VCOM Carolinas Research Day 2023

Biomedical Studies

The Effects of Tolylfluanid on Dopaminergic Cell Development and Dopamine Production Rayhan Karimi 1 , Shreya Shah 1 , Madison Battersby 1 , Yassine Lahlou 1 , Katherine Baumgarner 1 , Bradley Baumgarner, PhD 2 and Stephan Brown MD, PhD 1 1 VCOM CAROLINAS, Spartanburg, SC 29303 2 USC Upstate, Spartanburg, SC 29303

Abstract # BIOM-8

Abstract

Results

Conclusions

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. Lund human mesencephalic (LUHMES) cells are human embryonic neuronal precursor cells that can be maintained as proliferating cells due to the expression of a tetracycline-regulatable (Tet-off) v-myc transgene. LUHMES cells were used as the model for dopamine production and cell viability of dopaminergic cell after TF treatment. 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. Tolylfluanid (TF), (the active ingredient in agricultural pesticides/fungicides) is an endocrine disruptor that has been shown to increase anaerobic glycolysis and reduce oxygen consumption in human cells. TF is an environmental endocrine disrupting chemical (EDC) that decreases glucose oxidation in developing neurons causing metabolic stress by inhibiting the entry of pyruvate into the mitochondria. This constant stress alters neuron metabolism leading to gene expression alterations. Both gene expression and environmental input are essential for normal brain development and can fundamentally alter neural outcomes including the development of dopaminergic neurons. Chen et al. discovered that TF reduced glucose oxidation in human adipocytes by directly inhibiting the mitochondrial pyruvate carrier (MPC). A previous investigation by Vacanti et al. revealed that pharmacological inhibition (i.e. TF) or genetic deletion of MPC significantly reduced glucose oxidation in human skeletal muscle cells, which increased mitochondrial fatty acid glutamine oxidation. Therefore, it appears that by blocking mitochondrial pyruvate uptake, TF can increase the utilization of other oxidative fuels. However, the potential impact of TF exposure on cellular glycolytic capacity has yet to be fully investigated. Introduction or Methods

Continued

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 a reduced glycolytic rate of fully differentiated cortical neurons under basal and compensatory (anaerobic) conditions when compared to vehicle treated cells (not shown). Major findings revealed that treating induced-pluripotent stem cell derived cerebral organoids with TF throughout differentiation significantly altered organoid development and particularly affected subpopulations of neurons (ie. dopaminergic neurons and cortical) differently than other neural cell types. Metabolic studies showed a reduction in the glycolytic rate of fully differentiated cortical neurons under basal and compensatory (anaerobic) conditions when compared to vehicle treated cells. Chronic exposure to TF caused an increase in dopamine production with no significant impact on cell viability.

DAPI

MitoRed

Merge

Control

DAPI

MitoRed

Merge

TF-treated

DAPI

TTF

Merge

Control

DAPI

TTF

Merge

TF-treated

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

Vimentin

DAPI

Pax-6

Merge

Table 2 .

Seahorse XF Cell Mito Stress Test Bar Charts

Basal

Proton Leak

40.0

10.0

8.0

Control

30.0

6.0

TF-treated LUHMES cells. SeaHorse assay results for OCRs of Tet-treated LUHMES, in real time under basal conditions and in response to mitochondrial inhibitors. TF-treated DAPI Metabolic Profile of Tet-treated Vimentin DAPI Sox-6 DAPI Sox-6

Pax-6 20.0

Merge

4.0

10.0

OCR (pmol/min)

2.0

OCR (pmol/min)

0.0

0.0

Maximal Respiration

Spare RespiratoryCapacity

80.0

40.0

60.0

30.0

40.0

20.0

Ki67 20.0

Merge

10.0

OCR (pmol/min)

OCR (pmol/min)

0.0

0.0

ATP Production

Non-mitochondrial Oxygen Consumption

10.0 15.0 20.0 25.0 30.0 OCR (pmol/min)

25.0

20.0

References

Ki67 10.0 15.0

Merge

5.0 OCR (pmol/min)

0.0 5.0

0.0

Coupling Efficiency (%)

Spare RespiratoryCapacity (%)

64.0% 66.0% 68.0% 70.0% 72.0% 74.0% 76.0% OCR (pmol/min)

250.0%

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.

150.0% 200.0%

100.0%

0.0% 50.0%

OCR (pmol/min)

Dopamine Production

Table 3 .

Viability

Table 1 : Preliminary Data

5 μ M TF

Control

.5 μ M TF

Acknowledgements

Dopamine production from exogenousl-DOPA and its storage Tet-LUHMES cells. Time course of intracellular (A) and extracellular (B) dopamine levels in Tet-LUHMES cells (open circles with dotted line) and Untreated-LUHMES cells (filled circles with solid line) after 1 μ ml-DOPA incubation for various durations (0 – 48 hr). *p < 0.01 compared with the Untreated group and also with time 0 by Newman – Keuls test.

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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