Institute of Neuropathology, Bellvitge University Hospital-ICS, [Bellvitge Biomedical Research Institute-] IDIBELL, L'Hospitalet de Llobregat, Spain.
Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Ciencias y Tecnologías Químicas, Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Ciudad Real, Spain; Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Medicina de Ciudad Real, CRIB, Universidad de Castilla-La Mancha, Ciudad Real, Spain.
Neurobiol Dis. 2014 Sep;69:206-14. doi: 10.1016/j.nbd.2014.05.030. Epub 2014 Jun 2.
Adenosine A2A receptor (A2AR) is a G-protein coupled receptor that stimulates adenylyl cyclase activity. In the brain, A2ARs are found highly enriched in striatal GABAergic medium spiny neurons, related to the control of voluntary movement. Pharmacological modulation of A2ARs is particularly useful in Parkinson's disease (PD) due to their property of antagonizing dopamine D2 receptor activity. Increases in A2AR levels have been described in PD patients showing an important loss of dopaminergic denervation markers, but no data have been reported about A2AR levels in incidental PD brains. In the present report, we show that increased A2ARs protein levels were also detected in the putamen of incidental PD cases (Braak PD stages 1-2) with respect to age-matched controls. By contrast, A2ARs mRNA levels remained unchanged, suggesting that posttranslational mechanisms could be involved in the regulation of A2ARs. It has been described how miR-34b/c downregulation is an early event in PD cases. We found that miR-34b levels are also significantly reduced in the putamen of incidental PD cases and along disease progression. Given that 3'UTR of A2AR contains a predicted target site for miR-34b, the potential role of this miRNA in protein A2AR levels was assessed. In vitro studies revealed that endogenous A2AR protein levels increased when miR-34b function was blocked using a specific anti-miR-34b. Moreover, using a luciferase reporter assay with point mutations in a miR-34b predicted binding site within the 3'UTR region of A2AR mRNA abolished the effect of the miRNA using a miR-34b mimic. In addition, we showed a reduced percentage of DNA methylation in the 5'UTR region of ADORA2A in advanced PD cases. Overall, these findings reveal that increased A2AR protein levels occur in asymptomatic PD patients and provide new insights into the molecular mechanisms underlying A2AR expression levels along the progression of this neurodegenerative disease.
腺苷 A2A 受体（A2AR）是一种 G 蛋白偶联受体，可刺激腺苷酸环化酶活性。在大脑中，A2AR 高度富集于纹状体 GABA 能中间神经元中，与自主运动的控制有关。由于 A2AR 拮抗多巴胺 D2 受体活性的特性，对其进行药理学调节在帕金森病（PD）中特别有用。在表现出重要的多巴胺能去神经支配标志物丢失的 PD 患者中，已经描述了 A2AR 水平的增加，但尚未有关于偶然 PD 大脑中 A2AR 水平的报道。在本报告中，我们显示与年龄匹配的对照相比，偶然 PD 病例（Braak PD 阶段 1-2）的壳核中也检测到 A2AR 蛋白水平增加。相比之下，A2ARs mRNA 水平保持不变，这表明可能涉及翻译后机制来调节 A2ARs。已经描述了 miR-34b/c 的下调如何成为 PD 病例中的早期事件。我们发现，miR-34b 水平在偶然 PD 病例的壳核中以及疾病进展过程中也明显降低。鉴于 A2AR 的 3'UTR 包含 miR-34b 的一个预测靶标位点，因此评估了该 miRNA 在 A2AR 蛋白水平中的潜在作用。体外研究表明，当使用特定的抗 miR-34b 阻断 miR-34b 的功能时，内源性 A2AR 蛋白水平会增加。此外，使用带有 A2AR mRNA 3'UTR 区域中 miR-34b 预测结合位点点突变的荧光素酶报告基因测定，使用 miR-34b 模拟物消除了 miRNA 的作用。此外，我们显示在晚期 PD 病例中 ADORA2A 的 5'UTR 区域的 DNA 甲基化百分比降低。总体而言，这些发现表明，无症状 PD 患者中存在 A2AR 蛋白水平升高，并为沿这种神经退行性疾病进展的 A2AR 表达水平的分子机制提供了新的见解。
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