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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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, using LC-MS/MS-based protein analysis, confocal microscopy, RNAscope assays, and electrophysiological recordings, we show that β-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert-positive β-Nrxn variants, but not insert-negative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo Neuron-specific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4-positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits, including Schaffer collateral afferents., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Kim et al.)

The glutamate receptor delta2 (GluRdelta2) is predominantly expressed at parallel fiber-Purkinje cell postsynapses and plays crucial roles in synaptogenesis and synaptic plasticity. Although the mechanism by which GluRdelta2 functions remains unclear, its lack of channel activity and its role in controlling the endocytosis of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors have suggested that GluRdelta2 may convey signals by interacting with intracellular signaling molecules. Among several proteins that interact with GluRdelta2, delphilin is unique in that it is selectively expressed at parallel fiber-Purkinje cell synapses and that, in addition to a single PDZ domain, it contains a formin homology domain that is thought to regulate actin dynamics. Here, we report a new isoform of delphilin, designated as L-delphilin, that has alternatively spliced N-terminal exons encoding an additional PDZ domain. Although original delphilin, designated S-delphilin, was palmitoylated at the N terminus, this region was spliced out in L-delphilin. As a result, S-delphilin was associated with plasma membranes in COS cells and dendritic spines in hippocampal neurons, whereas L-delphilin formed clusters in soma and dendritic shafts. In addition, S-delphilin, but not L-delphilin, facilitated the expression of GluRdelta2 on the cell surface. These results indicate that, like PSD-95 and GRIP/ABP, delphilin isoforms with differential palmitoylation and clustering capabilities may provide two separate intracellular and surface GluRdelta2 pools and may control GluRdelta2 signaling in Purkinje cells.

The number of each subclass of ionotropic glutamate receptors (iGluRs) at the spines is differentially regulated either constitutively or in a neuronal activity-dependent manner. The delta2 glutamate receptor (GluRdelta2) is abundantly expressed at the spines of Purkinje cell dendrites and controls synaptic plasticity in the cerebellum. To obtain clues to the trafficking mechanism of the iGluRs, we expressed wild-type or mutant GluRdelta2 in cultured hippocampal and Purkinje neurons and analyzed their intracellular localization using immunocytochemical techniques. Quantitative analysis revealed that deletion of the 20 amino acids at the center of the C terminus (region E) significantly reduced the amount of GluRdelta2 protein at the spines in both types of neurons. This effect was partially antagonized by the inhibition of endocytosis by high dose sucrose treatment or coexpression of dominant negative dynamin. In addition, mutant GluRdelta2 lacking the E region (GluRdelta2DeltaE), but not wild-type GluRdelta2, was found to colocalize with the endosomal markers Rab4 and Rab7. Moreover, the antibody-feeding assay revealed that GluRdelta2DeltaE was internalized more rapidly than GluRdelta2wt. These results indicate that the E region (more specifically, a 12-amino-acid-long segment of the E2 region) is necessary for rendering GluRdelta2 resistant to endocytosis from the cell surface at the spines. Furthermore, insertion of the E2 region alone into the C terminus of the GluR1 subtype of iGluRs was sufficient to increase the amount of GluR1 proteins in the spines. Therefore, we propose that the E2 region of GluRdelta2 is necessary, and also sufficient, to inhibit endocytosis of the receptor from postsynaptic membranes.

The alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptors (iGluRs) mediates fast excitatory neurotransmission in the mammalian brain. Although the most N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain is suggested to play a role in the initial assembly of iGluR subunits, it is unclear how this domain is arranged and functions in intact iGluRs. Similarly, although recent crystallographic analyses indicate that the isolated ligand-binding lysine/arginine/ornithine-binding protein domain forms a 2-fold symmetric dimer, the subunit stoichiometry of intact iGluRs remains elusive. Here, we developed a new approach to address these issues. The LIVBP domain of the GluR1 subunit of AMPA receptors was replaced by leucine-zipper peptides designed to form stable symmetric dimers, trimers, tetramers, or pentamers. All these mutant GluR1s were expressed in human embryonic kidney 293 cells and were transported to the cell surface as well as wild type GluR1. Functional and biochemical analyses indicated that these oligomerizing peptides specifically controlled the formation of the expected number of subunits in a channel complex. However, the channel function was only restored by the tetramer-forming peptide. Although the purified LIVBP domain of GluR1 formed a dimmer in solution, a dimer-forming peptide could not restore the function of GluR1. Moreover, a cross-linking assay indicated that four LIVBP domains are located in proximity to each other. These results suggest that the function of the LIVBP domain is not simply to form initial dimers but to adopt a conformation compatible with the overall tetrameric arrangement of subunits in intact AMPA receptors.

The bcl-2 oncogene product delays apoptotic cell death and prolongs the cell survival. We cloned two bcl-2-related cDNAs from a rat thymus cDNA library by low stringency hybridization with a rat bcl-2 fragment as a probe. One of these, designated bcl-xalpha, was a counterpart of the human bcl-xL reported previously as a bcl-2-related gene (Boise, L. H., Gonzalez-Garcia, M., Postema, C. E. , Ding, L., Lindsten, T., Turka, L. A., Mao, M., Nunez, G., and Thompson, C. B. (1993) Cell 74, 597-608). The other, designated bcl-xbeta, was novel and found to be generated by an unspliced mRNA, whereas bcl-xalpha was generated from a spliced transcript. The splice junction exactly corresponded to that found in the bcl-2 gene. bcl-xbeta was specifically expressed in cerebellum, heart, and thymus. When bcl-xbeta directed by a strong promoter was introduced into an interleukin-3-dependent promyeloid cell line, FDC-P1, DNA fragmentation was observed even in the growing state in the presence of interleukin-3 although not in the control transfectants. This finding suggests that the rat bcl-xbeta gene product promotes apoptosis in the promyeloid cells.

We isolated a cDNA encoding an inositol 1,4,5-trisphosphate receptor (InsP3R) of Drosophila melanogaster. The predicted Drosophila InsP3R (2,833 amino acids) has extensive sequence similarity to the mouse InsP3R. The polypeptide encoded by the cDNA was functionally expressed and showed characteristic InsP3-binding activity. The Drosophila InsP3R gene is located at the region 83A5-9 on the third chromosome and expresses throughout development but predominantly in the adult. Localization of the InsP3R mRNA in adult tissues suggests strong expression in the retina and antenna, indicating the involvement of the InsP3R in visual and olfactory transduction. In addition, the InsP3R mRNA is abundant in the legs and thorax, which are enriched with a muscular system. Such localization is apparently consistent with the quantitatively predominant sites for [3H]InsP3 binding in Drosophila and the fleshfly (Boettcherisca peregrina). The present study points to the likely functional importance of the InsP3/Ca2+ signaling system in Drosophila.