Forebrain Development of Serotonin and Norepinephrine Neurotransmitter Systems is Abnormally Regulated in the Engrailed 2 (En2) Mutant Mouse

Background: ENGRAILED 2 (EN2) is a cerebellar patterning gene that has been reproducibly associated with autism spectrum disorders (ASD) in multiple datasets, and cerebella of En2 knock out mice (KO) phenocopy aspects of the human neuropathology. Prenatally, En2 is expressed in the mid- and hindbrain regions of the brainstem where monoamine-containing neurons that project to the forebrain develop. Since changes in serotonin levels, transmitter system maturation, and transmitter re-uptake genes are associated with ASD, we examined monoamine transmitters in this genetic model of autism.

Objectives: Define the effects of En2 deletion on development of monoamine neurotransmitters during brain development.

Methods: Brain regions including cerebral cortex, hippocampus, amygdala, midbrain and pons/medulla were dissected from postnatal day 21 (P21) wild type and En2 KO mice. Tissues were sonicated in buffer, acidified and centrifuged and levels of transmitters and metabolites were assessed by HPLC in supernatants in parallel with known standards. Other tissue homogenates were assayed by western blotting.

Results: At P21, the levels of serotonin and its metabolite, 5HIAA, were reduced 20-40% in the pons/medulla, midbrain, hippocampus and amygdala, but not cerebral cortex. Deficits in serotonin levels were paralleled by reductions in protein levels of its rate limiting biosynthetic enzyme, tryptophan hydroxylase, by western blotting. In contrast, levels of norepinephrine were elevated in the brainstem but markedly reduced in hippocampus and cortex, without change in amygdala. Finally, we observed no differences in GABA or glutamate in these regions, indicating selectivity of En2 effects on forebrain projecting systems.

Conclusions: En2 gene expression is required for normal development of brainstem monoamine-containing neurons that project to the forebrain, providing one mechanism by which this hindbrain patterning gene may contribute to altered brain growth and social, cognitive and repetitive behaviors observed in ASD.