Amyloid-␤ (A␤) has been implicated in memory loss and disruption of synaptic plasticity observed in early-stage Alzheimer's disease. Recently, it has been shown that soluble A␤ oligomers target synapses in cultured rat hippocampal neurons, suggesting a direct role of A␤ in the regulation of synaptic structure and function. Postsynaptic density-95 (PSD-95) is a postsynaptic scaffolding protein that plays a critical role in synaptic plasticity and the stabilization of AMPA (AMPARs) and NMDA (NMDARs) receptors at synapses. Here, we show that exposure of cultured cortical neurons to soluble oligomers of A␤ 1-40 reduces PSD-95 protein levels in a dose-and time-dependent manner and that the A␤1 1-40 -dependent decrease in PSD-95 requires NMDAR activity. We also show that the decrease in PSD-95 requires cyclin-dependent kinase 5 activity and involves the proteasome pathway. Immunostaining analysis of cortical cultured neurons revealed that A␤ treatment induces concomitant decreases in PSD-95 at synapses and in the surface expression of the AMPAR glutamate receptor subunit 2. Together, these data suggest a novel pathway by which A␤ triggers synaptic dysfunction, namely, by altering the molecular composition of glutamatergic synapses.

Sumoylation of α-synuclein decreases its rate of aggregation and its deleterious effects in vitro and in vivo.

SUMOylation, an essential posttranslational protein modification, is involved in many eukaryotic cellular signaling pathways. The identification of SUMOylated proteins is difficult, because SUMOylation sites in proteins are hard to predict, SUMOylated protein states are transient in vivo and labile in vitro, only a small substrate fraction is SUMOylated in vivo, and identification tools for natively SUMOylated proteins are rare. To solve these problems, we generated knock-in mice expressing His 6 -HA-SUMO1. By anti-HA immunostaining, we show that SUMO1 conjugates in neurons are only detectable in nuclei and annulate lamellae. By anti-HA affinity purification, we identified several hundred candidate SUMO1 substrates, of which we validated Smchd1, Ctip2, TIF1γ, and Zbtb20 as novel substrates. The knock-in mouse represents an excellent mammalian model for studies on SUMO1 localization and screens for SUMO1 conjugates in vivo.synapse | affinity purification S UMOylation is a conserved posttranslational protein modification in eukaryotes, akin to ubiquitylation, and can affect the localization, interactions, function, or stability of substrates (1). SUMOylation processes participate in many cellular signaling pathways, where they intersect with other posttranslational regulatory processes such as phosphorylation, ubiquitylation, or acetylation. Consequently, many cellular processes, from nuclear transport to neuronal synaptic transmission, are controlled by SUMOylation (2), and key SUMOylation substrates or altered SUMOylation are involved in many diseases, from cancer to neurological disorders (3).Reflecting the prominent nuclear role of SUMOylation, most known SUMOylation substrates are nuclear proteins (1, 2). However, SUMOylation appears to play a much more pervasive regulatory role in cells; recent studies have shown SUMOylation of ion channels, membrane-bound receptors, solute carriers, and mitochondrial or neuronal scaffolding and signaling proteins (4-6), leading to the notion that SUMOylation is a core regulatory process in all cellular subcompartments. This triggered substantial activities to develop tools for the discovery and validation of SUMOylation substrates in cells, which has proven difficult.Mammalian genomes contain four SUMO genes, encoding SUMO1, SUMO2, SUMO3 (7, 8), and SUMO4, of which SUMO4 is poorly characterized (9). SUMO2 and SUMO3 are almost identical, whereas SUMO1 is 50% homologous to SUMO2 and SUMO3. The 3D structure of SUMOs is similar to that of ubiquitin (10), and like ubiquitylation, SUMOylation involves an E1 activating enzyme, E2 conjugating enzymes, and E3 ligases (1,2,7,8).SUMOs are conjugated to lysine residues, often within a ϕKxD/E motif (ϕ, hydrophobic residue; x, any amino acid) (1), but in general SUMO acceptor lysines cannot be predicted, which prevents the identification of SUMO substrates by bioinformatics (1). Further, SUMOylated states of proteins are transient in vivo and labile in vitro because of isopeptidases that revert SUMOylation (1), and usually only a small f...

No abstract

SUMO1-conjugation of proteins at neuronal synapses is considered to be a major post-translational regulatory process in nerve cell and synapse function, but the published evidence for SUMO1-conjugation at synapses is contradictory. We employed multiple genetic mouse models for stringently controlled biochemical and immunostaining analyses of synaptic SUMO1-conjugation. By using a knock-in reporter mouse line expressing tagged SUMO1, we could not detect SUMO1-conjugation of seven previously proposed synaptic SUMO1-targets in the brain. Further, immunostaining of cultured neurons from wild-type and SUMO1 knock-out mice showed that anti-SUMO1 immunolabelling at synapses is non-specific. Our findings indicate that SUMO1-conjugation of synaptic proteins does not occur or is extremely rare and hence not detectable using current methodology. Based on our data, we discuss a set of experimental strategies and minimal consensus criteria for the validation of SUMOylation that can be applied to any SUMOylation substrate and SUMO isoform.DOI: http://dx.doi.org/10.7554/eLife.26338.001