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

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

OBJECTIVE: Few molecular targets are both consistently and selectively expressed in a majority of central nervous system (CNS) neoplasms. Receptor tyrosine kinases have been implicated in brain tumor oncogenesis. We previously isolated one such receptor, discoidin domain receptor-1 (DDR1), from high-grade pediatric brain tumors. Here, we analyze the cellular origin and distribution of DDR1 expression in human brain tumors and its expression in tumor cells relative to surrounding brain. METHODS: By use of a digoxigenin-labeled DDR1 riboprobe, we investigated the expression of DDR1 messenger ribonucleic acid in a prospective series of 30 resected human primary and metastatic brain neoplasms, nonneoplastic human brain, and mouse embryonic brain, as well as a mouse glioblastoma model, by in situ hybridization. RESULTS: All the high-grade primary brain and metastatic brain tumors showed unequivocal, intense DDR1 expression within the majority of tumor cells, whereas expression was not observed in hyperplastic tumor blood vessels, normal brain blood vessels, inflammatory cells, or in the normal brain tissue that surrounded the tumor. Receptor expression was limited to tumor cells located within solid tumor tissue. Overall, 27 of 29 resected CNS tumors exhibited tumor cell-specific DDR1 expression, whereas one specimen composed of isolated glioblastoma cells within invaded brain parenchyma showed no detectable staining for this receptor. DDR1 was also expressed preferentially in the ventricular zone (a region of highly proliferating precursor cells) of mice at embryonic Day 15.5. CONCLUSION: We found that DDR1 is consistently expressed in all high-grade brain neoplasms studied and is selective for tumor cells in the specimens analyzed. The expression of DDR1 by tumor cells of CNS neoplasms and by primitive cells of the embryonic ventricular zone suggests that DDR1 is a potentially useful marker of tumor cells within the CNS

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

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