A balance between excitatory and inhibitory synapses is controlled by PSD-95 and neuroligin.
ABSTRACT: Factors that control differentiation of presynaptic and postsynaptic elements into excitatory or inhibitory synapses are poorly defined. Here we show that the postsynaptic density (PSD) proteins PSD-95 and neuroligin-1 (NLG) are critical for dictating the ratio of excitatory-to-inhibitory synaptic contacts. Exogenous NLG increased both excitatory and inhibitory presynaptic contacts and the frequency of miniature excitatory and inhibitory synaptic currents. In contrast, PSD-95 overexpression enhanced excitatory synapse size and miniature frequency, but reduced the number of inhibitory synaptic contacts. Introduction of PSD-95 with NLG augmented synaptic clustering of NLG and abolished NLG effects on inhibitory synapses. Interfering with endogenous PSD-95 expression alone was sufficient to reduce the ratio of excitatory-to-inhibitory synapses. These findings elucidate a mechanism by which the amounts of specific elements critical for synapse formation control the ratio of excitatory-to-inhibitory synaptic input.
Project description:Presynaptic and postsynaptic differentiation occurs at axodendritic contacts between CNS neurons. Synaptic adhesion mediated by synaptic cell adhesion molecule (SynCAM) and beta-neurexins/neuroligins triggers presynaptic differentiation. The signals that trigger postsynaptic differentiation are, however, unknown. Here we report that beta-neurexin expressed in nonneuronal cells induced postsynaptic density (PSD)-95 clustering in contacting dendrites of hippocampal neurons. The effect is specific to beta-neurexin and was not observed with other synaptic cell adhesion molecules such as N-cadherin or SynCAM. NMDA receptors, but not alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs), were recruited to this beta-neurexin-induced PSD-95 scaffold. Remarkably, AMPARs were inserted into this scaffold upon glutamate application or expression of a constitutively active form of calmodulin kinase II in neurons. Expression of a dominant-negative neuroligin-1 in cultured neurons markedly reduced the sizes and densities of PSD-95 puncta and AMPAR clusters. In addition, excitatory, but not inhibitory, synaptic functions were impaired in these neurons, confirming that PSD-95/neuroligin-1 interaction is involved in postsynaptic assembly at glutamatergic synapses. These results demonstrate that postsynaptic assembly of the glutamatergic synapse may be initiated by presynaptic beta-neurexin and that glutamate release also is required for maturation of synapses.
Project description:We identify the leucine-rich repeat transmembrane protein LRRTM2 as a key regulator of excitatory synapse development and function. LRRTM2 localizes to excitatory synapses in transfected hippocampal neurons, and shRNA-mediated knockdown of LRRTM2 leads to a decrease in excitatory synapses without affecting inhibitory synapses. LRRTM2 interacts with PSD-95 and regulates surface expression of AMPA receptors, and lentivirus-mediated knockdown of LRRTM2 in vivo decreases the strength of evoked excitatory synaptic currents. Structure-function studies indicate that LRRTM2 induces presynaptic differentiation via the extracellular LRR domain. We identify Neurexin1 as a receptor for LRRTM2 based on affinity chromatography. LRRTM2 binds to both Neurexin 1alpha and Neurexin 1beta, and shRNA-mediated knockdown of Neurexin1 abrogates LRRTM2-induced presynaptic differentiation. These observations indicate that an LRRTM2-Neurexin1 interaction plays a critical role in regulating excitatory synapse development.
Project description:Synaptic cell adhesion molecules regulate signal transduction, synaptic function, and plasticity. However, their role in neuronal interactions with the extracellular matrix (ECM) is not well understood. Here we report that the CD44, a transmembrane receptor for hyaluronan, modulates synaptic plasticity. High-resolution ultrastructural analysis showed that CD44 was localized at mature synapses in the adult brain. The reduced expression of CD44 affected the synaptic excitatory transmission of primary hippocampal neurons, simultaneously modifying dendritic spine shape. The frequency of miniature excitatory postsynaptic currents decreased, accompanied by dendritic spine elongation and thinning. These structural and functional alterations went along with a decrease in the number of presynaptic Bassoon puncta, together with a reduction of PSD-95 levels at dendritic spines, suggesting a reduced number of functional synapses. Lack of CD44 also abrogated spine head enlargement upon neuronal stimulation. Moreover, our results indicate that CD44 contributes to proper dendritic spine shape and function by modulating the activity of actin cytoskeleton regulators, that is, Rho GTPases (RhoA, Rac1, and Cdc42). Thus CD44 appears to be a novel molecular player regulating functional and structural plasticity of dendritic spines.
Project description:As key functional units in neural circuits, different types of neuronal synapses play distinct roles in brain information processing, learning, and memory. Synaptic abnormalities are believed to underlie various neurological and psychiatric disorders. Here, by combining cryo-electron tomography and cryo-correlative light and electron microscopy, we distinguished intact excitatory and inhibitory synapses of cultured hippocampal neurons, and visualized the in situ 3D organization of synaptic organelles and macromolecules in their native state. Quantitative analyses of >100 synaptic tomograms reveal that excitatory synapses contain a mesh-like postsynaptic density (PSD) with thickness ranging from 20 to 50 nm. In contrast, the PSD in inhibitory synapses assumes a thin sheet-like structure ?12 nm from the postsynaptic membrane. On the presynaptic side, spherical synaptic vesicles (SVs) of 25-60 nm diameter and discus-shaped ellipsoidal SVs of various sizes coexist in both synaptic types, with more ellipsoidal ones in inhibitory synapses. High-resolution tomograms obtained using a Volta phase plate and electron filtering and counting reveal glutamate receptor-like and GABAA receptor-like structures that interact with putative scaffolding and adhesion molecules, reflecting details of receptor anchoring and PSD organization. These results provide an updated view of the ultrastructure of excitatory and inhibitory synapses, and demonstrate the potential of our approach to gain insight into the organizational principles of cellular architecture underlying distinct synaptic functions.SIGNIFICANCE STATEMENT To understand functional properties of neuronal synapses, it is desirable to analyze their structure at molecular resolution. We have developed an integrative approach combining cryo-electron tomography and correlative fluorescence microscopy to visualize 3D ultrastructural features of intact excitatory and inhibitory synapses in their native state. Our approach shows that inhibitory synapses contain uniform thin sheet-like postsynaptic densities (PSDs), while excitatory synapses contain previously known mesh-like PSDs. We discovered "discus-shaped" ellipsoidal synaptic vesicles, and their distributions along with regular spherical vesicles in synaptic types are characterized. High-resolution tomograms further allowed identification of putative neurotransmitter receptors and their heterogeneous interaction with synaptic scaffolding proteins. The specificity and resolution of our approach enables precise in situ analysis of ultrastructural organization underlying distinct synaptic functions.
Project description:Protein kinase C? (PKC?) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKC? induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKC? and PSD-95. We show that the PKC? activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKC?-induced membrane accumulation. Knockdown of either PKC? or JNK1 prevented PKC? activator-mediated membrane accumulation of PSD-95. PKC? directly phosphorylated PSD-95 and JNK1 in vitro Inhibiting PKC?, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKC? activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKC? and phosphorylated PSD-95 (p-PSD-95(S295)) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKC? also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKC? increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKC? promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin.
Project description:PSD-95 is a scaffolding protein that regulates the synaptic localization of many receptors, channels, and signaling proteins. The NLGN gene family encodes single-pass transmembrane postsynaptic cell adhesion molecules that are important for synapse assembly and function. At excitatory synapses, NLGN1 mediates transsynaptic binding with neurexin, a presynaptic cell adhesion molecule, and also binds to PSD-95, although the relevance of the PSD-95 interaction is not clear. We now show that disruption of the NLGN1 and PSD-95 interaction decreases surface expression of NLGN1 in cultured neurons. Furthermore, PKA phosphorylates NLGN1 on S839, near the PDZ ligand, and dynamically regulates PSD-95 binding. A phosphomimetic mutation of NLGN1 S839 significantly reduced PSD-95 binding. Impaired NLGN1/PSD-95 binding diminished synaptic NLGN1 expression and NLGN1-mediated synaptic enhancement. Our results establish a phosphorylation-dependent molecular mechanism that regulates NLGN1 and PSD-95 binding and provides insights into excitatory synaptic development and function.
Project description:At synapses, the presynaptic release machinery is precisely juxtaposed to the postsynaptic neurotransmitter receptors. We studied the molecular mechanisms underlying this exquisite alignment at the C. elegans inhibitory synapses. We found that the sole C. elegans neuroligin homolog, NLG-1, localizes specifically at GABAergic postsynapses and is required for clustering the GABA(A) receptor UNC-49. Two presynaptic factors, Punctin/MADD-4, an ADAMTS-like extracellular protein, and neurexin/NRX-1, act partially redundantly to recruit NLG-1 to synapses. In the absence of both MADD-4 and NRX-1, NLG-1 and GABA(A) receptors fail to cluster, and GABAergic synaptic transmission is severely compromised. Biochemically, we detect an interaction between MADD-4 and NLG-1, as well as between MADD-4 and NRX-1. Interestingly, the presence of NRX-1 potentiates binding between Punctin/MADD-4 and NLG-1, suggestive of a tripartite receptor ligand complex. We propose that presynaptic terminals induce postsynaptic receptor clustering through the action of both secreted ECM proteins and trans-synaptic adhesion complexes.
Project description:Homeostatic regulation of synaptic strength in response to persistent changes of neuronal activity plays an important role in maintaining the overall level of circuit activity within a normal range. Absence of miniature EPSCs (mEPSCs) for a few hours is known to cause upregulation of excitatory synaptic strength, suggesting that mEPSCs contribute to the maintenance of excitatory synaptic functions. In the present study, we found that the absence of mEPSCs for 1-3 h also resulted in homeostatic suppression of presynaptic functions of inhibitory synapses in acute cortical slices from juvenile rats, as suggested by the reduced frequency (but not amplitude) of miniature IPSCs (mIPSCs) as well as the reduced amplitude of IPSCs. This homeostatic regulation depended on endocannabinoid (eCB) signaling, because blockade of either the activation of cannabinoid type-1 receptors (CB1Rs) or the synthesis of its endogenous ligand 2-arachidonoylglycerol (2-AG) abolished the suppression of inhibitory synapses caused by the absence of mEPSCs. Blockade of group I metabotropic glutamate receptors (mGluR-I) also abolished the suppression of inhibitory synapses, consistent with the mGluR-I requirement for eCB synthesis and release in cortical synapses. Furthermore, this homeostatic regulation also required eukaryotic elongation factor-2 (eEF2)-dependent protein synthesis, but not gene transcription. Activation of eEF2 alone was sufficient to suppress the mIPSC frequency, an effect abolished by inhibiting CB1Rs. Thus, mEPSCs contribute to the maintenance of inhibitory synaptic function and the absence of mEPSCs results in presynaptic suppression of inhibitory synapses via protein synthesis-dependent elevation of eCB signaling.
Project description:We have recently shown that disrupting the expression and post-synaptic clustering of gephyrin in cultured hippocampal pyramidal cells, by either gephyrin RNAi (RNA interference) or over-expression of a dominant negative gephyrin-enhanced green fluorescent protein (EGFP) fusion protein, leads to decreased number of post-synaptic gephyrin and GABA(A) receptor clusters and to reduced GABAergic innervation of these cells. On the other hand, increasing gephyrin expression led to a small increase in the number of gephyrin and GABA(A) receptor clusters and to little or no effect on GABAergic innervation. We are now reporting that altering gephyrin expression and clustering affects the size but not the density of glutamatergic synaptic contacts. Knocking down gephyrin with gephyrin RNAi, or preventing gephyrin clustering by over-expression of the dominant negative gephyrin-enhanced green fluorescent protein fusion protein, leads to larger post-synaptic PSD-95 clusters and larger pre-synaptic glutamatergic terminals. On the other hand, over-expression of gephyrin leads to slightly smaller PSD-95 clusters and pre-synaptic glutamatergic terminals. The change in size of PSD-95 clusters were accompanied by a parallel change in the size of NR2-NMDA receptor clusters. It is concluded that the levels of expression and clustering of gephyrin, a protein that concentrates at the post-synaptic complex of the inhibitory synapses, not only has homotypic effects on GABAergic synaptic contacts, but also has heterotypic effects on glutamatergic synaptic contacts. We are proposing that gephyrin is a counterpart of the post-synaptic glutamatergic scaffold protein PSD-95 in regulating the number and/or size of the excitatory and inhibitory synaptic contacts.
Project description:The cell adhesion molecule pair neuroligin1 (Nlg1) and beta-neurexin (beta-NRX) is a powerful inducer of postsynaptic differentiation of glutamatergic synapses in vitro. Because Nlg1 induces accumulation of two essential components of the postsynaptic density (PSD) - PSD-95 and NMDA receptors (NMDARs) - and can physically bind PSD-95 and NMDARs at mature synapses, it has been proposed that Nlg1 recruits NMDARs to synapses through its interaction with PSD-95. However, PSD-95 and NMDARs are recruited to nascent synapses independently and it is not known if Nlg1 accumulates at synapses before these PSD proteins. Here, we investigate how a single type of cell adhesion molecule can recruit multiple types of synaptic proteins to new synapses with distinct mechanisms and time courses.Nlg1 was present in young cortical neurons in two distinct pools before synaptogenesis, diffuse and clustered. Time-lapse imaging revealed that the diffuse Nlg1 aggregated at, and the clustered Nlg1 moved to, sites of axodendritic contact with a rapid time course. Using a patching assay that artificially induced clusters of Nlg, the time course and mechanisms of recruitment of PSD-95 and NMDARs to those Nlg clusters were characterized. Patching Nlg induced clustering of PSD-95 via a slow palmitoylation-dependent step. In contrast, NMDARs directly associated with clusters of Nlg1 during trafficking. Nlg1 and NMDARs were highly colocalized in dendrites before synaptogenesis and they became enriched with a similar time course at synapses with age. Patching of Nlg1 dramatically decreased the mobility of NMDAR transport packets. Finally, Nlg1 was biochemically associated with NMDAR transport packets, presumably through binding of NMDARs to MAGUK proteins that, in turn, bind Nlg1. This interaction was essential for colocalization and co-transport of Nlg1 with NMDARs.Our results suggest that axodendritic contact leads to rapid accumulation of Nlg1, recruitment of NMDARs co-transported with Nlg1 soon thereafter, followed by a slower, independent recruitment of PSD-95 to those nascent synapses.