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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

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.

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.

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.

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.

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.

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.

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.

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