Dopamine D1-Like Receptor Stimulation Induces CREB, Arc, and BDNF Dynamic Changes in Differentiated SH-SY5Y Cells

Abstract

The dopamine D1-like receptor is a dopamine (DA) receptor regulating diverse brain functions. Once the dopamine D1-like receptor is activated, it induces activation of the Protein Kinase A (PKA) that phosphorylates the cAMP Response Element-Binding (CREB) transcription factor, which once active elicits the expression of the critical synaptic elements Activity-regulated cytoskeleton-associated (Arc) and the Brain-Derived Neurotrophic Factor (BDNF). The temporality and subcellular localization of proteins impact brain function. However, there is no information about the temporality of CREB activation and Arc and BDNF levels induced through dopamine D1-like receptor activation. In this study, we aimed to assess the specific effect of dopamine D1-like receptor activation on the temporality of CREB-phosphorylation (p-CREB^S133) and the spatiotemporal induction of Arc and BDNF. Using SY-SY5Y cells differentiated with Retinoic Acid (RA), the dopamine D1-like receptor activation with a specific agonist transiently increased p-CREB^S133 at 30 min of stimulation. It induced two spikes of Arc protein at 15 min and 6 h, forming clusters near the cell membrane. BDNF secretion temporarily increased, reaching a maximum at 6 h, while secretion was lower at 24 h compared to the unstimulated group. Our results provide new insight into the role of dopamine D1-like receptor activation on CREB activation, Arc, and BDNF increase, showing that these effects occur temporally and for Arc in subcellular specific sites. This study highlights the dopaminergic system as a critical regulator of subcellular events relevant to neuron plasticity. Future research should address the study of the implications for brain function and behavior.

Keywords: Arc; Brain-derived neurotrophic factor; Dopamine D1-like receptor; Protein Kinase A; cAMP response element-binding.

Fig. 1

Morphologic and molecular changes after seven days of differentiation with RA in SH-SY5Y cells. a) representative micrographs showing the emergent neurites (red arrow) of the SH-SY5Y cells treated (right) and untreated (left) with RA; b) each bar represents the neurite length mean ± SEM (n = 4 cells/per micrograph by triplicate) determined in pixels from soma to the end of each process from three independent experiments. c) A representative immunoblot of the Syn and β-Actin expression is shown; each bar represents the mean ± SEM of the Syn/Actin ratio from three independent experiments. A Student´s t-test was used to determine differences induced by RA treatment; ** p < 0.01 indicated statistical difference compared RA treatment to w/o treatment

Fig. 2

Effect of the RA-induced differentiation on DRD1 level, PKA activity, p-CREB^S133, Arc, and BDNF intracellular level/secretion in SH-SY5Y cells. In cultures treated or not with RA (10µM) were determined: a) The DRD1 level (immunoblot at the bottom); each bar represents the mean ± SEM of DRD1/β-actin ratio from three independent experiments. b) The PKA activity level; each bar represents the mean ± SEM of Relative activity kinase from three independent experiments in duplicates. c) The nuclear expression level of p-CREB^S133 (immunoblot at the bottom); each bar represents the mean ± SEM of p-CREB^S133/Lamin B ratio from three independent experiments, ***p < 0.001 statistical difference compared RA treatment to w/o treatment. d) The expression level of Arc (immunoblot at the bottom); each bar represents the mean ± SEM of Arc/β-actin ratio from three independent experiments, *p < 0.05 statistical difference compared RA treatment to w/o treatment. The BDNF (e) intracellular and (f) secreted level, each bar represents the mean ± SEM of BDNF from three independent experiments by duplicate, ** p < 0.01 and **** p < 0.0001 statistical differences compared RA treatment to w/o treatment. Data were analyzed by t-student test

Fig. 3

Effects of dopamine D1-like receptor activation on PKA activity and DRD1 level. a) the PKA activity was determined at indicated times of stimulation with SKF-38393 (10µM); each bar represents the mean ± SEM of Relative activity kinase from three independent experiments by duplicated, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 (0.5 h) to cells w/o stimulation (F = 3.071). b) A representative immunoblot result of DRD1 and β-Actin is at the bottom; each bar represents the mean ± SEM of DRD1/β-Actin ratio from three independent experiments (F = 0.2998). Data were analyzed using a one-way ANOVA and Tukey´s post hoc tests

Fig. 4

Effects of dopamine D1-like receptor activation on p-CREB ^S133level,Arc level,and BDNF secretion. a) A representative immunoblot of p-CREB^S133 and Lamin B is at the bottom of the graphic; each bar represents the mean ± SEM of the p-CREB^S133/Lamin B ratio from three independent experiments, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 (10 µM) (0.5 h) to cells w/o stimulation (F = 6.517). b) A representative immunoblot of the Arc and β-actin level is at the bottom of the graphic. Each bar represents the mean ± SEM of the Arc/Actin ratio from three independent experiments, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 (0.25 and 6 h) to cells non-stimulated (F = 4.378). c) ELISA determined the BDNF secretion; each bar represents the mean ± SEM of three independent experiments by duplicated, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 (6 and 24 h) to cells w/o stimulation. Results in a) and b) were analyzed using a one-way ANOVA test and Dunnet´s post hoc tests. Results of c) were analyzed using a two-way ANOVA test and Tukey´s post hoc test

Fig. 5

Blocking the dopamine D1-like receptor inhibits the Arc, BDNF, and p-CREB ^S133 induction. After the pretreatment with antagonist SCH-23390 (10µM), the cells were stimulated with agonist SKF-38393 (10 µM) at maximum induction time for each parameter. a) A representative immunoblot of p-CREB^S133 and Lamin B levels is at the bottom of the graphic; each bar represents the mean ± SEM of p-CREB^S133/Lamin B ratio from three independent experiments, ** p < 0.01 statistical difference compared cells stimulated with SKF-38393 (0.5 h) to cells w/o stimulation, and ^&&p < 0.01 cells treated with SCH-23390 + SKF-38393 to cells stimulated with SKF-38393. b) A representative immunoblot of Arc and β-Actin level is at the bottom of the graphic; each bar represents the mean ± SEM of the Arc/β-Actin ratio from three independent experiments, ** p < 0.01 statistical difference compared cells stimulated with SKF-38393 (0.25 and 6 h) to cells w/o stimulation, and ^&p < 0.05 statistical difference compared cells treated with SCH-23390 + SKF-38393 (6 h) to cells stimulated with SKF-38393 (6 h) (F = v4.564 for 0.25 min and F = 14.98 for 6 h). c) The BDNF secretion level determined by ELISA, each bar represents the mean ± SEM from three independent experiments by duplicated, * p < 0.05 statistical difference compared cells stimulated with SKF-38393 to cells w/o stimulation (F = 11.52). Data were analyzed using a t-test or one-way ANOVA test followed by a Tukey post hoc test

Fig. 6

Effects of dopamine D1-like receptor on p-CREB^S133and Arc location. After blocking (or not) the D1-like receptor, the cells were stimulated with SKF-38393 (10 µM) at indicated times. (a) After thirty minutes of stimulation, the p-CREB^S133 location was immunodetected with an antibody against phosphorylated CREB in Ser^− 133 (green); the F-actin was labeled with TRITC-phalloidin (red), and the nucleus was stained by DAPI (blue). For SKF-38,393 (10 µM), the yellow arrowheads indicate the prominent location of p-CREB^S133 concurring in the nucleus visualized by the white circles in the merge column. (b) After fifteen minutes or six hours of stimulation, the Arc location was detected by immunofluorescence labeling Arc protein (green); the F-actin was labeled using TRITC-phalloidin (red); the nucleus was stained by DAPI (blue). An area (white dotted square) was magnified. The white arrow/squares point out the clusters of Arc near the cell membrane and lamellipodial F-actin structures without colors overlapping in merge at 0.25 h. In contrast, at 6 h, the white arrowheads point out the yellow dots corresponding to Arc location (green) overlapping with F-actin (red)

Fig. 7

Schematical representation of dynamic changes by dopamine D1-like receptor activation on p-CREB ^S133, Arc, BDNF. PKA is activated once the dopamine D1-like receptor is stimulated, and CREB is phosphorylated (p-CREB^S133). Arc protein is increased at 15 min, suggesting preexistent mRNA translation; a second increase at 6 suggests a second cycle of transcription and translation. BDNF secretion fluctuates over time, suggesting that in addition to stimulation of secretion, at 24 h, the dopamine D1-like receptor would induce the BDNF endocytosis (scheme was created with BioRender.com)