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