Generation of Human iPSC-derived Entorhinal Cortex Neurons for Probing Selective Neuronal Vulnerability in Alzheimer's Disease

在阿尔茨海默病(AD)的早期阶段，只有特定类型的神经元受到病理损害的影响。 随着疾病的进展，更多的神经元类型逐渐开始表现出病理迹象和 整个大脑都在退化。治疗阿尔茨海默病(AD)的一个主要障碍是我们缺乏 了解这种选择性神经元脆弱性背后的分子机制。来自大脑的神经元 内嗅皮层的第二层(ECII)是大脑中最脆弱的神经元，但目前还没有 这些神经元的体外培养系统，阻止了对涉及神经元的基因的深入分析 脆弱性，以及寻找抑制影响这些神经元的早期神经退化的药物。在父级中 RF1授权，我们建议识别参与指定ECII神经元身份的转录因子(TF)，使用 ECII和对照神经元的详细分子图谱，我们在其不同的时间点生成 开发和尖端功能基因组分析。然后，这些TF将用于驱动 诱导多能干细胞向ECII神经元分化的研究虽然iPSC提供了一种多功能 平台可以分化为许多不同类型的神经元，有人担心神经元 来自ipscs的细胞在生物学上非常年轻，因为ipsc的生成协议重新设置了 一个牢房。AD相关的损害是年龄相关的，我们认为适合于这项研究的体外范式 衰老是至关重要的。因此，在本管理补充中，我们希望将此IPSC协议改编为直接 对人成纤维细胞进行重新编程，使用父母赠款中确定的TF。直接重新编程 成纤维细胞在生物学上不会重置，并呈现与成纤维细胞年龄相关的生物学年龄。 捐赠者。在优化了将成纤维细胞直接重新编程为ECII神经元的方案后，我们将提供 证明这些ECII神经元是通过以下方式探测选择性神经元脆弱性的良好模型 老年供者和AD供者成纤维细胞重编程神经元的Tau病理研究 突变。总的来说，这些直接从成纤维细胞重新编程的ECII神经元将是第一个体外模型 研究阿尔茨海默病选择性神经元脆弱性与衰老之间的相互作用。

In early phases of Alzheimer’s disease (AD) only specific types of neurons are affected by pathological lesions. As the disease progresses, additional neurons types gradually start expressing signs of pathology and degeneration throughout the brain. A major obstacle to treating Alzheimer’s disease (AD) is our lack of understanding of the molecular mechanisms underlying this selective neuronal vulnerability. Neurons from the layer II of the entorhinal cortex (ECII) are the most vulnerable neurons of the brain, but there are currently no in vitro culture system for these neurons, preventing both an in-depth analysis of genes involved in neuronal vulnerability, and the search for drugs inhibiting early neurodegeneration affecting these neurons. In the parent RF1 grant, we proposed to identify transcription factors (TFs) involved in specifying ECII neuron identity, using both detailed molecular profiles for ECII and control neurons, which we generated at different points of their development, and cutting-edge functional genomics analyses. These TFs will then be used to drive the differentiation of induced pluripotent stem cells (iPSC) into ECII neurons. While iPSCs provide a versatile platform that can be differentiated into many different neuron types, concerns have been raised that neurons derived from iPSCs are biologically very young, as the iPSC generation protocol “resets” the biological clock of a cell. AD-associated lesions are age-dependent, and we think that an in vitro paradigm adapted to this study of aging is crucial. In this administrative supplement, we thus want to adapt this iPSC protocol for the direct reprogramming of human fibroblasts, using the TFs identified in the parent grant. Directly reprogrammed fibroblasts are not reset biologically and present a biological age that correlates with the age of the fibroblast donor. After having optimized a protocol for direct reprogramming of fibroblasts into ECII neurons, we will provide a proof of concept that these ECII neurons are a good model for probing selective neuronal vulnerability by studying Tau pathology in neurons reprogrammed from fibroblasts of aging donors and of donors with AD mutations. Overall these ECII neurons directly reprogrammed from fibroblasts would be the first in vitro model for the study of the interplay between selective neuronal vulnerability in AD and aging.