Carolinas Research Day 2021

EXPOSURE TO THE ENVIRONMENTAL ENDOCRINE DISRUPTOR TOLYLFLUANID DURING CEREBRAL BRAIN DEVELOPMENT SIGNIFICANTLY ALTERS CORTICAL NEURON GENE EXPRESSION Amar Patel 1 , Ken Nguyen 1 , Rachel Winstead 1 , Olivia Mattner 1 , Rebecca Beaudry 1 , Katherine Baumgarner 1 , Bradley Baumgarner, PhD 2 and Stephan Brown MD, PhD 1 1 Gibbs/VCOM CAROLINAS, Spartanburg, SC 29303 2 USC Upstate, Spartanburg, SC 29303 Abstract Results Conclusions

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Tolylfluanid (TF), (the active ingredient in agricultural pesticides) is an endocrine disruptor that has been shown to increase anaerobic glycolysis and reduce oxygen consumption in human cells. Both gene expression and environmental input are essential for normal brain development, and disruption of either can fundamentally alter neural outcomes [1]. Brain formation begins with the development of neural progenitor cells, which are derived from the ectodermal germ layer [2]. The ensuing period of fetal development extends through the end of gestation. During this time there is rapid growth and elaboration of both cortical and subcortical structures, including the rudiments of the major fiber pathways (Kostovic and Jovanov-Milosevic 2006). Similar to pluripotent stem cells, neural progenitors rely almost exclusively on anaerobic glycolysis to meet their energy needs [4]. The differentiation of neural progenitor cells into functional cortical neurons coincides with a transition from anaerobic glycolysis to oxidative phosphorylation [4-5]. Neural differentiation increases the expression of genes associated with mitochondrial metabolism while simultaneously decreasing the expression of glycolysis-related genes [5]. TF is an environmental EDC that has been shown to reduce glucose oxidation in mammalian cells Cerebral organoids from iPSCs were grown and treated with varying concentrations of TF (0, 1 μM, 2 μM and 5 μM) to analyze global gene expression. Organoids were also cryo-sectioned to determine protein expression and morphological discrepancies between the treated and non- treated groups. Results Our results showed decreased proliferation and development of several regions of the brain. The expressivity of SATB2, vimentin, proliferation marker Ki-67 showed a reduction in growth of the superficial layers of neuron and subventricular structures as well as a decrease in development of the early ventricles. In contrast, SOX6 and TTF1 suggested increased growth in ventricle and dopaminergic neurons. Overall, the data showed developmental deviation from the early brain in TF (5 μM) treated cerebral organoids. Ultimately, our results demonstrated that TF causes global- suppression of proliferation and development of maturing central nervous system. Gene expression data via qPCR currently in being optimized. Introduction or Methods

Results reveal that treating induced-pluripotent stem cell derived cerebral organoids with TF throughout differentiation significantly altered organoid development and particularly effected subpopulations of neurons (ie. Dopaminergic and cortical neurons) differently than other neural cell types. Metabolic studies showed reduces the glycolytic rate of fully differentiated cortical neurons under basal and compensatory (anaerobic) conditions when compared to vehicle treated cells (not shown). RNAseq analysis shows altered dopaminergic neuron development which correlated with the immunohistochemical data showing increase in TTF labeled neuron. These neurons further develop into dopamine producing neurons which brings to question, how does TF effect dopamine production? The data shown in this presentation suggest TF is mediating metabolic stress causing structural cellular changes as well as gene alterations. Further evidence of metabolic manipulation leading to altered developmental patterns. These environmental factors play a significant role in effecting normal human physiology and can ultimately alter biological thresholds for disease.

DAPI

MitoRed

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Control

DAPI

MitoRed

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TF-treated

DAPI

TTF

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Control

DAPI

TTF

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TF-treated

TF had a minimal effect on Mitochondrial biogenesis but increased dopaminergic neuron development compared to control.

Vimentin

DAPI

Pax-6

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Control

Vimentin

DAPI

Pax-6

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TF-treated

Sox-6

DAPI

Ki67

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References

Sox-6

DAPI

Ki67

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TF-treated

1. Buckingham M, Meilhac S, Zaffran S. Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet. 2005; 6:826 – 835. 2. Parameswaran M, Tam PP. Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation. Dev Genet. 1995; 17:16 – 28. 3. Garry DJ, Olson EN. A common progenitor at the heart of development. Cell. 2006; 127:1101 – 1104. 4. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009; 324:1029 – 1033. 5. Chung S, Dzeja PP, Faustino RS, et al. Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells. Nat Clin Pract Cardiovasc Med. 2007;4(Suppl 1):S60 – S67.

TF-treated neurons showed a decrease in vimentin (neuronal scaffolding protein) and cell proliferation

Table 3 .

Gene Ontology

Table 1 .

5 μ M TF

Control

.5 μ M TF

RNA seq data from the TF-treated cerebral organoids showed altered gene expression in genes related to aging and dopaminergic neuron differentiation. Further analysis of the expression data linked many of the gene altered by TF are linked to forebrain development. Ͳ

I want to thank VCOM Research and the REAP Grant initiative for funding this project. I also want to thank the students that have participated in this research project.

Cerebral organoids exposed to TF showed a marked decrease in cortical neuron development. Red color represents cortical neuron marker.

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