Castro Patricio A, Ramirez Alejandra, Sepúlveda Fernando J, Peters Christian, Fierro Humberto, Waldron Javier, Luza Sandra, Fuentealba Jorge, Muñoz Francisco J, De Ferrari Giancarlo V, Bush Ashley I, Aguayo Luis G, Opazo Carlos M
Department of Physiology and Membrane Biology, Shriners Hospital for Children Northern California, University of California at Davis School of Medicine California, USA.
Laboratorio de Neurofisiología, Departamento de Fisiología, Facultad de Ciencias Biológicas, Universidad de Concepción Concepción, Chile ; Oxidation Biology Laboratory, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne Parkville, Melbourne, Victoria, Australia.
Front Aging Neurosci. 2014 Dec 3;6:319. doi: 10.3389/fnagi.2014.00319. eCollection 2014.
Extracellular and intracellular copper and zinc regulate synaptic activity and plasticity, which may impact brain functionality and human behavior. We have found that a metal coordinating molecule, Neocuproine, transiently increases free intracellular copper and zinc levels (i.e., min) in hippocampal neurons as monitored by Phen Green and FluoZin-3 fluorescence, respectively. The changes in free intracellular zinc induced by Neocuproine were abolished by the presence of a non-permeant copper chelator, Bathocuproine (BC), indicating that copper influx is needed for the action of Neocuproine on intracellular Zn levels. Moreover, Neocuproine decreased the mRNA levels of Synapsin and Dynamin, and did not affect the expression of Bassoon, tubulin or superoxide dismutase (SOD). Western blot analysis showed that protein levels of synapsin and dynamin were also down regulated in the presence of Neocuproine and that these changes were accompanied by a decrease in calcium transients and neuronal activity. Furthermore, Neocuproine decreased the number of active neurons, effect that was blocked by the presence of BC, indicating that copper influx is needed for the action of Neocuproine. We finally show that Neocuproine blocks the epileptiform-like activity induced by bicuculline in hippocampal neurons. Collectively, our data indicates that presynaptic protein configuration and function of primary hippocampal neurons is sensitive to transient changes in transition metal homeostasis. Therefore, small molecules able to coordinate transition metals and penetrate the blood-brain barrier might modify neurotransmission at the Central Nervous System (CNS). This might be useful to establish therapeutic approaches to control the neuronal hyperexcitabiltity observed in brain conditions that are associated to copper dyshomeotasis such as Alzheimer's and Menkes diseases. Our work also opens a new avenue to find novel and effective antiepilepsy drugs based in metal coordinating molecules.
细胞外和细胞内的铜和锌调节突触活动和可塑性，这可能会影响大脑功能和人类行为。我们发现，一种金属配位分子新铜试剂，分别通过吩嗪绿和FluoZin-3荧光监测，可使海马神经元内的游离铜和锌水平短暂升高（即分钟级）。新铜试剂诱导的细胞内游离锌的变化被非渗透性铜螯合剂bathocuproine（BC）的存在所消除，这表明铜内流是新铜试剂作用于细胞内锌水平所必需的。此外，新铜试剂降低了突触结合蛋白和发动蛋白的mRNA水平，并且不影响巴松管蛋白、微管蛋白或超氧化物歧化酶（SOD）的表达。蛋白质印迹分析表明，在新铜试剂存在的情况下，突触结合蛋白和发动蛋白的蛋白质水平也下调，并且这些变化伴随着钙瞬变和神经元活动的减少。此外，新铜试剂减少了活跃神经元的数量，这种效应被BC的存在所阻断，表明铜内流是新铜试剂发挥作用所必需的。我们最终表明，新铜试剂可阻断荷包牡丹碱在海马神经元中诱导的癫痫样活动。总体而言，我们的数据表明，原代海马神经元的突触前蛋白构型和功能对过渡金属稳态的短暂变化敏感。因此，能够配位过渡金属并穿透血脑屏障的小分子可能会改变中枢神经系统（CNS）的神经传递。这对于建立治疗方法以控制在与铜稳态失调相关的脑部疾病（如阿尔茨海默病和门克斯病）中观察到的神经元过度兴奋可能是有用的。我们的工作还开辟了一条新途径，以寻找基于金属配位分子的新型有效抗癫痫药物。
