A novel role for receptor tyrosine phosphatase in neuroblast migration

描述(申请人提供)：受体酪氨酸磷酸酶(RPTPs)在神经系统的神经发生、细胞增殖和神经元定位中发挥重要作用，但其作用机制尚不清楚。遗传分析因不同RPTP亚型1、2的重叠功能而变得复杂，而许多RPTP的真实配体仍不清楚3。最近使用模型系统Manduca sexta的研究发现了一个特定RPTP的新功能，可能会澄清这些问题。在Manduca发育的肠神经系统中，胎盘来源的神经母细胞在分化为实体神经元之前必须进行广泛的迁移，在此过程中，它们沿着预先形成的肌带通路移动，同时避开肠道中线。以前的工作表明，在这个系统中，中线肌肉表达唯一的Eph受体酪氨酸激酶(MsEph)，而神经母细胞表达其同源EPhin配体(MsEPhin；GPI连锁或A型EPhin)4。有趣的是，通过MsEPhin的“反向”信号阻止神经母细胞穿过表达MsEph的中线细胞，这一反应涉及到Src家族激酶(SFK)的局部激活和其主导过程的收缩5，6。相反，通过MsEph受体的传统“正向”信号在这一过程中没有作用，提供了第一个控制神经母细胞定位的EPhin-A反向信号的例子。然而，GPI连接的肾上腺素跨膜转导信号的机制仍然知之甚少。MsEphin共受体的亲和筛查确定了PTP10D，一种调节CNS2中线轴突反应的“III型”RPTP。初步研究表明，PTP10D在神经母细胞中与MsEph-rin共表达，而抑制PTP10D的表达可引起与阻断MsEphin信号相同的异位中线交叉反应。有趣的是，PDZ适配器蛋白ZO-1(zonula occludens-1)也在这个屏幕上被发现，并被证明与MsEPhin和PTP10D在神经母细胞中共定位。最初被归类为紧密连接蛋白7的ZO-1还可以调节细胞在体外的迁移，并可能 在各种情况下与RPTP和SFK互动10-12。这项建议的总体目标是确定PTP10D如何调节发育中的ENS中的神经母细胞迁移。中心假设是PTP10D作为MsEPhin的共同受体，将这种GPI连接的EPhin与ZO-1及其下游效应器偶联。SPICAL AIMS将测试PTP10D和ZO-1在依赖MSE的迁移方面的作用，使用已公布的方法来操纵胚胎培养中的基因表达和蛋白质相互作用。这些研究将为R01的应用提供必要的新数据，目标是全面定义神经系统中EPhin-A反向信号的机制。公共卫生相关性：展示RPTPs和ZO-1在EPhin-A信号中的新角色将提供新的工具，用于研究为什么人类影响EPhin的突变会导致大脑发育缺陷13，并为开发新的治疗策略来解决神经退行性疾病，在这种情况下EPhin-A反向信号可能被错误调节14-16。 公共卫生相关性：拟议的研究将调查受体酪氨酸磷酸酶(RPTPs)作为A型肾上腺素的辅助受体在控制神经母细胞迁移中的新作用。RPTPs和EPhin-as与正常的大脑发育和创伤后的神经源性反应独立相关，但它们与适配蛋白ZO1的功能相互作用此前尚未被认识到。在模型系统中确定RPTPs和ZO-1转导EPhin A依赖信号的机制将为这一信号通路如何调控胚胎和成人神经系统中的神经元分化以及在RPTP-Ewitin相互作用可能受损的神经疾病的背景下提供新的视角。

DESCRIPTION (provided by applicant): Receptor tyrosine phosphatases (RPTPs) play important roles during neurogenesis, cell proliferation, and ne ronal positioning in the nervous system, but their mechanisms of action remain poorly understood. Genetic analyses have been complicated by the overlapping functions of different RPTP subtypes1, 2, while authentic ligands for many RPTPs remain unknown3. Recent studies using the model system Manduca sexta have identified a novel function for a specific RPTP that may clarify these issues. In the developing enteric nervous sy tem (ENS) of Manduca, placode-derived neuroblasts must migrate extensively before differentiating into ma- ture neurons, during which they travel on pre-formed muscle band pathways while avoiding the enteric midline. Previous work showed that the midline muscles express the sole Eph receptor tyrosine kinase in this system (MsEph), while the neuroblasts express its cognate Ephrin ligand (MsEphrin; a GPI-linked or type-A Ephrin)4. Intriguingly, "reverse" signaling via MsEphrin prevents the neuroblasts from crossing the MsEph-expressing midline cells, a response that involves the local activation of a Src family kinase (SFK) and retraction of their leading processes5, 6. In contrast, conventional "forward" signaling via MsEph receptors plays no role in this process, providing the first example of Ephrin-A reverse signaling in the control of neuroblast positioning. However, the mechanisms by which GPI-linked Ephrins transduce signals across the membrane remain poorly understood. An affinity screen for MsEphrin co-receptors identified PTP10D, a "type-III" RPTP that regulates midline axonal responses in the CNS2. Preliminary studies showed that PTP10D is co-expressed with MsEph- rin by the neuroblasts, while inhibiting PTP10D expression induced the same pattern of ectopic midline cros- sovers caused by blocking MsEphrin signaling. Intriguingly, the PDZ adapter protein ZO-1 (zonula occludens- 1) was also identified in this screen, and shown to co-localize with MsEphrin and PTP10D in the neuroblasts. Originally classified as a tight junction protein7, ZO-1 can also regulate cell migration in vitro8, 9 and may interact with RPTPs and SFKs in a variety of contexts10-12. The overall objective of this proposal is to determine how PTP10D regulates neuroblast migration in the developing ENS. The central hypothesis is that PTP10D acts as a co-receptor for MsEphrin, coupling this GPI-linked Ephrin with ZO-1 and its downstream effectors. Specific aims will test the role of PTP10D and ZO-1 in MsEphrin-dependent aspects of migration, using published methods to manipulate gene expression and protein interactions in embryo culture. These studies will provide essential new data for an R01 application, with the goal of comprehensively defining the mechanisms of Ephrin-A reverse signaling in the nervous system. Public Heath Relevance: Demonstrating novel roles for RPTPs and ZO-1 in Ephrin-A signaling will provide new tools for investigating why human mutations that affect Ephrin-As result in defective brain growth13, and for developing new therapeutic strategies that address neuro- degenerative conditions in which Ephrin-A reverse signaling may be misregulated14-16. PUBLIC HEALTH RELEVANCE: The proposed research will investigate a novel role for receptor tyrosine phosphatases (RPTPs) as co- receptors for type-A Ephrins in the control of neuroblast migration. RPTPs and Ephrin-As have been independently linked with normal brain development and neurogenic responses to traumatic injury, but their functional interactions with the adapter protein ZO1 have not previously been recognized. Defining the mechanisms by which RPTPs and ZO-1 transduce Ephrin A-dependent signals in a model system will provide a new perspective on how this signaling pathway may regulate neuronal differentiation in both the embryonic an adult nervous system, and in the context of neurological disorders in which RPTP-Ephrin interactions may be impaired.