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. Author manuscript; available in PMC: 2009 May 7.
Published in final edited form as: J Neurochem. 2007 Jul 10;103(2):531–541. doi: 10.1111/j.1471-4159.2007.04749.x

SYNAPTIC TRANSLATION OF STRIATAL-ENRICHED TYROSINE PHOSPHATASE (STEP) AFTER β1-ADRENERGIC RECEPTOR STIMULATION

Yaer Hu 1,*, Yang Zhang 1,*, Deepa V Venkitaramani 1, Paul J Lombroso 1,1
PMCID: PMC2679031  NIHMSID: NIHMS107089  PMID: 17623046

Abstract

The β-adrenergic system is implicated in long-term synaptic plasticity in the central nervous system, a process that requires protein synthesis. To identify proteins that are translated in response to β-adrenergic receptor stimulation and the pathways that regulate this process, we investigated the effects of isoproterenol on the translation of striatal-enriched protein tyrosine phosphatase (STEP) in both cortico-striatal slices and primary neuronal cultures. Isoproterenol stimulation induced a rapid dose-dependent increase in STEP expression. Anisomycin blocked the increase in STEP expression while actinomycin D had no effect, suggesting a translation-dependent mechanism. Isoproterenol-induced STEP translation required activation of β1 receptors. Application of the MEK inhibitor SL327 blocked both isoproterenol-induced activation of pERK and subsequent STEP translation. Inhibitors of PI3K (LY294002) or mTOR (rapamycin) also completely blocked STEP translation. These results suggest that co-activation of both the ERK and PI3K-Akt-mTOR pathways are required for STEP translation. As the substrates of STEP include ERK itself, these results suggest that STEP is translated upon β-adrenergic activation as part of a negative feedback mechanism.

Noradrenergic activation of β-adrenergic receptors modulates learning and memory (1). Application of β-adrenergic agonists has been shown to enhance memory formation in various animal learning paradigms (2–5). On the other hand, β-receptor antagonists reduce the consolidation of memories associated with emotional experiences in humans (6–7), and decrease memory retrieval and reconsolidation in rodents (8–11). In addition, morphological and neurochemical studies reveal that degeneration of the noradrenergic system is associated with impaired memory in aged rodents and Alzheimer’s patients (12–14), and transplantation of norepinephrine neurons into aged rats improve certain types of learning paradigms (15).

β-adrenergic activation leads to the expression of a persistent form of long-term potentiation (LTP) in the hippocampus and amygdala. The generated LTP requires protein synthesis through activation of the cyclic AMP-dependent protein kinase (PKA) and mitogen-activated protein kinase (MAPK) signaling pathways (16–21). To determine the role of β-adrenergic activation in memory formation, it is important to identify proteins that are translated in response to β-adrenergic receptor stimulation and the pathways that regulate this process.

STEP, a striatal-enriched protein tyrosine phosphatase, is expressed in numerous brain regions involved in learning, including the striatum, hippocampus, amygdala, nucleus accumbens, and cortex (22–24). Previous studies have identified three substrates of STEP: the tyrosine kinase Fyn, the N-methyl-D-aspartate (NMDA) receptor subunit NR2B, and the extracellular–signal regulated kinase1/2 (ERK1/2). STEP binds to these substrates and dephosphorylates them on regulatory tyrosine residues. In the case of Fyn and ERK1/2, dephosphorylation leads to their inactivation (25–26), while dephosphorylation of the NR2B subunit stimulates endocytosis of NMDA receptors (27). Inactivation of Fyn and ERK1/2 as well as endocytosis of NMDA receptors allows STEP to oppose the development of synaptic plasticity.

Both ERK1/2 and NMDA receptors are involved in β-adrenergic-mediated LTP (17,21,28–29). As these proteins are substrates of STEP, it is possible that increased expression of STEP might act as a feedback mechanism to dampen β-adrenergic stimulation. This is supported by our recent finding that STEP is rapidly translated in the lateral amygdala in response to fear conditioning training, followed by the inactivation of pERK (30).

The present study tests the hypothesis that β-adrenergic receptor stimulation leads to increased STEP translation. We demonstrate that isoproterenol stimulation of β1-adrenergic receptors activates ERK1/2 and increases STEP expression. Isoproterenol-induced STEP expression is dependent on translation and requires activation of both the ERK1/2 and the phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathways. These findings are consistent with recent evidence that the ERK1/2 and PI3K-Akt-mTOR pathways promote the translation of proteins required for the expression and maintenance of LTP (21,31–32). Opposing this process, proteins such as STEP might be synthesized to closely regulate the development of synaptic plasticity. These findings address the signaling pathways that control the expression and activity of STEP during the development of synaptic plasticity.

EXPERIMENTAL PROCEDURES

Brain slice preparations and drug applications

All procedures were conducted in accordance to the NIH Guide for the Care and Use of Experimental Animals (http://oacu.od.nih.gov/regs/index.htm) and were approved by the Yale University Animal Care and Use Committee. Male Sprague-Dawley rats (6–7 weeks of age, Charles River Laboratories, Wilmington, MA) were sacrificed and the brains were removed rapidly and placed in ice-cold artificial cerebrospinal fluid (aCSF) (pH 7.4) containing the following (in mM): 124 NaCl, 4 KCl, 26 NaHCO3, 1.5 CaCl2, 1.25 KH2PO4, 1.5 MgSO4 and 10 D-glucose saturated with 95% O2/5% CO2). Coronal cortico-striatal slices (300 μm thick) were cut using a vibratome (1000 plus, Vibratome, St. Louis, MO). The slices were allowed to recover for at least 60 min in fresh aCSF at 30°C under constant oxygenation. Slices were treated with isoproterenol or vehicle in the absence or presence of various drugs as indicated in each experiment.

For bath application, the following drugs were stored as concentrated stock solutions and diluted 1000–2000 fold when applied (final concentrations): the β-adrenergic receptor agonist isoproterenol (10 μM); the protein synthesis inhibitors anisomycin (40 μM) and cycloheximide (60 μM); the transcriptional inhibitor actinomycin D (25 μM); the PKC inhibitor chelerythrine chloride (10 μM); the PKA inhibitors H89 (10 μM) and KT-5720 (4 μM); the β-adrenergic receptor antagonists propranolol (50 μM), atenolol (50μM) and ICI 118,551 (10 nM) (Sigma-Aldrich, St. Louis, MO); the MEK inhibitor SL327 (50 μM); the PI3K inhibitor LY294002 (50 μM); and the mTOR inhibitor rapamycin (200 nM) (Calbiochem, San Diego, CA). All antagonists and inhibitors were typically added 15–20 min before isoproterenol application and were present during the course of isoproterenol treatment. (All antagonists are present during isoproterenol treatment.)

Subcellular fractionation and western blotting

To obtain fractions enriched for synaptosomal proteins, subcellular fractionation was performed at 4°C, as previously described, with slight modifications (23,33). Slices were taken after drug treatments and homogenized in ice-cold TEVP buffer (pH 7.4) containing in mM: 10 Tris-HCl, pH 7.4, 5 NaF, 1 Na3VO4, 1 EDTA, 1 EGTA, and 320 sucrose. Homogenates were centrifuged at 800 × g for 10 min to remove nuclei and large debris. Supernatants were then centrifuged at 9,200 × g for 15 min to obtain a crude synaptosomal membrane fraction (P2). P2 fractions were subsequently resuspended and lysed in hypo-osmotic TEVP buffer containing 36 mM sucrose and centrifuged at 25,000 × g for 20 min to yield the synaptosomal membrane fractions (LP1). The resulting supernatants were centrifuged at 165,000 × g for 2 hours to obtain pellets that contained synaptic vesicle-enriched fractions (LP2). Both LP1 and LP2 pellets were resuspended and sonicated in TEVP buffer and stored at −80°C until used. Protein concentrations were determined by BCA protein assay (Pierce Biotechnology, Rockford, IL) using bovine serum albumin (BSA) as the standard. For western blotting, equal amounts of protein (5–10 μg) for each sample were resolved in 8–12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline containing Tween-20 (TBST) (in mM) (10 Tris-HCl buffer, pH 8.0, 150 NaCl and 0.1% Tween-20), incubated over night at 4°C with primary antibodies of interest. Primary antibodies used include: pERK1/2 (1:2000), pAkt (Ser 473) (1:1000), pmTOR (Ser-2448) (1:1000) (Cell Signaling Technologies, Danvers, MA), ERK2 (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA), and STEP (23E5, 1:1000) (Boulanger et al., 2005). Blots were washed three times in TBST and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary-antibody (1:5000–10,000; Amersham Biosciences, Piscataway, NJ). Bands were detected using enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL). Quantification of the immuno-positive bands was performed by using Image J 1.33 software supplied by NIH and densitometric values of each sample were normalized to total ERK2 from the same blot and presented as a percentage of the control.

Immunofluorescence staining

Cortico-striatal slices were treated for 10 min with isoproterenol (10 μM) or vehicle application in the absence or presence of various drugs, and treated immediately with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4, and fixed for 24 hours. The slices were sequentially cryoprotected in 15% sucrose and 30% sucrose in 0.1 M PBS overnight at 4°C and embedded in optimal cutting temperature compound (Electron Microscopy Sciences, Fort Washington, PA). Cryostat sections (12 μm) were mounted onto poly-L-lysine-coated slides (Sigma, St. Louis, MO) and blocked with 0.1 M PBST containing 10% normal goat serum (NGS), 3% BSA and 0.2% Triton X-100 for 1 hour at room temperature and incubated with anti-STEP antibody (23E5, 1:2000) overnight at 4°C. After washing three times with PBST, sections were incubated with Alexa Fluor 488 anti-mouse antibody (1:600) (Molecular Probes, Eugene, OR) diluted in PBST containing 1% NGS, 3% BSA and 0.2% Triton X-100 for 1 hour at room temperature. Sections were extensively washed and cover-slipped using Vectashield Hardmount with DAPI for analysis.

Quantification

Comparable sections were taken from each animal to allow for comparison among groups, and two images were taken in the desired regions with a 20X objective lens. Fluorescence levels were semiquantified. Two individuals blind to the experimental conditions measured the pixel intensity of a constant area in each image by using the measurement function provided by AxioVision. Measurements were made from four slides and from four different animals for each treatment group.

Cell culture

Cortico-striatal primary neuronal cultures were prepared from Sprague-Dawley rats (Charles River Laboratories, Willmington, MA) at embryonic day 18 or 19. Pregnant dams were euthanized with CO2 and embryos removed by Caesarean section. The brains were removed and the cortico-striatum dissected out under the microscope. The tissue was trypsinized with 0.025% trypsin-EDTA and dissociated in Hanks Balanced Salt Solution by tituration. Cells were resuspended in Neurobasal medium containing B27 supplement and 5% Fetal Bovine Serum (FBS) and plated in 12-well culture dishes containing 18 mm German-glass coverslips coated with poly-D-lysine. Cells were maintained in Neurobasal medium without FBS for 10–14 DIV at 37°C in a humidified 5% CO2 incubator.

Imunocytochemistry

Cortico-striatal cultures were fixed with 4% paraformaldehyde in 1× PBS for 15 min at room temperature following isoproterenol stimulation. The cells were permeabilized with 1× PBS-T (0.2% Triton-X-100) for 15 min and immunocytochemistry was performed with STEP (1:2000) and/or pERK1/2 (1:600) antibodies. The cells were blocked for 1 hr at room temperature with 1× PBS containing 10% NGS and 1% BSA, washed with 1× PBS and incubated with primary antibody in 1% BSA/1× PBS overnight. Following 3 washes in 1× PBS, the cells were incubated with goat anti-mouse Alexa Fluor 594 and/or goat anti-rabbit Alexa Fluor 488 secondary antibodies (1:600, Molecular Probes, Eugene, OR). The coverslips were washed extensively and mounted onto slides using Vectashield Hardmount with DAPI (Vector Labs, Burlingham, CA). Immunofluorescence was visualized using a Zeiss Axiovert 2000 microscope with an apotome (Applied Scientific Instruments, Eugene, OR).

Data analysis

All data were presented as means ± S.E. and the difference between two groups was determined by Student’s t-test. One-way ANOVA with post hoc Tukey test was applied where multiple comparisons were made against the control groups. A p<0.05 was considered statistically significant.

RESULTS

Isoproterenol stimulation leads to a dose-dependent increase in STEP expression

We previously demonstrated that STEP is translated within five minutes of fear conditioning training in the lateral amygdala (30), a brain region required for the consolidation of fear memories (34–35). Studies have also shown that β-adrenergic activation results in protein synthesis (21). Therefore, we first examined whether β-adrenergic activation alters the expression of STEP. Acute cortico-striatal slices were treated either with or without isoproterenol at three concentrations. Isoproterenol produced a dose-dependent increase in STEP expression within 10 minutes as determined by immunofluorescence labeling (1 μM - 131.48% ± 13.77, p > 0.10; 5 μM - 164.83% ± 5.19, p< 0.01; 10 μM - 168.8% ± 10.77; p < 0.05; Fig. 1A; n = 4).

Figure 1. Isoproterenol dose-dependently increases STEP translation.

Figure 1

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(A) Representative fluorescence images of acute striatal slices treated with different doses of isoproterenol (Iso) (n=4) for 10 minutes and stained for STEP and DAPI. Scale bar=100 μm. (B) STEP or ERK2 western blots of cortico-striatal slices treated with different doses of isoproterenol and processed to LP1 and LP2 fractions. Total ERK2 was used to normalize results (n=6). Data expressed as a percentage of control (mean ± S.E.M). Statistical analyses were carried out by using one-way ANOVA followed by post hoc Tukey test. * p<0.05; ** p<0.01 compared to control. n, number of animals. (C) Fluorescence staining of STEP DAPI staining for nuclei in cortico-striatal cell culture with different doses of isoproterenol. Scale bar=20 μm.

Alternative splicing produces both cytosolic and membrane-associated isoforms, including STEP46 and STEP61 (36). The cytosolic variant STEP46 is found throughout neuronal soma, dendrites and axons, while STEP61 is localized to the endoplasmic reticulum as well as being tightly associated with the postsynaptic density (23,36–37). We were interested in determining whether STEP expression increased within neurites after isoproterenol exposure. Cortico-striatal slice homogenates were processed by differential centrifugation to obtain fractions enriched for synaptosomal proteins (LP1 and LP2). A significant dose-dependent increase in STEP61 was observed in both LP1 and LP2 fractions by western blot analysis (Fig. 1B). Similar increases in STEP46 expression were also detected (data not shown).

To determine whether this increased STEP expression occurred in specific cellular compartments, we stimulated cortico-striatal neuronal cultures with increasing doses of isoproterenol and performed immunocyto-chemistry. As shown in Figure 1C, increased STEP expression was observed in cell bodies at lower concentrations of isoproterenol, while at the higher concentration (10 μM) STEP expression was enhanced in both cell bodies and dendrites. Taken together, the results suggest that β-adrenergic receptor stimulation enhances STEP expression in a dose-dependent fashion.

Isoproterenol-stimulated STEP expression is translationally dependent but transcriptionally independent

To determine whether the isoproterenol-induced increase in STEP expression was due to protein translation, we treated cortico-striatal slices with the translational inhibitor anisomycin (40 μM) for 15 min prior to isoproterenol stimulation (Fig 2A). In the absence of isoproterenol, anisomycin did not significantly affect basal STEP expression in LP2 fraction. However, anisomycin blocked the isoproterenol-induced increase in STEP expression. We confirmed the results by pretreating slices with a second protein synthesis inhibitor, cycloheximide. In the presence of cycloheximide (60 μM), isoproterenol-induced STEP expression was reduced from 296.5% to 112.6% (n = 4; p < 0.01) compared to the control group.

Figure 2. Isoproterenol-induced STEP expression is translationally dependent and transcriptionally independent.

Figure 2

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(A) Immunoblots with antibodies to STEP or ERK2 of cortico-striatal slices with or without isoproterenol treatments (10 μM for 10 min) in the absence or presence of actinomycin-D (ActD) or anisomysin (Aniso) (n=4). Both ActD and Aniso were added 20 min before Iso application and were present during the course of isoproterenol treatment (10 min). Histogram shows the summary of the quantitative analysis of STEP expression normalized with ERK2. (B) Fluorescence images of striatal region showing immunoreactivity of STEP in acute slices after isoproterenol treatments in the absence or presence of ActD or Aniso. Scale bar=100 μm. Histogram shows the cumulative data for immunostaining of STEP (n=4). Two images were taken in the desired regions with a 20X objective lens in the striatum. Fluorescence levels were determined by measurement of the pixel intensity of a constant area in each image. Measurements were made from four slides and from four different animals for each treatment group. Data expressed as percentage of control are the mean ± S.E.M. Statistical analysis was carried out by using one-way ANOVA followed by post hoc Tukey test. * p<0.05; ** p<0.01 compared to control. # p<0.05; ## p<0.01 compared to 10 μM isoproterenol.

To explore the possible involvement of transcription in this process, we performed separate experiments in the presence of the transcriptional inhibitor actinomycin-D (25 μM, added 20 min before isoproterenol treatment). Actinomycin-D showed no effect on either basal or isoproterenol-stimulated STEP expression (Figure 2A). Immunofluorescent staining was performed to extend these findings (Fig 2B). Quantification of the results indicate a nearly 2–fold increase in STEP expression after isoproterenol stimulation. Anisomycin blocked the increase in STEP expression after β-adrenergic activation while actinomycin D had no effect. These results suggest that isoproterenol increases STEP expression through a translation-dependent and transcription-independent mechanism.

Activation of β1 adrenergic receptors leads to isoproterenol-induced STEP translation

Studies have shown that both β1 and β2 adrenergic receptors are highly expressed in the cortex and striatum (38–39). To determine which receptor subtype might be involved in the increase in STEP expression, we treated slices with the β-adrenergic receptor antagonist propranolol, which blocks both β1 and β2 adrenergic receptors. We also treated with the antagonists atenolol and ICI-118,551, which are specific for β1 or β2 receptors, respectively. In the absence of isoproterenol, none of the antagonists by themselves had an effect on baseline STEP expression (Fig 3A). However, both propranolol and atenolol prevented the isoproterenol-stimulated STEP translation, while ICI-118,551 had no significant effect. Similar results were obtained by immunohistochemistry studies (isoproterenol 10 μM - 185.81% ± 13.33, p< 0.01, compared to the control group; propranolol + isoproterenol - 129.02% ± 21.82; p < 0.05; atenolol + isoproterenol - 122.01% ± 12.05; p < 0.05; ICI-118,551 + isoproterenol - 170.93% ± 8.39; p > 0.05, compared to the isoproterenol group; n = 4) (Fig 3B). These results suggest that the up-regulation of STEP expression by isoproterenol occurs through stimulation of β1-adrenergic receptors.

Figure 3. Isoproterenol-induced STEP translation requires activation of β1-adrenergic receptors.

Figure 3

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(A) Representative immunoblots of STEP and ERK2 immunoreactivity after isoproterenol treatments (10 μM for 10 min) in the absence or presence of propranolol (Pro), atenolol (Ate), and ICI-118,551 (ICI) (n=4). Scale bar=100 mm. The bar graph summarizes immunoblot data. (B) Immunofluorescent staining of STEP in striatal region of slices treated with Iso for 10 min with or without different β-adrenergic receptor antagonists (n=4). Histogram shows the cumulative data for immunostaining of STEP. Data expressed as percentage of control (mean ± S.E.M). Statistical analyses were carried out by using one-way ANOVA followed by post hoc Tukey test. ** p<0.01 compared to control. # p<0.05; ## p<0.01 compared to 10 μM isoproterenol.

Isoproterenol-induced translation of STEP requires activation of both MAPK and PI3K-Akt pathways

We next examined the signaling events downstream of β1-adrenergic receptor activation that could lead to the translation of STEP. As both the ERK1/2 and PI3K-Akt-mTOR signaling pathways are involved in protein synthesis (40), specific antagonists to these pathways were used. Cortico-striatal cell culture was pretreated with the MEK inhibitor SL327 (50 μM) 15 min before the addition of isoproterenol. As shown in the representative images of fluorescent staining of STEP (Figure 4A), SL327 completely blocked the isoproterenol-induced increase of STEP translation. We next used western blot analyses to quantify the changes suggested by immunostaining. In the control experiments, a 10 min incubation with isoproterenol induced an approximately 3-fold increase in ERK phosphorylation together with a 3-fold increase in STEP synthesis. SL327 alone did not significantly alter the levels of pERK1/2 and STEP, but significantly blocked the isoproterenol-induced increase of both pERK1/2 and STEP expression (Figure 4B). These results indicate that activation of ERK1/2 is required for the isoproterenol-induced STEP synthesis.

Figure 4. Isoproterenol-induced STEP synthesis requires activation of ERK.

Figure 4

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(A) Fluorescence staining of STEP in cortico-striatal cell culture after 10 min of isoproterenol treatments in the absence or presence of MEK inhibitor SL327. (B) Representative immunoblots of STEP61 and pERK1/2 immunoreactivity with the same treatments as in (A) (n=4). Results are summarized in the bar graph. Total ERK2 was used to normalize results. Data expressed as percentage of control (mean ± S.E.M). Statistical analyses were carried out by using one-way ANOVA followed by post hoc Tukey test. ** p<0.01 compared to control. # p<0.05 compared to 10 μM isoproterenol.

We next treated cortico-striatal neuronal culture with LY294002, a PI3K inhibitor, prior to the application of isoproterenol. As shown in Figure 5A, the isoproterenol-induced increase of STEP fluorescent staining was abolished by pretreatment of LY294002. We confirmed and quantified the changes by western blot analyses of cortico-striatal slices. Isoproterenol induced an increase in phosphorylation of both Akt and mTOR, two downstream effectors of PI3K, along with a 2-fold increase in STEP expression (Fig 5B). Pretreatment of LY294002 blocked the isoproterenol-stimulated phosphorylation of Akt and mTOR, as well as STEP synthesis. We also used rapamycin to specifically block mTOR activity. Treatment of slices with rapamycin abolished both the phosphorylation of mTOR at Ser 2448 and the increase in STEP expression (Fig 5C). Taken together, the data suggest that activation of the PI3K/mTOR signaling pathway is required for isoproterenol-stimulated STEP translation.

Figure 5. Isoproterenol-induced STEP synthesis requires activation of PI3K-Akt-mTOR pathway.

Figure 5

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(A) Fluorescence staining of STEP in cortico-striatal cell culture after 10 min of isoproterenol treatments in the absence or presence of PI3K inhibitor LY294002. (B) Western blots of STEP61, pAkt, pmTOR and ERK2 immunoreactivity in slices with the same treatments as in (A) (n=4). (C) Immunoblots of STEP61 and pmTOR immunoreactivity after isoproterenol treatments in the absence or presence of the mTOR inhibitor rapamycin (n=4). Summary of the quantitative analyses are in bar graphs. Total ERK2 was used to normalize results.. Data expressed as percentage of control (mean ± S.E.M). Statistical analyses were carried out by using one-way ANOVA followed by post hoc Tukey test. ** p<0.01 compared to control. # p<0.05; ## p<0.01 compared to 10 μM isoproterenol.

We also tested the possible roles for PKA and PKC in the isoproterenol-induced increases in STEP expression. The PKA inhibitors H89 and KT-5720 and the PKC inhibitor chelerythrine chloride had no effects on isoproterenol-induced elevation in STEP synthesis (data not shown), suggesting that activation of PKA and PKC is not required for the rapid STEP translation following β-adrenergic activation.

DISCUSSION

Considerable evidence from animal and human studies has suggested that the activation of the β-adrenergic system enhances memories associated with emotional arousal (6–7). Of the three β-adrenergic receptors, both the β1 and β2 subtypes are the most abundant in the CNS (38–39). Previous work has demonstrated that β1-adrenergic blockade (or stimulation) alters the consolidation, reconsolidation, or retrieval of memories (4,11,41). The mechanism by which β-adrenergic stimulation primes the CNS for the formation and use of memories is not well understood.

In the present study, isoproterenol-stimulation of β1-adrenergic receptors led to a rapid dose-dependent increase in STEP expression both in cortico-striatal slices and neuronal cell cultures. The isoproterenol-induced STEP translation was blocked by the β1 receptor antagonist atenolol, but not by the β2 receptor antagonist ICI-118,551. This is consistent with previous findings that atenolol blocked isoproterenol-induced enhancement of hippocampal CA3 network activity whereas the β2 receptor antagonist ICI-118,551 had little effect (42).

The increase in STEP expression was blocked by anisomycin but not by actinomycin D, suggesting an activity-dependent induction of protein translation. These results are consistent with the recent finding that STEP is rapidly translated in the lateral amygdala in an activity-dependent manner after fear-conditioning training (30). Moreover, the translation of STEP in the lateral amygdala required prior activation of the ERK1/2 pathway.

Previous studies have suggested that activation of both the cAMP-PKA and PKC pathways is involved in isoproterenol-induced mRNA and protein up-regulation (17,43). However, we found that the PKA inhibitors H89 and KT-5720 and the PKC inhibitor chelerythrine chloride failed to block the isoproterenol-induced STEP translation. Thus, both PKA and PKC activations are not required for the observed translation of STEP, but are likely to play other roles in the regulation of synaptic plasticity.

Our studies indicate that blockade of either the ERK1/2 or the PI3K signaling pathway abolishes STEP translation, and suggest that co-activation of both pathways are necessary for STEP synthesis. This is consistent with previous studies that have shown that these pathways are simultaneously activated to regulate local protein synthesis during the development of synaptic plasticity. For example, brain-derived neurotrophic factor (BDNF) increases protein translation in cortical neurons through multiple signaling cascades including PI3K, mTOR and MAPK (44). LTP induced by BDNF was inhibited either by the mTOR inhibitor rapamycin (31) or by MEK inhibitors (45).

Both the PI3K and ERK pathways regulate key proteins involved in the initiation of translation. Prior to translation initiation, the eukaryotic initiation factor 4E (eIF-4E) is inhibited through its interaction with 4E-binding proteins, and the latter proteins are phosphorylated at multiple sites by activated ERK1/2 and mTOR (46–47). Phosphorylation of 4E-binding proteins leads to the release of eIF-4E. eIF-4E is phosphorylated by MAP kinase-integrating kinase-1 (MNK1), which is itself activated by ERK1/2. eIF-4E can now bind to the 5′-cap of mRNAs, recruit eIF4G and the 40S ribosomal subunit, and initiate translation (40,48). Both ERK- and Akt/mTOR-dependent activation of ribosomal S6 kinase (S6K) and the subsequent phosphorylation of ribosomal S6 protein (S6) are also required for translation initiation. Moreover, ERK can directly phosphorylate S6 during L-LTP (40,49). A similar activation of the ERK1/2 and mTOR pathways is required for the protein translation that occurs during mGluR-dependent L-LTD (50–51).

Recent studies have found that many components of the translational machinery including polyribosomes, transfer RNA, and translational factors are present in the synaptosomal fractions and are proposed to participate in activity-dependent local protein synthesis (52). Using differential centrifugation, we observed an approximately 3-fold increase in STEP translation in synaptosomal fractions. Whether STEP is actually translated locally at dendritic spines in response to isoproterenol stimulation, or throughout the neuronal soma and dendritic arbor, remains to be determined. In this regard, however, there are two cytoplasmic polyadenylation elements (CPE) in the 3′ untranslated region of STEP. CPE sequences are present in the 3′ untranslated region (UTR) of αCaMKII and have been shown to be necessary for the transport of mRNAs to dendritic sites, where they are inhibited, until a signal arrives to initiate their translation (53–55).

The pathways from β-adrenergic receptor stimulation to activation of ERK1/2 and PI3K/Akt/mTOR have been determined. β-adrenergic receptor stimulation activates PKA through activation of Gs, and leads to phosphorylation of a number of substrates by PKA. One substrate is the β1-adrenergic receptor itself and phosphorylation alters the receptor’s G-protein specificity such that it now interacts with Gi rather than Gs (56–57). An important result of this switching is the activation of Ras and the ERK1/2 pathway. In addition, under more robust adrenergic stimulation, β-adrenergic receptors are phosphorylated by β-adrenergic receptor kinase at a site distinct from the PKA site. Phosphorylation at this site recruits β-arrestin that acts as a docking platform to recruit the tyrosine protein kinase Src, and initiates a cascade that leads to activation of ERK1/2 (58).

Isoproterenol also induced the phosphorylation of Akt and mTOR, two downstream effectors of PI3K. Brief isoproterenol stimulation induced phosphorylation of Akt at Ser 473 and mTOR at Ser 2448 in cortico-striatal slices. This finding is consistent with previous reports in neonatal cardiomyocytes in which isoproterenol phosphorylates Akt at Ser 473 through activation of the PI3K signaling pathway (59).

The data present here support our initial hypothesis that stimulation of β1-adrenergic receptors leads to the translation of STEP. There are two non-mutually exclusive models that might explain the function of newly translated STEP isoforms under these conditions. Previous work has shown that fear conditioning training leads to activation of ERK1/2 in the lateral amygdala within 5 minutes of training (30). In these experiments, activated ERK1/2 was required for the translation of STEP and was followed by a subsequent inactivation of ERK1/2. These data suggested that STEP might be functioning in a negative feedback mechanism to limit the duration of ERK1/2 activity. It is also possible that isoproterenol acts to initiate STEP translation as a priming mechanism. Subsequent stimulation of specific subsets of neurons by modulatory signals (e.g. dopamine) would lead to the phosphorylation and inactivation of STEP (26,60) and thereby inhibit STEP activity in those neurons where synaptic plasticity is needed. Distinguishing between these mechanisms is an important area for future research.

Acknowledgments

We thank members of the laboratory for their assistance and suggestions for this work. The National Association of Research on Schizophrenia and Depression (NARSAD), and NIH grants DA017360, MH01527, and MH52711 funded this work.

References

  • 1.Gold PE, McGaugh JL. In: Short-Term Memory. Deutsch D, Deutsch JA, editors. New York: Academic Press; 1975. pp. 355–378. [Google Scholar]
  • 2.Ferry B, McGaugh JL. Neurobiol Learn Mem. 1999;72:8–12. doi: 10.1006/nlme.1998.3904. [DOI] [PubMed] [Google Scholar]
  • 3.Gibbs ME, Summers RJ. Neurosci. 2000;95:913–922. doi: 10.1016/s0306-4522(99)00469-8. [DOI] [PubMed] [Google Scholar]
  • 4.Lalumiere RT, Buen TV, McGaugh JL. J Neurosci. 2003;23:6754–6758. doi: 10.1523/JNEUROSCI.23-17-06754.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Zarrindast MR, Haidari H, Jafari MR, Djahanguiri B. Behav Pharmacol. 2004;15:293–297. doi: 10.1097/01.fbp.0000137211.95623.07. [DOI] [PubMed] [Google Scholar]
  • 6.Cahill L, Prins B, Weber M, McGaugh J. Nature. 1994;371:702–704. doi: 10.1038/371702a0. [DOI] [PubMed] [Google Scholar]
  • 7.Hurlemann R, Hawellek B, Matusch A, Kolsch H, Wollersen H, Madea B, Vogeley K, Maier W, Dolan RJ. J Neurosci. 2005;25:6343–6349. doi: 10.1523/JNEUROSCI.0228-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Devauges V, Sara SJ. Behav Brain Res. 1991;43:93–97. doi: 10.1016/s0166-4328(05)80056-7. [DOI] [PubMed] [Google Scholar]
  • 9.Przybyslawski J, Roullet P, Sara SJ. J Neurosci. 1999;19:6623–6628. doi: 10.1523/JNEUROSCI.19-15-06623.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Tronel S, Feenstra MGP, Sara SJ. Learn Mem. 2004;11:453–458. doi: 10.1101/lm.74504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Debiec J, Ledoux JE. Ann NY Acad Sci. 2006;1071:521–524. doi: 10.1196/annals.1364.056. [DOI] [PubMed] [Google Scholar]
  • 12.Leslie FM, Loughlin SE, Sternberg DB, Mcgaugh JL, Young LE, Zornetzer SF. Brain Res. 1985;359:292–299. doi: 10.1016/0006-8993(85)91439-8. [DOI] [PubMed] [Google Scholar]
  • 13.Mann DM, Yates PO. Hum Neurobiol. 1986;5:147–158. [PubMed] [Google Scholar]
  • 14.Stemmelin J, Lazarus C, Cassel S, Kelche C, Cassel JC. Neurosci. 2000;96:275–289. doi: 10.1016/s0306-4522(99)00561-8. [DOI] [PubMed] [Google Scholar]
  • 15.Collier TJ, Gash DM, Sladek JR. Brain Res. 1988;448:77–87. doi: 10.1016/0006-8993(88)91103-1. [DOI] [PubMed] [Google Scholar]
  • 16.Huang YY, Kandel ER. Neuron. 1996;16:611–617. doi: 10.1016/s0896-6273(00)80080-x. [DOI] [PubMed] [Google Scholar]
  • 17.Huang YY, Martin KC, Kandel ER. J Neurosci. 2000;20:6317–6325. doi: 10.1523/JNEUROSCI.20-17-06317.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Watabe AM, Zaki PA, O’Dell TJ. J Neurosci. 2000;20:5924–5931. doi: 10.1523/JNEUROSCI.20-16-05924.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Lin YW, Min MY, Chiu TH, Yang HW. J Neurosci. 2003;23:4173–4181. doi: 10.1523/JNEUROSCI.23-10-04173.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Straube T, Korz V, Balschun D, Frey JU. J Phsiol. 2003;552:953–960. doi: 10.1113/jphysiol.2003.049452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Gelinas JN, Nguyen PV. J Neurosci. 2005;25:3294–3303. doi: 10.1523/JNEUROSCI.4175-04.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Lombroso PJ, Naegele JR, Sharma E, Lerner M. J Neurosci. 1993;13:3064– 3074. doi: 10.1523/JNEUROSCI.13-07-03064.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Boulanger LM, Lombroso PJ, Raghunathan A, During MJ, Wahle P, Naegele JR. J Neurosci. 1995;15:1532–1544. doi: 10.1523/JNEUROSCI.15-02-01532.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Pelkey K, Askalan R, Paul S, Kalia LV, Nguyen TH, Pitcher GM, Salter MW, Lombroso PJ. Neuron. 2002;34:127–138. doi: 10.1016/s0896-6273(02)00633-5. [DOI] [PubMed] [Google Scholar]
  • 25.Nguyen TH, Liu J, Lombroso PJ. J Biol Chem. 2002;277:24274–24279. doi: 10.1074/jbc.M111683200. [DOI] [PubMed] [Google Scholar]
  • 26.Paul S, Nairn A, Wang P, Lombroso PJ. Nature Neurosci. 2003;6:34–42. doi: 10.1038/nn989. [DOI] [PubMed] [Google Scholar]
  • 27.Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, Choi EY, Nairn AC, Salter MW, Lombroso PJ, Gouras GK, Greengard P. Nature Neurosci. 2005;8:1051–1058. doi: 10.1038/nn1503. [DOI] [PubMed] [Google Scholar]
  • 28.Raman IM, Tong G, Jahr CE. Neuron. 1996;16:415–421. doi: 10.1016/s0896-6273(00)80059-8. [DOI] [PubMed] [Google Scholar]
  • 29.Huang CC, Lin CH, Gean PW. Neurosci Lett. 1998;253:9–12. doi: 10.1016/s0304-3940(98)00571-0. [DOI] [PubMed] [Google Scholar]
  • 30.Paul S, Olausson P, Venkitaramani DV, Moran TD, Ruchkina I, Mills E, Tronson N, Hakim S, Salter MW, Taylor J, Lombroso PJ. Biol Psychiatry. 2006 doi: 10.1016/j.biopsych.2006.08.005. e-print. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Tang SJ, Reis G, Kang H, Gingras AC, Sonenberg N, Schuman EM. Proc Natl Acad Sci U S A. 2002;99:467–472. doi: 10.1073/pnas.012605299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Lee C, Huang C, Wu M, Hsu K. J Biol Chem. 2005;280:18543–18550. doi: 10.1074/jbc.M414112200. [DOI] [PubMed] [Google Scholar]
  • 33.Dunah AW, Sirianni AC, Fienberg AA, Bastia E, Schwarzschild MA, Standaert DG. Mol Pharmacol. 2004;65:121–129. doi: 10.1124/mol.65.1.121. [DOI] [PubMed] [Google Scholar]
  • 34.Blair HT, Schafe GE, Bauer EP, Rodrigues SM, LeDoux JE. Learn Mem. 2001;8:229–242. doi: 10.1101/lm.30901. [DOI] [PubMed] [Google Scholar]
  • 35.Rodrigues SM, Schafe GE, LeDoux JE. Neuron. 2004;44:75–91. doi: 10.1016/j.neuron.2004.09.014. [DOI] [PubMed] [Google Scholar]
  • 36.Bult A, Zhao F, Dirkx R, Sharma E, Lukacsi E, Solimena M, Naegele JR, Lombroso PJ. J Neurosci. 1996;16:7821–7831. doi: 10.1523/JNEUROSCI.16-24-07821.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Oyama T, Goto S, Nishi T, Sato K, Yamada K, Yoshikawa M, Ushio Y. Neurosci. 1995;69:869–880. doi: 10.1016/0306-4522(95)00278-q. [DOI] [PubMed] [Google Scholar]
  • 38.Goffinet AM, De Volder A. Acta Neurol Belg. 1985;85:82–109. [PubMed] [Google Scholar]
  • 39.Aoki C, Joh TH, Pickel VM. Brain Res. 1987;437:264–282. doi: 10.1016/0006-8993(87)91642-8. [DOI] [PubMed] [Google Scholar]
  • 40.Kelleher RJ, Govindarajan A, Jung HY, Kang H, Tonegawa S. Cell. 2004;116:467–469. doi: 10.1016/s0092-8674(04)00115-1. [DOI] [PubMed] [Google Scholar]
  • 41.Murchison CF, Zhang XY, Zhang WP, Ouyang M, Lee A, Thomas SA. Cell. 2004;117:131–143. doi: 10.1016/s0092-8674(04)00259-4. [DOI] [PubMed] [Google Scholar]
  • 42.Jurgens CWD, Rau KE, Knudson CA, King JD, Carr PA, Porter JE, Doze VA. J Pharmacol Exp Ther. 2005;314:552–560. doi: 10.1124/jpet.105.085332. [DOI] [PubMed] [Google Scholar]
  • 43.Kim SD, Lee WM, Suk K, Park SC, Kim SK, Cho JY, Rhee MH. Biochem Biophys Res Commun. 2006;349:408–415. doi: 10.1016/j.bbrc.2006.08.061. [DOI] [PubMed] [Google Scholar]
  • 44.Takei N, Kawamura M, Hara K, Yonezawa K, Nawa H. J Bio Chem. 2001;276:42818–42825. doi: 10.1074/jbc.M103237200. [DOI] [PubMed] [Google Scholar]
  • 45.Ying S, Futter M, Rosenblum K, Webber MJ, Hunt SP, Bliss TVP, Bramham CR. J Neurosci. 2002;22:1532–1540. doi: 10.1523/JNEUROSCI.22-05-01532.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Gingras AC, Raght B, Sonenberg N. Genes Dev. 2001;15:807–826. doi: 10.1101/gad.887201. [DOI] [PubMed] [Google Scholar]
  • 47.Herbert TP, Tee AR, Proud CG. J Biol Chem. 2002;277:11591–11596. doi: 10.1074/jbc.M110367200. [DOI] [PubMed] [Google Scholar]
  • 48.Banko JL, Hou L, Klann E. J Neurochem. 2004;91:462–470. doi: 10.1111/j.1471-4159.2004.02734.x. [DOI] [PubMed] [Google Scholar]
  • 49.Klann E, Antion MD, Banko JL, Hou L. Learn Mem. 2004;11:365–372. doi: 10.1101/lm.79004. [DOI] [PubMed] [Google Scholar]
  • 50.Gallagher SM, Daly CA, Bear MF, Huber KM. J Neurosci. 2004;24:4859– 4864. doi: 10.1523/JNEUROSCI.5407-03.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Hou L, Klann E. J Neurosci. 2004;24:6352–6361. doi: 10.1523/JNEUROSCI.0995-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Wang H, Tiedge H. Neuroscientist. 2004;10:456–466. doi: 10.1177/1073858404265866. [DOI] [PubMed] [Google Scholar]
  • 53.Huang YS, Jung MY, Sarkissian M, Richter JD. EMBO J. 2002;21:2139– 2148. doi: 10.1093/emboj/21.9.2139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Wells DG, Dong X, Quinlan EM, Huang YS, Bear MF, Richter JD, Fallon JR. J Neurosci. 2001;21:9541–9548. doi: 10.1523/JNEUROSCI.21-24-09541.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Wu L, Wells D, Tay J, Mendis D, Abbott MA, Barnitt A, Quinlan E, Heynen A, Fallon JR, Richter JD. Neuron. 1998;21:1129–1139. doi: 10.1016/s0896-6273(00)80630-3. [DOI] [PubMed] [Google Scholar]
  • 56.Daaka Y, Luttrell LM, Lefkowitz RJ. Nature. 1997;390:88–91. doi: 10.1038/36362. [DOI] [PubMed] [Google Scholar]
  • 57.Martin NP, Whalen EJ, Zamah MA, Pierce KL, Lefkowitz RJ. Cell Signal. 2004;16:1397–1403. doi: 10.1016/j.cellsig.2004.05.002. [DOI] [PubMed] [Google Scholar]
  • 58.Daaka Y, Luttrell LM, Ahn S, Della Rocca GJ, Ferguson SS, Caron MG, Lefkowitz RJ. J Biol Chem. 1998;273:685–688. doi: 10.1074/jbc.273.2.685. [DOI] [PubMed] [Google Scholar]
  • 59.Morisco C, Condorelli G, Trimarco V, Bellis A, Marrone C, Condorelli G, Sadoshima J, Trimarco B. Circ Res. 2005;96:180–188. doi: 10.1161/01.RES.0000152968.71868.c3. [DOI] [PubMed] [Google Scholar]
  • 60.Paul S, Snyder GL, Yokakura H, Picciotto MR, Nairn AC, Lombroso PJ. J Neurosci. 2000;20:5630–5638. doi: 10.1523/JNEUROSCI.20-15-05630.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]

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