Protein phosphatase 2A inhibition induces cerebellar long-term depression and declustering of synaptic AMPA receptor T. Launey*, S. Endo, R. Sakai, J. Harano, and M. Ito Laboratory for Memory and Learning, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Edited by Roger A. Nicoll, University of California, San Francisco, CA, and approved November 13, 2003 (received for review May 15, 2003)
Phosphorylation of synaptic (RS)-␣-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid) (AMPA) receptors (AMPARs) is an essential component of cerebellar long-term depression (LTD), a form of synaptic plasticity involved in motor learning. Here, we report that protein phosphatase 2A (PP-2A) plays a specific role in controlling synaptic strength and clustering of AMPARs at synapses between granule cells and Purkinje cells. In 22- to 35-day cerebellar cultures, specific inhibition of postsynaptic PP-2A by fostriecin (100 nM) or cytostatin (10 – 60 M) induced a gradual and use-dependent decrease of synaptic current evoked by the stimulation of a single granule cell, without altering receptor kinetics nor passive electrical properties. By contrast, PP-2A inhibition had no effect on immature Purkinje cells (12–15 days). Concurrent PP-2A inhibition and AMPAR stimulation induced a reduction of miniature synaptic currents and a reduction of AMPAR density at synapses. Either PP-2A inhibitor alone or AMPA stimulation alone had no significant effect. Inhibition of PP-1 by inhibitor 1 (10 –27 units兾l) had no effect on synaptic current. Synaptic depression induced by PP-2A inhibition occluded subsequent induction of LTD by conjunctive stimulation and was abolished by a calcium chelator or a protein kinase inhibitor, suggesting a shared molecular pathway and involvement of PP-2A in LTD induction. cerebellum 兩 Purkinje 兩 plasticity 兩 synapse
C
erebellar long-term depression (LTD) is a long-lasting attenuation of the synaptic transmission between granule cells (GCs) and Purkinje cells (PCs), which has been thought to be a major mechanism of motor learning (1, 2). Complex signal transduction mechanisms underlie this unique form of synaptic plasticity (reviewed in refs. 3–5) with several pathways eventually converging to alter (RS)- ␣ -amino-3-hydroxy-5-methyl-4isoxazolepropionic acid) (AMPA)-selective glutamate receptors (AMPARs), hence reducing GC–PC synaptic strength. Evidence indicate that phosphorylation of the AMPAR in its C-terminal domain plays a key role in this process (6–9), apparently triggering endocytotic removal of these receptors from the postsynaptic membrane (7, 10). Accordingly, several protein kinase-dependent signaling cascades have been shown to be essential for cerebellar LTD induction (reviewed in ref. 5). Although protein phosphatases (PPs) share with protein kinases the task of regulating protein phosphorylation level, they have long been considered mere housekeepers, with poorly understood substrate specificity and loose regulation. In recent years, however, studies using phosphatase inhibitors revealed that these enzymes exert a major influence on synaptic plasticity in the cerebellum (11–15) and other brain structures (16, 17). In the cerebellum, investigation of AMPAR regulation by PP at the GC–PC synapse represents a challenge as PCs abundantly express at least five major types of serine兾threonine PPs: PP-1, PP-2A, PP-2B, PP-2C, and PP-5 (18–22). In addition, PCs have the distinctive property of expressing G substrate, a PP-1, PP-2A (23–25), and possibly PP-5 inhibitory protein. Because G substrate is activated through the nitric oxide兾guanylyl cyclase兾 protein kinase G pathway known to be involved in LTD induc676 – 681 兩 PNAS 兩 January 13, 2004 兩 vol. 101 兩 no. 2
tion in mature cerebellum (26, 27), we undertook to clarify the relative influence of PP-2A, PP-1, and PP-5 on AMPAR regulation at the GC–PC synapse and as their interactions with the molecular mechanisms of pairing-induced LTD. Materials and Methods Cerebellar-Dissociated Culture. The experimental procedures and housing conditions for animals were approved by the RIKEN Institute’s Animal Experiment Committee. Dissociated rat cerebellar cultures were prepared as described (28) with minor modifications (see Supporting Text, which is published as supporting information on the PNAS web site). Reagents and Drugs. Microcystin-LR and fostriecin were pur-
chased from Calbiochem, and A MPA, D (-)-2-amino-5phosphonopentanoic acid, and 7-(hydroxyimino)-cyclopropachromen-1a-carboxylate ethyl ester were from Tocris Cookson (Bristol, U.K.). Cytostatin and dephosphocytostatin were generous gifts from T. Takeuchi (Institute for Chemotherapy, Shizuoka, Japan). The noncompetitive AMPAR antagonist (⫾)1-(4-aminophenyl)-3-methylcarbamyl-7,8-methylenedioxy3,4-dihydro-5H-2,3-benzodiazepin (GYKI-53655) was generously provided by I. Tarnawa (Gedeon Richter, Budapest). Thiophosphorylated inhibitor 1 was prepared as described (16). Electrophysiological Recording. Whole-cell voltage-clamp record-
ings were made from cultured PCs that were visually identified by using phase-contrast optics, occasionally confirmed by filling with fluorescent dye during recording (Fig. 1A). The extracellular solution contained 140 mM NaCl, 3 mM KCl, 3 mM CaCl2, 1 mM MgSO4, 0.5 mM Na2HPO4, 10 mM D-glucose, 10 mM Hepes, 3 mM pyruvate, 0.03 mM picrotoxin, and 0.03 mM D(-)-2-amino-5-phosphonopentanoic acid (pH 7.35, 330 ⫾ 5 mOsm, 33°C). Pipette-filling solution contained 60 mM K gluconate, 60 mM K methanesulfonate, 20 mM KCl, 3 mM MgCl2, 4 mM Na2ATP, 0.4 mM NaGTP, 15 mM Hepes, 2 mM EGTA, 0.8 mM CaCl2, and 1 mM reduced glutathione (pH 7.4, 310 ⫾ 5 mOsm, pCa ⫽ 7.3). We waited ⬎10 min after patch rupture to allow diffusion of the PP inhibitors into PC dendrites before starting experimental protocols. Seal resistance was 2–16 G⍀. Series resistance was 7–20 M⍀ and was not compensated. Recording was terminated if unrecoverable changes of series or input resistance exceeded 25%. For evoked excitatory postsynaptic current (eEPSC) a single GC was stimulated (0.3–4 V, 500 s) through a saline-filled, fire-polished glass pipette gently This paper was submitted directly (Track II) to the PNAS office. Abbreviations: AMPA, (RS)-␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPAR, AMPA receptor; LTD, long-term depression; PP, protein phosphatase; GC, granule cell; PC, Purkinje cell; EPSC, excitatory postsynaptic current; eEPSC, evoked EPSC; mEPSC, miniature EPSC; DIV, days in vitro; CS, conjunctive stimulation; GluR, glutamate receptor; GYKI-53655, (⫾)1-(4-aminophenyl)-3-methylcarbamyl-7,8-methylenedioxy-3,4-dihydro5H-2,3-benzodiazepin. *To whom correspondence should be addressed. E-mail: t㛭
[email protected]. © 2003 by The National Academy of Sciences of the USA
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Quantification of AMPAR Density. Expression of AMPAR at the surface of PCs was assessed by immunofluorescence, using an antibody that recognizes the N-terminal extracellular domain of its glutamate receptor 2 (GluR2) subunit (7), a gift from H. Hirai (Kobe University, Kobe, Japan). Briefly, after pharmacological treatment, fixation, and immunostaining, the cultures were imaged by using a charge-coupled device camera (Coolsnap, Photometrics, Tucson, AZ). We chose PCs for which most of the dendrites were within the focal plane (⫻100 objective, 1.4 numerical aperture). Digital images were high-pass filtered by convolution with a 5-by-5 pixels Laplace filter and thresholded to isolate receptor clusters. This mask was then applied to the original image. For each detected cluster, the mean fluorescence intensity and area were recorded. All images were acquired with the same exposure time and analyzed blindly with the same segmentation threshold. The data presented are from 36 cultures and 197 images. A minimum of 20,000 spines were analyzed for each experimental conditions. AMPA C-Terminal Domain Dephosphorylation Assay. Dephosphorylation of the cytoplasmic domain of AMPAR GluR2 subunit by 0.01 unit of PP-2A and PP-1 catalytic subunits was measured by using either the 51-aa C-terminal fragment of the protein (GluR2-CT) or a fusion protein made of this fragment together with GST (GST-GluR2-CT), as described (30). The procedure and unit definition have been reported in detail (31) and are provided in Supporting Text.
Fig. 1. Effect of PP inhibitors on GC–PC synapse. (A) (Left) A single GC is electrically stimulated, and eEPSCs are recorded in the postsynaptic PC infused with PP inhibitor. (Right) A PC infused with a fluorophore, from a 27-day cerebellar culture. (Scale bar: 15 m.) (B1) Time course of eEPSC depression induced by 100 nM fostriecin. Synaptic stimulation was started at least 10 min after establishment of the whole-cell configuration to allow for drug diffusion into distal dendrites. (B2) (Upper) eEPSCs from the experiment shown in B1, at the indicated time points a and b (average of 10 consecutive traces). The vertical transient preceding the eEPSC corresponds to the GC stimulation artifact. (Lower) Depressed eEPSC (b) peak-scaled to original amplitude. Note that the eEPSC time course did not change, nor did the passive electrical response to a 3-mV voltage pulse. (C) Average decay time course in control condition (n ⫽ 14) and in the presence of fostriecin (100 nM, culture 12–15 DIV, n ⫽ 8, and 22–35 DIV, n ⫽ 14), cytostatin (10 – 60 M, n ⫽ 12), microcystin-LR (10 M, n ⫽ 13), thiophosphorylated inhibitor 1 (10 –27 units兾l, n ⫽ 11), and dephosphocytostatin (30 M, n ⫽ 4).
Launey et al.
Results Synaptic activation was obtained by the stimulation of a single GC located 50–300 m from the recorded PC soma while PP inhibitors were infused into the recorded PC, allowing continuous monitoring of AMPAR alteration at functional synapses. The average amplitude of eEPSC was 139.2 ⫾ 6.2 pA, probably reflecting the presence of more than one synaptic contact between the stimulated GC and the PC (32), owing to the coplanar growth of GC axons and PC dendrites. These eEPSCs had brief and constant latencies of 2.16 ⫾ 0.11 msec (n ⫽ 91). The latency and amplitude did not change noticeably when the stimulus intensity was varied up to three times the threshold for eight GC–PC pairs examined in detail. The eEPSCs occurred in an all-or-none fashion (see failures in Fig. 1B1), the failures probably arising from spontaneous fluctuation of the GC soma excitability or axon refractoriness after spontaneous action potentials (33). PP-2A Inhibition Induces Synaptic Depression. Infusion of the specific PP-2A inhibitor fostriecin (100 nM) in PCs at 22–35 days in vitro (DIV) produced a gradual depression of eEPSC amplitude over 30–40 min (Fig. 1B1). The passive electrical properties of the recorded PC and the apparent kinetics of the stimulated AMPARs were unaffected (Fig. 1B2). On average, the depresPNAS 兩 January 13, 2004 兩 vol. 101 兩 no. 2 兩 677
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pressed against its soma. To record miniature EPSCs (mEPSCs), 1 M tetrodotoxin was added to the extracellular solution, and potassium was replaced by cesium in the internal solution. The mEPSCs were analyzed by using a custom semiautomatic algorithm (ref. 29 and see Supporting Text). When data from several experiments were combined, EPSC amplitudes for each experiment were normalized relative to the average amplitude at the beginning of the recording (first 12 stimulations). The eEPSC failures were not included in averaged traces. Statistical comparisons were done by using t test or Mann–Whitney U test when appropriate. All values are mean ⫾ SEM. The different experimental protocols were interleaved to avoid the influence of any heterogeneity between culture batches. The data presented were obtained from voltage-clamp recording of 161 PCs from 113 cultures (40 batches).
sion produced by 100 nM fostriecin and 10–60 M cytostatin (23–28 DIV), another specific PP-2A inhibitor, reached 35 ⫾ 5% and 46 ⫾ 9% of control amplitude, at 50 min (n ⫽ 14 and 12, respectively, Fig. 1C). No significant depression was observed in the absence of phosphatase inhibitor, with vehicle alone (0.1% DMSO) in the pipette (97 ⫾ 5%, n ⫽ 14, 14–32 DIV). Dephosphocytostatin (30 M), an inactive form of cytostatin, did not produce any marked effect on eEPSC amplitude (91 ⫾ 3%, n ⫽ 4). In contrast, infusion of 100 nM fostriecin into immature PCs (12–15 DIV) did not depress eEPSCs (93 ⫾ 5%, n ⫽ 8), confirming a recent report using young cerebellar cultures from mouse (15). All subsequent experiments were done on PCs aged 22–35 DIV (average 25.7 DIV). Being membrane permeable, both fostriecin and cytostatin may diffuse transsynaptically into GC axon terminals and affect glutamate release (34). Thus, the experiment was repeated with the membrane-impermeable PP inhibitor microcystin-LR (10 M) to confirm that the depression only resulted from postsynaptic inhibition of PP. Intracellular infusion microcystin-LR into PCs reproduced both the time course and magnitude (37 ⫾ 8% of control, at 50 min, n ⫽ 11) of the depression observed with fostriecin and cytostatin (Fig. 1C), whereas bath application had no effect (96 ⫾ 6%, n ⫽ 4, data not shown). To verify that PP-2A inhibitors were used at saturating concentration, 10, 30, and 60 M cytostatin was tested, and no differences were observed in time course or magnitude of depression (data not shown). At these concentrations, both fostriecin and cytostatin block PP-2A [EC50 ⬍5 nM and ⬍100 nM, respectively (35, 36), see also Fig. 6, which is published as supporting information on the PNAS web site], but do not affect PP-1, PP-5, PP-2B, or PP-2C. Intracellular infusion of thiophosphorylated inhibitor 1, a PP1-specific inhibitor, at 10 or 27 units兾l, did not significantly affect eEPSCs (90 ⫾ 7% of control, n ⫽ 11, P ⬎ 0.5). To ascertain the diffusion of this large peptide into the PC, some experiments were done with a fluorescent conjugate, showing diffusion to the distal dendrites within 15 min of infusion (data not shown). Occlusion with Classical LTD. To establish the relationship between PP inhibitor-induced depression and the classical LTD induced by conjunctive stimulation (CS), we performed an occlusion experiment (Fig. 2A). Once the depression induced by PP-2A inhibitor had reached steady state, further reduction of eEPSC amplitude by CS was attempted. Although the CS protocol used produces robust LTD at naive synapses (Fig. 2 A Inset), here no further depression was observed (n ⫽ 4). This finding suggests that both forms of synaptic depression share at least part of a common molecular pathway. This absence of depression after CS does not result from PC dialysis nor from the prior reduction of eEPSC amplitude because comparable partial reduction of eEPSC amplitude by a noncompetitive AMPAR antagonist (GYKI-53655) perfused for 20 min did not prevent CS-induced LTD (Fig. 2 A Inset), as recently shown (37). Note that we selected cell pairs for which the eEPSC amplitude was still at least 35 pA after the depression induced by fostriecin兾GYKI, to allow better detection of any further depression. Requirement for Ca2ⴙ and Protein Kinase but Not Metabotropic Receptors. We next examined whether the signaling cascades
underling classical LTD are shared by PP-2A inhibitor-induced depression. Replacement of calcium in the internal solution by the high-affinity calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetate acid (BAPTA) (2 mM) abolished the depression induced by microcystin-LR (94 ⫾ 7% of control at 50 min, n ⫽ 11, Fig. 2B), whereas superfusion of the metabotropic GluR (mGluR) antagonist 7-(hydroxyimino)cyclopropachromen-1a-carboxylate ethyl ester (100 M) had no effect on depression (44 ⫾ 14% at 50 min, n ⫽ 6). This finding 678 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0302914101
Fig. 2. Synaptic depression induced by PP-2A inhibitor and LTD share a common molecular pathway. (A) Occlusion experiment. Once depression induced by a PP-2A inhibitor (100 nM fostriecin) reached steady state, CS (vertical bar at 38 min) failed to induce further eEPSC depression. A gap was created to align the CS onsets, and eEPSC amplitudes were normalized to average amplitude within the 5-min period preceding the CS (n ⫽ 4). (Left Inset) eEPSC from such an experiment (average of 30 traces each) at 2 (a), 27 (b), and 57 (c) min. (Right Inset) LTD induced by CS in the absence of PP-2A inhibitor, with prior reduction of eEPSC amplitude by a glutamate antagonist, GYKI-53655 (500 – 800 nM, n ⫽ 6). (B) Replacement of intracellular calcium by 2 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetate acid (BAPTA) (n ⫽ 11) prevents depression whereas bath application of the metabotropic GluR inhibitor 7-(hydroxyimino)-cyclopropachromen-1a-carboxylate ethyl ester (CPCCOet, 100 M, n ⫽ 6) has no effect on microcystin-LR-induced depression (10 M, n ⫽ 13). (C) Requirement of protein kinase activity. Coinfusion of the broad-spectrum protein kinase inhibitor staurosporin (1 M) and fostriecin (n ⫽ 4) abolishes synaptic depression induced by fostriecin (100 nM) alone (n ⫽ 14, same data set as in Fig. 1C). (D) Use-dependent depression. Time course of synaptic depression for GC stimulation intervals of 3 and 30 s. Thin lines represent exponential fit to the data with decay time constants of 5.1 min (n ⫽ 5) and 28.9 min (n ⫽ 7), respectively. (E) Decay time constant as a function of stimulation interval. The numbers in brackets indicate the number of data points (E).
suggests that PP-2A inhibitors may cut into LTD signal transduction downstream of mGluRs. To ascertain that the depression induced by fostriecin兾cytostatin was specifically mediated by an alteration of the balance between phosphorylation and Launey et al.
Synaptic Depression Is Use-Dependent. It has been previously reported that depression induced by PP-1兾PP-2A inhibitors requires repetitive stimulation (12, 15). We extended this observation by using the PP-2A-specific cytostatin and further analyzed the effect of various stimulation frequencies on the depression time course (Fig. 2 D and E). Indeed, reducing the stimulation interval from 5 to 3 s resulted in a faster depression (time constant of 12.0 ⫾ 3.2 min and 5.9 ⫾ 0.9 min, respectively). Conversely, increasing the stimulation interval to 30 s resulted in an increase of rate constant to 33.8 ⫾ 4.5 min (Fig. 2D). Plotting decay rate constant for various stimulation intervals revealed a near-linear relationship, suggesting that the fractional depression per stimulation is independent of the stimulation interval within the 3- to 30-s range (Fig. 2E). On average, this fractional depression was 0.92 ⫾ 0.11% per stimulation and may correspond to the fraction of synaptic AMPARs rendered nonfunctional after each synaptic stimulation. Depression Requires Coincident PP-2A Inhibition and AMPAR Stimulation. Because PP-2A inhibitor-induced depression requires
synaptic stimulation but is independent of metabotropic GluR activation, we hypothesized that among the various synaptic signals, the postsynaptic action on AMPAR is the major component. Thus, we combined PP-2A inhibition and AMPA bath application. To avoid GC firing and consequent synaptic activation, we added 1 M tetrodotoxin. With all eEPSCs blocked, we turned to mEPSCs to evaluate alteration of synaptic transmission. After recording control mEPSCs during 15 min, we bath-applied the membrane permeable cytostatin (10 M) for 30 min with or without AMPA stimulation (10 M, 5 min). A second sample was then recorded after 15 min of AMPA wash-out (Fig. 3A). For each recorded PC (n ⫽ 19, 21–24 DIV), a minimum of 500 mEPSCs was analyzed by fitting to a template function to extract amplitude and shape parameters (29). Combination of cytostatin and AMPA resulted in a prominent decrease of average mEPSC amplitude and an apparent decrease of frequency (Fig. 3B). Because reduction of amplitude below noise level may masquerade as a decrease of frequency, this parameter was not further analyzed. For the representative PC shown in Fig. 3 B–D, comparison of amplitude and decay time constant distribution between control and cytostatin ⫹ AMPA conditions revealed that although the amplitude decreased (14.9 to 9.1 pA, Fig. 3E), there was no conspicuous change of decay time constant (3.2 vs. 3.8 ms, Fig. 3 C and D). Because amplitude distributions were often skewed (14) because of the imperfect space clamp and the heterogeneous population of synapses, we normalized all mEPSC amplitudes to the median amplitude measured during the control period. Perfusion of cytostatin alone (n ⫽ 5), AMPA alone (n ⫽ 4), or saline without drug (n ⫽ 5) did not produce any significant alteration of mEPSC amplitude or shape after 45 min of recording, with median amplitudes at 87.5%, 103.2%, and 89.7% of control, respectively (P ⬎ 0.1 in all cases, U test). When both AMPA and cytostatin were applied, mEPSC median amplitude was reduced to 53.7% of control amplitude (P ⬍ 0.001, n ⫽ 5, U test). Comparison of average Launey et al.
Fig. 3. Depression of mEPSCs induced by PP-2A inhibitor requires activation of AMPARs. (A) Drug application protocol, control mEPSCs were recorded during the first 15 min after patch rupture and compared to mEPSCs recorded at least 15 min after the end of AMPA perfusion. (B) mEPSCs at the beginning of the recording (Upper) and after perfusion of cytostatin plus AMPA (Lower). Superposition of five consecutive traces. (C) Average of mEPSCs before (488 traces, Left) and after AMPA plus cytostatin (429 traces, Right). The dashed blue line represents the average of the fitting function. Overlapping mEPSCs were not included in these averages. (Inset) Response to a 3-mV test pulse. (D) Trace from C, with the second average peak-scaled (dashed red line) to the average of the control mEPSC. (E) Cumulative distribution of mEPSC amplitude and decay time constant from the cell shown in B. (F) Change of mEPSC amplitude under the indicated experimental conditions, normalized to the median amplitude during the control period. Group size is indicated above each condition. ***, P ⬍ 0.001.
decay time constant between these different experimental conditions did not reveal any significant difference between control (4.9 ⫾ 0.7 ms) and cytostatin alone (5.5 ⫾ 1.0 ms), AMPA alone (3.9 ⫾ 0.8 ms), AMPA ⫹ cytostatin (4.4 ⫾ 1.1 ms), or 45-min recording without drug (4.7 ⫾ 1.0 ms), with P ⬎ 0.2 in all cases (U test). Thus, combined PP-2A inhibition and AMPAR stimulation are necessary and sufficient for inducing depression. PP-2A Activity Controls AMPAR Clustering. The comparable mag-
nitude of the cytostatin-induced depression between eEPSCs (53.1%) and mEPSCs (46.3%) suggests that site of expression of the depression is mostly postsynaptic. Thus, we examined the effect of a PP-2A inhibitor on AMPAR synaptic clustering because LTD and declustering have been shown to be correlated (7). To estimate the density of AMPARs at the surface of PCs (20.2 ⫾ 0.2 DIV, n ⫽ 197), we used an antibody recognizing the N-terminal extracellular domain of GluR2兾3 (GluR2-N), as in ref. 7. Cytostatin and兾or AMPA were added to the culture medium by using the same incubation times as for our mEPSC study, but with the cytostatin concentration raised to 60 M to counteract potential interaction with serum in the culture medium. Spontaneous action potentials were blocked by addition of 1 M tetrodotoxin. After incubation, the cultures were fixed and stained for GluR2-N. Control cultures not exposed to chemical stimulation exhibited bright, small fluorescence spots (Fig. 4A), previously shown to correspond to synaptic sites (7). Because PCs exhibited very high densities of GluR2兾3 compared to other cell types in the cerebellar cortex (38) and a very characteristic dendritic morphology, no PC-specific counterstain was necessary. After exposure to a combination of cytostatin (60 M, 30 min) and AMPA (10 M, 5 min), we observed a decrease in the PNAS 兩 January 13, 2004 兩 vol. 101 兩 no. 2 兩 679
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dephosphorylation, we examined whether a protein kinase inhibitor would prevent fostriecin-induced depression. A broadspectrum kinase inhibitor was used because at least five different protein kinases families (PKC, mitogen-activated protein kinase, cGMP-dependent protein kinase, tyrosine kinase, and Ca2⫹兾 calmodulin-dependent protein kinase) are essential for LTD induction (5). Coinfusion of 1 M staurosporin with 100 nM fostriecin abolished the depression induced by fostriecin alone (97 ⫾ 13% and 46 ⫾ 9% of control at 50 min, n ⫽ 14 and n ⫽ 4, respectively). Staurosporin alone produced a small, nonsignificant potentiation (105 ⫾ 6% of control at 50 min, n ⫽ 4, P ⬎ 0.5, Fig. 2C).
Fig. 5. In vitro assay of AMPA GluR2 C-terminal domain dephosphorylation by PP-2A (see Materials and Methods). Both GST-GluR2-CT and the thrombin cleaved GluR2-CT fragment are dephosphorylated. Thin line shows exponential fit to data points with indicated time constant. Average is from triplicate.
intensity, only puncta remaining ‘‘bright enough’’ after depression can be detected and quantified. Thus, the decrease of cluster intensity is probably underestimated. An apparent decrease of the number of clusters per unit of dendritic surface was also observed after AMPA plus cytostatin treatment but this reduction could not be reliably quantified. Although it would have been interesting to examine declustering in young cultures, most PCs before 15 DIV showed only short and spineless dendrites, precluding morphological analysis.
Fig. 4. Coapplication of AMPA and PP-2A inhibitor induces a declustering of GluR2兾3. (A) (Left) Digital fluoromicrography of a PC dendrite immunolabeled with an antibody binding the extracellular domain of GluR2兾3. (Upper) Control. Bright small puncta correspond to GluR2兾3 clustering at GC–PC synapses. (Lower) In a sister culture, cytostatin (60 M) plus AMPA (10 M) treatment caused a reduction of density, area, and fluorescence intensity of GluR2兾3 clusters. (Scale bar: 10 m.) (Right) Pseudocolored 3D elevation of pixel intensity. Scale values correspond to camera analog兾digital conversion units (12 bits). (B) Segmentation of cluster regions from fluorescence images. (Upper) Inverted fluorescence image. (Lower) Result of segmentation algorithm (see Materials and Methods). (C) Comparison of cluster alteration under various experimental conditions. A minimum of 25,000 clusters was analyzed per condition, and experiments were done in triplicate. *, P ⬍ 0.02; ***, P ⬍ 0.001.
number of clusters per dendrite and a decrease in spot intensity (Fig. 4A). For quantification, digital fluoromicroscopic images were acquired and analyzed blind to detect receptor clusters (Fig. 4B). Cluster fluorescence intensity and size were reduced after coapplication of cytostatin and AMPA to 78.7 ⫾ 2.8% and 69.0 ⫾ 4.2% of control, respectively (P ⬍ 0.05 and P ⬍ 0.001, Fig. 4C). By contrast, incubation with cytostatin alone, AMPA plus dephosphocytostatin, or AMPA plus cytostatin plus GYKI53655 (10 M) did not produce any significant alteration of clusters. Cluster intensities were 99.8 ⫾ 2.7%, 105.5 ⫾ 1.9%, and 103.6 ⫾ 3.9% of control for each condition, and areas were 104.5 ⫾ 5.2%, 97.3 ⫾ 5.8%, and 105.7 ⫾ 5.5% of control, respectively (Fig. 4C). The results suggest that PP-2A inhibition per se does not induce receptor declustering and that coactivation of AMPARs is required. Similar results were obtained when cytostatin was replaced by fostriecin (data not shown). It should be noted that because cluster detection is based on fluorescence 680 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0302914101
PP-2A Regulates Phosphorylation of GluR2 Cytoplasmic Domain. Cerebellar LTD correlates with an increased phosphorylation of the AMPAR GluR2 subunit cytoplasmic domain (7–9). Because PP-2A is known to dephosphorylate several membrane ion channels (39) and a GluR (40) there is a possibility that PP-2A may directly control AMPAR dephosphorylation. To examine this hypothesis, we conducted an in vitro assay with the PKCphosphorylated 32P-GluR2 C-terminal domain as a substrate and revealed that PP-2A dephosphorylated this substrate quickly (time constant: ⬍9.2 min) and efficiently (Fig. 5). Under the same conditions, dephosphorylation by PP-1 was also observed albeit with slower kinetic (time constant ⬎ 15.8 min, data not shown).
Discussion Our results demonstrate that selective inhibition of PP-2A in 22to 35-DIV cerebellar cultures produces a depression of GC–PC synaptic current resembling LTD induced by CS. This depression requires activation of AMPA-gated channels, protein kinase activity, and the presence of free cytoplasmic calcium (the estimated concentration in the pipette was ⬇50 nM). Combined PP-2A inhibition and AMPAR activation was sufficient to induce a depression of mEPSCs and a declustering of synaptic AMPA GluR2兾3 subunits. A selective PP-1 inhibitor did not induce any notable synaptic depression. By contrast, in immature PCs (12–15 DIV) a PP-2A inhibitor had no effect. This finding suggests that sustained PP-2A activity in mature PC contribute to the regulation AMPARs at GC–PC synapses (see below). The similar reduction of eEPSCs and mEPSCs amplitude, without alteration of kinetics, suggests that this depression is mostly postsynaptic, as described for LTD in similar preparations (41, 42). This notion is further confirmed by our receptor declustering experiments showing that the same stimulation protocol that induces mEPSC depression also induces a declustering of synaptic AMPARs. Taken together, these results suggest that PP-2A may regulate AMPAR density at synapses through a use-dependent mechanism involving protein kinase(s) activity, calcium signaling, and repetitive or sustained activation of synaptic AMPARs. Because cerebellar LTD depends on phosphorylation of GluR2 at Ser-880 (6–9), an attractive hypothesis Launey et al.
synaptic remodeling (disappearance of functional N-methyl-Daspartate receptors and of multiple climbing fiber innervation) and changes in electrical properties resulting from alteration of voltage-dependent channel populations (45, 46). The signaling cascade underlying cerebellar LTD also appears to undergo considerable maturation during this period, with neither IP3 nor NO required in immature PCs (47, 48), whereas these signals are necessary in the mature cerebellum (5). Furthermore, this period also corresponds to a drastic inversion of the relative abundance of PP-2AB and PP-2AB␥ in the brain, two PP-2A regulatory subunits highly expressed in PCs and involved in PP-2A targeting (21). Our results may provide a functional explanation for the recent clinical report that some forms of human spinocerebellar ataxia are associated with an alteration of the gene encoding the PP-2A regulatory subunit PP-2AB (49). In conclusion, our experiments shows that PP-2A plays a prominent role in the regulation of GC–PC synaptic efficacy. Unraveling the full extent of its involvement in cerebellar LTD will require a better understanding of its interactions with protein kinases and other proteins of the postsynaptic complex, probably through its regulatory subunits.
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We thank the late Dr. R. T. Kado for his helpful advice; Drs. T. Takeuchi, H. Hirai, I. Tarnawa, and M. Chinkers for their generous gifts of plasmids, antibodies, and reagents; Dr. N. Murphy for editorial comments; and Mr. T. Torashima for technical help. This work was supported in part by Japan Society for the Promotion of Science Grants-in-Aid 12780612 (to T.L.) and 12680790 and 13041064 (to S.E.).
PNAS 兩 January 13, 2004 兩 vol. 101 兩 no. 2 兩 681
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is that PP-2A directly regulates the phosphorylation level of this subunit, as shown for N-methyl-D-aspartate receptors in the hippocampus (40) and as suggested by our in vitro dephosphorylation result (Fig. 5). In addition, PP-2A is known as a key regulator of several types of protein kinases (reviewed in ref. 43), including some involved in LTD such as PKC, mitogen-activated protein kinase, and Ca2⫹兾calmodulin-dependent kinase (3–5). Reduction of PP-2A activity by an endogenous inhibitor would thus result in increased protein kinase activity, enabling LTD induction. Indeed, one such inhibitor is G substrate, which is interesting in the context of cerebellar LTD as it is expressed almost exclusively by PCs (23), it is phosphorylated through the NO-cGMP-protein kinase G signaling cascade known to be involved in LTD induction in cerebellar slices (4, 5), and its phosphorylation is increased after LTD-inducing stimuli (44). The phosphorylated form is a potent inhibitor of PP-2A and to a lesser extent of PP-1 (refs. 24 and 44, but see also ref. 25). Our study completes the demonstration by Eto et al. (15) that, in immature mouse PCs (9–16 DIV), a complex with PP-1 at its core, plays a major role in cerebellar LTD, whereas PP-2A inhibitors had little effect at this age (ref. 15 and this study). This finding raises the interesting hypothesis that the relative contribution of PP-2A and other phosphatases might change during cerebellar maturation, with PP-1-related phosphatase regulating synaptogenesis and plasticity at early developmental stages, whereas PP-2A would play a dominant role only at more mature stages. It should be noted that the period between the second and third postnatal week in vivo corresponds to an extensive
