Carsten Reissner

Caldendrin-Jacob: a protein liaison that couples NMDA receptor signalling to the nucleus.

NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacobs nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacobs nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacobs extranuclear localization by competing with the binding of Importin-α to Jacobs nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor–induced cellular degeneration.

Intramolecular interactions regulate SAP97 binding to GKAP

Membrane‐associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK‐associated protein). The GKAP–GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N‐terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U‐shaped conformation in which the N‐terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners.

Mutational analysis of the neurexin/neuroligin complex reveals essential and regulatory components

Neurexins are cell-surface molecules that bind neuroligins to form a heterophilic, Ca2+-dependent complex at central synapses. This transsynaptic complex is required for efficient neurotransmission and is involved in the formation of synaptic contacts. In addition, both molecules have been identified as candidate genes for autism. Here we performed mutagenesis experiments to probe for essential components of the neurexin/neuroligin binding interface at the single-amino acid level. We found that in neurexins the contact area is sharply delineated and consists of hydrophobic residues of the LNS domain that surround a Ca2+ binding pocket. Point mutations that changed electrostatic and shape properties leave Ca2+ coordination intact but completely inhibit neuroligin binding, whereas alternative splicing in α- and β-neurexins and in neuroligins has a weaker effect on complex formation. In neuroligins, the contact area appears less distinct because exchange of a more distant aspartate completely abolished binding to neurexin but many mutations of predicted interface residues had no strong effect on binding. Together with calculations of energy terms for presumed interface hot spots that complement and extend our mutagenesis and recent crystal structure data, this study presents a comprehensive structural basis for the complex formation of neurexins and neuroligins and their transsynaptic signaling between neurons.

Polarized Targeting of Neurexins to Synapses Is Regulated by their C-Terminal Sequences

Two families of cell-adhesion molecules, predominantly presynaptic neurexins and postsynaptic neuroligins, are important for the formation and functioning of synapses in the brain, and mutations in several genes encoding these transmembrane proteins have been found in autism patients. However, very little is known about how neurexins are targeted to synapses and which mechanisms regulate this process. Using various epitope-tagged neurexins in primary hippocampal neurons of wild-type and knock-out mice in vitro and in transgenic animals in vivo, we show that neurexins are trafficked throughout neurons via transport vesicles and the plasma membrane insertion of neurexins occurs preferentially in the axonal/synaptic compartment. We also observed that exit of neurexins from the ER/Golgi and correct targeting require their PDZ-binding motif at the C terminus, whereas two presumptive ER retention signals are inactive. The ubiquitous presence of neurexin-positive transport vesicles and absence of bassoon colabeling demonstrate that these carriers are not active zone precursor vesicles, but colocalization with CASK, RIM1α, and calcium channels suggests that they may carry additional components of the exocytotic machinery. Our data indicate that neurexins are delivered to synapses by a polarized and regulated targeting process that involves PDZ-domain mediated interactions, suggesting a novel pathway for the distribution of neurexins and other synaptic proteins.

Dendritic spine formation and synaptic function require neurobeachin

A challenge in neuroscience is to understand the mechanisms underlying synapse formation. Most excitatory synapses in the brain are built on spines, which are actin-rich protrusions from dendrites. Spines are a major substrate of brain plasticity, and spine pathologies are observed in various mental illnesses. Here we investigate the role of neurobeachin (Nbea), a multidomain protein previously linked to cases of autism, in synaptogenesis. We show that deletion of Nbea leads to reduced numbers of spinous synapses in cultured neurons from complete knockouts and in cortical tissue from heterozygous mice, accompanied by altered miniature postsynaptic currents. In addition, excitatory synapses terminate mostly at dendritic shafts instead of spine heads in Nbea mutants, and actin becomes less enriched synaptically. As actin and synaptopodin, a spine-associated protein with actin-bundling activity, accumulate ectopically near the Golgi apparatus of mutant neurons, a role emerges for Nbea in trafficking important cargo to pre- and postsynaptic compartments.

Role of Bassoon and Piccolo in Assembly and Molecular Organization of the Active Zone.

Bassoon and Piccolo are two very large scaffolding proteins of the cytomatrix assembled at the active zone (CAZ) where neurotransmitter is released. They share regions of high sequence similarity distributed along their entire length and seem to share both overlapping and distinct functions in organizing the CAZ. Here, we survey our present knowledge on protein-protein interactions and recent progress in understanding of molecular functions of these two giant proteins. These include roles in the assembly of active zones (AZ), the localization of voltage-gated Ca2+ channels (VGCCs) in the vicinity of release sites, synaptic vesicle (SV) priming and in the case of Piccolo, a role in the dynamic assembly of the actin cytoskeleton. Piccolo and Bassoon are also important for the maintenance of presynaptic structure and function, as well as for the assembly of CAZ specializations such as synaptic ribbons. Recent findings suggest that they are also involved in the regulation activity-dependent communication between presynaptic boutons and the neuronal nucleus. Together these observations suggest that Bassoon and Piccolo use their modular structure to organize super-molecular complexes essential for various aspects of presynaptic function.

Caldendrins in the inner retina.

Caldendrin is the first member of a novel family of Ca2+-binding proteins (CaBPs). Its unique two-domain structure is composed of a calmodulin-homologous teminus and an unrelated N-terminal part. The latter is thought to mediate the tight association of caldendrin with the subsynaptic cytoskeleton. Caldendrin is expressed in forebrain regions with a laminar cytoarchitecture as well as in the inner retina where it is localized to OFF cone bipolar and a subset of amacrine and ganlion cells. In addition, caldendrin is prominently present in processes and synapses of the inner plexiform layer. Thus, caldendrin-immunoreactivity is displayed by ubpopulations of most retinal cell classes, with the exception of glial cells. Caldendrin is most likely involved in dendritic Ca2+-signaling, one of the functions of its close relative, calmodulin. However, several lines of evidence suggest that due to its unique properties caldendrin might not merely substitute for calmodulin. t is speculated that either the specific enrichment in cellular micro-compartments like the postsynaptic cytomatrix, the unique two-domain structure or the altered distribution of surface charges renders caldendrin specific for distinct binding partners or certain Ca2+-triggered signaling events.

Dystroglycan binding to α-neurexin competes with neurexophilin-1 and neuroligin in the brain.

Background: Extracellular matrix dystroglycan has essential functions at the neuromuscular junction and at inhibitory synapses in the brain. Results: Brain dystroglycan competes with neurexophilin-1 and neuroligins for binding to presynaptic α-neurexins. Conclusion: Competition between α-neurexin ligands in combination with alternative splicing determines formation of important trans-synaptic complexes. Significance: This is the first analysis of binding interference in α-neurexin multiplexes. α-Neurexins (α-Nrxn) are mostly presynaptic cell surface molecules essential for neurotransmission that are linked to neuro-developmental disorders as autism or schizophrenia. Several interaction partners of α-Nrxn are identified that depend on alternative splicing, including neuroligins (Nlgn) and dystroglycan (αDAG). The trans-synaptic complex with Nlgn1 was extensively characterized and shown to partially mediate α-Nrxn function. However, the interactions of α-Nrxn with αDAG, neurexophilins (Nxph1) and Nlgn2, ligands that occur specifically at inhibitory synapses, are incompletely understood. Using site-directed mutagenesis, we demonstrate the exact binding epitopes of αDAG and Nxph1 on Nrxn1α and show that their binding is mutually exclusive. Identification of an unusual cysteine bridge pattern and complex type glycans in Nxph1 ensure binding to the second laminin/neurexin/sex hormone binding (LNS2) domain of Nrxn1α, but this association does not interfere with Nlgn binding at LNS6. αDAG, in contrast, interacts with both LNS2 and LNS6 domains without inserts in splice sites SS#2 or SS#4 mostly via LARGE (like-acetylglucosaminyltransferase)-dependent glycans attached to the mucin region. Unexpectedly, binding of αDAG at LNS2 prevents interaction of Nlgn at LNS6 with or without splice insert in SS#4, presumably by sterically hindering each other in the u-form conformation of α-Nrxn. Thus, expression of αDAG and Nxph1 together with alternative splicing in Nrxn1α may prevent or facilitate formation of distinct trans-synaptic Nrxn·Nlgn complexes, revealing an unanticipated way to contribute to the identity of synaptic subpopulations.

Promoter-like sequences regulating transcriptional activity in neurexin and neuroligin genes.

Synapse function requires the cell‐adhesion molecules neurexins (Nrxn) and neuroligins (Nlgn). Although these molecules are essential for neurotransmission and prefer distinct isoform combinations for interaction, little is known about their transcriptional regulation. Here, we started to explore this important aspect because expression of Nrxn1‐3 and Nlgn1‐3 genes is altered in mice lacking the transcriptional regulator methyl‐CpG‐binding protein2 (MeCP2). Since MeCP2 can bind to methylated CpG‐dinucleotides and Nrxn/Nlgn contain CpG‐islands, we tested genomic sequences for transcriptional activity in reporter gene assays. We found that their influence on transcription are differentially activating or inhibiting. As we observed an activity difference between heterologous and neuronal cell lines for distinct Nrxn1 and Nlgn2 sequences, we dissected their putative promoter regions. In both genes, we identify regions in exon1 that can induce transcription, in addition to the alternative transcriptional start points in exon2. While the 5′‐regions of Nrxn1 and Nlgn2 contain two CpG‐rich elements that show distinct methylation frequency and binding to MeCP2, other regions may act independently of this transcriptional regulator. These data provide first insights into regulatory sequences of Nrxn and Nlgn genes that may represent an important aspect of their function at synapses in health and disease.

Modulation of synaptic function through the α-neurexin–specific ligand neurexophilin-1

Significance Communication between neurons via synapses is essential for information processing and cognitive function in our brains and is found impaired in many neuropsychiatric disorders. Synaptic transmission is remarkably variable in strength, and cell-adhesion molecules as neurexins and their binding partners are candidates to regulate neurotransmission. This study changes our understanding of how neurotransmission can be adapted to local demands by investigating the previously undescribed functions of neurexophilins, arguably the most elusive ligands of α-neurexins. Neurexophilins are expressed only in subpopulations of synapses, and their presence is able to change short-term plasticity and molecular composition at these terminals. Neurotransmission at different synapses is highly variable, and cell-adhesion molecules like α-neurexins (α-Nrxn) and their extracellular binding partners determine synapse function. Although α-Nrxn affect transmission at excitatory and inhibitory synapses, the contribution of neurexophilin-1 (Nxph1), an α-Nrxn ligand with restricted expression in subpopulations of inhibitory neurons, is unclear. To reveal its role, we investigated mice that either lack or overexpress Nxph1. We found that genetic deletion of Nxph1 impaired GABAB receptor (GABABR)-dependent short-term depression of inhibitory synapses in the nucleus reticularis thalami, a region where Nxph1 is normally expressed at high levels. To test the conclusion that Nxph1 supports presynaptic GABABR, we expressed Nxph1 ectopically at excitatory terminals in the neocortex, which normally do not contain this molecule but can be modulated by GABABR. We generated Nxph1-GFP transgenic mice under control of the Thy1.2 promoter and observed a reduced short-term facilitation at these excitatory synapses, representing an inverse phenotype to the knockout. Consistently, the diminished facilitation could be reversed by pharmacologically blocking GABABR with CGP-55845. Moreover, a complete rescue was achieved by additional blocking of postsynaptic GABAAR with intracellular picrotoxin or gabazine, suggesting that Nxph1 is able to recruit or stabilize both presynaptic GABABR and postsynaptic GABAAR. In support, immunoelectron microscopy validated the localization of ectopic Nxph1 at the synaptic cleft of excitatory synapses in transgenic mice and revealed an enrichment of GABAAR and GABABR subunits compared with wild-type animals. Thus, our data propose that Nxph1 plays an instructive role in synaptic short-term plasticity and the configuration with GABA receptors.