Kimihiko Kameyama

Input- and subunit-specific AMPA receptor trafficking underlying long-term potentiation at hippocampal CA3 synapses

Hippocampal CA3 pyramidal neurons receive synaptic inputs from both mossy fibres (MFs) and associational fibres (AFs). Long‐term potentiation (LTP) at these synapses differs in its induction sites and N‐methyl‐D‐aspartate receptor (NMDAR) dependence. Most evidence favours the presynaptic and postsynaptic mechanisms for induction of MF LTP and AF LTP, respectively. This implies that molecular and functional properties differ between MF and AF synapses at both presynaptic and postsynaptic sites. In this study, we focused on the difference in the postsynaptic trafficking of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (AMPARs) between these synapses. To trace the subunit‐specific trafficking of AMPARs at each synapse, GluR1 and GluR2 subunits were introduced into CA3 pyramidal neurons in hippocampal organotypic cultures using the Sindbis viral expression system. The electrophysiologically‐tagged GluR2 AMPARs, produced by the viral‐mediated transfer of the unedited form of GluR2 (GluR2Q), were inserted into both MF and AF postsynaptic sites in a neuronal activity‐independent manner. Endogenous Ca2+‐impermeable AMPARs at these synapses were replaced with exogenous Ca2+‐permeable receptors, and Ca2+ influx via the newly expressed postsynaptic AMPARs induced NMDAR‐independent LTP at AF synapses. In contrast, no GluR1 AMPAR produced by the gene transfer was constitutively incorporated into AF postsynaptic sites, and only a small amount into MF postsynaptic sites. The synaptic trafficking of GluR1 AMPARs was triggered by the activity of Ca2+/calmodulin‐dependent kinase II or high‐frequency stimulation to induce LTP at AF synapses, but not at MF synapses. These results indicate that MF and AF postsynaptic sites possess distinct properties for AMPAR trafficking in CA3 pyramidal neurons.

Overexpression of Ca2+-permeable AMPA receptor promotes delayed cell death of hippocampal CA1 neurons following transient forebrain ischemia.

To examine the role of Ca(2+) entry through AMPA receptors in the pathogenesis of the ischemia-induced cell death of hippocampal neurons, we delivered cDNA of Q/R site-unedited form (GluR2Q) of AMPA receptor subunit GluR2 in the hippocampus by using an HVJ-liposome-mediated gene transfer technique. Two days prior to transient forebrain ischemia, we injected an HVJ-liposome containing cDNA of the GluR2Q-myc fusion gene into a rat unilateral hippocampus. In the absence of ischemic insult, overexpression of Ca(2+)-permeable GluR2Q did not cause any neurodegeneration in the cDNA-injected hippocampus. In ischemic rats, overexpression of Ca(2+)-permeable GluR2Q markedly promoted ischemic cell death of CA1 pyramidal neurons, while complete rescue of CA1 pyramidal neurons from ischemic damage occurred in the hippocampal hemisphere opposite the GluR2Q expression. Overexpression of the Q/R-site edited form (GluR2R) of subunit GluR2 did not affect the ischemia-induced damage of CA1 pyramidal neurons. From these results, we suggest that the Ca(2+)-permeability of AMPA receptors does not have a direct contribution to glutamate receptor-mediated neurotoxicity but has a promotive action in the evolution of ischemia-induced neurodegeneration of vulnerable neurons.

Melatonin signaling regulates locomotion behavior and homeostatic states through distinct receptor pathways in Caenorhabditis elegans

Melatonin is a hormone that controls circadian rhythms and seasonal behavioral changes in vertebrates. Recent studies indicate that melatonin participates in diverse physiological functions including the modulation of neural activities. Melatonin is also detected in many other organisms that do not exhibit obvious circadian rhythms, but their precise functions are not known. To understand the role of melatonin and its genetic pathway in vivo, we examined the effects of melatonin and its receptor antagonists on various behaviors in Caenorhabditis elegans. Exogenously applied melatonin specifically decreased locomotion rates in 15-min treatments, suggesting that melatonin directly regulates neural activities for locomotion. This melatonin signaling functions through MT1-like melatonin receptors, because the MT1/2 receptor antagonist luzindole effectively blocked the effect of melatonin on locomotion, while MT2-specific antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT) and MT3-selective antagonist prazosin had no effect. Alternatively, long-term treatment with prazosin specifically altered homeostatic states of the worm, suggesting another melatonin-signaling pathway through MT3-like receptors. We also found that two G-protein alpha subunit mutants and newly isolated five mutants exhibited defects in response to melatonin. Our findings imply that melatonin acts as a neuromodulator by regulating locomotion behavior and as a ligand for homeostatic control through distinct receptor pathways in C. elegans.

Regulation of AMPA receptor trafficking by δ-catenin

delta-catenin is a protein that binds to the classical cadherins and to synaptic scaffolding proteins in a manner which allows the protein to serve as a link between the adherens junction and the postsynaptic complex. Here we show the regulatory role of delta-catenin on synaptic transmission. delta-catenin increased the AMPA receptor-mediated EPSC, but had no significant effect on the NMDA receptor-mediated EPSC. The effect of delta-catenin on the AMPAR EPSC was mediated by its PDZ ligand. delta-catenin regulates the surface expression of GluR2 in the dendritic spines of neurons. Immunoprecipitation revealed that delta-catenin bound to GRIP-1. In COS7 cells, co-transfection of delta-catenin, GRIP and GluR2 showed that delta-catenin regulates the membrane localization of GRIP through its PDZ ligand and increased the surface expression of GluR2. This study directly shows that delta-catenin is essential for the trafficking and positioning GluR2 in the spine and thus further links delta-catenin to neuronal plasticity.

The C2A domain in dysferlin is important for association with MG53 (TRIM72)

In skeletal muscle, Mitsugumin 53 (MG53), also known as muscle-specific tripartite motif 72, reportedly interacts with dysferlin to regulate membrane repair. To better understand the interactions between dysferlin and MG53, we conducted immunoprecipitation (IP) and pull-down assays. Based on IP assays, the C2A domain in dysferlin associated with MG53. MG53 reportedly exists as a monomer, a homodimer, or an oligomer, depending on the redox state. Based on pull-down assays, wild-type dysferlin associated with MG53 dimers in a Ca2+-dependent manner, but MG53 oligomers associated with both wild-type and C2A-mutant dysferlin in a Ca2+-independent manner. In pull-down assays, a pathogenic missense mutation in the C2A domain (W52R-C2A) inhibited the association between dysferlin and MG53 dimers, but another missense mutation (V67D-C2A) altered the calcium sensitivity of the association between the C2A domain and MG53 dimers. In contrast to the multimers, the MG53 monomers did not interact with wild-type or C2A mutant dysferlin in pull-down assays. These results indicated that the C2A domain in dysferlin is important for the Ca2+-dependent association with MG53 dimers and that dysferlin may associate with MG53 dimers in response to the influx of Ca2+ that occurs during membrane injury. To examine the biological role of the association between dysferlin and MG53, we co-expressed EGFP-dysferlin with RFP-tagged wild-type MG53 or RFP-tagged mutant MG53 (RFP-C242A-MG53) in mouse skeletal muscle, and observed molecular behavior during sarcolemmal repair; it has been reported that the C242A-MG53 mutant forms dimers, but not oligomers. In response to membrane wounding, dysferlin accumulated at the injury site within 1 second; this dysferlin accumulation was followed by the accumulation of wild-type MG53. However, accumulation of RFP-C242A MG53 at the wounded site was impaired relative to that of RFP-wild-type MG53. Co-transfection of RFP-C242A MG53 inhibited the recruitment of dysferlin to the sarcolemmal injury site. We also examined the molecular behavior of GFP-wild-type MG53 during sarcolemmal repair in dysferlin-deficient mice which show progressive muscular dystrophy, and found that GFP-MG53 accumulated at the wound similar to wild-type mice. Our data indicate that the coordination between dysferlin and MG53 plays an important role in efficient sarcolemmal repair.

Affixin activates Rac1 via βPIX in C2C12 myoblast

— MINT‐6179203, MINT‐6179212, MINT‐6178859, MINT‐6178812, MINT‐6178832, MINT‐6178843: Affixin (uniprotkb:Q9HBI1) physically interacts (MI:0218) with βpix (uniprotkb:Q9ES28) by coimmunoprecipitation (MI:0019) — MINT‐6179221: Affixin (uniprotkb:Q9HBI1) physically interacts (MI:0218) with αpix (uniprotkb:Q8K4I3) by coimmunoprecipitation (MI:0019) — MINT‐6178962, MINT‐6178983: Affixin (uniprotkb:Q9HBI1) physically interacts (MI:0218) with βpix (uniprotkb:Q9ES28) by pull-down (MI:0096) — MINT‐6179002, MINT‐6179021: Affixin (uniprotkb:Q9HBI1) binds (MI:0407) βpix (uniprotkb:Q9ES28) by pull-down (MI:0096) — MINT‐6179039: PAK1 (uniprotkb:Q13153) physically interacts (MI:0218) with Rac1 (uniprotkb:P63001) by pull-down (MI:0096) — MINT‐6179054: PAK1 (uniprotkb:Q13153) physically interacts (MI:0218) with Cdc42 (uniprotkb:P70766) by pull-down (MI:0096) — MINT‐6178790: Affixin (uniprotkb:Q9HBI1) and αpix (uniprotkb:Q8K4I3) colocalize (MI:0403) by fluorescence microscopy (MI:0416) — MINT‐6178760: Affixin (uniprotkb:Q9HBI1) and βpix (uniprotkb:Q9ES28) colocalize (MI:0403) by fluorescence microscopy (MI:0416) — MINT‐6178801: Affixin (uniprotkb:Q9HBI1) and dysferlin (uniprotkb:Q9ESD7) colocalize (MI:0403) by fluorescence microscopy (MI:0416) — MINT‐6178779: Affixin (uniprotkb:Q9HBI1) and ILK (uniprotkb:O55222) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Recent advances in the study of AMPA receptors.

As glutamate is a dominant excitatory neurotransmitter in the central nervous system, glutamate receptors, and especially AMPA receptors, are located ubiquitously in all brain areas. In this paper, we reviewed recent advances of studies on AMPA receptor functions. AMPA receptors are cation-conducting complexes composed of various combinations of four subunits (GluR1 to GluR4). The glutamine residue located in the pore-forming segment of GluR2 subunit (Q/R site) is changed to arginine by RNA editing at the pre mRNA stage in normal adult mammalian animal. The edited GluR2 subunit is a major determination of Ca(2+) permeability of the AMPA receptor; only edited GluR2-lacking receptor shows high-Ca(2+) permeability. The assembly of glutamate AMPA receptor subunit is not completely according to the stochastic theory. The heteromeric subunits assembly is more rapid than the homomeric assembly is. The transfer of AMPA receptor subunit to the plasma membrane is conducted in multiple ways. Many molecules that interact with the intracellular domain of AMPA receptor subunits are reported as the modulators of AMPA receptor subunit transfer. In the motoneuron of sporadic amyotrophic lateral sclerosis (ALS) patients, the efficiency of RNA editing at the GluR2 Q/R site is significantly decreased. Relative low level of edited GluR2 subunit expression is likely responsible for motoneuronal death in ALS. Recently, AMPA receptors in glial cells have been studied. Bergmann glial cells in cerebellum express Ca(2+)-permeable AMPA receptors. Conversion of these AMPA receptors to Ca(2+)-impermeable type receptors induces morphological and functional changes. Glioblastoma cells also express Ca(2+)-permeable AMPA receptors, and their conversion to Ca(2+)-impermeable receptors inhibits cell locomotion and induces apoptosis.

Caenorhabditis elegans Rab escort protein (REP-1) differently regulates each Rab protein function and localization in a tissue-dependent manner.

Rab proteins play a critical role in intracellular vesicle trafficking and require post‐translational modification by adding lipids at the C‐terminus for proper functions. This modification is preceded by the formation of a trimeric protein complex with the Rab escort protein (REP) and the Rab geranylgeranyltransferase (RabGGTase). However, the genetic hierarchy among these proteins and the tissue‐specificity of each protein function are not yet clearly understood. Here we identified the Caenorhabditis elegans rep‐1 gene and found that a rep‐1 mutant showed a mild defect in synaptic transmission and defecation behaviors. Genetic analyses using the exocytic Rab mutants rab‐3 or rab‐27 suggested that rep‐1 functions only in the RAB‐27 pathway, and not in the RAB‐3 pathway, for synaptic transmission at neuromuscular junctions. However, the disruption of REP‐1 did not cause defecation defects compared to severe defects in either RAB‐27 or RabGGTase disruption, suggesting that REP‐1 is not essential for RAB‐27 signaling in defection. Some Rab proteins did not physically interact with REP‐1, and localization of these Rab proteins was not severely affected by REP‐1 disruption. These findings suggest that REP‐1 functions are required in specific Rab pathways and in specific tissues, and that some Rab proteins are functionally prenylated without REP‐1.

Directed evolution of three-finger toxin to produce serine protease inhibitors.

Abstract Directed evolution is a very popular strategy for improving biophysical properties and even for generating proteins with novel functions. Recent advances in combinatorial protein engineering mean it is now possible to develop protein scaffolds that could substitute for whole antibody-associated properties as emerging therapeutic proteins. In particular, disulfide-rich proteins are attractive templates for directed evolution in the search for novel molecules because they can regulate the activities of receptors, enzymes, and other molecules. Previously, we demonstrated that functional regulatory molecules against interleukin-6 receptor (IL-6R) could be obtained by directed evolution of the three-finger toxin (3F) scaffold. In the present study, trypsin was selected as a target for directed evolution to further explore the potential use of the 3F cDNA display library. After seven rounds of selection, the DNA sequences converged. The recombinant proteins produced by the selected candidates had inhibitory activity against trypsin (Ki of 33–450 nM). Three of the six groups had Ki values that were comparable to bovine pancreatic trypsin inhibitor and soybean trypsin inhibitor. Two of the candidates also had inhibitory effects against chymotrypsin and kallikrein. This study suggests that 3F protein is suitable for the preparation of high-diversity libraries that can be utilized to obtain protease inhibitors. In addition to our previous successful targeting of IL-6R, the technique developed in our studies may have wide applications in the generation of regulatory molecules for targets of interest, such as receptors, enzymes for research, diagnostic applications, and therapeutic uses.

G.P.48 Affixin is involved in sarcolemmal repair

Abstract Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and LGMD2B. In the presence of Ca2+, dysferlin accumulates around the damaged membrane site and is suggested to mediate sarcolemmal repair. We previously reported that affixin is a dysferlin-binding protein and co-localizes with dysferlin at the sarcolemma of normal human skeletal muscle. The association of dysferlin with affixin was confirmed by immunoprecipitation study using normal human skeletal muscles and COS-7 transfectants. The immunoreactivity of affixin was reduced in sarcolemma of dysferlinopathy muscles. We also reported that affixin activates Rac1 via GDP/GTP nucleotide exchange factor (GEF) and regulates the reorganization of cytoskeletal actin. The purpose of this study is to examine the effect of calcium on the association of dysferlin with affixin and to test the possibility that affixin is involved in sarcolemmal repair. The calcium-dependency of dysferlin–affixin association was examined by pull-down assay using lysates from COS-7 transfectants expressing human dysferlin and bacterially expressed affixin CH1 domain. To clarify molecular behavior of affixin in sarcolemmal repair, mCherry-tagged human affixin was expressed in FDB of mice (C57BL/6J and dysferlin-deficient A/J) by electroporation. Membrane wound-repair assay of single myofiber was performed using confocal microscope equipped two-photon laser. Pull-down assay revealed that dysferlin associates with affixin in a calcium-dependent manner. However, the association of dysferlin and caveolin-3 was not affected in different calcium concentrations. mCherry–affixin accumulates at the wounded site in C57BL/6J mice, however accumulation of affixin was not observed in A/J mice. These results suggest affixin involvement in sarcolemmal repair. We are analyzing movement of mCherry–affixin and dysferlin–GFP during sarcolemmal repair.