Identification of mechanisms by which cytisine and estrogen inhibit ER stress and exert neuroprotection in Parkinson's disease

帕金森病（PD）是第二常见的神经退行性疾病，预计影响超过12 到2040年，全球将有100万人。帕金森病无法治愈，目前的治疗方法仅仅是对症治疗， 这突出了对神经保护药物的迫切和未满足的需求。一个主要的障碍是， 开发有效的和临床上可转化的神经保护药物的一个重要原因是，没有单一的动物模型可以概括 导致PD中多巴胺能（DA）神经元损失的复杂过程。因此，大多数成功的药物 临床前动物模型在PD患者中不起作用。目前的建议通过以下方式解决这一障碍： 超活化内质网（ER）应激，这是一个既定的收敛机制， 多种PD相关毒性的下游。内质网应激由三个信号传导臂组成， 三种关键蛋白-ATF 6、XBP 1和CHOP易位到DA神经元的核中。中度 ATF 6和XBP 1的激活具有神经保护作用，而这些蛋白质的慢性、不受控制的激活导致 DA细胞死亡相反，CHOP的激活总是导致细胞死亡。实验表明 烟碱化合物金雀花碱引起称为ER出口位点（ERES）的特化结构的上调， 这与ATF 6和XBP 1的抑制有关。令人惊讶的是，在6-羟基多巴胺（6-OHDA）诱导的PD临床前毒素模型中的实验表明，金雀花碱仅在雌性小鼠中具有神经保护作用。 另外的初步实验表明，17-β-雌二醇抑制DA神经元中CHOP的表达， 与PD发病率和表型方面观察到的强烈性别差异相符，男性更容易发生PD 患帕金森病的人比女性多基于这些数据，该建议假设野靛碱和雌激素发挥作用， 在雌性小鼠中通过协同抑制所有三个臂（ATF 6、XBP 1和CHOP）来进行神经保护。 在DA神经元中过度激活的ER应激。提出实验来确定金雀花碱 需要ERES通过特异性删除DA神经元中的ERES或通过使用烟碱酸来发挥神经保护作用， 不能上调ERES的化合物。将切除雌性小鼠的卵巢，以通过以下方式确定机制： 雌激素受体的哪些特定亚型在雌性小鼠中发挥神经保护作用。于完成 在这项建议的实验中，金雀花碱和雌激素介导的关键信号靶标 将鉴定神经保护作用。这一提案的成功将为实现以下长期目标奠定基础： 发现临床上可转化的神经保护性PD药物，对男性和女性都有效。

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, predicted to affect over 12 million people worldwide by 2040. There is no cure for PD and current treatments are merely symptomatic, which underscores an urgent and unmet need for neuroprotective drugs. One major barrier for the inability to develop effective and clinically translatable neuroprotective drugs is that no single animal model recapitulates the complex process that leads to dopaminergic (DA) neuron loss in PD. Therefore, most drugs that succeed in preclinical animal models fail to work in PD patients. The current proposal addresses this barrier by focusing on hyperactivated endoplasmic reticulum (ER) stress, which is an established convergent mechanism downstream of multiple PD-related toxicities. ER stress consists of three signaling arms that culminate in the translocation of three key proteins – ATF6, XBP1, and CHOP into the nucleus of DA neurons. Moderate activation of ATF6 and XBP1 is neuroprotective, while chronic, uncontrolled activation of these proteins leads to DA cell death. By contrast, activation of CHOP invariably causes cell death. Experiments showed that the nicotinic compound cytisine causes an upregulation of specialized structures called ER exit sites (ERES), and this is associated with an inhibition of ATF6 and XBP1. Surprisingly, experiments in a 6-hydroxydopamine (6-OHDA)-induced preclinical toxin model of PD showed that cytisine is neuroprotective only in female mice. Additional preliminary experiments showed that 17-β-estradiol inhibits CHOP expression in DA neurons, which fits with strong sex-differences observed in the incidence and phenotype of PD, with men being more prone to developing PD than women. Based on these data, this proposal hypothesizes that cytisine and estrogen exert neuroprotection in female mice by synergistically inhibiting all three arms (ATF6, XBP1 and CHOP) of hyperactivated ER stress in DA neurons. Experiments are proposed to determine the extent to which cytisine requires ERES for exerting neuroprotection by specifically deleting ERES in DA neurons or by using nicotinic compounds that cannot upregulate ERES. Female mice will be ovariectomized to determine mechanisms by which specific subtypes of estrogen receptors exert neuroprotection in female mice. On completing experiments in this proposal, key signaling targets through which cytisine and estrogen mediate neuroprotective effects will be identified. Success in this proposal will lay groundwork for the long-term goal of discovering clinically translatable neuroprotective PD drugs that work in both men and women.