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Analysis of En2 Asd-Associated Haplotype on Spatiotemporal Expression and Neurobiological Effects in Primary Neurons

by
Mai Abdel Soliman
M.S., Montclair University- 2009

Thesis Advisor: James H. Millonig, Ph.D.
Graduate Program in Cell & Developmental Biology

CABM Room 010
Piscataway

Wednesday, January 18, 2017
1:30 p.m.


Abstract

Autism Spectrum Disorder (ASD) is a prevalent neurodevelopmental disorder that displays cognitive, developmental, behavioral and other deficits. The transcription factor ENGRAILED2 (EN2), which functions as a transcriptional repressor, is an established ASD susceptibility gene. Previous work in our lab demonstrated that the common EN2 alleles rs1861972 (A/G) and rs1861973 (C/T) are associated with ASD, both individually and as a haplotype (A-C). In vitro, in vivo, and human postmortem analysis revealed that the ASD-associated A-C haplotype is functional and increases EN2 expression by creating transcription factor binding. Here we investigate the impact of the A-C haplotype on spatiotemporal expression during development as well as the cellular and molecular effect on neurons. To determine the regulatory effects of the A-C and G-T haplotypes on the spatiotemporal expression of EN2, previously generated A-C and G-T DsRed transgenic mice carry the DsRed fluorescent reporter driven by 25kb of EN2 cis-regulatory sequence, were analyzed at four different developmental stages: E12.5, E17.5, P6, and adult. In situ hybridization (ISH) for DsRed revealed no consistent differences in expression in the P6 and adult brain. Additionally, ISH findings at E12.5 and E17.5 were not consistent with our DsRed qRT-PCR results. Therefore, our analysis of the transgenic mice did not provide any information regarding the spatiotemporal impact of the A-C haplotype. Next, to determine how ASD-relevant increased EN2 levels affect cellular and molecular processes in neurons, EN2 was overexpressed 2.5-fold in cerebellar granule cells (CGCs) in vitro and the effects on proliferation, differentiation, maturation and synaptogenesis investigated. We found significantly reduced EdU labeling and thymidine incorporation as well as decrease in expression of the proliferation markers MATH1, CyclinD2, and PCNA, demonstrating that ASD EN2 levels promote decreased proliferation. This was accompanied by early differentiation as evidenced by increased neurite length and number as well as increased NeuroD1 expression. Additionally, ASD-EN2 levels results in an increase in maturation marker Mef2a and the synaptogenesis markers Synapsin and PSD95. Previous studies looking at non-ASD EN2 levels of 250-fold have demonstrated a significant decrease in proliferation and increase in neuronal differentiation. Our analysis comparing ASD and non-ASD EN2 levels shows a similar phenotype in the EdU proliferation assay, indicating that both levels of EN2 overexpression equally impact proliferation in the cell cycle. However, we observed a more robust increase in neurite length and number in the non-ASD conditions, suggesting EN2 overexpressed to 250-fold may drive additional, non-ASD relevant interactions that further promote the differentiation phenotype. Studies of ASD-associated genes such as EN2 is essential in understanding the cellular and molecular processes leading to ASD and as such are critical in identifying novel targets for the development of new therapies and prevention.


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