PD-Ialpha/ATX's role for forebrain oligodendrocyte specification and migration

描述（由申请人提供）：成熟的哺乳动物中枢神经系统（CNS）仅具有有限的自我修复能力，主要是由于其产生和适当靶向新神经细胞类型的能力有限。拟议研究的长期目标是更好地了解刺激CNS驻留神经干细胞/祖细胞修复CNS所需的调节回路，特别是髓鞘，这是快速有效神经传导的先决条件。更具体地说，目前的建议调查的中心假设，脉络丛释放蛋白磷酸二酯酶-Ialfa/autotaxin（PD-Ialfa/ATX），通过其酶活性lyso-PLD网站，代表的因素之一，调节细胞命运和髓鞘细胞，即少突胶质细胞（OLG），在出生后的哺乳动物前脑的迁移。提案的两个具体目标中描述的研究以及解决上述中心假设的研究将主要在里约热内卢联邦大学（巴西）与Cecilia Hedin-Pereira合作完成，作为NIH资助5 R 01 NS 045883的扩展。在具体目标1中，我们将确定PD-Ialfa/ATX的作用，特别是其lysoPLD活性位点在啮齿动物前脑中对OLG细胞命运决定的作用。OLG特化已被证明发生在出生后前脑内的一个专门区域，即脑室下区（SVZ）。我们将使用SVZ外植体培养物研究溶血磷脂酸（LPA）（PD-Ialfa/ATX酶活性的终产物）、全功能PD-Ialfa/ATX和无酶活性PD-Ialfa/ATX对OLG细胞命运特化的影响。为了进一步表征LPA/PD-Ialfa/ATX介导的对OLG谱系的作用，我们将通过使用药理学和siRNA方法评估已知LPA受体的贡献。此外，我们将进行体内实验，其中SVZ的循环细胞将使用逆转录病毒注射进行标记。将额外注射PD-Ialfa/ATX和无酶活性的PD-Ialfa/ATX，并使用免疫组织化学和共聚焦成像确定对细胞命运规范的影响。在具体目标2中，我们将确定PD-Ialfa/ATX的作用，特别是其lysoPLD活性位点在啮齿动物前脑内新生成的OLG祖细胞（OPC）迁移中的作用。我们将使用与具体目标1研究中使用的相同的实验范式。然而，在这里，我们将重点分析OPCs的迁移途径。综上所述，所提出的一系列实验预计将产生新的洞察力的监管机制，控制1）OLG规范和2）OPC迁移在发育中的前脑。最重要的是，这些研究预计将导致后续的调查，旨在刺激内源性神经干细胞/祖细胞，以促进修复髓鞘在病理条件下。公共卫生相关性：成熟的哺乳动物中枢神经系统（CNS）在损伤后和病理条件下仅具有有限的自我修复能力。CNS驻留神经干细胞/祖细胞的刺激代表了恢复CNS功能的有希望的，但目前表征不佳的可能性，包括快速和有效的神经传导，其最终依赖于未受损的髓鞘的存在。为了更好地理解刺激髓鞘内源性修复所必需的调节回路，本授权申请研究了细胞外因子磷酸二酯酶-Ialfa/自分泌运动因子（PD-Ialfa/ATX）在刺激髓鞘形成细胞（即少突胶质细胞）的产生和迁移中的潜在作用。

DESCRIPTION (provided by applicant): The mature mammalian central nervous system (CNS) possesses only a restricted ability to repair itself, mainly due to its limited capacity in generating and properly targeting new neural cell types. The long-term goal of the proposed studies is to better understand the regulatory circuits that may be necessary for stimulating CNS-resident neural stem/progenitor cells for repair of the CNS and in particular the myelin sheath that is a prerequisite for fast and efficient nerve conduction. More specifically, the current proposal investigates the central hypothesis that the choroid plexus released protein phosphodiesterase-Ialfa/autotaxin (PD-Ialfa/ATX), via its enzymtically active lyso-PLD site, represents one of the factors regulating cell fate and migration of myelinating cells, i.e. oligodendrocytes (OLGs), in the postnatal mammalian forebrain. The studies described in the two Specific Aims of the proposal and addressing the above central hypothesis will be done primarily at the Federal University of Rio de Janeiro (Brazil) in collaboration with Cecilia Hedin-Pereira as an extension to NIH grant 5R01NS045883. In Specific Aim 1, we will determine the role of PD-Ialfa/ATX and in particular its lysoPLD-active site for OLG cell fate determination in the rodent forebrain. OLG specification has been shown to occur in the postnatal forebrain within a specialized region, namely the subventricular zone (SVZ). We will investigate the effects of lysophosphatidic acid (LPA), the endproduct of PD-Ialfa/ATX's enzymatic activity, fully functional PD-Ialfa/ATX and enzymatically inactive PD-Ialfa/ATX on OLG cell fate specification using SVZ explant cultures. To further characterize the LPA/PD-Ialfa/ATX-mediated effects on the OLG lineage, we will assess the contribution of the known LPA receptors by using pharmacological and siRNA approaches. In addition, we will perform in vivo experiments, in which cycling cells of the SVZ will be labeled using retroviral injections. PD-Ialfa/ATX and enzymatically inactive PD-Ialfa/ATX will additionally be injected and the effects on cell fate specification will be determined using immunohistochemistry and confocal imaging. In Specific Aim 2, we will determine the role of PD-Ialfa/ATX and in particular its lysoPLD-active site for migration of newly generated OLG progenitor cells (OPCs) within the rodent forebrain. We will use the same experimental paradigms as used in the studies to Specific Aim 1. Here we will, however, focus our analysis on the migratory pathways of OPCs. Taken together, the proposed sets of experiments are expected to yield novel insight into the regulatory mechanisms that control 1) OLG specification and 2) OPC migration in the developing forebrain. Most importantly, these studies are anticipated to lead to subsequent investigations aimed at stimulating endogenous neural stem/progenitor cells to contribute to repair of the myelin sheath under pathological conditions. Public Health Relevance: The mature mammalian central nervous system (CNS) possesses only a restricted ability to repair itself after injury and under pathological conditions. Stimulation of CNS-resident neural stem/progenitor cells represents a promising, yet currently poorly characterized, possibility for restoring CNS function including fast and efficient nerve conduction that is ultimately dependent on the presence of an undamaged myelin sheath. As an attempt to better understand the regulatory circuits that may be necessary for stimulating endogenous repair of the myelin sheath, the present grant application investigates the potential role of the extracellular factor phosphodiesterase-Ialfa/autotaxin (PD-Ialfa/ATX) in stimulating the generation and migration of myelinating cells, i.e. oligodendrocytes.