Role of Norrin in synaptic biology during development, adulthood, and disease

Norrin 在发育、成年和疾病期间突触生物学中的作用

• 批准号: 10348333
• 负责人: Emily G Thompson
• 金额: $ 6.64万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-07 至 2025-06-06
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10348333
• 关键词: Role; Norrin; synaptic; biology; during

PROJECT SUMMARY Neuronal synaptic connections are essential for executing the distinct tasks that are required for brain homeostasis and function. Disturbances to synaptic biology during development or in mature neuronal networks are a key hallmark of most neurodevelopmental disorders and neurodegenerative diseases, respectively. Thus, a better understanding of the mechanisms that govern the establishment and maintenance of synapses could provide new insights into the origins of these disorders and potential therapeutic opportunities. Extra-neuronal influences, whether via direct cellular contact or released proteins, have been demonstrated to be critical in governing synaptic physiology. Outside of the broad metabolic responsibilities that astrocytes fulfill in the brain during normal physiology, it is becoming increasingly clear that astrocytes are instrumental in modulating neuronal synaptic connections. Specifically, astrocytic interactions with neuronal synapses are increasingly implicated in 1) the establishment of proper synaptic connections and 2) dysfunctions that negatively impact synaptic biology. Our group was the first to document the involvement of alternative Wnt ligand, Norrin, in synaptic properties—a protein long associated with the X-linked genetic disorder Norrie Disease (ND). Namely, Norrin enhanced neuronal firing frequency and network connectivity in in vitro cultures and caused synaptic abnormalities in vivo in adult Norrin-null mice. However, the mechanistic basis of these findings remains unknown, as well as whether Norrin is important in synaptogenesis. Thus, the overall goal of this proposal is to define the role that Norrin plays in synaptic biology, including the establishment of these connections during development and maintenance in adulthood. Additionally, this study will be the first to use a humanized mouse model of ND to provide characterization of the inherent and synaptic properties of ND cortical neurons. These objectives will be pursued with murine models, including Norrin-null mice and human Norrin gene point mutation (V45E) mice. First, I will determine the physiological mechanism by which Norrin mediates enhanced neuronal firing frequency and connectivity using whole-cell patch clamp electrophysiology in in vitro and ex vivo preparations. Additionally, I will use molecular biology and imaging techniques to evaluate Norrin’s effect on synaptic neurotransmitter receptor expression. Secondly, I will investigate the involvement of Norrin in synaptogenesis by utilizing iDISCO, single-cell RNAseq, and RNAscope in the postnatal mouse brain. Using the V45E mice I will characterize the electrophysiological properties of cortical neurons in the mature ND brain. Lastly, utilizing high-content in vitro and in vivo imaging, real-time synaptic dynamics will be evaluated in conditions of functional and dysfunctional Norrin across the synaptogenesis period. This proposal will provide novel insights into Norrin-mediated synaptic modulation during development, normal physiology, and ND pathology. The fundamental understanding of this facet of synaptic biology will have broad implications for other neurodevelopmental and neurodegenerative diseases.

项目摘要 神经元突触连接对于执行大脑所需的不同任务至关重要 稳态和功能。发育期间或成熟神经元网络中突触生物学的干扰 分别是大多数神经发育障碍和神经变性疾病的关键标志。因此，在本发明中， 更好地理解控制突触建立和维持的机制， 为这些疾病的起源和潜在的治疗机会提供了新的见解。神经元外的 无论是通过直接的细胞接触还是释放的蛋白质， 控制着突触生理学除了星形胶质细胞在大脑中履行的广泛的代谢职责之外， 在正常生理过程中，越来越清楚的是星形胶质细胞在调节 神经元突触连接具体来说，星形胶质细胞与神经元突触的相互作用越来越多， 涉及1）建立适当的突触连接和2）功能障碍， 突触生物学我们的小组是第一个记录替代Wnt配体Norrin参与的人， 突触特性-一种长期与X连锁遗传疾病诺里病（ND）相关的蛋白质。也就是说， Norrin在体外培养中增强神经元的放电频率和网络连接，并引起突触 在成年Norrin缺失小鼠中的体内异常。然而，这些发现的机制基础仍然存在， 未知，以及Norrin是否在突触发生中很重要。因此，本提案的总体目标是 来定义Norrin在突触生物学中的作用，包括这些连接的建立 在成年期的发育和维持过程中。此外，这项研究将是第一个使用 人源化的ND小鼠模型，以提供ND的固有和突触性质的表征 皮质神经元这些目标将用鼠模型来追求，包括Norrin缺失的小鼠和人模型。 Norrin基因点突变（V45 E）小鼠。首先，我将确定诺林的生理机制 使用全细胞膜片钳电生理学介导增强的神经元放电频率和连接 在体外和离体制备中。此外，我将使用分子生物学和成像技术来评估 诺林对突触神经递质受体表达的影响。其次，我会调查 Norrin在突触发生中的作用：利用iDISCO、单细胞RNAseq和RNAscope在出生后小鼠大脑中进行研究。 使用V45 E小鼠，我将描述成熟ND中皮质神经元的电生理特性。 个脑袋最后，利用高含量的体外和体内成像，实时突触动力学将被评估， 条件的功能和功能障碍Norrin在突触发生期间。该提案将提供 对发育、正常生理和ND过程中Norrin介导的突触调节的新见解 病理对突触生物学这一方面的基本理解将对其他研究产生广泛的影响。 神经发育和神经变性疾病。