Abbreviations used in the paper: CCV, clathrin-coated vesicle; GEF, guanine nucleotide exchange factor; PP2A, protein phosphatase 2A; PtdIns(4,5)P 2 , phosphoinositide 4,5-bisphosphate; Tfn, transferrin; TfnR, tranferrin receptor.The online version of this article contains supplemental material.

BackgroundIt is becoming increasingly evident that deficits in the cortex and hippocampus at early stages of dementia in Alzheimer’s disease (AD) are associated with synaptic damage caused by oligomers of the toxic amyloid-β peptide (Aβ42). However, the underlying molecular and cellular mechanisms behind these deficits are not fully understood. Here we provide evidence of a mechanism by which Aβ42 affects synaptic transmission regulating neurotransmitter release.Methodology/FindingsWe first showed that application of 50 nM Aβ42 in cultured neurones is followed by its internalisation and translocation to synaptic contacts. Interestingly, our results demonstrate that with time, Aβ42 can be detected at the presynaptic terminals where it interacts with Synaptophysin. Furthermore, data from dissociated hippocampal neurons as well as biochemical data provide evidence that Aβ42 disrupts the complex formed between Synaptophysin and VAMP2 increasing the amount of primed vesicles and exocytosis. Finally, electrophysiology recordings in brain slices confirmed that Aβ42 affects baseline transmission.Conclusions/SignificanceOur observations provide a necessary and timely insight into cellular mechanisms that underlie the initial pathological events that lead to synaptic dysfunction in Alzheimer’s disease. Our results demonstrate a new mechanism by which Aβ42 affects synaptic activity.

bRegulation of the activity of N-methyl-D-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors. In an effort to identify genes involved in cortical development, we have previously cloned slap, which encodes a Src-like adaptor protein (Slap) (1). slap is expressed strongly in the forebrain, but its function there remains unknown. slap encodes a 34-kDa protein containing SH2 and SH3 domains followed by a unique 104-amino-acid COOH terminal. Similar to results seen with members of the Src family kinases (SFKs), myristoylation at the NH2 terminus targets Slap to cellular membranes (2). Slap has been studied in the immune system, where it inhibits T-cell receptor (TCR) signaling (3) and is involved in the internalization and degradation of the TCR subunit (4). Furthermore, Slap abrogates the mitogenic response to platelet-derived growth factors (PDGFs) in NIH 3T3 fibroblasts, antagonizing the mitotic activity of Src kinase (5).Slap was initially identified in a yeast two-hybrid screen using the cytoplasmic tail of EphrinA2 as bait (6). Ephrin (Eph) receptors constitute the largest known family of receptor tyrosine kinases and, together with their membrane-bound ligands (eph ligands), have been implicated in a variety of patterning events during the development of the central nervous system (7-21). Ephs and their ligands act as contact-dependent adhesive molecules and are implicated in the development and function of synapses. They are divided into two classes (EphA and EphB), based on sequence homologies and binding specificity (22). EphBs have established roles in the formation of synapses, transforming dendritic filopodia to spines and clustering and phosphorylating Nmethyl-D-aspartate receptors (NMDARs) (7,9,23), and are implicated in...

Reversible phosphorylation has long been an attractive mechanism to control cycles of coat assembly and disassembly during clathrin-mediated endocytosis. Many of the coat proteins are phosphorylated in vivo and in vitro. Our work has focused on the role of phosphorylation of the $#x03BC;2 subunit of AP-2 (adaptor protein 2), which appears to be necessary for efficient cargo recruitment. Studies to probe the regulation of $#x03BC;2 phosphorylation demonstrated that clathrin is a specific activator of the $#x03BC;2 kinase, and, in permeabilized cells, cargo sequestration, driven by exogenously added clathrin, results in elevated levels of $#x03BC;2 phosphorylation. Furthermore, phosphorylated $#x03BC;2 is mainly associated with assembled clathrin in vivo and its steady-state level is strongly reduced in cells depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via modulation of phospho-$#x03BC;2 levels. This is therefore a novel regulatory role for clathrin that is independent of its structural role and that provides elegant spatial control of AP-2 and cargo interactions, ensuring that AP-2 is only activated at the correct cellular location and in the correct functional context. Ongoing studies are exploring further the roles of reversible phosphorylation in the coated vesicle cycle.