Association of patterning gene EN2 with ASD has been replicated independently in multiple datasets, supporting EN2 as an ASD susceptibility gene. We are using mouse models to define En2 developmental functions. Since En2 is expressed in embryonic mid/hindbrain regions where forebrain-projecting monoamine neurons originate, we examined neurotransmitters norepinephrine (NE), serotonin (5HT) and dopamine in En2 knock out (KO) and wild type (WT) mice. In previous study on postnatal day 21 (P21), KOs exhibited reduced forebrain monoamines, with changes in NE greater than 5HT, and increases in hindbrain. These results raise two questions: what are the developmental mechanisms of forebrain deficits? What are the behavior consequences?
Objectives:
Identify mechanisms mediating reduced forebrain monoamine neurotransmitters and characterize behavioral effects.
Methods:
Neurotransmitter levels were assessed using HPLC. Levels of TH protein were detected using western blotting, and axonal fibers were quantified using TH immunohistochemistry and Z-stack image analysis. Porsolt forced swim: Mice were placed in 20cm Plexiglas cylinder containing 12cm water. Immobility was measured every 5s during the last 4min of a 6min test, with observer blind to genotype. Tail suspension: Mice were suspended over a visually isolated area by the distal tail and immobility scored every 5s for 6min.
Results:
Overall, during development, monoamine neurotransmitters in En2 KO mice were increased in mid/hindbrain regions but decreased in forebrain structures. NE exhibited the greatest changes, with levels elevated 40-70% in brainstem/cerebellum and reduced 30-60% in hippocampus from P7 to P21 (N=15). However, by adulthood, P60, NE deficits were diminished, with 23% reductions (p<0.05) in hippocampus and no differences detected in hindbrain, suggesting partial developmental recovery. Regarding mechanisms, changes in NE levels were paralleled by those in biosynthetic enzyme protein, TH, which was decreased 50% in hippocampus and increased 60% in cerebellum at P21 (p<0.05). Further, TH protein deficits were paralleled by changes in axonal innervation: TH fibers were reduced 73.2% in the KO at P21 (p=2.74E-12), and also displayed mild recovery by P60 (to 59.2%). Significantly, we also detected sex-dependent differences in both neurotransmitters and behaviors. While NE levels in adult cerebral cortex did not differ between KO and WT littermates, sex-specific analysis revealed that male KOs retained a 26.5% reduction (p<0.05). Behaviorally, the male but not the female En2 KOs displayed significantly higher levels of immobility than WT littermates on the depression-related forced swim test, coinciding with sex dependent NE deficits, whereas tail suspension was unaffected.
Conclusions:
Development of forebrain-projecting monoamine systems is disturbed in the En2 KO, producing elevated levels in mid/hindbrain regions and reduced levels and axonal fibers in forebrain structures. These forebrain deficits, reminiscent of human ASD, partially recover as development proceeds. Thus En2 may regulate axonal innervation and/or neurotransmitter system development. Further, with age, transmitter system abnormalities and depression related behaviors were sex-dependent, potentially relevant to the male preponderance of ASD. These observations provide one mechanism by which hindbrain patterning gene abnormalities may affect development of and connectivity to the forebrain, likely reflecting known monoamine effects on proliferation, survival and differentiation.