Date of Award
Doctor of Philosophy (PhD)
Dr. Lisa Bain
Dr. William Baldwin
Dr. Charles Rice
Dr. Yanzhang Wei
Arsenic is an environmental contaminant commonly found in food and drinking water. Exposure to arsenic during embryonic development has been linked to reduced muscle growth, disrupted muscle development and locomotor activity, impaired neurodevelopment, reduced IQ, impaired memory and learning deficits. While the mechanisms responsible for developmental changes following in utero exposure to arsenic are not well known, one possibility is that arsenic might disrupt proper cellular differentiation. Therefore, we aimed to investigate the mechanisms by which arsenic exposure could alter stem cell differentiation into neurons.
First, we continuously exposed P19 mouse embryonic stem (ES) cells to 0.1 μM (7.5 ppb) arsenic for 28 weeks to assess if chronic, low level arsenic exposure would delay cellular differentiation into neuronal cells. Importantly, this concentration is below the current drinking water standard of 10 ppb. The results show temporal changes of genes associated with pluripotency and cellular differentiation. Specifically, starting at week 12, transcript levels of the pluripotency markers Sox2 and Oct4 were increased by 1.9- to 2.5- fold in arsenic-exposed cells. By week 16, SOX2 protein expression was increased, and starting at week 20, the expression of a SOX2 target protein, N-cadherin, was also increased. Concurrently, by week 16, levels of the differentiation marker Gdf3 were decreased by 3.4- fold, along with the reduced phosphorylation of the GDF3 target protein SMAD2/3.
To investigate the mechanisms responsible for maintaining pluripotency and hindering cellular differentiation into neurons, RNA sequencing was performed in control and arsenic-exposed cells at week 8, 16 and 24. This analysis revealed significant exposure-dependent changes in gene expression starting at week 16. Pathway analysis showed that arsenic exposure disrupts the Hippo signaling pathway, which is involved in pluripotency maintenance and embryonic development. Immunohistochemistry revealed that the ratios between nuclear (active) and cytoplasmic (inactive) expression of the main effector YAP and the main transcription factor TEAD were significantly increased in arsenic-exposed cells at week 16 and 28. Consistently, expression of the Hippo pathway target genes Ctgf and c-Myc were also significantly upregulated following arsenic exposure. These results indicate that chronic arsenic exposure impairs the Hippo signaling pathway resulting in increased YAP activation, thereby reducing neuronal differentiation.
Previous studies have shown that P19 cells differentiate into sensory neurons, so we also wanted to investigate whether arsenic impaired differentiation into motor neurons. Thus, we switched to using human induced pluripotent stem (iPS) cells, which can differentiate into day 6 neuroepithelial progenitors (NEPs), day 12 motor neuron progenitors (MNPs), day 18 early motor neurons (MNs) and day 28 mature MNs. During this process, cells were exposed to arsenic concentrations up to 0.75 μM (56.25 ppb), and morphological alterations along with pluripotency and stage-specific neuronal markers were assessed. Day 6 NEPs exposed to arsenic had reduced levels of the neural progenitor/stem cell marker NES and neuroepithelial progenitor marker SOX1, while levels of these transcripts were increased in MNPs at day 12. Additionally, levels of the motor neuron progenitor marker OLIG2 were increased in day 12 MNPs while levels of the cholinergic neuron marker CHAT were reduced by 2.5- fold in MNPs exposed arsenic. RNA sequencing and pathway analysis showed that the cholinergic synapse pathway was impaired following exposure to 0.5 μM arsenic, and that transcript levels of genes involved in acetylcholine synthesis (CHAT), transport (SLC18A3 and SLC5A7) and degradation (ACHE) were all downregulated in early motor neurons at day 18. In mature motor neurons at day 28, expression of MAP2 and ChAT protein was significantly downregulated by 2.8- and 2.1- fold, respectively, concomitantly with a reduction in neurite length by 1.8- fold following exposure to 0.5 μM arsenic. Similarly, adult mice exposed to 100 ppb arsenic for five weeks had significantly reduced hippocampal ChAT levels. Taken all together, the results of the dissertation show that environmentally relevant levels of arsenic have detrimental effects on neuronal differentiation.
Perego, M. Chiara, "Developmental Effects of Chronic Low-Level Arsenic Exposure in Mouse Embryonic Stem Cells and in Human Induced Pluripotent Stem Cells" (2022). All Dissertations. 3237.