Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, Florida, United States of America.
PLoS One. 2012;7(9):e44207. doi: 10.1371/journal.pone.0044207. Epub 2012 Sep 6.
To better understand the short and long-term effects of stress on the developing cerebral cortex, it is necessary to understand how early stress response genes protect or permanently alter cells. One family of highly conserved, stress response genes is the growth arrest and DNA damage-45 (Gadd45) genes. The expression of these genes is induced by a host of genotoxic, drug, and environmental stressors. Here we examined the impact of altering the expression of Gadd45alpha (Gadd45a), a member of the Gadd45 protein family that is expressed throughout the developing cortices of mice and humans. To manipulate levels of Gadd45a protein in developing mouse cortex, we electroporated cDNA plasmids encoding either Gadd45a or Gadd45a shRNA to either overexpress or knockdown Gadd45a levels in the developing cortices of mice, respectively. The effects of these manipulations were assessed by examining the fates and morphologies of the labeled neurons. Gadd45a overexpression both in vitro and in vivo significantly impaired the morphology of neurons, decreasing neurite complexity, inducing soma hypertrophy and increasing cell death. Knockdown of Gadd45a partially inhibited neuronal migration and reduced neurite complexity, an effect that was reversed in the presence of an shRNA-resistant Gadd45a. Finally, we found that shRNA against MEKK4, a direct target of Gadd45a, also stunted neurite outgrowth. Our findings suggest that the expression of Gadd45a in normal, developing brain is tightly regulated and that treatments or environmental stimuli that alter its expression could produce significant changes in neuronal circuitry development.
为了更好地理解压力对发育中的大脑皮层的短期和长期影响，有必要了解早期应激反应基因是如何保护或永久改变细胞的。一组高度保守的应激反应基因是生长停滞和 DNA 损伤 45（Gadd45）基因。这些基因的表达受多种遗传毒性、药物和环境应激源的诱导。在这里，我们研究了改变 Gadd45alpha（Gadd45a）表达的影响，Gadd45a 是 Gadd45 蛋白家族的一员，在小鼠和人类发育中的皮质中广泛表达。为了在发育中的小鼠皮质中操纵 Gadd45a 蛋白的水平，我们用电穿孔法将编码 Gadd45a 或 Gadd45a shRNA 的 cDNA 质粒转染到小鼠发育中的皮质中，分别过表达或敲低 Gadd45a 的水平。通过检查标记神经元的命运和形态来评估这些操作的影响。Gadd45a 的过表达无论是在体外还是体内都显著损害了神经元的形态，降低了神经突的复杂性，诱导了体肥大并增加了细胞死亡。Gadd45a 的敲低部分抑制了神经元的迁移并减少了神经突的复杂性，而在存在 shRNA 抗性 Gadd45a 的情况下，这种效应被逆转。最后，我们发现针对 MEKK4 的 shRNA，Gadd45a 的直接靶标，也抑制了神经突的生长。我们的研究结果表明，Gadd45a 在正常发育的大脑中的表达受到严格调控，而改变其表达的治疗或环境刺激可能会对神经元回路发育产生重大影响。
