基于患者C9ORF72-ALS-iPS细胞模型研究PTEN-knockdown的神经保护作用的机制

Amyotrophic lateral sclerosis (ALS) is characterized by loss of motoneurons and there is no effective treatment for this disease. AMPA receptor-dependent excitotoxicity is believed to be one of the key factors leading to the cell death in this disease. It is known that PTEN knockdown can exert significant neural protective effects in various pathological conditions. Our previous results have found that PTEN knockdown may protect human iPS-derived motoneurons by down-regulation of AMPA receptor expression which leads to increased cell viability through inhibiting excitatory transmission. In this project, we will differentiate the C9ORF72-ALS patient-specific induced pluripotent stem cells (iPS) into functional motoneurons and try to generate a human cell-based C9ORF72-ALS disease model which can facilitate its mechanistic study. We will try to unravel the protective effect of PTEN knockdown in C9ORF72-ALS patient’s iPS-derived motoneurons by studying the cell morphology, electrophysiological properties, RNA foci and protein expression of patient-iPS derived motorneurons. And we will also demonstrate whether PTEN knockdown could protect patient-iPS-derived motoneurons by decreasing the AMPA receptor-dependent excitotoxicity. Furthermore, we will confirm that PTEN knockdown will also reduce the apoptosis through PI3K/AKT signaling pathway by detecting the expression level of pAKT and pBAD. Lastly, we will manipulate the PI3K/AKT signaling pathway by inhibiting its upstream and downstream targets and test whether it plays any role in the expression and function of AMPA receptors. Based on aforementioned analysis, we might be able to reveal that PTEN knockdown may protect C9ORF72-ALS patient’s iPS-derived motoneurons by down-regulation of AMPA receptor via PI3K/AKT signaling pathway. Our study may provide a theoretical basis for therapeutic treatment of ALS.

ALS以运动神经元（MN）选择性病变为特征，尚无有效疗法。AMPA介导的兴奋毒作用是重要致病因素。已知PTEN下调可保护神经系统。本课题组前已证实：PTEN下调可抑制AMPA介导的兴奋毒作用，保护正常人iPS分化的MN。本项目拟将C9ORF72-ALS患者iPS分化为MN，建立疾病细胞模型。通过基于细胞形态、电生理功能和分子生物学的实验手段检测PTEN下调对患者MN形态、功能、RNA聚集、蛋白表达等疾病表型的改善作用；刺激或抑制AMPA受体，检测MN凋亡，验证PTEN下调可抑制AMPA介导的兴奋毒作用；通过检测pAKT和pBAD表达水平，验证PTEN下调可调节PI3K/AKT/BAD凋亡信号通路；分别抑制PI3K/AKT上下游信号通路，检测AMPA功能，验证PTEN可通过PI3K/AKT信号通路调控AMPA受体，对患者MN发挥神经保护作用。本项目拟为今后抗ALS新药研发提供理论基础。

肌萎缩侧索硬化症(Amyotrophic Lateral Sclerosis，ALS)是运动神经元选择性病变的神经退行性疾病，尚无有效的治疗办法。本项目完成了ALS患者特异 iPS 细胞系及对照者的细胞系的诱导建系工作，并在此基础上开展了运动神经元定向分化方法的新探索、基因治疗、基因编辑、细胞模型的应激反应等研究工作。我们所建立的多种细胞系为下一步研究ALS储备了丰富的种子资源；得到的运动神经元可以用于体外研究运动神经元细胞谱系决定的模型，为今后开展神经细胞移植治疗运动神经元疾病的研究奠定了基础；我们的研究能够为ALS的机制研究和药物治疗提供理论基础。.我们还将研究领域拓展到其他运动神经元疾病领域——构建了 3 种基于 DNA 构象转变的生物传感器，既实现了对 SMN1和 SMN2 基因的检测，又实现了荧光和可视化区分 SMN1 和 SMN2 的单碱基差异。该方法可应用于对 SMA 的基因诊断，从而为 SMA 的临床诊断和产前基因诊断带来帮助和突破。

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