CAREER: Uncovering Mechanisms of Filopodia-Based Synaptogenesis

职业：揭示基于丝状伪足的突触发生机制

• 批准号: 2144912
• 负责人: Karen Litwa
• 金额: $ 123.87万
• 依托单位: East Carolina University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2144912&HistoricalAwards=false
• 关键词: CAREER; Uncovering; Mechanisms; Filopodia; Based

The brain supports complex cognitive processes, such as learning and memory. These complex processes require information transfer within the brain. Synapses mediate this information transfer. Yet, important details about the exact ways that synapses form in brain development are still unknown. To address this critical knowledge gap, this project examines synapse formation in rodent and human brain development using cutting-edge techniques for growing developing brain circuits in tissue culture preparations, which allow us unrivalled experimental access to developing synapses. Using these tools to uncover the detailed mechanisms of synapse formation will enable us to gain new insights into the emergence of complex cognitive processes. Since this project studies both rodent and human synapse formation, it will lead to the identification of shared and unique mechanisms underlying synapse formation in these two. By integrating this research with scientific outreach and educational activities, this project will also increase research opportunities for high school, undergraduate and graduate students. Students will be exposed to diverse scientific disciplines, including neuroscience and stem cell biology. Through the use of human brain models, students will develop skills in tissue engineering. This project will develop a microscopy course for undergraduates to learn advanced microscopy techniques. Furthermore, the use of microscopy in this research will result in both informative and beautiful images. This project will share the beauty of scientific discovery with the community by publicly displaying microscopy images in the community. Art will also be used as a medium to explore scientific concepts with K-12 students. Thus, this project will cultivate student and community science appreciation and interest, while also equipping a future generation of scientists with the knowledge and technical skills to tackle difficult biological questions.Excitatory synapses are an important basis of information transfer in neural circuits which give rise to complex cognitive functions. However, the mechanisms that initiate excitatory synapse formation in developing neural circuits are unclear. To address this gap, this research focuses on two fundamental questions: 1) what are the requirements for pre- and post- synaptic compartments to initially adhere? and 2) what determines whether this association persists and matures? The set of experiments will test the hypothesis that post-synaptic dendritic filopodia promote synapse formation in developing neural circuits. These experiments are designed to test the molecular requirements for filopodia to participate in distinct stages of excitatory synapse formation: axon association, contact stabilization, and morphological maturation. Neurons will be co-cultured in microfluidic systems to isolate the contributions of pre- and post-synaptic compartments to synaptogenesis. CRISPR interference will be used to alter the expression of specific molecules either separately or in combination and assess the resulting impact to synapse formation with confocal and STORM microscopy. Fluorescent biosensors will be used to perturb and monitor filopodia-mediated signaling events. Synapse formation will be observed in live neuron cultures and fixed brain tissue from both mice and humans. This research will also compare regional differences between synapse formation in the hippocampus and cortex. Successful completion of this research will result in the identification of conserved mechanisms of filopodia-mediated synaptogenesis, while also uncovering regional and species-specific differences in filopodial populations.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

大脑支持复杂的认知过程，如学习和记忆。这些复杂的过程需要大脑内的信息传递。突触介导这种信息传递。然而，关于突触在大脑发育中形成的确切方式的重要细节仍然是未知的。为了解决这一关键的知识差距，该项目研究了啮齿动物和人类大脑发育中的突触形成，使用尖端技术在组织培养制剂中生长发育中的大脑回路，这使我们能够无与伦比地获得发育中的突触。利用这些工具来揭示突触形成的详细机制将使我们能够对复杂认知过程的出现获得新的见解。由于该项目同时研究啮齿动物和人类的突触形成，它将导致识别这两种突触形成的共同和独特机制。通过将这项研究与科学推广和教育活动相结合，该项目还将增加高中生、本科生和研究生的研究机会。学生将接触到不同的科学学科，包括神经科学和干细胞生物学。通过使用人脑模型，学生将发展组织工程的技能。本计画将发展一个显微学课程，让大学生学习先进的显微技术。此外，在这项研究中使用显微镜将产生信息丰富和美丽的图像。该项目将通过在社区公开展示显微镜图像与社区分享科学发现的美丽。艺术也将被用来作为一种媒介，探索与K-12学生的科学概念。因此，这项计划将培养学生和社会对科学的欣赏和兴趣，同时也为下一代的科学家提供知识和技术，以解决困难的生物学问题。兴奋性突触是神经回路中信息传递的重要基础，产生复杂的认知功能。 然而，在发育中的神经回路中启动兴奋性突触形成的机制尚不清楚。为了解决这一差距，本研究集中在两个基本问题：1）突触前和突触后区室最初粘附的要求是什么？2）是什么决定了这种关联是否持续和成熟？这组实验将检验突触后树突状丝状伪足促进发育中神经回路突触形成的假设。这些实验的目的是测试参与兴奋性突触形成的不同阶段的丝状伪足的分子要求：轴突协会，接触稳定，形态成熟。神经元将在微流体系统中共培养，以分离突触前和突触后区室对突触发生的贡献。CRISPR干扰将用于单独或组合改变特定分子的表达，并通过共聚焦和STORM显微镜评估对突触形成的影响。荧光生物传感器将用于干扰和监测丝状伪足介导的信号事件。将在小鼠和人类的活神经元培养物和固定脑组织中观察突触形成。这项研究还将比较海马和皮层中突触形成的区域差异。这项研究的成功完成将导致丝状伪足介导的突触发生的保守机制的鉴定，同时也揭示了丝状伪足种群的区域和物种特异性差异。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。