Metabotropic action of postsynaptic kainate receptors triggers hippocampal long-term potentiation

Long-term potentiation (LTP) in the rat hippocampus is the most extensively studied cellular model for learning and memory. Induction of classical LTP involves an NMDA receptor- and calcium-dependent increase in functional synaptic AMPA receptors mediated by enhanced recycling of internalized AMPA receptors back to the postsynaptic membrane. Here we report a novel, physiologically relevant NMDA receptor-independent mechanism that drives increased AMPA receptor recycling and LTP. This pathway requires the metabotropic action of kainate receptors and activation of G-protein, protein kinase C and phospholipase C. Like classical LTP, kainate receptor-dependent LTP recruits recycling endosomes to spines, enhances synaptic recycling of AMPA receptors to increase their surface expression and elicits structural changes in spines, including increased growth and maturation. These data reveal a new and previously unsuspected role for postsynaptic kainate receptors in the induction of functional and structural plasticity in the hippocampus

A proteomic analysis reveals the interaction of GluK1 ionotropic kainate receptor subunits with go proteins

Kainate receptors (KARs) are found ubiquitously in the CNS and are present presynaptically and postsynaptically regulating synaptic transmission and excitability. Functional studies have proven that KARs act as ion channels as well as potentially activating G-proteins, thus indicating the existance of a dual signaling system for KARs. Nevertheless, it is not clear how these ion channels activate G-proteins and which of the KAR subunits is involved. Here we performed a proteomic analysis to define proteins that interact with the C-terminal domain of GluK1 and we identified a variety of proteins with many different functions, including a Go α subunit. These interactions were verified through distinct in vitro and in vivo assays, and the activation of the Go protein by GluK1 was validated in bioluminescence resonance energy transfer experiments, while the specificity of this association was confirmed in GluK1-deficient mice. These data reveal components of the KAR interactome, and they show that GluK1 and Go proteins are natural partners, accounting for the metabotropic effects of KARs.This work was supported by grants to J.L. from the Spanish Ministry of Science and Innovation (BFU2006-07138 and BFU2011-24084), CONSOLIDER (CSD2007-00023), and Prometeo/2011/086, and by the EU FP7 Capacities Project BIO-IMAGINE (Grant Agreement No. 264173).Peer reviewe

DP-b99 modulates matrix metalloproteinase activity and neuronal plasticity.

DP-b99 is a membrane-activated chelator of zinc and calcium ions, recently proposed as a therapeutic agent. Matrix metalloproteinases (MMPs) are zinc-dependent extracellularly operating proteases that might contribute to synaptic plasticity, learning and memory under physiological conditions. In excessive amounts these enzymes contribute to a number of neuronal pathologies ranging from the stroke to neurodegeneration and epileptogenesis. In the present study, we report that DP-b99 delays onset and severity of PTZ-induced seizures in mice, as well as displays neuroprotective effect on kainate excitotoxicity in hippocampal organotypic slices and furthermore blocks morphological reorganization of the dendritic spines evoked by a major neuronal MMP, MMP-9. Taken together, our findings suggest that DP-b99 may inhibit neuronal plasticity driven by MMPs, in particular MMP-9, and thus may be considered as a therapeutic agent under conditions of aberrant plasticity, such as those subserving epileptogenesis

Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families

Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/ Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/ specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid

Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families

Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/ Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/ specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid

DP-b99 diminishes neurodegenerative effect of kainate in hippocampal slice cultures.

<p><b>A</b> – Representative fluorescence photomicrographs of organotypic hippocampal slices (16 DIV) showing uptake of PI 24 h after various treatments: CTRL (untreated cultures); DP-b99 20 µM and 0.12 µM; KA (5 µM); KA+DP-b99 20 µM or 0.12 µM. <b>B -</b> Quantification done by ImageJ software and represented in arbitrary units of fluorescence shows a significant decrease in neuronal death in all groups treated with DP-b99 (n = 3, mean ± SE; * - p<0.05). <b>C -</b> Western blot analysis of β-dystroglycan cleavage. Organotypic hippocampal cultures were treated with 50 µM KA for 10 min. DP-b99 (0.12 µM or 20 µM) was added to the culture media 1 h before KA administration. <b>D –</b> Quantification of β-dystroglycan 30 kDa product of MMP-9 cleavage intensity from three independent experiments (n = 3, mean ± SE; * - p<0.05).</p

Effect of DP-b99 on PTZ kindling induced seizures.

<p><b>A</b>- Seizure scores of PTZ-kindled vehicle- and DP-b99 treated mice during the course of the experiment (mean ± SE). Note the delay of seizures in DP-b99 treated group (n = 7 per group, repeated measures ANOVA:F (1, 10) = 5.6382, p<0.05). <b>B</b> – Schematic diagram of a hippocampal section showing suprapyramidal (SP) and infrapyramidal (IP) bundles of mossy fiber pathway arising from hilus of dentate gyrus (DG) to area of CA 3 pyramidal cells. Representative images of coronal sections immunostained for ZnT-3 (white) in CA3 hippocampal region of vehicle- and DP-b99 treated kindled animals. Vehicle treated animals demonstrated much longer IP length, indicating a robust mossy fibers sprouting (red asterisks). SR, stratum radiatum; SL, stratum lucidum; SP, stratum pyramidale; SO, stratum orient. Scale bar = 100 µm. <b>C</b>– Quantitative analysis of Zn-T3 immunofluorescence in vehicle- and DP-b99 treated kindled animals. ZN-T3 expression was evaluated in IP mossy fibers using the ratio of IP length to the length of SP (n = 4 animals for each group, mean ± SE; * - indicates p<0.05). <b>D, E -</b> Western blot analysis and quantification of β-dystroglycan cleavage product in the hippocampus of kindled animals (n = 4 per group, mean ± SE; * - indicates p<0.05).</p