(J-362) Modeling Parkinson’s Disease using a Chemical Neurotoxin, MPTP, in Zebrafish

Introduction:: Dopamine (DA) is a neuroprotective catecholamine and neurotransmitter that works to regulate cognitive functions such as movement, breathing, and heart rate, as well as emotional and memory regulation. Patients affected by Parkinson’s Disease (PD) experience a loss of dopaminergic neurons, lower levels of DA, and downregulated DA production in the brain. This leads to cognitive and physical decline which is the hallmark of PD². Currently, no cure exists for this prevalent neurodegenerative disease. Danio rerio, or zebrafish, have become an increasingly popular disease model used in PD research and pharmaceutical development¹. Specifically, neurotoxins, like MPTP, have been used to alter D. rerio brain function as a mimic of PD. Though D. rerio exhibits high genetic homology with humans and a similar catecholamine production cascade, work on the MPTP model in zebrafish began as recently as 2011 and the literature on this model is very limited. This model still requires extensive development in order to fully recapitulate the many biochemical mechanisms associated with PD, as the great majority of these neurochemical models are developed in zebrafish embryos. In this project, we aim to create a more age-appropriate model of PD in zebrafish using intraperitoneal injections of MPTP in sexually mature fish. We are working to provide extensive characterization of the model’s similarities and differences with human PD to enable more appropriate drug discovery and development for this devastating disease.


Materials and Methods:: Mature D. rerio are treated with the neurotoxic prodrug MPTP through intraperitoneal injection to induce Parkinsonian bradykinesia caused by decreased DA levels¹. Behavioral analysis through video motion tracking over the course of seven days was performed to confirm the disparity in swim distance between saline-injected control organisms and the MPTP-injected experimental group. We also monitored cognitive differences through y-maze analysis. Seven days post injection animals are sacrificed for neural analysis. Cryosectioning whole-mount organisms and immunofluorescence assays throughout the brain are used to confirm dopaminergic behavior and presentation. qPCR and western blot techniques are used to determine transcription and translation of Parkinsonian genetic markers dopamine transporter (dat), tyrosine hydroxylase (th1), and a-synuclein-related gene (scna). All zebrafish data is compared to available human PD data.


Results, Conclusions, and Discussions:: We observed a significant decrease in velocity and distance traveled, and an increase in freezing periods D. rerio post intraperitoneal injection of 3.5 µg/ml of MPTP. Specifically, at day 7 post injection, zebrafish injected with saline traveled an average distance of 78.3 ± 12.7 cm at an average speed of 5.56 ± 0.89 cm/s, which dropped to 43.7 ± 25.1 cm traveled at an average speed of 3.16 ± 1.8 cm/s in MPTP treated fish. MPTP injected zebrafish also demonstrated significant levels of cognitive decline, with the number of novel arms in the Y-Maze traveled to over a 3 minute period decreasing from 39.6 ± 17.9 to 9 ± 3.6. qPCR has confirmed decreased dat transcription and is expected to confirm decreased th1 levels in MPTP-injected fish, with upregulation of scna gene. Western blot and immunofluorescence are expected to quantify the decrease in DA and TH present in D. rerio neurological tissues. These techniques and protocols will help to establish an animal model of Parkinson’s Disease, a prevalent disease that lacks a uniform animal model. This novel work with adult D. rerio aims to recapitulate the many complex aspects of the disease and can be used in pharmaceutical research and development in future research.


Acknowledgements (Optional): : This work was partially supported by Clemson University’s Core Incentivized Access Program, and Clemson Creative Inquiry.

References (Optional): :

1. 
   Kinkel, M. D., Eames, S. C., Philipson, L. H., & Prince, V. E. (2010, August 30). Intraperitoneal injection into adult zebrafish. Journal of visualized experiments: JoVE.
   
   


2. 
   Triarhou, L. C. (n.d.). Dopamine and Parkinson's disease - NCBI bookshelf. Madame Curie Biosciences Database.