細胞膜膽固醇耗竭增強Aβ與α-Synuclein誘導神經毒性促進路易氏體失智症病理變化

路易氏體失智症(Dementia with Lewy bodies, DLB)是一種神經退化性疾病，主要由突觸核蛋白(α-synuclein, α-syn)的錯誤折疊堆積造成神經細胞的損傷。當前對DLB的治療主要集中在緩解症狀，尚無法完全清除異常的蛋白質堆積。近年研究指出，周邊或神經系統膽固醇異常可能與認知功能下降有關；然而，細胞膜膽固醇穩定性是否會影響 Aβ 與 α-synuclein 共同誘導的 DLB-like 神經毒性，目前仍未明確。因此，我們假設膽固醇耗竭環境可能通過誘發氧化壓力，導致脂質過氧化並損傷細胞膜，進而增加DLB風險。在我們的實驗中為了重現DLB中觀察到Amyloid-beta (Aβ)和α-synuclein病理共存的現象，我們利用mifepristone誘導HT22 (Mouse Hippocampal Neuronal Cell Line)轉基因細胞使α-synuclein條件性過度表達，去建構一個結合α-synuclein的過度表達和Aβ暴露的體外模型。首先利用MTT assay去測量我們細胞實驗所需要的Aβ濃度，實驗結果顯示，當Aβ濃度為2.5 μM時，細胞存活率顯著下降。基於此，我們選擇了2.5μM的Aβ濃度來進行後續實驗。為了降低細胞膜中的有效膽固醇含量，我們使用了Methyl-β-cyclodextrin （MβCD）進行處理，並在處理24小時後再加入Aβ，觀察細胞的死亡情況。可以發現和加入Aβ相比，在加入MβCD後，細胞存活率明顯降低。接著我們會利用Dichlorodihydrofluorescein diacetate (DCFH-DA)染色來測量細胞內的活性氧物質多寡，有發現在加入MβCD後，活性氧物質增加。最後我們利用西方墨點法檢測細胞內的抗氧化功能。從目前的實驗結果顯示，在加入Aβ和MβCD後，細胞存活率明顯降低。我們也發現經MβCD處理後，細胞內活性氧增多，進一步加劇氧化壓力對細胞的損害，證實了膽固醇耗竭環境可能會通過氧化壓力機制，增加DLB的發病風險。綜合我們的研究結果顯示，膽固醇耗竭會使 Aβ加劇 α-synuclein的毒性使細胞存活率下降，另外我們也發現膽固醇耗竭會使粒線體膜電位下降，造成活性氧物質增加，氧化壓力上升，這些結果可以證明，膽固醇耗竭會增強Aβ和α-synuclein的神經毒性。因此，維持膽固醇穩定可能是預防或延緩與Aβ和α-synuclein病理相關的神經退化性疾病的重要因子。

Dementia with Lewy bodies (DLB) is a neurodegenerative disease characterized by neuronal damage caused by the misfolding and aggregation of α-synuclein (α-syn). Current treatments for DLB are largely limited to symptomatic treatment, as effective approaches to eliminate pathological protein aggregates are still lacking. Although low cholesterol levels have been confirmed to be associated with cognitive impairment, it remains unclear whether a similar relationship exists in the context of DLB. Accordingly, we hypothesized that a low-cholesterol environment may enhance oxidative stress, leading to lipid peroxidation and cell membrane damage, thereby increasing the risk of DLB. To test this hypothesis, HT22 mouse hippocampal neuronal cells were used as an in vitro model. To recapitulate the coexistence of Aβ and α-syn pathology observed in DLB, a conditional α-syn overexpression system was established using mifepristone induction in transgenic cells, thereby creating an in vitro model combining α-syn overexpression with Aβ exposure. An MTT assay was first performed to determine the appropriate concentration of amyloid-beta (Aβ), revealing a significant reduction in cell viability at 2.5 μM. This concentration was therefore selected for subsequent experiments. To reduce cholesterol content in cell membranes, cells were treated with methyl-β-cyclodextrin (MβCD), Aβ was added after 24 hours of treatment. Compared with Aβ treatment alone, cell viability was significantly reduced following MβCD treatment. Intracellular reactive oxygen species (ROS) levels were assessed using Dichlorodihydrofluorescein diacetate (DCFH-DA) staining, which demonstrated increased ROS production following cholesterol depletion. In addition, western blotting analysis was performed to examine cellular antioxidant capacity. Taken together, our results demonstrate that cholesterol depletion exacerbates Aβ-enhanced α-synuclein toxicity, leading to a significant reduction in cell viability. Furthermore, cholesterol depletion was found to decrease mitochondrial membrane potential, increase reactive oxygen species (ROS) production, and elevate oxidative stress. These findings suggest that cholesterol depletion plays a critical role in potentiating the neurotoxic effects of Aβ and α-synuclein. Therefore, maintaining cholesterol homeostasis may represent an important strategy for preventing or slowing the progression of neurodegenerative disorders associated with Aβ and α-synuclein pathology.