Pax6 转录因子对老年痴呆(Alzheimer's)病脑神经元凋亡调控机制的研究

Alzheimer's disease (AD) is a progressive neurological disorder which is characterized by the neuronal dysfunction and loss in AD brain. Previous studies has shown that cell cycle components such as Cyclin dependent kinase 4 (Cdk4), pRb and transcription factor E2F1 play critical roles in various models of AD. However, the mechanisms by which cell cycle proteins regulate neuronal loss, especially the downstream events of this pathway, are largely unknown. In this study, we, for the first time, identified a novel E2F1 target gene Pax6 although it is known as a key transcription factor essential for the development of brain, eye and pancreas, as an important regulator of neuronal cell death evoked by Beta-amyloid (1-42) (Aβ1-42) treatment. Our preliminary data showed that, in murine embryonic cortical neurons, C-myb mRNA/protein levels，Pax6 mRNA/protein levels and transcription activity, increase with Aβ treatment. We also found that siRNA-mediated knockdown of Pax6 or C-myb protects neurons from Aβ induced neuronal death. Both E2F1 and C-myb can interact with Pax6 promoter region. We hypothesize that C-myb and Pax6 regulate downstream of cell cycle pathway, and are directly regulated by transcription factor E2F1. C-myb and Pax6 maybe required for Aβ induced neuronal apoptosis and play an important role in AD pathogenesis. We plan to use pax6 deficient mice/AD transgenic mouse modes to test this hypothesis in vivo and explore putative mechanism of C-myb and Pax6 involved in cell cycle mediated neuronal death pathway. Furthermore, based on our RNA-seq data of Pax6 silence in AD neurons, we shall assess the function of downstream novel targets of Pax6 in AD models. This study may provide insights into development of therapeutic agents and approaches for AD through inhibition of Pax6 in AD brain.

大脑中神经元缺失是阿尔茨海默症(AD)的一个重要特征。细胞周期相关分子-CDK4/pRb/E2F1等，在AD病变中，均起关键作用。 但其调控神经元凋亡的机制，特别是该通路下游调控，尚不清楚。我们的先期实验首次发现了在Aβ1-42诱导凋亡的小鼠胚胎皮层神经元中，C-myb和Pax6的mRNA、蛋白质水平以及转录活性均随着Aβ诱导而增加。如沉默Pax6或c-Myb的表达则保护皮层神经元，抵制Aβ对神经元的死亡诱导作用。E2F1和C-myb均可与Pax6的启动子区域结合。虽然Pax6已知在大脑、眼等发育中是一个重要的转录因子，Pax6 却是一个E2F1的新下游靶点起着重要作用。为证实这一新机制，我们将用Pax6突变小鼠和AD模型及分子和细胞技术究研这一新的通路；同时用RNA-seq先期结果发现pax6 新的下游靶点。这项研究可能为AD的治疗药物的开发提供新的思路，即抑制Pax6在AD中的表达。

阿尔兹海默病（AD）的两个主要病理学特征：淀粉样板块和神经纤维缠结（NFT）的分子作用关系是一个长期存在争议的问题。越来越多的证据显示，β淀粉样蛋白（Aβ）可诱导众多细胞周期调控因子的异常活化，包括Cdc25、Cdks和E2F1。本研究在AD的细胞模型中，发现Pax6是E2F1及其下游靶标c-Myb的直接作用靶点，这三个转录因子在培养皮层神经细胞中均可被Aβ上调。siRNA介导的E2F1沉默可阻断Aβ诱导的Pax6和c-Myb过表达，这表明E2F1在Pax6和c-Myb的上调中发挥了重要作用。重要的是，c-Myb或Pax6的下调可保护神经元，抵御Aβ多肽诱发的神经元凋亡，这说明c-Myb和Pax6是该信号通路中必需的下游介导因子。我们还发现Pax6在Aβ过表达的转基因小鼠中也有所升高。GSK-3β是在tau蛋白过度磷酸化和NFT的形成中起重要作用的激酶。Pax6可调控GSK-3β的转录。下调Pax6可阻断Aβ诱导的GSK-3β上调以及tau蛋白在Ser356, Ser396和Ser404位点的磷酸化。综上所述，我们的研究表明，Cdk/pRb/E2F1所在的信号通路通过激活c-Myb和Pax6这两个下游转录因子以及上调GSK3β来调控神经元死亡信号。Aβ诱发的神经毒性通过激活细胞周期以及随后的c-Myb/Pax6/GSK-3β，最终导致tau蛋白的过度磷酸化。该信号级联反应为进一步的药物干预提供了多个潜在靶标。