We have found the EN2 gene to be associated with ASD in 3 different datasets, and disease associations have been reported by 6 other groups. En2 is a transcription factor that is expressed in and patterns the mid/hindbrain region where monoamines neurons originate. Importantly, hindbrain monoamine neuron projections are the sole source of forebrain norepinephrine (NE) and serotonin. Significantly, ASD patients exhibit 1) many behaviors influenced by monoamines, 2) abnormal development of serotonin systems, 3) positive responses to monoamine antagonist, risperidol.
Previously, we found that En2 knock out (KO) mice exhibited decreased monoamines, especially NE, in cerebral cortex and hippocampus due to reduced innervation by the locus coeruleus. Monoamines impact development, and in humans as in mice, monoamine levels correlate with brain growth. In En2 KO, 35% reductions in forebrain NE levels were associated with smaller hippocampus (-12%) and striatum (-13%). To explore relationships of reduced monoamines with brain growth, we defined neurogenesis in hippocampal dentate gyrus (DG) and subventricular zone (SVZ).
Objectives:
We aim to define changes in brain growth, neurogenesis and cell death in En2 KO mice.
Methods:
Immunohistochemical markers of neural progenitors (Sox2, Dcx), apoptosis (Cleaced-caspase3, CCP3; pyknotic bodies) and proliferation (BrdU, PCNA) were assessed on brain sections. To define survival, newly born cells labeled by BrdU injection at P21 were quantified 3 weeks later at P42.
Results:
At P21, En2 KOs exhibited increases in proliferation (BrdU +127%, PCNA +86%) and apoptosis (CCP3 +77%, Pyknotic body +66%) in DG. Similar changes were also observed in the SVZ (BrdU +65%, CCP3 +46%), indicating general dysregulation of neurogenesis. While proliferation was increased 2-fold at P21, the excess cells failed to survive 3 weeks later. At P21, there is no increase in pools of early (Sox2+) or late (Dcx+) neural precursors. However, we expect that more precursors will be in the cell cycle, which we will define by performing double immunostaining for markers of cell identity (Sox2+, Dcx+, nestin+), proliferation (BrdU+) and apoptosis (CCP3+).
Conclusions:
Eventhough En2 expression is restricted to the mid/hindbrain region, its mutation induces reductions in forebrain size and monoamine neurotransmitters. In En2 KO hippocampus, both cell death and proliferation are increased, suggesting that neurogenesis is dysregulated. While underlying mechanisms remain to be defined, these studies suggest that altered En2 expression in the hindbrain produces abnormal forebrain growth, one feature of ASD. In separate studies in collaboration with JN Crwaley, these mice exhibit reduced social interactions, deficits in contextual fear conditioning and abnormal cognition. These studies now lay a foundation to explore therapeutic interventions using monoaminergic drugs to rescue the ASD associated structural and behavioral phenotypes present in this model.