Development of a screening strategy to identify A-beta resistance genes

项目总结 遗传因素在阿尔茨海默病(AD)的发病和发展中起着重要作用。一 阿尔茨海默病的特征是淀粉样蛋白(A？)神经症性斑块和纤维的发展，据信这在 在神经细胞丢失中起中心作用。大多数老年人在体内形成淀粉样斑块和纤维。 大脑，但只有一小部分老龄化人口会出现阿尔茨海默病的临床症状。这一发现表明 存在控制疾病发展和潜在疾病严重程度的基因调控途径。 对阿尔茨海默病的保护机制似乎存在，但尚未在遗传或 分子水平。在这里，我们建议在哺乳动物细胞中筛选提供保护的基因/通路。 以A？细胞毒抗性作为替代抗AD的方法。我们的实验室已经建立了一个图书馆 突变的小鼠胚胎干细胞(ES)，包含42,000个独立突变的小鼠胚胎干细胞 (PB)转座子介导的基因捕获。我们将利用A？42低聚物进行抗性筛选 通过高通量ES细胞平台进行正向遗传筛选中的突变。这种新颖的方法将允许 对基因组的无偏见询问，潜在地揭示了预防AD的新的功能途径， 这可能为AD治疗的治疗靶点提供新的见解。我们的长期计划是筛选 整个文库(42,000个突变体)，以分离A？抗性突变体，并鉴定潜在的基因和 小路。在本应用程序中，我们将建立必要的技术来选择A？抗性和 以确定抗性基因；将进行试点筛查，作为原则证明。A？会影响神经元，所以 我们将把突变的ES细胞分化为神经元，然后在96孔板上进行A？抗性筛选。 在Aü存在下存活率显著增加的突变株将被广泛隔离 特征，包括突变基因的鉴定(S)，突变事件的性质(空， 功能的部分丧失或功能的获得)，以及确认 基因突变和A？抗性表型。一旦这些基因被识别出来，它们的功能也可以 通过使用CRISPR对突变进行工程设计，在人类神经元中得到证实。本研究追求的是一种创新 包括使用干细胞、转座子和正向遗传学来识别基因/途径的方法 预防老年痴呆症。此外，携带AD保护性突变的ES细胞可以用于后续的 实验产生相应的小鼠用于活体研究。

PROJECT SUMMARY Genetic factors play an important role in the pathogenesis and progression of Alzheimer's disease (AD). One hallmark of AD is the development of amyloid ß (Aß) neuritic plagues and fibrils, which is believed to play a central role in neuronal cell loss. Most people of advanced age develop amyloid plagues and fibrils in their brains, but only a subset of the aging population develops clinical signs of AD. This finding suggests the existence of gene regulatory pathways controlling disease development and potentially disease severity. Protective mechanisms against AD appear to exist, but have not been characterized at the genetic or molecular level. Herein, we propose to screen for genes/pathways in mammalian cells that confer protection against AD by using resistance to Aß cytotoxicity as a surrogate. Our laboratory has generated a library of mouse mutant embryonic stem (ES) cells, comprising 42,000 independent mutants mutagenized by piggyBac (PB) transposon-mediated gene entrapment. We will utilize Aß42 oligomers for the selection of resistance mutants in a forward genetic screen via a high-throughput ES cell platform. This novel approach will allow for an unbiased interrogation of the genome, potentially revealing novel functional pathways protective against AD, which may provide new insights into therapeutic targets for AD treatment. Our long-term plan is to screen the entire library (42,000 mutants) to isolate Aß resistance mutants and identify the underlying genes and pathways. In this application, we will establish the necessary techniques required to select Aß resistance and to identify resistance genes; a pilot screening will be conducted as a proof of principle. Aß affects neurons, so we will differentiate our mutant ES cells into neurons, followed by selection for Aß resistance in 96-well plates. Mutants showing a significant increase in survival in the presence of Aß will be isolated for extensive characterizations, including the identification of the mutated gene(s), the nature of the mutational events (null, partial loss of function, or gain of function), and the confirmation of the causal relationship between the disrupted gene and the Aß resistance phenotype. Once those genes are identified, their functions can also be confirmed in human neurons by engineering the mutations using CRISPR. This study pursues an innovative approach encompassing the use of stem cells, a transposon, and forward genetics to identify genes/pathways protective against AD. Furthermore, the ES cells carrying AD-protective mutations may be used in subsequent experiments to generate the corresponding mice for in vivo studies.