Tokuyama Y, Reddy A P, Bethea C L
Division of Reproductive Sciences, Oregon National Primate Research Center, Beaverton, OR 97006, USA.
Neuroscience. 2008 Jun 23;154(2):720-31. doi: 10.1016/j.neuroscience.2008.03.056. Epub 2008 Apr 3.
Using a nonhuman primate model of surgical menopause, our laboratory has shown that ovarian hormone treatment (HT) improves 5-HT neural function in the dorsal raphe nucleus (DRN). We further hypothesize that HT may increase 5-HT neuronal resilience. Recent data from microarray analysis indicated that HT regulates gene expression in pathways that lead to apoptosis. In this study, we questioned whether HT alters protein expression in caspase-dependent and independent pathways. Ovariectomized monkeys received Silastic implants containing placebo (empty), estrogen (E) or E+ progesterone (P). A small block of the midbrain containing the DRN was dissected and subjected to subcellular fractionation, yielding cytosolic, nuclear and mitochondrial fractions (n=4/group). The pro-apoptotic protein, c-jun n-terminal kinase (JNK1) and its phosphorylation were decreased by E+P treatment in the cytosolic fraction. Downstream of JNK are proteins in the caspase-dependent and -independent pathways. First, in the caspase-dependent pathway, cytoplasmic and mitochondrial fractions were immunoblotted for Bcl-2 family members, cytochrome c, Apaf1 and XIAP. However, the expression of these proteins did not differ among treatments. Pro-caspase 3 was decreased by E+P, but there was no evidence of active caspase in any group. Then, we examined the involvement of a protein in the caspase-independent pathway, called apoptosis-inducing factor (AIF). AIF mRNA (n=3/group) and AIF mitochondrial protein tended to decrease with hormone treatment. However, AIF protein in the nuclear fraction in E+P treated monkeys was significantly reduced. This indicates that HT is reducing the translocation of AIF from mitochondria to nucleus, thus inhibiting AIF-mediated apoptosis. AIF was immunocytochemically localized to large 5-HT-like neurons of the dorsal raphe. These data suggest that in the absence of global trauma or ischemia, HT may act through the caspase-independent pathway to promote neuroprotection in the 5-HT system.
利用手术绝经的非人灵长类动物模型，我们实验室已表明卵巢激素治疗（HT）可改善中缝背核（DRN）中的5-羟色胺（5-HT）神经功能。我们进一步推测HT可能会增加5-HT神经元的恢复力。来自微阵列分析的最新数据表明，HT可调节导致细胞凋亡的信号通路中的基因表达。在本研究中，我们探讨了HT是否会改变半胱天冬酶依赖性和非依赖性信号通路中的蛋白质表达。将去卵巢的猴子植入含安慰剂（空管）、雌激素（E）或E+孕酮（P）的硅橡胶植入物。解剖包含DRN的一小片中脑并进行亚细胞分级分离，得到胞质、核和线粒体级分（每组n = 4）。在胞质级分中，E+P治疗可降低促凋亡蛋白c-Jun氨基末端激酶（JNK1）及其磷酸化水平。JNK的下游是半胱天冬酶依赖性和非依赖性信号通路中的蛋白质。首先，在半胱天冬酶依赖性信号通路中，对胞质和线粒体级分进行免疫印迹检测Bcl-2家族成员、细胞色素c、凋亡蛋白酶激活因子1（Apaf1）和X连锁凋亡抑制蛋白（XIAP）。然而，这些蛋白质的表达在各治疗组之间没有差异。E+P可降低前半胱天冬酶3水平，但在任何组中均未发现有活性的半胱天冬酶。然后，我们检测了一种参与半胱天冬酶非依赖性信号通路的蛋白质——凋亡诱导因子（AIF）的作用。AIF信使核糖核酸（每组n = 3）和AIF线粒体蛋白水平倾向于随激素治疗而降低。然而，E+P处理的猴子核级分中的AIF蛋白显著减少。这表明HT正在减少AIF从线粒体向细胞核的转位，从而抑制AIF介导的细胞凋亡。AIF通过免疫细胞化学方法定位到中缝背核的大型5-HT样神经元。这些数据表明，在没有全身性创伤或缺血的情况下，HT可能通过半胱天冬酶非依赖性信号通路在5-HT系统中发挥神经保护作用。
