Dopamine Toxicity Induces ROS-Dependent Death of Murine Neuroblastoma Cells: Impact on the Interactions of Cofilin...

The death of dopaminergic neurons, a hallmark event during Parkinson's disease (PD), leads to increased dopamine concentration in the neuronal micro-environment. Keeping this in mind, we intend to understand the impact of elevated dopamine concentration on molecular interactions among proteins and the stability of the neuronal cytoskeleton. We used differentiated N2A cells and exposed them to 100 µM DA for 24 h. Evaluations of cell death, measurement of the concentration of DA oxidation products and reactive oxygen species (ROS), conventional RT-PCR, western blotting, zymography, reverse zymography, co-immunoprecipitation, mitochondrial transmembrane potential, confocal imaging, and in-silico studies were performed thereon. We observed that a significant number of viable N2A cells underwent ROS-dependent apoptotic cell death under elevated media DA concentrations. An altered transcriptional pattern of alpha-synuclein, UCHL1, and cofilin genes and their respective gene products were also observed. The activity and expression of matrix metalloproteinases9 (MMP9), involved in neuro-inflammation, was enhanced upon DA-exposure. Further, DA exposure also led to degradation of actin cytoskeleton. In silico studies revealed that interactions of Cofilin with UCHL1 and MMP9 were altered in dopamine-rich microenvironment. This result was further validated by co-immunoprecipitation experiments. Collectively our observations with murine neuroblastoma cells suggest that DA toxicity alters interaction patterns among intracellular proteins and degrades neuronal cytoskeleton that finally leads to cell death. Our study unveils a new frontier in PD treatment by paving the way for the development of specific drugs targeting the DA altered protein interactions.