Patient-derived iPS cells as model systems for AxD

摘要 Alexander病(AxD)是由GFAP基因突变引起的星形胶质细胞原发疾病。 AxD突变如何导致蛋白质聚集和星形胶质细胞功能障碍 GFAP表达的星形胶质细胞导致神经元变性的机制尚不清楚。从进化的角度来看， 星形胶质细胞在人脑功能中发挥着越来越重要和复杂的作用。因此， 直接检测AxD患者星形胶质细胞的能力不仅是对现有模型的补充，而且 也揭示了人类星形胶质细胞在AxD发病机制中潜在的独特方面。我们已经建立了诱导式 三种不同突变的AxD患者的多能干细胞(IPSCs) 与梅辛实验室合作(项目3)。我们还开发了一种可复制的策略来 引导hPSCs分化为富含星形胶质细胞。我们的初步研究揭示了AxD中RFS的存在 患者的星形胶质细胞在培养中和移植到小鼠脑内后，突出了 重述我们IPSC系统中的关键AxD病理。然而，星形胶质细胞来源于AxD 患者的GFAP水平与非AxD患者相似，这表明GFAP可能不是 AxD的早期增加和GFAP蛋白的简单增加可能不是主要触发因素 用于人类细胞中RF的形成和星形胶质细胞功能障碍，如为模型动物所建议的。我们的能力 从AxD患者中培养丰富的星形胶质细胞也导致了AxD星形胶质细胞的发现 改变了GFAP-d/a亚型的比例，这导致了在 转基因小鼠。我们将使用先进的基因编辑技术来确定GFAP的变化 异构体介导GFAP突变对蛋白质聚集和/或星形胶质细胞功能障碍的影响。 通过神经元-星形胶质细胞共培养和神经移植，我们将确定AxD星形胶质细胞是否以及如何 导致神经元变性。作为计划项目的一部分，我们将验证学习到的AxD的修改器 从我们的AxD患者星形胶质细胞/神经元的动物模型中，为未来奠定基础 翻译。

ABSTRACT Alexander disease (AxD) is a primary disease of astrocytes caused by mutations in the gfap gene. How AxD mutations lead to protein aggregation and astrocyte dysfunction as well as how mutant GFAP-expressing astrocytes result in neuronal degeneration remain unknown. Evolutionarily, astrocytes play increasingly more important and complex roles in human brain functions. Hence, the ability to directly examine AxD patients' astrocytes will not only complement the existing models but also reveal potential unique aspects of human astrocytes in AxD pathogenesis. We have built induced pluripotent stem cells (iPSCs) from AxD patients with three different mutations through close collaboration with the Messing lab (project 3). We have also developed a reproducible strategy to guide hPSCs to enriched astrocytes. Our preliminary study revealed the presence of RFs in AxD patients' astrocytes in culture and following transplantation into the mouse brain, highlighting the recapitulation of key AxD pathology in our iPSC system. However, astrocytes derived from AxD patients exhibit comparable GFAP levels as non-AxD individuals, suggesting that GFAP may not increase at early stages of AxD and a simple increase in GFAP protein might not be the major trigger for RF formation and astrocyte dysfunction in human cells as proposed for model animals. Our ability to produce enriched astrocytes from AxD patients has also led to the discovery that AxD astrocytes have altered ratio of GFAP-d/a isoforms, which has led to the “rediscovery” of a similar ratio change in transgenic mice. We will use advanced gene editing technology to determine if change of GFAP isoforms mediates the effects of gfap mutations on protein aggregation and/or astrocyte dysfunction. By neuron-astrocyte co-culture and neural transplantation, we will determine if and how AxD astrocytes cause neuronal degeneration. As part of the program project, we will validate modifiers of AxD learned from animal models in our AxD patients' astrocytes/neurons, setting up the foundation for future translation.