| 摘要: | 阿茲海默症是最常見的一種漸進式腦神經退化疾病，在病理學上的特徵是神經細胞外的類澱粉胜肽堆積，被認為是造成阿茲海默症的主因。然而AD的致病機轉至今仍有許多爭議，並無有效地治癒藥物與策略。為了明白類澱粉胜肽導致神經細胞死亡的機制，因此提出『Recruiting Hypothesis』，用來描述類澱粉胜肽與細胞膜之間交互作用機轉。 本研究為了驗證『Recruiting Hypothesis』，利用實驗設計分別以動力學及熱力學分析Aβ在溶液中聚集機轉及Aβ與人工合成脂膜交互作用，最終再以動物細胞模式驗證Aβ與類神經細胞(PC12)之交互作用。 (1) Aβ在溶液中聚集機轉： Aβ在溶液中是以random coil結構存在，在聚集的過程中Aβ的結構會由random coil轉變為β-sheet，且Aβ是以疏水作用力來穩定β-sheet之二級結構。另，Aβ聚集的動力學分析發現成核反應為Aβ聚集的速率決定步驟；而成核反應主要是以疏水作用力主導。在離子強度效應會導致Aβ具有相異的反應機制，形成不同型態的Aβ聚集體。 (2) Aβ與人工合成脂膜交互作用： Aβ與細胞膜作用會加速Aβ聚集形成纖維狀。Aβ在生理環境中會以靜電作用被吸引到細胞膜表面，隨後誘發GM1與膽固醇聚集形成raft-like。當中的Aβ會調整其構型為β-sheet，並吸引溶液中其他的Aβ聚集。最後raft-like間彼此聚集，形成富含Aβ、GM1及膽固醇的相，因此造成細胞膜不穩定，導致細胞功能降解甚至死亡。 (3) Aβ與類神經細胞(PC12)之交互作用： Aβ的細胞毒性隨著Aβ的濃度與C端序列長度增加；fresh Aβ在與PC12細胞作用時，較纖維狀的Aβ有較高的細胞毒性。降低細胞膜中膽固醇及GM1含量能夠有效地降低Aβ細胞毒性。由Aβ與PC12交互作用的熱量分析，也得到與細胞活性分析相符合的結果。 研究結果與文獻報導都支持所提出的『Recruiting Hypothesis』。因此，可藉由『Recruiting Hypothesis』中對Aβ與細胞膜間交互作用行為的描述，設計出治療AD的策略或藥物。另，研究中利用ITC即時地偵測Aβ與細胞交互作用時，PC12細胞的代謝熱量變化。此生物熱力學量測的方法具有快速且直接量測等優點，可用來觀測藥物/生物分子與活體細胞交互作用的熱量變化，甚至可得到細胞生理學的有益資訊，是極具發展潛力的偵測方法。 AD is the most common neurodegenerative disease. The pathological hallmark of extra-cellular β-amyloid (Aβ) deposit is considered as one of the primary factors in inducing Alzheimer’s disease (AD). However, the mechanism of Aβ deposition on the cell membrane and the induced cytotoxicity is still unclear. The major obstacle in designing an effective therapeutic or strategy lies in our incomplete understanding of the mechanism of AD. On the basis of the previous reports and results, the “Recruiting Hypothesis” was proposed on the interaction between the plasma membrane and Aβ. For “Recruiting Hypothesis” verification , this study is analyzed by kinetic and thermodynamic methods which divided into three parts which shown as follows. (1) Investigation of the mechanism of beta-amyloid fibril formation In the aggregation process, the secondary structure of Aβ (1-40) transforms to the β-sheet conformation, which could be described as a two-state model. As the temperature and ionic strength increase, the conformation of Aβ converts to the β-sheet structure with an increased rate. Results of circular dichroism monitoring demonstrate that the rate constant of nucleation is smaller than that of elongation, and the nucleation is the rate-determining step during the overall Aβ aggregation. The β-sheet structure was stabilized by hydrophobic force as revealed by the ITC measurements. The different structural aggregates and forming pathways could be identified and discriminated at high and low ionic strengths, resulting in distinctive fibril conformations. Furthermore, the thermodynamic analysis shows that hydrophobic interaction is the major driving force in the nucleation step. (2)Studying the interaction between beta-amyloid and artificial cell membranes Results from SPR and lipid monolayer trough studies showed that the rate of Aβ adsorption onto lipid monolayer/liposome is mainly due to the electrostatic effect which is sensitive to the lipid monolayer/liposome composition. Due to the electrostatic attraction of more number of GM1 by the Aβ leads to the formation of GM1 clusters. The GM1 clusters incurred cholesterol recruitment and form raft-like structure Consequently, the Aβ conformation changed to β-sheet, which acts as a seed and initiates a chain reaction, in that it attract other Aβs to interact with the GM1. This resulted in the accumulation of Aβ on the plasma membrane. At the same time, both GM1 and cholesterol accumulate more and form larger clusters. Finally, each clusters aggregate with each other and form Aβ, GM1 and cholesterol rich phase which resulted in the function of membrane degradation. (3)Examining the interaction between beta-amyloid and PC12 cell Monomeric Aβ could attack the plasma membrane resulting in cytotoxicity, however, fibrillar Aβ was found to be less toxic. Aβ (1-40) was more toxic than Aβ (25-35) and the cytotoxcity of Aβ was proportional to its concentration. Besides, the depletion of GM1 from plasma membrane, it would block the Aβ-induced cytotoxicity. Decreasing the cholesterol level by around 30 % could attenuate the cytotoxicity of Aβ. These findings validate our idea that the cholesterol could stabilize the lateral pressure derived from the formation of GM1-Aβ complex on the membrane surface. Furthermore, both GM1 and cholesterol are essential in mechanism of Aβ accumulation and could modulate the cytotoxicity of monomeric Aβ. All these results list above and published references are support “Recruiting Hypothesis”. Understanding the insight of the interaction between Aβ and cell membrane which provide by “Recruiting Hypothesis” could be helpful in developing medicines and strategies aimed to cure AD. In addition, a biothermodynamic approach to real-time monitor the heat of metabolism by isothermal titration calorimetry (ITC) during PC12 cell-Aβ (1-40) interaction was provided by this study. This approach with rapid and directly measurement may provide not only real-time information for the interaction between Aβ and live cell but also more options for candidate drug development. |
