Signaling Roles of Intermediate Filament Isoforms in Neural Development

中间丝亚型在神经发育中的信号作用

• 批准号: 2215401
• 负责人: Bettina Winckler
• 金额: $ 130万
• 依托单位: University of Virginia Main Campus
• 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: 2022-05-15 至 2026-04-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2215401&HistoricalAwards=false
• 关键词: Signaling; Roles; Intermediate; Filament; Isoforms

This project aims to advance scientific knowledge by addressing an unanswered question in biology: what are the molecular principles that create different types of cells? The research focuses on a class of proteins which are expressed in strikingly cell-type specific patterns, the intermediate filament (IF) proteins. The project focuses on neurons in the brain which are known to switch IF subtype as they develop their mature shape, including extending long axons to the correct location in the brain. The research aims to determine why neurons switch from one IF subtype to another as they grow. The project will also broaden the impact by contributing to desired societal outcomes: 1) broadening education in STEM fields to support a better informed and more scientifically literate public, and 2) increasing the diversity and inclusivity of the STEM workforce. Especially, the lab aims for full participation of women, persons with disabilities, and underrepresented minorities in STEM at all levels. The project funds research experiences for undergraduates (REU) for one underrepresented minority student during each summer. The REU funded by this project provides hands-on experience in laboratory research by investigating the roles of diverse intermediate filaments in growing axons. In addition, the selected REU student will participate in the institutionally established SRIP (summer research internship program) at the University of Virginia which provides professional skill training and guidance for pursuing a career in STEM in individual and group settings. SRIP has a history of successfully placing participating undergraduate researchers into STEM careers.This project seeks to discover how developmental IF subtype switching from nestin to neurofilaments in neurons affects signaling to the microtubule cytoskeleton to regulate growth cone structure and motility. The PI’s lab recently discovered that the IF protein nestin regulates growth cone morphology. In addition, nestin changes the response of growth cones to the guidance cue Sema3a. It does so by binding the kinase Cdk5 and its substrate, the microtubule associated protein DCX, thereby increasing DCX phosphorylation. As neurons mature, they switch IF subtype from nestin to neurofilaments, which bind DCX but not Cdk5. The project will test the novel hypothesis that neuronal IFs regulate microtubule dynamics and bundling in axonal growth cones via DCX downstream of Sema3a signaling. Aim 1 will discover how experimental IF switching affects axonal microtubules and growth cone behavior. Aim 2 will discover how experimental IF switching affects Sema3a responses in cortical neurons. The project will use state-of-the-art imaging approaches in cultured cortical neurons in combination with downregulation and overexpression of different IF subtypes to uncover the mechanisms by which different IF subtypes modulate microtubule dynamics in growth cones during axon outgrowth. Responsiveness to extracellular cues by growth cones during development is influenced not only by the cue available, the receptors expressed on the surface, the intracellular substrates present, but also by the IF subtypes expressed. The PI proposes that neuronal IF subtypes can act as either amplifiers or dampeners of ubiquitous kinase cascades to provide local and cell-type specific regulation of effector responses.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.

该项目旨在通过解决生物学中一个悬而未决的问题来推进科学知识：创造不同类型细胞的分子原理是什么？该研究重点关注一类以惊人的细胞类型特异性模式表达的蛋白质，即中间丝 (IF) 蛋白质。该项目重点关注大脑中的神经元，这些神经元在形成成熟形状时会切换 IF 亚型，包括将长轴突延伸到大脑中的正确位置。该研究旨在确定神经元在生长过程中从一种 IF 亚型转变为另一种 IF 亚型的原因。该项目还将通过促进期望的社会成果来扩大影响：1）扩大 STEM 领域的教育，以支持更了解情况和更具科学素养的公众，2）增加 STEM 劳动力的多样性和包容性。特别是，该实验室的目标是让妇女、残疾人和代表性不足的少数群体充分参与各级 STEM。该项目每年夏天为一名代表性不足的少数族裔学生提供本科生研究经历 (REU) 资助。该项目资助的 REU 通过研究不同中间丝在轴突生长中的作用，提供实验室研究的实践经验。此外，入选的 REU 学生将参加弗吉尼亚大学机构设立的 SRIP（夏季研究实习计划），该计划为个人和团体环境中的 STEM 职业提供专业技能培训和指导。 SRIP 拥有成功将参与的本科研究人员纳入 STEM 职业的历史。该项目旨在发现神经元中从巢蛋白到神经丝的发育 IF 亚型转换如何影响微管细胞骨架的信号传导，从而调节生长锥结构和运动。 PI 的实验室最近发现 IF 蛋白巢蛋白调节生长锥形态。此外，巢蛋白改变生长锥对引导信号 Sema3a 的反应。它通过结合激酶 Cdk5 及其底物（微管相关蛋白 DCX）来实现这一点，从而增加 DCX 磷酸化。随着神经元成熟，它们将 IF 亚型从巢蛋白转变为神经丝，后者结合 DCX，但不结合 Cdk5。该项目将测试新的假设，即神经元 IF 通过 Sema3a 信号下游的 DCX 调节微管动力学和轴突生长锥的捆绑。目标 1 将发现实验性 IF 切换如何影响轴突微管和生长锥行为。目标 2 将发现实验性 IF 切换如何影响皮质神经元中的 Sema3a 反应。该项目将在培养的皮质神经元中使用最先进的成像方法，结合不同 IF 亚型的下调和过表达，以揭示不同 IF 亚型在轴突生长过程中调节生长锥微管动力学的机制。 发育过程中生长锥对细胞外信号的反应不仅受到可用信号、表面表达的受体、存在的细胞内底物的影响，而且还受到表达的 IF 亚型的影响。 PI 提出，神经元 IF 亚型可以充当普遍存在的激酶级联的放大器或抑制器，以提供效应器反应的局部和细胞类型特异性调节。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力优点和更广泛的影响审查标准进行评估，被认为值得支持。