The Role of MEF2A in the Selective Vulnerability of Dopamine Neurons in Parkinson's Disease - ABSTRACT The motor symptoms associated with Parkinson’s Disease (PD) are caused by progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Currently, there is no explanation for what causes this selective cell vulnerability. In addition to age, environmental factors (e.g. exposure to neurotoxins) also increase the risk for PD, but the mechanisms contributing to vulnerability are poorly understood. Further, there are no available treatment options that effectively prevent or slow DA neuron loss or the progression of related symptoms. Thus, there is a significant need for molecular dissection of the key pathways involved in regulating susceptibility to degeneration in order to identify novel potential drug targets and develop improved treatment options for PD patients. Myocyte enhancer factor 2A (MEF2A) was identified as a genetic master regulator whose activity is decreased in the DA neurons of MPTP treated mice. Dysregulation of MEF2A activity is thought to underlie DA neuron vulnerability. Inactivation of MEF2A can occur through phosphorylation of serine 408 (S408) in the carboxy terminal of the protein. The kinases involved in this phosphorylation are known to be activated by environmental exposure to neurotoxins which disrupt mitochondrial function and energy production. My preliminary data in stem cell-derived midbrain DA neurons indicates that in the absence of MEF2A, these cells experience significant changes in the expression of genes associated with synaptic signaling, maintenance of membrane potential, regulation of cell cycle processes, and DNA metabolic processes. My central hypothesis is that MEF2A is a critical regulator of vulnerability because it regulates the expression of other genes necessary for proper DA neuronal function, and that dysregulation of MEF2A activity underlies the DA neuron vulnerability associated with PD pathogenesis. To test this hypothesis, I will carry out two Aims. In Aim 1, I will determine how MEF2A affects intrinsic DA neuron vulnerability. Using in vitro human embryonic stem cell (hESC)-derived midbrain DA neurons, I have generated an inducible MEF2A knockout cell line. I will use this line, along with wild type (WT) DA neurons, to define the role of MEF2A in cell vulnerability using cell death assays, immunohistochemistry, senescence assays, patch clamp electrophysiology, live Ca2+ imaging, and fluorescent mitochondrial ROS assays. In Aim 2, I will look at how environmental factors contribute to MEF2A activity dysregulation and SNpc DA neuron vulnerability. For this, I will use in vitro neurotoxin and kinase inhibitor treatments along with cell death assays and quantitative western blotting to look at changes in survival as well as levels of phosphorylated MEF2A (p-MEF2A) in both WT and MEF2A-KO DA neurons. This proposed research will provide crucial insights into the molecular and cellular mechanism underlying SNpc DA neuron vulnerability and inform future avenues of investigation for the development of preventative treatments for PD patients.
