Loss of GABAergic neurons in the hippocampus and cerebral cortex of Engrailed-2 null mutant mice: Implications for autism spectrum disorders

Abstract

The homeobox-containing transcription factor Engrailed-2 (En2) is involved in patterning and neuronal differentiation of the midbrain/hindbrain region, where it is prominently expressed. En2 mRNA is also expressed in the adult mouse hippocampus and cerebral cortex, indicating that it might also function in these brain areas. Genome-wide association studies revealed that En2 is a candidate gene for autism spectrum disorders (ASD), and mice devoid of its expression (En2−/− mice) display anatomical, behavioural and clinical “autistic-like” features. Since reduced GABAergic inhibition has been proposed as a possible pathogenic mechanism of ASD, we hypothesized that the phenotype of En2−/− mice might include defective GABAergic innervation in the forebrain. Here we show that the Engrailed proteins are present in postnatal GABAergic neurons of the mouse hippocampus and cerebral cortex, and adult En2−/− mice show reduced expression of GABAergic marker mRNAs in these areas. In addition, reduction in parvalbumin (PV), somatostatin (SOM) and neuropeptide Y (NPY) expressing interneurons is detected in the hippocampus and cerebral cortex of adult En2−/− mice. Our results raise the possibility of a link between altered function of En2, anatomical deficits of GABAergic forebrain neurons and the pathogenesis of ASD.

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

“…Animals were housed in a 12 h light/dark cycle with food and water available ad libitum. En2 mutants were originally generated on a mixed 129Sv ϫ C57BL/6 genetic background (Joyner et al, 1991) and then backcrossed at least five times into a C57BL/6 background (Sgadò et al, 2013a). Male and female WT and En2 Ϫ/Ϫ agematched adult littermates (3-5 months old; weight 25-35 g) obtained from heterozygous matings were used.…”

“…In situ hybridization was performed as previously described (Sgadò et al, 2013a) using an Arc-specific digoxigenin-labeled riboprobe (nucleotides 358 -1355 of the rat cDNA sequence NM_019361; 96% homologous to the mouse Arc sequence NM_018790.3). Digital images of three dorsal hippocampus sections per animal were analyzed using ImageJ (http://rsb.info.nih.gov/ij/).…”

“…Deficits in neurofibromin signaling pathways have been associated with im-paired learning: mice with an Nf1 heterozygous null mutation showed enhanced phosphorylation of ERK, leading to impaired long-term potentiation and hippocampal-dependent learning (Cui et al, 2008). Interestingly, these effects seem to be mediated by loss of Nf1 in inhibitory neurons (Cui et al, 2008), whose number is markedly reduced in the hippocampus of En2 Ϫ/Ϫ mice (Sgadò et al, 2013a). We therefore investigated the neurofibromin/ ERK pathway in the hippocampus of WT and En2 Ϫ/Ϫ mice following a spatial learning test in the MWM.…”

“…Recent studies demonstrate that En2 is also widely expressed in the postnatal brain and suggest that it might control the structure and function of learning-related circuits (Tripathi et al, 2009;Brielmaier et al, 2012;Sgadò et al, 2013a). Genetic studies associated En2 to autism spectrum disorders (ASD; Gharani et al, 2004;Benayed et al, 2009), and an abnormal expression and methylation of the En2 gene was found in the cerebellum of ASD patients (James et al, 2013).…”

“…Genetic studies associated En2 to autism spectrum disorders (ASD; Gharani et al, 2004;Benayed et al, 2009), and an abnormal expression and methylation of the En2 gene was found in the cerebellum of ASD patients (James et al, 2013). En2 Ϫ/Ϫ mice display neuropathological changes related to ASD, including cerebellar hypoplasia (Joyner et al, 1991;Kuemerle et al, 1997) and loss of forebrain GABAergic interneurons (Tripathi et al, 2009;Sgadò et al, 2013a). ASD-related behavioral deficits, including increased seizure susceptibility (Tripathi et al, 2009), reduced sociability, and impaired cognitive flexibility (Cheh et al, 2006;Brielmaier et al, 2012) were also detected in En2 Ϫ/Ϫ mice.…”