新型HDAC6抑制劑在神經保護及改善阿茲海默症模式大鼠認知能力之研究

Abstract

實驗方法：本研究利用細胞轉染技術，將神經細胞株過量表達β-amyloid前身蛋白與易磷酸化之突變Tau蛋白，建立阿茲海默症模式神經細胞株，以螢光顯微鏡及西方點墨法建立及檢定轉染條件，同樣以西方點墨法觀察compound A在神經細胞株中抑制HDAC6酵素活性之能力及減少Tau蛋白磷酸化累積之情形；利用流式細胞儀觀察細胞週期變化；免疫沉澱法觀察compound A影響熱休克蛋白90與HDAC6蛋白結合程度、乙醯化HSP90累積量與磷酸化Tau蛋白與泛素結合之情形。在活體實驗中，利用腹腔注射scopolamine建立大鼠阿茲海默症大鼠模型，並藉由舉臂式十字迷宮、莫氏水迷宮及海馬迴之免疫組織化學染色確認神經保護及改善認知能力之效果。 實驗結果：在兩株不同的神經細胞株中，轉染後可顯著誘導出Tau蛋白在Ser396位置之磷酸化，compound A藉由抑制HDAC6活性，增加細胞內乙醯化α微管表現量，且與臨床二期HDAC6抑制劑 (Rocilinostat, ACY1215) 相比更為顯著。同時compound A也抑制HDAC6與HSP90的結合作用，促使HSP90乙醯化，進而與其下游Tau蛋白分離，失去保護的磷酸化Tau會經由蛋白酶體路徑被降解，藉由減少神經細胞內累積的磷酸化Tau蛋白堆積，達到神經保護功效。在動物實驗中口服給予compound A可增加海馬迴CA1區域之Ac-α-tubulin表現量，減少海馬迴中經由scopolamine誘導出的磷酸化Tau表現，並且改善AD模式大鼠的行為能力：舉臂式十字迷宮實驗中的記憶學習能力及莫氏水迷宮的空間認知能力，證實compound A有良好的神經保護功效。 結論：實驗結果顯示，compound A具有良好的神經保護效果，透過抑制HDAC6活性可以減少磷酸化Tau蛋白在神經細胞中的累積，抑制細胞細胞凋亡，在活體實驗中也有明顯改善AD模式大鼠記憶學習及空間認知能力，因此compound A是一個有潛力可以發展成為神經性疾病治療藥物的新型HDAC6抑制劑。

Objective：Alzheimer’s disease (AD) is a neurodegenerative disorder and the most common cause of dementia. Pathological features of AD are senile plaques (SPs), comprised of aggregated Aβ and intracellular neurofibrillary tangles (NTFs) that consist of aggregated and hyperphosphorylated tau protein in the hippocampus of AD patients. Previous studies showed that HDAC6 was overexpressed in AD patient brain. Overexpressing intracellular HDAC6 induces the phosphorylated tau aggregate and cognitive deficits in neuron and AD rodent model. The aim of this study is to investigate the effect and mechanism of the novel HDAC6 inhibitor, compound A, on neuronal protection and cognitive function of AD rat model. Methods：In this study, we overexpressed two kinds of plasmids, hAPP695 and hTauP301L, to establish the AD cell model and using the fluorescence microscope and western blot to examine the transfection effect. The inhibition effect of HDAC6 and the accumulation of phosphorylated tau protein were evaluated by western blot. Cell cycle was studied by flow cytometry. Immunoprecipitation was used to evaluate acetylation of heat shock protein 90 (HSP90), the interaction between HDAC6 and HSP90, and the ubiquitinated tau protein. We established the AD rat model by the scopolamine intraperitoneal injection. Morris water maze, elevated plus maze and immunohistochemistry stain (IHC) were used to determine the effect of neuronal protection and cognitive function of test compounds in AD rat model. Result：We overexpressed two kinds of plasmids, hAPP695 and hTauP301L, in neuron cells to induce the phosphorylated tau (Ser396). Comparing with ACY1215 (Rocilinostat), a HDAC6 inhibitor in phase II clinical trial, compound A significantly increased intracellular acetyl-α-tubulin level by inhibiting the activity of HDAC6 in the low dosage (0.1μM). In addition, compound A decreased the HDAC6/HSP90 binding and then increased HSP90 acetylation. The acetyl-HSP90 decrease the affinity of HSP90 and phosphorylated tau, leading to chaperone function impairment and phosphorylated tau degradation by proteasome pathway. Therefore, compound A show the neuron protective effect by reducing tau aggregates. In animal studies, compound A not only significantly improved the cognition in elevated plus maze and Morris water maze tests, but also ameliorated the pathological features in the animal brain. Conclusion：Our results demonstrate that compound A has potent neuron protective effect by inhibiting HDAC6 activity and down regulation phosphorylated tau aggregation, then reducing cell apoptosis. The in vivo results also shown compound A ameliorated cognitive deficits. Therefore, it suggests that compound A has a potential as a therapeutic ahent in AD treatment.