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

项目总结/摘要 阿尔茨海默氏病（AD）是一种复杂的高度异质性疾病，其特征在于 细胞外和细胞内蛋白质沉积。越来越多的资料表明自噬-溶酶体途径 (ALP)在人类AD中严重受损，ALP的激活可能具有治疗AD的潜力。 对抗AD中的神经变性。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ALP的主要调节因子。有趣的是，我们发现 在AD患者中，以Braak分期依赖的方式选择性丢失核TFEB蛋白水平， 原代神经元暴露于Aβ寡聚体也显著降低TFEB免疫反应性。到目前为止， 世界各地的实验室已经反复证实TFEB在显着减少蛋白质中的作用， 在APP/PS1中的聚集体以及使用病毒载体用于TFEB表达的tau蛋白病模型。通过 首次产生flag-TFEB转基因小鼠，我们发现TFEB表达显著降低， 在tau蛋白病的P301 S模型中过度磷酸化的tau。 由于TFEB在AD模型中表现出有希望的效果，我们的继续研究取得了进展。 令人惊讶的发现是，TFEB增强了α-分泌酶ADAM 10的表达和sAPPα的产生， 多个细胞系和原代神经元。更重要的是，在这里我们第一次表明TFEB表达 增加体内成熟的ADAM 10蛋白水平。sAPPα和ADAM 10都具有许多有益的特性 包括神经保护、神经突延伸、预防树突变性、记忆增强 抑制tau磷酸化和增加营养支持。更重要的是，ADAM 10和sAPPα 在神经前体细胞（NPC）的增殖和成体神经发生中显示出无可争议的作用。的 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 AD，可能需要一种机制来增加额外的好处。因此，在本提案中， 我们新产生的flag-TFEB小鼠，我们想评估TFEB是否可以减轻年龄相关的 通过定量BrdU+、NeuN+、DCX+和它们的双磷酸化， positives.in通过测量空间记忆，运动活动， ADAM 10活性和sAPPα水平。更重要的是，我们还设计了实验来识别 TFEB在神经发生中作用的机制基础。我们还将测试TFEB是否能提供显著的 在器官型培养物中对兴奋性毒性和Aβ寡聚体的保护。使用抗 ADAM 10/sAPPα，我们还将讨论ADAM 10/sAPPα是否负责神经保护。如果呈阳性 这些结果的获得，可能会导致对TFEB及其作用途径的新的研究路线的启动 在AD模型中的神经发生和最终TFEB可能成为AD的极好治疗靶点。

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.