Faculty Bibliography

Stimulation of NMDA receptors activates neuronal nitric oxide synthase (nNOS) and the production of nitric oxide (NO). Dephosphorylation of nNOS increases nNOS enzymatic activity. We have examined the regulation of nNOS phosphorylation in rat cortical neurons following NMDA receptor activation. We show that nNOS is constitutively phosphorylated and that NMDA receptor activation decreases the level of nNOS phosphorylation by a mechanism that is blocked specifically by NMDA receptor antagonists and inhibitors of the Ca2+-regulated phosphatases calcineurin and PP1/PP2A. Using quantitative digital microscopy, we show that NMDA receptor activation induces the accumulation of nitrotyrosine, a measure of nNOS activity, and TdT-mediated fluorescein-dUTP nick end labeling (TUNEL) positivity, a measure of cell death. A calcineurin inhibitor blocked the increase in both TUNEL and nitrotyrosine positivity. Notably, TUNEL was increased in those neurons that were most strongly positive for nitrotyrosine. We conclude that NMDA receptor activation induces death of neurons by a cell autonomous pathway involving nNOS dephosphorylation by a calcineurin-dependent mechanism

AMPA-type glutamate receptors (AMPARs) play a major role in excitatory synaptic transmission and plasticity. Channel properties are largely dictated by their composition of the four subunits, GluR1-4 (or A-D). Here we show that AMPAR assembly and subunit stoichiometry are determined by RNA editing in the pore loop. We demonstrate that editing at the GluR2 Q/R site regulates AMPAR assembly at the step of tetramerization. Specifically, edited R subunits are largely unassembled and ER retained, whereas unedited Q subunits readily tetramerize and traffic to synapses. This assembly mechanism restricts the number of the functionally critical R subunits in AMPAR tetramers. Therefore, a single amino acid residue affects channel composition and, in turn, controls ion conduction through the majority of AMPARs in the brain

AMPA receptor-binding protein (ABP) is a multi-postsynaptic density-95/discs large/zona occludens (PDZ) protein that binds to the glutamate receptor 2/3 (GluR2/3) subunits of the AMPA receptor and is implicated in receptor membrane anchorage. A palmitoylated form of ABP localizes to spine heads, whereas a nonpalmitoylated form is found in intracellular clusters. Here, we investigate intracellular cluster formation by ABP and the ability of ABP to associate with GluR2 while in these clusters. We show that ABP interacts with intracellular membranes via the ABP linker I (LI)-set II (SII) subdomain, a region consisting of ABP linker 1 and PDZ4, -5, and -6. This suggests that cluster formation results from LI-SII ABP association with the membrane of a vesicular structure. We present evidence that ABP can self-associate at intracellular membrane surfaces via interactions involving SII. ABP in such membrane clusters can bind and retain GluR2 that has trafficked endocytotically from the plasma membrane. Phosphorylation of GluR2 at serine 880, proximal to the ABP binding site, has been implicated by others in the release of ABP from GluR2 and the mobilization of AMPA receptors for trafficking. We show that binding of GluR2 to ABP blocks phosphorylation of serine 880. This suggests that ABP can stabilize its own association with GluR2. We discuss a model in which ABP can form a protein scaffold at a vesicular membrane that is capable of binding GluR2, leading to formation of an intracellular AMPA receptor pool. Receptors in such a pool may contribute to receptor endocytotic and exocytotic trafficking and recycling

Neuropeptide FF (NPFF) is an RF-amide peptide with pleiotropic functions in the mammalian central nervous system, including pain modulation, opiate interactions, cardiovascular regulation and neuroendocrine effects. To gain further insights into the transcriptional mechanisms that regulate NPFF gene expression, we performed extensive homology analyses on the NPFF 5'-flanking region cloned from mouse, rat and human. Regions with high sequence homology between mouse, rat and human were expected to have higher probability to interact with regulatory proteins and were studied further. Electromobility shift assays revealed one region that may interact with homeobox proteins and two consensus DRE sites that bind a nuclear protein, which was identified as the downstream regulatory element antagonistic modulator DREAM by supershift assays. The region containing the DRE elements was found to repress reporter gene transcription as studied with chimeric luciferase reporter gene constructs. Co-transfections with DREAM expression vectors and chimeric NPFF promoter/luciferase gene constructs are in progress. We postulate that DREAM, a known transcriptional regulator of prodynorphin gene expression, is also a likely transcriptional regulator of NPFF gene expression

AMPA receptors are tetrameric cation channels that mediate the majority of fast excitatory transmission in the brain. Four subunits, GluR1-4 (or A-D) assemble in various stoichiometries, resulting in a spectrum of functionally distinct channels. Rules that govern assembly are largely unknown. The majority of AMPARs contain GluR2, which dominates transmission properties via Arg607, introduced into the pore loop by RNA editing (at the Q/R-site). Here we report that Arg607 also determines AMPAR assembly. Sedimentation analysis reveals that edited GluR2-R channels remain incompletely assembled and ER-retained, whereas unedited GluR2-Q channels readily tetramerize and exit from the ER, in neurons and HeLa cells. Mutagenesis reveals that the structure of the pore loop affects tetramerization. Therefore, by modulating a critical assembly surface, Q/R-editing determines AMPAR assembly and subunit stoichiometry

How the synapse is organized and how its organization changes during events that result in long-term changes in synaptic efficacy is the subject of intense study. Various anchoring proteins work in concert to organize the postsynaptic side of the membrane, and the interactions of these proteins can be altered by synaptic activity. DeSouza and Ziff discuss the evidence that the reversible palmitoylation of the postsynaptic density protein PSD-95 may result in the movement of AMPA-type glutamate receptors into and out of lipid raft domains, ultimately controlling AMPA receptor accumulation at the postsynaptic membrane

As an initial step to study the function of the gene encoding the human neuropeptide FF (NPFF), we cloned a 4.7-kb sequence from the promoter region. Primer extension and 5'-rapid amplification of cDNA ends revealed multiple transcription initiation sites. Northern blot analysis of the mRNA expression revealed a specific signal only in poly(A) + RNA from medulla and spinal cord. Chimeric luciferase reporter gene constructs were transiently transfected in A549, U-251 MG, SK-N-SH, SK-N-AS and PC12 cells. The promoter activity was directly comparable with the level of endogenous NPFF mRNA as determined by real-time quantitative RT-PCR. The highest promoter activity was measured when a region from - 552 to - 830 bp of the 5'-flanking region was fused to the constructs, and a potential silencer element was localized between nucleotides -220 and -551. A twofold increase in NPFF mRNA was observed after 72 h of nerve growth factor stimulation of PC12 cells and the region between - 61 and - 214 bp of the 5'-flanking region was found to be responsive to this stimulation. We postulate that control of human NPFF gene expression is the result of both positive and negative regulatory elements and the use of multiple transcription initiation sites

We used specific antibodies against two postsynaptic density proteins, GRIP (glutamate receptor interacting protein) and ABP (AMPA receptor-binding protein), to study their distribution in the rat retina. In the central nervous system, it has been shown that both proteins bind strongly to the AMPA glutamate receptor (GluR) 2/3 subunits, but not other GluRs, through a set of three PDZ domains. Western blots detected a single GRIP protein that was virtually identical in retina and brain, whereas retinal ABP corresponded to only one of three ABP peptides found in brain. The retinal distributions of GluR2/3, GRIP, and ABP immunoreactivity (IR) were similar but not identical. GluR2/3 immunoreactivity (IR) was abundant in both plexiform layers and in large perikarya. ABP IR was concentrated in large perikarya but was sparse in the plexiform layers, whereas GRIP IR was relatively more abundant in the plexiform layers than in perikarya. Immunolabel for these three antibodies consisted of puncta < or = 0.2 microm in diameter. The cellular localization of GRIP and ABP IR was examined by double labeling subclasses of retinal neuron with characteristic marker proteins, e.g., calbindin. GRIP, ABP, and GluR2/3 IR were detected in horizontal cells, dopaminergic and glycinergic AII amacrine cells and large ganglion cells. Immunolabel was absent in rod bipolar and weak or absent in cholinergic amacrine cells. By using the tyramide method of signal amplification, a colocalization of GluR2/3 was found with either GRIP or ABP in horizontal cell terminals, and perikarya of amacrine and ganglion cells. Our results show that ABP and GRIP colocalize with GluR2/3 in particular subsets of retinal neuron, as was previously established for certain neurons in the brain

Newly discovered features of the trafficking of AMPA receptors to and from the postsynaptic membrane of excitatory synapses are now bringing the mechanisms of synaptic plasticity into focus. Recent advances, including the existence of slots, anchors, transport factors and pathways for activity-dependent control, have elucidated the role of the individual AMPA receptor subunits and their binding partners. The latest views describe how subunit type dictates the assembly of heteromeric receptors, and how these heteromers interact with the receptor trafficking machinery and synaptic anchorage factors. Moreover, phosphorylation may play an important role in receptor transport and synaptic turnover

AMPA-receptor (AMPAR) transport to synapses plays a critical role in the modulation of synaptic strength. We show that the functionally critical GluR2 subunit stably resides in an intracellular pool in the endoplasmic reticulum (ER). GluR2 in this pool is extensively complexed with GluR3 but not with GluR1, which is mainly confined to the cell surface. Mutagenesis revealed that elements in the C terminus including the PDZ motif are required for GluR2 forward-transport from the ER. Surprisingly, ER retention of GluR2 is controlled by Arg607 at the Q/R-editing site. Reversion to Gln (R607Q) resulted in rapid release from the pool and elevated surface expression of GluR2 in neurons. Therefore, Arg607 is a central regulator. In addition to channel gating, it also controls ER exit and may thereby ensure the availability of GluR2 for assembly into AMPARs