Proteomic mapping of oligodendrocyte-associated proteins

描述(申请人提供)：最近研究了少突胶质细胞在发育和疾病或创伤后的作用，暴露了我们对少突胶质细胞功能的有限了解，并强调了建立新工具的必要性，这些工具将使我们能够揭示由少突胶质细胞调控的全部蛋白质库并确定它们的功能。不幸的是，缺乏来自髓鞘少突胶质细胞的cDNA文库，阻碍了对来自这些细胞或受这些细胞调控的蛋白质的鉴定。因此，我们建立了一种结合X-射线、双向凝胶电泳法和质谱学技术来分析这类蛋白质的新方法。具体地说，在以前的研究中，我们已经证明，通过在出生时对动物进行单侧X射线照射，可以选择性地从大鼠的一条视神经中消除少突胶质细胞。我们进一步表明，随着时间的推移，X射线照射的视神经从出生后(P)14天开始重新填充第二批少突胶质细胞。这种细胞重新聚集到X射线照射的神经中，几乎恢复了正常情况下在P28之前在视神经中发现的少突胶质细胞的全部数量。因此，利用这种方法，我们在同一动物体内实验创建了一个不含少突胶质细胞及其祖细胞的视神经(X射线照射侧)和一个含有正常少突胶质细胞群体的视神经(未处理侧)。我们现在提供的数据表明，可以比较正常和X射线照射的大鼠视神经的2-D凝胶蛋白质谱，以便从体内模型系统中快速分离由少突胶质细胞表达的蛋白质以及由少突胶质细胞调控的其他细胞表达的蛋白质。这些少突胶质细胞相关蛋白的后续鉴定是通过质谱学完成的。这个实验模型系统特别强大和独特，因为它保留了对髓鞘形成至关重要的所有细胞-细胞相互作用。综上所述，这些实验应该会让我们更清楚地了解少突胶质细胞在中枢神经系统发育中的作用，以及与髓鞘形成相关的一系列蛋白质。

DESCRIPTION (provided by applicant): Recent studies examining the role of oligodendrocytes in development, and following disease or trauma, have exposed our limited understanding of oligodendrocyte function and underscore the need to establish new tools which will allow us to uncover the full repertoire of proteins regulated by oligodendrocytes and ascertain their function. Unfortunately, the lack of cDNA libraries derived from myelinating oligodendrocytes has hampered attempts to identify proteins derived from or regulated by these cells. We therefore established a new method to elucidate such proteins by coupling the techniques of X-irradiation, 2-D gel electrophoresis, and mass spectrometry. Specifically, in previous studies, we had demonstrated that oligodendrocytes could be selectively eliminated from one optic nerve of a rat by treating the animal to a unilateral exposure of X-irradiation at the time of birth. We further showed that, with time, the X-irradiated optic nerve became repopulated with a second cohort of oligodendrocytes from postnatal day (P) 14 on. This repopulation of cells into the X-irradiated nerve nearly restores the full complement of oligodendrocytes normally found in the optic nerve by P28. Therefore, utilizing this approach, we experimentally created, within the same animal, one optic nerve devoid of oligodendrocytes and their progenitors (the X-irradiated side) and one optic nerve containing the normal oligodendrocyte population (the untreated side). We now provide data demonstrating that 2-D gel protein profiles of normal and X-irradiated rat optic nerves can be compared to quickly isolate, from an in vivo model system, those proteins expressed by oligodendrocytes as well as those expressed by other cells which are regulated by oligendrocytes. The subsequent identification of these oligodendrocyte-associated proteins is accomplished by mass spectrometry. This experimental model system is especially powerful and unique since it retains all of the cell-cell interactions crucial to myelination. Taken together, these experiments should lead to a clearer understanding of the role of oligodendrocytes in CNS development and of the array of proteins associated with myelination.