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First published online 19 August 2008
doi: 10.1242/jcs.032987

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Cytosolic PLA₂ activation in Purkinje neurons and its role in AMPA-receptor trafficking

¹ Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chuo-ku, Chiba 260-8675, Japan
² Biomembrane Signaling Project, The Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan
³ Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

^* Author for correspondence (e-mail: hirabayashi-tt{at}igakuken.org.jp)

Accepted 18 June 2008

	Summary

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Cytosolic phospholipase A₂ (cPLA₂) selectively releases arachidonic acid from membrane phospholipids and has been proposed to be involved in the induction of long-term depression (LTD), a form of synaptic plasticity in the cerebellum. This enzyme requires two events for its full activation: Ca²⁺-dependent translocation from the cytosol to organelle membranes in order to access phospholipids as substrates, and phosphorylation by several kinases. However, the subcellular distribution and activation of cPLA₂ in Purkinje cells and the role of arachidonic acid in cerebellar LTD have not been fully elucidated. In cultured Purkinje cells, stimulation of AMPA receptors, but not metabotropic glutamate receptors, triggered translocation of cPLA₂ to the somatic and dendritic Golgi compartments. This translocation required Ca²⁺ influx through P-type Ca²⁺ channels. AMPA plus PMA, a chemical method for inducing LTD, released arachidonic acid via phosphorylation of cPLA₂. AMPA plus PMA induced a decrease in surface GluR2 for more than 2 hours. Interestingly, this reduction was occluded by a cPLA₂-specific inhibitor. Furthermore, PMA plus arachidonic acid caused the prolonged internalization of GluR2 without activating AMPA receptors. These results suggest that cPLA₂ regulates the persistent decrease in the expression of AMPA receptors, underscoring the role of cPLA₂ in cerebellar LTD.

Key words: cPLA₂, Purkinje neuron, LTD, Translocation, AMPA receptor, Ca²⁺

	Introduction

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Long-term depression (LTD) of synaptic transmission at excitatory parallel fiber Purkinje-cell synapses in the cerebellum is induced when parallel-fiber and climbing-fiber inputs to a Purkinje cell are repeatedly coactivated, which contributes to certain forms of motor learning and coordination (Derkach et al., 2007; Ito, 2001; Steinberg et al., 2006; Tanaka et al., 2007). The induction of LTD is triggered by a large postsynaptic Ca²⁺ influx through voltage-sensitive Ca²⁺ channels and by the activation of two groups of glutamate receptors: ionotropic -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and type-I G-protein-coupled metabotropic glutamate (mGlu) receptors. Over the past two decades, some of the signal-transduction mechanisms controlling the induction of this form of synaptic plasticity have been established (Evans, 2007; Ito, 2002). Much evidence suggests that the depression of synaptic activity in parallel fiber Purkinje-cell synapses is primarily expressed through the activity-dependent removal of AMPA receptors containing the subunit GluR2 from the postsynaptic membrane of Purkinje-cell dendrites (Derkach et al., 2007; Evans, 2007; Matsuda et al., 2000; Xia et al., 2000). AMPA receptors form hetero-oligomeric complexes composed of the subunits GluR1-GluR4, and GluR2-GluR3 receptors are predominant in Purkinje cells. A number of signaling pathways converge on AMPA-receptor trafficking to regulate the stabilization of AMPA-receptor clusters at synapses and determine whether there is a net increase or decrease of synaptic AMPA receptors. A large increase in dendritic Ca²⁺ due to the activation of climbing fibers, combined with production of inositol-(1,4,5)-trisphosphate [Ins(1,4,5)P₃] and diacylglycerol downstream of the type-I mGlu receptor, activates protein kinase C (PKC). The removal of AMPA receptors during LTD correlates with phosphorylation of GluR2 at Ser880 in the C-terminus by PKC (Chung et al., 2003; Leitges et al., 2004). Unphosphorylated GluR2 on the plasma membrane preferentially interacts with the PDZ-domain-containing protein GRIP1 (glutamate receptor-interacting protein 1); this interaction favors the presence of AMPA receptors at synapses. After phosphorylation by PKC, GluR2 dissociates from GRIP1 and binds PICK1 (protein interacting with C kinase 1), and this leads to a decrease in the number of synaptic AMPA receptors, via clathrin-mediated endocytosis (Matsuda et al., 2000; Wang and Linden, 2000).

Although many other signaling molecules, such as mitogen-activated protein kinase (MAPK) (Endo and Launey, 2003a; Endo and Launey, 2003b; Kawasaki et al., 1999; Tatsukawa et al., 2006), Ca²⁺/calmodulin-dependent protein kinase (CaMK)II and CaMKIV (Ahn et al., 1999; Hansel et al., 2006), and nitric oxide (NO) synthase (Boxall and Garthwaite, 1996), are involved in the induction of cerebellar LTD, phospholipase A₂ (PLA₂) has obtained particular attention because there is substantial evidence that its product, arachidonic acid, serves as an intracellular messenger that modulates synaptic transmission (Kuroda et al., 2001; Linden, 1995; Massicotte, 2000; Reynolds and Hartell, 2001). First, arachidonic acid is produced in a variety of neuronal cell preparations in response to the activation of glutamate receptors under certain conditions (Stella et al., 1995), and the application of arachidonic acid to cerebellar slices depresses the synaptic current at parallel fiber Purkinje-cell synapses (Kovalchuk et al., 1994). Second, inhibitors of PLA₂ converts LTD to short-term depression, and this effect is compensated by exogenously applied arachidonic acid (Linden, 1995; Reynolds and Hartell, 2001). Third, arachidonic acid or unsaturated fatty acids can produce a large, synergistic activation of PKC and consequent phosphorylation of its substrate proteins when present together with diacylglycerol and Ca²⁺ (Lopez-Nicolas et al., 2006; McPhail et al., 1984; O'Flaherty et al., 2001). In addition, roles for Ca²⁺-activated PLA₂ and/or arachidonic acid in cerebellar LTD were assumed in a computational simulation based on a positive-feedback signal-transduction cycle that includes Ca²⁺, PKC, Raf, MAPK/ERK kinase (MEK), extracellular signal-regulated kinase (ERK), Ca²⁺-dependent PLA₂ and arachidonic acid (Kuroda et al., 2001; Tanaka et al., 2007). This model can reproduce the properties of Ca²⁺-triggered LTD through stable phosphorylation of GluR2 by sustained activation of PKC.

Because the production of free arachidonic acid from membrane phospholipids is tightly controlled by PLA₂ activity, elucidating the function and regulation of PLA₂ that is involved in the mobilization of arachidonic acid in Purkinje cells is important to better understand the molecular mechanism underlying LTD. The PLA₂ family comprises more than 20 isozymes and is classified into three main groups: cytosolic Ca²⁺-dependent PLA₂ (cPLA₂), Ca²⁺-independent PLA₂ and secretory PLA₂. Among these PLA₂ proteins, group IVA PLA₂ [also known as cytosolic phospholipase A₂ (cPLA₂)] is abundant in Purkinje neurons (Kishimoto et al., 1999; Kuroda et al., 2001; Shirai and Ito, 2004). This enzyme preferentially catalyzes the hydrolysis of the sn-2 position of glycerophospholipids containing arachidonic acid, and its activation is regulated by an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and phosphorylation at serine residues (Ghosh et al., 2006; Kudo, 2004; Leslie, 1997). In many non-neuronal cells, the binding of Ca²⁺ to an N-terminal C2 domain induces translocation of cPLA₂ from the cytosol to the perinuclear region [containing the Golgi complex, endoplasmic reticulum (ER) and nuclear membranes] so that it can access glycerophospholipid substrates (Evans et al., 2001; Hirabayashi et al., 1999). Serine phosphorylation is mediated by ERK, CaMKII, and MAPK-interacting kinase Mnk1 (Hefner et al., 2000; Kramer et al., 1996; Pavicevic et al., 2008; Sano et al., 2001). Although cPLA₂ has been shown to play physiological and pathophysiological roles in many tissues and cell types (Clark et al., 1995; Hirabayashi et al., 2004; Kita et al., 2006), the function of this enzyme in cerebellar plasticity has not been fully addressed. Here, we used live imaging and immunocytochemistry to monitor the translocation and phosphorylation of cPLA₂, mobilization of Ca²⁺ and surface expression of AMPA receptors in cultured cerebellar Purkinje cells. Our results suggest that dynamic translocation of cPLA₂ to the somatic and dendritic Golgi compartments in Purkinje neurons is controlled by AMPA-receptor-mediated Ca²⁺ influx through voltage-gated Ca²⁺ channels, and that full activation of this enzyme and consequent liberation of arachidonic acid are required for the prolonged decrease in the synaptic-surface expression of AMPA receptors, which might underscore the key regulatory role of cPLA₂ in cerebellar LTD.

	Results

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Glutamate stimulates cPLA₂ translocation to the somatic and dendritic Golgi compartments in Purkinje cells
We first examined the distribution of endogenous cPLA₂ in cultured mouse cerebellar neurons by immunocytochemistry. Although Purkinje cells could be readily identified by their unique morphology with prominent dendritic trees, and large and round soma, their identity was confirmed by immunofluorescent co-staining for calbindin-D28K (calbindin-D), a marker of Purkinje cells (Celio, 1990). Endogenous cPLA₂ was present uniformly in both the soma and dendrites of calbindin-D-positive Purkinje cells (Fig. 1), whereas significant immunoreactivity was only occasionally observed in the long extended axons. Immunostaining for cPLA₂ was rather weak in other cell types. The predominant distribution of cPLA₂ in the soma and dendrites in Purkinje cells is consistent with a recent study using rat cerebellar slices (Shirai and Ito, 2004).

View larger version (37K): [in this window] [in a new window]   Fig. 1. Endogenous expression of cPLA2 in Purkinje cells. Immunofluorescent detection of the Purkinje marker calbindin D (A) and endogenous cPLA2 (B). The merged image (C) shows colocalization of the two signals (yellow). The immunostaining for cPLA2 in surrounding cells was rather weak. Scale bar: 20 µm.

To visualize the subcellular localization of cPLA₂ in live Purkinje cells in response to glutamate, green-fluorescent-protein-tagged cPLA₂ (GFP-cPLA₂) was expressed in cultured cerebellar neurons by use of the Sindbis-virus-based expression system. Sindbis viral vectors were used because of their preference for neurons over glial cells and high expression levels of exogenous recombinant genes (Ehrengruber et al., 1999). In unstimulated Purkinje cells, GFP-cPLA₂ was present uniformly in the soma and dendrites (Fig. 2A), which was similar to the distribution of endogenous cPLA₂. The puff application of 30 µM glutamate to Purkinje cells induced a rapid redistribution of GFP-cPLA₂ to a perinuclear convoluted structure in the soma and to discrete intracellular regions of dendritic shafts (Fig. 2A). The sites of GFP-cPLA₂ translocation in dendrites could be clearly visualized in the ratio image showing relative fluorescence intensity [F/F₀, i.e. background-corrected increases in fluorescence (F) divided by the pre-stimulus fluorescence (F₀)] (Fig. 2B; supplementary material Movie 1). The time course of F/F₀ in the most distal spiny dendrites exhibited a peak within a few seconds after the addition of glutamate, followed by a rapid decline (Fig. 2B,C). A concomitant transient decrease in fluorescence in the adjacent distal spine suggests the relocation of cPLA₂ from spines to dendrites. The values of F/F₀ at the dendritic branch points and at the target sites in the proximal dendrites and soma rose and fell with slower kinetics, depending on the distance from the soma (Fig. 2C). When cPLA₂ was fused with monomeric red fluorescent protein (RFP), the redistribution of RFP-cPLA₂ in response to glutamate was essentially the same as that of GFP-cPLA₂ (Fig. 3). In contrast to these chimeric proteins, unconjugated GFP, RFP and RFP from Discosoma sp. (DsRed) were present homogenously in the soma and dendrites of Purkinje cells, and their distribution was not affected by the application of glutamate (Fig. 2A; and data not shown), indicating specific targeting of cPLA₂ in response to glutamate.

View larger version (66K): [in this window] [in a new window]   Fig. 2. Translocation of cPLA2 in living Purkinje cells in response to glutamate. (A) An example of a Purkinje cell co-expressing GFP-cPLA2 and DsRed. GFP-cPLA2 showed a uniform cytosolic distribution in unstimulated cells (upper panels, t=0 seconds), whereas a marked translocation of GFP-cPLA2 to restricted sites was observed after stimulation with 30 µM glutamate (Glu, lower panels, t=20 seconds). DsRed was expressed to visualize the morphology of Purkinje cells simultaneously (inserts). Note that all z-series sections at 2-µm intervals were combined in two-dimensional xy images. Scale bar, 20 µm. Boxed regions corresponding to the dendrites and soma in the merged images were magnified with orthogonal sections viewing axial directions (zx and zy). Scale bars, 5 µm. (B) Time-lapse images of GFP-cPLA2 fluorescence following the local application of a 30 µM glutamate solution (t=1-5 seconds, indicated by the horizontal bar). Relative fluorescence intensity (F/F0) is displayed according to a pseudocolor scale. Scale bar: 20 µm. (C) Traces represent the time course of F/F0 measured in the indicated areas in B. Five small rectangles delimit the distal spiny dendrite (1), the distal spine (2), the dendritic branch point (3), the proximal main dendrite (4) and the soma (5). The peak time of F/F0 depends on the distance from the soma. Note that fluorescence is reduced gradually by bleaching.

View larger version (49K): [in this window] [in a new window]   Fig. 3. Glutamate-induced translocation of cPLA2 to the somatic and dendritic Golgi compartments. (A) A whole Purkinje cell co-expressing RFP-cPLA2 and GFP-GalT (a marker of the Golgi complex). Scale bar: 20 µm. Boxed areas are magnified in B. (B) The target sites of RFP-cPLA2 in response to 30 µM glutamate identified by subtraction analysis (left, the fluorescence image after glutamate addition was subtracted by the image before addition) colocalized with the Golgi marker GFP-GalT (middle) in the dendrites (upper panels) and soma (lower panels). Colocalization is apparent as a yellow in the merged image (right). Scale bars: 5 µm.

To specify the site of cPLA₂ translocation after stimulation with glutamate, we compared the localization of cPLA₂ to that of β-1,4-galactosyltransferase (GalT), a well-characterized resident Golgi enzyme, tagged with GFP (GFP-GalT). The convoluted and discrete distribution of RFP-cPLA₂ in the cell soma and dendrites, respectively, after glutamate application showed a marked colocalization with GFP-GalT (Fig. 3B). By contrast, no significant colocalization of GFP-cPLA₂ and RFP-calreticulin, a marker for ER, was observed in either the soma or dendrites of Purkinje cells treated with glutamate (data not shown). These data indicate that glutamate stimulates cPLA₂ translocation from the cytosol to the somatic and dendritic Golgi compartments in Purkinje cells.

AMPA-receptor activation and Ca²⁺ influx through P-type Ca²⁺ channels regulate cPLA₂ translocation
Antagonists and agonists for glutamate receptors were used to determine which class of glutamate receptor is responsible for cPLA₂ translocation. When glutamate-receptor antagonists were examined, the glutamate-induced translocation of GFP-cPLA₂ to the dendritic and somatic Golgi was found to be completely inhibited in the presence of 30 µM 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX), a selective AMPA-receptor blocker (Fig. 4A,B), but not by treatment with 1 mM (RS)--methyl-4-carboxyphenylglycine (MCPG), a non-selective antagonist for mGlu receptors (Fig. 4C). Moreover, when AMPA receptors, NMDA receptors or mGlu receptors were selectively stimulated with a local application of AMPA, NMDA or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), respectively, only AMPA induced the Golgi-based distribution of GFP-cPLA₂ (Fig. 4D,E; and data not shown). These results indicate that activation of AMPA receptors is not only necessary but also sufficient for cPLA₂ translocation. A comparison of peak values for F/F₀ in dendrites in response to the application of glutamate-receptor agonists and antagonists confirmed that the translocation of cPLA₂ can be largely attributed to the activation of AMPA receptors (Fig. 4I). The translocation and activation of cPLA₂ are exquisitely regulated by the amount and duration of increase in [Ca²⁺]_i in many cell types (Clark et al., 1995; Hirabayashi et al., 1999; Hirabayashi et al., 2004; Leslie, 2004). Because both AMPA receptors and mGlu receptors mediate the mobilization of [Ca²⁺]_i in Purkinje cells, we investigated how cPLA₂ translocation is synchronized with the agonist-induced responses. Purkinje cells expressing RFP-cPLA₂ were loaded with Oregon green 488-BAPTA-1/AM for simultaneous imaging of the distribution of cPLA₂ and the change in [Ca²⁺]_i. When the same cells were treated with 30 µM AMPA, a drastic increase in both [Ca²⁺]_i and RFP-cPLA₂ translocation occurred (Fig. 5A). This detailed simultaneous analysis revealed that the increase in [Ca²⁺]_i that was induced by activation of AMPA receptors was mirrored by the translocation of cPLA₂ (Fig. 5A; supplementary material Movie 2). The rise in [Ca²⁺]_i in dendrites slightly preceded that in soma. Similarly, the AMPA-induced translocation of cPLA₂ in dendrites was also followed by a subtle delay in translocation in soma. By contrast, stimulation of mGlu receptors with 300 µM ACPD caused a small increase in [Ca²⁺]_i in both the dendrites and soma without remarkable changes in the subcellular localization of RFP-cPLA₂ (Fig. 5B). The temporal correlation between the increase in Ca²⁺ and the translocation of cPLA₂ suggest that cPLA₂ redistribution in Purkinje cells is regulated by the mobilization of Ca²⁺ evoked by the activation of AMPA receptors (supplementary material Movie 2).

View larger version (30K): [in this window] [in a new window]   Fig. 4. Regulation of cPLA2 translocation by AMPA receptors via P-type Ca2+ channels. (A-H) Time course of F/F0 of GFP-cPLA2 in the dendritic (solid lines) and somatic (broken lines) Golgi structures in Purkinje cells stimulated with a transient application of glutamate-receptor agonists (A-C, 100 µM glutamate; D, 1 mM ACPD; E-G, 30 µM AMPA) in the absence or presence of glutamate-receptor antagonists (B, 30 µM CNQX; C, 1 mM MCPG) and KCl (H, 50 mM). Agonists and KCl were administered by a local application near the Purkinje cells for the indicated period (horizontal solid bar). Vehicle (A), antagonists (B,C), 5 mM EGTA (F) and 200 nM -agatoxin IVA (AgaIVA, G) were bath-applied 5 minutes prior to the addition of agonists. (I) Peak values of F/F0 in the dendrites after stimulation. The values are means ± s.e.m. (n=7-17). ***P<0.001.

View larger version (91K): [in this window] [in a new window]   Fig. 5. Simultaneous observation of RFP-cPLA2 translocation and the Ca2+ response. (A,B) Time-lapse images of RFP-cPLA2 (upper panels) and Oregon green 488 BAPTA-1 (OGB-1, lower panels) after agonist stimulation. The same Purkinje cell was stimulated with 30 µM AMPA (A) or 300 µM ACPD (B). The agonists were added at t=0 seconds. Scale bar: 20 µm.

The requirement for an influx of Ca²⁺ in triggering cPLA₂ translocation was tested by chelating extracellular Ca²⁺ in the bathing medium and in the presence of a Ca²⁺-channel blocker. Application of 4 mM EGTA abolished the translocation of GFP-cPLA₂ induced by AMPA (Fig. 4F). Because Purkinje cells consistently express high levels of the AMPA-receptor GluR2 subunits that have low Ca²⁺ permeability (Tempia et al., 1996), the pathway for AMPA-triggered Ca²⁺ influx is mainly through the P-type voltage-dependent Ca²⁺ channels that open in response to the depolarization that results from the activation of AMPA receptors (Gruol et al., 1996). Indeed, the redistribution of GFP-cPLA₂ that was elicited by application of AMPA was completely suppressed in the presence of 200 nM -agatoxin IVA, a P-type voltage-gated Ca²⁺-channel blocker (Fig. 4G). These observations indicate that the influx of Ca²⁺ from the extracellular space through the P-type voltage-gated Ca²⁺ channels is required for the AMPA-stimulated translocation of cPLA₂. Furthermore, depolarization of Purkinje cells with high K⁺ levels (50 mM KCl) induced the translocation of GFP-cPLA₂ (Fig. 4H), suggesting that a large increase in [Ca²⁺]_i, via depolarization-evoked Ca²⁺ entry, promotes cPLA₂ translocation in Purkinje cells.

Phosphorylation by PMA leads to full activation of cPLA₂
In addition to the Ca²⁺-induced translocation of cPLA₂ from the cytosol to organelle membranes, phosphorylation at Ser residues is a key event for the optimal activation of this enzyme (Hirabayashi et al., 2004; Leslie, 2004). The phosphorylation of cPLA₂ in Purkinje cells was examined by staining with an antibody against Ser505-phosphorylated cPLA₂. The application of glutamate, AMPA, ACPD, or AMPA plus ACPD had no measurable effect in dendrites (Fig. 6; and data not shown), suggesting that the simple stimulation of glutamate receptors is not sufficient to fully activate cPLA₂. Because the simultaneous application of AMPA and 4β-phorbol 12-myristate 13-acetate (PMA) causes LTD of synaptic transmission in Purkinje cells (Ito, 2001; Matsuda et al., 2000; Xia et al., 2000), the effect of PMA on cPLA₂ phosphorylation was investigated. Treatment with 200 nM of PMA alone or with AMPA induced marked phosphorylation of cPLA₂ in proximal dendrites of Purkinje cells (Fig. 6). This phosphorylation was maintained for at least 2 hours following the PMA treatment (PMA, 502.4±84.1%, n=10; PMA + AMPA, 407.9±63.3%, n=8; supplementary material Fig. S1). The phosphorylation of cPLA₂ at Ser505 by PMA plus AMPA was completely suppressed in the presence of 10 µM U0126, a MEK inhibitor. This suggests that the MEK-ERK pathway is involved in the PMA-induced phosphorylation of cPLA₂ in Purkinje cells. Treatment with PMA alone did not cause cPLA₂ translocation or a change in Ca²⁺ levels in Purkinje cells (data not shown), as previously reported for other cell types (Shimizu et al., 2004).

View larger version (39K): [in this window] [in a new window]   Fig. 6. Phosphorylation of cPLA2 at Ser505. (A) Immunocytochemistry with antibody to cPLA2 phosphorylated at Ser505 and to calbindin D. Treatment with 200 nM PMA for 20 minutes triggered marked phosphorylation of cPLA2, whereas stimulation with glutamate-receptor agonists alone (100 µM glutamate or 30 µM AMPA for 3 minutes) had little effect. Pre-treatment with 10 µM U0126 for 30 minutes completely blocked the phosphorylation of cPLA2 that was induced by the combination of PMA and AMPA. Scale bar: 10 µm. (B) Quantification of experiments. The average fluorescence intensity of phosphorylated cPLA2 in proximal dendrites after treatment with glutamate-receptor agonists and/or PMA was calculated. The values are means ± s.e.m. (n=13-30 for each condition). ***P<0.001.

We then examined the impact of cPLA₂ phosphorylation on the enzymatic activity of cPLA₂. In cerebellar cells that were prelabeled by incubation with [³H]arachidonic acid, co-stimulation with AMPA and PMA produced a significant increase in radioactivity in culture supernatants, taking only a small portion of Purkinje cells in the cultures into consideration (Fig. 7). This elevation was abolished by pre-treatment of the cells with 10 µM pyrrophenone, a potent and selective inhibitor of cPLA₂ (Fig. 7). These results suggest that endogenous cPLA₂ is functional and activated to release arachidonic acid by the combination of AMPA and PMA. In contrast to the stimulatory effect of AMPA and PMA, neither AMPA, ACPD nor PMA alone had significant effects on arachidonic-acid liberation, indicating that full activation of cPLA₂ in Purkinje cells requires its phosphorylation induced by PMA treatment in addition to its Ca²⁺-mediated translocation evoked by the stimulation of AMPA receptors.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Pyrrophenone-sensitive release of arachidonic acid induced by AMPA and PMA. Cultured cerebellar cells were labeled with [3H]arachidonic acid, preincubated with 200 nM PMA or vehicle for 20 minutes and stimulated for 3 minutes with vehicle or glutamate receptor agonists (30 µM AMPA, 1 mM ACPD) by bath application. Pre-treatment with 10 µM pyrrophenone (Pyrro) for 30 minutes completely blocked the increase in the release of arachidonic acid elicited by AMPA and PMA. The values are means ± s.e.m. (n=4-9). ***P<0.001.

cPLA₂ and arachidonic acid regulate prolonged internalization of GluR2
To explore the functional implication of cPLA₂ activation in AMPA-receptor trafficking within Purkinje cells, we assessed the distribution of AMPA receptors by immunostaining with an anti-GluR2 N-terminal antibody in non-permeabilized conditions. Cerebellar LTD is expressed primarily as a reduction of the number of synaptic AMPA receptors, which occurs via clathrin-mediated endocytosis, without a change in the properties of the receptor, such as conductance and kinetics (Leitges et al., 2004; Linden, 2001). Brief exposure of Purkinje cells to AMPA (30 µM, 1 minute) in the presence of PMA (200 nM, 20 minutes) led to a sustained reduction of surface GluR2 intensity in the dendrites for at least 2 hours after removal of the reagents (Fig. 8A,B), as reported previously (Matsuda et al., 2000). Treatment with AMPA alone for 1 minute did not change the surface expression of GluR2 (data not shown). Although pre-treatment with pyrrophenone did not affect the decrease in surface GluR2 levels that were induced by PMA and AMPA at 10 minutes after the wash, it did block the prolonged loss of the receptor from the surface at 2 hours (Fig. 8B). These findings suggest that cPLA₂ activation is necessary for the prolonged internalization of GluR2 induced by the activation of AMPA receptors and PKC, but not for the induction of the receptor endocytosis. As a corollary to these experiments, we tested the effect of arachidonic acid on GluR2 distribution. PMA alone caused a loss of surface GluR2 expression under our conditions, as reported previously (Matsuda et al., 2000), but this reduction did not continue for 2 hours after the removal of PMA (Fig. 8C,D). The application of 200 µM arachidonic acid in addition to PMA resulted in a prolonged decrease in surface GluR2 intensity at 2 hours, to a similar level caused by combined stimulation with PMA and AMPA (Fig. 8C). This suggests that a transient application of arachidonic acid could substitute for the activation of AMPA receptors to sustain the PMA-induced internalization of GluR2. Taken together, our study suggests that the activation of cPLA₂ and resultant release of arachidonic acid have a crucial role in regulating AMPA-receptor trafficking by inducing persistent loss of surface GluR2 expression for the LTD of synaptic transmission in Purkinje cells.

View larger version (44K): [in this window] [in a new window]   Fig. 8. Effect of a cPLA2 inhibitor and arachidonic acid on surface GluR2 expression in Purkinje dendrites. (A) Immunocytochemistry with polyclonal antibodies to the GluR2 N-terminus revealed the surface expression of GluR2 receptors on dendrites with deconvolution microscopy. Cerebellar cultures were stimulated with 200 nM PMA for 20 minutes and 30 µM AMPA for 1 minute in the absence or presence of 10 µM pyrrophenone, and were then washed with HBSS. At 120 minutes after the wash, the cells were processed for immunostaining. Scale bar: 5 µm. (B) Time course of the change in mean intensity of GluR2 immunoreactivity in dendrites. Data are shown as a percentage of control samples. The cPLA2 inhibitor restored the prolonged reduction of postsynaptic GluR2 expression on Purkinje dendrites that were stimulated with PMA and AMPA. The values were means ± s.e.m. (n=6-9). *P<0.05, ***P<0.001. (C,D) Purkinje cells were stimulated with 200 nM PMA for 20 minutes in the absence or presence of 200 µM arachidonic acid (AA) and then washed with HBSS. At 120 minutes after the wash, the cells were processed for immunostaining of the expression of GluR2 on the dendrites. Scale bar: 10 µm. The values are means ± s.e.m. (n=6-9). **P<0.01.

	Discussion

Top Summary Introduction Results Discussion Materials and Methods References

 
The questions addressed here were how are the translocation and activation of cPLA₂ controlled by stimuli involved in inducing cerebellar LTD, and what is the possible functional consequence of this activation during the process of cerebellar LTD? We found that the AMPA-receptor-mediated influx of Ca²⁺ (Figs 4 and 5) regulates a dynamic redistribution of cPLA₂ to the somatic and dendritic Golgi structures (Figs 2 and 3), and the concomitant phosphorylation at Ser505 by ERK (Fig. 6) leads to activation of this enzyme to liberate arachidonic acid in Purkinje cells (Fig. 7). Importantly, cPLA₂ activity is required for the sustained decrease in surface GluR2 expression induced by AMPA and PMA but is not necessary for the initial internalization of GluR2 (Fig. 8A,B). The transient application of PMA and arachidonic acid causes a persistent reduction in the amount of GluR2 at the cell surface (Fig. 8C,D), suggesting a pivotal role for cPLA₂ and arachidonic acid in AMPA-receptor trafficking.

cPLA₂ translocation to the somatic and dendritic Golgi compartments
The translocation of cPLA₂ to the dendritic Golgi structures in response to the activation of AMPA receptors (Fig. 3) strongly supports the localized production of arachidonic acid near postsynaptic compartments. Previous studies have shown that many neurons possess both somatic Golgi and discrete, discontinuous Golgi-type structures located in dendrites (Horton and Ehlers, 2003). The distribution of GFP-GalT in the dendrites in our study was in accord with the results of immunocytochemistry and live-cell confocal imaging to identify the Golgi complexes in neuronal dendrites (Horton and Ehlers, 2003). Although dendritic spines in most neurons contain organelles such as the smooth ER and stack-like structures that resemble the Golgi complex, called the spine apparatus (Job and Eberwine, 2001), no spine apparatus is present in Purkinje cells (Spacek, 1985). Indeed, GFP-cPLA₂ fluorescence in dendritic spines showed a rapid decrease upon stimulation with glutamate (Fig. 2C). Thus, the target sites for cPLA₂ in response to the stimulation of AMPA receptors are consistent with these characteristic features of Golgi structures in Purkinje neurons.

The temporal correlation between the increase in Ca²⁺ and cPLA₂ translocation after activation of AMPA receptors (Fig. 5), and the requirement for Ca²⁺ influx in triggering this translocation (Fig. 4F,G), indicate that the Ca²⁺ signal is a crucial determinant for regulating subcellular distribution of cPLA₂ in stimulated Purkinje cells. A role for Ca²⁺ binding to the C2 domain of cPLA₂ as an electrostatic switch has been proposed in previous studies (Murray and Honig, 2002). Ca²⁺ binding decreases the polarity on the surface of the C2 domain by neutralizing negative charges and promotes penetration of the hydrophobic region at one end of the domain into the membrane interior. By contrast, phosphorylation of cPLA₂ is required to increase its intrinsic enzymatic activity rather than to promote its translocation to membranes because the rate of membrane translocation is indistinguishable between wild-type cPLA₂ and the S505A mutant in stimulated non-neuronal cells (Ghosh et al., 2006; Hirabayashi and Shimizu, 2000; Schievella et al., 1995). One report, however, indicates that Ser505 phosphorylation improves the affinity of cPLA₂ for the membrane at submicromolar Ca²⁺ levels (Das et al., 2003). The promotion of cPLA₂ phosphorylation by treatment with PMA produced no detectable alternation in AMPA-induced translocation of cPLA₂ in Purkinje cells in our settings (supplementary material Fig. S2). Several other factors, such as anionic phospholipids and cytoskeletal proteins, regulate the translocation of cPLA₂ in non-neuronal cells (Cybulsky et al., 2004; Fatima et al., 2005; Ghosh et al., 2006; Hirabayashi et al., 2004; Mosior et al., 1998; Subramanian et al., 2007). The interaction between cPLA₂ and actin is especially interesting because actin polymerization is suggested to be involved in the surface expression of AMPA receptors in Purkinje cells (Tatsukawa et al., 2006).

Role of cPLA₂ in regulating surface expression of AMPA receptors
The inhibition of cPLA₂ activity shortened the duration of decrease in surface GluR2 expression that was triggered by the transient application of AMPA and PMA (Fig. 8B). This indicates that cPLA₂ activation and the resultant release of arachidonic acid regulate the recycling rather than the internalization of GluR2. The number of synaptic AMPA receptors is determined by the balance between insertion and removal of receptors at the postsynaptic site (Derkach et al., 2007; Jörntell and Hansel, 2006; Tatsukawa et al., 2006). These processes are highly dynamic and tightly regulated, probably involving exocytosis, endocytosis, and lateral diffusion in the plasma membrane. Following clathrin-mediated endocytosis, the endocytosed AMPA receptors can be sorted into early endosomal compartments and then either recycled back to the plasma membrane or recruited to the lysosome for degradation, depending on the synaptic activity (Ehlers, 2000; Martin and Henley, 2004). Thus, a likely explanation for the effect of cPLA₂ inhibition on GluR2 surface expression is that cPLA₂ activation switches AMPA receptors from the recycling pathway to the degradative pathway (Fig. 9). In fact, PLA₂ activity has been proposed to play a role in endosome trafficking and lysosome activity in non-neuronal cells (Brown et al., 2003; Burlando et al., 2002; Mayorga et al., 1993).

View larger version (32K): [in this window] [in a new window]   Fig. 9. Model for a role of cPLA2 in LTD induction. AMPA and PMA can mimic the stimuli that induce the depression of synaptic transmission in Purkinje cells. LTD of synaptic activity is primarily expressed by the reduction in the number of postsynaptic AMPA receptors containing the GluR2 subunit. Phosphorylation of GluR2 at Ser880 by PKC reduces its stabilizing interaction with GRIP and causes GluR2 to preferentially bind PICK1, leading to its subsequent internalization to early endosomes. Stimulation of AMPA receptors promotes a large Ca2+ influx through voltage-gated P-type Ca2+ channels, which in turn triggers cPLA2 translocation to the dendritic and somatic Golgi membranes. Simultaneous phosphorylation of cPLA2 at Ser505 by ERK leads to an increase in its enzymatic activity. Following the coincidence detection of the large Ca2+ signaling and activation of the PKC-MEK-ERK signaling pathway, cPLA2 is activated to produce free arachidonic acid. This switches AMPA receptors from the recycling pathway to the degradative pathway by regulating endosome trafficking or lysosomal activity and ensures the persistent decrease in surface expression of AMPA receptors. Regulatory molecules that provide a direct link between arachidonic acid and the sorting of internalized AMPA receptors remain to be identified. AA, arachidonic acid; DAG, diacylglycerol; IP3, Ins(1,4,5)P3.

Inhibition of the phosphorylation of cPLA₂ at Ser505 by the MEK inhibitor U0126 indicates the presence of the PKC-ERK-cPLA₂ signaling cascade in Purkinje cells after stimulation with AMPA and PMA (Fig. 9). Previous studies have proposed a computational model based on a positive-feedback signal-transduction cycle that includes PKC, Raf, MEK, ERK, PLA₂ and arachidonic acid, and that this cycle is capable of reproducing the properties of Ca²⁺-triggered LTD through stable phosphorylation of AMPA receptors by sustained activation of PKC (Bhalla and Iyengar, 1999; Kuroda et al., 2001; Tanaka et al., 2007). In this model, the initial activation of PKC due to the influx of Ca²⁺ and phospholipase-Cβ-mediated production of diacylglycerol results in activation of the Raf-MEK-ERK pathway. ERK phosphorylates and activates cPLA₂, and then the arachidonic acid produced by cPLA₂ acts synergistically with diacylglycerol to reactivate PKC, establishing a positive-feedback loop. When the feedback loop is active, sustained activation of ERK and PKC can occur even after the extracellular signal is withdrawn. Regarding the isoform of PKC that is responsible for LTD, PKC, β and are all highly expressed in Purkinje cells, but strong evidence exists in favor of PKC, which, when knocked out in mice or knocked down in cultured Purkinje cells by RNA interference, abolishes LTD, which can only be rescued by PKC and not by other isoforms (Leitges et al., 2004).

Although kinetic simulation supports this positive-feedback hypothesis and most of our data are not inconsistent with this predictive model, experimental data from some recent studies do not support the model. For example, the conjunctional application of glutamate and depolarization pulses that induce LTD in cultured Purkinje cells was found to trigger a transient translocation of PKC to the plasma membrane (Tsuruno and Hirano, 2007). This suggests that persistent activation of PKC is not necessary for the expression of LTD. In addition, the MEK inhibitor PD98059 suppressed the declustering of GluR2-GluR3 that was induced by PMA or the co-application of high K⁺ and glutamate, without significant inhibition of phosphorylation of GluR2 at Ser880 (Endo and Launey, 2003a), implying that GluR2 phosphorylation is not necessarily required for the prolonged internalization of AMPA receptors. Therefore, activation of PKC and subsequent phosphorylation of GluR2 at Ser880 are both crucial for the initial depression of synaptic efficacy, but their persistence might not be necessary for the maintenance of LTD. In our proposed model, by controlling the degree of AMPA-receptor recycling and/or by targeting internalized AMPA receptors for lysosomal degradation, the activation of cPLA₂ and the release of arachidonic acid enable a sustained reduction in synaptic AMPA-receptor expression independent of or parallel to the positive-feedback loop (Fig. 9).

There are several issues to be resolved in the future. First, further research is needed to uncover the mechanism by which arachidonic acid regulates AMPA-receptor trafficking. It is also important to determine whether transformation of arachidonic acid into its metabolites (prostaglandins, leukotrienes and hydroxyeicosanoic acids) is required. For example, 12(S)-hydroperoxyeicosa-5Z,8Z, 10E,14Z-tetraenoic acid [12(S)-HPETE], a 12-lipoxygenase metabolite of arachidonic acid, mediates mGlu-receptor-dependent LTD at hippocampal CA3-CA1 synapses (Feinmark et al., 2003). Second, it is necessary to confirm the role of cPLA₂ in electrophysiology and examine the phenotype associated with cerebellar LTD in cPLA₂-deficent mice. Finally, the apparently contradictory role of cPLA₂ in synaptic plasticity remains to be explained (Ménard et al., 2005). In the mouse hippocampus, inhibition of cPLA₂ by caveolin-1-related peptide decreases the binding of AMPA to AMPA receptors containing GluR2, and might interfere with long-term potentiation in the mouse hippocampus (Gaudreault et al., 2004).

In summary, we have characterized the subcellular targeting and activation of cPLA₂ in Purkinje cells in response to stimuli that induce cerebellar LTD. The former is achieved by the activation of AMPA receptors that generate a large Ca²⁺ signal. The target sites of cPLA₂ are the dendritic and somatic Golgi structures. However, this targeting is insufficient to stimulate enzymatic activity. Additional phosphorylation at Ser505 is required to liberate arachidonic acid from membrane phospholipids. A persistent decrease in the number of synaptic surface GluR2 AMPA receptors requires the activity of this enzyme. The potent ability of arachidonic acid to modulate GluR2 trafficking through selective sorting of internalized GluR2 between recycling and degradative pathways might provide a basis for ensuring the induction of cerebellar LTD.

	Materials and Methods

Top Summary Introduction Results Discussion Materials and Methods References

 
Cerebellar cultures
Primary cerebellar cell cultures were prepared from neonatal ICR mice and Wistar rats (P0), as previously described (Okubo et al., 2001) with minor modifications. Briefly, the cerebellum was treated with the cell-dispersion solution (Sumitomo Bakelite, Japan), and the dispersed cells were plated at a density of 2.0x10⁵ cells/cm² on glass-bottom 35-mm dishes (=12 mm, Iwaki, Japan) coated with 0.01% poly-D-lysine (Sigma, St Louis, MO) and grown for 24 hours at 37°C in neuronal culture medium (Sumilon Nerve Culture System, Sumitomo Bakelite) containing 10 mM HEPES, pH 7.4, under a humidified atmosphere containing 5% CO₂. Then, the medium was changed to Neurobasal A medium (Invitrogen, Carlsbad, CA) supplemented with B-27 (Invitrogen), 2 mM L-glutamate and 10 mM HEPES, pH 7.4. Half of the medium was replaced every 5 days. Cells cultured for 3-4 weeks were used for the experiments. Animals were treated in accordance with the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society and the Experimental Animal Committee at the authors' institution approved the experiments.

Immunocytochemistry
For staining of endogenous cPLA₂, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 20 minutes and blocked with 10% normal goat serum (Sigma). The polyclonal anti-cPLA₂ (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100 dilution) and monoclonal anti-calbindin-D (Sigma; 1:1000) antibodies were revealed using Alexa-Fluor-488- and Alexa-Fluor-546-labeled secondary antibodies (Invitrogen), and neurons were imaged using an LSM510 Laser Scanning Microscope (Carl Zeiss, Germany). For phosphorylated cPLA₂, cells were fixed with 4% paraformaldehyde in Tris-buffered saline (TBS), pH 7.4, for 10 minutes at room temperature and treated with methanol for 10 minutes at –20°C. Then, the preparations were blocked with 10% normal goat serum (DAKO, Germany) with 1% BSA in TBS. For the staining of GluR2 on the cell surface, rat cerebellar cultures were prepared because the anti-GluR2 antibody (a gift from Hirokazu Hirai, Gunma University, Japan) reacts with GluR2 from rats much greater than it does with that from mice. Although the immunoreactivity of mouse GluR2 was very weak, similar trends were also observed in mouse Purkinje cells. Cells were fixed with 4% paraformaldehyde in PBS, and the preparations were blocked with 3% BSA without the permeabilization step. Antibodies to phospho-Ser505-cPLA₂ (Cell Signaling, Beverly, MA, 1:200), calbindin D (1:1000) and GluR2 (1:200) were used, followed by Alexa-Fluor-488- and Alexa-Fluor-546-conjugated secondary antibodies (Molecular Probes). Fluorescent images were taken using a Fluoview 500 laser-scanning microscope IX60 equipped with a 60x water-immersion objective (NA=1.2), 488- and 543-nm laser lines for excitation, 515-540-nm band-pass and 560-nm long-pass filters for emission, or ZEISS Axiovert S100 with a 40x objective (NA=0.17).

Recombinant Sindbis viral vectors
The cDNA fragments encoding full-length human cPLA₂ were inserted into the expression vector pEGFP-C1 (Clontech, Palo Alto, CA) to construct GFP-cPLA₂ as previously described (Hirabayashi et al., 1999). The region coding for GFP was replaced by monomeric RFP (a gift from Roger Tsien, University of California, San Diego, CA) to obtain RFP-cPLA₂. GFP-GalT, a Golgi marker encoding the N-terminal 82 residues of human GalT, was constructed by modification of CFP-Golgi (Clontech). The cDNA fragments encoding GFP, DsRed-Express (Clontech), GFP-cPLA₂, RFP-cPLA₂, GFP-GalT and RFP-calreticulin, a marker for ER, were subcloned into the vector pSinRep5 (Invitrogen). All constructs were confirmed by sequencing. These vectors were linearized and subsequently transcribed in vitro into capped mRNA with the mMessage mMachine SP6 kit (Ambion, Austin, TX) according to the manufacturer's instructions. The RNA transcripts and the helper RNA from the DH(26S) cDNA template (Invitrogen), which encodes the genes for structural proteins of the Sindbis virus, were electroporated into baby hamster kidney cells with a Gene Pulser II (Bio-Rad, Hercules, CA) in the presence of 1 µM RNasin (Promega, Madison, WI). After 36-48 hours of incubation in -modified Eagle's medium supplemented with 5% fetal bovine serum, the culture supernatant containing the infectious particles of replication-incompetent pseudovirus was harvested. The viral particles were concentrated by centrifugation at 10,000g for 1 hour and preserved at –80°C until used.

Live-cell confocal microscopy and Ca²⁺ imaging
The infectious pseudovirions were diluted into the culture medium so that approximately 40-80% of cultured Purkinje cells were infected. The cerebellar cultures were incubated in this condition for 16-24 hours at 37°C in the presence of 1 µM tetrodotoxin (Wako, Osaka, Japan) to block spontaneous action potentials. Sindbis virus did not cause any apparent toxicity in cerebellar neurons for at least 2 days. The cultures were washed three times with Hank's balanced salt solution (HBSS) containing 10 mM HEPES, pH 7.4, and incubated in the same buffer for 30 minutes at 37°C in the presence of tetrodotoxin. Confocal microscopy was performed on a Fluoview 500 laser-scanning microscope IX60 equipped with a 60x water-immersion objective (NA=1.2), 488- and 543-nm laser lines for excitation, 515-540-nm band-pass and 560-nm long-pass filters for emission, and an incubation chamber to keep the temperature constant at 37°C (Olympus, Tokyo, Japan). Time-lapse sequences were recorded with FluoView Tiempo, and fluorescence intensity was quantified using FluoView version 4.2 software. For the imaging of [Ca²⁺]_i, the cells were preloaded for 45 minutes at 37°C with 6 µM Oregon Green 488 BAPTA-1 AM (a green-emitting Ca²⁺-sensitive fluorescent probe, Invitrogen) in HBSS containing 0.1% BSA and 10 mM HEPES, pH 7.4.

Release of arachidonic acid
The cerebellar cells were seeded onto 48-well plates at a density of 3x10⁵ cells/well in the growth medium. After a 3-week culture, the cells were labeled by overnight incubation with Neurobasal medium containing 0.05 µCi/well of [³H]arachidonic acid (Amersham, Buckinghamshire, UK), B-27 supplement, 0.1% BSA and 10 mM HEPES, pH 7.4. The cells were washed three times with HBSS containing 0.1% BSA and 10 mM HEPES, pH 7.4, and stimulated with the indicated reagents at 37°C in the same buffer. The radioactivity of the supernatant and cell lysates (in 1% Triton X-100) was measured by liquid scintillation counting and the amount of radioactivity released into the supernatant was expressed as a percentage of the total amount incorporated (Hirabayashi et al., 1999).

Reagents
Glutamate and PMA were obtained from Sigma (St Louis, MO). AMPA, CNQX, ACPD and MCPG were from Research Biochemical International (Natick, MA). -agatoxin IVA was from PEPTIDE Institute (Osaka, Japan). Pyrrophenone was kindly provided by Shionogi Pharma (Osaka, Japan). U0126 was obtained from Calbiochem (San Diego, CA). To prepare stock solutions, reagents other than CNQX were dissolved in distilled water, and CNQX was dissolved in dimethylsulfoxide. Stock solutions of reagents were diluted with the buffer and then adjusted to pH 7.4. Administration of glutamate-receptor agonists was performed for 10-15 seconds by gravity flow through a micropipette positioned beside the cells, which enabled a local application of the reagents to the targeted cells. The administration rate was about 1 µl/second.

Data analysis
Fluorescence data were processed with ImageJ 1.38 (National Institutes of Health) and expressed as F/F₀, i.e. as background-corrected increases in fluorescence (F) divided by the pre-stimulus fluorescence (F₀). The highest value of F/F₀ in each trace was defined as the peak value. Values are means ± s.e.m. from more than three independent experiments. In the case of multiple comparisons, the significance of differences was determined using a one-way analysis of variance followed by the Bonferroni test. For pairwise comparisons, the Student's two-tailed t-test was used. P values <0.05 were considered significant.

	Acknowledgments

 
We thank Sho Kakizawa (Graduate School of Medicine, The University of Tokyo) and Kenzo Hirose (Nagoya University Graduate School of Medicine) and Sinji Matsuda (Keio University School of Medicine) for their technical suggestions. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

	Footnotes

 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/18/3015/DC1

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