Kogo Takamiya

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.

Emotion Enhances Learning via Norepinephrine Regulation of AMPA-Receptor Trafficking

Emotion enhances our ability to form vivid memories of even trivial events. Norepinephrine (NE), a neuromodulator released during emotional arousal, plays a central role in the emotional regulation of memory. However, the underlying molecular mechanism remains elusive. Toward this aim, we have examined the role of NE in contextual memory formation and in the synaptic delivery of GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors during long-term potentiation (LTP), a candidate synaptic mechanism for learning. We found that NE, as well as emotional stress, induces phosphorylation of GluR1 at sites critical for its synaptic delivery. Phosphorylation at these sites is necessary and sufficient to lower the threshold for GluR1 synaptic incorporation during LTP. In behavioral experiments, NE can lower the threshold for memory formation in wild-type mice but not in mice carrying mutations in the GluR1 phosphorylation sites. Our results indicate that NE-driven phosphorylation of GluR1 facilitates the synaptic delivery of GluR1-containing AMPARs, lowering the threshold for LTP, thereby providing a molecular mechanism for how emotion enhances learning and memory.

Synapse-specific regulation of AMPA receptor function by PSD-95

PSD-95 is a major protein found in virtually all mature excitatory glutamatergic synapses in the brain. Here, we have addressed the role of PSD-95 in controlling glutamatergic synapse function by generating and characterizing a PSD-95 KO mouse. We found that the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)subtype of glutamate receptor (AMPAR)-mediated synaptic transmission was reduced in these mice. Two-photon (2P) uncaging of MNI-glutamate onto individual spines suggested that the decrease in AMPAR function in the PSD-95 KO mouse stems from an increase in the proportion of “silent” synapses i.e., synapses containing N-methyl-d-aspartate (NMDA) receptors (NMDARs) but no AMPARs. Unexpectedly, the silent synapses in the KO mouse were located onto morphologically mature spines. We also observed that a significant population of synapses appeared unaffected by PSD-95 gene deletion, suggesting that the functional role of PSD-95 displays synapse-specificity. In addition, we report that the decay of NMDAR-mediated current was slower in KO mice: The contribution of NR2B subunit containing receptors to the NMDAR-mediated synaptic current was greater in KO mice. The greater occurrence of silent synapses might be related to the greater magnitude of potentiation after long-term potentiation induction observed in these mice. Together, these results suggest a synapse-specific role for PSD-95 in controlling synaptic function that is independent of spine morphology.

Calcium-permeable AMPA receptor plasticity is mediated by subunit-specific interactions with PICK1 and NSF

A recently described form of synaptic plasticity results in dynamic changes in the calcium permeability of synaptic AMPA receptors. Since the AMPA receptor GluR2 subunit confers calcium permeability, this plasticity is thought to occur through the dynamic exchange of synaptic GluR2-lacking and GluR2-containing receptors. To investigate the molecular mechanisms underlying this calcium-permeable AMPA receptor plasticity (CARP), we examined whether AMPA receptor exchange was mediated by subunit-specific protein-protein interactions. We found that two GluR2-interacting proteins, the PDZ domain-containing Protein interacting with C kinase (PICK1) and N-ethylmaleimide sensitive fusion protein (NSF), are specifically required for CARP. Furthermore, PICK1, but not NSF, regulates the formation of extrasynaptic plasma membrane pools of GluR2-containing receptors that may be laterally mobilized into synapses during CARP. These results demonstrate that PICK1 and NSF dynamically regulate the synaptic delivery of GluR2-containing receptors during CARP and thus regulate the calcium permeability of AMPA receptors at excitatory synapses.

Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and palmitoylation

The insertion of AMPA receptors (AMPARs) into the plasma membrane is an important step in the synaptic delivery of AMPARs during the expression of synaptic plasticity. However, the molecular mechanisms regulating AMPAR insertion remain elusive. By directly visualizing individual insertion events of the AMPAR subunit GluR1 in rodents, we found that the protein 4.1N was required for activity-dependent GluR1 insertion. Protein kinase C (PKC) phosphorylation of the serine 816 (S816) and S818 residues of GluR1 enhanced 4.1N binding to GluR1 and facilitated GluR1 insertion. In addition, palmitoylation of GluR1 C811 residue modulated PKC phosphorylation and GluR1 insertion. Finally, disrupting 4.1N-dependent GluR1 insertion decreased surface expression of GluR1 and the expression of long-term potentiation. Our study uncovers a previously unknown mechanism that governs activity-dependent GluR1 trafficking, reveals an interaction between AMPAR palmitoylation and phosphorylation, and underscores the functional importance of 4.1N in AMPAR trafficking and synaptic plasticity.

Targeted In Vivo Mutations of the AMPA Receptor Subunit GluR2 and Its Interacting Protein PICK1 Eliminate Cerebellar Long-Term Depression

Cerebellar long-term depression (LTD) is a major form of synaptic plasticity that is thought to be critical for certain types of motor learning. Phosphorylation of the AMPA receptor subunit GluR2 on serine-880 as well as interaction of GluR2 with PICK1 have been suggested to contribute to the endocytic removal of postsynaptic AMPA receptors during LTD. Here, we show that targeted mutation of PICK1, the GluR2 C-terminal PDZ ligand, or the GluR2 PKC phosphorylation site eliminates cerebellar LTD in mice. LTD can be rescued in cerebellar cultures from mice lacking PICK1 by transfection of wild-type PICK1 but not by a PDZ mutant or a BAR domain mutant deficient in lipid binding, indicating the importance of these domains in PICK1 function. These results demonstrate that PICK1-GluR2 PDZ-based interactions and GluR2 phosphorylation are required for LTD expression in the cerebellum.

Reevaluating the Role of LTD in Cerebellar Motor Learning

Long-term depression at parallel fiber-Purkinje cell synapses (PF-PC LTD) has been proposed to be required for cerebellar motor learning. To date, tests of this hypothesis have sought to interfere with receptors (mGluR1) and enzymes (PKC, PKG, or αCamKII) necessary for induction of PF-PC LTD and thereby determine if cerebellar motor learning is impaired. Here, we tested three mutant mice that target the expression of PF-PC LTD by blocking internalization of AMPA receptors. Using three different cerebellar coordination tasks (adaptation of the vestibulo-ocular reflex, eyeblink conditioning, and locomotion learning on the Erasmus Ladder), we show that there is no motor learning impairment in these mutant mice that lack PF-PC LTD. These findings demonstrate that PF-PC LTD is not essential for cerebellar motor learning.

Persistent inflammation induces GluR2 internalization via NMDA receptor-triggered PKC activation in dorsal horn neurons

Spinal cord GluR2-lacking AMPA receptors (AMPARs) contribute to nociceptive hypersensitivity in persistent pain, but the molecular mechanisms underlying this event are not completely understood. We report that complete Freunds adjuvant (CFA)-induced peripheral inflammation induces synaptic GluR2 internalization in dorsal horn neurons during the maintenance of CFA-evoked nociceptive hypersensitivity. This internalization is initiated by GluR2 phosphorylation at Ser880 and subsequent disruption of GluR2 binding to its synaptic anchoring protein (GRIP), resulting in a switch of GluR2-containing AMPARs to GluR2-lacking AMPARs and an increase of AMPAR Ca2+ permeability at the synapses in dorsal horn neurons. Spinal cord NMDA receptor-mediated triggering of protein kinase C (PKC) activation is required for the induction and maintenance of CFA-induced dorsal horn GluR2 internalization. Moreover, preventing CFA-induced spinal GluR2 internalization through targeted mutation of the GluR2 PKC phosphorylation site impairs CFA-evoked nociceptive hypersensitivity during the maintenance period. These results suggest that dorsal horn GluR2 internalization might participate in the maintenance of NMDA receptor/PKC-dependent nociceptive hypersensitivity in persistent inflammatory pain.

A direct functional link between the multi-PDZ domain protein GRIP1 and the Fraser syndrome protein Fras1

Cell adhesion to extracellular matrix (ECM) proteins is crucial for the structural integrity of tissues and epithelial-mesenchymal interactions mediating organ morphogenesis. Here we describe how the loss of a cytoplasmic multi-PDZ scaffolding protein, glutamate receptor interacting protein 1 (GRIP1), leads to the formation of subepidermal hemorrhagic blisters, renal agenesis, syndactyly or polydactyly and permanent fusion of eyelids (cryptophthalmos). Similar malformations are characteristic of individuals with Fraser syndrome and animal models of this human genetic disorder, such as mice carrying the blebbed mutation (bl) in the gene encoding the Fras1 ECM protein. GRIP1 can physically interact with Fras1 and is required for the localization of Fras1 to the basal side of cells. In one animal model of Fraser syndrome, the eye-blebs (eb) mouse, Grip1 is disrupted by a deletion of two coding exons. Our data indicate that GRIP1 is required for normal cell-matrix interactions during early embryonic development and that inactivation of Grip1 causes Fraser syndrome–like defects in mice.

Involvement of AMPA receptor phosphorylation in antidepressant actions with special reference to tianeptine

Depression is associated with abnormal neuronal plasticity. AMPA receptors mediate transmission and plasticity at excitatory synapses in a manner which is positively regulated by phosphorylation at Ser831‐GluR1, a CaMKII/PKC site, and Ser845‐GluR1, a PKA site. Treatment with the selective serotonin (5‐hydroxytryptamine; 5‐HT) reuptake inhibitor fluoxetine increases P‐Ser845‐GluR1 but not P‐Ser831‐GluR1. Here, it was found that treatment with another antidepressant, tianeptine, increased P‐Ser831‐GluR1 in the frontal cortex and the CA3 region of hippocampus and P‐Ser845‐GluR1 in the CA3 region of hippocampus. A receptorome profile detected no affinity for tianeptine at any monaminergic receptors or transporters, confirming an atypical profile for this compound. Behavioural analyses showed that mice bearing point mutations at both Ser831‐ and Ser845‐GluR1, treated with saline, exhibited increased latency to enter the centre of an open field and increased immobility in the tail‐suspension test compared to their wild‐type counterparts. Chronic tianeptine treatment increased open‐field locomotion and reduced immobility in wild‐type mice but not in phosphomutant GluR1 mice. P‐Ser133‐CREB was reduced in the CA3 region of hippocampus in phosphomutant mice, and tianeptine decreased P‐Ser133‐CREB in this region in wild‐type, but not in phosphomutant, mice. Tianeptine increased P‐Ser133‐CREB in the CA1 region in wild‐type mice but not in phosphomutant GluR1 mice. There were higher basal P‐Ser133‐CREB and c‐fos levels in frontal and cingulate cortex in phosphomutant GluR1 mice; these changes in level were counteracted by tianeptine in a GluR1‐independent manner. Using phosphorylation assays and phosphomutant GluR1 mice, this study provides evidence that AMPA receptor phosphorylation mediates certain explorative and antidepressant‐like actions under basal conditions and following tianeptine treatment.