SUMOylation Is Required for Glycine-Induced Increases in AMPA Receptor Surface Expression (ChemLTP) in Hippocampal Neurons

et al. (2013) SUMOylation Is Required for Glycine-Induced Increases in AMPA Receptor Surface Expression (ChemLTP) in Hippocampal Neurons. PLoS ONE 8(1): e52345. doi:10.1371/journal.pone.0052345 SUMOylation Is Required for Glycine-Induced Increases in AMPA Receptor Surface Expression (ChemLTP) in Hippocampal Neurons Nadia Jaafari. 0 Filip A. Konopacki. 0 Thomas F. Owen 0 Sriharsha Kantamneni 0 Philip Rubin 0 Tim J. Craig 0 Kevin A. Wilkinson 0 Jeremy M. Henley 0 Anna Dunaevsky, University of Nebraska Medical Center, United States of America 0 School of Biochemistry, University of Bristol , Bristol , United Kingdom Multiple pathways participate in the AMPA receptor trafficking that underlies long-term potentiation (LTP) of synaptic transmission. Here we demonstrate that protein SUMOylation is required for insertion of the GluA1 AMPAR subunit following transient glycine-evoked increase in AMPA receptor surface expression (ChemLTP) in dispersed neuronal cultures. ChemLTP increases co-localisation of SUMO-1 and the SUMO conjugating enzyme Ubc9 and with PSD95 consistent with the recruitment of SUMOylated proteins to dendritic spines. In addition, we show that ChemLTP increases dendritic levels of SUMO-1 and Ubc9 mRNA. Consistent with activity dependent translocation of these mRNAs to sites near synapses, levels of the mRNA binding and dendritic transport protein CPEB are also increased by ChemLTP. Importantly, reducing the extent of substrate protein SUMOylation by overexpressing the deSUMOylating enzyme SENP-1 or inhibiting SUMOylation by expressing dominant negative Ubc9 prevent the ChemLTP-induced increase in both AMPAR surface expression and dendritic SUMO-1 mRNA. Taken together these data demonstrate that SUMOylation of synaptic protein(s) involved in AMPA receptor trafficking is necessary for activity-dependent increases in AMPAR surface expression. - . These authors contributed equally to this work. AMPA receptors (AMPARs) mediate most fast excitatory neurotransmission in the CNS and are key determinants of neuronal function and dysfunction [1,2]. Increases in the number and changes in the composition and/or properties of synaptic AMPARs mediate long-term potentiation (LTP) of synaptic efficacy whereas their removal leads to long-term depression (LTD). AMPAR-mediated synaptic plasticity occurs at synapses throughout the CNS and underlies many neuronal, network and systems level functions ranging from sensory perception to intellectual processing [3]. AMPAR trafficking is complex and stringently regulated, and it is now clear that there are distinct pathways and multiple layers of control for the delivery, residence time and removal of synaptic AMPARs. The contribution of each pathway depends on specific signaling cues and the precise AMPAR subunit composition [1,2]. Although remarkable progress has been achieved towards more complete understanding of the core mechanisms, fundamental questions remain about the function and dysfunction of AMPAR trafficking and synaptic plasticity in health and disease. Posttranslational modification can direct protein folding, distribution, stability, activity and function. Consequently, posttranslational modifications are integral to signalling cascades, especially in the CNS where the processes affecting synaptic communication between neurons are highly orchestrated. For example both phosphorylation and ubiquitination have been extensively studied and play complex roles in postsynaptic protein regulation [4]. Protein modification by Small Ubiquitin-like MOdifier (SUMO) has emerged as an important regulator of neuronal function and dysfunction [5,6]. SUMO is conjugated to lysine residues in target proteins by a three-enzyme pathway analogous to, but distinct from, ubiquinitation. A major difference is that unlike for ubiquitin where there are many E2 conjugating enzymes, Ubc9 is the only SUMO E2. There are 3 validated SUMO isoforms in mammals, designated SUMO-1-3. SUMO-2 and -3 differ by only 3 residues, and are collectively known as SUMO-2/3. SUMOylation is reversed via the actions of a family of SUMO-specific deconjugating enzymes, SENPs [7]. In general, SUMO acts by altering the protein interactions of the substrate protein. We have shown previously that SUMOylation of the kainate receptor subunit GluK2a at K886 is required for agonist-induced endocytosis of GluK2a-containing kainate receptors [8]. More recently, we have demonstrated that GluK2a SUMOylation is evoked by agonistinduced PKC phosphorylation of GluK2, and that this phosphoSUMOylation switch is a crucial determinant of kainate receptor LTD at mossy fibre synapses [9,10,11]. We, and others have also identified other synaptic SUMO substrates e.g. [12,13] and SUMOylation has been implicated in several clinically important neurological and neurodegenerative diseases [14]. Interestingly, although there is currently no evidence to suggest that AMPARs are direct targets of SUMOylation [8], we have recently demonstrated that SUMOylation can regulate AMPAR trafficking during homeostatic synaptic plasticity [13]. We therefore investigated a potential role for SUMOylation in AMPAR surface expression mediated by more acute forms of plasticity using a well-characterised glycine stimulation protocol (ChemLTP) on cultured neurons [15,16]. Our data show that ChemLTP stimulation recruited SUMO-1 to synapses and increased co-localisation of SUMO-1 with Ubc9. SUMO-1 and Ubc9 mRNAs were also increased and trafficked to dendrites following ChemLTP. Importantly, reducing global levels of protein SUMOylation by overexpression of a dominant negative Ubc9 or the catalytic domain of SENP-1 prevented the ChemLTP-induced increase in GluA1-containing AMPAR surface expression. These data indicate that SUMOylation of synaptic protein(s) involved in AMPAR trafficking is necessary for activity-dependent increases in AMPAR surface expression. Materials and Methods Primary neuronal culture All experiments in this study were performed in accordance with UK Home Office Schedule 1 guidelines. Animals were sacrificed by cervical dislocation using procedures approved by the Home Office Licensing Team at the University of Bristol (UIN UB/12/008). Rat embryonic hippocampal neuronal cultures were prepared from E18 Wistar rats using standard procedures. The culture medium was Neurobasal medium (Gibco) supplemented with B27 (Gibco) and 2 mM glutamine. For some experiments, neurons were transduced with Sindbis virus at DIV 1720 and used 18 24 h later. Antibodies The following antibodies are used: N-terminal anti-GluA1 (Millipore), anti PSD95 and anti- b-tubulin (Sigma), SUMO-1 and SUMO2 (hybridoma bank), UBC9, CPEB, MAP-2 (Sigma). Chemical LTP (Chem-LTP) Chem-LTP was induced as described previously [15,17,18]. Briefly, neuronal cultures were transferred from Neurobasal growth medium to extracellular solution (ECS) containing: 150 mM NaCl, 2 mM CaCl2, 5 mM KCl, 10 mM HEPES (pH 7.4), 30 mM glucose, (...truncated)