The role of KCNS3 in Dopaminergic Neuron Pacemaking and Parkinson's Disease

About This Grant

ABSTRACT The motor symptoms associated with Parkinson’s Disease (PD) are caused by a progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc), which act as key modulators in the nigrostriatal circuitry. Currently, there is no explanation for what causes this cell vulnerability. A key physiological feature of DA neurons is that they pacemake – they fire action potentials (AP) autonomously without the need for synaptic input. Pacemaking is essential for their functionality but is also energetically demanding, which puts them on an ‘energetic cliff’ that may increase their likelihood of cell death. KCNS3 is a high-risk PD gene that is highly expressed in the highly vulnerable SNpc DA neurons. KCNS3 encodes KCNS3, a K+ channel modulatory subunit. In other brain regions, KCNS3 has been shown to regulate action potential firing, but its role in SNpc DA neurons and how it might contribute to SNpc vulnerability is completely unknown. My preliminary data in stem-cell derived midbrain DA neurons indicate that in the absence of KCNS3, these cells have changes in action potential shape and pacemaking frequency. Additionally, these DA neurons experience greater cell death. Based on these results, my central hypothesis is that KCNS3 is a key regulator of SNpc DA neuron pacemaking and that its downregulation increases their vulnerability and PD pathophysiology. To test this hypothesis, I will carry out two Aims. In Aim 1, I will determine how KCNS3 affects DA neuron intrinsic excitability and vulnerability. Using in vitro human embryonic stem cell (hESC) derived midbrain DA neurons, I have generated a knockout of KCNS3, and I will use this construct, along with wild type DA neurons, to define the role of KCNS3 in regulating pacemaking using patch clamp electrophysiology, cell vulnerability using cell death assays, and pacemaking-driven Ca2+ influx using 2-photon calcium imaging. I will also test comparable factors in native SNpc neurons in mice, using stereotaxic injections to virally knock down KCNS3 expression in SNpc DA neurons. In Aim 2, I will look at how the loss of KCNS3 in SNpc DA neurons affects downstream nigrostriatal circuitry and the generation of a parkinsonian behavioral phenotype. For this, I will use the in vivo mouse model mentioned above to look at changes in basal dopamine release and subsequent changes to striatal circuitry. In addition, I will conduct motor behavioral paradigms in these mice to study KCNS3’s impact on motor control. This proposed research will uncover crucial insights into how the functionality of SNpc DA neurons influence the pathophysiology of PD.