PKA-GluA1 coupling via AKAP5 controls AMPA receptor phosphorylation and cell-surface targeting during bidirectional homeostatic plasticity

Abstract

Bidirectional synaptic plasticity occurs locally at individual synapses during long-term potentiation (LTP) or long-term depression (LTD), or globally during homeostatic scaling. LTP, LTD, and homeostatic scaling alter synaptic strength through changes in postsynaptic AMPA-type glutamate receptors (AMPARs), suggesting the existence of overlapping molecular mechanisms. Phosphorylation controls AMPAR trafficking during LTP/LTD. We addressed the role of AMPAR phosphorylation during homeostatic scaling. We observed bidirectional changes of the levels of phosphorylated GluA1 S845 during scaling, resulting from a loss of protein kinase A (PKA) from synapses during scaling down and enhanced activity of PKA in synapses during scaling up. Increased phosphorylation of S845 drove scaling up, while a knockin mutation of S845, or knockdown of the scaffold AKAP5, blocked scaling up. Finally, we show that AMPARs scale differentially based on their phosphorylation status at S845. These results show that rearrangement in PKA signaling controls AMPAR phosphorylation and surface targeting during homeostatic plasticity.

Figure 1

Levels of phosphorylated AMPAR change during homeostatic scaling. (A) Cortical neurons (13DIV) were treated 24 or 48hrs with control media (Con), bicuculline (Bic, 20µM) or tetrodotoxin (TTX, 1µM), followed by surface biotinylation and Western blot. (B) Quantification of cell-surface levels of the AMPAR subunits GluA1, GluA2 or GluA3. (C) Quantification of total levels of phosphorylated GluA1-S845 or S831 or GluA2-S880. Values relative to control, indicated by the dashed line. * and ** (p<0.05 and p<0.01 respectively). Error bars indicate ±sem. N=4–6 independent experiments. See also figure S1.

Figure 2

Reorganization of synaptic PKA signaling during scaling. (A–B) Cortical neurons (13-14DIV) were treated with bicuculline/TTX for 48hrs and indicated molecules were quantified by Western blot, N=5. (C) Cortical neurons (13-14DIV) were treated with bicuculline/TTX for 48hrs followed by isoproterenol (Iso) 10nM for 5min. (D) Changes in pS845 were quantified by Western blot. Iso-induced increases in pS845 were reduced by bicuculline and enhanced by TTX treatment, N=6. (E–H) Hippocampal neurons (14DIV) transfected with GFP and treated with bicuculline or TTX. The localization of PKA catalytic subunit (E) or AKAP5 (G) to dendritic spines was observed and quantified (F) and (H) respectively, N=50–75 spines each from 11–17 neurons. Scale bar 5µm. PKA has strong dendritic shaft localization but is observed in dendritic spines. Following bicuculline treatment PKA moves out of spines. (I) AKAP5 was immunoprecipitated from bicuculline/TTX treated cortical neurons (13-14DIV). Co- immunoprecipitation of PKA is reduced following bicuculline treatment, N=3. * and ** (p<0.05 and p<0.01 respectively). Error bars indicate ±sem. See also figures S1 and S3.

Figure 3

Levels and cellular distribution of PKA activity during scaling. (A) Cortical neurons (13-14DIV) were treated with bicuculline/TTX for 48hrs. Global PKA or PKC activity was assessed by Western blot of whole cell lysates using pan phospho-substrate antibodies. (B) Quantification of pan phospho-substrate blots indicates a significant reduction in PKA activity in whole cell lysate after TTX treatment, N=6. (C) PKA activity was visualized using a FRET-based A-Kinase Activity Reporter, AKAR4, transfected into cortical neurons (13-15DIV) treated with bicuculline/TTX for 48hrs. The false color scale is indicated. Scale bar (5µm). (D) FRET spine enrichment value, N=50–70 spines each from 9–18 neurons. (E) PSD preparations from cultured cortical neurons. Homogenate (Homo.), cytosol fraction (S2), membrane fraction (P2), PSD fraction (PSD). (F) Western blot for PKA phospho-substrate or AKAP5 in PSD samples from control, bicuculline or TTX treated neurons. (G) PKA substrate blot indicates an increase in PKA activity in the PSD following TTX treatment, N=4. (H) AKAP5 in the PSD was significantly increased following TTX treatment, N=4. * and ** (p<0.05 and p<0.01 respectively). Error bars indicate ±sem. See also figures S2 and S3.

Figure 4

AKAP5 knock-down partly occludes scaling-down and blocks scaling-up. (A–C) Cortical neurons (13-14DIV) electroporated with control vector (pSuper) or shRNA against rat AKAP5 (shAKAP5), and treated with bicuculline/TTX for 48hrs followed by surface biotinylation and Western blot. Surface levels of GluA1, GluA2 or GluA3 (B), or total levels of phosphorylated GluA1 S845 or S831, or GluA2 S880 (C) were quantified, N=5. Data are normalized and compared statistically to untreated vector transfected controls. (D) Cortical neurons (13-14DIV) were co-transfected with GFP together with control vector, shAKAP5, or shAKAP5/human myctagged AKAP5 (sh+rescue) and treated with bicuculline/TTX for 48hrs. Surface GluA1 was labeled with an anti-N-terminal antibody followed by immunofluorescence microscopy. GFP (green), surface GluA1 (magenta). Scale bar 20µm, inset 5µm. (E) Surface GluA1 signal from transfected cells was quantified, N=approximately 10 dendrite segments each from 20–35 transfected neurons. Data are normalized and compared statistically to untreated vector transfected controls. * and ** (p<0.05 and p<0.01 respectively). Error bars indicate ±sem. See also figure S4.

Figure 5

S845 of GluA1 is required for TTX-induced scaling up. (A–F) Mouse cortical neurons (14-15DIV) cultured from wild-type (WT) or S831A or S845A GluA1 knock-in mice were treated for 48hrs with bicuculline/TTX. (A-B) Surface-biotinylation and Western blot. Scaling up is blocked in S845A neurons, N=7. Data from each genotype are normalized to untreated controls. (C) mEPSC recordings from WT, S831A or S845A cortical neurons following 48hrs of drug treatment. (D) Quantification of mEPSC amplitude, N=5–11. Data from each genotype are normalized to untreated controls. (E) Surface GluA1 was labeled using an anti-N terminal antibody under non-permeabilized conditions following 48hrs of drug treatment. Scale bar 20µm, inset 5µm. (F) Surface GluA1 levels were quantified on 10–15 dendritic segments each from 20–24 neurons. Data from each genotype are normalized to untreated controls. * and ** (p<0.05 and p<0.01 respectively). ns, no statistical significance. Error bars indicate ± sem. See also figure S5.

Figure 6

PKA activity drives scaling-up. (A–F) Cultured cortical neurons (13-14DIV) were treated for 48 hours with control media or combinations of bicuculline (20µM), TTX (1µM), forskolin + rolipram (FR, 2.5µM/100nM), or FK506 (2.5µM). (A) Drug treatment was followed by surface biotinylation and Western blot. (B) Quantification of surface GluA1 or total phospho-S845. Treatment with FR or FK506 increased GluA1 surface levels occluding the effects of TTX. FR causes a high level of S845 phosphorylation that is maintained throughout scaling. N=6. (C) Following drug treatments, surface GluA1 (magenta) was labeled followed by total VGlut1 (green). Scale bar 5µm. (D) Surface GluA1 fluorescence intensity was quantified only at areas that overlapped with the synapse marker VGlut1. N=24 fields. FR treatment increased synaptic surface GluA1 to a similar degree as TTX. FR and TTX showed an additive effect. (E) mEPSC recording from cortical neurons following 48hrs of drug treatment. (F) Quantification of mEPSC amplitude. FR treatment increased mEPSC amplitude to a similar degree as TTX. FR and TTX showed an additive effect. N=7–15. * and ** (p<0.05 and p<0.01 respectively). ns, no statistical significance. Error bars indicate ±sem. See also figure S6.

Figure 7

Phosphorylated S845 AMPARs are resistant to scaling-down. (A–E) Cortical neurons (13-14DIV) were treated with bicuculline (20µM), TTX (1µM), or forskolin/rolipram (FR, 2.5µM/100nM) for 48hrs. (A) Surface biotinylation and Western blot. Blots were probed for surface GluA1 and then re-probed for surface phospho-S845. (B) Quantification of surface phospho-S845. Surface phospho-S845 receptors are maintained at a high level in the presence of FR throughout the scaling process despite the observation that total surface GluA1 levels are reduced in the presence of bicuculline. N=4. (C) PSD preparation from drug treated neurons. PSD samples were probed for GluA1 and phospho-S845 by Western blot. (D) Phospho-S845 receptors are retained in the PSD during scaling under conditions of high PKA activity (FR), N=3. (E) phospho-S845 receptors were localized by comparing their distribution to total GluA1 and VGlut1. Scale bar 5µm. Phospho-S845 receptors are localized to synapses as seen from the triple co-localization and are maintained at the synapse in the presence of bicuculline. Note that phospho-S845 receptors are only visible by immunofluorescence in the presence of FR. * and ** (p<0.05 and p<0.01 respectively). ns, no statistical significance. Error bars indicate ±sem. See also figures S7.

Figure 8

Scaling and chemical LTP. (A–H) Cortical neurons (13-14DIV) were treated with bicuculline (20µM)/TTX (1µM) for 48hrs, followed by chemical LTP (cLTP) using glycine (Gly, 200µM, 5min treatment, 20min chase). (A) Neurons were then surface-biotinylated and lysed for Western blot. (B) Quantification of surface GluA1 and phospho-S845 Western blots. Glycine treatment in control neurons results in increased surface GluA1 and phospho-S845 levels. N=5. Increases are blocked by treatment with bicuculline or occluded by treatment with TTX. (C) Neurons were treated as in A, followed by fixation and surface GluA1 labeling with an anti-N terminal antibody. Scale bar 20µm, inset 5µm. (D) Surface GluA1 levels were quantified on 10–15 dendritic segments each from 28–32 neurons per condition. (E) Following scaling and cLTP treatments cortical neurons were fractionated to obtain PSD samples. PSD material was analyzed by Western blot. (F) cLTP results in increased levels of synaptic GluA1 and GluA2 in control neurons but not in neurons pre-treated with bicuculline or TTX. For each scaling condition, data are presented as glycine treated relative to untreated, indicated by the dashed line. N=3. (G) Surface GluA1 (magenta) and total VGlut1 (green) were labeled following scaling and cLTP. Scale bar 5µm. (H) Surface GluA1 fluorescence intensity was quantified only at areas that overlapped with the synapse marker VGlut1. For each scaling condition data are presented as glycine treated relative to untreated. N=35 fields. * and ** (p<0.05 and p<0.01 respectively). ns, no statistical significance. Error bars indicate ±sem.

Figure 9

Model of signaling mechanisms affecting AMPA receptor phosphorylation during scaling, LTP, and LTD. Scaling involves a change in the coupling of basal PKA with AMPA receptors via AKAP5, resulting in increased or decreased AMPA receptor phosphorylation. LTP and LTD involve the activation and recruitment of kinases or phosphatases respectively to increase or decrease AMPA receptor phosphorylation respectively. Note that AKAP5 also participates in LTP and LTD.