Doi:10.1016/j.mcn.2005.11.01

Mol. Cell. Neurosci. 31 (2006) 574 – 585 NMDA receptors mediate calcium-dependent, bidirectional changes indendritic PICK1 clustering K.G. Sossa, B.L. Court, and R.C. Carroll* Department of Neuroscience, Rose Kennedy Center for Mental Retardation, 1410 Pelham Parkway,Albert Einstein College of Medicine, Bronx, NY 10461, USA Received 16 August 2005; revised 7 November 2005; accepted 22 November 2005Available online 10 January 2006 AMPA receptor (AMPAR) trafficking at CNS synapses is regulated by rearrangements underlying synaptic depression ( several receptor-binding proteins. One model of AMPAR endocytosis Malenka, 2002).
entails the cotargeting of the GluR2-interacting protein PICK1 and Several studies have established a requirement for GRIP/ABP activated PKC to synapses. We demonstrate that NMDA receptor and/or PICK1 binding to GluR2 in the regulated endocytosis of (NMDAR) activation mediates bidirectional changes in surface AMPARs, which mediates LTD in the hippocampus and cerebellum AMPARs through two additional forms of PICK1 redistribution. In neurons, NMDAR activation, which induces AMPAR endocytosis, 2000). In one prominent scheme for this process, GRIP/ABP, via a increases endosomal PICK1 clustering. In contrast, stronger NMDARactivation rapidly reduces PICK1 clustering accompanied by decreases PDZ domain, anchors AMPARs at synaptic membranes ( in PICK1/GluR2 association and increases in surface AMPAR levels.
2003), while PICK1, binding to the same C-terminal site of GluR2, PICK1-siRNA similarly increases surface AMPARs and occludes the can mediate the mobilization of AMPARs. For example, PICK1 NMDAR-mediated effect, demonstrating the role of PICK1 in this interacts with activated C-protein kinase a (PKCa) and synaptically process. Bidirectional NMDAR-mediated changes in PICK1 localiza- localizes the kinase, enabling phosphorylation of GluR2-Ser880 tion are determined by the magnitude of receptor-activated dendritic (This phosphorylation decreases GRIP-GluR2 calcium signals. Our results show that PICK1 localization in dendrites association and facilitates AMPAR internalization ( is subject to multiple forms of regulation that contribute to surface 1999; Chung et al., 2000; Perez et al., 2001). A recent report further AMPAR expression, likely by modulating the numbers of AMPARs demonstrates that the direct interaction between GRIP/ABP and maintained in intracellular compartments.
D PICK1 is important for Ser880 phosphorylation and GluR2 2005 Elsevier Inc. All rights reserved.
trafficking (Although this evidence provides acompelling model for regulated AMPAR endocytosis, questionsremain as to how signaling pathways that drive receptor endocytosis in the absence of PKC activation modulate PICK1.
The role of PICK1 in AMPAR trafficking has proven more Neurotransmission at many CNS synapses is regulated by the complex than simply to enhance receptor endocytosis. Knockout dynamic trafficking of AMPA-type glutamate receptors (AMPARs) mice revealed PICK1's importance in the recycling of AMPARs to into and out of the membrane surface. The modulation of this the membrane surface in cerebellar stellate cells ( process occurs via the interaction of AMPAR subunits (i.e. GluR1 – 2005). Disruption of GRIP and PICK1 interaction interrupts the 4) and cytosolic proteins. In recent years, much attention has been recycling of AMPARs in hippocampal neurons ( given to the protein – protein interactions of GluR2-containing As GRIP/ABP may be targeted to intracellular as well as synaptic AMPARs, which have been shown to influence both surface membranes (recycling may be dependent on the receptor maintenance and activity-driven changes in synaptic PICK1-mediated disruption of internalized AMPARs stabilized by strength (In particular, investigations of the GluR2 cytoplasmic tail interacting proteins glutamate receptor interacting protein (GRIP/ABP) and PICK1 protein interacting with PICK1 itself may play a role in stabilizing receptors away from C-kinase have brought a closer understanding of the synaptic surface synaptic sites. The disruption of the interaction of PICK1and GluR2 with intracellularly introduced peptides results in therapid delivery of AMPARs to synapses ( * Corresponding author. Fax: +1 718 430 2721.
E-mail address: (R.C. Carroll).
et al., 2004). Additionally, overexpression of PICK1 causes a Available online on ScienceDirect reduction in the surface levels of GluR2 ( 1044-7431/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
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K.G. Sossa et al. / Mol. Cell. Neurosci. 31 (2006) 574 – 585 Together, these results suggest that PICK1 is retaining GluR2- The functions proposed for PICK1 in AMPAR delivery, containing AMPARs away from the synapse. It remains to be recycling, and intracellular stabilization suggest the importance determined whether the interaction of PICK1 and GluR2 can be of localizing PICK1 to appropriate compartments. The ability of modulated by the activation of cellular signaling pathways as a PICK1 to mediate the destabilization of GRIP-bound AMPARs means for elevating surface AMPARs. Interestingly, both electro- from synaptic or intracellular compartments necessitates the physiological (and imaging ( selective targeting of PICK1 to these sites. Furthermore, if PICK1 analyses have demonstrated that NMDA-type glutamate receptor mediates the stabilization of a population of intracellular AMPARs, (NMDAR) signaling can trigger the synaptic insertion of previously its clustering with these receptors must be subject to regulation. In internalized AMPARs: a population of receptors modulated by this way, mechanisms that regulate the subcellular distribution and clustering of PICK1 could locally and bidirectionally influence Henley, 2003). Thus, NMDAR activation may alter GluR2-PICK1 functional AMPAR levels.
association in order to increase as well as decrease surface AMPAR Here, we show that the extent and localization of PICK1 clustering are modulated by receptor signaling pathways. Activa- Fig. 1. NMDA receptor stimulation enhances dendritic PICK1 clustering in endosomal but not in synaptic compartments. (A) Imaged neurons immunolabeledfor PICK1 show strong somatic (inset) and punctate dendritic staining (left). NMDA treatment (50 AM, 2 min) results in increased dendritic PICK1 labeling(right). (B) Quantitation of the intensity of PICK1 labeling in soma and dendrites of NMDA treated neurons. Values are presented as a percent of control. (C)Imaged dendrites of neurons colabeled for PICK1 (red) and EEA1 (green). NMDA activation causes increased dendritic PICK1 puncta including thosecolocalized with EEA1 (arrowheads). (D) Quantitation of dendritic PICK1, and EEA1 puncta per unit length in untreated control, NMDA-treated (50 AM, 1min), and TPA treated (100 nM, 15 min) neurons. (E) Quantitation of percent of EEA1 puncta colocalized with PICK1 in control, NMDA-, and TPA-treatedneurons. (F) Dendrites immunolabeled for PICK1 (red) and synaptophysin (green) are shown for control, NMDA, and TPA treated neurons. (G) The percent ofPICK1 clusters localized at synapses was quantified for neurons that were untreated (control), NMDA-treated for 1 min and immediately fixed, NMDA-treatedfor 1 min and fixed after 15 min, and TPA-treated for 15 min. Phorbol esters induce an increase in the synaptic localization of PICK1, as reported previously,while NMDA has no effect. Scale bar: A, 5 AM; C, 3 AM. *P < 0.05, ***P < 0.001.

K.G. Sossa et al. / Mol. Cell. Neurosci. 31 (2006) 574 – 585 tion of NMDARs triggers rapid, bidirectional changes in PICK1 PICK1 similar to that induced by activation of PKCa ( clustering in dendritic endosomes, differing from the phorbol ester- 2000; Perez et al., 2001). In cultured hippocampal neurons, 2 min activated translocation of PICK1 to synaptic sites. Moderate exposure to NMDA (resulted in an increase in NMDAR signaling increases PICK1 clustering in dendritic endo- the dendritic immunolabeling of PICK1 after 15 min, both in somes. In contrast, stronger NMDAR activation decreases den- overall intensity and in number of puncta (B, D, 64 T dritic PICK1 clustering. Interestingly, the robust activation of 13% increase in intensity, control 4.9 T 0.14 PICK1 clusters/10 Am, NMDARs, which reduces PICK1/GluR2 interactions, increases NMDA 6.6 T 0.18 clusters/10 Am, n = 3, P < 0.001).
dendritic surface AMPAR expression, demonstrating the impor- PICK1 clusters were found largely colocalized with transferrin- tance of this process in modulating AMPAR trafficking. These positive as well as with EEA1-labeled early endosomes (76.1 T 3.1% differential NMDAR-mediated effects can be attributed to dendritic PICK1 vesicles colocalized with transferrin, data not shown; calcium levels activated under each condition: large calcium 1C, 43.9 T 3.5% PICK1 vesicles colocalized with EEA1). The signals drive a reduction in PICK1, while smaller calcium signals numbers of EEA1 positive clusters were unchanged following induce increases in PICK1. Our results suggest that NMDARs can NMDA treatments (However, the proportion of transferrin bidirectionally regulate the clustering of PICK1 at endosomes, and EEA1 clusters colocalized with PICK1 was enhanced by thereby modulating the retention of intracellular AMPARs and treatment with the NMDA (transferrin: control 67.3 T 3.6%, consequently affecting surface AMPARs levels.
colocalized, NMDA 82.1 T 2.3% n = 4, P < 0.05, data not shown;EEA1: control 47.7 T 2.0%, NMDA 56.1 T 2.1%, n = 3, P <0.05). In contrast, 15-min treatment with phorbol esters did not increase the numbers of dendritic PICK1 puncta (4.5 T 0.34puncta/10 Am), and decreased the extent of EEA1 vesicles NMDAR activation reduces dendritic PICK1 clustering colocalized with PICK1 (phorbol ester 39.4 T 1.3%colocalized, n = 4, P < 0.05), indicating that NMDAR and PKC We initially tested whether or not activation of NMDARs, activation has differential effects on the localization of dendritic which induces AMPAR endocytosis, causes a relocalization of Fig. 2. NMDA-induced PICK1 clustering occurs preferentially at vesicles expressing activated rab5 and does not require endocytosis. (A) PICK1 clusterstogether preferentially with activated but not inactivated rab5. Neurons transfected with activated (Q79L) or inactivated (S34N) rab5-GFP (green) were fixedafter 36 h and immunolabeled for PICK1 (red). (B) Quantitation of the percent of PICK1 clusters colocalized with either inactivated or activated rab5. (C)PICK1 is coclustered with internalized AMPARs. Live neurons were labeled with an anti-N terminal GluR2 antibody and subsequently treated with NMDA toinduce AMPAR internalization. Following fixation, PICK1 (left) and internalized AMPARs (center) were immunocytochemically detected. Merge (right)demonstrates coclustering of internalized receptors with PICK1. (D) Blockade of clathrin-mediated endocytosis by hypertonicity (350 mM sucrose duringtreatment) does not block the NMDA-induced increase in PICK1 clustering. (E) Quantitation of the effects of hypertonic extracellular media on the NMDA-mediated changes in dendritic PICK1 levels. Scale bars: A and C, 3 AM.

K.G. Sossa et al. / Mol. Cell. Neurosci. 31 (2006) 574 – 585 Fig. 3. Increased NMDAR activation stimulates rapid unclustering of dendritic PICK1. (A) Enhanced activation of NMDARs by treating with NMDA (50 AM,1 min) in reduced extracellular magnesium (0.13 mM) induces a reduction in dendritic PICK1 clustering 15 min after treatment. Representative images areshown of PICK1 immunolabeled neurons that were either untreated (left) or NMDA/low Mg2+-treated (right). Insets show soma with a brightness level toenable visualization of labeling. (B) Quantitation of the intensity of PICK1 labeling in soma and dendrites of NMDA/low Mg2+-treated neurons. Values arepresented as percent of control. (C) Time course demonstrates that NMDA/low Mg2+ treatment produces considerable changes in dendritic PICK1 within 1min, which stabilizes after 5 min. Somatic labeling shows little change. (D) Imaged dendrites of neurons colabeled for PICK1 (red) and EEA1 (green). NMDA/low Mg2+ activation causes decreased dendritic PICK1 puncta, with a lower proportion colocalized with EEA1 (arrowheads). (E) Quantitation of dendriticPICK1, and EEA1 puncta per unit length in untreated control and NMDA/low Mg2+-treated (50 AM, 1 min) neurons. (F) Quantitation of percent of EEA1puncta colocalized with PICK1 in control and NMDA/low Mg2+-treated neurons. Scale bar: A, 5 Am, **P < 0.01, *P < 0.05.
Similar to previous studies ( activation rapidly increases the dendritic clustering of PICK1 2001), we found that phorbol ester treatment (100 nM TPA, 15 including its association with early endosomes, while PKC min) resulted in an increased localization of PICK1 at synaptic activation redistributes PICK1 from endosomes to synaptic sites.
sites (, G, control 23.7 T 1.8%, TPA 45.2 T 3.1% PICK1clusters colocalized with synaptophysin, n = 5, P < 0.001). NMDA PICK1 clustering in endosomes is influenced by the activation treatment, however, had no effect on the proportion of PICK1 puncta at synapses after 15 min (, G, 18.7 T 0.6%colocalized, n = 4). Even 1 min after NMDA treatment, there was As rab5 is localized at early endosomes where PICK1 an increase in the number of dendritic PICK1 puncta (control 4.1 T accumulates, and is activated by NMDAR stimulation ( 0.11, NMDA 1 min 5.1 T 0.11 puncta/10 Am, n = 5), with no effect al., 2005), we tested whether the activation state of rab5 could on the proportion of PICK1 at synapses (24.9 T 1.9% affect the localization of PICK1. Hippocampal neurons were colocalized, n = 5). Together, these data suggest that NMDAR transfected either with an activated (GTP hydrolysis deficient,

K.G. Sossa et al. / Mol. Cell. Neurosci. 31 (2006) 574 – 585 Q79L) or an inactivated (low affinity for GTP, S34N) form of rab5(and after 36 h were immunolabeled forPICK. In transfected neurons, 61.0 T 4% of PICK1 clusters werecolocalized with the activated form of rab5 while only 33.5 T 2%of PICK1 colocalized with the inactive form (B).
Dendritic PICK1 accumulation occurs independently of AMPARendocytosis Both NMDAR and rab5 activations are known to increase AMPAR endocytosis (Furthermore, we found that NMDAR signaling induces acoclustering of internalized AMPARs with PICK1 in dendrites(We, therefore, examined whether the clustering ofPICK1 is dependent on its association with internalized AMPARs.
Hypertonic media (350 mM sucrose) block clathrin-mediatedendocytosis (and have been shown to inhibitglutamate receptor-mediated AMPAR internalization (1999). Incubating neurons in hypertonic media during NMDAtreatment did not block the increased clustering of PICK1 (2D, E, NMDA 83.6 T 21.2% increase from control, NMDA/sucrose 77.5 T 13.7%). This result indicates that the accumulationof internalized AMPARs is not necessary for NMDAR-mediatedPICK1 clustering.
Strong NMDAR activation drives the loss of clustered dendriticPICK1 We further sought to determine whether activity that might drive the insertion of AMPARs would have any effect ondendritic PICK1 levels. Traditional models of NMDAR plastic-ity suggest that while weaker activation of NMDARs triggersLTD, stronger activation of NMDARs couples to LTP or de-depression, presumably through increased calcium influx (man, 1989). We therefore enhanced the activation of NMDARs by Fig. 4. NMDA/low Mg2+-induced loss of PICK1 levels requires applying the agonist (1 min) in low magnesium ACSF (NMDA/ microtubules. (A) Labeling of PICK1 in cells treated T colchicine (10 low Mg2+, 0.13 mM Mg2+) to reduce the extracellular blockade of AM for 30 min) prior to NMDA/low Mg2+-exposure. (B) Quantitation the channel by magnesium. Following NMDA/low Mg2+ treat- of colchicine's effects on NMDA- and NMDA/low Mg2+-induced ment, we observed a marked decrease in the dendritic labeling of changes in dendritic PICK1 labeling. The NMDA/low Mg2+-meditated PICK1 (Changes were observed both in the overall reduction, but not the NMDA-dependent increase in PICK1 labeling is labeling intensity and in the number of dendritic puncta ( E, intensity: soma 0.8 T 2.6%, dendrites 48.7 T 4.8%, n = 14, puncta: control 4.6 T 0.4%, NMDA/low Mg2+ 2.5 T 0.6 puncta/10Am, n = 3, P < 0.05). The changes were rapid and near maximal 0.05). This result is consistent with a role for microtubule- within 1 min of agonist exposure (The lower PICK1 dependent vesicle trafficking in the unclustering of PICK1 in clustering reduced the extent of EEA1 positive endosomes coclustered with PICK1 (F, control 57.5 T 4.2%, NMDAlow Mg2+ 35.4 T 4.6% colocalized, n = 3, P < 0.01).
Increased NMDAR activation reduces the association of GluR2and PICK1 Regulated PICK1 loss in dendrites is microtubule-dependent The unclustering of PICK1 in hippocampal dendrites suggests The later steps of endosome processing, including sorting, are that the activation of the NMDAR results in the uncoupling of dependent on microtubules (There- PICK1 from GluR2 at endosomes. We tested this biochemically in fore, we tested whether the redistribution of PICK1 away from untreated and NMDA/low Mg2+-treated hippocampal slices.
endosomes requires microtubule-based transport. NMDAR-in- Lysates were immunoprecipitated with GluR2 antibodies, and duced PICK1 clustering was not inhibited by the pretreatment of assayed for PICK1 association by Western blot. Probing with anti- cultures with colchicine (30 min pretreatment, 10 AM; PICK1 antibodies demonstrated a significant decrease ( 25.4 T NMDA, 145.1 T 34.5%, NMDA/colchicine, 105.8 T 33.0%, n = 4).
6.9% change from untreated control, n = 4, P < 0.05) of GluR2/ However, colchicine treatment eliminated NMDA/low Mg2+- PICK1 association in NMDA/low Mg2+-treated slices ( induced loss of PICK1 (B, NMDA/low Mg2+, Immunoprecipitation with a nonspecific mouse IgG revealed no 5.5%, NMDA/low Mg2+/colchicine, 12.6 T 11.9%, n = 4, P < staining for PICK1 (

K.G. Sossa et al. / Mol. Cell. Neurosci. 31 (2006) 574 – 585 Fig. 5. NMDA/low Mg2+ induces a PICK1-dependent increase in surface GluR2-containing AMPARs. (A) NMDA/low Mg2+ treatment reduces PICK1/GluR2interaction in hippocampal slices. Lysates from treated and untreated hippocampal slices were immunoprecipitated with an anti-GluR2 or a nonspecific mouseIgG antibody. Total lysates and immunoprecipitated proteins were subjected to SDS-PAGE and probed by Western blot for PICK1 (top). Quantitation of PICK1coimmunoprecipitated with GluR2 (bottom) shows a reduction in PICK1/GluR2 association with NMDA/low Mg2+ treatment. The ratio of immunoprecipitatedPICK1 to total PICK1 levels in the lysate was determined, and is presented as a percent of control (n = 4, *P < 0.05, paired Student's t test). (B) Quantitation ofthe intensity of surface GluR2 immunocytochemical labeling demonstrates an increase in surface receptor levels following NMDA/low Mg2+ treatment.
Somatic GluR2 levels show little change while dendrites exhibit a large increase. (C) Introduction of PICK1-siRNA mediates a decrease inimmunocytochemically detected PICK1 (left panels) in hippocampal neurons compared to neurons treated with scrambled siRNA. Levels of GRIP1 (rightpanels) were unaffected. (D) PICK1 – siRNA decreases dendritic PICK1 and increases surface GluR2 levels. The graph depicts analysis of the intensities ofimmunocytochemically detected PICK1, GRIP, and surface GluR2 in PICK1-siRNA versus scrambled siRNA-treated neurons (PICK1 and GluR2, n = 6;GRIP, n = 3). (E) Immunolabeling of surface GluR2 in control (left) and NMDA/low Mg2+-treated (right) neurons. Cells were exposed to control scrambledsiRNA (top) or PICK1 – siRNA bottom. Control siRNA cells treated with NMDA/low Mg2+ show an increase in surface GluR2 labeling. PICK1-siRNA cellsexhibit higher basal surface GluR2 levels that are not further enhanced by NMDA/low Mg2+ treatment. (F) Quantitation of siRNA experiments shown in panelE. Results are presented as percent change in fluorescent intensity of GluR2 labeling from control. *P < 0.05.
Reduction of clustered PICK1 in dendrites mediates an increase in increase in surface dendritic GluR2s (58.1 T 10.5% surface AMPAR expression increase from control, n = 7, P < 0.001). GluR1 receptor levelsincreased in a similar manner although with a lesser magnitude of To test whether NMDAR-mediated reduction in PICK1 change (40.8 T 8.2% increase from control, n = 4, P < 0.05, data clustering and GluR2 binding directly influences surface AMPARs, we measured changes in surface AMPARs following The increase in surface AMPARs following NMDA/low Mg2+ strong activation of NMDARs. Immunocytochemical analysis treatment is consistent with, though not necessarily the direct result demonstrated that NMDA/low Mg2+ treatment results in an of, PICK1 redistribution. To test the role of reducing dendritic

K.G. Sossa et al. / Mol. Cell. Neurosci. 31 (2006) 574 – 585 PICK1 on surface AMPARs, we used an RNAi approach to as compared to cells treated with control-siRNA, but to a lesser knockdown the expression of PICK1. After treatment with PICK1- extent (52.0 T 20.5% change from control, n = 5, data not shown).
siRNA for 72 h, immunocytochemically detected PICK1 was To test whether this PICK1-mediated change in surface GluR2 reduced to 36 T 4% (n = 6, P = 0.01) of levels in cultures loaded is the likely mechanism behind the increase in surface AMPARs with a control scrambled-siRNA (D). Levels of the following strong NMDAR activation, we applied NMDA/low AMPAR interacting protein GRIP1, in contrast, were unaffected Mg2+ to siRNA treated cells (F). While in control- indicating the specificity of the siRNA effect (+2.8 T 6.0%, n = 4 siRNA-treated cells there was an increase in surface AMPAR ns, D). Analysis of siRNA treated neurons showed that following treatment (126.5 T 17.4%, n = 4), siRNA-treated cells the reduction of PICK1 levels was also accompanied by a showed no further increases in surface receptor levels following significant increase in surface GluR2-containing AMPARs ( NMDA/low Mg2+ treatment ( 9.1 T 12.7% siRNA-NMDA/low 5D, E, 168.4 T 37% change from control, n = 6, P < 0.05). Surface Mg2+-treated/siRNA alone, n = 4). This occlusion supports the GluR1 levels were also higher in cells treated with PICK1-siRNA conclusion that the loss of dendritic PICK1 with NMDA/low Mg2+ Fig. 6. Bidirectional receptor-induced changes in PICK1 clustering are determined by the magnitude of intracellular calcium signals. (A) NMDA/low Mg2+-and NMDA-mediated changes in PICK1 are blocked in neurons loaded with BAPTA-AM (25 AM) or treated in the absence of extracellular calcium (0 Ca2+ Prevention of intracellular calcium release with CPA (25 AM) reveals that the effect of NMDA/low Mg2+ treatment, and not that of NMDA treatment, isdependent on the release of calcium from intracellular stores. (B) Fluo-3-AM (1 AM) calcium imaging of hippocampal neurons treated with NMDA/low Mg2+ TCPA (black and red, respectively), and NMDA in regular (green). NMDA/Low Mg2+ produces a large calcium response, while lower calcium signals areevident for NMDA alone and NMDA/low Mg2+/CPA. (C) Photolytic uncaging of caged calcium generates elevations in dendritic calcium comparable to thoseelicited by activation of receptors with NMDA/low Mg2+ and NMDA. Hippocampal neurons loaded with o-nitrophenyl-EGTA-AM were stimulated with either7 (red) or 15 (black) 2 s flashes of dim UV light to elicit small or large calcium responses as measured by fluo-3-AM fluorescence. Cells loaded with Fluo-3-AM alone show no changes in calcium following stimulation with 7 (green) or 15 (blue) flashes. (D) Stimulation with 15 flashes (long) results in a markeddecrease in dendritic PICK1 levels, while the short stimulation (7 flashes) results in increased dendritic PICK1 labeling. In the absence of caged calcium, thereis little effect on PICK1 levels. (E) The magnitude of cytosolic calcium levels differentially influences surface GluR2 levels in dendrites. Neurons weresubjected to photolytic uncaging of calcium as described. Five minutes following uncaging flash, neurons were fixed and labeled for surface GluR2. Exposureto a long flash that elicits a large calcium elevation resulting in a net, albeit variable, increase in surface AMPARs (+88 T 40%, n = 6). Neurons from five of sixexperiments showed an increase in surface AMPARs. A short flash, which elicited a smaller calcium response, resulted in a decrease in surface AMPARs( 46 T 10%, n = 4 compared to unflashed controls). *P < 0.05, **P < 0.01, ***P < 0.001.
K.G. Sossa et al. / Mol. Cell. Neurosci. 31 (2006) 574 – 585 activation accounts for the observed receptor-mediated increases in of control levels, n = 9). Control flashes in the absence of loaded surface AMPARs.
calcium cage showed no net change in basal PICK1 levels (6C, D). These data demonstrate that the bidirectional regulation of Differential PICK1 redistribution is calcium-dependent dendritic PICK1 clustering is dependent on the magnitude ofcytosolic calcium signals. Consistent with a link between changes The triggering of bidirectional NMDAR-dependent plasticity has in the clustering of dendritic PICK1 and surface AMPAR been suggested to be determined by extent of calcium entry through expression, higher levels of uncaged calcium resulted in an the channel: small elevations in cytosolic calcium drive depression, increase in surface AMPAR levels in 4 of 6 experiments, whereas and large increases induce potentiation ( lower calcium levels reduced surface AMPARs (4 of 4 1999). Therefore, we investigated whether calcium signals might be responsible for the opposing changes seen in the dendritic clusteringof PICK1. Both the NMDAR-mediated increases and decreases indendritic PICK1 were blocked by preincubating cultured neurons with a cell permeable calcium chelator, BAPTA-AM (30 minpretreatment, 25 AM; NMDA, 62.7 T 21.4%, NMDA/ We report here, for the first time, that levels of clustered PICK1 10.4 T 5.8%, n = 4, P < 0.05, NMDA/low Mg2+, 63.6 T in dendrites can be directly modulated by the activation of a 10.9%, NMDA/low Mg2+/BAPTA, 2.7 T 9.6%, n = 4, P < 0.01).
neurotransmitter receptor. We find that moderate NMDAR Similarly, removal of extracellular calcium blocked the effects, stimulation increases the clustering of PICK1 in dendrites. In supporting the possibility that calcium influx through the NMDARs contrast, strong NMDAR activation drives the loss of dendritic mediates the changes (NMDA/0 Ca2+ ext 4.7 T 18%, n = 5, P < PICK1 clusters, via microtubule-dependent relocalization. This 0.05, NMDA/low Mg2+/0 Ca2+ ext 6.1 T 8.5%, n = 4, P < 0.01). We also differential effect of NMDAR activation on PICK1 clustering is investigated whether the release of calcium from intracellular stores directly dependent on the magnitude of the calcium response plays a role in PICK1 redistribution. We found that while the generated in the dendrites. As PICK1 is implicated in AMPAR preincubation of cultured neurons with cyclopiazonic acid (CPA; an trafficking, its regulation by NMDAR activation could be expected inhibitor of intracellular calcium ATPases) had no effect on to impact the expression of surface dendritic AMPARs. Consistent NMDAR-mediated increases in dendritic PICK1, this treatment with this, moderate NMDAR stimulation, which results in PICK1 blocked, and partially reversed, the NMDA/low Mg2+-mediated accumulation in dendrites, is known to decrease surface AMPAR reductions in PICK1 (NMDA, 50.2 T 25.5%, NMDA/low levels (Strong NMDAR activation, on the other Mg2+/CPA, +26.1 T 7.9%). This suggests that the calcium influx hand, couples to increases in surface dendritic AMPARs via a through NMDARs under conditions of strong receptor activation PICK1-dependent process. Collectively, our data provide evidence results in calcium-induced calcium release that contributes to the that dendritic PICK1 clustering is bidirectionally regulated and loss of PICK1 labeling in dendrites.
influences local levels of surface AMPARs, likely by modulating Since both increases and decreases in PICK1 can be mediated the numbers of receptors found in intracellular pools.
by NMDAR-induced changes in calcium, we sought to measure Previous studies showed that phorbol esters and basal PKC the calcium responses that mediate these effects. In fluo-3-AM activity induce a synaptic localization of PICK1 and decreased loaded neurons, NMDA induced a moderate increase in the surface AMPAR levels ( dendritic levels of calcium (However, NMDA applied Terashima et al., 2004). We likewise see in our system that PKC in low extracellular magnesium induced a response nearly twice activation leads to a shift in the localization of PICK1 clusters from the size (The same treatment in the presence of CPA early endosomes to synaptic sites without an increase in the overall induced a calcium response slightly smaller than the NMDA number of dendritic PICK1 clusters. It has been suggested that PICK1, under these conditions, may be important for triggering the These results raise the question of whether the magnitude or the events that initialize AMPAR endocytosis, including the facilita- source of calcium may dictate the differential effects on PICK1 tion of PKC-mediated GluR2 phosphorylation. We find that a localization. To directly test whether calcium levels alone may second pathway linked to AMPAR endocytosis, NMDAR activa- influence the distribution of dendritic PICK1, we employed tion, has a contrasting effect on dendritic PICK1. Activation of this photolytic uncaging of calcium to directly manipulate cytosolic pathway leads to an increased number of PICK1 clusters in levels of the ion and monitor its effect on PICK1. Dim light dendrites without an evident shift towards synaptic sites. While it is stimulation of neurons loaded with caged calcium (o-nitrophenyl- possible that PICK1 moves transiently to synapses following EGTA-AM) was adjusted to elicit calcium levels (as measured by NMDAR activation, even at very early time points (1 – 2 min), we fluo-3-AM fluorescence) comparable to those achieved by do not see any evidence for this redistribution. Another possibility activation of NMDARs (compare C). Control cells not is that NMDAR activation triggers increased AMPAR endocytosis loaded with caged calcium were exposed to the same stimuli to test through AP2/dynamin-mediated mechanisms ( for nonspecific effects of photolytic damage. These controls Lee et al., 2002), and PICK1 clusters by binding to these newly showed no elevations in basal calcium levels (Five internalized receptors. In fact, it has been demonstrated that minutes following a ‘‘long'' stimulation (15 flashes) to generate NMDAR activation increases the binding of PICK1 and GluR2 higher ‘‘NMDA/low Mg2+-like'' calcium responses, immunocyto- (Interestingly, we find that PICK1 clustering chemical labeling of PICK1 in neurons demonstrated a large occurs under conditions that block GluR2 endocytosis. This decrease in dendritic PICK1 levels (D, suggests that AMPAR endocytosis is triggered in parallel with, control levels, n = 8). In contrast, a shorter stimulus (7 flashes) or as a result of, the recruitment of PICK1 to endosomes. In any of generating ‘‘NMDA-like'' calcium responses resulted in an these cases, our data demonstrating the localization of PICK1 away increase in the net clustering of PICK1 (D, +65 T 9% from synaptic sites support a model in which the primary role of K.G. Sossa et al. / Mol. Cell. Neurosci. 31 (2006) 574 – 585 PICK1, under these signaling conditions, lies away from the neurons (and because GluR2 is synapse, and likely plays a role in maintaining AMPA receptor dominant in the regulated endocytosis of AMPARs ( retention at endosomes.
al., 2004), it is not completely surprising that over the short We find that PICK1 preferentially colocalizes with activated time course of our experiments they behave similarly. Previous rab5 over inactive rab5. Activated rab5 increases basal endocytosis experiments that have demonstrated subtype-specific changes in and promotes membrane fusion during endocytosis ( hippocampal neurons in response to elevated PICK1 levels al., 1994). PICK1 clustering could reflect an association of PICK1 analyzed receptor expression at significantly later time points with AMPARs, internalized by rab5 overexpression ( following manipulations (i.e. following 22 h of PICK1 over- 2005). If so, this may indicate that internalized AMPARs cluster expression; Thus, the subtype-specific PICK1 in the absence of activation of any other signaling differences in expression may represent either delayed, com- pathways. However, as we observe accumulation of PICK1 in pensatory, or homeostatic changes that develop in response to endosomes in the absence of GluR2 endocytosis, PICK1 may be initial reductions in surface GluR2 levels. Consistent with this, recruited to early endosomes independent of AMPAR expression in we found that following a more prolonged reduction of PICK1 those vesicles. While the mechanism of this targeting remains to be levels with siRNA treatment, there is a much greater effect on determined, PICK1 has a BAR domain (a motif that enables the GluR2 than GluR1.
binding of proteins to curved membranes), which in some proteins The differential effects of NMDAR signaling on PICK1 has been found to interact with small GTPases that reside in distribution appear to be linked to the absolute magnitude of the trafficking endosomes ( calcium response elicited. Stimuli that induce large elevations in PICK1 itself, through its PDZ domain, interacts with ARF1, a calcium levels trigger decreases in dendritic PICK1 levels, while GTPase that modulates vesicle trafficking at the Golgi ( stimuli that generate lesser calcium responses couple to increases al., 2000). Further studies will be necessary to determine whether in PICK1 clustering. Furthermore, direct manipulation of calcium additional PICK1 interactions may serve to direct its localization levels in neurons with flash photolysis demonstrates the impor- under various signaling conditions.
tance of the magnitude of calcium response in mediating increases We present a novel finding that a receptor-mediated signaling and decreases in dendritic PICK1 levels. While the duration of the pathway can initiate the unclustering of dendritic PICK1. Previous calcium response may also be a factor in determining the studies have reported that activation of NMDARs coupled to redistribution of PICK1, the fact that a one-min exposure to AMPAR endocytosis results in an increase in the binding of PICK1 NMDA was sufficient to drive the bidirectional changes in PICK1 and GluR2. In contrast, we find that strong NMDAR activation clustering suggests that the size of the response plays a greater role.
reduces PICK1 labeling in dendrites and mediates the unbinding of Additionally, we find that with strong activation of NMDARs, an PICK1 and GluR2. This suggests that PICK1 unclustering might intracellular calcium release component becomes important for serve to initiate changes in the trafficking of AMPARs. In fact, we effects on PICK1 distribution. Calcium-induced calcium release find that the loss of PICK1 is accompanied by an increase in (CICR) could simply provide a source of calcium necessary to detectable surface AMPARs. That this change is dependent on generate a sufficient magnitude of response, or may rather PICK1 was confirmed by the observation that knockdown of PICK1 represent a spatially localized calcium signal that selectively induces an increase in surface GluR2 that occludes any further couples to PICK1 unclustering. Interestingly, direct activation of NMDAR-mediated changes in surface AMPARs. PICK1 has been calcium release has been reported to induce an increase in surface proposed to contribute to the recycling of internalized AMPARs AMPARs in a PICK1-dependent manner ( back to the membrane surface by mediating the dissociation of However, our photolysis experiment, in which the magnitude GluR2 from intracellular GRIP ( rather than the source of calcium is manipulated, suggests that the 2005). In this model, PICK1, together with activated PKC, is magnitude of the response is the dominant factor. Postsynaptic targeted to the site of GRIP bound AMPARs which then mediate depolarization and direct or synaptic activation of NMDARs have GluR2 Ser880 phophorylation and GRIP/GluR2 unbinding. How- been shown to trigger potentiation or depression of synaptic ever, our result, in which PICK1 is rapidly removed from endocytic transmission in hippocampal pyramidal neurons, depending on the vesicles, is more consistent with a separate mechanism by which a extent of calcium entry through calcium channels ( population of PICK1-bound receptors is released for delivery to the 1992; Malgaroli and Tsien, 1992; Cummings et al., 1996; Lee et membrane surface. The means by which this dissociation occurs is al., 1998). The calcium-dependent redistribution of PICK1 unclear; however, studies have previously demonstrated the role of provides one means by which differing levels of postsynaptic the ATPase activity of NSF in mediating the unbinding of PICK1 activation could be coupled to forms of synaptic plasticity.
(enabling the synaptic insertion of AMPARs We have investigated mechanisms that control surface AMPAR (The activity of NSF itself may, therefore, be a expression in response to glutamate receptor activation. We point of regulation by NMDA receptors.
propose that activation of NMDARs differentially affects the The heterologous overexpression of PICK1 in hippocampal localization and clustering of dendritic PICK1. Through regulating neurons results in a shift in the composition of synaptic dendritic PICK1 expression, neurons can modulate surface AMPARs; the surface levels of GluR2 being reduced while AMPAR levels by mediating the numbers of internalized levels of GluR1 elevated (In our AMPARs. Future studies will determine the significance of PICK1 experiments, the surface GluR1 and GluR2 levels were both clustering in the regulation of other proteins, given that PICK1 altered following glutamate receptor activation and PICK1 interacts with a number of pre- and postsynaptic proteins, including redistribution, albeit the effects on GluR2 were slightly greater.
mGluR7a and EphBRs (While our results However, due to differences in antibody characteristics, it is have clear implications for the maintenance and regulation of difficult to interpret these differences. Also, since GluR1 is synaptic transmission, the further impact of PICK1 distribution on thought to exist as a heteromer with GluR2 in hippocampal overall neuronal physiology is likely.
K.G. Sossa et al. / Mol. Cell. Neurosci. 31 (2006) 574 – 585 Experimental methods receptors were labeled with a fluorescently conjugated secondary antibody(1:300; 1 hr). Neurons were permeabilized in TBS/2%BSA/0.1% Triton- Primary hippocampal cultures X100 (30 min) and internalized AMPA receptors were labeled with adifferent secondary antibody (1:300; 1 h). Neurons were washed with 1 Cultured primary hippocampal neurons were prepared as described TBS before mounting using Vectashield (Vector Laboratories, Burlingame, previously (Hippocampi were isolated from P0 rats (Sprague – Dawley) and the dentate gyrus was removed. Papain-treatedtissue was dissociated and plated on poly-l-lysine coated glass cover- Assays for PICK1 Redistribution slips. Cells were maintained in Neurobasal media (GIBCO) supple- Cultures were treated with NMDA or NMDA/low Mg2+ (as described mented with B27 (GIBCO) and glutamax (GIBCO, Invitrogen Corp., above) followed by fixation and permeabilization. PICK1 was labeled with Grand Island, NY). All experiments were performed using cultures at 14 an N-terminal polyclonal antibody (1:1000, 1 h) and detected with a Cy3- to 21 days in vitro.
conjugated secondary antibody.
Antibodies and reagents Colocalization studies Colocalization experiments employed various monoclonal markers of Antibodies for immunocytochemistry included monoclonal anti-N- sorting and trafficking machinery in conjunction with polyclonal PICK1 to terminal GluR2 (Chemicon International, Temecula, CA), polyclonal double label neurons. For early endosomal labeling, EEA1 (1:500; 1 h; BD GluR1 (Calbiochem, La Jolla, CA), polyclonal PICK1 (Affinity Biore- Transduction Laboratories, San Diego, CA) was used. For transferrin agents, Golden, CO), EEA1 (Pharmingen, San Diego, CA), and labeling, cultures were loaded with transferrin-tetramethylrhodamine (20 synaptophysin (Chemicon International, Temecula, CA). Secondary Ag/ml; Molecular Probes, Inc., Eugene, OR) for 15 min before and during antibodies (donkey anti-mouse or rabbit, Cy-3 or FITC-conjugated) treatment with NMDA or NMDA/Low Mg2+. Following treatments, were purchased from Jackson ImmunoResearch Laboratories (West neurons were fixed, permeabilized, and labeled for PICK1 and above markers as described. Positive colocalization was scored for discreteclusters of comparable size that were greater than 50% overlapping.
Analysis of EEA1 and transferrin experiments focused on dendrites 20 –100 Am away from the soma and was performed by investigators blind to Glutamate receptors were activated using NMDA (50 AM; Tocris Cookson, Inc., Ellisville, MO, with 10 AM CNQX also from Tocris) in thepresence of normal (1.3 mM) or of low (0.13 mM) magnesium for 1 to 2 min as specified in each experiment. TTX was not included in most our Immunolabeled neurons were imaged using a Hamamatsu Orca ER experiments. A subset of experiments including TTX exhibited comparable camera attached to an inverted Nikon fluorescent microscope with a 60 results. Neurons were washed in media and returned to 37-C for a total of 15 Plan Apo lens. Exposures were adjusted in each experiment to ensure that min from the initiation of drug treatment. Cells were then fixed and processed signals throughout the neuron were not saturated and fell within the linear for immunocytochemistry. TPA (100 nM, Calbiochem) was applied to range of the camera. Images were analyzed using Metamorph (Universal neurons for 15 min prior to fixation and immunostaining. To determine the Imaging, Inc., Downingtown, PA). Images were background subtracted and calcium requirements of PICK1 redistribution, cells were incubated prior to thresholded to include only signals about 2-fold greater than the diffuse agonist application in BAPTA-AM (25 AM for 30 min; Sigma, St. Louis, labeling in dendritic shafts. This process effectively highlighted the MO) or cyclopiazonic acid (CPA; 25 AM for 15 min; Sigma, St. Louis, MO).
punctate labeling of PICK1 and surface AMPARs in dendrites. Integrated To disrupt microtubule structure and elongation, cultures were incubated in signal intensity values of PICK1 and surface AMPARs were determined for colchicine (10 AM for 30 min, Tocris Cookson, Inc., Ellisville, MO) before soma or dendrites, normalized to area, and were graphed as a percent NMDA or NMDA/low Mg2+ treatment. In order to block endocytosis, a change from labeling in untreated controls. For all experiments, except hyperosmotic solution of sucrose (350 mM) was applied to neurons for 10 where noted, n values represent individual experiments in which 7 – 20 cells min. For treatments where calcium concentrations were changed, artificial are imaged in each.
cerebral spinal fluid (ACSF, contained (in mM): 119 NaCl, 26 NaHCO3, 10glucose, 2.5 KCl, 1 NaH Mutant Rab5 overexpression 2PO4, 1.3 MgSO4, and 2.5 CaCl2) was used.
Nominally, zero calcium concentration was obtained by omitting the CaCl Cultured neurons (11 – 14 DIV) were transfected with GFP-tagged from ACSF. In calcium imaging and uncaging experiments, HEPES (10 mM) Rab5 (Q79L = activated or S34N = inactivated) using lipofectamine was used instead of NaHCO 2000 (Invitrogen Corp., Carlsbad, CA). Rab5 constructs were generously 3. Cultures were fixed and processed for immunocytochemical analysis 15 min following initiation of agonist donated by Brian P. Ceresa, Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (Ceresa, 2004). Neurons were incubated with the DNA-lipofectamine 2000 complex for 2 h before washing with conditioned media.
Following a 36-h incubation, PICK1 was immunocytochemically Assay of surface receptors detected. Colocalization analysis of PICK1 and GFP-Rab5 (mutants) Mature (14 – 21 DIV) neuronal cultures were treated with NMDA/low was done as previously described.
Mg2+ prior to live primary GluR2 antibody labeling (35 min at 4-C).
Following AMPAR labeling, neurons were fixed and blocked under Coimmunoprecipitation and Western blot analysis nonpermeabilizing conditions (TBS/4%BSA). Surface receptors were Hippocampal slices (from 2 – 3 week old immunocytochemically labeled with fluorescently conjugated secondary rat pups were prepared and separated into 2 groups: control and treated with NMDA/low Mg2+ (2 min followed by 13 min recovery period). Bothconditions were homogenized on ice in 25 mM HEPES-KOH (pH 7.8), 150 Assay of receptor endocytosis mM NaCl, 3 mM MgCl2, 1 mM DTT plus ATPa˜S (Sigma-Aldrich Co., St.
Live cultures were labeled in conditioned growth media with GluR2 N- Louis, MO) plus protease inhibitors (Complete Mini, EDTA-free, Roche terminal antibody (10 Ag/ml) for 30 min. After antibody removal, cells were Diagnostics, Mannheim, Germany). Following two cycles of homogeniza- stimulated with glutamate receptor agonists. Fifteen minutes after initial tion and centrifugation (1000 rpm, 5 min, 4-C), 1% TX-100 was added for drug exposure, cells were fixed in paraformaldehyde (4%). Following 20 min while rocking at 4-C. Five micrograms of GluR2 antibody blockade of nonspecific proteins in TBS, 2% BSA (30 min), surface AMPA (Chemicon International, Temecula, CA) or nonspecific mouse IgG along K.G. Sossa et al. / Mol. Cell. Neurosci. 31 (2006) 574 – 585 with 500 Ag of extract per condition was incubated overnight while rocking Resources Program for Medical Schools and the NIH (NINDS) at 4-C. After three washes with the above buffer, protein G agarose beads (Sigma-Aldrich Co.) were added for 2 h to the extract/antibody mixture.
Bound proteins were separated from the beads by boiling for 5 min insample buffer. Immunoprecipitated and total lysate proteins were separatedon a 12.5% SDS/PAGE gel and PICK1 was detected by Western blotting using PICK1 N-18 goat antibody (Santa Cruz Biotech, Santa Cruz, CA) andperoxidase-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Beattie, E.C., Carroll, R.C., et al., 2000. Regulation of AMPA receptor Laboratories, West Grove, PA). Proteins were detected by enhanced endocytosis by a signaling mechanism shared with LTD. Nat. Neurosci.
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