Composition of the synaptic PSD-95 complex

Postsynaptic density protein 95 (PSD-95), a specialized scaffold protein with multiple protein interaction domains, forms the backbone of an extensive postsynaptic protein complex that organizes receptors and signal transduction molecules at the synaptic contact zone. Large, detergent-insoluble PSD-95-based postsynaptic complexes can be affinity-purified from conventional PSD fractions using magnetic beads coated with a PSD-95 antibody. In the present study purified PSD-95 complexes were analyzed by LC/MS/MS. A semiquantitative measure of the relative abundances of proteins in the purified PSD-95 complexes and the parent PSD fraction was estimated based on the cumulative ion current intensities of corresponding peptides. The affinity-purified preparation was largely depleted of presynaptic proteins, spectrin, intermediate filaments, and other contaminants prominent in the parent PSD fraction. We identified 525 of the proteins previously reported in parent PSD fractions, but only 288 of these were detected after affinity purification. We discuss 26 proteins that are major components in the PSD-95 complex based upon abundance ranking and affinity co-purification with PSD-95. This subset represents a minimal list of constituent proteins of the PSD-95 complex and includes, in addition to the specialized scaffolds and N-methyl-d-aspartate (NMDA) receptors, an abundance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, small G-protein regulators, cell adhesion molecules, and hypothetical proteins. The identification of two Arf regulators, BRAG1 and BRAG2b, as co-purifying components of the complex implies pivotal functions in spine plasticity such as the reorganization of the actin cytoskeleton and insertion and retrieval of proteins to and from the plasma membrane. Another co-purifying protein (Q8BZM2) with two sterile alpha motif domains may represent a novel structural core element of the PSD.     

PSD-95 promotes the stabilization of young synaptic contacts

Maintaining a population of stable synaptic connections is probably of critical importance for the preservation of memories and functional circuitry, but the molecular dynamics that underlie synapse stabilization is poorly understood. Here, we use simultaneous time-lapse imaging of post synaptic density-95 (PSD-95) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) to investigate the dynamics of protein composition at axodendritic (AD) contacts. Our data reveal that this composition is highly dynamic, with both proteins moving into and out of the same synapse independently, so that synapses cycle rapidly between states in which they are enriched for none, one or both proteins. We assessed how PSD-95 and CaMKII interact at stable and transient AD sites and found that both phospho-CaMKII and PSD-95 are present more often at stable than labile contacts. Finally, we found that synaptic contacts are more stable in older neurons, and this process can be mimicked in younger neurons by overexpression of PSD-95. Taken together, these data show that synaptic protein composition is highly variable over a time-scale of hours, and that PSD-95 is probably a key synaptic protein that promotes synapse stability.     

Synaptic accumulation of PSD-95 and synaptic function regulated by phosphorylation of serine-295 of PSD-95

The scaffold protein PSD-95 promotes the maturation and strengthening of excitatory synapses, functions that require proper localization of PSD-95 in the postsynaptic density (PSD). Here we report that phosphorylation of ser-295 enhances the synaptic accumulation of PSD-95 and the ability of PSD-95 to recruit surface AMPA receptors and potentiate excitatory postsynaptic currents. We present evidence that a Rac1-JNK1 signaling pathway mediates ser-295 phosphorylation and regulates synaptic content of PSD-95. Ser-295 phosphorylation is suppressed by chronic elevation, and increased by chronic silencing, of synaptic activity. Rapid dephosphorylation of ser-295 occurs in response to NMDA treatment that causes chemical long-term depression (LTD). Overexpression of a phosphomimicking mutant (S295D) of PSD-95 inhibited NMDA-induced AMPA receptor internalization and blocked the induction of LTD. The data suggest that synaptic strength can be regulated by phosphorylation-dephosphorylation of ser-295 of PSD-95 and that synaptic depression requires the dephosphorylation of ser-295.     

Rapid redistribution of synaptic PSD-95 in the neocortex in vivo

Most excitatory synapses terminate on dendritic spines. Spines vary in size, and their volumes are proportional to the area of the postsynaptic density (PSD) and synaptic strength. PSD-95 is an abundant multi-domain postsynaptic scaffolding protein that clusters glutamate receptors and organizes the associated signaling complexes. PSD-95 is thought to determine the size and strength of synapses. Although spines and their synapses can persist for months in vivo, PSD-95 and other PSD proteins have shorter half-lives in vitro, on the order of hours. To probe the mechanisms underlying synapse stability, we measured the dynamics of synaptic PSD-95 clusters in vivo. Using two-photon microscopy, we imaged PSD-95 tagged with GFP in layer 2/3 dendrites in the developing (postnatal day 10–21) barrel cortex. A subset of PSD-95 clusters was stable for days. Using two-photon photoactivation of PSD-95 tagged with photoactivatable GFP (paGFP), we measured the time over which PSD-95 molecules were retained in individual spines. Synaptic PSD-95 turned over rapidly (median retention times τ_r ~ 22–63 min from P10–P21) and exchanged with PSD-95 in neighboring spines by diffusion. PSDs therefore share a dynamic pool of PSD-95. Large PSDs in large spines captured more diffusing PSD-95 and also retained PSD-95 longer than small PSDs. Changes in the sizes of individual PSDs over days were associated with concomitant changes in PSD-95 retention times. Furthermore, retention times increased with developmental age (τ_r ~ 100 min at postnatal day 70) and decreased dramatically following sensory deprivation. Our data suggest that individual PSDs compete for PSD-95 and that the kinetic interactions between PSD molecules and PSDs are tuned to regulate PSD size.     

S-nitrosylation and S-palmitoylation reciprocally regulate synaptic targeting of PSD-95

PSD-95, a principal scaffolding component of the postsynaptic density, is targeted to synapses by palmitoylation, where it couples NMDA receptor stimulation to production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS). Here, we show that PSD-95 is physiologically S-nitrosylated. We identify cysteines 3 and 5, which are palmitoylated, as sites of nitrosylation, suggesting a competition between these two modifications. In support of this hypothesis, physiologically produced NO inhibits PSD-95 palmitoylation in granule cells of the cerebellum, decreasing the number of PSD-95 clusters at synaptic sites. Further, decreased palmitoylation, as seen in heterologous cells treated with 2-bromopalmitate or in ZDHHC8 knockout mice deficient in a PSD-95 palmitoyltransferase, results in increased PSD-95 nitrosylation. These data support a model in which NMDA-mediated production of NO regulates targeting of PSD-95 to synapses via mutually competitive cysteine modifications. Thus, differential modification of cysteines may represent a general paradigm in signal transduction.     

Synaptic strength regulated by palmitate cycling on PSD-95

Dynamic regulation of AMPA-type glutamate receptors represents a primary mechanism for controlling synaptic strength, though mechanisms for this process are poorly understood. The palmitoylated postsynaptic density protein, PSD-95, regulates synaptic plasticity and associates with the AMPA receptor trafficking protein, stargazin. Here, we identify palmitate cycling on PSD-95 at the synapse and find that palmitate turnover on PSD-95 is regulated by glutamate receptor activity. Acutely blocking palmitoylation disperses synaptic clusters of PSD-95 and causes a selective loss of synaptic AMPA receptors. We also find that rapid glutamate-mediated AMPA receptor internalization requires depalmitoylation of PSD-95. In a nonneuronal model system, clustering of PSD-95, stargazin, and AMPA receptors is also regulated by ongoing palmitoylation of PSD-95 at the plasma membrane. These studies suggest that palmitate cycling on PSD-95 can regulate synaptic strength and regulates aspects of activity-dependent plasticity.     

Molecular mechanisms regulating the differential association of kainate receptor subunits with SAP90/PSD-95 and SAP97

Recent studies have demonstrated that kainate receptors are associated with members of the SAP90/PSD-95 family (synapse-associated proteins (SAPs)) in neurons and that SAP90 can cluster and modify the electrophysiological properties of GluR6/KA2 kainate receptors when co-expressed in transfected cells. In vivo, SAP90 tightly binds kainate receptor subunits, while SAP97 is only weakly associated, suggesting that this glutamate receptor differentially associates with SAP90/PSD-95 family members. Here, green fluorescent protein (GFP)-tagged chimeras and deletion mutants of SAP97 and SAP90 were employed to define the molecular mechanism underlying their differential association with kainate receptors. Our results show that a weak interaction between GluR6 and the PDZ1 domain of SAP97 can account for the weak association of GluR6 with the full-length SAP97 observed in vivo. Expression studies in HEK293 cells and in vitro binding studies further show that although the individual Src homology 3 and guanylate kinase domains in SAP97 can interact with the C-terminal tail of KA2 subunit, specific intramolecular interactions in SAP97 (e.g. the SAP97 N terminus (S97N) binding to the Src homology 3 domain) interfere with KA2 binding to the full-length molecule. Because receptor subunits are known to segregate to different parts of the neuron, our results imply that differential association of kainate receptors with SAP family proteins may be one mechanism of subcellular localization.     

PSD-95与神经精神疾病

PSD-95（postsynaptic density protein 95）是在兴奋性突触后密集区中纯化鉴定出的一种脚手架蛋白。通过PDZ（1-3）、SH3和GK结构域,PSD-95募集多种信号分子,在谷氨酸受体的信号整合和转导中具有关键性作用。PSD-95的功能异常与多种神经精神疾病密切相关,是多种重大脑病防治的新的药物作用靶点。     

PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs) and synaptic plasticity

Activity-dependent modification of excitatory synaptic transmission is a fundamental mechanism for developmental plasticity of the neural circuits and experience-dependent plasticity. Synaptic glutamatergic receptors including AMPA receptors and NMDA receptors (AMPARs and NMDARs) are embedded in the postsynaptic density, a highly organized protein network. Overwhelming data have shown that PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs), a major family of scaffold proteins at glutamatergic synapses, regulate basal synaptic AMPAR function and trafficking. It is now clear that PSD-MAGUKs have multifaceted functions in regulating both basal synaptic transmission and synaptic plasticity. Here we discuss recent advancements in understanding the roles of PSD-95 and other family members of PSD-MAGUKs in synaptic plasticity, both as an anchoring protein for synaptic AMPARs and as a signaling scaffold for mediating the interaction of the signaling complex and NMDARs.     

Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD

Long-term depression (LTD) is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR)–dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs) via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.     

Spatial memory formation induces recruitment of NMDA receptor and PSD-95 to synaptic lipid rafts

NMDA receptors (NMDARs) activation in the hippocampus and insular cortex is necessary for spatial memory formation. Recent studies suggest that localization of NMDARs to lipid rafts enhance their signalization, since the kinases that phosphorylate its subunits are present in larger proportion in lipid raft membrane microdomains. We sought to determine the possibility that NMDAR translocation to synaptic lipid rafts occurs during plasticity processes such as memory formation. Our results show that water maze training induces a rapid recruitment of NMDAR subunits (NR1, NR2A, NR2B) and PSD-95 to synaptic lipid rafts and decrease in the post-synaptic density plus an increase of NR2B phosphorylation at tyrosine 1472 in the rat insular cortex. In the hippocampus, spatial training induces selective translocation of NR1 and NR2A subunits to lipid rafts. These results suggest that NMDARs translocate from the soluble fraction of post-synaptic membrane (non-raft PSD) to synaptic lipid raft during spatial memory formation. The recruitment of NMDA receptors and other proteins to lipid rafts could be an important mechanism for increasing the efficiency of synaptic transmission during synaptic plasticity process.     

Identification of PSD-95 palmitoylating enzymes

Palmitoylation is a lipid modification that plays a critical role in protein trafficking and function throughout the nervous system. Palmitoylation of PSD-95 is essential for its regulation of AMPA receptors and synaptic plasticity. The enzymes that mediate palmitoyl acyl transfer to PSD-95 have not yet been identified; however, proteins containing a DHHC cysteine-rich domain mediate palmitoyl acyl transferase activity in yeast. Here, we isolated 23 mammalian DHHC proteins and found that a subset specifically palmitoylated PSD-95 in vitro and in vivo. These PSD-95 palmitoyl transferases (P-PATs) showed substrate specificity, as they did not all enhance palmitoylation of Lck, SNAP-25b, Galpha(s), or H-Ras in cultured cells. Inhibition of P-PAT activity in neurons reduced palmitoylation and synaptic clustering of PSD-95 and diminished AMPA receptor-mediated neurotransmission. This study suggests that P-PATs regulate synaptic function through PSD-95 palmitoylation.     

Interaction between SAP97 and PSD-95, two Maguk proteins involved in synaptic trafficking of AMPA receptors

Synapse-associated protein 97 (SAP97) and postsynaptic density 95 (PSD-95) are closely related membrane-associated guanylate kinase homologs (Maguks) implicated in the synaptic targeting and anchoring of alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid (AMPA)-selective glutamate receptors. Prompted by accumulating evidence for an oligomeric nature of Maguks, we examined the potential of SAP97 and PSD-95 to form heteromeric complexes. SAP97 and PSD-95 coimmunoprecipitated from rat brain detergent extracts and subsequent glutathione S-transferase pull-down and immunoprecipitation experiments showed that the interaction is mediated by binding of the N-terminal segment of SAP97 (SAP97(NTD)) to the Src homology 3 domain of PSD-95 (PSD-95(SH3)). In cultured hippocampal neurons, expression of green fluorescent protein-tagged PSD-95 triggered accumulation of SAP97 in synaptic spines, which was totally inhibited by coexpression of PSD-95(SH3). Furthermore, overexpression of green fluorescent protein-PSD-95 induced dendritic clustering of GluR-A subunit-containing AMPA receptors, which was strongly inhibited by cotransfection with SAP97(NTD) and PSD-95(SH3) constructs. Our results demonstrated a direct interaction between SAP97 and PSD-95 and suggested that this association may play a functional role in the trafficking and clustering of AMPA receptors.     

Alternative N-terminal domains of PSD-95 and SAP97 govern activity-dependent regulation of synaptic AMPA receptor function

PSD-95 and SAP97 are scaffolding proteins that have been implicated in regulating AMPA receptor incorporation and function at synapses. Gain- and loss-of-function approaches, however, have generated conflicting results. To minimize adaptations during development and potential dominant-negative effects of overexpression, we have combined silencing of endogenous PSD-95 in mature neurons with heterologous expression of specific SAP97 or PSD-95 isoforms. We find that both PSD-95 and SAP97 contain alternative N termini expressing either double cysteines that normally are palmitoylated (alpha-isoforms) or an L27 domain (beta-isoforms). Whereas alpha-isoforms of PSD-95 and SAP97 influence AMPA receptor-mediated synaptic strength independent of activity, the effects of beta-isoforms are regulated by activity in a CaMKII-dependent manner. Importantly, the synaptic effects of the beta-isoforms are masked by the endogenous alpha-isoform of PSD-95. These results demonstrate that the different N termini of the predominant endogenous forms of PSD-95 (alpha-isoform) and SAP97 (beta-isoform) govern their role in regulating synaptic function.     

PSD-95 and SAP97 exhibit distinct mechanisms for regulating K(+) channel surface expression and clustering

Mechanisms of ion channel clustering by cytoplasmic membrane-associated guanylate kinases such as postsynaptic density 95 (PSD-95) and synapse-associated protein 97 (SAP97) are poorly understood. Here, we investigated the interaction of PSD-95 and SAP97 with voltage-gated or Kv K(+) channels. Using Kv channels with different surface expression properties, we found that clustering by PSD-95 depended on channel cell surface expression. Moreover, PSD-95-induced clusters of Kv1 K(+) channels were present on the cell surface. This was most dramatically demonstrated for Kv1.2 K(+) channels, where surface expression and clustering by PSD-95 were coincidentally promoted by coexpression with cytoplasmic Kvbeta subunits. Consistent with a mechanism of plasma membrane channel-PSD-95 binding, coexpression with PSD-95 did not affect the intrinsic surface expression characteristics of the different Kv channels. In contrast, the interaction of Kv1 channels with SAP97 was independent of Kv1 surface expression, occurred intracellularly, and prevented further biosynthetic trafficking of Kv1 channels. As such, SAP97 binding caused an intracellular accumulation of each Kv1 channel tested, through the accretion of SAP97 channel clusters in large (3-5 microm) ER-derived intracellular membrane vesicles. Together, these data show that ion channel clustering by PSD-95 and SAP97 occurs by distinct mechanisms, and suggests that these channel-clustering proteins may play diverse roles in regulating the abundance and distribution of channels at synapses and other neuronal membrane specializations.     

Distribution of the scaffolding proteins PSD-95, PSD-93, and SAP97 in isolated PSDs

We compared the distribution of three scaffolding proteins, all belonging to a family of membrane-associated guanylate kinases, thought to have key roles in the organization of the postsynaptic density (PSD). Isolated PSDs readily adhered to treated glass coverslips where they were labeled with immunogold and rotary shadowed for analysis by EM. The distribution of proteins within individual PSDs were measured by counting and mapping individual immunogold particles. PSD-95, as previously described, is distributed evenly throughout the PSD. We find here that PSD-93 has a nearly identical distribution suggesting that PSD-95 and PSD-93 could perform similar roles. SAP97, in contrast, is concentrated near edges of cleft sides of the PSDs, and in small clumps on their cytoplasmic sides. The homogenous distribution of PSD-95 and PSD-93 throughout the PSD is consistent with their being part of a backbone that stabilizes their various binding partners within the PSD. The distribution of SAP97 confirms that this protein is actually an integral component of the PSD, and suggests that it may have a role in inserting or stabilizing its main binding partner, Glu-R1, at the edge of the PSD.     

Neuroligins mediate excitatory and inhibitory synapse formation: involvement of PSD-95 and neurexin-1beta in neuroligin-induced synaptic specificity

The balance between excitatory and inhibitory synapses is a tightly regulated process that requires differential recruitment of proteins that dictate the specificity of newly formed contacts. However, factors that control this process remain unidentified. Here we show that members of the neuroligin (NLG) family, including NLG1, NLG2, and NLG3, drive the formation of both excitatory and inhibitory presynaptic contacts. The enrichment of endogenous NLG1 at excitatory contacts and NLG2 at inhibitory synapses supports an important in vivo role for these proteins in the development of both types of contacts. Immunocytochemical and electrophysiological analysis showed that the effects on excitatory and inhibitory synapses can be blocked by treatment with a fusion protein containing the extracellular domain of neurexin-1beta. We also found that overexpression of PSD-95, a postsynaptic binding partner of NLGs, resulted in a shift in the distribution of NLG2 from inhibitory to excitatory synapses. These findings reveal a critical role for NLGs and their synaptic partners in controlling the number of inhibitory and excitatory synapses. Furthermore, relative levels of PSD-95 alter the ratio of excitatory to inhibitory synaptic contacts by sequestering members of the NLG family to excitatory synapses.