Hippocampal Dysregulation of Neurofibromin-Dependent Pathways Is Associated with Impaired Spatial Learning in Engrailed 2 Knockout Mice

Background: Genome-wide association studies indicated the homeobox-containing transcription factor Engrailed-2 (En2) as a candidate gene for autism spectrum disorders (ASD). Accordingly, En2 knockout (En2-/-) mice show anatomical and behavioural “ASD-like” features, including decreased sociability and learning deficits. The molecular pathways underlying these deficits in En2-/- mice are not known. Deficits in signaling pathways involving neurofibromin and extracellular-regulated kinase (ERK) have been associated to impaired learning.

Objectives: Here we investigated the neurofibromin-ERK cascade in the hippocampus of wild-type (WT) and En2-/- mice before and after spatial learning testing.

Methods: Twenty-two mice (11 per genotype) were used for the MWM. Animals were killed at the end of MWM and brains dissected: for RT-PCR and immunoblotting, 4 hippocampi per genotype were dissected and frozen in dry ice; for immunohistochemistry, 4 brains per genotype were fixed by 4% paraformaldehyde perfusion; for in situ hybridization, 3 brains per genotype were frozen on dry ice. An additional group of age-matched, naïve mice (8 per genotype) were not subjected to MWM and used a controls.

Results: When compared to WT littermates, En2-/- mice showed impaired performance in the Morris water maze (MWM), which was accompanied by lower expression of the activity-dependent gene Arc. Quantitative RT-PCR, immunoblotting and immunohistochemistry experiments showed a marked downregulation of neurofibromin expression in the dentate gyrus of both naïve and MWM-treated En2-/- mice. ERK phosphorylation, known to be induced in the presence of neurofibromin deficiency, was increased in the dentate gyrus of En2-/- mice after MWM.

Conclusions: Dysregulation of the neurofibromin/ERK pathway in the hippocampus may contribute to spatial learning deficits in the En2 mouse model of ASD.