Eye opening induces a rapid dendritic localization of PSD-95 in central visual neurons.
ABSTRACT: The membrane-associated guanylate kinase PSD-95 scaffolds N-methyl-d-aspartate receptors to cytoplasmic signaling molecules, and associates with other glutamate receptors at central synapses. However, regulation of PSD-95 in vivo is poorly understood. We provide evidence of an activity-dependent redistribution of PSD-95 to dendrites in central visual neurons that is tied to eye opening. Six hours after eye opening, increased dendritic PSD-95 coimmunoprecipitates with the same proportions of stargazin, increased proportions of the N-methyl-d-aspartate receptor subunit NR2A, and decreased proportions of NR2B. Sustained high levels of PSD-95 in dendrites are dependent on continued pattern vision in juvenile but not mature animals, suggesting that the stabilization of PSD-95 at synapses may be involved in the control of developmental plasticity.
Project description:Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like membrane-associated guanylate kinases. Among the three classes of ionotropic glutamate receptors (AMPA, NMDA, and kainate type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively charged lipid bilayers in a phosphorylation-dependent manner and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction.
Project description:The mechanisms by which experience guides refinement of converging afferent pathways are poorly understood. We describe a vision-driven refinement of corticocollicular inputs that determines the consolidation of retinal and visual cortical (VC) synapses on individual neurons in the superficial superior colliculus (sSC). Highly refined corticocollicular terminals form 1-2 days after eye-opening (EO), accompanied by VC-dependent filopodia sprouting on proximal dendrites, and PSD-95 and VC-dependent quadrupling of functional synapses. Delayed EO eliminates synapses, corticocollicular terminals, and spines on VC-recipient dendrites. Awake recordings after EO show that VC and retina cooperate to activate sSC neurons, and VC light responses precede sSC responses within intervals promoting potentiation. Eyelid closure is associated with more protracted cortical visual responses, causing the majority of VC spikes to follow those of the colliculus. These data implicate spike-timing plasticity as a mechanism for cortical input survival, and support a cooperative strategy for retinal and cortical coinnervation of the sSC.
Project description:Stargazin is a transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory protein that controls the surface and synaptic expression of AMPA-type glutamate receptors (AMPARs). Synaptic anchoring of AMPARs is influenced by the interaction between stargazin's C-terminal post-synaptic density-95 (PSD-95)/discs large/zona occludens-1 (PDZ) ligand and the synaptic scaffolding protein PSD-95. Phosphorylation of the stargazin PDZ ligand by protein kinase A (PKA) disrupts stargazin's interaction with PSD-95, but whether this phosphorylation plays a role in activity-dependent regulation of stargazin/AMPAR synaptic trafficking is unknown. Here, we show that stargazin is phosphorylated within the PDZ ligand at threonine residue 321 (T321) by mitogen-activated protein kinases (MAPKs) as well as PKA. By expressing constructs that selectively block T321 phosphorylation by either PKA or MAPKs, we show that stargazin T321 phosphorylation is required for activity-dependent changes in stargazin synaptic clustering in dissociated rat hippocampal neuron cultures. Specifically, we find that mutations that block stargazin T321 phosphorylation by PKA prevent activity-dependent increases in stargazin synaptic clustering, whereas a point mutant that blocks MAPK phosphorylation of T321 prevents activity-dependent decreases in stargazin synaptic clustering. Taken together, our studies implicate phosphorylation of stargazin T321 by PKA and MAPKs in bidirectional control of stargazin/AMPAR synaptic clustering during synaptic plasticity.
Project description:The plasticity of excitatory synapses is an essential brain process involved in cognitive functions, and dysfunctions of such adaptations have been linked to psychiatric disorders such as depression. Although the intracellular cascades that are altered in models of depression and stress-related disorders have been under considerable scrutiny, the molecular interplay between antidepressants and glutamatergic signaling remains elusive. Using a combination of electrophysiological and single nanoparticle tracking approaches, we here report that the cognitive enhancer and antidepressant tianeptine (S 1574, [3-chloro-6-methyl-5,5-dioxo-6,11-dihydro-(c,f)-dibenzo-(1,2-thiazepine)-11-yl) amino]-7 heptanoic acid, sodium salt) favors synaptic plasticity in hippocampal neurons both under basal conditions and after acute stress. Strikingly, tianeptine rapidly reduces the surface diffusion of AMPA receptor (AMPAR) through a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent mechanism that enhances the binding of AMPAR auxiliary subunit stargazin with PSD-95. This prevents corticosterone-induced AMPAR surface dispersal and restores long-term potentiation of acutely stressed mice. Collectively, these data provide the first evidence that a therapeutically used drug targets the surface diffusion of AMPAR through a CaMKII-stargazin-PSD-95 pathway, to promote long-term synaptic plasticity.
Project description:The N-methyl-d-aspartate (NMDA) glutamate receptor (NMDAR), long implicated in developmental plasticity, shows decay time kinetics that shorten postnatally as NR2A subunits are added to the receptor. Neither the mechanism nor immediate effect of this change is known. We studied developing NMDAR currents by using visual neurons in slices from NR2A knockout (NR2AKO) and WT mice. Both strains show increased dendritic levels of synaptic density scaffolding protein PSD-95 with age. Dendritic levels of NR2A increased at the same time in WT and immunoprecipitated with PSD-95. PSD-95NMDAR binding was significantly decreased in the NR2AKO. Moreover, NMDAR miniature currents (minis) were lost and rise times of NMDAR evoked currents increased in mutant mice. Age-matched WT cells showed NR2A-rich receptors predominating in minis, yet slow NR2B mediated currents persisted in evoked currents. Disrupting photoreceptor activation of retinal ganglion cells eliminated increases in PSD-95 and NR2A in superior collicular dendrites of WT mice and slowed the loss of miniature NMDAR currents in NR2AKOs. These data demonstrate that NMDARs that respond to single quantal events mature faster during development by expressing the NR2A subunit earlier than NMDARs that respond to evoked release. We hypothesize that NR2A-rich NMDARs may be localized to the center of developing synapses by an activity-dependent process that involves the targeting of PSD-95 to the postsynaptic density. Neonatal receptors become restricted to perisynpatic or extrasynaptic sites, where they participate primarily in evoked currents.
Project description: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 alpha-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.
Project description:The spatial coordination of neurotransmitter receptors with other postsynaptic signaling and structural molecules is regulated by a diverse array of cell-specific scaffolding proteins. The synaptic trafficking of AMPA receptors by the stargazin protein in some neurons, for example, depends on specific interactions between the C terminus of stargazin and the PDZ [postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] domains of membrane-associated guanylate kinase scaffolding proteins PSD-93 or PSD-95. Stargazin [Cacng2 (Ca2+ channel gamma2 subunit)] is one of four closely related proteins recently categorized as transmembrane AMPA receptor regulating proteins (TARPs) that appear to share similar functions but exhibit distinct expression patterns in the CNS. We used yeast two-hybrid screening to identify MAGI-2 (membrane associated guanylate kinase, WW and PDZ domain containing 2) as a novel candidate interactor with the cytoplasmic C termini of the TARPs. MAGI-2 [also known as S-SCAM (synaptic scaffolding molecule)] is a multi-PDZ domain scaffolding protein that interacts with several different ligands in brain, including PTEN (phosphatase and tensin homolog), dasm1 (dendrite arborization and synapse maturation 1), dendrin, axin, beta- and delta-catenin, neuroligin, hyperpolarization-activated cation channels, beta1-adrenergic receptors, and NMDA receptors. We confirmed that MAGI-2 coimmunoprecipitated with stargazin in vivo from mouse cerebral cortex and used in vitro assays to localize the interaction to the C-terminal -TTPV amino acid motif of stargazin and the PDZ1, PDZ3, and PDZ5 domains of MAGI-2. Expression of stargazin recruited MAGI-2 to cell membranes and cell-cell contact sites in transfected HEK-293T cells dependent on the presence of the stargazin -TTPV motif. These experiments identify MAGI-2 as a strong candidate for linking TARP/AMPA receptor complexes to a wide range of other postsynaptic molecules and pathways and advance our knowledge of protein interactions at mammalian CNS synapses.
Project description:Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs) modulate AMPAR synaptic trafficking and transmission via disc-large (DLG) subfamily of membrane-associated guanylate kinases (MAGUKs). Despite extensive studies, the molecular mechanism governing specific TARP/MAGUK interaction remains elusive. Using stargazin and PSD-95 as the representatives, we discover that the entire tail of stargazin (Stg_CT) is required for binding to PSD-95. The PDZ binding motif (PBM) and an Arg-rich motif upstream of PBM conserved in TARPs bind to multiple sites on PSD-95, thus resulting in a highly specific and multivalent stargazin/PSD-95 complex. Stargazin in complex with PSD-95 or PSD-95-assembled postsynaptic complexes form highly concentrated and dynamic condensates via phase separation, reminiscent of stargazin/PSD-95-mediated AMPAR synaptic clustering and trapping. Importantly, charge neutralization mutations in TARP_CT Arg-rich motif weakened TARP's condensation with PSD-95 and impaired TARP-mediated AMPAR synaptic transmission in mice hippocampal neurons. The TARP_CT/PSD-95 interaction mode may have implications for understanding clustering of other synaptic transmembrane proteins.
Project description:PSD-95 is a major scaffolding protein of the post-synaptic density (PSD) of a glutamatergic synapse. PSD-95, via interactions with stargazin, anchors AMPA receptors at the synapse and regulates AMPAR currents. The expression of PSD-95 is regulated during synaptic plasticity. It is, however, unknown whether this regulation is required for induction of functional plasticity of glutamatergic synapses. Here, we show that NMDA-induced long-term depression of synaptic transmission (NMDA-LTD) is accompanied by downregulation of PSD-95 protein levels. Using pharmacologic and molecular manipulations, we further demonstrate that the NMDA-induced downregulation of PSD-95 depends on the activation of CaMKII and CaMKII-driven phosphorylation of PSD-95 serine 73. Surprisingly, neither CaMKII activity nor CaMKII-dependent phosphorylation of PSD-95 serine 73 are required for the expression of NMDA-LTD. These results support the hypothesis that synaptic plasticity of AMPARs may occur without dynamic regulation of PSD-95 protein levels.
Project description:The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95-like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.