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

Date Published: August 15, 2017

Publisher: Public Library of Science

Author(s): Asma Soltani, Solène Lebrun, Gilles Carpentier, Giulia Zunino, Sandrine Chantepie, Auriane Maïza, Yuri Bozzi, Claire Desnos, François Darchen, Olivier Stettler, Michelle M. Adams.

http://doi.org/10.1371/journal.pone.0181350

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.

Partial Text

Autism spectrum disorders (ASDs) comprise heterogeneous neurodevelopmental disorders characterized by significant social and communication deficits and by repetitive and stereotyped behaviors. In syndromic ASDs (e.g., fragile X syndrome, Angelman syndrome, tuberous sclerosis), autism is one component of a broader phenotype [1]. ASDs manifest at key steps of brain development when sensory experience is modifying synapse maturation and when inhibitory synapses become functional [2]. Targeted mutations of several susceptibility genes for ASDs with seemingly unrelated functions often lead to similar abnormalities of the development of dendrites and synapses of excitatory glutamatergic neurons in mice [3]. This phenotypical convergence suggests that ASD may originate in altered synaptic development leading to imbalance between excitation and inhibition (E/I), and defects in brain connectivity [2–4]. In this respect, many genes linked to ASDs have been shown to control the morphology and signaling of dendrites and dendritic spines of excitatory neurons [3], by encoding synaptic adhesion or scaffolding proteins (e.g., Shank3, Neuregulin, Mecp2, Neuroligin) [3], or molecules regulating protein synthesis (e.g., PTEN, TSC1/2, NF1, and FMRP) [3,5].

We found here that excessive signaling by En alters dendritogenesis and spinogenesis and thus the accurate development of hippocampal cell network. It is well established that En drives important aspects of the development of midbrain and cerebellum [12,16]. However, the possibility of a role of En in the forebrain, which could better reflect the cognitive impairments observed in ASDs and En2-/- mice [1,25], has been disregarded, possibly because of the highest expression level of En in the midbrain/hindbrain regions (http://www.gensat.org). We revisited the expression of En in the hippocampus, confirmed precedent reports showing that En are expressed in the adult hippocampus and hippocampal cells [18,23–25], and further showed that the expression of En1 and En2 is subjected to a tight regulation during the development of this structure. Particularly striking is the downregulation of En2 mRNA during the first postnatal week, a period associated with a sudden increase in synaptogenesis in rodents [33]. A similar downregulation of En2 at birth has been reported in the cerebellum [40]. This downregulation is functionally important as maintenance of En2 overexpression postnatally in Purkinje cells delays their differentiation [41], a phenomenon hypothesized to occur during ASD [42]. Considering the ability of En2 to reduce excitatory synaptic contacts, the sharp downregulation of En2 mRNA at P0-P5 may usefully prevent interference between En2 effects and glutamatergic synaptogenesis. Differential expression of En1 and En2 during hippocampal development suggests that these two genes have distinct activities in relation to neuronal differentiation and maturation. Accordingly, we found that En2 increased GABAergic dendritic complexity and reduced glutamatergic connectivity, while En1 did not. On the other hand, En1 was more effective than En2 to increase spinogenesis on pyramidal neurons in vitro. This functional partitioning is maintained in vivo in the adult hippocampus since En2 KO but not En1+/- mice display a loss of GABAergic neurons [15, 23], while spine density is reduced in the hippocampus of adult En1+/- but not in En2 KO mice (present results). Still, the functional specificity of En proteins does not exclude some redundancy as evidenced by the finding that, in vitro, both En1 and En2 promote dendritic complexity and spinogenesis in glutamatergic cells.

 

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http://doi.org/10.1371/journal.pone.0181350

 

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