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The Engrailed-2 (En2) gene codes for a homeobox-containing transcription factor, involved in midbrain-hindbrain embryonic development. In postnatal brain, En2 is expressed in the ventral mesencephalon, cerebellum, hippocampus and neocortex. Two single-nucleotide polymorphisms (SNPs) that are associated to autism spectrum disorders (ASD) have been identified in the human EN2 gene. Accordingly, mice lacking the En2 homeodomain (En2hd/hd, referred to as En2-/-) show molecular, anatomical and behavioral "ASD-like" features. Among these, we previously showed a partial loss of GABAergic interneurons in the En2-/- postnatal hippocampus and neocortex, accompanied by a marked decrease of brain-derived neurotrophic factor (BDNF) signaling, a crucial determinant of GABAergic differentiation. In order to better investigate the role of En2 in GABAergic interneuron differentiation, we generated and subsequently differentiated neural stem cells (NSCs) from basal ganglia and neocortex of En2+/+ and En2-/- mouse embryos. Wild-type NSCs from both basal ganglia and neocortex express En2, while mutant ones do not, as expected. As compared to En2+/+ NSCs, En2-/- NSCs derived from basal ganglia show impaired GABAergic differentiation accompanied by a reduced expression of the BDNF receptor trkB. Conversely, En2-/- NSCs derived from the neocortex expressed high levels of trkB and readily differentiated into neurons, as En2+/+ NSCs. Our results suggest that En2 contributes to GABAergic neuron differentiation from basal ganglia NSCs through a trkB-dependent BDNF signaling, thus providing a possible explanation for the reduced number of GABAergic interneurons detected in the En2-/- postnatal forebrain.

Mice lacking the homeodomain transcription factor Engrailed-2 (En2(-/-) mice) are a well-characterized model for autism spectrum disorders (ASD). En2(-/-) mice present molecular, neuropathological and behavioral deficits related to ASD, including down-regulation of ASD-associated genes, cerebellar hypoplasia, interneuron loss, enhanced seizure susceptibility, decreased sociability and impaired cognition. Specifically, impaired spatial learning in the Morris water maze (MWM) is associated with reduced expression of neurofibromin and increased phosphorylation of extracellular-regulated kinase (ERK) in the hippocampus of En2(-/-) adult mice. In the attempt to better understand the molecular cascades underlying neurofibromin-dependent cognitive deficits in En2 mutant mice, we investigated the expression and phosphorylation of synapsin I (SynI; a major target of neurofibromin-dependent signaling) in the hippocampus of wild-type (WT) and En2(-/-) mice before and after MWM. Here we show that SynI mRNA and protein levels are down-regulated in the hippocampus of naïve and MWM-treated En2(-/-) mice, as compared to WT controls. This down-regulation is paralleled by reduced levels of SynI phosphorylation at Ser549 and Ser553 residues in the hilus of mutant mice, before and after MWM. These data indicate that in En2(-/-) hippocampus, neurofibromin-dependent pathways converging on SynI phosphorylation might underlie hippocampal-dependent learning deficits observed in En2(-/-) mice.