Dey Swatee, Snow Diane M
Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536, USA.
J Neurochem. 2007 Oct;103(2):542-56. doi: 10.1111/j.1471-4159.2007.04750.x. Epub 2007 Jul 17.
Cocaine exposure results in aberrant outgrowth and decreased survival for locus coeruleus (LC), a noradrenergic population of neurons that putatively regulates attentional function; however, the underlying mechanisms for these events are not known. We previously showed that cocaine exposure in vitro activates pro-apoptotic Bax, caspase-9, and caspase-3 in LC neurons dissected from embryonic day 14 rats, implicating that apoptosis may be orchestrated via signal transduction events. In the current study in vitro, we examined upstream events to determine the role of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), on LC signal transduction, because cocaine exposure to LC neurons triggered TNF-alpha expression at 30 min as measured by ELISA. Exposure of LC neurons to recombinant-TNF-alpha resulted in decreased metabolic activity, an indicator of reduced neuron viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], and increased apoptosis (terminal deoxynucleotidyl transferase-mediated DNA nick end labeling assay). Pro-apoptotic caspase-3 was induced by cocaine starting at 30 min. Recombinant-TNF-alpha induced caspase-3 activity earlier than cocaine (15 and 20 min). The caspase-3 levels were significantly reduced when cocaine and TNF-alpha were combined with neutralizing-TNF-alpha (nTNF-alpha), respectively. Further, cocaine alone elevated phospho-p38-mitogen-activated protein kinases that persisted when combined with nTNF-alpha. However, both cocaine and TNF-alpha independently increased phospho-c-Jun NH(2)-terminal kinase and Bax levels at concurrent time periods (30 min and 1 h), and this elevation was attenuated in the presence of nTNF-alpha. These simultaneous molecular events triggered by cocaine and TNF-alpha implicate a potential apoptotic signal transduction pathway via induction of phospho-c-Jun NH(2)-terminal kinase and Bax that may lead to caspase-3 activation and apoptosis in cocaine-exposed fetal LC neurons.
可卡因暴露会导致蓝斑核（LC）出现异常生长并降低其存活率，蓝斑核是一群去甲肾上腺素能神经元，据推测可调节注意力功能；然而，这些现象背后的机制尚不清楚。我们之前表明，体外给予可卡因会激活从胚胎第14天大鼠分离出的LC神经元中的促凋亡蛋白Bax、半胱天冬酶-9和半胱天冬酶-3，这表明细胞凋亡可能是通过信号转导事件来调控的。在当前的体外研究中，我们研究了上游事件，以确定促炎细胞因子肿瘤坏死因子α（TNF-α）在LC信号转导中的作用，因为通过酶联免疫吸附测定法（ELISA）检测发现，可卡因暴露于LC神经元后30分钟会触发TNF-α表达。将LC神经元暴露于重组TNF-α会导致代谢活性降低，这是神经元活力降低的一个指标[3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐测定法]，并增加细胞凋亡（末端脱氧核苷酸转移酶介导的DNA缺口末端标记测定法）。可卡因从30分钟开始诱导促凋亡的半胱天冬酶-3。重组TNF-α比可卡因更早诱导半胱天冬酶-3活性（15分钟和20分钟）。当可卡因和TNF-α分别与中和性TNF-α（nTNF-α）联合使用时，半胱天冬酶-3水平显著降低。此外，单独使用可卡因会使磷酸化p38丝裂原活化蛋白激酶升高，与nTNF-α联合使用时该升高持续存在。然而，可卡因和TNF-α在同时期（30分钟和1小时）均独立增加磷酸化c-Jun氨基末端激酶和Bax水平，并且在存在nTNF-α的情况下这种升高会减弱。可卡因和TNF-α引发的这些同时发生的分子事件表明，通过诱导磷酸化c-Jun氨基末端激酶和Bax可能存在一条潜在的凋亡信号转导途径，这可能导致可卡因暴露的胎儿LC神经元中的半胱天冬酶-3激活和细胞凋亡。
