SCCM Resource Library-50-A Zebrafish Model to Develop Mitochondrial Targeted Therapeutics for Sodium Fluoroacetate Toxicity

Video Transcription

Hello. My name is Carly Klayman. I'm a research scientist working at the Children's Hospital  of Philadelphia in the lab of Dr. Todd Kilball. Today, I'll be speaking to you on the topic  of a zebrafish model to develop mitochondrial-targeted therapeutics for sodium fluoroxetate toxicity.  I have no actual or potential conflict of interest in relation to this presentation.  Funding sources include National Counteract Program, NINDS R21 Fund, the Kilball Endowed Chair Fund, and the Center of Excellence in Environmental Toxicology grant from the  University of Pennsylvania. Many of the studies in our lab at Children's Hospital of Philadelphia  focus on mitochondrial-targeted therapeutics as countermeasures against chemical threats.  This work is funded by the sources displayed here.  Our project scope includes assessing mechanisms of chemical toxins, studying therapeutic development, and utilizing high-fidelity animal models, including pigs, rats, and zebrafish.  These model systems each have clinical and translational relevance.  This presentation will specifically focus on our lab's development of a high-throughput translational zebrafish model for advancing mitochondrial-targeted therapeutic development  with application toward acute sodium fluoroxetate intoxication.  Sodium fluoroxetate, also known as SF, or compound 1080, is a pesticide and cardiac toxin, as well as a potential neurotoxin. Its mechanisms of action include the following.  SF inhibits the tricarboxylic acid cycle, also known as the Krebs cycle. This inhibition of the  TCA cycle reduces cellular energy production. This also inhibits glycolysis as citrate accumulates  due to TCA cycle inhibition. Treatments for exposure to SF include supportive care for  maintenance of cardiopulmonary function. For this reason, further therapeutic development is needed to address additional effects of SF.  Methods for our project include exposing embryonic zebrafish for 24 hours to sodium fluoroxetate dissolved in E3 embryo medium, starting at one day post-fertilization, DPF,  until 2DPF. SF doses range from 0.1 to 1.0 millimolars of SF, or control E3 medium.  At 2DPF, heart rates are assessed. In addition, oxygen consumption, OCR, in the seahorse machine  are also assessed. At 5DPF, behavior was specifically investigated using DanyoVision, a behavioral tracking system, in order to examine larval zebrafish swimming activity.  Parameters assessed include thigmotaxis and movement upon light stimulus, as well as  C-start reflexes in response to a tapping stimulus. These each serve as a measure of intact neurobehavioral functions. Along with these behaviors, locomotor control activity  was also measured. Heart rate results from SF exposure are displayed below. The figure on the  left shows that heart rates decrease dose-dependently with exposure to 0.5 to 1.0 millimolar SF.  If you look at the figure on the right, you can see a dose-dependent recovery of heart rates by  5DPF. We then went to look at OCR, or oxygen consumption rate, using the seahorse machine.  We saw that SF induced a dose-dependent decrease in OCR, shown in the figure on the left.  In the figure on the right, you can observe a reduction in OCR that is specific to embryos that possessed differential heart rates. Zebrafish were divided into groups with  fish that showed low or high heart rates according to a median split.  And the results show that zebrafish with low heart rates displayed less oxygen consumption  compared to zebrafish with high heart rates at each SF dose from 0.25 millimolar to 1.0 millimolar.  In the Ouroboros Respirometry Assay, measurements of the electron transport chain at 2DPF indicate  reduced complex 1 and 2 mitochondrial function after 24 hours of SF exposure. You can observe  a significant reduction in oxygen flow per embryo in the group that received 0.5 millimolar SF  compared to the control group when assessing each of the following. Oxidative phosphorylation,  or OxFos, C1, OxFos C1 and 2, as well as electron transport chain ETS for complex 1 and 2,  along with ETS C2.  When we looked at hatching and survival, we saw that both were reduced with a high SF dose.  Velocity as well as distance traveled were impaired  when zebrafish were exposed to both low and high SF doses.  Zebrafish exposed to SF maintained their responses to light and tapping stimuli with an intact startle response. The figure on the top is a picture of a zebrafish with  an intact startle response. The figure on the top shows that the light stimulus response was  enhanced in SF-exposed larvae. Here you can see increased directional turning at a medium and  high SF dose specifically. In the bottom figure, you can see decreased time spent in the center of the well with a light stimulus. In the schematic on the left here, you can see a  depiction of the mechanism of sodium fluoroacetate intoxication in which citrate from the TCA cycle accumulates as glucose metabolism into pyruvate is disrupted.  The schematic on the right, however, depicts potential for bioenergetic bypass with substances such as fructose 1,6-bisphosphate. This restores glucose metabolism so that  succinate levels are restored and electron flow to complex 1 and 2 is restored.  Here you can see that when larvae are co-treated with NV354, the succinate pro-drug, along with  0.5 millimolar sodium fluoroacetate from one to two days post-fertilization, this does not prevent an acute decrease in oxygen consumption rate and heart rate at 2 dPF.  However, you did see a recovery in heart rates by 4 dPF. When we looked at fructose 1,6-bisphosphate  we saw, in the figure on the left, a trend of increased heart rates in the presence of SF,  along with fructose 1,6-bisphosphate at 2 dPF. After removing both SF and the co-treatment,  the trend of increased heart rate with 1,6-bisphosphate is also observed at 3 dPF. This indicates the ability of 1,6-bisphosphate to bypass the bioenergetic impairment caused by SF.  This study has several conclusions. We established a high-throughput zebrafish model of SF poisoning,  showing mitochondrial and cardiac dysfunction, as well as altered neurobehavioral responses  as a result of SF exposure. Zebrafish may be utilized as a model organism for assessing the  impact of toxins on mitochondrial, cardiac, and lymphomotor function. We are currently investigating  different dosing regimens of zebrafish, and we believe that zebrafish can be used to  assess different dosing regimens of the cell-permeable succinate prodrug, with specific focus on improving recovery following exposure.  This platform will be expanded to assess the effects of other clinically relevant toxins on mitochondrial, cardiac, and neurological function, as well as to evaluate and develop  other mitochondrial-targeted therapies. I would like to acknowledge our funding sources.  The CEET pilot project program from the Center of Excellence in Environmental Toxicology at the University of Pennsylvania, the NIH Environmental Health Sciences Division,  and the R21 funding source from the NIH CounterACT program.  I would also like to acknowledge the members of our team shown here. This work was performed by our team at the Children's Hospital of Philadelphia,  including myself, Dr. Sarah Peel, and Katie Berg. In addition, other members of our team supported our work, including our principal investigator, Dr. Todd Kilball, as well as  collaborators at Lund University Sweden and NeuroViv Pharmaceutical Sweden.  My email is shown below, along with that of Sarah Peel and Todd Kilball.  Please reach out to us with any questions or comments on our project.  This concludes this presentation. I hope you found this presentation to be informative.  Again, our team would be happy to respond to any questions that you may have. Thank you for listening.

Video Summary

Scientists at the Children's Hospital of Philadelphia have developed a zebrafish model to study and develop mitochondrial-targeted therapeutics for sodium fluoroxetate (SF) toxicity. SF is a pesticide and potential neurotoxin that inhibits cellular energy production. The researchers exposed embryonic zebrafish to SF and observed various effects, including reduced heart rates and oxygen consumption, impaired hatching and survival, and altered neurobehavioral responses. They also tested potential treatments, such as fructose 1,6-bisphosphate, and found that it could bypass the bioenergetic impairment caused by SF. This zebrafish model has potential for studying the impact of toxins on mitochondrial, cardiac, and neurological function, and for evaluating and developing targeted therapies.

Asset Subtitle

Research, Professional Development and Education, 2021

Asset Caption

The Society of Critical Care Medicine's Critical Care Congress features internationally renowned faculty and content sessions highlighting the most up-to-date, evidence-based developments in critical care medicine. This is a presentation from the 2021 Critical Care Congress held virtually from January 31-February 12, 2021.

Carly L. Clayman

Introduction/Hypothesis: The pesticide sodium fluoroacetate (SF, Compound 1080) is an established cardiac toxin. Its primary mechanism is inhibition of the tricarboxylic acid cycle, which reduces cellular energy production. Since treatment strategies consist of supportive care to maintain cardiopulmonary function, further therapeutic development is needed. The aim is to develop a high-throughput translational zebrafish model system to advance mitochondrial targeted therapeutic development for acute SF intoxication.

Methods: Zebrafish were randomly assigned to an acute exposure dose of SF dissolved in E3 embryo medium (0, 0.1, 0.25, 0.50, 0.75, or 1.0mM SF) for 24 hours starting at 1 day post fertilization. At the end of exposure, heart rates and respirometry of embryonic zebrafish were assessed. Fish were raised for daily assessments of heart rate and behavioral parameters. DanioVision, a behavioral tracking system, was used to track larval zebrafish swimming activity. Thigmotaxis and reduced exploration upon light stimulus and c-start reflexes in response to a tapping stimulus are assessed as an index of intact neurobehavioral functions along with locomotor function.

Results: SF induced a dose-dependent decrease in oxygen consumption, a measure of mitochondrial function (p<0.0001). Heart rates decreased dose-dependently with exposure to 0.5 to 1.0mM SF (p<0.001) and also recovered dose-dependently. Locomotor activity was impaired, with reduced velocity and distance traveled by fish exposed to low and high doses of SF (p<0.001). Zebrafish exposed to SF maintained responses to light and tapping stimuli, with an intact startle response. Light stimulus response was enhanced in SF-exposed larvae, which decreased time spent in the center of the well and increased directional turning (p<0.001).

Conclusions: We established a high-throughput zebrafish model of SF poisoning showing mitochondrial and cardiac dysfunction and altered neurobehavioral responses as a result of SF exposure. This indicates that zebrafish may be utilized as a model organism for assessing the impact of toxins on mitochondrial, cardiac, and locomotor function. This platform for assessing toxins may be developed by assessing recovery through applying mitochondrial targeted therapeutic cell-permeable succinate prodrugs.

Meta Tag

Content Type Presentation

Knowledge Area Research

Knowledge Area Professional Development and Education

Knowledge Level Advanced

Membership Level Select

Tag Veterinary Medicine

Year 2021

Keywords

Children's Hospital of Philadelphia

zebrafish model

mitochondrial-targeted therapeutics

sodium fluoroxetate toxicity

neurotoxin

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