Engrailed Expression in Hippocampus and Its Effects on Dendritic Complexity: Implication for Autism Spectrum Disorders

Background:   Engrailed (En) is a transcription factor that plays essential roles in the establishment of embryo anterior-posterior axis during early embryogenesis. It also contributes to midbrain and hindbrain patterning, to development and maintenance of monoaminergic pathways, and to retinotectal wiring. En2 is an autism susceptibility gene but the mechanisms remain unclear. As several homeoproteins, En can also act as an extracellular signaling molecule being secreted and internalized in target cells. As a signaling molecule En acts through transcriptional and/or translational dependent mechanisms. 

Objectives:   Cognitive disorders are encountered in En1+/- mice and En2-/- mice. The present work was aimed at addressing the effect of En on synaptogenesis in the forebrain.   

Methods:   To address a putative role for En1 and En2 in the forebrain we measured their expression in hippocampus in wild-type mice using RT-qPCR, and performed immunochemistry on hippocampal primary culture cells to characterize cell type expression and localization. Dendritic spine density was measured in hippocampal neurons in En2-/- and En1+/- mice as well as in cultured hippocampal neurons treated with bath application of recombinant En protein.   

Results:  Both mRNAs were readily detected in late mouse embryos. Levels of En1 declined after postnatal day 14 down to 50% of initial values while En2 mRNAs abruptly dropped during the first postnatal week before rising back to initial values, suggesting a temporal coupling between En2 expression and synaptogenesis. Engrailed was detected by immunocytochemistry in most of the neurons from hippocampal cultures and was markedly enriched in GABAergic cells. Adding recombinant Engrailed to cultured neurons increased dendritic branching and dendritic spine density. Stubby, thin and branched spines were particularly affected, suggesting increased spine plasticity. In contrast, the number of presynaptic terminals and synapses remained unchanged. However, an En mutant defective in eIF4E binding reduced presynaptic terminal density and synaptic matching, suggesting that Engrailed impacts on synaptogenesis. The latter results indicate that eIF4E interaction with Engrailed is, at least in part, responsible for its effects on spinogenesis and suggest a role of En in presynaptic button formation/stabilization. Because eIF4E has a key function in translation, it is possible to speculate that some of En effects reported here could be translation dependent. Consistently, our results show that Engrailed is capable to increase protein synthesis in hippocampal neurons. In vivo, spine density was increased in the hippocampus of young En1+/- mice but reduced in adults, whereas the opposite was found in En2-/- mice, confirming that Engrailed controls spinogenesis either directly or indirectly.  

Conclusions: Taken together, our results identify Engrailed as a novel actor in dendritic plasticity. They reveal that an excess of En during synaptogenesis may alter the characteristics of dendritic plasticity that could ultimately lead to synaptic network dysfunction. Our observations open new perspectives on the relationship between Engrailed and ASD.