Autism-Relevant Social Abnormalities and Cognitive Deficits in Engrailed-2 Knockout Mice

Background: Several association studies have identified the homeobox transcription factor ENGRAILED 2 (EN2) as a likely autism susceptibility gene (Gharani et al. 2004; Benayed et al. 2005; Wang et al. 2008; Sen et al. 2010; Yang et al., 2010). In the mouse, En2 serves as a patterning gene of hindbrain and cerebellum, and impacts neurogenesis and development of monoamine systems (Joyner, 1996; Simon et al., 2005; Cheh et al., 2006). We previously reported that monoamine system development is disrupted in mice with a deletion in En2, producing elevated levels of these transmitters in midbrain and hindbrain regions and reduced levels and axonal fibers in forebrain structures, with the greatest changes occurring in norepinephrine and its biosynthetic enzyme tyrosine hydroxylase (Lin et al., 2010; Genestine et al., 2011).  These monoamine system abnormalities were accompanied by increased depression-relevant behavior in the forced swim test (Lin et al., 2010).

Objectives: To understand additional consequences of En2 mutations on behaviors relevant to autism, we conducted comprehensive behavioral phenotyping of En2 mutant mice, employing social, communication, repetitive, and cognitive behavioral assays, and a series of control measures for physical abilities.

Methods: Male and female wildtype (+/+), heterozygote (+/-) and null mutant (-/-) littermate offspring were tested on multiple measures of social interactions and social approach, communication, repetitive behaviors, cognitive abilities, anxiety and depression-relevant behaviors, sensory and motor functions,  exploratory locomotor activity, general health parameters and a sequence of neurological reflexes. To evaluate the robustness of the behavioral abnormalities detected, two separate cohorts representing all three genotypes were evaluated.

Results: En2 -/- mice exhibited robust deficits in reciprocal social interactions as juveniles and adults, and absence of sociability in adults, replicated in two independent cohorts.  No genotype differences were detected on measures of ultrasonic vocalizations in social contexts, and no stereotyped or repetitive behaviors were observed. Fear conditioning and water maze learning and memory were impaired in En2 -/- mice.  High immobility in the forced swim test, reduced prepulse inhibition, mild motor coordination impairments and reduced grip strength were additionally detected in En2 -/-.  Parameters of general health, olfactory abilities, exploratory locomotor activity, anxiety-like behaviors and pain responses did not differ across genotypes, indicating that the social and cognitive deficits detected in En2 -/- mice were not attributable to physical or procedural confounds.

Conclusions: Our results are consistent with the subset of assays previously reported in En2 -/- mice (Cheh et al. 2006). Higher immobility on forced swim replicates our previous report (Lin et al. 2010) and suggests a depression-like phenotype in En2 -/- mice. Our findings support the interpretation that deletion of the neurodevelopmental gene En2 in mice results in multiple behavioral phenotypes relevant to the diagnostic and associated symptoms of autism, offering a translational model for investigating mechanistically-driven therapeutics. We are currently assessing the potential of chronic oral treatment with desipramine, which selectively inhibits norepinephrine reuptake, to reverse the autism-relevant behavioral abnormalities and reduced forebrain norepinephrine levels detected in En2 -/- mice.