State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR, China.
Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Hainan University, Haikou, Hainan 570228, China.
Phytomedicine. 2022 Jul;101:154125. doi: 10.1016/j.phymed.2022.154125. Epub 2022 Apr 25.
Parkinson's disease (PD) is a multi-factorial neurodegenerative disease affecting motor function of patients. The hall markers of PD are dopaminergic neuron loss in the midbrain and the presence of intra-neuronal inclusion bodies mainly composed of aggregation-prone protein alpha-synuclein (α-syn). Ubiquitin-proteasome system (UPS) is a multi-step reaction process responsible for more than 80% intracellular protein degradation. Impairment of UPS function has been observed in the brain tissue of PD patients. PDE4 inhibitors have been shown to activate cAMP-PKA pathway and promote UPS activity in Alzheimer's disease model. α-mangostin is a natural xanthonoid with broad biological activities, such as antioxidant, antimicrobial and antitumour activities. Structure-based optimizations based on α-mangostin produced a potent PDE4 inhibitor, 4e. Herein, we studied whether 4e could promote proteasomal degradation of α-syn in Parkinson's disease models through PKA activation.
cAMP Assay was conducted to quantify cAMP levels in samples. Model UPS substrates (Ub-G76V-GFP and Ub-R-GFP) were used to monitor UPS-dependent activity. Proteasome activity was investigated by short peptide substrate, Suc-LLVY-AMC, cleavage of which by the proteasome increases fluorescence sensitivity. Tet-on WT, A30P, and A53T α-syn-inducible PC12 cells and primary mouse cortical neurons from A53T transgenic mice were used to evaluate the effect of 4e against α-syn in vitro. Heterozygous A53T transgenic mice were employed to assess the effect of 4e on the clearance of α-syn in vivo, and further validations were applied by western blotting and immunohistochemistry.
Taken together, α-mangostin derivative 4e, a PDE4 inhibitor, efficiently activated the cAMP/PKA pathway in neuronal cells, and promoted UPS activity as evidenced by enhanced degradation of UPS substrate Ub-G76V-GFP and Ub-R-GFP, as well as elevated proteasomal enzyme activity. Interestingly, 4e dramatically accelerated degradation of inducibly-expressed WT and mutant α-syn in PC12 cells, in a UPS dependent manner. Besides, 4e consistently activated PKA in primary neuron and A53T mice brain, restored UPS inhibition and alleviated α-syn accumulation in the A53T mice brain.
4e is a natural compound derived highly potent PDE4 inhibitor. We revealed its potential effect in promoting UPS activity to degrade pathogenic proteins associated with PD.
帕金森病（PD）是一种多因素神经退行性疾病，影响患者的运动功能。PD 的标志性特征是中脑多巴胺能神经元丧失和主要由聚集倾向蛋白α-突触核蛋白（α-syn）组成的神经元内包涵体的存在。泛素蛋白酶体系统（UPS）是负责超过 80%的细胞内蛋白降解的多步反应过程。在 PD 患者的脑组织中观察到 UPS 功能受损。磷酸二酯酶 4 抑制剂已被证明可激活 cAMP-PKA 途径并促进阿尔茨海默病模型中的 UPS 活性。α-密蒙素是一种具有广泛生物学活性的天然黄烷酮，具有抗氧化、抗菌和抗肿瘤活性。基于 α-密蒙素的结构优化产生了一种有效的 PDE4 抑制剂 4e。在此，我们研究了 4e 是否可以通过激活 PKA 促进帕金森病模型中 α-syn 的蛋白酶体降解。
通过 cAMP 测定法定量样品中的 cAMP 水平。使用模型 UPS 底物（Ub-G76V-GFP 和 Ub-R-GFP）监测 UPS 依赖性活性。通过短肽底物 Suc-LLVY-AMC 研究蛋白酶体活性，该底物被蛋白酶体切割可增加荧光敏感性。使用 Tet-on WT、A30P 和 A53T α-syn 诱导型 PC12 细胞和来自 A53T 转基因小鼠的原代皮质神经元在体外评估 4e 对 α-syn 的作用。杂合 A53T 转基因小鼠用于评估 4e 在体内清除 α-syn 的作用，并通过 Western blot 和免疫组织化学进一步验证。
综上所述，磷酸二酯酶 4 抑制剂 α-密蒙素衍生物 4e 可有效激活神经元细胞中的 cAMP/PKA 途径，并通过增强 UPS 底物 Ub-G76V-GFP 和 Ub-R-GFP 的降解以及提高蛋白酶体酶活性来促进 UPS 活性。有趣的是，4e 可显著加速 PC12 细胞中诱导表达的 WT 和突变型 α-syn 的降解，这是一种依赖 UPS 的方式。此外，4e 可在原代神经元和 A53T 小鼠脑中持续激活 PKA，恢复 UPS 抑制并减轻 A53T 小鼠脑中的 α-syn 积累。
4e 是一种源自高度有效的 PDE4 抑制剂的天然化合物。我们揭示了其促进 UPS 活性以降解与 PD 相关的致病性蛋白的潜在作用。
