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The postsynaptic density is an electron dense meshwork composed of a variety of molecules facilitating neuronal signal transmission. ProSAP2/Shank3 represents a crucial player at postsynaptic sites, assembling large multimeric platforms and anchoring numerous other molecules, thereby linking the functional synapse with the cytoskeleton. ProSAP2/Shank3 is also implicated in the pathogenesis of numerous diseases, including autism spectrum disorders. KvBeta2 (Kvβ2) on the other hand serves as a regulatory subunit of voltage-gated potassium channels. Kvβ2 is located at various sites in the neuron including the axon (binding to Kv1.2), the dendrites (binding to Kv4.2) and the synapse. Binding of Kvβ2 to either Kv1.2 or Kv4 modulates not only the channel conformation but directs targeting of the channel protein complex to distinct loci within the cell. Thus an interaction between ProSAP2 and Kvβ2 could have important roles at diverse cellular compartments and moreover during maturation stages. We report here on the direct protein–protein interaction of the postsynaptic density anchoring molecule ProSAP2 and the potassium channel subunit Kvβ2, initially identified in a yeast-two-hybrid-screen. Furthermore, we characterize this interaction at synapses using primary hippocampal neurons in vitro .

Abelson interactor protein 1 (Abi-1) localizes to postsynaptic densities (PSDs) of excitatory synapses and was shown to be transported from the PSD to the nucleus and back depending upon synaptic activation. We employed a yeast-two-hybrid screen to search for putative transport molecules. We found Kif26B a member of the Kif family of transport proteins that has not been characterized in the central nervous system as a direct interaction partner of Abi-1. We delineated a proline-rich motif within the cargo-binding domain of Kif26B to be responsible for this protein–protein interaction. Kif26B was able to recruit Abi-1 to the microtubule network and we found that the expression of Kif26B is responsible for the localization of Abi-1 to PSDs in maturing neurons. Taken together we report that Abi-1 is a cargo of Kif26B in primary hippocampal neurons, pointing to a role of this transport molecule in the movement of Abi-1 between different cell compartments. Additionally, we provide the first detailed investigation of Kif26B and its cargo molecules in neuronal cells.

Proline-rich synapse-associated protein-1 and 2 (ProSAP1/Shank2 and ProSAP2/Shank3) were originally found as synapse-associated protein 90/postsynaptic density protein-95-associated protein (SAPAP)/guanylate-kinase-associated protein (GKAP) interaction partners and also isolated from synaptic junctional protein preparations of rat brain. They are essential components of the postsynaptic density (PSD) and are specifically targeted to excitatory asymmetric type 1 synapses. Functionally, the members of the ProSAP/Shank family are one of the postsynaptic key elements since they link and attach the postsynaptic signaling apparatus, for example N-methyl-d-aspartic acid (NMDA)-receptors via direct and indirect protein interactions to the actin-based cytoskeleton. The functional significance of ProSAP1/2 for synaptic transmission and the paucity of data with respect to the molecular composition of PSDs of the peripheral nervous system (PNS) stimulated us to investigate neuromuscular junctions (NMJs), synapses of the superior cervical ganglion (SCG), and synapses in myenteric ganglia as representative synaptic junctions of the PNS. Confocal imaging revealed ProSAP1/2-immunoreactivity (-iry) in NMJs of rat and mouse sternomastoid and tibialis anterior muscles. In contrast, ProSAP1/2-iry was only negligibly found in motor endplates of striated esophageal muscle probably caused by antigen masking or a different postsynaptic molecular anatomy at these synapses. ProSAP1/2-iry was furthermore detected in cell bodies and dendrites of superior cervical ganglion neurons and myenteric neurons in esophagus and stomach. Ultrastructural analysis of ProSAP1/2 expression in myenteric ganglia demonstrated that ProSAP1 and ProSAP2 antibodies specifically labelled PSDs of myenteric neurons. Thus, scaffolding proteins ProSAP1/2 were found within the postsynaptic specializations of synapses within the PNS, indicating a similar molecular assembly of central and peripheral postsynapses.

The ability of the mammalian brain to store and recall information is based on synaptic plasticity due to constant remodeling of synaptic contacts. Although various classes of proteins such as neurotransmitter receptors, cytoskeletal components and protein kinases were already identified as modulators of memory formation, their specific interactions and crosstalks are still poorly understood. Genetic variants of the scaffolding protein KIBRA (kidney brain) a substrate of the memory-related protein kinase C zeta and component of the neuronal cytoskeleton, were recently shown to be associated with human memory performance. However, the function of KIBRA on the cellular and physiological level is still unclear. To gain more insights into the temporal and spatial expression of KIBRA, we performed in situ hybridization assays and immunohistological staining of human and rodent (rat) brain. Our data demonstrate that KIBRA is mainly expressed in memory-related regions of the brain (hippocampus, cortex) but is also found in the cerebellum and the hypothalamus. In primary hippocampal neurons, KIBRA displays a somatodendritic distribution and an enrichment at the postsynaptic density. Binding studies further show that KIBRA is able to form head-to-tail homodimers and that dimerization is mediated by the internal C2-like domain. Our data indicate that KIBRA is involved in brain development and memory formation as a postsynaptic scaffold protein connecting cytoskeletal and signaling molecules.

The postsynaptic density is a highly dynamic structure, which is reorganized in an activity-dependent manner. An animal model for temporal lobe epilepsy, i.e. kainate-induced limbic seizures in rats, was used to study changes in postsynaptic density composition after extensive synaptic activity. Six hours after kainate injection, the protein content of the postsynaptic density fractions from rats that developed strong seizures was increased three-fold compared to saline-treated controls. Immunoblot analysis revealed that the relative amounts of metabotropic glutamate receptor 1alpha, N-ethylmaleimide-sensitive fusion protein, protein kinases C, Fyn and TrkB, as well as the neuronal nitric oxide synthase, were significantly higher in seizure-developing than in control rats. In contrast, the relative contents of the kainate receptor KA2 subunit, beta-actin, alpha-adducin and the membrane-associated guanylate kinase homolog SAP90/PSD-95 were decreased. The relative amounts of additional postsynaptic density proteins, including alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-D-aspartate receptor subunits, calcium/calmodulin-dependent kinase type II, casein kinase 2, tubulin, microtubule-associated protein 2B, the membrane-associated guanylate kinase homolog SAP102, and proline-rich synapse-associated protein 1/cortactin binding protein 1/Shank2 remained essentially unchanged. To assess possible changes in postsynaptic performance, postsynaptic densities were isolated from control and epileptic rats, incorporated into giant liposomes and N-methyl-D-aspartate receptor currents were recorded. A significant reduction in the mean conductance was observed in patches containing postsynaptic densities from animals with high seizure activity. This was due to the presence of reduced conductance levels in each membrane patch compared to control postsynaptic density preparations. From these data, we suggest that intense synaptic activity associated with seizures modifies the composition of postsynaptic densities and has profound consequences on the function of the N-methyl-D-aspartate receptors present in them. This rearrangement may accompany impairment of synaptic plasticity.