Neural Precursor Cells (NPCs) from 16p11.2 Deletion Patients Exhibit Enhanced Proliferation, and Alterations in FGF Mitogenic Signaling

Poster Presentation
Friday, May 11, 2018: 5:30 PM-7:00 PM
Hall Grote Zaal (de Doelen ICC Rotterdam)
R. J. Connacher1, M. Williams2, S. Prem2, M. Mehta3, P. G. Matteson3, J. H. Millonig3 and E. DiCicco-Bloom2, (1)Rutgers University- New Jersey Medical School, Piscataway, NJ, (2)Rutgers Robert Wood Johnson Medical School, Piscataway, NJ, (3)Center for Advanced Biotechnology and Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ
Autism spectrum disorder (ASD) genetic etiology is complex and heterogeneous. Within ASD, ~1% contain a copy number variation (CNV) within the 16p11.2 chromosomal region, exhibiting varying phenotypes. This CNV affects one copy of 27 genes, including MAPK3 encoding for ERK-1. Extracellular factors signal through ERK to stimulate proliferation and growth while alterations in ERK-1 impact brain development, as observed in 16p11.2 deletion (16p) mice that exhibit ERK dysregulation. Humans with 16p often have macrocephaly, suggesting developmental growth phenotypes due to this deletion.

We investigated induced pluripotent stem cell (iPSC) derived neural precursor cells (NPCs) from 3 16p individuals (2:1; M:F) compared to 3 age matched controls. 16p individuals were diagnosed with ASD to various degree, as well as comorbid macrocephaly and cognitive impairment. We hypothesized there is a proliferation phenotype in 16p NPC and altered ERK signaling.

iPSCs were induced into NPCs which expressed markers Sox2, Pax6, and Nestin, and differentiated into neurons and glial subtypes. All proliferation and signaling assays utilized NPCs from passages 3-8 of 1-3 clones per individual cultured in control media +/- fibroblast growth factor (FGF). Experiments were conducted in 24-well plates or 35mm dishes and harvested after 2 days unless otherwise specified. DNA synthesis was assayed by measuring tritiated thymidine 3[H] incorporation following a 2hr pulse. NPCs cell counts were performed at 2, 4 and 6 days using hemocytometer. Signaling proteins levels from two male 16p compared to 3 controls were visualized by western blot.

CNV 16p patient NPCs exhibited 2-fold greater DNA synthesis than controls at 2 days. Exposure to extracellular factor FGF, a well-known neurogenic regulator and stimulator of ERK, elicited increased DNA synthesis in both 16p and unaffected NPCs. Interestingly, the magnitude of the FGF-induced stimulation of DNA synthesis was blunted in 16p NPCs. While cell counts were not different between groups at 2 days, there were 2-fold increases in 16p NPC numbers by day 6, reflective of earlier increases in DNA synthesis. Preliminary analysis of total ERK-1 protein in 16p NPCs revealed a 30% reduction, while P-ERK protein normalized to total ERK revealed a non-significant increase compared to control. Interestingly, mitogenic signals downstream of ERK, including Cyclin D1 and P-S6, were increased by 30 and 80%, respectively in 16p NPCs, suggesting differential activation of ERK-1 pathway.

Using iPSC technologies, we observe that 16p NPCs exhibit greater DNA synthesis and proliferation, as well as alterations in signaling compared to unaffected controls. 16p NPCs may also exhibit a blunted response to FGF, though further studies are warranted. The increased proliferation and activation of mitotic regulators, cyclins and P-S6, may provide a mechanism that contributes to the macrocephaly observed in these two male 16p patients. These studies suggest that analyses of ASD NPCs may provide insights into developmental dysregulation that contributes to these disorders, as well as define underlying mechanisms that may ultimately be targeted to address these complex disorders.