Dopamine Signaling In Neurons Research Articles

Simulation of nonlinear system dynamics of calcium and dopamine signaling in neurons

Simulation of nonlinear system dynamics of calcium and dopamine signaling in neurons

The SUMO-Conjugase Ubc9 Prevents the Degradation of the Dopamine Transporter, Enhancing Its Cell Surface Level and Dopamine Uptake.

The dopamine transporter (DAT) is a plasma membrane protein responsible for the uptake of released dopamine back to the presynaptic terminal and ending dopamine neurotransmission. The DAT is the molecular target for cocaine and amphetamine as well as a number of pathological conditions including autism spectrum disorders, attention-deficit hyperactivity disorder (ADHD), dopamine transporter deficiency syndrome (DTDS), and Parkinson’s disease. The DAT uptake capacity is dependent on its level in the plasma membrane. In vitro studies show that DAT functional expression is regulated by a balance of endocytosis, recycling, and lysosomal degradation. However, recent reports suggest that DAT regulation by endocytosis in neurons is less significant than previously reported. Therefore, additional mechanisms appear to determine DAT steady-state level and functional expression in the neuronal plasma membrane. Here, we hypothesize that the ubiquitin-like protein small ubiquitin-like modifier 1 (SUMO1) increases the DAT steady-state level in the plasma membrane. In confocal microscopy, fluorescent resonance energy transfer (FRET), and Western blot analyses, we demonstrate that DAT is associated with SUMO1 in the rat dopaminergic N27 and DAT overexpressing Human Embryonic Kidney cells (HEK)-293 cells. The overexpression of SUMO1 and the Ubc9 SUMO-conjugase induces DAT SUMOylation, reduces DAT ubiquitination and degradation, enhancing DAT steady-state level. In addition, the Ubc9 knock-down by interference RNA (RNAi) increases DAT degradation and reduces DAT steady-state level. Remarkably, the Ubc9-mediated SUMOylation increases the expression of DAT in the plasma membrane and dopamine uptake capacity. Our results strongly suggest that SUMOylation is a novel mechanism that plays a central role in regulating DAT proteostasis, dopamine uptake, and dopamine signaling in neurons. For that reason, the SUMO pathway including SUMO1, SUMO2, Ubc9, and DAT SUMOylation, can be critical therapeutic targets in regulating DAT stability and dopamine clearance in health and pathological states.

A glutathione deficit alters dopamine modulation of L-type calcium channels via D2 and ryanodine receptors in neurons

Synthesis of glutathione, a major redox regulator, is compromised in schizophrenia. We postulated that the resulting glutathione deficit via its effect on redox-sensitive proteins could contribute to dysfunction of some neurotransmitter systems in schizophrenia. We investigated whether a glutathione deficit, induced by a blocker of glutathione synthesis, L-buthionine-(S,R)-sulfoximine, affects intracellular pathways implicated in dopamine signaling in neurons, namely dopamine modulation of calcium responses to NMDA. Such a glutathione deficit changed the modulation of responses by dopamine, from enhanced responses in control neurons (likely via D1-type receptors) to decreased responses in low-glutathione neurons (via D2-type receptors). This difference in dopamine modulation was due to a different modulation of L-type calcium channels activated during NMDA stimulation: dopamine enhanced function of these channels in control neurons but decreased it in low-glutathione neurons. The effect of a glutathione deficit on dopamine signaling was dependent on the redox-sensitive ryanodine receptors (RyRs), whose function was enhanced in low-glutathione neurons. This suggests that enhanced RyRs in low-glutathione neurons strengthens intracellular calcium-dependent pathways following activation of D2-type receptors and causes a decrease in function of L-type channels. This represents a mechanism by which dopaminergic systems could be dysfunctional under conditions of impaired glutathione synthesis as in schizophrenia.

GC box-binding transcription factors control the neuronal specific transcription of the cyclin-dependent kinase 5 regulator p35.

Cyclin-dependent kinase 5 (cdk5)/p35 kinase activity is highest in post-mitotic neurons of the central nervous system and is critical for development and function of the brain. The neuronal specific activity of the cdk5/p35 kinase is achieved through the regulated expression of p35 mRNA. We have identified a small 200-bp fragment of the p35 promoter that is sufficient for high levels of neuronal specific expression. Mutational analysis of this TATA-less promoter has identified a 17-bp GC-rich element, present twice, that is both required for promoter activity and sufficient for neuronal specific transcription. A GC box within the 17-bp element is critical for both promoter activity and protein-DNA complex formation. The related transcription factors Sp1, Sp3, and Sp4 constitute most of the GC box DNA binding activity in neurons. We have found that both the relative contribution of the Sp family proteins to GC box binding and the transcriptional activity of these proteins is regulated during neuronal differentiation. Thus, our data show that the GC box-binding Sp proteins contribute to the regulation of p35 expression in neurons, suggesting changes in the Sp transcription factors level and activity may contribute to cell type-specific expression of many genes in the central nervous system.

Cyclin-dependent Kinase 5 Promotes Insulin Exocytosis

Cyclin-dependent kinase 5 (Cdk5) is widely expressed although kinase activity has been described preferentially in neuronal systems. Cdk5 has an impact on actin polymerization during neuronal migration and neurite outgrowth and deregulation of the kinase has been implicated in the promotion of neurodegeneration. Recently it was shown that Cdk5 modulates dopamine signaling in neurons by regulating DARPP-32 function. In addition, Cdk5 phosphorylates munc-18 and synapsin I, two essential components of the exocytotic machinery. We have shown by reverse transcriptase-polymerase chain reaction, immunocytochemistry, and Western blotting that Cdk5 is present in the insulin-secreting pancreatic beta-cell. Subcellular fractionation of isolated beta-cells revealed a glucose-induced translocation of membrane-bound Cdk5 protein to lower density fractions. Inhibition of Cdk5 with roscovitine reduced insulin secretion with approximately 35% compared with control after glucose stimulation and with approximately 65% after depolarization with glucose and KCl. Capacitance measurements performed on single beta-cells that expressed a dominant-negative Cdk5 mutant showed impaired exocytosis. The effect on exocytosis by Cdk5 appeared to be independent of changes in free cytoplasmic Ca(2+) concentration. Taken together these results show that Cdk5 is present in beta-cells and acts as a positive regulator of insulin exocytosis.