Translational potential of the internalization of Nogo-A receptor to enhance axonal regeneration after stroke

项目摘要/摘要 中风是致残的主要原因，也是导致死亡的主要原因。慢性中风治疗需要 通过克服轴突生长抑制物如Nogo-A来恢复功能。尽管毒品正在被 开发，目前，没有临床证明的药物来从中风和其他神经元损伤中恢复。 评估安全、神经保护、神经再生和廉价的天然产品的疗效 将是对正在进行的努力的补充。缺血性损伤后，轴突生长可通过常规的 “外源性”阻断轴突生长抑制物的方法。我们的假设是，轴突生长也可以是 这是通过“本质上”降低神经元对轴突生长抑制剂的敏感性来实现的。将这一点结合起来 具有神经营养活性的内在方法可能会更有效。在低纳摩尔浓度下， 绿茶多酚，如表没食子儿茶素没食子酸酯(EGCG)，提高cAMP-EPAC(交换 由cAMP直接激活的蛋白)途径和诱导Nogo-A受体(NgR1)等的内化 相关受体。因此，EGCG不仅阻止Nogo-A的活动，而且还阻止不同的轴突生长 抑制剂。同时，egcg还激活了活性氧物种(Ros)-蛋白激酶ce途径。 并增强神经营养因子的作用，如脑源性神经营养因子(BDNF)。这个 EGCG(神经元对轴突生长抑制剂的脱敏和增强)的联合作用 神经营养因子)导致长轴突生长和功能恢复，这可能被用于慢性卒中 心理治疗。在第一个目标中，我们将使用原代皮质神经元来确定EGCG诱导的cAMP- EPAC途径可导致NgR1及其辅受体及其他相关受体的内化和降解 感受器。我们将确定这是否与EGCG诱导的Nogo-A活性下降有关 和其他轴突生长抑制药。我们还将确定EGCG诱导的ROS是否平行激活- PKCE途径可增强BDNF等神经营养因子的作用，促进长轴突生长。 在第二个目的中，我们将使用MCAO(大脑中动脉阻塞/再灌注)的小鼠模型来 诱发中风。对于慢性中风治疗，我们将每天通过饮酒给予安全剂量的EGCG 水。我们将确定EGCG是否减少神经元表面相关的NgR1和其他相关的 大脑中的感受器。然后我们将确定这种下降是否与NOGO-A的减少有关 抑制途径以及cAMP、轴突生长、BDNF、神经可塑性和功能的增加 通过行为研究评估的恢复情况。我们将确定使用EGCG的方法(哪种 阻断轴突生长抑制剂以及增强神经营养因子)可以与 比NgR1拮抗肽Np1-40(鼻腔给药到大脑)更有效。成功者 这一临床前研究的结果可能证明，细胞表面NgR1的内化，由 EGCG或其他药物可使神经元对轴突生长抑制剂不敏感。

Project Summary/Abstract Stroke is a major cause of disability and a leading cause of death. Chronic stroke therapy requires a functional recovery by overcoming axonal growth inhibitors such as Nogo-A. Although drugs are being developed, currently, there are no clinically proven drugs for recovery from stroke and other neuronal injuries. Evaluating the efficacy of natural products that are safe, neuroprotective, neuroregenerative, and inexpensive would complement ongoing efforts. Following ischemic injury, axonal growth is enhanced by the conventional approach of “extrinsically” blocking axonal growth inhibitors. Our hypothesis is that axonal growth can also be achieved by “intrinsically” decreasing the susceptibility of neurons to axonal growth inhibitors. Combining this intrinsic approach with neurotrophic activity could be even more effective. At low nanomolar concentrations, green tea polyphenols, such as epigallocatechin-3-gallate (EGCG), elevate the cAMP-Epac (exchange protein directly activated by cAMP) pathway and induce internalization of Nogo-A receptor (NgR1) and other related receptors. EGCG thereby blocks the actions of not only Nogo-A, but also diverse axonal growth inhibitors. In parallel, EGCG also activates the reactive oxygen species (ROS)-protein kinase Ce pathway and potentiates the actions of neurotrophins, such as brain-derived neurotrophic factor (BDNF). The combined effects of EGCG (desensitization of neurons to axonal growth inhibitors and potentiation of neurotrophins) lead to long axonal growth and functional recovery, which may be exploited for chronic stroke therapy. In the first aim, we will use primary cortical neurons to determine whether the EGCG-induced cAMP- Epac pathway can cause internalization and degradation of NgR1 and its coreceptors as well as other related receptors. We will ascertain whether this correlates with the EGCG-induced decrease in the action of Nogo-A and other axonal growth inhibitors. We will also determine if EGCG-induced parallel activation of the ROS- PKCe pathway can potentiate the actions of neurotrophins, such as BDNF, and enhance long neurite growth. In the second aim, we will use the mouse model of MCAO (middle cerebral artery occlusion/reperfusion) to induce stroke. For chronic stroke therapy, we will administer a safe dose of EGCG daily through drinking water. We will determine whether EGCG decreases the neuronal surface-associated NgR1 and other related receptors in the brain. We will then ascertain whether this decrease correlates with a reduction in the Nogo-A inhibitory pathway as well as with an increase in cAMP, axonal growth, BDNF, neuroplasticity, and functional recovery as assessed by behavioral studies. We will determine whether the approach of using EGCG (which blocks axonal growth inhibitors as well as potentiates neurotrophins) can be as efficient as or even more efficient than the NgR1 antagonistic peptide NEP1-40 (intranasal delivery to the brain). The successful outcome of this preclinical exploration may prove that the internalization of cell-surface NgR1, induced by EGCG or other agents, desensitizes neurons to axonal growth inhibitors.