Disruption of AMPA receptor GluR2 clusters following long-term depression induction in cerebellar Purkinje neurons

Cerebellar long-term depression (LTD) is thought to play an important role in certain types of motor learning. However, the molecular mechanisms underlying this event have not been clarified. Here, using cultured Purkinje cells, we show that stimulations inducing cerebellar LTD cause phosphorylation of Ser880 in the intracellular C-terminal domain of the AMPA receptor subunit GluR2. This phosphorylation is accompanied by both a reduction in the affinity of GluR2 to glutamate receptor interacting protein (GRIP), a molecule known to be critical for AMPA receptor clustering, and a significant disruption of postsynaptic GluR2 clusters. Moreover, GluR2 protein released from GRIP is shown to be internalized. These results suggest that the dissociation of postsynaptic GluR2 clusters and subsequent internalization of the receptor protein, initiated by the phosphorylation of Ser880, are the mechanisms underlying the induction of cerebellar LTD.     

NSF binds calcium to regulate its interaction with AMPA receptor subunit GluR2

N-ethylmaleimide-sensitive fusion protein (NSF) is essential for numerous Ca(2+)-triggered vesicle trafficking events. It functions as a molecular chaperone to regulate trafficking protein complexes such as the soluble NSF attachment protein (SNAP) receptor complex and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-protein interacting with C-kinase (PICK1) complex. AMPAR trafficking is fundamental to processes of synaptic plasticity, which may underlie learning and memory. Changes in synaptic strength brought about by AMPAR trafficking are triggered by a post-synaptic influx of Ca(2+), which may have numerous molecular targets including PICK1. NSF binds AMPAR subunit glutamate receptor subunit 2 (GluR2) and functions to maintain receptors at the synapse. In this study, it was showed that NSF is a Ca(2+)-binding protein and that GluR2-NSF interactions are inhibited by the presence of 15 mumol/L Ca(2+). NSF Ca(2+)-binding is reciprocally inhibited by the presence of GluR2 C-terminus. Mutant of NSF that binds Ca(2+) with reduced affinity and binds GluR2 with reduced sensitivity to Ca(2+) was identied. In addition, the interaction of betaSNAP with PICK1 is sensitive to Ca(2+). This study demonstrates that the GluR2-NSF-betaSNAP-PICK1 complex is regulated directly by Ca(2+), allowing for the transduction of Ca(2+) signals into concerted alterations in protein-protein interactions to bring about changes in AMPAR trafficking during synaptic plasticity.     

AMPA receptor subunit GluR2 gates injurious signals in ischemic stroke

Ischemic stroke, or a brain attack, is the third leading cause of death in developed countries. A critical feature of the disease is a highly selective pattern of neuronal loss; certain identifiable subsets of neurons--particularly CA1 pyramidal neurons in the hippocampus are severely damaged, whereas others remain intact. A key step in this selective neuronal injury is Ca2+/Zn2+ entry into vulnerable neurons through alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor channels, a principle subtype of glutamate receptors. AMPA receptor channels are assembled from glutamate receptor (GluR)1, -2, -3, and -4 subunits. Circumstance data have indicated that the GluR2 subunits dictate Ca2+/Zn2+ permeability of AMPA receptor channels and gate injurious Ca2+/Zn2+ signals in vulnerable neurons. Therefore, targeting to the AMPA receptor subunit GluR2 can be considered a practical strategy for stroke therapy.     

The PDZ domain of PICK1 differentially accepts protein kinase C-alpha and GluR2 as interacting ligands

The C terminus (ct) of protein kinase C-alpha (PKCalpha) has a type I PDZ binding motif, whereas GluR2 has a type II PDZ binding motif. Both motifs are recognized by the PDZ domain of protein interacting with protein kinase C (PICK1), and PICK1-PKCalpha-controlled phosphorylation regulates the synaptic expression and function of GluR2. Here, we show that a specific mutation within the carboxylate-binding loop of the PDZ domain of PICK1 (K27E; PICK1-KE) results in a loss of interaction with GluR2 but not with PKCalpha. In GST pull-down studies, PICK1-WT (wild type) but not PICK1-KE was retained by GST-ct-GluR2. Furthermore, PICK1-WT co-immunoprecipitated both PKCalpha and GluR2, whereas PICK1-KE only co-immunoprecipitated PKCalpha. In heterologous cells, PICK1-WT, but not PICK1-KE, clustered GluR2 and also clustered GluR1 in a GluR2-dependent manner. However, neither PICK1-WT nor PICK1-KE altered the distribution of PKCalpha, even after phorbol ester-induced redistribution of PKCalpha to the membrane. Finally, PICK1-KE showed no mislocalization when compared with PICK1-WT in neurons. Taken together, it appears that the PDZ domain of PICK1 is less sensitive to mutations for PKCalpha when compared with GluR2 binding. These results suggest that the PDZ domain of PICK1 has distinct PKCalpha and GluR2 binding subsite(s).     

The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity

The AMPA receptor (AMPAR) GluR2 subunit dictates the critical biophysical properties of the receptor, strongly influences receptor assembly and trafficking, and plays pivotal roles in a number of forms of long-term synaptic plasticity. Most neuronal AMPARs contain this critical subunit; however, in certain restricted neuronal populations and under certain physiological or pathological conditions, AMPARs that lack this subunit are expressed. There is a current surge of interest in such GluR2-lacking Ca2+-permeable AMPARs in how they affect the regulation of synaptic transmission. Here, we bring together recent data highlighting the novel and important roles of GluR2 in synaptic function and plasticity.     

Phosphorylation-dependent interactions of alpha-Actinin-1/IQGAP1 with the AMPA receptor subunit GluR4

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors play key roles in excitatory synaptic transmission and synaptic plasticity in the CNS. Although a variety of proteins has been characterized to interact with AMPA receptors and regulate their function, little is known about the regulation of the AMPA receptor subunit GluR4. To understand the molecular mechanisms of GluR4 functional regulation, the yeast two-hybrid system was used to identify GluR4-interacting molecules. alpha-Actinin-1 and IQGAP1 were identified to be GluR4-specific binding partners. Both proteins interact specifically with GluR4 and co-cluster with GluR4 individually in neurons. Mapping experiments revealed that alpha-Actinin-1 and IQGAP1 bind to the same region within the C-terminus of GluR4 that contains a previously identified PKA phosphorylation site, Ser842, phosphorylation of which is regulated by synaptic activity. Interestingly, the phosphorylation of Ser842 differentially regulates interactions of GluR4 with alpha-Actinin-1 and IQGAP1; phosphorylation strongly inhibits interaction of GluR4 with alpha-Actinin-1 but has little effect on its interaction with IQGAP1. These results suggest that alpha-Actinin-1 and IQGAP1 regulate GluR4 functions via their specific associations with GluR4. In addition, our data indicate that activity-dependent phosphorylation of GluR4 may regulate its synaptic targeting through phosphorylation-dependent interactions with alpha-Actinin-1 and IQGAP1.     

Long-term depression requires postsynaptic AMPA GluR2 receptor in adult mouse cingulate cortex

Synaptic long-term depression (LTD) is thought to be important for various brain functions such as learning, memory, and development. Although anterior cingulated cortex (ACC) has been demonstrated to contribute to learning and memory, no studies has been reported about the synaptic mechanisms for cingulate LTD. Here, we used integrative genetic, pharmacological and electrophysiological approaches to demonstrate that AMPA GluR2, but not GluR3, subunit is critical for cingulate LTD. We found that LTD was absent in adult cingulate slices of GluR2 knockout mice. Furthermore, postsynaptic injections of peptides that inhibit AMPA GluR2-PDZ interactions blocked the induction of LTD. To determine if the requirement for AMPA receptor-PDZ interaction is time-dependent, we injected the same inhibiting peptide into the postsynaptic cells 5 min after the induction of LTD. We found that LTD was not affected by the peptide, providing the first evidence that postsynaptic AMPA GluR2-mediated depression occurs rapidly (within t = 5 min). Genetic deletion of GluR3 did not affect cingulate LTD. Our results provide the first study of cingulate LTD mechanism using whole-cell patch-clamp recording in adult cortical slices and demonstrate that postsynaptic AMPA GluR2 subunit is crucial for synaptic depression in the ACC of adult mice.     

Differential regulation of dendrite complexity by AMPA receptor subunits GluR1 and GluR2 in motor neurons

Activity-dependent developmental mechanisms in many regions of the central nervous system are thought to be responsible for shaping dendritic architecture and connectivity, although the molecular mechanisms underlying these events remain obscure. Since AMPA glutamate receptors are developmentally regulated in spinal motor neurons, we have investigated the role of activation of AMPA receptors in dendritic outgrowth of spinal motor neurons by overexpression of two subunits, GluR1 and GluR2, and find that dendrite outgrowth is differentially controlled by expression of these subunits. Overexpression of GluR1 was associated with greater numbers of filopodia, and an increase in the length and complexity of dendritic arbor. In contrast, GluR2 expression did not alter dendritic complexity, but was associated with a moderate increase in length of arbor, and decreased numbers of filopodia. Neither GluR1 nor GluR2 had any effect on the motility of filopodia. In addition, GluR1 but not GluR2 expression increased the density of dendritic puncta incorporating a GFP-labeled PSD95, suggesting that GluR1 may mediate its effect in part by augmenting the number of excitatory synapses within motor neuron dendrites. Together these results suggest that in spinal motor neurons, AMPA receptors composed of GluR1 subunits may facilitate neurotrophic mechanisms in these neurons, permitting sustained dendrite outgrowth and synaptogenesis, whereas expression of AMPA receptors containing GluR2 acts to preserve existing dendritic arbor. Thus, the observed downregulation of GluR1 in motor neurons during postnatal development may limit the formation of new dendrite segments and synapses, promoting stabilized synaptic connectivity.     

Evidence for low GluR2 AMPA receptor subunit expression at synapses in the rat basolateral amygdala

Fast excitatory synaptic responses in basolateral amygdala (BLA) neurons are mainly mediated by ionotropic glutamate receptors of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype. AMPA receptors containing an edited GluR2 subunit are calcium impermeable, whereas those that lack this subunit are calcium permeable and also inwardly rectifying. Here, we sought to determine the extent to which synapses in the rat BLA have AMPA receptors with GluR2 subunits. We assessed GluR2 protein expression in the BLA by immunocytochemistry with a GluR2 subunit-specific antiserum at the light and electron microscopic level; for comparison, a parallel examination was carried out in the hippocampus. We also recorded from amygdala brain slices to examine the voltage-dependent properties of AMPA receptor- mediated evoked synaptic currents in BLA principal neurons. At the light microscopic level, GluR2 immunoreactivity was localized to the perikarya and proximal dendrites of BLA neurons; dense labeling was also present over the pyramidal cell layer of hippocampal subfields CA1 and CA3. In electron micrographs from the BLA, most of the synapses were asymmetrical with pronounced postsynaptic densities (PSD). They contained clear, spherical vesicles apposed to the PSD and were predominantly onto spines (86%), indicating that they are mainly with BLA principal neurons. Only 11% of morphological synapses in the BLA were onto postsynaptic elements that showed GluR2 immunoreactivity, in contrast to hippocampal subfields CA1 and CA3 in which 76% and 71% of postsynaptic elements were labeled (p < 0.001). Synaptic staining in the BLA and hippocampus, when it occurred, was exclusively postsynaptic, and particularly heavy over the PSD. In whole-cell voltage clamp recordings, 72% of BLA principal neurons exhibited AMPA receptor-mediated synaptic currents evoked by external capsule stimulation that were inwardly rectifying. Although BLA principal neurons express perikaryal and proximal dendritic GluR2 immunoreactivity, few synapses onto these neurons express GluR2, and a preponderance of principal neurons have inwardly rectifying AMPA-mediated synaptic currents, suggesting that targeting of GluR2 to synapses is restricted. Many BLA synaptic AMPA receptors are likely to be calcium permeable and could play roles in synaptic plasticity, epileptogenesis and excitoxicity.     

Synaptic transmission and plasticity in the absence of AMPA glutamate receptor GluR2 and GluR3

The AMPA glutamate receptor (AMPAR) subunits GluR2 and GluR3 are thought to be important for synaptic targeting/stabilization of AMPARs and the expression of hippocampal long-term depression (LTD). In order to address this hypothesis genetically, we generated and analyzed knockout mice deficient in the expression of both GluR2 and GluR3. We show here that the double knockout mice are severely impaired in basal synaptic transmission, demonstrating that GluR2/3 are essential to maintain adequate synaptic transmission in vivo. However, these mutant mice are competent in establishing several forms of long-lasting synaptic changes in the CA1 region of the hippocampus, including LTD, long-term potentiation (LTP), depotentiation, and dedepression, indicating the presence of GluR2/3-independent mechanisms of LTD expression and suggesting that AMPA receptor GluR1 alone is capable of various forms of synaptic plasticity.     

Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory

Plasticity of the nervous system is dependent on mechanisms that regulate the strength of synaptic transmission. Excitatory synapses in the brain undergo long-term potentiation (LTP) and long-term depression (LTD), cellular models of learning and memory. Protein phosphorylation is required for the induction of many forms of synaptic plasticity, including LTP and LTD. However, the critical kinase substrates that mediate plasticity have not been identified. We previously reported that phosphorylation of the GluR1 subunit of AMPA receptors, which mediate rapid excitatory transmission in the brain, is modulated during LTP and LTD. To test if GluR1 phosphorylation is necessary for plasticity and learning and memory, we generated mice with knockin mutations in the GluR1 phosphorylation sites. The phosphomutant mice show deficits in LTD and LTP and have memory defects in spatial learning tasks. These results demonstrate that phosphorylation of GluR1 is critical for LTD and LTP expression and the retention of memories.     

ADAR2-dependent RNA editing of AMPA receptor subunit GluR2 determines vulnerability of neurons in forebrain ischemia

ADAR2 is a nuclear enzyme essential for GluR2 pre-mRNA editing at Q/R site-607, which gates Ca2+ entry through AMPA receptor channels. Here, we show that forebrain ischemia in adult rats selectively reduces expression of ADAR2 enzyme and, hence, disrupts RNA Q/R site editing of GluR2 subunit in vulnerable neurons. Recovery of GluR2 Q/R site editing by expression of exogenous ADAR2b gene or a constitutively active CREB, VP16-CREB, which induces expression of endogenous ADAR2, protects vulnerable neurons in the rat hippocampus from forebrain ischemic insult. Generation of a stable ADAR2 gene silencing by delivering small interfering RNA (siRNA) inhibits GluR2 Q/R site editing, leading to degeneration of ischemia-insensitive neurons. Direct introduction of the Q/R site edited GluR2 gene, GluR2(R607), rescues ADAR2 degeneration. Thus, ADAR2-dependent GluR2 Q/R site editing determines vulnerability of neurons in the rat hippocampus to forebrain ischemia.     

Interaction of the N-terminal domain of the AMPA receptor GluR4 subunit with the neuronal pentraxin NP1 mediates GluR4 synaptic recruitment

Synaptogenesis requires recruitment of neurotransmitter receptors to developing postsynaptic specializations. We developed a coculture system reconstituting artificial synapses between neurons and nonneuronal cells to investigate the molecular components required for AMPA-receptor recruitment to synapses. With this system, we find that excitatory axons specifically express factors that recruit the AMPA receptor GluR4 subunit to sites of contact between axons and GluR4-transfected nonneuronal cells. Furthermore, the N-terminal domain (NTD) of GluR4 is necessary and sufficient for its recruitment to these artificial synapses and also for GluR4 recruitment to native synapses. Moreover, we show that axonally derived neuronal pentraxins NP1 and NPR are required for GluR4 recruitment to artificial and native synapses. RNAi knockdown and knockout of the neuronal pentraxins significantly decreases GluR4 targeting to synapses. Our results indicate that NP1 and NPR secreted from presynaptic neurons bind to the GluR4 NTD and are critical trans-synaptic factors for GluR4 recruitment to synapses.     

Regulation of phosphorylation of the GluR1 AMPA receptor by dopamine D2 receptors

In the striatum, stimulation of dopamine D2 receptors results in attenuation of glutamate responses. This effect is exerted in large part via negative regulation of AMPA glutamate receptors. Phosphorylation of the GluR1 subunit of the AMPA receptor has been proposed to play a critical role in the modulation of glutamate transmission, in striatal medium spiny neurons. Here, we have examined the effects of blockade of dopamine D2-like receptors on the phosphorylation of GluR1 at the cAMP-dependent protein kinase (PKA) site, Ser845, and at the protein kinase C and calcium/calmodulin-dependent protein kinase II site, Ser831. Administration of haloperidol, an antipsychotic drug with dopamine D2 receptor antagonistic properties, increases the phosphorylation of GluR1 at Ser845, without affecting phosphorylation at Ser831. The same effect is observed using eticlopride, a selective dopamine D2 receptor antagonist. In contrast, administration of the dopamine D2-like agonist, quinpirole, decreases GluR1 phosphorylation at Ser845. The increase in Ser845 phosphorylation produced by haloperidol is abolished in dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) knockout mice, or in mice in which the PKA phosphorylation site on DARPP-32 (i.e. Thr34) has been mutated (Thr34-->Ala mutant mice), and requires tonic activation of adenosine A2A receptors. These results demonstrate that dopamine D2 antagonists increase GluR1 phosphorylation at Ser845 by removing the inhibitory tone exerted by dopamine D2 receptors on the PKA/DARPP-32 cascade.     

A long-term depression of AMPA currents in cultured cerebellar Purkinje neurons.

Cerebellar long-term depression (LTD) is a model of synaptic plasticity in which conjunctive stimulation of parallel fiber and climbing fiber inputs to a Purkinje neuron induces a persistent depression of the parallel fiber-Purkinje neuron synapse. We report that an analogous phenomenon may be elicited in the cultured mouse Purkinje neuron when iontophoretic glutamate application and depolarization of the Purkinje neurons are substituted for parallel fiber and climbing fiber stimulation, respectively. The induction of LTD in these cerebellar cultures requires activation of both ionotropic (AMPA) and metabotropic quisqualate receptors, together with depolarization in the presence of external Ca2+. This postsynaptic alteration is manifest as a depression of glutamate or AMPA currents, but not aspartate or NMDA currents. These results strengthen the contention that the expression of cerebellar LTD is at least in part postsynaptic and provide evidence that activation of both ionotropic and metabotropic quisqualate receptors are necessary for LTD induction.     

Clustering of AMPA receptors by the synaptic PDZ domain-containing protein PICK1

Synaptic clustering of neurotransmitter receptors is crucial for efficient signal transduction and integration in neurons. PDZ domain-containing proteins such as PSD-95/SAP90 interact with the intracellular C termini of a variety of receptors and are thought to be important in the targeting and anchoring of receptors to specific synapses. Here, we show that PICK1 (protein interacting with C kinase), a PDZ domain-containing protein, interacts with the C termini of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors in vitro and in vivo. In neurons, PICK1 specifically colocalizes with AMPA receptors at excitatory synapses. Furthermore, PICK1 induces clustering of AMPA receptors in heterologous expression systems. These results suggest that PICK1 may play an important role in the modulation of synaptic transmission by regulating the synaptic targeting of AMPA receptors.     

Time-dependent postsynaptic AMPA GluR1 receptor recruitment in the cingulate synaptic potentiation

The anterior cingulate cortex (ACC) is critical for brain functions including learning, memory, fear and pain. Long-term synaptic potentiation (LTP), a cellular model for learning and memory, has been reported in the ACC neurons. Unlike LTP in the hippocampus and amygdala, two key structures for memory and fear, little is known about the synaptic mechanism for the expression of LTP in the ACC. Here we use whole-cell patch clamp recordings to demonstrate that cingulate LTP requires the functional recruitment of GluR1 AMPA receptors; and such events are rapid and completed within 5-10 min after LTP induction. Our results demonstrate that the GluR1 subunit is essential for synaptic plasticity in the ACC and may play critical roles under physiological and pathological conditions.     

Interactions between NEEP21, GRIP1 and GluR2 regulate sorting and recycling of the glutamate receptor subunit GluR2

Trafficking of AMPA-type glutamate receptors (AMPAR) between endosomes and the postsynaptic plasma membrane of neurons plays a central role in the control of synaptic strength associated with learning and memory. The molecular mechanisms of its regulation remain poorly understood, however. Here we show by biochemical and atomic force microscopy analyses that NEEP21, a neuronal endosomal protein necessary for receptor recycling including AMPAR, is associated with the scaffolding protein GRIP1 and the AMPAR subunit GluR2. Moreover, the interaction between NEEP21 and GRIP1 is regulated by neuronal activity. Expression of a NEEP21 fragment containing the GRIP1-binding site decreases surface GluR2 levels and delays recycling of internalized GluR2, which accumulates in early endosomes and lysosomes. Infusion of this fragment into pyramidal neurons of hippocampal slices induces inward rectification of AMPAR-mediated synaptic responses, suggesting decreased GluR2 expression at synapses. These results indicate that NEEP21-GRIP1 binding is crucial for GluR2-AMPAR sorting through endosomes and their recruitment to the plasma membrane, providing a first molecular mechanism to differentially regulate AMPAR subunit cycling in internal compartments.     

Differences in AMPA and kainate receptor interactomes facilitate identification of AMPA receptor auxiliary subunit GSG1L

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Highlights? Mass spectrometry identified proteins copurifying with AMPA and kainate receptors ? GSG1L is an AMPA-receptor-binding transmembrane protein ? GSG1L uniquely modulates AMPA receptor trafficking and channel gating ? GSG1L colocalizes with AMPA receptors at synapses     

Immunocytochemical localization of neurons containing the AMPA GluR2/3 subunit in the hamster visual cortex

AMPA glutamate receptors play a crucial role in brain functions such as synaptic plasticity and development. We have studied the chemo-architecture of the AMPA glutamate receptor subtype GluR2/3 in the hamster visual cortex by immunocytochemistry and compared it with the distribution of the calcium-binding proteins, calbindin D28K and calretinin. Anti-GluR2/3-immunoreactive (IR) neurons were predominantly located in layers II/III, V, and VI, and the majority of the labeled neurons were round or oval. However, many pyramidal cells in layer V were also labeled. Two-color immunofluorescence revealed that none of the GluR2/3-IR neurons contained calbindin D28 K or calretinin. Thus specific layers of neurons express the GluR2/3 subunit and these do not correlate with expression of calbindin D28K and calretinin.