Division of Reproductive Sciences, Oregon National Primate Research Center, Beaverton, OR97006, USA.
Mol Psychiatry. 2010 Oct;15(10):1034-44. doi: 10.1038/mp.2009.78. Epub 2009 Aug 18.
Dendritic spines are the elementary structural units of neuronal plasticity and the cascades that promote dendritic spine remodeling center on Rho GTPases and downstream effectors of actin dynamics. In a model of hormone replacement therapy, we sought the effect of estradiol (E) and progesterone (P) on gene expression in these cascades in laser-captured serotonin neurons from rhesus macaques with complementary DNA array analysis. Ovariectomized rhesus macaques were treated with either placebo, E or E+P through Silastic implant for 1 month before euthanasia. The midbrain was obtained, sectioned and immunostained for tryptophan hydroxylase (TPH). TPH-positive neurons were laser captured using an Arcturus Laser Dissection Microscope (PixCell II). RNA from laser-captured serotonin neurons (n=2 animals/treatment) was hybridized to Rhesus Affymetrix GeneChips. With E±P treatment, there was a significant change in 744 probe sets (analysis of variance, P<0.05), but 10,493 probe sets exhibited a twofold or greater change. Pivotal changes in pathways leading to dendritic spine proliferation and transformation included twofold or greater increases in expression of the Rho GTPases called CDC42, Rac1 and RhoA. In addition, twofold or greater increases occurred in downstream effectors of actin dynamics, including p21-activated kinase (PAK1), Rho-associated coiled-coil-containing protein kinase (ROCK), PIP5K, IRSp53, Wiskott-Aldrich syndrome protein (WASP), WASP family Verprolin-homologous protein (WAVE), MLC, cofilin, gelsolin, profilin and three subunits of actin-related protein (ARP2/3). Finally, twofold or greater decreases occurred in CRIPAK, LIMK2 and myosin light chain kinase (MLCK). The regulation of RhoA, Rac1, CDC42, ROCK, PIP5k, IRSp53, WASP, WAVE, LIMK2, CRIPAK1, MLCK, ARP2/3 subunit 3, gelsolin, profilin and cofilin was confirmed with nested quantitative reverse transcriptase-PCR on laser-captured RNA (n=3 animals/treatment). The data indicate that ovarian steroids target gene expression of the Rho GTPases and pivotal downstream proteins, that in turn would promote dendritic spine proliferation and stabilization on serotonin neurons of the dorsal raphe nucleus.
树突棘是神经元可塑性的基本结构单元，促进树突棘重塑的级联反应集中在 Rho GTPases 和肌动蛋白动力学的下游效应物上。在激素替代疗法的模型中，我们通过 cDNA 阵列分析，研究了雌二醇 (E) 和孕酮 (P) 对恒河猴激光捕获 5-羟色胺神经元中这些级联反应的基因表达的影响。对去卵巢恒河猴用硅酮植入物进行安慰剂、E 或 E+P 处理 1 个月，然后安乐死。获得中脑，切片并用色氨酸羟化酶 (TPH) 免疫染色。使用 Arcturus 激光解剖显微镜 (PixCell II) 激光捕获 TPH 阳性神经元。来自激光捕获的 5-羟色胺神经元的 RNA（n=2 只动物/处理）与恒河猴 Affymetrix GeneChips 杂交。用 E±P 处理时，有 744 个探针集发生了显著变化（方差分析，P<0.05），但有 10493 个探针集发生了两倍或更多倍的变化。导致树突棘增殖和转化的途径的关键变化包括 Rho GTPases 称为 CDC42、Rac1 和 RhoA 的表达增加两倍或更多倍。此外，肌动蛋白动力学的下游效应物也增加了两倍或更多倍，包括 p21 激活激酶 (PAK1)、Rho 相关卷曲螺旋蛋白激酶 (ROCK)、PIP5K、IRSp53、Wiskott-Aldrich 综合征蛋白 (WASP)、WASP 家族卷曲螺旋同源蛋白 (WAVE)、肌球蛋白轻链 (MLC)、丝切蛋白、凝胶蛋白、原肌球蛋白和肌动蛋白相关蛋白 (ARP) 的三个亚基 2/3。最后，CRIPAK、LIMK2 和肌球蛋白轻链激酶 (MLCK) 的表达减少了两倍或更多倍。用激光捕获的 RNA（n=3 只动物/处理）进行嵌套定量逆转录-PCR 证实了 RhoA、Rac1、CDC42、ROCK、PIP5k、IRSp53、WASP、WAVE、LIMK2、CRIPAK1、MLCK、ARP2/3 亚基 3、凝胶蛋白、原肌球蛋白和丝切蛋白的调节。数据表明，卵巢类固醇靶向 Rho GTPases 和关键下游蛋白的基因表达，这反过来又会促进背缝核 5-羟色胺神经元的树突棘增殖和稳定。
