Probing Alzheimer synaptopathy in neurons derived from engineered human iPS cells

描述（由申请人提供）：在本项目中，我们将表征源自iPS细胞的人诱导神经元（iN）细胞中高风险阿尔茨海默病（AD）突变的突触、细胞和生化表型。我们相信，多能干细胞生物学和表观遗传重编程的新进展将提供一个重要的突破，因为它们允许人类神经元的遗传修饰和功能评估。因此，现在有可能在功能上询问风险突变并研究它们在人类神经元中的细胞生物学效应。特别是，人类诱导多能干细胞（iPS）中基因靶向工具的最新进展以及我们最近开发的从iPS细胞产生全功能诱导神经元（iN）细胞的快速方法为开始将该技术应用于脑疾病（如AD）的疾病建模提供了理想的条件。我们将向对照iPS细胞系中引入来自充分表征的健康正常受试者的条件突变，其赋予AD的高风险。在我们在初步研究中开发的方案中，将使用同源重组引入突变，然后将条件突变iPS细胞转化为精确匹配的野生型和突变iN细胞。突变体和对照细胞将表征Ab和Tau生物化学，重要的是详细的突触特性。我们相信，对精确突触表征的关注代表了我们提案的关键创新因素，因为突触功能障碍可能比其他细胞生物学测定（如细胞死亡）更敏感。最后，我们已经在我们的iN细胞/星形胶质细胞共培养系统中证实，ApoE主要由神经胶质产生，并且ApoE是神经胶质诱导的原代神经元和人iN细胞的突触成熟的关键介质，ApoE 3和ApoE 4具有可能不同的作用。在这些结果的基础上，我们建议在这个特定的目标中评估ApoE 3和ApoE 4对Aim 1中产生的野生型和APP突变iN细胞中突触成熟的精确作用，目的是深入了解ApoE 4在AD病理学中的作用。这些特定的目标结合在一起，将使我们能够对AD相关APP突变对人类神经元及其突触特性的影响进行良好的对照评估。

DESCRIPTION (provided by applicant): In this Project, we will characterize synaptic, cellular and biochemical phenotypes of high-risk Alzheimer Disease (AD) mutations in human induced neuronal (iN) cells derived from iPS cells. We believe that the new advances in pluripotent stem cell biology and epigenetic reprogramming will provide an important breakthrough as they allow the genetic modification and functional evaluation of human neurons. Therefore, it is now possible to functionally interrogate risk mutations and study their cell biological effects in huma neurons. In particular, recent advances of gene targeting tools in human induced pluripotent stem (iPS) cells and our recent development of rapid methods that generate fully functional induced neuronal (iN) cells from iPS cells provide ideal conditions to begin to apply this technology to disease modeling for brain diseases such as AD. We will introduce into control iPS cell line derived from a well-characterized healthy normal subject conditional mutations that confer high risk for AD. Mutations will be introduced using homologous recombination in a protocol that we have developed in preliminary studies, and the conditionally mutant iPS cells will then be converted into precisely matched wild-type and mutant iN cells. Mutant and control cells will be characterized for Ab and Tau biochemistry and importantly for detailed synaptic properties. We believe the focus on the precise synaptic characterization represents a key innovative factor of our proposal as synaptic dysfunction may be much more sensitive than other cell biological assays such as cell death. Finally, we have confirmed in our iN cell/ astrocyte co-culture system that ApoE is primarily produced by the glia and that ApoE is a critical mediator of the glia-induced synaptic maturation of primary neurons and human iN cells, with possibly different effects of ApoE3 and ApoE4. Building on these results, we propose to evaluate in this specific aim the precise effects of ApoE3 and ApoE4 on synaptic maturation in wild type and APP-mutant iN cells generated in Aim 1, with the goal of gaining insight into the role of ApoE4 in AD pathology. Applied together, these specific aims will allow us to perform a well-controlled assessment of the effect of AD-associated APP mutations on the properties of human neurons and their synapses.