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

Friday, May 16, 2014
Atrium Ballroom (Marriott Marquis Atlanta)
G. Provenzano1, L. Pangrazzi1, A. Poli2, P. Sgad1, S. Genovesi1, G. Zunino1, N. Berardi2, S. Casarosa3 and Y. Bozzi1, (1)Molecular Neuropathology Laboratory, Centre for Integrative Biology (CIBIO), University of Trento, Italy, Trento, Italy, (2)C.N.R. Neuroscience Institute, Pisa, Italy, Pisa, Italy, (3)Developmental Neurobiology Laboratory, Centre for Integrative Biology (CIBIO), University of Trento, Italy, Trento, Italy
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.

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See more of: Animal Models