The membrane-associated guanylate kinases [Chapsyn-110/postsynaptic density-93 (PSD-93), synapse-associated protein-90 (SAP-90)/PSD-95, and SAP-102] are believed to cluster and anchor NMDA receptors at the synapse and to play a role in signal transduction. We have investigated the developmental changes in expression of these proteins in rat hippocampus using biochemical analyses and quantitative immunogold electron microscopy. At postnatal day 2 (P2), SAP-102 was highly expressed, whereas PSD-93 and PSD-95 were low. SAP-102 expression increased during the first week, stayed stable through P35, and showed a reduced expression at 6 months. From P2 through 6 months, PSD-93 and PSD-95 increased. For PSD-95, the percent of labeled synapses increased almost threefold with age, whereas the number of gold particles per labeled synapse did not change significantly, suggesting that the increase in PSD-95 is attributable primarily to an increase in the number of synapses containing PSD-95. In contrast, for SAP-102, both percent labeled synapses and the number of gold particles per labeled synapse decreased during this time. From Western blots of hippocampus and immunogold analysis of CA1 synapses, the high expression of NR2B at P2 coincides with the high level of SAP-102 at synapses, whereas the later expression of NR2A coincides with that of PSD-93 and PSD-95. To determine whether the changes in PSD-93/95 and SAP-102 reflect preferred associations with NR2A and NR2B, respectively, we measured co-immunoprecipitation in the adult hippocampus. These studies suggest that there is a preference for complexes of NR2A/PSD-93/95 and NR2B/SAP-102. These results indicate that individual receptor-associated proteins may have specific functions that are critical to synapse development.

Planar cell polarity (PCP) is a process in which cells develop with uniform orientation within the plane of an epithelium. To begin to elucidate the mechanisms of PCP in vertebrates, the localization of the protein Vangl2 (Van Gogh-like) was determined during the development of the mammalian cochlea. Results indicate that Vangl2 becomes asymmetrically localized to specific cell–cell boundaries along the axis of polarization and that this asymmetry is lost in PCP mutants. In addition, PDZ2 (postsynaptic density/Discs large/zona occludens 1), PDZ3, and PDZ4 of the PCP protein Scrb1 (Scribble) are shown to bind to the C-terminal PDZ binding domain of Vangl2, suggesting that Scrb1 plays a direct role in asymmetric targeting of Vangl2. Finally, Fz3 (Frizzled), a newly demonstrated mediator of PCP, is also asymmetrically localized in a pattern that matches that of Vangl2. The presence and asymmetry of Fz3 at the membrane is shown to be dependent on Vangl2. This result suggests a role for Vangl2 in the targeting or anchoring of Fz3, a hypothesis strengthened by the existence of a physical interaction between the two proteins. Together, our data support the idea that protein asymmetry plays an important role in the development of PCP, but the colocalization and interaction of Fz3 and Vangl2 suggests that novel PCP mechanisms exist in vertebrates.

Genetic mutations of SHANK3 have been reported in patients with intellectual disability, autism spectrum disorder (ASD) and schizophrenia. At the synapse, Shank3/ProSAP2 is a scaffolding protein that connects glutamate receptors to the actin cytoskeleton via a chain of intermediary elements. Although genetic studies have repeatedly confirmed the association of SHANK3 mutations with susceptibility to psychiatric disorders, very little is known about the neuronal consequences of these mutations. Here, we report the functional effects of two de novo mutations (STOP and Q321R) and two inherited variations (R12C and R300C) identified in patients with ASD. We show that Shank3 is located at the tip of actin filaments and enhances its polymerization. Shank3 also participates in growth cone motility in developing neurons. The truncating mutation (STOP) strongly affects the development and morphology of dendritic spines, reduces synaptic transmission in mature neurons and also inhibits the effect of Shank3 on growth cone motility. The de novo mutation in the ankyrin domain (Q321R) modifies the roles of Shank3 in spine induction and morphology, and actin accumulation in spines and affects growth cone motility. Finally, the two inherited mutations (R12C and R300C) have intermediate effects on spine density and synaptic transmission. Therefore, although inherited by healthy parents, the functional effects of these mutations strongly suggest that they could represent risk factors for ASD. Altogether, these data provide new insights into the synaptic alterations caused by SHANK3 mutations in humans and provide a robust cellular readout for the development of knowledge-based therapies.