cap端半乳糖苷酶表現的情形下,篩選可以抑制IRES活性之化合物。我們利用轉染載體至神經纖維瘤細胞 (N2A)中,測試了過去研究中發現在治療阿茲海默症中具潛力之化合物,包括薑黃素(curcumin)、去甲氧基薑黃素(demethoxycurcumin)、白藜蘆醇 (resveratrol)以及維生素B12 (vitamin B12)。我們發現薑黃素相較於去甲氧基薑黃素,較能有效抑制APP或Tau IRES的轉譯活性。在西方墨點法的分析中,我們也確認薑黃素可以降低APP C端蛋白、人類Tau蛋白以及磷酸化的Tau蛋白 (pS262、pS396) 的表現。結果顯示薑黃素的確可以透過抑制APP及Tau上游之IRES的活性來達到緩和阿茲海默症的病理徵兆。此外在酵素活性測試及西方墨點法分析中也發現白藜蘆醇 (resveratrol),能在較大藥物濃度差異 (1-40 M) 的處理下,亦能不影響cap端的轉譯,且對IRES也達到一樣的抑制效果。然而,維生素B12則在IRES活性的抑制上較無差異,再加上儘管對N2A細胞處理高濃度之維生素B12 (1000 M),APP C端蛋白也僅些微的降低,因此推測維生素B12極可能並非直接的透過APP-IRES活性抑制的機制來達到預防或治療阿茲海默症。另一方面,在細胞活性測試中發現維生素B12以及甲氧基薑黃素皆不會對細胞活性有影響,而薑黃素以及白藜蘆醇以高濃度下處理,對細胞存活則會有輕微影響。而本研究另一部分實驗也利用此藥物篩選平台,證實維生素B12具有抑制腸病毒71型 (EV-71)以及腦心肌炎病毒 (EMCV) 之IRES活性。 綜合上述,我們建立了以IRES為基礎的藥物篩選平台,成功的篩選出可以抑制APP、Tau蛋白、以及腸病毒之IRES活性的化合物,未來這些化合物極有潛力發展成為治療阿茲海默症及抗腸病毒感染之藥物。

並列摘要


ABSTRACT   This is a two-part study presenting the incorporation of internal ribosome entry site (IRES) elements in the construction of bi-cistronic plasmid vectors used as screening tool for the identification of compounds that may inhibit the cap-independent translation initiation without affecting the canonical cap-dependent mechanism of protein translation in eukaryotic cells.   Alzheimer’s disease (AD) is an alarming neurodegenerative disease that if not given the correct diagnosis and proper management could be life threatening. This disease is characterized primarily by the progressive loss in memory due to deposition of senile plaques mainly composed of -amyloid fragments and neurofibrillary tangles in AD patient’s brain. The bi-cistronic reporter assay was performed wherein the expression of first cistron, -galactosidase gene under the control of a cytomegalovirus (CMV) promoter, represent the canonical cap-dependent mechanism while the second cistron involves the utilization of the APP or the tau IRES elements to drive the expression of secreted alkaline phosphatase (SEAP) under the cap-independent mechanism. Selected bioactive compounds previously reported to have therapeutic potential for AD namely curcumin, demethoxycurcumin, resveratrol and vitamin B12 were screened using murine neuroblastoma (N2A) cells. Bi-cistronic reporter assay revealed that curcumin was more effective than demethoxycurcumin, a structural analog of curcumin, in inhibiting both APP and Tau IRES-dependent translation. This result was confirmed by Western blot analysis for the expression of APP, C-terminal protein, human Tau-1, phosphorylated Tau at Serine 262 (pS262) and Serine 396 (pS396) suggesting that curcumin may play a role in AD pathology alleviation through the inhibition of APP and Tau IRES-mediated translation mechanism. On the other hand, demethoxycurcumin was observed to inhibit the phosphorylation of both tau pS262 and pS396. Resveratrol either inhibited both the APP and Tau -dependent translation initiation over a wide concentration range (1 – 40 M) without affecting the cap-dependent translation and Western blot analyses confirmed the prior results. Furthermore, vitamin B12 stalled both the APP and Tau IRES activities in a dose-dependent fashion (100 – 1000 M). However, it seemed that vitamin B12 may not directly play a role in the prevention or treatment of AD through the APP-IRES dependent mechanism but may be via its involvement in other pathways. This is because there was only a minimal effect observed on the APP-C-terminal protein expression even after treatment of N2A cells with high concentration of 1000 M vitamin B12. There was marginal decrease in the expressions of phosphorylated tau pS262 and pS396 upon treatment with vitamin B12 starting at 300 – 1000 M. Based on the MTT assay results, vitamin B12 did not elicit any toxic effect on N2A cells while demethoxycurcumin was able to promote viability in N2A cells. Furthermore, curcumin and resveratrol slightly decreased the cell’s viability at higher concentrations (20 and 40 M). On a different note, the bi-cistronic reporter assay was also utilized to determine the anti-enterovirus 71 activity of vitamin B12. It is reported herein that vitamin B12 active inhibited the translation ability of IRESes derived from EV-71 and EMCV, although the potency of inhibition was less than that of the HCV IRES.   A novel assay system using the bi-cistronic reporter constructs for the identification of compounds with activity against the translation directed by APP, Tau and EV-71 IRES was developed. Results obtained provided suggestive insights for the potential use of the mentioned compounds as a prophylactic and therapeutic anti-AD and anti-viral (vitamin B12) agents.

並列關鍵字

IRES Alzheimer's disease

參考文獻


Chen, C.Y., Chang, Y.C., Huang, C.C., Lui, C.C., Lee, K.W., Huang, S.C., 2001. Acute flaccid paralysis in infants and young children with enterovirus 71 infection: MR imaging findings and clinical correlates. AJNR Am J Neuroradiol. 22, 200-5.
Clark, R.F., Hutton, M., Fuldner, M., Froelich, S., Karran, E., Talbot, C., Crook, R., Lendon, C., Prihar, G., He, C., Korenblat, K., Martinez, A., Wragg, M., Busfield, F., Behrens, M.I., Myers, A., Norton, J., Morris, J., Mehta, N., Pearson, C., Lincoln, S., Baker, M., Duff, K., Zehr, C., Perez-Tur, J., Houlden, H., Ruiz, A., Ossa, J., Lopera, F., Arcos, M., Madrigal, L., Collinge, J., Humphreys, C., Ashworth, A., Sarner, S., Fox, N., Harvey, R., Kennedy, A., Roques, P., Cline, R.T., Philips, C.A., Venter, J.C., Forsell, L., Axelman, K., Lilius, L., Johnston, J., Cowburn, R., Viitanen, M., Winblad, B., Kosik, K., Haltia, M., Poyhonen, M., Dickson, D., Mann, D., Neary, D., Snowden, J., Lantos, P., Lannfelt, L., Rossor, M., Roberts, G.W., Adams, M.D., Hardy, J., Goate, A., 1995. The structure of the presenilin 1 (S182) gene and identification of six novel mutations in early onset AD families. Nat Genet. 11, 219-22.
Abe, Y., Hashimoto, S.H.U., Horie, T., 1999. Curcumin inhibition inflammatory cytokine production by human peripheral blood monocytes and alveolar macrophages. Pharmacol Res. 39, 41-47.
AbuBakar, S., Chee, H.-Y., Al-Kobaisi, M.F., Xiaoshan, J., Bing Chua, K., Kit Lam, S., 1999. Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res. 61, 1-9.
Adams, B.K., Ferstl, E.M., Davis, M.C., Herold, M., Kurtkaya, S., Camalier, R.F., Hollingshead, M.G., Kaur, G., Sausville, E.A., Rickles, F.R., Snyder, J.P., Liotta, D.C., Shoji, M., 2004. Synthesis and biological evaluation of novel curcumin analogs as anti-cancer and anti-angiogenesis agents. Bioorgan Med Chem. 12, 3871-83.

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