The developing brain is sensitive to chemical exposures, however, currently there is a lack of good, regulatory accepted methods to study developmental neurotoxicity (DNT). The cell line C17.2 can be used as a model for DNT studies since it has the capacity to differentiate into neuronal and neuroglial populations. However, in a recent experiment, single cell RNA sequencing (scRNAseq) indicated that the cell line might contain a good proportion of fibroblasts. The aim of this project was to complement the scRNAseq with staining for protein markers for specific cell types in order to elucidate the cellular composition of differentiated and undifferentiated C17.2 cultures. The markers Hyaluronan-Mediated Motility Receptor (HMMR), vimentin and Platelet Derived Growth Factor Receptor A (PDGFRA) were identified as promising markers for radial glial cells (RGC) and subsequently fibroblast and validated by immunofluorescence. HMMR by itself was used as a marker for RGC and was, as expected, decreasing during the differentiation process. Co-localization of vimentin and PDGFRA was used as markers for fibroblast. However, the conditions for the PDGFRA-antibody could not be optimized due to time restraints. Thus no specific staining could be obtained and no conclusions could be drawn regarding the presence or absence of fibroblasts in the culture. The results emphasize the need for more optimisation or the selection of more specific markers, e.g. Collagen type I alpha 1 (Col1a1). Further, these findings highlight the complexity of the cellular composition and the need for other methods to characterize C17.2.

In recent times, endocrine disrupting chemicals (EDCs) have been associated with the rise in neurodevelopmental disorders such as autism, attention deficit hyperactivity disorder (ADHD) and decreased intelligence quotient (IQ) in children. This effect is suspected to be induced at pre-/peri-natal development, via an alteration in hormonal signaling, thus interfering with neuronal differentiation, with subsequent effect on normal brain development and function in exposed children. This issue increases the need for chemical screening for potential developmental neurotoxicity (DNT) effect. The current available EDC induced DNT test guideline is based on in-vivo testing that requires animal use. Here, a multipotent neural progenitor cell line, the C17.2 cell-line, generated from neural stem cells of the external germinal layer of mouse cerebellum, with potential to differentiate to neurons or astrocytes, is introduced for in-vitro EDC induced DNT testing. This project focused on optimizing the C17.2 cell-line for the detection of EDC-induced DNT with emphasis on the disruption of the oestrogen, androgen, and thyroid hormone systems. It aimed at validating the involvement of oestrogen, androgen, and thyroid hormone on molecular and cellular endpoints relevant for the differentiation of the C17.2 cells.  Herein, the cells were exposed to the hormonal agonist and antagonist at a range of concentrations for a 10-day differentiation period. After exposure, LDH, viability assay and morphological changes (percentage of neurons in culture and neurite outgrowth) were evaluated. The results showed no morphological changes induced by androgen receptor (AR) agonist/antagonist at relevant physiological concentrations. The thyroid receptor (TR) agonist and antagonist on the other hand showed a response in the form of increased neurite outgrowth in relation to the negative control at a concentration range of 40-200 nM and 40 nM respectively. The oestrogen receptor (ER) antagonist at 100 nM also increased percentage neuron in culture. Additionally, in-silico analysis of microarray and RNA sequencing data were used to map out target genes regulated by ER, AR and TR and involved in neurodevelopment. With this approach, 29 marker genes were identified. Validation of the marker genes by means of gene expression (qPCR) was carried out, ER and TR agonist/antagonist were observed to modulate the expression of examined genes. In summary, the model could not be established for detecting EDC induced DNT via androgenic and oestrogenic pathway, while it is a promising model for identifying DNT induced by thyroid hormone signalling disruption.

Retinoic acid (RA) is a well-known neurodevelopmental signaling molecule. It is reported to induce effects on neurite formation in differentiating neurons and to interfere with the actin cytoskeleton. Therefore, this project aimed to investigate the mechanisms behind effects of RA on the actin cytoskeleton of developing neurons using the C17.2 neural progenitor cells (NPCs) in vitro model. The goal was to evaluate the morphological effects the growth cone had upon exposure to RA agonist and antagonist, and to analyze the expression of three genes: Coronin actin-binding protein 1C(Coro1c), Cdc42 effector protein 4 gene (Cdc42), and Fibronectin (Fn1). These genes were selected because of their relation to actin dynamics and/or their regulation by the Wnt pathway, which regulates/affects actin reorganization. Since the Wnt pathway was also shown to be affected by RA, this study aimed to investigate the relationship between RA and actin through the Wnt pathway. Cdc42 and Fn1 are related to both the Wnt pathway and actin dynamics, whereas Coro1cis a known actin-related protein. The expressions showed significant increase with Coro1c, while Cdc42 and Fn1 had a similar overall trend increase with the RA agonist. The RA antagonist showed no significant effect, except a trend decrease in all the genetic expressions. All genetic expression effects subside with the increase of RA agonist and antagonist concentrations. The results suggest the changes in actin filaments are related to a low dose effect of RA. The findings indicate a possibility of a regulation mechanism that controls actin-related gene expression in response to RA. This mechanism is possibly not restricted to the Wnt pathway seeing that a non-Wnt related gene was affected as well.