Trk Ligands for Treatment of Traumatic Brain Injury

描述(由申请人提供)： 在退伍军人中，创伤性脑损伤(TBI)占伤害的比例很大，而且比例还在不断上升；爆炸、机动车事故和跌倒的后遗症。脑外伤也是美国总人口死亡和残疾的主要原因，特别是在40岁以下的人群中。大约2%的人口患有慢性创伤性脑损伤相关残疾。目前临床上还没有有效的保护性或恢复性疗法。在几种中枢神经系统创伤模型中，应用神经营养因子(如BDNF、NGF)可以有效地保护组织并促进长期恢复。然而，神经营养因子是劣质药物，因为它们不稳定，表现出较差的中枢神经系统穿透性，并可能增加细胞死亡和疼痛通路。这些特性是由于它们的多肽组成和通过激活多个受体刺激交叉的信号通路。这些问题至少可以通过使用最近发现的小的、稳定的非肽类药物化合物(命名为LM22)来部分避免，这些化合物通过与Trk神经营养素受体(与TrkB的LM22A-4和与TrkB的LM22B-10以及与TrkC的LM22A-10)的选择性相互作用来促进神经元存活。这些化合物抑制神经元死亡，促进神经前体细胞的增殖和分化，并增强功能和形态上的神经可塑性。我们假设LM22化合物将：通过激活特定的Trk偶联存活和分化信号通路的不同机制来增加神经前体细胞的存活和分化；在脑损伤后增加TrkB信号将促进神经元和神经前体细胞的早期存活和长期可塑性，对神经元突起和脊椎产生影响；TrkB和TrkC一起激活将比单独激活TrkB和TrkC更有效，并且 这些化合物应该会改善行为结果，长期或过度增强的可塑性可能是有害的。具体目的是：1.测定LM22化合物对体外培养的海马神经前体细胞的作用：A)检测LM22A-4和LM22B-10对体外培养的海马神经前体细胞增殖、存活、神经和/或神经胶质分化及形态的影响，并与神经营养素蛋白配体进行比较；B)检测LM22A-4和LM22B-10对体外培养的海马神经前体细胞增殖、存活、神经和/或胶质分化及形态的影响； 使用TrkB、TrkC或这两种受体的选择性可抑制酪氨酸激酶突变体和：C)使用选择性化学抑制剂、miRNA介导的下调和病毒载体介导的强制表达，确定PI3K/AKT、ERKs1、2和5、PLC�、miRNA9和NRSF/REST在复合效应中的作用；2.在大鼠受控皮质撞击(CCI)模型中，确定LM22化合物对细胞反应的影响，与给药剂量和给药时机有关：评估A)神经元和神经前体细胞死亡；B)Trk激活和下游信号；C)神经元/胶质发生和神经元形态；D)炎症；以及3)确定LM22化合物对大鼠CCI后行为的影响：检查A)：空间/海马记忆(Morris水迷宫)；B)焦虑/抑郁(开场测试，高架+迷宫)；C)运动学习(旋转/步态分析)这些研究将为这些或相关化合物在临床脑创伤中的最终应用提供重要信息，并将促进我们对TrkB和TrkC在病理状态中的作用的了解。这项研究的总体目标是推进这些神经营养化合物在脑创伤和其他疾病治疗中的应用。

DESCRIPTION (provided by applicant): Traumatic brain injuries (TBIs) constitute a significant and growing percentage of injuries in the veteran population; sequelae of explosions, motor vehicle accidents and falls. TBI is also a major cause of death and disability in the general population of the US, particularly in those under 40. Approximately 2% of the population is living with a chronic TBI-related disability. There are currently no effective protective or restorative therapies available clinically. In severl models of CNS trauma, administration of a neurotrophin (e.g. BDNF, NGF) protects tissues acutely and promotes longer term recovery. However, the neurotrophins are poor drugs as they are labile, exhibit poor CNS penetration and may augment cell death and pain pathways. These properties are due to their polypeptide composition and the stimulation of intersecting signalling pathways through the activation of multiple receptors. These problems may be at least partially circumvented through the use of recently discovered small, stable, non-peptidyl drug-like compounds (designated LM22) that promote neuronal survival through selective interactions with the Trk neurotrophin receptors (LM22A-4 with TrkB and LM22B-10 with TrkB and TrkC). The compounds inhibit neuronal death, promote neural progenitor cell proliferation and differentiation and augment functional and morphologic neuroplasticity. We hypothesize that the LM22 compounds will: increase neural progenitor survival and differentiation through distinct mechanisms involving activation of specific Trk-coupled survival and differentiative signalling pathways; that increasing TrkB signaling following TBI will promote early neuronal and neural progenitor survival and longer term plasticity, with effects on neuronal processes and spines; that activation of TrkB and TrkC together will be more effective than either alone, and; that while the compounds should improve behavioral outcomes, prolonged or excessive increased plasticity may be detrimental. Specific Aims are to: 1. Determine LM22 compound effects on hippocampal progenitors in vitro: A) Examine the effects of LM22A-4 and LM22B-10 on proliferation, survival, neuro- and/or gliogenic differentiation and morphology of hippocampal neural progenitor cells in vitro, in comparison with neurotrophin protein ligands; B) determine the Trk specificity of the compounds for these effects using selectively inhibitable tyrosine kinase mutants of TrkB, TrkC or both receptors and; C) determine the role of PI3K/AKT, ERKs 1, 2 and 5, PLC�, miRNA9 and NRSF/REST in compound effects, using selective chemical inhibitors, miRNA-mediated downregulation and viral construct-mediated enforced expression; 2. Determine LM22 compound effects on cellular responses relative to dose and timing of administration in the rat controlled cortical impact (CCI) model of TBI: assess A) neuronal and neural progenitor cell death; B) Trk activation and downstream signaling; C) neuro/gliogenesis and neuronal morphology and; D) inflammation, and; 3. Determine LM22 compound effects on behavior following rat CCI: Examine A): spatial/hippocampal memory (Morris Water Maze); B) anxiety/depression (open field testing, elevated plus maze) and; C) motor learning (rotorod/gait analysis) These studies will provide information important for the eventual application of these or related compounds to clinical head trauma, and will advance our knowledge of the roles of TrkB and TrkC in pathologic states. The overall goal of this research is to advance the application of these neurotrophic compounds to the treatment of brain trauma and other conditions.