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

调节星形胶质细胞半通道以延迟 ALS 的空间和时间进展。

• 批准号: 10033432
• 负责人: NICHOLAS J MARAGAKIS
• 金额: $ 39.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10033432
• 关键词: ALS patients; Affect; Amyotrophic Lateral Sclerosis; Anatomy; Astrocytes; Astrocytosis; Autopsy; Behavioral; Biological Markers; Biology; Blood - brain barrier anatomy; Blood Vessels; Body part; Brain; Buffers; Cell model; Cells; Cessation of life; Chronic; Clinical; Clinical Trials; Coculture Techniques; Complement; Connexin 43; Connexins; Coupled; Data; Development; Diagnosis; Disease; Disease Progression; Dose; Electrophysiology (science); Environment; Event; Eye; Functional disorder; Gap Junctions; Glutamates; Human; Hyperactive behavior; In Situ; In Vitro; Knock-out; Learning; Libraries; Link; Measures; Mediating; Membrane; Metabolic; Modeling; Motor Neurons; Mus; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Neurons; Oral; Pathologic; Pathway interactions; Patient observation; Patients; Pharmacology; Physiology; Play; Probability; Property; Proteins; Proteomics; Publications; Publishing; Regulation; Reporting; Rodent Model; Role; Safety; Spinal Cord; Symptoms; Synapses; Testing; Therapeutic; Time; Tissues; Toxic effect; Translating; Treatment Efficacy; clinical phenotype; design; disease phenotype; epigenomics; extracellular; frontal lobe; human tissue; in vitro Model; in vivo; induced pluripotent stem cell; insight; metabolomics; mouse model; multi-electrode arrays; neuron loss; neuroprotection; neurotoxic; neurotoxicity; novel; patient population; release factor; repository; superoxide dismutase 1; transcriptomics

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.

项目总结/摘要 了解为什么大多数肌萎缩侧索硬化症（ALS）患者的疾病进展 随着时间的推移发生在相邻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ALS后的疾病传播中发挥作用。 发病然而，星形胶质细胞功能障碍并不仅仅局限于ALS啮齿动物模型， 重要的是，当在原位检查时， 以及在体外使用人类细胞。 星形胶质细胞在中枢神经系统（CNS）中通过间隙形成高度耦合的细胞间网络 连接点（GJ）和半通道（HC），其由围绕中心孔排列的6个连接蛋白亚基组成。 连接蛋白在星形胶质细胞中具有关键作用：稳态缓冲，星形胶质细胞网络的同步化，代谢， 支持神经元和调节血管生理。它们还可以传播Ca ~（2+）波， 突触事件或释放胶质递质，包括谷氨酸和ATP，通过半通道。连接蛋白43 Cx43是星形胶质细胞中主要的连接蛋白，在CNS中广泛表达。 我们最近发表的研究表明，星形胶质细胞Cx43的表达增加， ALS患者的额叶皮层和脊髓，这一观察反映在ALS的SOD 1小鼠模型中。这 这种现象不仅仅是反应性星形胶质细胞增多症的非特异性效应，正如我们也能够证明的那样， 体外和体内小鼠模型中，SOD 1星形胶质细胞介导运动神经元毒性，至少部分， 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，通过使用一个完全人性化的，脊髓特异性的，ALS iPSC-星形胶质细胞/运动神经元平台以研究hiPSC-星形胶质细胞膜处的Cx43 HC定位， 检查Cx43 HC开放在ALS背景下调节的机制，并了解这是如何发生的。 影响Cx43 HC介导的ALS hiPSC-星形胶质细胞对运动神经元的毒性。使用新报告的具体 HC活性的阻断剂，我们现在将能够剖析Cx43 HC对运动神经元的特异性作用。 死亡当我们考虑翻译潜力时，我们将测试这些Cx43 HC阻断剂，它们可以穿透细胞膜， 血脑屏障，耐受性良好，可口服，在人iSPC衍生的星形胶质细胞介导的 MN毒性平台和两种ALS啮齿动物模型。我们从这项建议中学到的东西将使我们能够发展 将人体组织数据与新型体外建模相结合的ALS治疗的系统方法，以及 最后涉及体内翻译应用。