Akihiko Kato

Novel Members of the Vesl/Homer Family of PDZ Proteins That Bind Metabotropic Glutamate Receptors

Vesl-1S (186 amino acids, also called Homer) is a protein containing EVH1- and PDZ-like domains whose expression in the hippocampus is regulated during long term potentiation (LTP), one form of synaptic plasticity thought to underlie memory formation (Kato, A., Ozawa, F., Saitoh, Y., Hirai, K., and Inokuchi, K. (1997) FEBS Lett. 412, 183–189; Brakeman, P. R., Lanahan, A. A., O’Brien, R., Roche, K., Barnes, C. A., Huganir, R. L., and Worley, P. F. (1997)Nature 386, 284–288). Here we report additional members of the Vesl/Homer family of proteins, Vesl-1L and Vesl-2. Vesl-1L (366 amino acids), a splicing variant of Vesl-1S, shares N-terminal 175 amino acids with Vesl-1S and contains additional amino acids at the C terminus. Vesl-2 (354 amino acids) was highly related to Vesl-1L in that both contain EVH1- and PDZ-like domains at the N terminus (86% conservation) and an MCC (mutated in colorectal cancer)-like domain and a leucine zipper at the C terminus. In contrast to vesl-1S, we observed no changes in the levels of vesl-1L andvesl-2 mRNAs during dentate gyrus LTP. All these proteins interacted with metabotropic glutamate receptors (mGluR1 and mGluR5) as well as several hippocampal proteins in vitro. Vesl-1L and Vesl-2, but not Vesl-1S, interacted with each other through the C-terminal portion that was absent in Vesl-1S. Vesl-1L and Vesl-2 may mediate clustering of mGluRs at synaptic junctions. We propose that Vesl-1S may be involved in the structural changes that occur at metabotropic glutamatergic synapses during the maintenance phase of LTP by modulating the redistribution of synaptic components.

New Transmembrane AMPA Receptor Regulatory Protein Isoform, γ-7, Differentially Regulates AMPA Receptors

AMPA-type glutamate receptors (GluRs) mediate most excitatory signaling in the brain and are composed of GluR principal subunits and transmembrane AMPA receptor regulatory protein (TARP) auxiliary subunits. Previous studies identified four mammalian TARPs, γ-2 (or stargazin), γ-3, γ-4, and γ-8, that control AMPA receptor trafficking, gating, and pharmacology. Here, we explore roles for the homologous γ-5 and γ-7 proteins, which were previously suggested not to serve as TARPs. Western blotting reveals high levels of γ-5 and γ-7 in the cerebellum, where γ-7 is enriched in Purkinje neurons in the molecular layer and glomerular synapses in the granule cell layer. Immunoprecipitation proteomics shows that cerebellar γ-7 avidly and selectively binds to AMPA receptor GluR subunits and also binds to the AMPA receptor clustering protein, postsynaptic density-95 (PSD-95). Furthermore, γ-7 occurs together with PSD-95 and AMPA receptor subunits in purified postsynaptic densities. In heterologous cells, γ-7 but not γ-5 greatly enhances AMPA receptor glutamate-evoked currents and modulates channel gating. In granule cells from stargazer mice, transfection of γ-7 but not γ-5 increases AMPA receptor-mediated currents. Compared with stargazin, γ-7 differentially modulates AMPA receptor glutamate affinity and kainate efficacy. These studies define γ-7 as a new member of the TARP family that can differentially influence AMPA receptors in cerebellar neurons.

TARPs differentially decorate AMPA receptors to specify neuropharmacology.

Transmembrane AMPA receptor regulatory proteins (TARPs) are the first identified auxiliary subunits for a neurotransmitter-gated ion channel. Although initial studies found that stargazin, the prototypical TARP, principally chaperones AMPA receptors, subsequent research demonstrated that it also regulates AMPA receptor kinetics and synaptic waveforms. Recent studies have identified a diverse collection of TARP isoforms--types Ia, Ib II--that distinctly regulate AMPA receptor trafficking, gating and neuropharmacology. These TARP isoforms are heterogeneously expressed in specific neuronal populations and can differentially sculpt synaptic transmission and plasticity. Whole-genome analyses also link multiple TARP loci to childhood epilepsy, schizophrenia and bipolar disorder. TARPs emerge as vital components of excitatory synapses that participate both in signal transduction and in neuropsychiatric disorders.

Homer/Vesl proteins and their roles in CNS neurons.

Since their initial discovery in 1997, Homer/Vesl proteins have become increasingly investigated as putative regulators of receptor and ion-channel function in the central nervous system. Within a relatively brief period, numerous research reports have described manifold effects of Homer proteins, including the modulation of the trafficking of type I metabotropic glutamate receptors (mGluRs), axonal pathfinding, mGluR coupling to calcium and potassium channels, agonist-independent mGluR activity, ryanodine receptor regulation, locomotor activity, and behavioral plasticity. This review summarizes our current knowledge on the induction, expression, and structure of the various forms of Homer proteins, as well as their roles in neuronal function. In addition, we provide an outlook on novel developments with regard to the involvement of Homer-1a in hippocampal synaptic function.

Homer-1a/Vesl-1s enhances hippocampal synaptic transmission

Homer/Vesl proteins are involved in regulating metabotropic glutamate receptors, synaptogenesis, dendritic spine development and axonal pathfinding. We investigated the potential modulation of glutamatergic synaptic transmission by the immediate early gene product Homer‐1a/Vesl‐1S and by the constitutively expressed long‐form Homer‐1c/Vesl‐1L in CA1 pyramidal cells from cultured rat hippocampal slices. Semliki Forest virus vector‐mediated overexpression of Homer‐1a enhanced alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor function, but did not detectably affect N‐methyl‐d‐aspartate (NMDA) receptor function and presynaptic glutamate release. Overexpression of Homer‐1c, by contrast, did not alter synaptic transmission. To corroborate our electrophysiological results obtained in slice cultures, we performed quantitative immunocytochemistry in cultures of dissociated hippocampal neurons. Homer‐1a also increased synaptic clustering of AMPA but not NMDA receptors, whereas Homer‐1c had no detectable effect. Our results show that Homer‐1a potentiates synaptic AMPA receptor function, supporting a critical role for Homer‐1a in hippocampal synaptic plasticity.

Cornichon-2 Modulates AMPA Receptor–Transmembrane AMPA Receptor Regulatory Protein Assembly to Dictate Gating and Pharmacology

Neuronal AMPA receptor complexes comprise a tetramer of GluA pore-forming subunits as well as accessory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon-2/3 (CNIH-2/3). The mechanisms that control AMPA receptor complex assembly remain unclear. AMPA receptor responses in neurons differ from those in cell lines transfected with GluA plus TARPs γ-8 or γ-7, which show unusual resensitization kinetics and non-native AMPA receptor pharmacologies. Using tandem GluA/TARP constructs to constrain stoichiometry, we show here that these peculiar kinetic and pharmacological signatures occur in channels with four TARP subunits per complex. Reducing the number of TARPs per complex produces AMPA receptors with neuron-like kinetics and pharmacologies, suggesting a neuronal mechanism controls GluA/TARP assembly. Importantly, we find that coexpression of CNIH-2 with GluA/TARP complexes reduces TARP stoichiometry within AMPA receptors. In both rat and mouse hippocampal neurons, CNIH-2 also associates with AMPA receptors on the neuronal surface in a γ-8-dependent manner to dictate receptor pharmacology. In the cerebellum, however, CNIH-2 expressed in Purkinje neurons does not reach the neuronal surface. In concordance, stargazer Purkinje neurons, which express CNIH-2 and γ-7, display AMPA receptor kinetics/pharmacologies that can only be recapitulated recombinantly by a low γ-7/GluA stoichiometry. Together, these data suggest that CNIH-2 modulates neuronal AMPA receptor auxiliary subunit assembly by regulating the number of TARPs within an AMPA receptor complex to modulate receptor gating and pharmacology.

PDZ binding of TARPγ-8 controls synaptic transmission but not synaptic plasticity.

The reduction in synaptic transmission and plasticity in mice lacking the hippocampus-enriched AMPA receptor (AMPAR) auxiliary subunit TARPγ-8 could be a result of a reduction in AMPAR expression or of the direct action of γ-8. We generated TARPγ-8Δ4 knock-in mice lacking the C-terminal PDZ ligand. We found that synaptic transmission and AMPARs were reduced in the mutant mice, but extrasynaptic AMPAR expression and long-term potentiation (LTP) were unaltered. Our findings suggest that there are distinct TARP-dependent mechanisms for synaptic transmission and LTP.

Glutamate Receptor δ2 Associates with Metabotropic Glutamate Receptor 1 (mGluR1), Protein Kinase Cγ, and Canonical Transient Receptor Potential 3 and Regulates mGluR1-Mediated Synaptic Transmission in Cerebellar Purkinje Neurons

Cerebellar motor coordination and cerebellar Purkinje cell synaptic function require metabotropic glutamate receptor 1 (mGluR1, Grm1). We used an unbiased proteomic approach to identify protein partners for mGluR1 in cerebellum and discovered glutamate receptor δ2 (GluRδ2, Grid2, GluΔ2) and protein kinase Cγ (PKCγ) as major interactors. We also found canonical transient receptor potential 3 (TRPC3), which is also needed for mGluR1-dependent slow EPSCs and motor coordination and associates with mGluR1, GluRδ2, and PKCγ. Mutation of GluRδ2 changes subcellular fractionation of mGluR1 and TRPC3 to increase their surface expression. Fitting with this, mGluR1-evoked inward currents are increased in GluRδ2 mutant mice. Moreover, loss of GluRδ2 disrupts the time course of mGluR1-dependent synaptic transmission at parallel fiber–Purkinje cells synapses. Thus, GluRδ2 is part of the mGluR1 signaling complex needed for cerebellar synaptic function and motor coordination, explaining the shared cerebellar motor phenotype that manifests in mutants of the mGluR1 and GluRδ2 signaling pathways.

Activity-inducible protein Homer1a/Vesl-1S promotes redistribution of postsynaptic protein Homer1c/Vesl-1L in cultured rat hippocampal neurons

In cultured rat hippocampal neurons, overexpression of Homer1a/Vesl-1S, an inducible protein upregulated by seizure or long-term potentiation, caused a reduction of punctate distribution of a postsynaptic protein Homer1c/Vesl-1L, without significant decrease in its total amount. Clusters of F-actin were also decreased. Treatments of cells with BDNF or a proteasome inhibitor, which cause increase in the expression level of endogenous Homer1a, also resulted in the reduction of Homer1c puncta. These results indicate that the accumulation of Homer1a, either exogenously expressed or endogenously induced, caused redistribution and dispersion of postsynaptic clusters of Homer1c and F-actin, suggesting an important role of Homer1a in synaptic remodeling.

Phorbol esters promote postsynaptic accumulation of Vesl-1S/Homer-1a protein: Phorbol esters promote postsynaptic accumulation of Vesl-1S

We examined effects of phorbol esters on the amount and the subcellular distribution of the activity‐regulated protein Vesl‐1S/Homer‐1a in cultured hippocampal neurons. Major Vesl‐1S immunoreactivity (IR) was detected throughout neuronal somata under control conditions. Bath application of phorbol esters, PMA and PDBu resulted in the increase in the amount of Vesl‐1S proteins and promoted punctate distribution of Vesl‐1S IR at the cortical regions of the neuronal somata. Immunofluorescent observations using antisynaptophysin and anti‐Vesl‐1S antibodies, and electron microscopic observations, revealed that Vesl‐1S accumulated at postsynaptic regions following PMA application. Membrane depolarization with high concentrations of external potassium also promoted the punctate distribution of Vesl‐1S IR. These results demonstrate that phorbol‐triggered reaction cascades result in the accumulation of Vesl‐1S protein at postsynaptic regions, and suggest that these phorbol effects may mimic those caused by synaptic activities.