Increased signaling by the autism-related Engrailed-2 protein enhances dendritic branching and spine density, alters synaptic structural matching, and exaggerates protein synthesis

PLoS One. 2017 Aug 15;12(8):e0181350. doi: 10.1371/journal.pone.0181350. eCollection 2017.

Abstract

Engrailed 1 (En1) and 2 (En2) code for closely related homeoproteins acting as transcription factors and as signaling molecules that contribute to midbrain and hindbrain patterning, to development and maintenance of monoaminergic pathways, and to retinotectal wiring. En2 has been suggested to be an autism susceptibility gene and individuals with autism display an overexpression of this homeogene but the mechanisms remain unclear. We addressed in the present study the effect of exogenously added En2 on the morphology of hippocampal cells that normally express only low levels of Engrailed proteins. By means of RT-qPCR, we confirmed that En1 and En2 were expressed at low levels in hippocampus and hippocampal neurons, and observed a pronounced decrease in En2 expression at birth and during the first postnatal week, a period characterized by intense synaptogenesis. To address a putative effect of Engrailed in dendritogenesis or synaptogenesis, we added recombinant En1 or En2 proteins to hippocampal cell cultures. Both En1 and En2 treatment increased the complexity of the dendritic tree of glutamatergic neurons, but only En2 increased that of GABAergic cells. En1 increased the density of dendritic spines both in vitro and in vivo. En2 had similar but less pronounced effect on spine density. The number of mature synapses remained unchanged upon En1 treatment but was reduced by En2 treatment, as well as the area of post-synaptic densities. Finally, both En1 and En2 elevated mTORC1 activity and protein synthesis in hippocampal cells, suggesting that some effects of Engrailed proteins may require mRNA translation. Our results indicate that Engrailed proteins can play, even at low concentrations, an active role in the morphogenesis of hippocampal cells. Further, they emphasize the over-regulation of GABA cell morphology and the vulnerability of excitatory synapses in a pathological context of En2 overexpression.

MeSH terms

  • Animals
  • Autistic Disorder / metabolism*
  • Cells, Cultured
  • Dendrites / genetics*
  • Fluorescent Antibody Technique
  • Gene Expression Regulation / genetics*
  • Hippocampus / cytology
  • Hippocampus / metabolism
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism*
  • Mechanistic Target of Rapamycin Complex 1
  • Mice
  • Microscopy, Confocal
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Neuroglia / cytology
  • Neuroglia / metabolism
  • Signal Transduction*
  • Synapses / metabolism
  • TOR Serine-Threonine Kinases / genetics
  • TOR Serine-Threonine Kinases / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • En1 protein, mouse
  • Homeodomain Proteins
  • Multiprotein Complexes
  • Nerve Tissue Proteins
  • Transcription Factors
  • engrailed 2 protein
  • TOR Serine-Threonine Kinases
  • Mechanistic Target of Rapamycin Complex 1

Grant support

This work was supported by France Alzheimer, Fondation Louis D. and the Agence Nationale pour la Recherche (ANR-12-Polybex) (to F.D.) and grants from the Italian Ministry of University and Research (PRIN 2010-2011 grant #2010N8PBAA_002 to Y.B.), University of Trento (CIBIO start-up grant to Y.B.). F.D. is supported by Inserm. A.S. was supported by a fellowship from the French ministry of higher education and research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.