Abstract
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.
Highlights
Like all eukaryotic cells, developing neurons possess molecular pathways that either protect or eliminate them in the presence of cellular stressors
To determine if Gadd45a is expressed in developing forebrain, we analyzed forebrain tissue extracts isolated from fetal mouse and human using a combination of RT-PCR and western blot analyses
The results of our in vitro and in vivo experiments provide strong evidence that Gadd45a plays a role in shaping the formation of the soma and dendrites of neurons in developing cortex and that either abnormal increases or decreases in the levels of Gadd45a can disrupt these developmental processes
Summary
Like all eukaryotic cells, developing neurons possess molecular pathways that either protect or eliminate them in the presence of cellular stressors. One family of highly conserved genes that contributes to these pathways is the growth arrest and DNA damage 45 (Gadd45) family which includes three members: alpha (Gadd45a), beta (Gadd45b) and gamma (Gadd45g) [1,2]. Gadd45a shares ,50% sequence homology with Gadd45b and Gadd45g [3]. The downstream pathways through which Gadd45a signals are diverse and include activation of various mitogen activated protein kinase (MAPK) signaling cascades, transcription factors, cell cycle regulators, and DNA repair processes (for review see: [3]). While Gadd45a expression has been detected in the early stages of mouse forebrain development [5], it remains unclear as to whether its expression persists or is inducible in developing and postnatal cerebral cortex
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