Juergen Bockmann

Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders

SHANK3 (also known as ProSAP2) regulates the structural organization of dendritic spines and is a binding partner of neuroligins; genes encoding neuroligins are mutated in autism and Asperger syndrome. Here, we report that a mutation of a single copy of SHANK3 on chromosome 22q13 can result in language and/or social communication disorders. These mutations concern only a small number of individuals, but they shed light on one gene dosage–sensitive synaptic pathway that is involved in autism spectrum disorders.

Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2

Autism spectrum disorders comprise a range of neurodevelopmental disorders characterized by deficits in social interaction and communication, and by repetitive behaviour. Mutations in synaptic proteins such as neuroligins, neurexins, GKAPs/SAPAPs and ProSAPs/Shanks were identified in patients with autism spectrum disorder, but the causative mechanisms remain largely unknown. ProSAPs/Shanks build large homo- and heteromeric protein complexes at excitatory synapses and organize the complex protein machinery of the postsynaptic density in a laminar fashion. Here we demonstrate that genetic deletion of ProSAP1/Shank2 results in an early, brain-region-specific upregulation of ionotropic glutamate receptors at the synapse and increased levels of ProSAP2/Shank3. Moreover, ProSAP1/Shank2−/− mutants exhibit fewer dendritic spines and show reduced basal synaptic transmission, a reduced frequency of miniature excitatory postsynaptic currents and enhanced N-methyl-d-aspartate receptor-mediated excitatory currents at the physiological level. Mutants are extremely hyperactive and display profound autistic-like behavioural alterations including repetitive grooming as well as abnormalities in vocal and social behaviours. By comparing the data on ProSAP1/Shank2−/− mutants with ProSAP2/Shank3αβ−/− mice, we show that different abnormalities in synaptic glutamate receptor expression can cause alterations in social interactions and communication. Accordingly, we propose that appropriate therapies for autism spectrum disorders are to be carefully matched to the underlying synaptopathic phenotype.

Proline-Rich Synapse-Associated Protein-1/Cortactin Binding Protein 1 (ProSAP1/CortBP1) Is a PDZ-Domain Protein Highly Enriched in the Postsynaptic Density

The postsynaptic density (PSD) is crucially involved in the structural and functional organization of the postsynaptic neurotransmitter reception apparatus. Using antisera against rat brain synaptic junctional protein preparations, we isolated cDNAs coding for proline-rich synapse-associated protein-1 (ProSAP1), a PDZ-domain protein. This protein was found to be identical to the recently described cortactin-binding protein-1 (CortBP1). Homology screening identified a related protein, ProSAP2. Specific antisera raised against a C-terminal fusion construct and a central part of ProSAP1 detect a cluster of immunoreactive bands of 180 kDa in the particulate fraction of rat brain homogenates that copurify with the PSD fraction. Transcripts and immunoreactivity are widely distributed in the brain and are upregulated during the period of synapse formation in the brain. In addition, two short N-terminal insertions are detected; they are differentially regulated during brain development. Confocal microscopy of hippocampal neurons showed that ProSAP1 is predominantly localized in synapses, and immunoelectron microscopy in situ revealed a strong association with PSDs of hippocampal excitatory synapses. The accumulation of ProSAP1 at synaptic structures was analyzed in the developing cerebral cortex. During early postnatal development, strong immunoreactivity is detectable in neurites and somata, whereas from postnatal day 10 (P10) onward a punctate staining is observed. At the ultrastructural level, the immunoreactivity accumulates at developing PSDs starting from P8. Both interaction with the actin-binding protein cortactin and early appearance at postsynaptic sites suggest that ProSAP1/CortBP1 may be involved in the assembly of the PSD during neuronal differentiation.

Concerted action of zinc and ProSAP/Shank in synaptogenesis and synapse maturation

Neuronal morphology and number of synapses is not static, but can change in response to a variety of factors, a process called synaptic plasticity. These structural and molecular changes are believed to represent the basis for learning and memory, thereby underling both the developmental and activity‐dependent remodelling of excitatory synapses. Here, we report that Zn2+ ions, which are highly enriched within the postsynaptic density (PSD), are able to influence the recruitment of ProSAP/Shank proteins to PSDs in a family member‐specific manner during the course of synaptogenesis and synapse maturation. Through selectively overexpressing each family member at excitatory postsynapses and comparing this to shRNA‐mediated knockdown, we could demonstrate that only the overexpression of zinc‐sensitive ProSAP1/Shank2 or ProSAP2/Shank3 leads to increased synapse density, although all of them cause a decrease upon knockdown. Furthermore, depletion of synaptic Zn2+ along with the knockdown of zinc‐insensitive Shank1 causes the rapid disintegration of PSDs and the loss of several postsynaptic molecules including Homer1, PSD‐95 and NMDA receptors. These findings lead to the model that the concerted action of ProSAP/Shank and Zn2+ is essential for the structural integrity of PSDs and moreover that it is an important element of synapse formation, maturation and structural plasticity.

Abelson interacting protein 1 (Abi-1) is essential for dendrite morphogenesis and synapse formation

Synaptogenesis and synaptic plasticity depend crucially on the dynamic and locally specific regulation of the actin cytoskeleton. We identified an important component for controlled actin assembly, abelson interacting protein‐1 (Abi‐1), as a binding partner for the postsynaptic density (PSD) protein ProSAP2/Shank3. During early neuronal development, Abi‐1 is localized in neurites and growth cones; at later stages, the protein is enriched in dendritic spines and PSDs, as are components of a trimeric complex consisting of Abi‐1, Eps8 and Sos‐1. Abi‐1 translocates upon NMDA application from PSDs to nuclei. Nuclear entry depends on abelson kinase activity. Abi‐1 co‐immunoprecipitates with the transcription factor complex of Myc/Max proteins and enhances E‐box‐regulated gene transcription. Downregulation of Abi‐1 by small interfering RNA results in excessive dendrite branching, immature spine and synapse morphology and a reduction of synapses, whereas overexpression of Abi‐1 has the opposite effect. Data show that Abi‐1 can act as a specific synapto‐nuclear messenger and is essentially involved in dendrite and synapse formation.

Synaptic Cross-talk between N-Methyl-D-aspartate Receptors and LAPSER1-β-Catenin at Excitatory Synapses

Memory formation in the brain is thought to be depending upon long lasting plastic changes of synaptic contacts that require alterations on the transcriptional level. Here, we characterize LAPSER1, a putative cytokinetic tumor suppressor that binds directly to ProSAP2/Shank3 and the synaptic Rap-Gap protein SPAR1 as a novel postsynaptic density component. Postsynaptic LAPSER1 is in complex with all important members of the canonical Wnt pathway including β-catenin. Upon N-methyl-d-aspartate receptor-dependent activation, LAPSER1 and β-catenin comigrate from the postsynaptic density to the nucleus and induce the transcription and translation of known β-catenin target genes, including Tcfe2a and c-Myc. The nuclear export and cytoplasmic redistribution of β-catenin is tightly regulated by LAPSER1. We postulate a postsynaptic cross-talk between N-methyl-d-aspartate receptors and a LAPSER1-β-catenin complex that results in a self-regulated, synaptic activity-dependent expression of β-catenin target genes. This calls for a novel role of Tcfe2a and c-Myc in plastic changes of neural tissue.

ProSAP-interacting Protein 1 (ProSAPiP1), a Novel Protein of the Postsynaptic Density That Links the Spine-associated Rap-Gap (SPAR) to the Scaffolding Protein ProSAP2/Shank3

ProSAPs/Shanks are a family of proteins that have a major scaffolding function for components of the postsynaptic density (PSD) of excitatory brain synapses. Members of the family harbor a variety of domains for protein-protein interactions, one of which is a unique PDZ domain that differs significantly from those of other proteins. We have identified a novel binding partner for this PDZ domain, termed ProSAPiP1, that is highly enriched in the PSD and shares significant sequence homology with the PSD protein PSD-Zip70. Both molecules code for a Fez1 domain that can be found in a total of four related proteins. ProSAPiP1 is widely expressed in rat brain and co-localizes with ProSAP2/Shank3 in excitatory spines and synapses. ProSAP2/Shank3 co-immunoprecipitates with ProSAPiP1 but not with PSD-Zip70. Both proteins, however, bind and recruit SPAR to synapses with a central coiled-coil region that harbors a leucine zipper motif. This region is also responsible for homo- and heteromultimerization of ProSAPiP1 and PSD-Zip70. Thus, ProSAPiP1 and PSD-Zip70 are founders of a novel family of scaffolding proteins, the “Fezzins,” which adds further complexity to the organization of the PSD protein network.

An SK3 Channel/nWASP/Abi-1 Complex Is Involved in Early Neurogenesis

Background The stabilization or regulated reorganization of the actin cytoskeleton is essential for cellular structure and function. Recently, we could show that the activation of the SK3-channel that represents the predominant SK-channel in neural stem cells, leads to a rapid local outgrowth of long filopodial processes. This observation indicates that the rearrangement of the actin based cytoskeleton via membrane bound SK3-channels might selectively be controlled in defined micro compartments of the cell. Principal Findings We found two important proteins for cytoskeletal rearrangement, the Abelson interacting protein 1, Abi-1 and the neural Wiskott Aldrich Syndrome Protein, nWASP, to be in complex with SK3- channels in neural stem cells (NSCs). Moreover, this interaction is also found in spines and postsynaptic compartments of developing primary hippocampal neurons and regulates neurite outgrowth during early phases of differentiation. Overexpression of the proteins or pharmacological activation of SK3 channels induces obvious structural changes in NSCs and hippocampal neurons. In both neuronal cell systems SK3 channels and nWASP act synergistic by strongly inducing filopodial outgrowth while Abi-1 behaves antagonistic to its interaction partners. Conclusions Our results give good evidence for a functional interplay of a trimeric complex that transforms incoming signals via SK3-channel activation into the local rearrangement of the cytoskeleton in early steps of neuronal differentiation involving nWASP and Abi-1 actin binding proteins.

Heterogeneous nuclear ribonucleoprotein k interacts with Abi-1 at postsynaptic sites and modulates dendritic spine morphology.

Background Abelson-interacting protein 1 (Abi-1) plays an important role for dendritic branching and synapse formation in the central nervous system. It is localized at the postsynaptic density (PSD) and rapidly translocates to the nucleus upon synaptic stimulation. At PSDs Abi-1 is in a complex with several other proteins including WASP/WAVE or cortactin thereby regulating the actin cytoskeleton via the Arp 2/3 complex. Principal Findings We identified heterogeneous nuclear ribonucleoprotein K (hnRNPK), a 65 kDa ssDNA/RNA-binding-protein that is involved in multiple intracellular signaling cascades, as a binding partner of Abi-1 at postsynaptic sites. The interaction with the Abi-1 SH3 domain is mediated by the hnRNPK-interaction (KI) domain. We further show that during brain development, hnRNPK expression becomes more and more restricted to granule cells of the cerebellum and hippocampal neurons where it localizes in the cell nucleus as well as in the spine/dendritic compartment. The downregulation of hnRNPK in cultured hippocampal neurons by RNAi results in an enlarged dendritic tree and a significant increase in filopodia formation. This is accompanied by a decrease in the number of mature synapses. Both effects therefore mimic the neuronal morphology after downregulation of Abi-1 mRNA in neurons. Conclusions Our findings demonstrate a novel interplay between hnRNPK and Abi-1 in the nucleus and at synaptic sites and show obvious similarities regarding both protein knockdown phenotypes. This indicates that hnRNPK and Abi-1 act synergistic in a multiprotein complex that regulates the crucial balance between filopodia formation and synaptic maturation in neurons.

ProSAPiP2, a novel postsynaptic density protein that interacts with ProSAP2/Shank3.

The postsynaptic density (PSD) is a highly specialized structure that is located juxtaposed to the presynaptic active zone of excitatory synapses. It is composed of a variety of proteins that include receptors, signaling molecules, cytoskeletal components and scaffolding proteins. ProSAP/Shank proteins are large multidomain proteins that facilitate multiple functions within the PSD. They build large scaffolds that are the structural basis for the direct and/or indirect connection between receptor proteins and the actin based cytoskeleton. Here, we characterize a novel interaction partner of ProSAP2/Shank3, named ProSAP interacting protein 2 (ProSAPiP2) that does not show any close homology to other known proteins. It binds to the PDZ domain of ProSAP2/Shank3 and is highly expressed in the neuronal system. ProSAPiP2 is located in dendrites and spines, is enriched in the PSD and interacts with actin. Therefore ProSAPiP2 could be involved in the linkage between molecules of the PSD and the cytoskeleton.