Investigating mechanisms of neuronal growth and synapse formation

研究神经元生长和突触形成的机制

• 批准号: RGPIN-2019-06332
• 负责人: Singh, Karun
• 金额: $ 2.33万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744183
• 关键词: Investigating; mechanisms; neuronal; growth; synapse

The formation of neuronal connections (synapses) between nerve cells (neurons) is a fundamental hallmark in the nervous system. Synapses are critical for brain function as they mediate cognitive processes. Synapses form by the coordinated actions of signaling molecules that carryout structural and functional changes to promote growth and maturation. Our long-term goal is to uncover novel signaling mechanisms regulating mammalian synapse formation, to better understand how the nervous system develops. Typically, only a single isoform of a given protein is studied within a signaling pathway. However, proteins can have multiple isoforms that have very different activities in neurons. Therefore, our understanding of whether different isoforms of the same protein family have distinct or similar signaling networks is significantly under developed. This knowledge would strenghthen our basic understanding of nervous system development . Therefore in the short term, we are studying a member of the Ste family of kinases with multiple isoforms that have been poorly studied during synapse development, to identify isoform-specific signaling mechanisms. Our previously work on the Ste family member, thousand and one amino acid 2 (TAOK2) revealed that mice lacking TAOK2 have structural and functional defects in cortical excitatory synapses. However, TAOK2 has two predominant protein isoforms (alpha and beta), but the details of when and how these isoforms regulate synapse development, maturation and plasticity are unknown. We have generated proteomics reagents to study this problem and identify binding partners of each TAOK2 isoform using BioID, a method for screening protein-protein interactions. We will use proteomic data in combination with our TAOK2 mouse model to perform functional studies to understand how the novel TAOK2 isoform-specific signaling pathways regulate synapse development. Our functional studies include the use of state-of-the-art approaches including electrophysiology on individual neurons, and electrophysiology on populations of neurons with new multielectrode array technology. Our program will address previous limitations and provide novel mechanistic insight into how kinases regulate synapse development. The novel and unbiased proteomic techniques will reveal unappreciated details into how a kinase with multiple isoforms controls different aspects of synapse formation and maturation. If we are successful, our future program will perform isoform-specific proteomics and functional validation experiments on other synaptic proteins, including the novel TAOK2-interacting proteins, which will significantly advance our fundamental understanding of neuronal signaling. Lastly, our program will make use of emerging and state-of-the-art experimental approaches including molecular and cellular biology, proteomics, biochemistry, gene editing and electrophysiology, providing HQP a cutting-edge and dynamic training environment.

神经细胞（神经元）之间的神经元连接（突触）的形成是神经系统中的基本标志。突触对大脑功能至关重要，因为它们介导认知过程。突触是通过信号分子的协调作用形成的，这些信号分子进行结构和功能变化以促进生长和成熟。我们的长期目标是发现调节哺乳动物突触形成的新信号机制，以更好地了解神经系统如何发育。 通常，在信号传导通路内仅研究给定蛋白质的单一同种型。然而，蛋白质可以有多种亚型，在神经元中具有非常不同的活性。因此，我们对同一蛋白质家族的不同亚型是否具有不同或相似的信号网络的了解还远远不够。这些知识将加强我们对神经系统发育的基本理解。因此，在短期内，我们正在研究Ste激酶家族的一个成员，该家族具有在突触发育过程中研究较少的多种亚型，以确定亚型特异性信号传导机制。我们之前对Ste家族成员，第一千零一个氨基酸2（TAOK 2）的研究表明，缺乏TAOK 2的小鼠在皮质兴奋性突触中存在结构和功能缺陷。然而，TAOK 2有两种主要的蛋白质亚型（α和β），但这些亚型何时以及如何调节突触发育，成熟和可塑性的细节尚不清楚。我们已经产生了蛋白质组学试剂来研究这个问题，并使用BioID（一种筛选蛋白质-蛋白质相互作用的方法）鉴定每个TAOK 2亚型的结合伴侣。我们将使用蛋白质组学数据结合我们的TAOK 2小鼠模型进行功能研究，以了解新的TAOK 2亚型特异性信号通路如何调节突触发育。我们的功能研究包括使用最先进的方法，包括对单个神经元的电生理学，以及使用新的多电极阵列技术对神经元群体的电生理学。我们的计划将解决以前的局限性，并提供新的机制如何激酶调节突触发育的见解。新的和公正的蛋白质组学技术将揭示未知的细节如何激酶与多种亚型控制突触的形成和成熟的不同方面。如果我们成功了，我们未来的计划将对其他突触蛋白进行亚型特异性蛋白质组学和功能验证实验，包括新型TAOK 2相互作用蛋白，这将大大推进我们对神经元信号传导的基本理解。最后，我们的计划将利用新兴和最先进的实验方法，包括分子和细胞生物学，蛋白质组学，生物化学，基因编辑和电生理学，为HQP提供一个尖端和动态的培训环境。