Absence of Engrailed 2 (En2), the Autism Spectrum Disorder (ASD) Associated Gene, Alters Monoamine Transmitter Systems, Forebrain Structure and Developmental Neurogenesis and Apoptosis

Background:

We have found the homeobox gene EN2 is associated with ASD in 3 different datasets, and disease associations have been reported by 4 other groups. It is perhaps surprising that EN2, a transcription factor that patterns mid/hindbrain and cerebellum, contributes to ASD symptoms that depend more on forebrain structures and functions. However, ASD has been characterized functionally by abnormal neurotransmission between hindbrain and forebrain, and structurally by brain enlargement. Furthermore, ASD is associated with changes in monoamines, especially serotonin (5HT), including increased plasma 5HT, disturbed central 5HT development and responsiveness to related drugs, as well as symptoms influenced by monoamines including attention, mood and aggression. Is it possible changes in patterning genes in hindbrain secondarily affect forebrain development?

We are using En2 knock out (KO) mice as a model system to define roles of En2 in development of monoamine transmitters, including 5HT and norepinephrine (NE) and effects on forebrain structure. Previous work indicates NE levels are reduced 30-40% in hippocampus and cortex, with parallel changes in biosynthetic enzyme tyrosine hydroxylase protein and axonal fibers, and smaller changes in 5HT. Transmitter reductions are associated with smaller structures including 12% decreases in hippocampus and 4% in cortex. Stereological analysis indicates a 16% reduction in hippocampal dentate gyrus (DG) granule neurons. Since the DG exhibits lifelong neurogenesis that might contribute to changes in DG structure, we examined changes in cell proliferation and cell death.

Objectives:

Define the consequences of En2 deletion on forebrain neurogenesis and apoptosis.

Methods:

Wild type (WT) and En2 KO mice were injected on P21 with BrdU, a marker of cells in mitotic S phase, and sacrificed 2hrs later. Fixed brains were frozen sectioned and immunostained for nuclear BrdU labeling or activated caspase 3, a marker of apoptosis.

Results:

In P21 hippocampal DG, there was a 77% increase in caspase 3+ cells compared to WT mice (p<0.004), suggesting cell death was increased, a potential mechanism contributing to smaller structure. However, BrdU labeling was increased >2-fold (p<0.01), raising the possibility of compensatory responses. To compare changes in DG to another neurogenetic region, we assessed the subventricular zone (SVZ) lining the lateral ventricles. Caspase 3 was increased by 46% (p<0.05), which was also accompanied by 66% increase in BrdU labeling (p<0.03), results paralleling those in DG.

Conclusions:

Our observations indicate that in the absence of hindbrain En2, forebrain cell death and neurogenesis are both increased. An excess of cell death over neuron production may account for smaller forebrain structures and neuron numbers. These observations suggest that reduced monoamines may lead to reduced cell survival, with compensatory cell production, yet underlying mechanisms remain to be defined. More generally, changes in the levels or timing of EN2 expression may contribute to human ASD by altering forebrain development through changes in neuronal production and/or survival.