Autism-Associated Gene Engrailed-2 Plays a Cell Autonomous Role in Regulating Proliferation and Cell Death in Hippocampal Neural Stem Cells in Vitro

Background: Engrailed-2 (En2) is a homeodomain transcription factor important in the proper development of the cerebellum and midbrain. Two intronic single nucleotide polymorphisms were shown to be over-transmitted to individuals with autism spectrum disorder (ASD) compared to unaffected siblings. Similar neuropathology is observed in the cerebellum of En2 knockout (En2-KO) animals and some ASD patients, including reduced cerebellar vermis size, decreased Purkinje neuron numbers, and defects in foliation. More recently, there is increased attention to changes in the forebrain, in part because ASD-related behavioral phenotypes in the En2-KO, such as deficits in social interactions, fear conditioning and learning, depend on forebrain circuits. These behaviors are accompanied by structural changes, including reduced weight, size and cell number in several areas including hippocampus, likely reflecting abnormalities in neurogenesis. Indeed, En2-KO exhibits ~2-fold increases in stem cell proliferation and immature neuron apoptosis in the dentate gyrus, which implies that En2 activity in the brain can regulate proliferation and cell survival in the hippocampus. While these differences have been attributed to reductions in norepinephrine innervation, very low levels of En2are detected in the hippocampus, raising the possibility of cell autonomous functions.

Objectives: This study seeks to examine cell autonomous roles for En2 in regulating hippocampal neurogenesis by showing that (1) stem cells cultured in vitro as neurospheres express En2, and (2) the absence of En2in cultured cells leads to increased proliferation and apoptosis.  

Methods: Hippocampi from P7 En2-KO and WT mice were dissociated and cells were plated in defined media containing 2% B27 supplement, 20 ng/ml EGF and 10 ng/ml FGF. En2 mRNA expression was assessed using quantitative PCR. Primary neurospheres were assayed for neurosphere numbers and size after 7 days in culture. Immunofluorescent labeling of whole neurospheres was performed to determine rates of proliferation (BrdU incorporation) and apoptosis (cleaved caspase-3, pyknosis). Experiments were performed at least 3 times, with each experiment consisting of at least one WT and En2-KO culture.

Results: Expression of En2 mRNA as well as its homolog Engrailed-1 was detected in WT neurospheres. While primary neurosphere assays revealed no change in numbers of primary spheres, measurement of sphere diameter showed increased frequency of larger spheres in En2-KO, suggesting potential effects on proliferation and/or survival. Compared to WT neurospheres, the En2-KO exhibited a 2-fold increase in BrdU labeling (p<0.005) as well as apoptosis (p<0.05).

Conclusions: This study suggests that En2 plays a role in regulating proliferation and apoptosis in hippocampal neural stem cells. While evidence of En2 expression in hippocampal dentate gyrus cells in vivo remains uncertain, we were able to show expression of En2 in neural precursor cultures. Furthermore, increased proliferation and cell death observed in En2-KO neurosphere cultures recapitulates what is observed in vivo, suggesting that En2 may inhibit proliferation and promote survival in a cell autonomous manner. More broadly, these studies indicate how the activities of developmental regulatory genes in diverse brain regions impact brain structure and function, and may contribute to our understanding of how ASD-related behaviors occur in the En2-KO mouse.