Characterization of the Myelin-Associated Glycoprotein Receptor NgR2 in vivo

描述（由申请人提供）：描述：髓鞘相关糖蛋白（MAG）是一种双功能分子，参与稳定中枢和外周神经系统中的轴突-神经胶质相互作用。此外，MAG已被鉴定为体外神经突生长的有效抑制剂。我们实验室的工作将NgR 2鉴定为MAG的高亲和力受体，足以赋予MAG反应性。本提案中概述的研究旨在研究NgR 2是否是体内功能性MAG受体。为了研究NgR 2功能，将采用小鼠遗传方法。我已经通过将GFP报告盒引入NgR 2基因的外显子III产生了NgR 2缺陷小鼠。与MAG突变体类似，NgR 2缺失小鼠可以存活至成年，因此，将使我们能够研究NgR 2在成熟中枢神经系统中的作用。将使用抗NgR 2和抗MAG免疫荧光的组合来绘制NgR 2的时空表达模式，与MAG表达直接比较。将使用体外神经突生长测定来检验以下假设：NgR 2是MAG介导的抑制体内视神经损伤后的神经突生长和轴突再生所必需的MAG受体。MAG的缺失导致髓鞘的缺陷和轴突的变性。为了检验NgR 2是体内MAG受体的假设，将进行髓鞘的电子显微镜研究以检查NgR 2的损失是否导致与先前MAG缺陷小鼠的报告者类似的缺陷。我的初步结果表明，NgR 2基因敲除小鼠坐骨神经髓鞘形成缺陷。作为NgR 2小鼠体内视神经再生研究的另一种方法，我建议使用NgR 1/NgR 2双突变小鼠，因为NgR 1可以补偿NgR 2缺失小鼠中NgR 2的缺失。总的来说，所提出的实验预计提供的见解在MAG介导的生长抑制和轴突-神经胶质细胞的相互作用在体内的NgR 2功能的作用。与公共卫生的关系：创伤性脑损伤（TBI）、脊髓损伤或中风中发生的损伤通常会导致永久性神经功能缺损。这主要是由于缺乏自发轴突再生和/或髓鞘再生。对NgR 2在介导MAG功能中所起作用的完整理解将有助于开发可以减弱MAG抑制同时保留其对轴突的保护和稳定作用的治疗剂。

DESCRIPTION (provided by applicant): Description: Myelin-associated glycoprotein (MAG) is a bi-functional molecule that has been implicated in stabilizing axon-glial interactions in both the central and peripheral nervous system. In addition, MAG has been identified as a potent inhibitor of neurite outgrowth in vitro. Work from our laboratory identified NgR2 as a high affinity receptor for MAG sufficient to confer MAG responsiveness. The studies outlined in this proposal are aimed at investigating whether NgR2 is a functional MAG receptor in vivo. To study NgR2 function a mouse genetic approach will be pursued. I have generated mice deficient for NgR2 by introducing a GFP reporter cassette into exon III of the NgR2 gene. Similar to MAG mutants, NgR2 null mice are viable into adulthood and thus, will allow us to study the role of NgR2 in the mature central nervous system. A combination of anti-NgR2 and anti-MAG immunofluorescence will be used to map the tempero-spatial expression pattern of NgR2 in direct comparison to MAG expression. In vitro neurite outgrowth assays will be used to test the hypothesis that NgR2 is a MAG receptor necessary for MAG-mediated inhibition of neurite outgrowth and axonal regeneration following optic nerve injury in vivo. Loss of MAG leads to defects in myelin sheaths and the degeneration of axons. To test the hypothesis that NgR2 is a MAG receptor in vivo, electron microscopical studies of myelin sheaths will be performed to examine whether loss of NgR2 leads to defects reminiscent to the ones previously reporter for MAG deficient mice. My preliminary results suggest that NgR2 null mice show myelination defects in the sciatic nerve. As an alternate approach for optic nerve regeneration studies of NgR2 mice in vivo, I propose to use NgR1/NgR2 double mutant mice, as NgR1 might compensate for the loss of NgR2 in NgR2 null mice. Collectively, the experiments proposed are anticipated to provide insights in the role of NgR2 function in MAG mediated growth inhibition and axon-glia interactions in vivo. RELEVANCE TO PUBLIC HEALTH: The damage that occurs in traumatic brain injury (TBI), spinal cord injury or stroke, often leads to permanent neurological deficits. This is primarily due to the lack of spontaneous axonal regeneration and/or remyelination. A complete understanding of the role NgR2 plays in mediating MAG's function, will aid in the development of therapeutic agents that can attentuate MAG inhibition while preserving its protective and stabilizing effects on axons.