Modulating an Astrocyte Hemichannel to Delay Spatial and Temporal Progression in ALS.

项目摘要/摘要 了解肌萎缩侧索硬化症(ALS)患者病情进展的原因 随着时间的推移发生在连续的解剖区域是设计疾病的基本限制之一 修改诊断后可以使用的治疗方法。 在ALS啮齿动物模型中的几项研究表明，星形胶质细胞在疾病传播中发挥作用 开始了。然而，星形胶质细胞功能障碍并不仅限于ALS啮齿动物模型， 重要的是，在ALS患者中进行原位检查时，这是最一致的观察结果之一 以及在体外使用人类细胞。 星形胶质细胞通过GAP在中枢神经系统形成高度耦合的细胞间网络 连接(GJS)和半通道(HC)由6个连接蛋白亚基组成，排列在中央气孔周围。 星形胶质细胞中的连接蛋白具有关键作用：动态平衡缓冲、星形胶质细胞网络的同步化、代谢 对神经元的支持，以及对血管生理的调节。它们还可以传播钙电波并调制 突触活动或通过半脑沟释放包括谷氨酸和三磷酸腺苷在内的神经胶质递质。缝隙连接蛋白43 Cx43是星形胶质细胞中主要的连接蛋白，在中枢神经系统中广泛表达。 我们最近发表的研究表明，星形胶质细胞Cx43的表达在 ALS患者的额叶皮质和脊髓，这一观察反映在ALS的SOD1小鼠模型中。这 现象不仅仅是反应性星形细胞增多症的非特异性影响，正如我们也能够展示的那样，使用 在体外和体内小鼠模型中，SOD1星形胶质细胞介导运动神经元毒性至少部分地， 通过Cx43半通道介导。 这项建议建立在这些初步观察的基础上，通过使用完全人性化的、脊髓特异性的ALS IPSC-星形胶质细胞/运动神经元平台研究Cx43 HC在HiPSC-星形胶质细胞膜上的定位 在肌萎缩侧索硬化症的背景下，研究Cx43 HC开放的调节机制，并理解这是如何 影响Cx43HC介导的ALS-HiPSC-星形胶质细胞对运动神经元的毒性。使用最新报告的特定 HC活性的阻滞剂现在我们将能够剖析Cx43 HC对运动神经元的特定贡献 死亡。当我们考虑翻译潜力时，我们将测试这些Cx43 HC阻滞剂可以穿透 血脑屏障具有良好的耐受性和口服可利用性，可在人iSPC来源的星形胶质细胞中发挥作用 在两种肌萎缩侧索硬化症啮齿动物模型中，锰中毒平台。我们从这项提案中学到的东西将使我们能够开发 将人体组织数据与新的体外建模相结合的ALS疗法的系统方法，以及 最后是体内的翻译应用。

Project Summary/Abstract Understanding why disease progression in the majority of patients with Amyotrophic Lateral Sclerosis (ALS) occurs in contiguous anatomic regions over time is one of the fundamental limitations to designing disease modifying therapies that can be utilized after a diagnosis. Several studies in ALS rodent models suggest that astrocytes play a role in disease propagation after onset. However, astrocyte dysfunction is not an observation merely limited to ALS rodent models but, importantly, has been one of the most consistent observations in humans with ALS when examined in situ as well as using human cells in vitro. Astrocytes form a highly coupled intercellular network in the central nervous system (CNS) through gap junctions (GJs) and hemichannels (HC) composed of 6 connexin subunits arranged around a central pore. Connexins in astrocytes have key roles: homeostatic buffering, synchronization of astrocyte networks, metabolic support for neurons, and regulation of vascular physiology. They can also propagate Ca2+ waves and modulate synaptic events or release gliotransmitters, including glutamate and ATP, through hemichannels. Connexin 43 (Cx43) is the predominant connexin in astrocytes and is expressed ubiquitously in the CNS. Our recently published studies have demonstrated that astrocyte expression of Cx43 is increased in the frontal cortex and spinal cords of ALS patients, an observation mirrored in the SOD1 mouse model of ALS. This phenomenon is not merely a non-specific effect of reactive astrocytosis as we were also able to show, using in vitro and in vivo mouse modeling, that SOD1 astrocyte-mediated motor neuron toxicity was, at least in part, mediated through Cx43 hemichannels. This proposal builds upon these initial observations by using a fully humanized, spinal cord-specific, ALS iPSC-astrocyte/motor neuron platform to investigate Cx43 HC localization at the hiPSC-astrocyte membrane, examine mechanisms by which Cx43 HC opening is modulated in the context of ALS, and understand how this affects Cx43 HC-mediated ALS hiPSC-astrocyte toxicity to motor neurons. Using newly reported specific blockers of HC activity we will now be able to dissect the specific contributions of Cx43 HC to motor neuron death. As we think about translational potential, we will test these Cx43 HC blockers that can penetrate the blood brain barrier, are well tolerated and orally available, for efficacy in human iSPC-derived astrocyte mediated MN toxicity platforms and in two ALS rodent models. What we learn from this proposal will allow us to develop a systematic approach to ALS therapeutics that combines human tissue data with novel in vitro modeling, and finally to in vivo translational applications.