Abstract
Cardiotonic steroids (CTS) are specific inhibitors and endogenous ligands of a key enzyme in the CNS—the Na+, K+-ATPase, which maintains and creates an ion gradient on the plasma membrane of neurons. CTS cause the activation of various signaling cascades and changes in gene expression in neurons and other cell types. It is known that intracerebroventricular injection of cardiotonic steroid ouabain causes mania-like behavior in rodents, in part due to activation of dopamine-related signaling cascades in the dopamine and cAMP-regulated phosphoprotein 32 (DARPP-32) expressing medium spiny neurons in the striatum. Dopaminergic projections in the striatum innervate these GABAergic medium spiny neurons. The objective of this study was to assess changes in the expression of all genes in human iPSC-derived expressing DARPP-32 and GABA receptors neurons under the influence of ouabain. We noted a large number of statistically significant upregulated and downregulated genes after a 16-h incubation with non-toxic concentration (30 nM) of ouabain. These changes in the transcriptional activity were accomplished with activation of MAP-kinase ERK1/2 and transcriptional factor cAMP response element-binding protein (CREB). Thus, it can be concluded that 30 nM ouabain incubated for 16 h with human iPSC-derived expressing DARPP-32 and GABA receptors neurons activates genes associated with neuronal maturation and synapse formation, by increasing the expression of genes associated with translation, vesicular transport, and increased electron transport chain function. At the same time, the expression of genes associated with proliferation, migration, and early development of neurons decreases. These data indicate that non-toxic concentrations of ouabain may induce neuronal maturation, neurite growth, and increased synaptogenesis in dopamine-receptive GABAergic neurons, suggesting formation of plasticity and the establishment of new neuronal junctions.
Highlights
Over the past few years, our understanding of the physiological role of the Na+,K+ATPase and its endogenous regulators, cardiotonic steroids (CTS), in the central nervous system (CNS) has broadened significantly [1,2]
To confirm neuronal differentiation of the iPSC culture, colonies from the same passage as the ones used during the rest of the experiments presented in this work were stained with antibodies to β3 tubulin, Microtubule Associated Protein 2 (MAP2), Dopamine and CAMP-Regulated Neuronal Phosphoprotein 32 (DARPP-32), and Glial Fibrillary Acidic Protein (GFAP) иtyrosine hydroxylase (Th)
Sixteen hours of incubation with 30 nM ouabain caused a 1.2-fold (p < 0.05) decrease in expression of 81 genes, which were sorted in the same way as upregulated genes: biological processes (GOTERM_BP_DIRECT)—15 groups, cellular components (GOTERM_CC_DIRECT)—12 Brain Sci. 2021, 11, x FOR PEEgRroREuVpIEsW(groups of interest shown in Figure 6B, Supplementary Table S6), m1o3loefc2u5lar function (GOTERM_MF_DIRECT)—7 groups
Summary
Over the past few years, our understanding of the physiological role of the Na+,K+ATPase and its endogenous regulators, cardiotonic steroids (CTS), in the central nervous system (CNS) has broadened significantly [1,2]. It is known that synergistically interacting dopamine D1 and NMDA receptors mediate non-vesicular transporter-dependent GABA release from rat striatal medium spiny neurons [11]. The effects of CTS, mediated both by interaction with partner proteins and by changes in the ion gradient, are enacted through the activation of intracellular signaling pathways, such as MAP-kinase (ERK1/2, p38, JNK) [29], IP3K, PKC, and Akt [30,31,32,33,34,35] These signaling pathways, in turn, can affect both properties of various proteins via phosphorylation, and the expression of genes via the activation of various transcription factors. Brain Sci. 2021, 11, 203 ouabain on gene expression profiles of human iPSC-derived neurons expressing DARPP-32 and GABA receptors
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