SUBCELLULAR DOMAINS OF MYELINATING-GLIA: CAPTURING AXONAL CONTACT.

描述(申请人提供)：神经胶质细胞必须在多个轴上极化。例如，星形胶质细胞、放射状胶质细胞、髓鞘形成胶质细胞或终末雪旺细胞参与多种细胞-细胞和细胞-基质相互作用(即与神经元、内皮细胞、肌肉和基底膜)。这种复杂的细胞结构对胶质细胞的功能至关重要，但在空间上也是不可接近的。因此，很难分离出专门的亚细胞隔室用于生化研究。因此，研究神经胶质细胞的一个主要障碍是分子的不可及性。 发生在相关亚细胞隔间的事件。我们已经调整了一个系统，通常用于分离极化细胞突起，以响应可溶性刺激，以适应神经元-雪旺细胞的相互作用。我们介绍了使用神经元细胞膜作为刺激，而不是可溶性或细胞外基质分子，来模拟神经胶质细胞中细胞与细胞之间的相互作用的创新。为此，我们将雪旺细胞放置在带有微孔过滤器的改良Boyden小室中，并将它们暴露在底部小室的神经细胞膜中。这会导致雪旺细胞极化和片状伪足的延伸。然后，伪足和细胞体可以在物理上分开，并对它们的内容进行比较。我们对这些伪足进行了蛋白质组学和蛋白质印迹分析，发现了位于轴突-神经胶质相互作用部位的已知分子，验证了该系统。我们现在建议使用这个系统来识别轴突-神经胶质相互作用中的新参与者，使用大型感觉神经元，并在雪旺细胞中添加第二个极化线索(细胞外基质)。接下来，我们将询问该系统是否可以用于研究神经元和其他胶质细胞之间的相互作用，即少突胶质细胞和星形胶质细胞。该系统可以适应多种野生型或突变型胶质细胞与细胞的相互作用，以探讨它们在蛋白质或RNA极化、形成特定分子复合体或蛋白质修饰方面的作用。这些现象与神经胶质细胞的生理和病理功能有关。这种变革性的资源可以克服在专门的细胞连接上研究重要的神经胶质功能的困难。好了！ 与公共健康相关：我们正在开发一种创新的系统，在培养皿中研究胶质细胞的相互作用和功能。该系统可用于研究神经胶质细胞特定区域的功能，这些区域对正常大脑、周围神经功能和几种神经疾病具有重要意义。

DESCRIPTION (provided by applicant): Glial cells must polarize on multiple axes. For example astrocytes, radial glia, myelin-forming glia or terminal Schwann cells engage in multiple cell-cell and cell-matrix interactions (i.e., with neurons, endothelial cells, muscle and basal lamina). This complex cytoarchitecture is crucial for glial function, but is also spatially inaccessible. As a result it is difficult to isolate specialized subcellular compartments for biochemical studies. Thus a major obstacle to the study of glia is the inaccessibility of molecular events occurring in relevant subcellular compartments. We have adapted a system, normally used to isolate polarized cell protrusion formed in response to soluble stimuli, to neuronal-Schwann cell interactions. We introduced the innovation of using neuronal cell membranes as stimulus, instead of soluble or extracellular matrix molecules, to mimic cell-cell interactions in glial cells. To this end we placed Schwann cells on a modified Boyden chamber with microporous filters, and exposed them to neuronal cell membranes in the bottom chamber. This causes Schwann cells polarization and extension of lamellipodia-like pseudopodia. Pseudopodia and cell bodies can then be physically separated and their contents compared. We performed proteomic and western blot analysis on these pseudopods, and found known molecules located at sites of axo-glial interactions, validating the system. We now propose to use this system to identify novel players in axo-glial interactions, using large sensory neurons and after addition of a second polarizing cue (extracellular matrix) to the Schwann cells. Next we will ask if the system can be used to study interactions between neurons and other glia, namely oligodendrocytes and astrocytes. The system can be adapted to multiple wild-type or mutant glia-cell interactions, to probe their role on protein or RNA polarization, formation of specific molecular complex or protein modification. These phenomena are relevant to physiological and pathological glial cell functions. This transformative resource could overcome the difficulty to study important glial undertakings at specialized cell junctions. ! PUBLIC HEALTH RELEVANCE: We are developing an innovative system to study glial cell interactions and function in a culture dish. This system can be used to study the function of specialized domains of glial cells, which are important for normal brain, and peripheral nerve function and several neurological diseases.