Bidirectional regulation of cytoplasmic polyadenylation element-binding protein phosphorylation by Ca2+/calmodulin-dependent protein kinase II and protein phosphatase 1 during hippocampal long-term potentiation

Abstract

Induction of hippocampal long-term potentiation (LTP) requires activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), whereas maintenance of LTP additionally requires protein synthesis. We recently reported that CaMKII stimulates protein synthesis in depolarized hippocampal neurons through phosphorylation of the mRNA translation factor cytoplasmic polyadenylation element-binding protein (CPEB), and this phosphorylation is rapidly reversed by protein phosphatase 1 (PP1). Protein synthesis-dependent late-phase LTP (L-LTP) in the hippocampus requires calcium influx through the NMDA-type glutamate receptor (NMDA-R) to activate CaMKII as well as concomitant inhibition of PP1 mediated by protein kinase A. Therefore, we investigated the regulation of CPEB phosphorylation during L-LTP. Pharmacological stimulation of the NMDA-R in hippocampal slices to produce chemical long-term depression induced a brief dephosphorylation of CPEB. Modest LTP induction (once at 100 Hz), which induces a protein synthesis-independent early-phase LTP (E-LTP), resulted in a transient phosphorylation of CPEB. However, stronger stimulation (four times at 100 Hz), known to induce protein synthesis-dependent L-LTP, elicited a prolonged phosphorylation of CPEB. Furthermore, CPEB phosphorylation correlated with phosphorylation of PP1 inhibitor dopamine- and cAMP-regulated phosphoprotein, a known substrate for protein kinase A. These results evoke the hypothesis that bidirectional regulation of CPEB phosphorylation by CaMKII and protein phosphatases may serve as a mechanism to convert E-LTP into protein synthesis-dependent L-LTP by stimulating protein synthesis and thereby stabilizing synaptic enhancement.

Figure 1.

CPEB and CaMKII phosphorylation and dephosphorylation during NMDA-R activation. A, Representative Western blots of pCPEB, total CPEB (CPEB), autophosphorylated CaMKII (pCaMKII), and total CaMKII from hippocampal slices stimulated with NMDA (25 μm) for the indicated time points. B, Densitized results of Western blot analysis for pCPEB indicated that pCPEB modestly and significantly increased only after 30 s or 1 min of NMDA stimulation (n = 16; ^*p < 0.05 for each). Ten minutes of NMDA stimulation led to a significant dephosphorylation of CPEB (n = 12; ^**p < 0.01). C, Densitized results of phospho-Thr²⁸⁶ CaMKII (pCaMKII) revealed a significant increase at 10 s (n = 15; ^*p < 0.05), 30 s (n = 16; ^**p < 0.01), and 1 min (n = 16; ^**p < 0.01). Although CPEB was dephosphorylated at 10 min of stimulation, phospho-CaMKII was still nonsignificantly elevated over baseline at 10 min (n = 12). D, Representative Western blots of phospho-Thr¹⁷¹ CPEB (pCPEB), total CPEB (CPEB), and a total protein control (Tubulin) during an NMDA dose-response curve. E, At all concentrations tested, NMDA application for 10 min resulted in dephosphorylation of CPEB (pCPEB; n = 8 for each concentration; ^***p < 0.001). No changes in total CPEB levels were seen (CPEB). F, Dephosphorylation of CPEB with NMDA (n = 8; ^***p < 0.001) was blocked by the NMDA-R antagonist APV (50 μm; n = 4) but not by the AMPA-R antagonist DNQX (20 μm; n = 4; ^**p < 0.01) or the sodium channel antagonist TTX (1 μm; n = 4; ^***p < 0.001).

Figure 2.

NMDA-induced Chem-LTD dephosphorylates CPEB. A, Control slices treated with vehicle (DMSO; 0.01%) for 3 min had modest rundown, whereas slices treated with NMDA (25 μm) for 3 min showed prolonged depression between 40 and 70 min after washout (t = 66 min; control fEPSP slope, 0.90 ± 0.3, n = 6; NMDA fEPSP slope, 0.74 ± 0.4, n = 12; p < 0.05). The bar indicates when NMDA was applied. B, Representative Western blots of hippocampal slices probed with phospho-CPEB and total CPEB treated with either vehicle or NMDA. Three minutes of NMDA treatment (n = 5; ^***p < 0.001) significantly decreased CPEB phosphorylation with a slow recovery back to baseline levels at 20 min (n = 5) and 60 min (n = 7) after drug treatment.

Figure 3.

CPEB phosphorylation depends on the LTP induction paradigm. A, Hippocampal slice physiology of slices tetanized with either 1 × 100 Hz or 4 × 100 Hz stimulation. Both paradigms induced hippocampal LTP as measured by the fEPSP slope (t = 45 min; 1 × 100 Hz LTP fEPSP slope, 1.42 ± 0.1, n = 4; 4 × 100 Hz LTP fEPSP slope, 1.71 ± 0.11, n = 9). Arrows indicate when the tetanization was applied. B, Representative Western blots of hippocampal CA1 regions probed with phospho-CPEB, total CPEB, phospho-DARPP-32, total DARPP-32, and tubulin antibodies. Control slices (C) versus LTP slices (L) are compared. C, Densitized results of Western blots from slices stimulated with 1 × 100 Hz. CPEB phosphorylation significantly increased only at 5 min (n = 11; p < 0.001) and returned to baseline by 10 min (n = 7). DARPP-32 phosphorylation did not increase at any time point. D, Densitized results of Western blots from slices stimulated with 4 × 100 Hz. This protocol elicited significant increases in CPEB phosphorylation at 5 min (n = 10; ^***p < 0.001), 10 min (n = 7; ^***p < 0.001), and 30 min (n = 11; ^*p < 0.05) after tetanization. DARPP-32 phosphorylation also increased with this protocol at 5 min (n = 9; ^***p < 0.001) and 10 min (n = 7; ^**p < 0.01) after LTP induction.

Figure 4.

Increased CPEB phosphorylation during hippocampal LTP was blocked by the NMDA-R and CaM-kinase antagonists APV and KN-93, respectively. Arrows indicate when the tetanization was applied. A, Hippocampal slice recordings with 4 × 100 Hz tetanization in the presence of APV (50 μm; t = 10 min; normalized LTP fEPSP slope, 2.03 ± 0.38, n = 7; normalized LTP+APV fEPSP slope, 0.93 ± 0.1, n = 6; p < 0.05) or KN-93 (10 μm; normalized LTP+KN-93 fEPSP slope, 1.17 ± 0.13; n = 7). B, Densitized results from Western blots of slices stimulated with 4 × 100 Hz with APV (n = 6) or KN-93 (n = 7). Neither CPEB nor DARPP-32 phosphorylation increased 10 min after LTP tetanization when APV or KN-93 were applied. ^***p < 0.001.

Figure 5.

Model for bidirectional regulation by CaMKII and PP1 of CPEB-mediated protein synthesis. Modest LTP induction (1 × 100 Hz) stimulates Ca²⁺ influx through the NMDA-R (1). Ca²⁺ binds calmodulin (CaM) to activate CaMKII (2) through autophosphorylation. Activated CaMKII phosphorylates CPEB (3) to stimulate protein synthesis (4). The elevated Ca²⁺ also activates PP2B (5), which dephosphorylates DARPP-32, thereby activating PP1 to dephosphorylate CPEB (6) and limiting protein synthesis. However, robust LTP (4 × 100 Hz) further elevates Ca²⁺, stimulating adenylyl cyclase (AC) (7) to generate cAMP. This activates PKA (8), which phosphorylates DARPP-32, thereby inhibiting PP1 to prolong CPEB phosphorylation and protein synthesis. Green represents events that enhance protein synthesis, and red denotes inhibitory events.