Srinivasan Rahul, Henley Beverley M, Henderson Brandon J, Indersmitten Tim, Cohen Bruce N, Kim Charlene H, McKinney Sheri, Deshpande Purnima, Xiao Cheng, Lester Henry A
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125.
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125
J Neurosci. 2016 Jan 6;36(1):65-79. doi: 10.1523/JNEUROSCI.2126-15.2016.
Retrospective epidemiological studies show an inverse correlation between susceptibility to Parkinson's disease and a person's history of tobacco use. Animal model studies suggest nicotine as a neuroprotective agent and nicotinic acetylcholine (ACh) receptors (nAChRs) as targets for neuroprotection, but the underlying neuroprotective mechanism(s) are unknown. We cultured mouse ventral midbrain neurons for 3 weeks. Ten to 20% of neurons were dopaminergic (DA), revealed by tyrosine hydroxylase (TH) immunoreactivity. We evoked mild endoplasmic reticulum (ER) stress with tunicamycin (Tu), producing modest increases in the level of nuclear ATF6, phosphorylated eukaryotic initiation factor 2α, nuclear XBP1, and the downstream proapoptotic effector nuclear C/EBP homologous protein. We incubated cultures for 2 weeks with 200 nm nicotine, the approximate steady-state concentration between cigarette smoking or vaping, or during nicotine patch use. Nicotine incubation suppressed Tu-induced ER stress and the unfolded protein response (UPR). Study of mice with fluorescent nAChR subunits showed that the cultured TH+ neurons displayed α4, α6, and β3 nAChR subunit expression and ACh-evoked currents. Gene expression profile in cultures from TH-eGFP mice showed that the TH+ neurons also express several other genes associated with DA release. Nicotine also upregulated ACh-induced currents in DA neurons by ∼2.5-fold. Thus, nicotine, at a concentration too low to activate an appreciable fraction of plasma membrane nAChRs, induces two sequelae of pharmacological chaperoning in the ER: UPR suppression and nAChR upregulation. Therefore, one mechanism of neuroprotection by nicotine is pharmacological chaperoning, leading to UPR suppression. Measuring this pathway may help in assessing neuroprotection.
Parkinson's disease (PD) cannot yet be cured or prevented. However, many retrospective epidemiological studies reveal that PD is diagnosed less frequently in tobacco users. Existing programs attempting to develop nicotinic drugs that might exert this apparent neuroprotective effect are asking whether agonists, antagonists, partial agonists, or channel blockers show the most promise. The underlying logic resembles the previous development of varenicline for smoking cessation. We studied whether, and how, nicotine produces neuroprotective effects in cultured dopaminergic neurons, an experimentally tractable, mechanistically revealing neuronal system. We show that nicotine, operating via nicotinic receptors, does protect these neurons against endoplasmic reticulum stress. However, the mechanism is probably "inside-out": pharmacological chaperoning in the endoplasmic reticulum. This cellular-level insight could help to guide neuroprotective strategies.
回顾性流行病学研究表明，帕金森病易感性与个人吸烟史之间存在负相关。动物模型研究表明尼古丁是一种神经保护剂，烟碱型乙酰胆碱（ACh）受体（nAChRs）是神经保护的靶点，但其潜在的神经保护机制尚不清楚。我们将小鼠腹侧中脑神经元培养3周。酪氨酸羟化酶（TH）免疫反应显示10%至20%的神经元为多巴胺能（DA）神经元。我们用衣霉素（Tu）引发轻度内质网（ER）应激，使核ATF6、磷酸化真核起始因子2α、核XBP1和下游促凋亡效应因子核C/EBP同源蛋白水平适度升高。我们将培养物与200 nM尼古丁孵育2周，这一浓度接近吸烟、吸电子烟或使用尼古丁贴片时的稳态浓度。尼古丁孵育可抑制Tu诱导的ER应激和未折叠蛋白反应（UPR）。对带有荧光nAChR亚基的小鼠研究表明，培养的TH+神经元表达α4、α6和β3 nAChR亚基，并产生ACh诱发电流。来自TH-eGFP小鼠培养物的基因表达谱显示，TH+神经元还表达其他几种与DA释放相关的基因。尼古丁还使DA神经元中ACh诱导的电流上调约2.5倍。因此，尼古丁在浓度过低而无法激活相当一部分质膜nAChRs的情况下，在内质网中诱导了药理学伴侣作用的两个后果：UPR抑制和nAChR上调。因此，尼古丁神经保护的一种机制是药理学伴侣作用，导致UPR抑制。检测这一途径可能有助于评估神经保护作用。
帕金森病（PD）目前尚无法治愈或预防。然而，许多回顾性流行病学研究表明，吸烟者中PD的诊断频率较低。现有的试图开发可能具有这种明显神经保护作用的烟碱类药物的项目正在探讨激动剂、拮抗剂、部分激动剂或通道阻滞剂是否最有前景。其基本逻辑类似于先前开发伐尼克兰用于戒烟的过程。我们研究了尼古丁是否以及如何在培养的多巴胺能神经元中产生神经保护作用，这是一个实验上易于处理、能揭示机制的神经元系统。我们表明，尼古丁通过烟碱型受体发挥作用，确实能保护这些神经元免受内质网应激。然而，其机制可能是“由内而外”的：内质网中的药理学伴侣作用。这种细胞水平的见解有助于指导神经保护策略。
