探討在路易氏體失智症中β類澱粉蛋白對α-Synuclein誘導之神經毒性

路易氏體失智症(Dementia with Lewy bodies, DLB)是僅次於阿茲海默症(Alzheimer’s disease,AD)第二常見的神經退化性疾病，而α-Synuclein與β類澱粉蛋白(beta-Amyloid, Aβ)分別為此兩種疾病患者腦部中的個別主要病理特徵，其機制皆為該蛋白質不正常摺疊堆積在神經細胞當中。由於不少DLB患者腦內會同時合併AD的病理特徵，故以往文獻中多以LB variant of AD命名，然而相較於AD患者，DLB患者的病程進展非常快速，但目前對於Aβ究竟是如何促使α-Synuclein沈積與造成神經毒性之機轉則尚未完全明瞭。為了解決這問題，本研究的目的在於探討DLB的病理發展過程中，Aβ是否影響了α-Synuclein的聚集，甚至是干擾了清除機制因而產生神經毒性。為了進行本研究，我們建立一株條件式調控α-Synuclein大量表現的神經細胞株，再對此細胞處理Aβ來模擬DLB患者腦部的情況，並藉此這個模式來研究可能造成DLB的病理機制；此外我們也同時建立了路易氏體失智症的大鼠動物模式，用以探討Aβ是否促進α-Synuclein的聚集並干擾清除機制而產生神經毒性，在此研究中使用12週齡之雄性Wistar大鼠，以立體定位手術給予大鼠腦室內注射α-Synuclein基因載體（10 μg/10 μL/rat），並於雙側大腦皮質注射Aβ（10 μg/5 μL/side），動物手術後接受各種行為試驗，包括：短期記憶、學習能力及物件辨識能力試驗，並行為實驗結束後摘取動物全腦進行神經組織免疫染色，半腦用以測量腦組織內α-Synuclein之含量，並收集血液及腦組織以西方點墨法測量生化參數以檢驗神經毒性及細胞自噬活性。結果發現，在細胞實驗中以MTT觀察到在α-Synuclein大量表現的細胞中，Aβ毒性顯著增強，以西方墨點法及螢光染色也發現細胞凋亡的比例上升，且細胞自噬有被抑制的現象，而對抗老化及對抗ROS相關蛋白表現則降低，此外利用螢光染色觀察到細胞出現粒線體功能障礙(mitochondrial dysfunction)；動物實驗中也驗證了細胞實驗的結果，此外行為試驗中也觀察到，DLB模擬疾病組不論在記憶或是學習能力上相較正常組大鼠表現較差。總體來說，本研究認為Aβ可能藉由直接影響α-Synuclein聚集，或是抑制清除錯誤α-Synuclein的細胞自噬機制來參與DLB疾病的致病過程。

Dementia with Lewy bodies (DLB) is the second most common cause of dementia, trailing Alzheimer’s disease (AD), which possessing a large proportion of demented population. In these two diseases, α-Synucleinand beta-Amyloid (Aβ) are the main pathologies in DLB and ADpatients brain respectively, and both proteins involved the mechanism of misfolded proteins in neurodegeneration. In the past, patients with DLB revealed similar pathology with AD in their brains; thus, the name LB variant of AD was used in previous documents. Comparing to AD patients,DLB patientsdevelop a much sooner process in disease progression; however, the mechanism of deposition and neurotoxicity of α-Synuclein by Aβis not yet clarified. In order to understand the effects of Aβ on α-Synucleinaccumulation, clearance, and production of neurotoxicity, we investigated the mechanisms of DLB pathology through established a conditionaloverexpressing α-Synuclein cell line. Furthermore, we treated the cells with Aβto mimic the pathological condition within DLB brains. In addition, tofurther understanding neurotoxicity of Aβin α-Synuclein accumulation and clearance, twelve weeks old male Wistar rats werei.c.v. injected by α-Synucleinvector (10 μg/10μL/rat) into brain ventricle, and/orAβ（10 μg/5 μL/side）into double sides of cortex by stereotactic surgery. After the brain lesion, animal behavioral tests were performed including short-term memory, learning, and object recognition test. Finally, rats were scarified in order to observe the level of α-Synuclein through Immunohistochemistry; moreover, blood and brain tissue were also collected and analyzed to understand neurotoxicity and autophagy through western blotting. Our data showed that overexpression of α-Synuclein increasesneurotoxicityby Aβ co-treatment. According to western blotting and fluorescent staining, the results displayed an increasing in apoptosisbut inhibition in autophagy. Furthermore, proteins which related to aging and ROS were also decreased. In addition,mitochondrial dysfunction was shown by fluorescent staining. More interestingly, DLB animal showed deficits both in memory and learning ability comparing to normal control through behavioral tests. In summary, our results suggest that Aβ maycontribute to the DLB pathogenesis by affecting the accumulation of α-Synuclein or interfering autophagic clearancemechanism.