Balancing Arc synthesis, mRNA decay, and proteasomal degradation: maximal protein expression triggered by rapid eye movement sleep-like bursts of muscarinic cholinergic receptor stimulation

Abstract

Cholinergic signaling induces Arc/Arg3.1, an immediate early gene crucial for synaptic plasticity. However, the molecular mechanisms that dictate Arc mRNA and protein dynamics during and after cholinergic epochs are little understood. Using human SH-SY5Y neuroblastoma cells, we show that muscarinic cholinergic receptor (mAchR) stimulation triggers Arc synthesis, whereas translation-dependent RNA decay and proteasomal degradation strictly limit the amount and duration of Arc expression. Chronic application of the mAchR agonist, carbachol (Cch), induces Arc transcription via ERK signaling and release of calcium from IP(3)-sensitive stores. Arc translation requires ERK activation, but not changes in intracellular calcium. Proteasomal degradation of Arc (half-life ∼37 min) was enhanced by thapsigargin, an inhibitor of the endoplasmic calcium-ATPase pump. Similar mechanisms of Arc protein regulation were observed in cultured rat hippocampal slices. Functionally, we studied the impact of cholinergic epoch duration and temporal pattern on Arc protein expression. Acute Cch treatment (as short as 2 min) induces transient, moderate Arc expression, whereas continuous treatment of more than 30 min induces maximal expression, followed by rapid decline. Cholinergic activity associated with rapid eye movement sleep may function to facilitate long term synaptic plasticity and memory. Employing a paradigm designed to mimic intermittent rapid eye movement sleep epochs, we show that application of Cch in a series of short bursts generates persistent and maximal Arc protein expression. The results demonstrate dynamic, multifaceted control of Arc synthesis during mAchR signaling, and implicate cholinergic epoch duration and repetition as critical determinants of Arc expression and function in synaptic plasticity and behavior.

FIGURE 1.

Carbachol induces mAchR-mediated rapid, transient Arc mRNA and protein expression in human SH-SY5Y neuroblastoma cells. A, representative Western blots show expression of Arc protein in SH-SY5Y cells pretreated for 30 min with ACD, anisomycin (ANI), atropine (ATR), or dimethyl sulfoxide vehicle (Veh), then stimulated with carbachol (Cch, 50 μm) for 2 h (Ctrl: untreated cells). GAPDH served as a loading control. B, representative Western blots show Arc expression at 30, 60, 120, 240, and 360 min after the start of chronic Cch treatment (upper panel) or acute, 10-min long Cch treatment followed by washout (lower panel). C, quantitative analysis of Western blots shows expression of Arc protein resulting from acute (black boxes) or prolonged (gray boxes) Cch treatment. Arc levels are normalized to GAPDH and expressed in percent of the maximum Arc expression relative to untreated control cells. Asterisks indicate significant increase relative to Ctrl or between time groups (p < 0.05; n = 3–4). D, quantitative analysis of Arc mRNA expression by real-time semiquantitative PCR in SH-SY5Y cells acutely (black boxes) or continuously (gray boxes) treated with Cch at time points ranging from 15 to 360 min after Cch application. Arc levels are normalized to both hypoxanthine-guanine phosphoribosyltransferase and cyclopholin and expressed in percent of the maximum Arc expression relative to untreated cells. Asterisks indicate a significant increase relative to Ctrl (p < 0.05; n = 5–6).

FIGURE 2.

Chronic Cch treatment boosts Arc protein expression via a transcription-dependent mechanism. A, representative Western blots and quantitative analysis show Arc protein expression in cells treated with Cch for 2, 10, 30, 60, or 120 min (black boxes) followed by washout (white boxes) with or without atropine until lysate preparation. All lysates were prepared 120 min after beginning the Cch treatment. Arc levels are normalized to GAPDH and expressed in percent of Arc expression induced by chronic Cch treatment. Asterisks indicate significant increase relative to Ctrl (p < 0.05; n = 5). B, representative Western blot shows expression of Arc protein in SH-SY5Y cells stimulated with Cch for 30 or 120 min. Cells stimulated for 120 min additionally received ACD or vehicle 30 min after the onset of stimulation (Ctrl, unstimulated cells).

FIGURE 3.

Repeated, acute Cch treatment maximizes Arc protein expression. Representative Western blot and quantitative analysis show Arc protein expression in cells treated repeatedly (up to 6 times) with Cch followed by washout. Each Cch application lasted for 2-min, and 10-min intervals separated the onset of consecutive applications. All lysates were prepared 120 min after beginning the initial Cch application. Arc levels are normalized to GAPDH and expressed in percent of Arc expression induced by chronic Cch treatment. Asterisks indicate a significant increase relative to single, acute Cch stimulation (p < 0.05; n = 4).

FIGURE 4.

ERK signaling and intracellular calcium release differentially control Cch-induced Arc protein expression at transcriptional and translational levels. A, representative Western blot and quantitative analysis show Arc protein expression in SH-SY5Y cells pretreated for 30 min with U0126, rapamycin (rapa), wortmannin (wort), or vehicle (veh), then stimulated with Cch for 120 min (n = 4). B, representative Western blot and quantitative analysis show Arc protein expression in cells pretreated for 30 min with BAPTA-AM (bapta), thapsigargin (thapsi), or vehicle (veh), then stimulated with Cch for 120 min (n = 4). Arc levels are normalized to GAPDH and expressed in percent of vehicle pretreatment. C, real-time semiquantitative PCR shows Arc mRNA expression in cells pretreated for 30 min with U0126, BAPTA-AM, thapsigargin, anisomycin (ANI), or vehicle, then stimulated with Cch for 30 min (n = 5–6). Arc levels are normalized to both hypoxanthine-guanine phosphoribosyltransferase and cyclopholin and expressed in percent of vehicle pretreatment. Asterisks indicate a significant change relative to vehicle pretreatment (p < 0.05). D, representative Western blot and quantitative analysis show Arc protein expression in Cch-stimulated cells treated with U0126, BAPTA-AM, thapsigargin, ACD, atropine (ATR), or vehicle 30 min after the start of Cch treatment (n = 4). Arc levels are normalized to GAPDH and expressed in percent of vehicle pretreatment. Asterisks indicate a significant change relative to vehicle pretreatment or between treatment groups (p < 0.05).

FIGURE 5.

Thapsigargin triggers rapid proteasomal degradation of Cch-induced Arc protein. A, representative blot and quantitative analysis show Arc protein expression in cells stimulated with Cch and treated simultaneously with MG-132 (MG) or vehicle (veh). Cells additionally received thapsigargin (thapsi) or vehicle 30 min after the start of Cch stimulation. B, representative blot and quantitative analysis show Arc protein expression in cells stimulated with Cch and treated simultaneously with MG-132 or vehicle. Cells additionally received BAPTA-AM (bapta) or vehicle 30 min after the start of Cch stimulation. Arc levels are normalized to GAPDH and expressed in percent of vehicle treatment. Asterisks indicate a significant change relative to vehicle or between groups (p < 0.05; n = 4). C, representative blot shows Arc protein expression in cells stimulated with Cch, then treated with BAPTA-AM and thapsigargin, respectively. Additional controls are provided in Fig. S5.

FIGURE 6.

Cch-induced Arc protein expression is strictly limited by ubiquitin proteasome-mediated degradation. A, representative Western blot and quantitative analysis show Arc protein expression 60 or 240 min after the start of continuous Cch treatment. Cells stimulated for 240 min additionally received MG-132 (MG) or vehicle 60 min after Cch application. Arc protein levels are normalized to GAPDH and expressed in percent of Arc expression at 60 min post-Cch application. Asterisks indicate a significant change between groups (n = 4, p < 0.05). B, cells were stimulated with Cch for 2 and 4 h in the presence of MG-132 or for 2 h in the presence of vehicle. Lysates were processed for Arc immunoprecipitation. Western blots (WB) using a polyubiquitin antibody (pUbi, upper panel) or an alternative Arc antibody (lower panel) reveal the accumulation of both Arc (dark gray arrowhead) and polyubiquitinated Arc protein (pU-Arc, light gray arrowhead) in MG-132-treated cells. Ctrl lane corresponds to a negative control where SH-SY5Y cell lysate was omitted during the immunoprecipitation (IP). C, representative Western blot and quantitative analysis show a time course of Arc protein expression induced by chronic Cch treatment and where anisomycin is applied onto the cells after 60 min of stimulation. Arc protein levels are normalized to GAPDH and expressed in percent of Arc expression at 60 min of post-Cch stimulation. Asterisks indicate a significant change relative to maximum Arc expression (n = 4, p < 0.05). Solid line represents the corresponding regression curve calculated from the last 3 h of the experiment. Additional controls are provided in Fig. S5.

FIGURE 7.

Arc mRNA is destabilized by translation-dependent RNA decay. A, quantitative analysis of Arc mRNA expression by real-time semiquantitative PCR shows Arc levels in cells stimulated with Cch for 3 h. Actinomycin D was applied in combination with wortmannin (wort) or vehicle (veh) 60 min after the start of continuous Cch treatment. Arc mRNA levels are normalized to both hypoxanthine-guanine phosphoribosyltransferase and cyclopholin and expressed as fold-change relative to vehicle treatment (p < 0.05; n = 6). B, representative Western blot and quantitative analysis show Arc protein expression 60 or 240 min after the start of continuous Cch treatment. Cells received wortmannin or vehicle 60 min after the start of Cch treatment. Arc protein levels are normalized to GAPDH and expressed in percent of Arc expression at 60 min post-Cch stimulation. Asterisks indicate a significant change between groups (n = 4, p < 0.05). C, representative Western blot shows Arc protein expression in cells stimulated with Cch for 240 min. Cells additionally received wortmannin and/or anisomycin (ANI) 1 h after onset of stimulation. Additional controls are provided in Fig. S5.

FIGURE 8.

Cch induces Arc protein expression in rat organotypic hippocampal slice culture. A and B, immunohistochemistry reveals Arc protein expression in untreated slices (A, ctrl) and slices stimulated with Cch (50 μm) for 2 h (B, Cch). Arc-positive cells are visible in CA1 (white arrowhead) and dentate gyrus (DG, black arrowhead). Dotted lines indicate the DG- and CA1 cell layers. C and D, high magnification reveals Arc-positive CA1 pyramidal cells (C, white arrowheads) and dentate granule cells (D, black arrowheads). Note that Arc protein is found in dendrites, the somatic cytoplasm, and nucleus of granule cells and CA1 pyramidal cells. E, quantitative analysis of Western blots shows Arc protein expression in slices pretreated with U0126, thapsigargin (thapsi), or vehicle (veh) and then stimulated with Cch for 2 h. Arc protein levels are normalized to GAPDH and expressed in percent of Arc expression in untreated slices (Ctrl). Asterisks indicate a significant change relative to Ctrl and between groups (n = 4–5, p < 0.05).

FIGURE 9.

Model of mAchR-mediated regulation of Arc expression. A, this model shows the major cellular pathways and regulatory mechanisms governing synthesis and degradation of Arc mRNA and protein. Stimulation of mAchR by Cch activates Arc transcription via two independent pathways involving ERK phosphorylation and release of calcium from IP₃-sensitive intracellular stores. Cch-induced ERK activation also modulates translation of Arc mRNA. Destabilization of Arc mRNA is mediated by wortmannin-sensitive, translation-dependent decay. Finally, Arc protein is ubiquitinated and targeted for proteasomal degradation. Thapsigargin induces Arc degradation, suggesting regulation of Arc degradation during ER stress. B, sketch of Arc mRNA and protein dynamics and regulatory mechanisms. Acute Cch treatment (<30 min) and chronic Cch treatment both trigger rapid and transient Arc mRNA expression. However, chronic treatment triggers a delayed boost in transcription (thick solid line) resulting in enhanced protein expression. Translation of Arc transcripts is also rapidly initiated as Cch-induced Arc protein is detectable as soon as 30 min of treatment. Arc protein expression peaks after 1–2 h and returns to basal levels after 4 h. The reversal of Arc levels is the result of combined destabilization of Arc mRNA and protein by translation-dependent decay and proteasomal degradation, respectively. Solid lines show the time course based on the data obtained. Dotted lines are estimates of the complete time course.