Prostaglandin E2 Increases Neurite Length and the Formation of Axonal Loops, and Regulates Cone Turning in Differentiating NE4C Cells Via PKA

Abstract

Prostaglandin E2 (PGE2) is a membrane-derived lipid signaling molecule important in neuronal development. Abnormal levels of PGE2, due to environmental insults prenatal development, have been linked to brain pathologies. We have previously shown that the addition of PGE2 to neuroectodermal (NE4C) stem cells affects early stages of neuronal differentiation (day 0-8) including increased stem cell motility, accelerated formation of neurospheres, and elevated calcium levels in growth cones. In this study, we further examine whether PGE2 can influence actin-dependent neuronal morphology in later stages (day 8-12) of NE4C cell differentiation. We show that exposure to PGE2 from the initiation of differentiation increased neurite length and the proportion of neurites that formed axonal loops. We also observed changes in the proportion of turning growth cones as the differentiation progressed, with a reduced likelihood of observing turning (or asymmetrical) growth cones on day 8 and increased odds on days 10 and 12. Moreover, we showed for the first time that the observed changes in cytoskeletal morphology were PGE2/PKA dependent. Interestingly, we also found that PGE2 decreased the total protein levels of the actin-bound form of spinophilin and increased levels of unbound PKA-phosphorylated ser94-spinophilin. Hence, we propose that exposure to PGE2 can destabilize the actin cytoskeleton at various stages of neuronal differentiation due to dissociation of ser94-spinophilin causing changes in neuronal morphology.

Keywords: Autism; Lipid signaling; Neurodevelopment; Neuronal differentiation; Prostaglandin E2; Spinophilin.

Fig. 1

Average neurite length of differentiated NE4C cells. NE4C cells were differentiated over 12 days and neurite length was quantified in µM (± SD). N values shown represent the number of neurites measured for each treatment. a Representative images of neurite length measurement in a differentiated cell on day 12. b Treatment with PGE2 across days 8, 10, and 12 shows that PGE2 increases neurite length on days 8 and 12 compared to control cells. Significance is indicated as ***(p < 0.001). c, d Cells treated with PGE2 (c) and Forskolin (d) were treated with KT5720 to determine PKA dependence. Significant differences are indicated with different letters. Cells treated with PGE2 showed similar trends to those treated with Forskolin on day 8. The effects of both treatments were attenuated by the addition of KT5720

Fig. 2

Measurement of turning growth cones. NE4C cells were imaged and the symmetry of each growth cone was used to classify each as turning (asymmetrical) or non-turning (symmetrical)

Fig. 3

Percentage of axonal loops formed out of total neurites. NE4C cells were imaged during days 8, 10, and 12 during differentiation and the proportion of neurites forming loops of all cells measured was determined. Results are shown as a percentage of neurites forming loops out of the total number of neurites measured (± SD). N values, representing the total number of neurites measured for each condition are shown below the x-axis. a Representative images of control cells and PGE2-treated cells on day 8 of differentiation showing an example of typical neurites and neurites forming a loop, respectively. b Cells treated with PGE2 had a higher percentage of neurites form loops on day 12 compared to control cells. c Cells treated with PGE2 formed significantly more loops than control treated cells as well as cells treated with KT5720. d Cells treated with forskolin showed a similar trend to those treated with PGE2, forming significantly more loops than control cells or cells treated with KT5720

Fig. 4

PGE₂-PKA effect on total and ser94 phosphorylated spinophilin expression. Protein was isolated from cells on day 8 following the induction of differentiation after treatment with PGE2 or PGE2 + KT5720 and the a total spinophilin protein and b P-Ser94 phosphorylated spinophilin were quantified relative to control cells through western blotting. Results are shown as fold change to control cells (± SD)

Fig. 5

Proposed model of PGE2 effect on spinophilin. PGE2 is synthesized from arachidonic acid which is released from the cell membrane in response to various environmental factors. Literature has demonstrated that PGE2 can increase the activity of PKA through E-Prostanoid receptors. Here we have demonstrated that through PGE2, PKA can phosphorylate spinophilin. We propose that this results in dysregulation of actin stability, leading to morphologies including increased axonal loop formation and neurite length, and a decrease in growth cone turning