Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Debresha Shelton - Fisk University
Co-Author(s): Bryan Cawthon, Vanderbilt University, Nashville, TN; Brian Nelms, Fisk University, Nashville, TN
Rationale: Dysregulation of dopamine(DA) signaling is a common hallmark of Parkinson’s disease, addiction and depression. Studies of aberrant DA signaling linked to human disease have shown that systemic loss of dopaminergic neurons, mutations in the dopamine transport protein (DAT-1), and loss of DA receptors are key in disease onset and progression. Less is known about how mechanisms controlling DA metabolism contribute to disease pathogenesis. Understanding molecular controls mediating the balance between DA metabolism and DA neuron function are critical in setting up the molecular systems that precisely regulate DA levels. Thus, there is a need to further study the impact neurotransmitter metabolism plays in DA-dependent behavior and identify targets that regulate metabolism and signaling. Using the model organism Caenorhabditis elegans (C.elegans), we will assess the role of DA metabolism machinery in the regulation of DA-mediated behavior.
Hypothesis: We hypothesize that enzymes involved in DA metabolism, AMX-2 (a putative monoamine oxidase in C.elegans) and several COMTs (catechol-o-methyltransferases), are necessary for DA-mediated behavior. We predict that their expression is mediated by the forkhead-8(FKH-8) winged-helix transcription factor, which is expressed in DA neurons from embryonic stages throughout development.
Methods: Using genetic deletions and pharmacological inhibition, we assess changes in the DA-facilitated behavior, swimming induced paralysis (SWIP), as an extension of DA regulation. Clorgyline (100M) and tolcapone (100M), MAO and COMT inhibitors, respectively, are feed to synchronized C.elegans for 48 hours before worm collection and use in SWIP analysis. To study the importance of MAOs and COMTs, we are using N2(wildtype worms), dat-1, fkh-8, amx-2, amx-2; dop3, and dop3 (lack DA receptor-3) mutants.
Results and Discussion: Preliminary data show that N2 and amx-2 have increased SWIP during clorgyline treatment. While only, amx-2 showed sensitivity to tolcapone exposure. Tolcapone treatment did not significantly affect N2 worms compared to untreated. Neither treatment significantly changed SWIP of dat-1 or fkh-8.
Conclusion and Future Questions: There is still an increased SWIP response during clorgyline exposure, even with AMX-2 deleted; this may suggest clorgyline is acting on other molecules important for DA regulation. Data also show that loss of COMTs can be compensated for if there is functional MAOs present. This suggests that MAOs may have a more prominent role in DA metabolism than COMTs. Finally, neither treatment significantly changed fkh-8 SWIP over untreated. However, further study is needed to verify that FKH-8 does not work upstream of MAO and COMTs. Does loss of FKH-8 change levels of MAO and COMT protein levels? Does inhibition or deletion of these enzymes cause a loss of structural integrity in DA neurons? What other players/pathways could clorgyline and tolcapone be interacting with?
Funder Acknowledgement(s): Acknowledgements: NSF CREST grant #1547757
Faculty Advisor: Brian Nelms, email@example.com
Role: I conducted the experiments for most of the data that will be presented. I conducted all of the drug inhibition SWIP and generated the amx-2;dop3 double mutant. I also significantly contributed to the untreated SWIP data that will be shown.