探討KPI與Heparin-binding Domains對APP之Homodimerization及代謝之影響

Abstract

阿茲海默氏症(Alzheimer’s disease，AD)是一種漸進式的神經退化疾病，其組織病理學上的特徵之一為APP蛋白經酵素循序切割水解後產生的Aβ在細胞間隙堆積的老年斑。過去已經有許多研究顯示APP dimerization會對APP processing產生影響，而也有許多研究顯示heparin及其類似物會促進APP的dimer產生並且對processing也有所影響，但影響的效果依然存在爭議。 在本研究中，我們使用BiFC系統觀察活細胞中APP dimerization的程度，比較APP不同isoforms：APP751和APP695，以及外加不同濃度之heparin或fondaparinux對dimerization造成之影響。從螢光顯微鏡觀察以及流式細胞儀分析的結果發現，在此系統下的APP產生之螢光強度和control組並無差異，顯示在本研究之實驗條件下，BiFC系統似乎並不適合用於觀測APP dimerization。另外，Western blot分析的結果發現，只有少部分APP自然形成之dimer及其他oligomer可以在含SDS的環境下穩定存在，因此利用DSS將dimer鏈結在一起，以便能直接用SDS-PAGE/Western blot觀測APP的聚合反應，結果顯示含有KPI domain的APP751比不含KPI domain的APP695更容易形成dimer。在加入heparin後，APP751和APP695形成dimer及oligomer的比例似乎都有些微的提高，增加的比例在兩種isoforms間並沒有顯著差異；而在加入fondaparinux後，對APP751的聚合反應沒有影響，至於APP695，dimer及oligomer則都有增加，增加的程度和heparin作用時相似。 APP processing的研究則是直接用Western blot分析APP不同水解路徑的產物C99與C83間的比例，結果發現APP751的C99/C83比例較APP695為低，顯示APP751似乎更傾向走non-amyloidogenic pathway。在加入heparin後，隨著濃度的上升，APP751和APP695的C99/C83比值都有下降的趨勢，減少的比例在兩種isoforms間並沒有差異；而在加入fondaparinux後，APP751和APP695的C99/C83比值雖然都有下降，但似乎和fondaparinux濃度的變化無關，且影響程度較heparin為弱。 此研究結果顯示dimerization會促使APP傾向進行non-amyloidogenic pathway。而KPI domain會促使APP dimerization，且造成APP對不同鏈長的heparin促使之dimerization反應不同，另外，heparin可能藉由多種不同的機轉影響APP processing。

Alzheimer’s disease (AD) is a progressive neurodegenerative disease. One histopathological feature of AD is the intercellular senile plaques (SPs) formed by aggregation of amyloid β (Aβ), which is derived from sequential proteolytic processing of amyloid precursor protein (APP). Many previous studies have indicated that APP dimerization may have an impact on APP processing. Furthermore, heparin and its analogs have been reported to induce APP dimerization and influence its processing. However, the exact effect of these polysaccharide is still controversial. In this study, a bimolecular fluorescence complementation (BiFC) system is used to measure the dimerization level of two isoforms of APP, APP751 and APP695, in the absence or presence of various concentrations of heparin or fondaparinux. Fluorescent microscopy and flow cytometry data revealed that the fluorescence signals obtained from APP-positive cells cannot be distinguished from the control groups. This result suggests that our BiFC system is not suitable to measure the dimerization of APP under the conditions of our experiment. Besides, the results of Western blotting analysis indicate that only a small portion of APP dimers and oligomers are stable in the presence of SDS. Therefore, disuccinimidyl suberate (DSS) is used to crosslink adjacent protein molecules to observe APP oligomerization directly by SDS-PAGE/Western blot analysis. Our data indicate that KPI domain-containing APP751 forms more dimers than KPI domain-lacking APP695. After heparin treatment, dimer/monomer and oligomer/monomer ratio in APP751 and APP695 are both slightly enhanced. There is no significant difference in the increase in ratio between these two isoforms. However, fondaparinux does not affect APP751 oligomerization, but increases the levels of APP695 dimer and oligomer to the same extent as heparin does. The preference of the proteolytic pathway of APP is represented by the ratio of the levels of proteolytic products of APP from different processing pathway, C99 to C83, determined by Western blot analysis. Our data indicates that the C99/C83 ratio of APP751 is lower than APP695. It suggests that APP751 favors the non-amyloidogenic processing. After heparin treatment, C99/C83 ratio of APP751 and APP695 are both declined with the increase of heparin concentration. There is no significant difference in the decrease in ratio between these two isoforms. On the other hand, fondaparinux treatment leads to an decrease in the C99/C83 ratio of APP751 and APP695, but to a lesser extent than heparin in a dose-independent manner. In conclusion, results from this study indicate that increased APP dimerization favors non-amyloidogenic processing. KPI domain promotes APP dimerization, and results in different response of heparin-induced dimerization of various lengths of heparin chain. On the other hand, heparin may affect APP processing by diverse mechanisms.