Role for A kinase-anchoring proteins (AKAPS) in glutamate receptor trafficking and long term synaptic depression

Abstract

Expression of N-methyl d-aspartate (NMDA) receptor-dependent homosynaptic long term depression at synapses in the hippocampus and neocortex requires the persistent dephosphorylation of postsynaptic protein kinase A substrates. An attractive mechanism for expression of long term depression is the loss of surface AMPA (alpha-amino-3-hydroxy-5-methylisoxazale-4-propionate) receptors at synapses. Here we show that a threshold level of NMDA receptor activation must be exceeded to trigger a stable loss of AMPA receptors from the surface of cultured hippocampal neurons. NMDA also causes displacement of protein kinase A from the synapse, and inhibiting protein kinase A (PKA) activity mimics the NMDA-induced loss of surface AMPA receptors. PKA is targeted to the synapse by an interaction with the A kinase-anchoring protein, AKAP79/150. Disruption of the PKA-AKAP interaction is sufficient to cause a long-lasting reduction in synaptic AMPA receptors in cultured neurons. In addition, we demonstrate in hippocampal slices that displacement of PKA from AKADs occludes synaptically induced long term depression. These data indicate that synaptic anchoring of PKA through association with AKAPs plays an important role in the regulation of AMPA receptor surface expression and synaptic plasticity.

Fig. 1. ChemLTD NMDA treatment causes a rapid and long-lasting decrease in surface GluR1

A, cultured hippocampal neurons were treated with NMDA (20 μm, 3 min), and surface receptors were biotinylated 5, 30, or 60 min later. NMDA causes a rapid loss of surface (S) GluR1 but no change in total (T) receptor levels. Levels of the transferrin receptor (TfR) were not affected. B, NMDA reduced surface/total GluR1 ratio to 63 ± 7% control levels within 5 min (n = 5), to 53 ± 6% at 30 min (n = 5), and to 48 ± 6% by 60 min (n = 12).

Fig. 2. A high threshold of NMDA receptor activation is required to induce a long term reduction in surface GluR1

A, total (T) and surface (S) GluR1 levels were measured 60 min following NMDA (20 μm). A 1-min NMDA treatment induces little or no change, whereas a 3-min treatment greatly reduces surface GluR1. B, surface GluR1 was decreased 60 min following the chemLTD NMDA treatment (20 μm, 3 min; 45 ± 11% controls; n = 4; *, p < 0.04 versus control, Student's t test). In contrast, GluR1 levels were unaffected following a more brief (20 μm, 1 min) NMDA treatment (92 ± 8% controls; n = 4) or treatment with lower concentration of NMDA (10 μm, 3 min; 88 ± 11% control; n = 4).

Fig. 3. PKA regulates surface AMPA receptor levels

A, schematic of the experimental protocol. Cultures were treated with NMDA for 3 min, returned to the incubator for 27 min, and then treated with forskolin (forsk) to activate PKA. B, representative immunoblot for surface GluR1 showing that forskolin reverses the NMDA-induced loss of surface GluR1. C, NMDA reduces surface GluR1 to 48 ± 7% controls. Forskolin significantly reverses this effect (83 ± 8%; n = 8; *, p < 0.05 versus NMDA alone, Student's t test). D, blocking PKA activity with Rp-cAMPS (Rp) causes a reduction in surface GluR1. E, Rp-cAMPs significantly reduces surface GluR1 to 82 ± 6% control levels. (n = 6; *, p < 0.05 versus control, Student's t test).

Fig. 4. ChemLTD NMDA treatment causes displacement of PKA from synaptosomal membrane fractions

A, NMDA treatment causes no change in total levels of PKA regulatory subunit IIβ (PKA-RIIβ) in neuronal homogenates (93 ± 18% control, cont). B, in contrast, NMDA causes a significant reduction in levels of PKA-RIIβ in synaptic (P3) fractions (72 ± 5% control; *, p < 0.01 versus control, Student's t test). C, similarly, NMDA treatment causes no change in levels of catalytic subunit of PKA (PKA-cat) in total homogenates (94 ± 14% control). D, in synaptic fractions, however, NMDA reduces PKA-cat to 71 ± 9% control levels (*, p < 0.05 versus control (cont), Student's t test).

Fig. 5. Disruption of PKA anchoring to AKAPs is sufficient to cause a reduction in surface GluR1

A, representative immunoblots demonstrating that disrupting PKA-AKAP binding with stearated Ht31 peptide decreases surface GluR1. Control (cont) peptide Ht31p has no effect on surface GluR1. B, quantification revealed that Ht31 (50 μm, 60 min) reduces surface GluR1 to 65 ± 5% control levels (n = 8; *, p < 0.03 versus control, Student's t test). Ht31p has no effect (96 ± 4%; n = 4). C, the effect of Ht31 on AMPA receptor surface expression is blocked by the PP2B inhibitor ascomycin (asc; 2 μm, 60 min). D, ascomycin alone has no effect on surface GluR1 levels (108 ± 4% control; n = 3) but blocks the Ht31-induced loss of surface GluR1 (102 ± 10%, n = 3).

Fig. 6. Disruption of PKA anchoring reduces synaptic AMPA receptors

Cultures were treated with Ht31 or control Ht31p peptides and stained with antibodies against synapsin 1 (A–D) or synaptophysin (F–I) to mark synapses along with antibodies to the AMPA receptor subunit GluR2 (A–D) or GluR1 (F–I). In cultures treated with the control peptide Ht31p, synapsin and GluR2 staining overlapped frequently (A and B). Similarly, synaptophysin and GluR1 staining overlapped following Ht31p treatment (F and G). In contrast, in Ht31-treated cultures, staining for synaptic GluR2 was dramatically reduced (C and D). Ht31 similarly reduced staining for synaptic GluR1 (H and I). E, quantification of 150 synapses in 7 cells revealed that, in control-untreated cultures, 74 ± 8% synapsin puncta colocalized with GluR2. One hour following Ht31 treatment, 28 ± 6% synapsin puncta were associated with GluR2 staining (*, p < 0.01, versus control, Student's t test). Neurons treated with Ht31p exhibited GluR2 staining at 70 ± 6% synapses. J, quantification of synaptophysin and GluR1 staining revealed that, in control cultures, 64 ± 10% synapses were associated with GluR1, whereas Ht31 treatment reduced GluR1 staining to 15 ± 9% synapses (*, p < 0.01 versus control, Student's t test). In neurons treated with Ht31p, GluR1 was present at 60 ± 10% synapses.

Fig. 7. Ht31 produces EPSC rundown that occludes pairing-induced LTD in hippocampal slices

A, sample sweeps before (1) and after (2) pairing-induced LTD under control conditions and during intracellular introduction of Ht31. Sweeps are the average of consecutive responses taken 5 min prior to pairing onset and 25–30 min following completion of the pairing protocol. For clarity, stimulus artifacts were blanked. Scale bars: 25 pA and 25 ms. B, overlay of grouped data time courses demonstrating blockade of LTD by Ht31. No peptide (n = 5) and Ht31p (n = 5) conditions manifested LTD, whereas (n = 8) prevented LTD. Each point represents data averaged into 2-min bins. Plotted on the leftward y axis is EPSC amplitude normalized to a 15-min base-line period. Plotted at the bottom of the graph are series resistance points that refer to the rightward y axis. The black bar indicates the duration of pairing of a 1-Hz stimulation with –40-mV membrane depolarization. C, experiments with a higher resistance pipette tip revealed a rundown of EPSCs that was not detected with larger tip pipettes (B). However, in both cases, the effect of Ht31 on LTD was the same.