Deficiency of Engrailed 2 (En2), the Autism Spectrum Disorder (ASD) Associated Gene, Produces Abnormal Development of Forebrain-Projecting, Monoamine Neurotransmitters Systems

Background:

We have found that EN2 is associated with ASD in 3 datasets, and 4 other groups report associations in datasets of different ethnicities that exhibit allelic differences, observations that support EN2 as an ASD susceptibility gene. We are using the En2 mutant mouse (knock out, KO) as a genetically valid model to define the roles of En2 during development. Significantly, En2 is expressed prenatally in mid- and hindbrain regions where monoamine-containing neurons that project to the forebrain originate. In ASD, abnormalities in monoamines, especially serotonin (5HT), are well known and include increased blood levels in 1/3 of patients, altered rates of 5HT synthesis detected by PET scanning, and serotonin transporter gene (5-HTT) linkage. Furthermore, forebrain-projecting monoamine neurons play major roles in behaviors altered in ASD, including attention, mood, repetitive movements and self-injury. The therapeutic efficacy of monoamine modulating drugs provides additional evidence of altered monoamine function in ASD. A possible link between EN2 and development of monoamine neurotransmitter systems prompted us to examine development of the major monoamine transmitters, norepinephrine (NE), dopamine (DA), 5HT and their metabolites, in En2 KO and wild type (WT) mice.

Objectives:

Identify the role of En2 in monoamine neurotransmitter system development using En2 KO mutant as animal model.

Methods:

Cerebral cortex, striatum, hippocampus, amygdala, cerebellum, midbrain and pons/medulla were dissected from postnatal day 0 (P0), P7, P14, P21 and P60 WT and En2 KO mice. Neurotransmitter levels were assessed by HPLC and tyrosine hydroxylase (TH) protein levels were assayed by Western blotting.

Results:

Overall, during development, monoamine transmitters were increased in mid/hindbrain regions but decreased in forebrain structures of En2 KO mice. The greatest changes were in NE, with levels elevated 40-70% in cerebellum but reduced 30-60% in hippocampus from P7 to P21. Most forebrain regions displayed decreased NE levels at P14 and P21, while all mid/hindbrain regions showed increases. Reduction of NE levels in forebrain started as early as P0. However, by adulthood, P60, NE deficits were diminished, with 16% and 23% reductions in amygdala and hippocampus respectively, without changes in other regions, suggesting partial recovery as development proceeded. One potential mechanism, a deficit in NE synthesizing enzyme, TH, was supported by Western blot, which demonstrated a parallel 50% reduction in protein levels at P21 in KO hippocampus. 5HT and DA exhibited smaller changes in brain regions at several ages, with similar mid/hindbrain increases and forebrain decreases, suggesting an overall regional delay/deficit in monoamine neurogenesis and/or axonal process elaboration. In contrast, there was no difference in regional GABA content.

Conclusions:

Development of forebrain-projecting monoamine neurotransmitter systems is disturbed by deletion of En2, producing elevated levels in mid/hindbrain regions and reduced levels in forebrain structures. These forebrain deficits partially recover as development proceeds. These observations provide one mechanism by which patterning gene abnormalities in the hindbrain can secondarily affect forebrain development, especially given known effects of monoamines on proliferation, survival and differentiation. We plan to further characterize brain region structure and function to identify the roles of En2 during development.