TrkB激动剂对帕金森猴多巴胺神经元的保护与功能恢复机制的研究

Parkinson disease (PD) is a neurodegenerative disease. Although some of current dopaminergic medications provide symptomatic relief, the eventual development of severe side effects limit their utility and, at present, there is no therapy that can slow the onset and progression of the disease. Via binding to TrkB, BDNF mediates neuron maintenance and survival in several neurodegenerative diseases, including: PD. However, BDNF has the inability to penetrate brain blood barrier (BBB). To search for BDNF mimetic small molecules, we have recently discovered a TrkB agonist 7,8-dihydroxyflavone (7,8-DHF), which mimics BDNF. Administration of 7,8-DHF into mouse attenuates MPTP-induced damage and promotes dopamine synthesis in surviving neurons, supporting the idea that TrkB agonists have therapeutic potential for PD. 7,8-DHF is orally bioactive and penetrates BBB. Due to a significant difference between mouse and human PD, there are three unclear questions: 1) whether 7,8-DHF has the ability to neuroprotect dopamine neurons resistance to damage cues; 2) whether 7,8-DHF can restore surviving dopamine neuron functions in primate PD as an effective drug; 3) if work, what are the mechanisms? The MPTP-treated monkey is considered as the "gold standard" model of PD because of the close similarities in motor behavioral changes and basal ganglia pathophysiology between this model and the human PD condition. Various models of MPTP toxicity in nonhuman primates are being used in different laboratories. The main differences between these models relate to the regimen of MPTP administration and the progressive development of PD motor signs. In the previous works, we directly injected MPP+ (one metabolite of MPTP) into lateral ventricle of monkey brain and get ideal PD models. Because of the inability to penetrate BBB, MPP+ is unable to damage gastrointestinal dopamine neurons. Thus, these PD monkeys have no other health problems caused by conventional administration of MPTP. In the study, we shall use the PD monkeys developed by us to test the functions of 7,8-DHF. The first aim is to determine the in vivo PKs and subchronic toxicity of 7,8-DHF in monkeys. The second aim is to determine the neuroprotection of the 7,8-DHF by preventing the death of nigral neurons in primate PD model with immunohistochemistry and behavioral testing. The third aim is to determine the restoration by testing the efficacy of 7,8-DHF for its ability to restore neurochemical and behavioral function in animals with established lesions of their dopamine system in PD model. The primary endpoints will be an increase in striatal dopamine and terminal markers and improvement of behavioral deficits. The forth aim is to figure out 7,8-DHF how to exert on the neuroprotection and restoration functions in PD monkeys. Based on the study, we hope to gain more insights into the potential therapeutic efficacy in primates, before we move into clinical trials to test their efficacy in patients with PD.

帕金森氏病（PD）是一类重大的神经系统退行性疾病。目前治疗PD的药物并不能阻止病变的恶化，而且长期服用导致对药物产生耐受性和副作用。BDNF作为神经营养因子具有保护和治疗PD的作用，不能通过血脑屏障，无法在临床上使用。为寻求能够模拟BDNF功能的小分子物质，在前期小鼠上的研究上，我们成功地发现7,8-二羟基黄酮 （7,8-DHF）作为TrkB激动剂，具有BDNF对神经细胞的功能。由于小鼠和人的PD存在巨大差异，因此两个重要问题仍不清楚： 7,8-DHF在灵长类动物上是否具有多巴胺神经元（DA）的保护和治疗作用以及相应的作用机制。在本项目中，我们利用自身发展的PD猴模型，研究7,8-DHF对DA的保护和治疗作用，通过SPECT多巴胺转运体检测技术、行为学分析、组织病理分析、显微电镜分析以及转录组分析等技术，评价7,8-DHF对DA的保护和对PD的治疗效果，并理解其作用机制，推动该药进入临床。

帕金森氏病（PD）是一类重大的神经系统退行性疾病，而无治疗的特效药物。BDNF作为神经营养因子具有保护和治疗PD的作用，但无法通过血脑屏障，限制了临床使用。在前期小鼠上的研究上发现7,8-二羟基黄酮 （7,8-DHF）作为TrkB激动剂，具有BDNF 对神经细胞的功能。由于小鼠和人的PD存在巨大差异，因此两个重要问题仍不清楚：7, 8-DHF 在灵长类动物上是否具有多巴胺神经元（DA）的保护和治疗作用以及相应的作用机制。针对这两个问题，本项目为了能获得更好的帕金森猴模型，项目组成员首先在现有帕金森疾病猴模型制备方法的基础上进行了改进，建立了双侧脑室给MPP+药，该方法能产生更为稳定的帕金森猴模型，为项目后继的研究以及将来帕金森疾病的致病机制研究和治疗帕金森疾病方法的探索等方面提供理想的模型。利用该PD模型，我们阐明了7, 8-DHF在灵长类动物体内的代谢学，通过对灵长类动物生理生化以及病理的系统分析，证明了长期、高剂量服用7, 8-DHF对机体无毒副作用，表明该药的安全性；揭示了7, 8-DHF在对多巴胺神经元具有明显的保护作用，可以抑制其多巴胺神经元凋亡，证明7, 8-DHF治疗帕金森疾病的有效性；发现7, 8-DHF对损伤的多巴胺神经元的作用机制，是通过直接促进多巴胺神经元存活，但与体内的小胶质细胞和星型胶质细胞的激活无相关性。这些结果为促进该药进入临床应用奠定了重要的基础。课题任务全部完成，发表了两篇论文，一篇发表于Scientific Reports (2016) ；一篇发表于Acta Physiologica Sinica（2017）。目前有其资助下的两篇SCI论文正在投稿。培养博士1名，硕士4名。另根据学科的发展，我们增加了干细胞治疗帕金森疾病的内容。发现利用基因修饰后的干细胞治疗帕金森疾病具有长期的安全性和有效性，为干细胞的转化提供了重要的临床前研究基础。