Novel role of TFEB in ADAM10 potentiation and proliferation of neural precursor cells relevant to Alzheimer's disease

项目摘要/摘要 阿尔茨海默病(AD)是一种复杂且高度异质性的疾病，其特征是 胞外和胞内蛋白质沉积。越来越多的数据表明，自噬-溶酶体途径 (ALP)在人类AD中严重受损，激活ALP可能具有治疗阿尔茨海默病的潜力 对抗阿尔茨海默病的神经退行性变。最近发现转录因子EB(TFEB)，一种 碱性螺旋-环-螺旋转录因子是整个碱性磷酸酶的主要调节者。有趣的是，我们发现 以Braak分期依赖的方式选择性丢失AD患者核TFEB蛋白水平 原代神经元暴露于Aβ寡聚体也显著降低了TFEB免疫反应性。到目前为止有多个 世界各地的实验室一再证实TFEB在显著降低蛋白质含量方面的作用 聚集在APP/PS1以及使用病毒载体表达TFEB的tautation模型中。通过 首次建立FLAG-TFEB转基因小鼠，我们发现TFEB的表达显著降低 在肌萎缩侧索硬化症的P301S模型中，tau过度磷酸化。 由于TFEB已在AD模型中显示出良好的效果，我们的持续研究取得了 令人惊讶的发现：TFEB增强α-分泌酶ADAM10的表达和sAPPα的产生 多个细胞系和原代神经元。更重要的是，这里我们第一次展示了TFEB表达式 增加体内成熟的ADAM10蛋白水平。Sappα和ADAM10都有许多有益的特性 包括神经保护，神经突起延伸，防止树突变性，增强记忆， 抑制tau的磷酸化和增加营养支持。更重要的是，ADAM10和SAPα 在神经前体细胞的增殖和成体神经发生中发挥着无可争辩的作用。这个 大多数AD临床试验的失败可能表明，仅仅降低Aβ水平可能不足以对抗 因此，可能需要建立一种增加额外福利的机制。因此，在本提案中使用 我们新产生的FLAG-TFEB小鼠，我们想评估TFEB是否可以缓解与年龄相关的 BrdU+、NeuN+、DCX+及其倍体定量检测神经前体细胞数量下降与神经再生 在脑室下区和颗粒区通过测量空间记忆，运动活动， ADAM10活性和SAPα水平。更关键的是，我们还设计了实验，以确定 TFEB在神经发生中作用的机制基础。我们还将测试TFEB是否能够提供显著的 器官培养中对兴奋性毒性和A-β寡聚体的保护。使用抗体对抗 ADAM10/SAPPα，我们还将讨论ADAM10/SAPPα是否负责神经保护。如果是肯定的 这些结果可能会开启TFEB及其作用途径研究的新思路 研究AD模型中的神经发生，最终TFEB可能被证明是治疗AD的良好靶点。

PROJECT SUMMARY/ABSTRACT Alzheimer’s disease (AD) is a complex and a highly heterogeneous disease characterized by the deposition of extracellular and intracellular protein deposits. Accumulating data suggest that autophagy-lysosome pathway (ALP) is severely compromised in human AD and activation of ALP is likely to have therapeutic potential for combating neurodegeneration in the AD. It was recently discovered that the transcription factor EB (TFEB), a basic helix-loop-helix transcription factor is a master regulator of the entire ALP. Interestingly, we found the selective loss of nuclear TFEB protein levels in Braak stage-dependent manner in AD patients and that exposure of primary neurons to Aβ oligomers also markedly reduced TFEB immunoreactivity. So far multiple laboratories around the world have repeatedly confirmed the role of TFEB in markedly reducing the protein aggregates in the APP/PS1 as well as tauopathy models using viral vectors for TFEB expression. By generating flag-TFEB transgenic mice for the first time, we showed that TFEB expression significantly reduces hyperphosphorylated tau in the P301S model of tauopathy. Since TFEB has demonstrated promising effects in models of AD, our continued investigations made a surprising discovery that TFEB potentiates the α-secretase ADAM10 expression and sAPPα generation in multiple cell lines and primary neurons. More importantly, here for the first time we show that TFEB expression increases mature ADAM10 protein levels in vivo. Both sAPPα and ADAM10 have many beneficial properties including neuroprotection, neurite extension, prevention of dendritic degeneration, memory enhancement, inhibition of tau phosphorylation and increased trophic support. More importantly, both ADAM10 and sAPPα have shown indisputable role in the proliferation of neural precursor cells (NPCs) and adult neurogenesis. The failure of most AD clinical trials may suggest that mere reductions in Aβ levels may be insufficient to combat AD and that a mechanism to increase additional benefits may be needed. Therefore in this proposal by using our newly generated flag-TFEB mice we want to assess whether TFEB can mitigate the age-associated decline in the number of NPCs and neurogenesis by quantifying BrdU+, NeuN+, DCX+ and their double positives.in the subventricular zone and subgranular zone by measuring spatial memory, motor activity, ADAM10 activity, and sAPPα levels. More crucially, we have also designed experiments to identify the mechanistic basis for TFEB’s role in neurogenesis. We will also test whether TFEB can provide significant protection against excitotoxicity and Aβ oligomers in the organotypic cultures. Using antibodies against ADAM10/sAPPα, we will also address whether ADAM10/sAPPα are responsible for neuroprotection. If positive results are obtained, it may lead to the initiation of a new line of research on TFEB and its pathway on the role of neurogenesis in models of AD and ultimately TFEB may turn out to be excellent therapeutic target for AD.