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Autism associated gene Engrailed-2 affects forebrain structure via direct and indirect pathways

by
Madel Durens
B.S. University of Philippines, 2004

Thesis advisor: Dr. Emanuel DiCicco-Bloom

Graduate Program in Neuroscience

CABM,Conference Room 010

Piscataway

Tuesday, July 11, 2017
1:00 p.m.


Abstract

Engrailed-2 (En2) is a homeodomain transcription factor that has been shown to be a risk factor contributing to autism spectrum disorder (ASD). The En2 knockout (KO) mouse exhibits behavioral abnormalities analogous to the human syndrome, making it a relevant model that may help uncover underlying biological mechanisms of ASD. The major neuroanatomical deficits in the En2-KO mouse localize to the mid-hindbrain where En2 is highly expressed and plays a role in regulating cerebellar growth by controlling the proliferation and differentiation of cerebellar granule cells. However, our previous studies on the En2-KO revealed low levels of En2 mRNA expression in some forebrain regions, as well as abnormalities in forebrain structure, function, and forebrain-dependent behaviors. In this study, we investigated development of several forebrain regions, including the hippocampus, striatum, and nucleus accumbens (NAc), and explored direct and indirect effects of the absence of En2. We hypothesized that behavioral phenotypes in the En2-KO are correlated with structural and cellular abnormalities in these regions, and that the absence of En2 causes abnormalities in two ways, (1) by direct or cell-autonomous regulation of cell cycle progression and differentiation during postnatal neurogenesis and (2) by affecting monoamine populations of the mid-hindbrain that project to and influence forebrain structures, thereby acting in indirect or non-cell autonomous fashion.

In previous studies we found that the En2-KO hippocampus displayed reductions in total volume and cell numbers due to dysregulation of postnatal neurogenesis, including proliferation and cell death in the dentate gyrus. To begin defining underlying mechanisms, we now investigated the role of En2 in modulating proliferation, survival, and differentiation in hippocampal neural stem cells (NSCs). We used an in vitro neurosphere culture model to establish possible cell autonomous functions of En2 in these cells. We detected En2 expression in neurosphere cultures derived from wild type (WT) postnatal day 7 (P7) hippocampus but not in En2-KO. After 7 days in culture, En2-KO neurospheres were larger on average than WT spheres, and exhibited 2.5-fold greater proliferation. Additionally, there was an increase in apoptotic cells in En2-KO neurospheres. Both these observations are consistent with the En2-KO phenotype

observed in the hippocampus in vivo. Furthermore, expression of En2 protein in En2-KO NSCs was sufficient to rescue excess proliferation, suggesting that En2 acts as a negative regulator of proliferation. Lastly, En2-KO cultures show a 40% reduction in cells with neuritic processes. These results indicate that En2 may regulate the cell cycle in hippocampal stem cells, serving to inhibit proliferation and promote survival and differentiation in a cell autonomous manner.

The absence of En2 also causes forebrain abnormalities through indirect mechanisms, by altering development and projections of monoamine systems that originate in the mid-hindbrain region. We explored possible abnormalities in the nucleus accumbens (NAc) and striatum, two areas that may have roles in both ASD symptoms and the abnormalities observed in the En2-KO. The NAc is thought to play a role in social motivation and reward, and the striatum facilitates motor function and aspects of repetitive behavior. Both regions are innervated by midbrain dopaminergic nuclei. Using an En2tauLacZ reporter mouse line, we colocalized En2 expression specifically to midbrain dopaminergic neurons in the substantia nigra (SN) and ventral tegmental area (VTA) at different timepoints from development to adulthood. We also demonstrated that En2 expressing neurons innervate the NAc and striatum starting from around E16.5, and continue to innervate both regions in adults. However, we did not detect changes in dopamine innervation and expression of dopaminergic receptors to the striatum and NAc of the En2-KO. Because increases in the sizes of the NAc and striatum in the En2-KO were observed in MRI studies conducted previously, we investigated the contribution of cell number and morphology to these increases. Stereological analysis confirmed changes in volume, which was increased by 16% in the NAc and 26% in the striatum, but did not detect changes in cell number. Rather, significant changes in dendritic spine density in medium spiny neurons were found in both areas, increasing by 12.5% in the NAc and 16.7% in the striatum. We also detected a >2-fold increase in the levels of GFAP in both regions, which suggests an increase in the reactive astrocyte population. Both factors could account for increased volume in the NAc and striatum. Overall, although En2 is expressed in the VTA and SN, the absence of En2 did not produce detectable changes in

dopaminergic innervation. However, the absence of En2 resulted in structural alterations in the NAc and striatum due to changes in dendritic morphology and cellular composition.

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-


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