Characterization of the Myelin-Associated Glycoprotein Receptor NgR2 in vivo

描述（由申请人提供）：描述：髓磷脂相关糖蛋白（MAG）是一种双功能分子，涉及稳定中枢和周围神经系统的轴突-胶质相互作用。此外，MAG已被确定为体外神经突生长的有效抑制剂。我们实验室的工作发现NgR2是MAG的高亲和力受体，足以赋予MAG反应性。本研究旨在探讨NgR2在体内是否是一种功能性MAG受体。为了研究NgR2的功能，我们将采用小鼠遗传学方法。我通过在NgR2基因的外显子III中引入GFP报告盒，产生了缺乏NgR2的小鼠。与MAG突变体类似，NgR2缺失的小鼠可以存活到成年，因此，我们可以研究NgR2在成熟中枢神经系统中的作用。结合抗NgR2和抗MAG的免疫荧光将用于绘制NgR2与MAG表达的时空表达模式。体外神经突生长试验将用于验证假设，即NgR2是MAG受体，在体内视神经损伤后，MAG介导的神经突生长和轴突再生抑制是必需的。MAG的缺失导致髓鞘缺损和轴突变性。为了验证NgR2在体内是MAG受体的假设，将对髓鞘进行电子显微镜研究，以检查NgR2的缺失是否会导致与先前报道的MAG缺陷小鼠相似的缺陷。我的初步结果表明，NgR2缺失小鼠在坐骨神经中表现出髓鞘缺损。作为NgR2小鼠体内视神经再生研究的另一种方法，我建议使用NgR1/NgR2双突变小鼠，因为NgR1可能弥补NgR2缺失小鼠中NgR2的缺失。总的来说，所提出的实验有望在体内提供NgR2功能在MAG介导的生长抑制和轴突-胶质细胞相互作用中的作用。与公共卫生的相关性：外伤性脑损伤（TBI）、脊髓损伤或中风造成的损害往往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。全面了解NgR2在MAG功能调节中的作用，将有助于开发出能够减弱MAG抑制作用的治疗药物，同时保留其对轴突的保护和稳定作用。

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.