Feature characteristics and signaling mechanisms involved in SGN neurite guidance by engineered topographical and biochemical micropatterns

项目摘要/摘要 耳蜗利用一种复杂的传入神经组织来高效地处理 复杂的听觉刺激。为了创造这种精确组织的模式，螺旋神经节神经元的神经突起 (SGN)在细胞、细胞外基质和生化梯度的复杂环境中导航，以达到其 Corti器和耳蜗核分别有外周和中央靶点。轴突的这个过程 在环境中成长被称为寻路。在寻路过程中，轴突的尖端，生长锥体， 对生物化学和生物物理信号作出反应，感知、转向并向目标生长。这项建议 研究的重点是了解不同的底物线索(地形、化学吸引力和 化学斥力)可以促进SGN轴突转向，也可以通过信号通路促进生长锥 来执行这一基本的生物过程。 以前的工作已经证实，工程微图案化的衬底可以用于(1)直接SGN (2)研究这一现象在体外激活的信号通路。我们已经证明了 神经元将它们的突起与各种工程化的图案化底物(地形、化学吸引力和 化学脉冲剂)。此外，我们还发现RhoA-GTP酶和钙信号与SGN中的 随着地形的变化而生长。我建议使用这种工程化的微图案基板来研究(1) 决定轴突转折的地形和生化线索的层次性和交互作用 生长锥体对这些信号做出反应所必需的生物化学途径。尤其是，我 将研究化学吸引力(层粘连蛋白)和化学排斥性(EphA4)如何与地形生长线索相互作用 当被放置在互补(在低谷中吸引人)或对立(在低谷中令人厌恶)的模式时。另外， 我将利用药理学和生物学方法研究Rho/ROCK和IP3信号在这一基本生物过程中的作用 实时成像在各种图案化的衬底上生长时这些通路的激活。 总体而言，这项研究的目标是更好地了解SGN神经突起如何形成的关键的基本生物学过程 对底物暗示作出反应的感觉和转弯。我希望通过利用小说为这一领域贡献知识 3D组合微图案化衬底、感兴趣路径的实时成像方法以及 机器学习图像分类模型在评估神经突起行为反应中的无偏方法 到微图案化的基板。这些新颖的见解将为SGN寻路的许多方面提供信息(1) 确定SGN在转弯时是否使用类似的基本信号通路 生物物理和生化线索，(2)阐明耳蜗声直视组织的机制 发展，以及(3)虽然这不是一个直接针对翻译的建议，但调查结果也将推动目标的实现 诱导有组织的轴突生长到接近脑梗塞的位置。

Project Summary/Abstract The cochlea exploits an intricate tonotopic organization of afferent innervation to effectively process highly complex auditory stimuli. To create this precisely organized pattern, the neurites from spiral ganglion neurons (SGNs) navigate a complex milieu of cells, extracellular matrix, and biochemical gradients to reach their peripheral and central targets in the organ of Corti and cochlear nuclei, respectively. This process of a neurite growing through the environment is called pathfinding. In pathfinding, the tip of the neurite, the growth cone, senses, turns, and grows toward a target in response to biochemical and biophysical cues. This proposed research focuses on understanding how different substrate cues (topography, chemoattractive, and chemorepulsive) can promote an SGN neurite to turn as well as by which by signaling pathways a growth cone relies on to execute this basic biological process. Previous work has identified that engineered micropatterned substrates can be used to both (1) direct SGN growth and (2) study the signaling pathways activated by this phenomenon in vitro. We have demonstrated that neurons align their outgrowth to various engineered patterned substrates (topographical, chemoattractive, and chemorepulstive). Additionally, we have implicated RhoA-GTPase and calcium signaling in SGNs aligning their outgrowth to topographical cues. I propose to use this engineered micropatterned substrates to study (1) the hierarchy and interaction of topographical and biochemical cues in dictating neurite turning and (2) the biochemical pathways necessary for a growth cone to sense and turn in response to these cues. In particular, I will study how chemoattractive (laminin) and chemorepulsive (EphA4) interact with topographical growth cues when placed in complimentary (attractive in troughs) or antagonistic (repulsive in troughs) patterns. Additionally, I will research the role of Rho/ROCK and IP3 signaling in this basic biological process using pharmacology and imaging the activation of these pathways in real time when growing on various patterned substrates. Overall, the goal of this research is to better understand the key, basic biological process of how an SGN neurite senses and turns in response to substrate cues. I expect to contribute knowledge to this field by utilizing novel 3D combination micropatterned substrates, real time imaging approaches of the pathways of interest, and a machine learning image sorting model to use an unbiased approach in assessing neurite behavior in response to the micropatterned substrates. These novel insights will inform many aspects of SGN pathfinding through (1) determining if similar fundamental signaling pathways are used by SGNs when turning in response to both biophysical and biochemical cues, (2) clarifying the mechanisms of how the tonotopic organization of the cochlea develops, and (3) though this is not directly a translationally aimed proposal, the findings will also further the goal of inducing organized neurite growth into close proximity to a CI.