NSF ATPase and alpha-/beta-SNAPs disassemble the AMPA receptor-PICK1 complex

AMPA receptor (AMPAR) trafficking is crucial for synaptic plasticity that may be important for learning and memory. NSF and PICK1 bind the AMPAR GluR2 subunit and are involved in trafficking of AMPARs. Here, we show that GluR2, PICK1, NSF, and alpha-/beta-SNAPs form a complex in the presence of ATPgammaS. Similar to SNARE complex disassembly, NSF ATPase activity disrupts PICK1-GluR2 interactions in this complex. Alpha- and beta-SNAP have differential effects on this reaction. SNAP overexpression in hippocampal neurons leads to corresponding changes in AMPAR trafficking by acting on GluR2-PICK1 complexes. This demonstrates that the previously reported synaptic stabilization of AMPARs by NSF involves disruption of GluR2-PICK1 interactions. Furthermore, we are reporting a non-SNARE substrate for NSF disassembly activity.     

Role of TARP interaction in S-SCAM-mediated regulation of AMPA receptors

Scaffolding proteins are involved in the incorporation, anchoring, maintenance, and removal of AMPA receptors (AMPARs) at synapses, either through a direct interaction with AMPARs or via indirect association through auxiliary subunits of transmembrane AMPAR regulatory proteins (TARPs). Synaptic scaffolding molecule (S-SCAM) is a newly characterized member of the scaffolding proteins critical for the regulation and maintenance of AMPAR levels at synapses, and directly binds to TARPs through a PDZ interaction. However, the functional significance of S-SCAM–TARP interaction in the regulation of AMPARs has not been tested. Here we show that overexpression of the C-terminal peptide of TARP-γ2 fused to EGFP abolished the S-SCAM-mediated enhancement of surface GluA2 expression. Conversely, the deletion of the PDZ-5 domain of S-SCAM that binds TARPs greatly attenuated the S-SCAM-induced increase of surface GluA2 expression. In contrast, the deletion of the guanylate kinase domain of S-SCAM did not show a significant effect on the regulation of AMPARs. Together, these results suggest that S-SCAM is regulating AMPARs through TARPs.     

Transmembrane and ubiquitin-like domain-containing protein 1 (Tmub1/HOPS) facilitates surface expression of GluR2-containing AMPA receptors

Some ubiquitin-like (UBL) domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported.Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1) protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS) protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP) were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.     

Regulation of neuronal PKA signaling through AKAP targeting dynamics

Central to organization of signaling pathways are scaffolding, anchoring and adaptor proteins that mediate localized assembly of multi-protein complexes containing receptors, second messenger-generating enzymes, kinases, phosphatases, and substrates. At the postsynaptic density (PSD) of excitatory synapses, AMPA (AMPAR) and NMDA (NMDAR) glutamate receptors are linked to signaling proteins, the actin cytoskeleton, and synaptic adhesion molecules on dendritic spines through a network of scaffolding proteins that may play important roles regulating synaptic structure and receptor functions in synaptic plasticity underlying learning and memory. AMPARs are rapidly recruited to dendritic spines through NMDAR activation during induction of long-term potentiation (LTP) through pathways that also increase the size and F-actin content of spines. Phosphorylation of AMPAR-GluR1 subunits by the cAMP-dependent protein kinase (PKA) helps stabilize AMPARs recruited during LTP. In contrast, induction of long-term depression (LTD) leads to rapid calcineurin-protein phosphatase 2B (CaN) mediated dephosphorylation of PKA-phosphorylated GluR1 receptors, endocytic removal of AMPAR from synapses, and a reduction in spine size. However, mechanisms for coordinately regulating AMPAR localization, phosphorylation, and synaptic structure by PKA and CaN are not well understood. A kinase-anchoring protein (AKAP) 79/150 is a PKA- and CaN-anchoring protein that is linked to NMDARs and AMPARs through PSD-95 and SAP97 membrane-associated guanylate kinase (MAGUK) scaffolds. Importantly, disruption of PKA-anchoring in neurons and functional analysis of GluR1-MAGUK-AKAP79 complexes in heterologous cells suggests that AKAP79/150-anchored PKA and CaN may regulate AMPARs in LTD. In the work presented at the "First International Meeting on Anchored cAMP Signaling Pathways" (Berlin-Buch, Germany, October 15-16, 2005), we demonstrate that AKAP79/150 is targeted to dendritic spines by an N-terminal basic region that binds phosphatidylinositol-4,5-bisphosphate (PIP(2)), F-actin, and actin-linked cadherin adhesion molecules. Thus, anchoring of PKA and CaN as well as physical linkage of the AKAP to both cadherin-cytoskeletal and MAGUK-receptor complexes could play roles in coordinating changes in synaptic structure and receptor signaling functions underlying plasticity. Importantly, we provide evidence showing that NMDAR-CaN signaling pathways implicated in AMPAR regulation during LTD lead to a disruption of AKAP79/150 interactions with actin, MAGUKs, and cadherins and lead to a loss of the AKAP and anchored PKA from postsynapses. Our studies thus far indicate that this AKAP79/150 translocation depends on activation of CaN, F-actin reorganization, and possibly Ca(2+)-CaM binding to the N-terminal basic regions. Importantly, this tranlocation of the AKAP79/150-PKA complex from spines may shift the balance of PKA kinase and CaN/PP1 phosphatase activity at the postsynapse in favor of the phosphatases. This loss of PKA could then promote actions of CaN and PP1 during induction of LTD including maintaining AMPAR dephosphorylation, promoting AMPAR endocytosis, and preventing AMPAR recycling. Overall, these findings challenge the accepted notion that AKAPs are static anchors that position signaling proteins near fixed target substrates and instead suggest that AKAPs can function in more dynamic manners to regulate local signaling events.     

TARPs and the AMPA receptor trafficking paradox

AMPA receptors (AMPARs) conduct fast, excitatory currents that depolarize neurons and trigger action potentials. AMPARs took on new importance when it was shown that AMPAR transport can increase or decrease the number of AMPARs at synapses and give rise to synapse plasticity, including long-term potentiation (LTP) and long-term depression (LTD). This review considers how transmembrane AMPAR regulatory proteins (TARPs), a novel family of AMPAR auxiliary subunits, have changed our view of AMPAR transport and raised some perplexing questions.     

Subunit-dependent and subunit-independent rules of AMPA receptor trafficking during chemical long-term depression in hippocampal neurons

Long-term potentiation (LTP) and long-term depression (LTD) of excitatory neurotransmission are believed to be the neuronal basis of learning and memory. Both processes are primarily mediated by neuronal activity–induced transport of postsynaptic AMPA-type glutamate receptors (AMPARs). While AMPAR subunits and their specific phosphorylation sites mediate differential AMPAR trafficking, LTP and LTD could also occur in a subunit-independent manner. Thus, it remains unclear whether and how certain AMPAR subunits with phosphorylation sites are preferentially recruited to or removed from synapses during LTP and LTD. Using immunoblot and immunocytochemical analysis, we show that phosphomimetic mutations of the membrane-proximal region (MPR) in GluA1 AMPAR subunits affect the subunit-dependent endosomal transport of AMPARs during chemical LTD. AP-2 and AP-3, adaptor protein complexes necessary for clathrin-mediated endocytosis and late endosomal/lysosomal trafficking, respectively, are reported to be recruited to AMPARs by binding to the AMPAR auxiliary subunit, stargazin (STG), in an AMPAR subunit–independent manner. However, the association of AP-3, but not AP-2, with STG was indirectly inhibited by the phosphomimetic mutation in the MPR of GluA1. Thus, although AMPARs containing the phosphomimetic mutation at the MPR of GluA1 were endocytosed by a chemical LTD-inducing stimulus, they were quickly recycled back to the cell surface in hippocampal neurons. These results could explain how the phosphorylation status of GluA1-MPR plays a dominant role in subunit-independent STG-mediated AMPAR trafficking during LTD.     

A role for cGMP-dependent protein kinase II in AMPA receptor trafficking and synaptic plasticity

Regulated trafficking of AMPA receptors (AMPARs) is an important mechanism that underlies the activity-dependent modification of synaptic strength. Trafficking of AMPARs is regulated by specific interactions of their subunits with other proteins. Recently, we have reported that the AMPAR subunit GluR1 binds the cGMP-dependent kinase type II (cGKII) adjacent to the kinase catalytic site, and that this interaction is increased by cGMP. In this complex, cGKII phosphorylates GluR1 at serine 845 (S845), a site known to be phosphorylated also by PKA. S845 phosphorylation leads to an increase of GluR1 on the plasma membrane. In neurons, cGMP is produced by soluble guanylate cyclase (sGC), which is activated by nitric oxide (NO). Calcium flux through the NMDA receptor (NMDAR) activates neuronal nitric oxide synthase (nNOS), which produces NO. Using a combination of biochemical and electrophysiological experiments, we have shown that trafficking of GluR1 is under the regulation of NO, cGMP and cGKII. Moreover, our study indicates that the interaction of cGKII with GluR1, which is under the regulation of the NMDAR and NO, plays an important role in hippocampal plasticity.     

A role for cGMP-dependent protein kinase II in AMPA receptor trafficking and synaptic plasticity

Regulated trafficking of AMPA receptors (AMPARs) is an important mechanism that underlies the activity-dependent modification of synaptic strength. Trafficking of AMPARs is regulated by specific interactions of their subunits with other proteins. Recently, we have reported that the AMPAR subunit GluR1 binds the cGMP-dependent kinase type II (cGKII) adjacent to the kinase catalytic site, and that this interaction is increased by cGMP. In this complex, cGKII phosphorylates GluR1 at serine 845 (S845), a site known to be phosphorylated also by PKA. S845 phosphorylation leads to an increase of GluR1 on the plasma membrane. In neurons, cGMP is produced by soluble guanylate cyclase (sGC), which is activated by nitric oxide (NO). Calcium flux through the NMDA receptor (NMDAR) activates neuronal nitric oxide synthase (nNOS), which produces NO. Using a combination of biochemical and electrophysiological experiments, we have shown that trafficking of GluR1 is under the regulation of NO, cGMP and cGKII. Moreover, our study indicates that the interaction of cGKII with GluR1, which is under the regulation of the NMDAR and NO, plays an important role in hippocampal plasticity.     

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 of glutamate receptor interacting protein 1 regulates surface expression of glutamate receptors

The number of synaptic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPARs) controls the strength of excitatory transmission. AMPARs cycle between internal endosomal compartments and the plasma membrane. Interactions between the AMPAR subunit GluR2, glutamate receptor interacting protein 1 (GRIP1), and the endosomal protein NEEP21 are essential for correct GluR2 recycling. Here we show that an about 85-kDa protein kinase phosphorylates GRIP1 on serine 917. This kinase is present in NEEP21 immunocomplexes and is activated in okadaic acid-treated neurons. Pulldown assays and atomic force microscopy indicate that phosphorylated GRIP shows reduced binding to NEEP21. AMPA or N-methyl-D-aspartate stimulation of hippocampal neurons induces delayed phosphorylation of the same serine 917. A wild type carboxy-terminal GRIP1 fragment expressed in hippocampal neurons interferes with GluR2 surface expression. On the contrary, a S917D mutant fragment does not interfere with GluR2 surface expression. Likewise, coexpression of GluR2 together with full-length wild type GRIP1 enhances GluR2 surface expression in fibroblasts, whereas full-length GRIP1-S917D had no effect. This indicates that this serine residue is implicated in AMPAR cycling. Our results identify an important regulatory mechanism in the trafficking of AMPAR subunits between internal compartments and the plasma membrane.     

The phosphorylation state of GluR1 subunits determines the susceptibility of AMPA receptors to calpain cleavage

The alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) is an ionotropic glutamate receptor that governs most of excitatory synaptic transmission in neurons. In vitro biochemical assay has shown that calpain, a Ca2+-activated protease, can cleave AMPAR GluR1 subunits. Our physiological study found that calpain, which was activated by prolonged stimulation of the N-methyl-D-aspartate receptor (100 microM, 10 min), caused a substantial suppression of AMPAR currents in cortical neurons. Since the phosphorylation sites of GluR1 by several protein kinases are located in close proximity to the calpain cleavage sites, we investigated the effect of phosphorylation on the susceptibility of GluR1 to calpain cleavage. Interestingly, we found that the calpain regulation of AMPAR currents was diminished by inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) but was augmented by inhibition of protein phosphatase 1/2A (PP1/2A). In agreement with this, in vitro assay showed that the calpain-induced proteolytic cleavage of GluR1 C-terminal fusion protein was strongly potentiated by adding the purified active CaMKII, and GluR1 phosphorylated at Ser831 by CaMKII is much more sensitive to calpain cleavage. Taken together, our data suggest that calpain activation suppresses AMPA receptor currents via proteolytic cleavage of GluR1 subunits, and the susceptibility of AMPARs to calpain cleavage is determined by the phosphorylation state of GluR1 subunits, which is mediated by CaMKII-PP1/2A activity.     

High-resolution proteomics unravel architecture and molecular diversity of native AMPA receptor complexes

AMPA-type glutamate receptors (AMPARs) are responsible for a variety of processes in the mammalian brain including fast excitatory neurotransmission, postsynaptic plasticity, or synapse development. Here, with comprehensive and quantitative proteomic analyses, we demonstrate that native AMPARs are macromolecular complexes with a large molecular diversity. This diversity results from coassembly of the known AMPAR subunits, pore-forming GluA and three types of auxiliary proteins, with 21 additional constituents, mostly secreted proteins or transmembrane proteins of different classes. Their integration at distinct abundance and stability establishes the heteromultimeric architecture of native AMPAR complexes: a defined core with a variable periphery resulting in an apparent molecular mass between 0.6 and 1 MDa. The additional constituents change the gating properties of AMPARs and provide links to the protein dynamics fundamental for the complex role of AMPARs in formation and operation of glutamatergic synapses.     

Prenatal cocaine exposure increases synaptic localization of a neuronal RasGEF, GRASP-1 via hyperphosphorylation of AMPAR anchoring protein, GRIP

Prenatal cocaine exposure causes sustained phosphorylation of the synaptic anchoring protein, glutamate receptor interacting protein (GRIP1/2), preventing synaptic targeting of the GluR2/3-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs; J. Neurosci. 29: 6308-6319, 2009). Because overexpression of GRIP-associated neuronal rasGEF protein (GRASP-1) specifically reduces the synaptic targeting of AMPARs, we hypothesized that prenatal cocaine exposure enhances GRASP-1 synaptic membrane localization leading to hyper-activation of ras family proteins and heightened actin polymerization. Our results show a markedly increased GRIP1-associated GRASP-1 content with approximately 40% reduction in its rasGEF activity in frontal cortices (FCX) of 21-day-old (P21) prenatal cocaine-exposed rats. This cocaine effect is the result of a persistent protein kinase C (PKC)- and downstream Src tyrosine kinase-mediated GRIP phosphorylation. The hyperactivated PKC also increased membrane-associated GRASP-1 and activated small G-proteins RhoA, cdc42/Rac1 and Rap1 as well as filamentous actin (F-actin) levels without an effect on the phosphorylation state of actin. Since increased F-actin facilitates protein transport, our results suggest that increased GRASP-1 synaptic localization in prenatal cocaine-exposed brains is an adaptive response to restoring the synaptic expression of AMPA-GluR2/3. Our earlier data demonstrated that persistent PKC-mediated GRIP phosphorylation reduces GluR2/3 synaptic targeting in prenatal cocaine-exposed brains, we now show that the increased GRIP-associated GRASP-1 may contribute to the reduction in GluR2/3 synaptic expression and AMPAR signaling defects.     

The role of transmembrane AMPA receptor regulatory proteins (TARPs) in neurotransmission and receptor trafficking (Review)

AMPA receptors (AMPAR) mediate the majority of fast excitatory neurotransmission in the central nervous system (CNS). Transmembrane AMPAR regulatory proteins (TARPs) have been identified as a novel family of proteins which act as auxiliary subunits of AMPARs to modulate AMPAR trafficking and function. The trafficking of AMPARs to regulate the number of receptors at the synapse plays a key role in various forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Expression of the prototypical TARP, stargazin/TARPgamma2, is ablated in the stargazer mutant mouse, an animal model of absence epilepsy and cerebellar ataxia. Studies on the stargazer mutant mouse have revealed that failure to express TARPgamma2 has widespread effects on the balance of expression of both excitatory (AMPAR) and inhibitory receptors (GABA(A) receptors, GABAR). The understanding of TARP function has implications for the future development of AMPAR potentiators, which have been shown to have therapeutic potential in both psychological and neurological disorders such as schizophrenia, depression and Parkinson's disease.     

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.     

Ca2+/Calmodulin-dependent Protein Kinase II Binds to and Phosphorylates a Specific SAP97 Splice Variant to Disrupt Association with AKAP79/150 and Modulate ��-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid-type Glutamate Receptor (AMPAR) Activity

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) promotes trafficking and activation of the GluR1 subunit of α-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) during synaptic plasticity. GluR1 is also modulated in parallel by multiprotein complexes coordinated by synapse-associated protein 97 (SAP97) that contain A-kinase anchoring protein 79/150 (AKAP79/150), protein kinase A, and protein phosphatase 2B. Here we show that SAP97 is present in CaMKII immune complexes isolated from rodent brain as well as from HEK293 cells co-expressing CaMKIIα and SAP97. CaMKIIα phosphorylated recombinant SAP97 within immune complexes in vitro and in intact cells. Four alternative mRNA splice variants of SAP97 expressing combinations of four inserts (I2, I3, I4, I5) in the U5 region between Src homology 3 (SH3) and guanylyl kinase-like (GK) domains were identified in rat brain at postnatal day 21. CaMKIIα preferentially phosphorylated a full-length SAP97 and a glutathione S-transferase (GST) fusion protein containing the I3 and I5 inserts (SAP97-I3I5 and GST-SH3-I3I5-GK, respectively) and also specifically interacted with GST-SH3-I3I5-GK compared with GST proteins containing other naturally occurring insert combinations. AKAP79/150 also directly and specifically bound only to GST-SH3-I3I5-GK, but CaMKII phosphorylation of GST-SH3-I3I5-GK prevented this interaction. AKAP79-dependent down-regulation of GluR1 AMPAR currents was ablated by overexpression of SAP97-I2I5 (which does not bind AKAP79) or by infusion of active CaMKIIα. Collectively, the data suggest that CaMKIIα targets a specific SAP97 splice variant to disengage AKAP79/150 from regulating GluR1 AMPARs, providing new insight into protein-protein interactions and phosphorylation events that are required for normal regulation of glutamatergic synaptic transmission, learning, and memory.     

PICK1-mediated Glutamate Receptor Subunit 2 (GluR2) Trafficking Contributes to Cell Death in Oxygen/Glucose-deprived Hippocampal Neurons

Oxygen and glucose deprivation (OGD) induces delayed cell death in hippocampal CA1 neurons via Ca²⁺/Zn²⁺-permeable, GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents in hippocampal neurons show marked inward rectification and increased sensitivity to channel blockers selective for GluR2-lacking AMPARs. This occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression and a rapid internalization of GluR2-containing AMPARs during the OGD insult, which are replaced by GluR2-lacking receptors. The mechanisms that underlie this rapid change in subunit composition are unknown. Here, we demonstrate that this trafficking event shares features in common with events that mediate long term depression and long term potentiation and is initiated by the activation of N-methyl-d-aspartic acid receptors. Using biochemical and electrophysiological approaches, we show that peptides that interfere with PICK1 PDZ domain interactions block the OGD-induced switch in subunit composition, implicating PICK1 in restricting GluR2 from synapses during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at synapses during OGD as a result of PICK1 PDZ interactions are involved in OGD-induced delayed cell death. This work demonstrates that PICK1 plays a crucial role in the response to OGD that results in altered synaptic transmission and neuronal death and has implications for our understanding of the molecular mechanisms that underlie cell death during stroke.Oxygen and glucose deprivation (OGD)³ associated with transient global ischemia induces delayed cell death, particularly in hippocampal CA1 pyramidal cells (1–3), a phenomenon that involves Ca²⁺/Zn²⁺-permeable, GluR2-lacking AMPARs (4). AMPARs are heteromeric complexes of subunits GluR1–4 (5), and most AMPARs in the hippocampus contain GluR2, which renders them calcium-impermeable and results in a marked inward rectification in their current-voltage relationship (6–8). Ischemia induces a delayed down-regulation of GluR2 mRNA and protein expression (4, 9–11), resulting in enhanced AMPAR-mediated Ca²⁺ and Zn²⁺ influx into CA1 neurons (10, 12). In these neurons, AMPAR-mediated postsynaptic currents (EPSCs) show marked inward rectification 1–2 days following ischemia and increased sensitivity to 1-naphthyl acetyl spermine (NASPM), a channel blocker selective for GluR2-lacking AMPARs (13–16). Blockade of these channels at 9–40 h following ischemia is neuroprotective, indicating a crucial role for Ca²⁺-permeable AMPARs in ischemic cell death (16).In addition to delayed changes in AMPAR subunit composition as a result of altered mRNA expression, it was recently reported that Ca²⁺-permable, GluR2-lacking AMPARs are targeted to synaptic sites via membrane trafficking at much earlier times during OGD (17). This subunit rearrangement involves endocytosis of AMPARs containing GluR2 complexed with GluR1/3, followed by exocytosis of GluR2-lacking receptors containing GluR1/3 (17). However, the molecular mechanisms behind this trafficking event are unknown, and furthermore, it is not known whether these trafficking-mediated changes in AMPAR subunit composition contribute to delayed cell death.AMPAR trafficking is a well studied phenomenon because of its crucial involvement in long term depression (LTD) and long term potentiation (LTP), activity-dependent forms of synaptic plasticity thought to underlie learning and memory. AMPAR endocytosis, exocytosis, and more recently subunit-switching events (brought about by trafficking that involves endo/exocytosis) are central to the necessary changes in synaptic receptor complement (7, 18–20). It is possible that similar mechanisms regulate AMPAR trafficking during OGD.PICK1 is a PDZ and BAR (Bin-amphiphysin-Rus) domain-containing protein that binds, via the PDZ domain, to a number of membrane proteins including AMPAR subunits GluR2/3. This interaction is required for AMPAR internalization from the synaptic plasma membrane in response to Ca²⁺ influx via NMDAR activation in hippocampal neurons (21–23). This process is the major mechanism that underlies the reduction in synaptic strength in LTD. Furthermore, PICK1-mediated trafficking has recently emerged as a mechanism that regulates the GluR2 content of synaptic receptors, which in turn determines their Ca²⁺ permeability (7, 20). This is likely to be of profound importance in both plasticity and pathological mechanisms. Importantly, PICK1 overexpression has been shown to induce a shift in synaptic AMPAR subunit composition in hippocampal CA1 neurons, resulting in inwardly rectifying AMPAR EPSCs via reduced surface GluR2 and no change in GluR1 (24). This suggests that PICK1 may mediate the rapid switch in subunit composition occurring during OGD (17). Here, we demonstrate that the OGD-induced switch in AMPAR subunit composition is dependent on PICK1 PDZ interactions, and importantly, that this early trafficking event that occurs during OGD contributes to the signaling that results in delayed neuronal death.     

Sequential Delivery of Synaptic GluA1- and GluA4-containing AMPA Receptors (AMPARs) by SAP97 Anchored Protein Complexes in Classical Conditioning

Multiple signaling pathways are involved in AMPAR trafficking to synapses during synaptic plasticity and learning. The mechanisms for how these pathways are coordinated in parallel but maintain their functional specificity involves subcellular compartmentalization of kinase function by scaffolding proteins, but how this is accomplished is not well understood. Here, we focused on characterizing the molecular machinery that functions in the sequential synaptic delivery of GluA1- and GluA4-containing AMPARs using an in vitro model of eyeblink classical conditioning. We show that conditioning induces the interaction of selective protein complexes with the key structural protein SAP97, which tightly regulates the synaptic delivery of GluA1 and GluA4 AMPAR subunits. The results demonstrate that in the early stages of conditioning the initial activation of PKA stimulates the formation of a SAP97-AKAP/PKA-GluA1 protein complex leading to synaptic delivery of GluA1-containing AMPARs through a SAP97-PSD95 interaction. This is followed shortly thereafter by generation of a SAP97-KSR1/PKC-GluA4 complex for GluA4 AMPAR subunit delivery again through a SAP97-PSD95 interaction. These data suggest that SAP97 forms the molecular backbone of a protein scaffold critical for delivery of AMPARs to the PSD during conditioning. Together, the findings reveal a cooperative interaction of multiple scaffolding proteins for appropriately timed delivery of subunit-specific AMPARs to synapses and support a sequential two-stage model of AMPAR synaptic delivery during classical conditioning.     

The interaction between Stargazin and PSD-95 regulates AMPA receptor surface trafficking

Accumulation of AMPA receptors at synapses is a fundamental feature of glutamatergic synaptic transmission. Stargazin, a member of the TARP family, is an AMPAR auxiliary subunit allowing interaction of the receptor with scaffold proteins of the postsynaptic density, such as PSD-95. How PSD-95 and Stargazin regulate AMPAR number in synaptic membranes remains elusive. We show, using single quantum dot and FRAP imaging in live hippocampal neurons, that exchange of AMPAR by lateral diffusion between extrasynaptic and synaptic sites mostly depends on the interaction of Stargazin with PSD-95 and not upon the GluR2 AMPAR subunit C terminus. Disruption of interactions between Stargazin and PSD-95 strongly increases AMPAR surface diffusion, preventing AMPAR accumulation at postsynaptic sites. Furthermore, AMPARs and Stargazin diffuse as complexes in and out synapses. These results propose a model in which the Stargazin-PSD-95 interaction plays a key role to trap and transiently stabilize diffusing AMPARs in the postsynaptic density.     

Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting

Both acute and chronic changes in AMPA receptor (AMPAR) localization are critical for synaptic formation, maturation, and plasticity. Here I report that AMPARs are differentially sorted between recycling and degradative pathways following endocytosis. AMPAR sorting occurs in early endosomes and is regulated by synaptic activity and activation of AMPA and NMDA receptors. AMPAR intemalization triggered by NMDAR activation is Ca2+-dependent, requires protein phosphatases, and is followed by rapid membrane reinsertion. Furthermore, NMDAR-mediated AMPAR trafficking is regulated by PKA and accompanied by dephosphorylation and rephosphorylation of GluR1 subunits at a PKA site. In contrast, activation of AMPARs without NMDAR activation targets AMPARs to late endosomes and lysosomes, independent of Ca2+, protein phosphatases, or PKA. These results demonstrate that activity regulates AMPAR endocytic sorting, providing a potential mechanistic link between rapid and chronic changes in synaptic strength.