Faculty Bibliography

The GTPase dynamin I is required for synaptic vesicle (SV) endocytosis. Our observation that dynamin binds to the SV protein synaptophysin in a Ca(2+)-dependent fashion suggested the possibility that a dynamin/synaptophysin complex functions in SV recycling. In this paper we show that disruption of the dynamin/synaptophysin interaction by peptide injection into the squid giant synapse preterminal results in a decrease in transmitter release during high-frequency stimulation, indicating an inhibition of SV recycling. Electron microscopy of these synapses reveals a depletion of SVs, demonstrating a block of vesicle retrieval after fusion. In addition, we observed an increase in clathrin-coated vesicles, indicating that the peptide does not block clathrin-dependent endocytosis. We conclude that the dynamin/synaptophysin complex functions in a clathrin-independent mechanism of SV endocytosis that is required for efficient synaptic transmission

AMPA receptors mediate the majority of fast synaptic neurotransmission at excitatory synapses within the CNS. One approach to studying these receptors is to construct a blueprint of the proteins located within the PSD which interact with AMPA receptor subunits. Towards this goal, a number of PDZ-containing proteins have been shown to interact with the cytoplasmic C-terminus of GluR2, the dominant AMPA receptor subunit. ABP is one such protein that contains six or seven PDZ domains, depending on its form. PDZ domain 5 of ABP shows the highest binding affinity for GluR2, and therefore it is likely that this domain mediates the interaction with AMPA receptors in vivo. Presumably, the rest of the PDZ domains of ABP are free to bind other proteins. These interactions may be responsible for anchoring AMPA receptors at the synapse, or alternatively, may be involved with the transport of AMPA receptors into and out of the postsynaptic membrane. Therefore, in order to understand the processing of AMPA receptors it is necessary to decipher the identity of additional proteins that interact with ABP. Using the Yeast Two-Hybrid system, a rat brain cDNA library was screened for potential ABP binding partners. Specifically, the first three PDZ domains of ABP were used as bait to discover novel interactions. Additional biochemical methods have been used to prove the specificity of binding and the function of these novel interacting proteins

The GTPase dynamin I is required for synaptic vesicle (SV) endocytosis. Our observation that dynamin binds to the SV protein synaptophysin in a Ca2+-dependent fashion suggested the possibility that a dynamin/synaptophysin complex functions in SV recycling. Here we show that disruption of the dynamin/synaptophysin interaction by peptide injection into the squid giant synapse preterminal results in a decrease in transmitter release during high-frequency stimulation, indicating an inhibition of SV recycling. Electron microscopy of these synapses reveals a depletion of SVs, demonstrating a block of vesicle retrieval after fusion. In addition, we observed an increase in clathrin-coated vesicles, indicating that the peptide does not block clathrin-dependent endocytosis. We conclude that the dynamin/synaptophysin complex functions in a clathrin-independent mechanism of SV endocytosis that is activated by the high Ca2+ concentration at SV release sites

PDZ domains are structural motifs that interact with the C-termini of proteins. Several labs, including ours, have identified two homologous proteins that interact with the C-terminus of the GluR2 subunit of the glutamate receptor: AMPA receptor Binding Protein (ABP - containing 6 PDZ domains) and Glutamate Receptor Interacting Protein (GRIP - containing 7 PDZ domains). The role that ABP, GRIP and other molecules that interact with glutamate receptors, such as PICK1 and NSF, play in the precise synaptic localization, targeting, clustering and anchoring of the receptor is still not clear. Several splice variants of ABP were cloned from a rat brain cDNA library, including a form that contains 7 PDZ domains (ABP2) and two ABP forms with alternatively spliced N-termini. We have characterized the expression and localization of these different ABP forms both in Hela cells and hippocampal neurons. Currently, we are investigating the molecular basis underlying the differential localization of these ABP splice variants

NMDA-induced apoptosis in cultured primary neurons was characterized by analysis of DNA laddering and formation of TUNEL positive cells. Western blot analyses showed the appearance of activated caspase-3 immediately following NMDA stimulation of apoptotic cell death of cultured cortical neurons. The activation of caspase-3 was correlated with increased NO., levels detected in real time using the cell permeable DAF2-DA fluorophore. To determine the role of activated caspaases in NMDA receptor mediated apoptotic death we used the luciferase cell assay system (Rameau et al., (2000) Neuropharmacology, in press) to investigate the ability of cell survival factors and a broad inhibitor of caspases to reduce NMDA induced cell death. Cortical neurons were sensitive to both the baculovirus p35 caspase inhibitor expressed by transfection, and the Z-DEVD-FMK peptide, another inhibitor of caspases. Although the inhibitory effects of p35 and Z-DEVD-FMK are not specific to caspase-3 alone, these results indicate a primary role for caspases and in particular the action of activated caspase-3 in the mechanism of NMDA-induced excitotoxicity. Furthermore, the overexpression of Bcl-2, a cell survival factor also reduced NMDA induced cell death. These data add further support to a role for an apoptotic mechanism of NMDA receptor induced cell death and identify physiological effects of NMDA induced activation of caspases in the death of cultured primary cortical neurons

Peptide growth factors such as the neurotrophins and fibroblast growth factors have potent effects on synaptic transmission, development, and cell survival. We report that chronic (hours) treatment with basic fibroblast growth factor (FGF-2) potentiates Ca(2+)-dependent N-methyl-D-aspartate (NMDA) receptor inactivation in cultured hippocampal neurons. This effect is specific for the NMDA-subtype of ionotropic glutamate receptor and FGF-2. The potentiated inactivation requires ongoing protein synthesis during growth factor treatment and the activity of protein phosphatase 2B (PP2B or calcineurin) during agonist application. These results suggest a mechanism by which FGF-2 receptor signaling may regulate neuronal survival and synaptic plasticity

Neuropeptides FF (NPFF), AF (NPAF), and SF (NPSF) are homologous amidated peptides that were originally identified on the basis of similarity to the molluscan neuropeptide FMRF-amide. They have been hypothesized to have wide-ranging functions in the mammalian central nervous system, including pain modulation, opiate function, cardiovascular regulation, and neuroendocrine function. We have cloned the NPFF gene from human, bovine, rat, and mouse, and show that the precursor mRNA encodes for all three of the biochemically identified peptides (NPFF, NPAF, and NPSF). We demonstrate that NPFF precursor mRNA expression by Northern analysis and map sites of expression by in situ hybridization. We confirm the validity of the in situ hybridization by showing that its distribution in the brain and spinal cord matches the distribution of NPFF and NPSF immunoreactivity. We go on to show that the mRNA levels (as measured by in situ hybridization) in the spinal cord can be up-regulated by a model for inflammatory pain (carrageenan injection), but not by a model for neuropathic pain (lumbar nerve ligation). Our results confirm the evolutionary conservation of NPFF, NPAF, and NPSF neuropeptide expression in mammalian brain. They also provide a context for the interpretation of the pain-sensitizing effects of injections of these peptides that have been previously reported. Our results support a model for the role of these peptides in pain regulation at the level of the spinal cord

We review the cloning of a novel AMPA receptor binding protein (ABP) that interacts with GluR2/3 and is homologous to GRIP. ABP is enriched in the PSD with GluR2 and is localized to the PSD by EM. ABP binds GluR2 via the C-terminal VXI motif through a Class I PDZ interaction. ABP and GRIP can also homo- and heteromultimerize. Thus, ABP and GRIP may be involved in AMPA receptor regulation and localization, by linking it to other cytoskeletal or signaling molecules. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors. We are currently investigating proteins that bind ABP and that may regulate the AMPA receptor

The synapse is a specialized cellular junction with an elaborate and highly evolved capacity for signal transduction. At excitatory synapses, the neurotransmitter glutamate is released from the presynaptic nerve terminal and stimulates several types of glutamate receptors in the postsynaptic membrane. These include the ionotropic receptors, which are glutamate-gated cation channels, and the metabotropic receptors, which are G protein-coupled seven-transmembrane receptors. The ionotropic glutamate receptors have received special attention because of growing evidence that changes in their synaptic abundance, posttranslational modification, or molecular interactions can provide long-term changes in synaptic strength. This review summarizes new information about the ionotropic glutamate receptors and relates receptor function to the organization of the postsynaptic membrane and the regulation of electrophysiologic and biochemical signaling at the synapse