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David S Bredt

Publications and source records attributed to David S Bredt.

At least 37 records · Page 2Linked to original sources
PubMed2004

Differential localization of mammalian Lin-7 (MALS/Veli) PDZ proteins in the kidney.

Lin-7 PDZ proteins, also called MALS or Velis, have been shown to coordinate basolateral membrane expression of various target proteins in renal epithelial cell models. Three different Lin-7/MALS/Veli isoforms, encoded by separate genes, have been identified. Here, we show that each Lin-7/MALS/Veli isoform is expressed in the kidney. Using MALS isoform-specific antibodies in combination with cell-specific marker antibodies, we found the products of the three mammalian Lin-7/MALS/Veli genes are differentially expressed along the length of the nephron. MALS/Veli 1 is predominately expressed in the glomerulus, thick ascending limb of Henle's loop (TAL), and the distal convoluted tubule (DCT). MALS/Veli 2 is exclusively expressed in the vasa recta. MALS/Veli 3 is largely located in the DCT and collecting duct. The subcellular localization of MALS/Veli proteins can vary, depending on the isoform and the cell type. In contrast to the predominate basolateral location of MALS/Veli 1 in the TAL and DCT and MALS/Veli 3 in the DCT, MALS/Veli 1 is found diffusely throughout the cytosol of intercalated cells. In the collecting duct, MALS/Veli 3 is chiefly located on the basal membrane. Collectively, these results suggest that different MALS/Veli isoforms may carry out cell type-specific functions. The TAL and distal segments appear to have the most significant capacity for a basolateral membrane-targeting mechanism involving different MALS/Veli isoforms.

Animals↗
PubMed2004

AMPA receptor synaptic targeting regulated by stargazin interactions with the Golgi-resident PDZ protein nPIST.

Regulation of AMPA receptors (AMPARs) at synapses plays a critical role in alterations of synaptic strength in the brain. Stargazin, an AMPAR-interacting protein, is critical for clustering and regulation of synaptic AMPARs. Stargazin interacts with AMPARs via its extracellular domain and with PDZ [postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)] proteins via its C-terminal PDZ-binding motif, and these interactions are necessary for stargazin and AMPAR synaptic targeting. By studying the expression of stargazin mutant constructs in cultured hippocampal neurons, we identified a novel domain corresponding to residues 243-283 within the cytoplasmic C terminus of stargazin that is also required for stargazin and AMPAR synaptic clustering. To identify proteins that interact with this stargazin synaptic clustering domain, we performed a yeast two-hybrid assay and found that this stargazin domain binds to nPIST (neuronal isoform of protein-interacting specifically with TC10), a Golgi-enriched protein implicated in trafficking of transmembrane proteins. Using in situ hybridization, immunohistochemistry, coimmunoprecipitation studies, and biochemical fractionation, we found that stargazin and nPIST colocalize and interact in the brain. Finally, by studying AMPAR clustering in transfected hippocampal neurons, we found that overexpression of nPIST enhances AMPAR synaptic clustering, whereas transfection of a dominant-negative nPIST construct attenuates AMPAR synaptic clustering. These studies identify a novel stargazin domain necessary for synaptic clustering of AMPARs and suggest that nPIST and stargazin interactions play a critical role in AMPAR trafficking to the synapse.

Animals↗
PubMed2004

Calcium channel function regulated by the SH3-GK module in beta subunits.

beta subunits of voltage-gated calcium channels (VGCCs) regulate channel trafficking and function, thereby shaping the intensity and duration of intracellular changes in calcium. beta subunits share limited sequence homology with the Src homology 3-guanylate kinase (SH3-GK) module of membrane-associated guanylate kinases (MAGUKs). Here, we show biochemical similarities between beta subunits and MAGUKs, revealing important aspects of beta subunit structure and function. Similar to MAGUKs, an SH3-GK interaction within beta subunits can occur both intramolecularly and intermolecularly. Mutations that disrupt the SH3-GK interaction in beta subunits alter channel inactivation and can inhibit binding between the alpha(1) and beta subunits. Coexpression of beta subunits with complementary mutations in their SH3 and GK domains rescues these deficits through intermolecular beta subunit assembly. In MAGUKs, the SH3-GK module controls protein scaffolding. In beta subunits, this module regulates the inactivation of VGCCs and provides an additional mechanism for tuning calcium responsiveness.

Animals↗
PubMed2004

Palmitoylation controls trafficking of GAD65 from Golgi membranes to axon-specific endosomes and a Rab5a-dependent pathway to presynaptic clusters.

The GABA-synthesizing enzyme GAD65 is synthesized as a soluble cytosolic protein but undergoes post-translational modification(s) to become anchored to the cytosolic face of Golgi membranes before targeting to synaptic vesicle membranes in neuroendocrine cells. Palmitoylation of cysteines 30 and 45 in GAD65 is not required for targeting to Golgi membranes but is crucial for post-Golgi trafficking to presynaptic clusters in neurons. Here, we show that palmitoylated GAD65 colocalizes with the small GTP-binding protein Rab5a in Golgi membranes and in axons but not in dendrites. In the presence of the constitutively positive mutant Rab5(Q79L) palmitoylation resulted in polarized targeting of GAD65 to giant Rab5a-positive axonal endosomes, characterized by the absence of the Rab5a-effector molecule EEA1 and the transferrin receptor. By contrast, Rab5a-positive/EEA1-positive somatodendritic giant endosomes containing the transferrin receptor were devoid of GAD65. Palmitoylation-deficient GAD65 was excluded from endosomal compartments. A dominant negative mutant of Rab5a, Rab5a(S34N), specifically blocked axonal trafficking and presynaptic clustering of palmitoylated GAD65, but did not affect axonal trafficking of mutants of GAD65 that fail to traffic to giant axonal endosomes containing Rab5a(Q79L). Two transmembrane synaptic vesicle proteins, VAMP2 and VGAT also localized to the axonal giant endosomes, and their axonal trafficking and presynaptic clustering was blocked by Rab5a(S34N). The results suggest that palmitoylation of GAD65 regulates the trafficking of the protein from Golgi membranes to an endosomal trafficking pathway in axons that is dependent on Rab5a and is required for the targeting of several synaptic vesicle proteins to presynaptic clusters.

Animals↗
PubMed2004

Dynamic interaction of stargazin-like TARPs with cycling AMPA receptors at synapses.

Activity-dependent plasticity in the brain arises in part from changes in the number of synaptic AMPA receptors. Synaptic trafficking of AMPA receptors is controlled by stargazin and homologous transmembrane AMPA receptor regulatory proteins (TARPs). We found that TARPs were stable at the plasma membrane, whereas AMPA receptors were internalized in a glutamate-regulated manner. Interaction with AMPA receptors involved both extra- and intracellular determinants of TARPs. Upon binding to glutamate, AMPA receptors detached from TARPs. This did not require ion flux or intracellular second messengers. This allosteric mechanism for AMPA receptor dissociation from TARPs may participate in glutamate-mediated internalization of receptors in synaptic plasticity.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2004

Regulation of dendritic branching and filopodia formation in hippocampal neurons by specific acylated protein motifs.

Although neuronal axons and dendrites with their associated filopodia and spines exhibit a profound cell polarity, the mechanism by which they develop is largely unknown. Here, we demonstrate that specific palmitoylated protein motifs, characterized by two adjacent cysteines and nearby basic residues, are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and the branching of dendrites and axons in neurons. Such motifs are present at the N-terminus of GAP-43 and the C-terminus of paralemmin, two neuronal proteins implicated in cytoskeletal organization and filopodial outgrowth. Filopodia induction is blocked by mutations of the palmitoylated sites or by treatment with 2-bromopalmitate, an agent that inhibits protein palmitoylation. Moreover, overexpression of a constitutively active form of ARF6, a GTPase that regulates membrane cycling and dendritic branching reversed the effects of the acylated protein motifs. Filopodia induction by the specific palmitoylated motifs was also reduced upon overexpression of a dominant negative form of the GTPase cdc42. These results demonstrate that select dually lipidated protein motifs trigger changes in the development and growth of neuronal processes.

ADP-Ribosylation Factor 6↗
PubMed2004

PSD93 regulates synaptic stability at neuronal cholinergic synapses.

Neuronal cholinergic synapses play important roles in both the PNS and CNS. However, the mechanisms that regulate the formation, maturation, and stability of neuronal cholinergic synapses are poorly understood. In this study, we use the readily accessible mouse superior cervical ganglion (SCG) and submandibular ganglion (SMG) to examine the assembly of the postsynaptic complex of neuronal cholinergic synapses. We find that novel splicing forms of PSD93 (postsynaptic density 93) are expressed in SCG. By immunostaining, we show that PSD93 proteins precisely colocalize with neuronal nicotinic acetylcholine receptors (nAChRs) at synapses of the SCG and SMG. Subcellular fractionation demonstrates that PSD93 is enriched in the PSD fraction of SCG, and coimmunoprecipitation shows that PSD93 and neuronal nAChRs form a complex in vivo. Furthermore, two additional components of the well characterized glutamatergic postsynaptic complex, GKAP/SAPAP (guanylate kinase domain-associated protein/synapse-associated protein-associated protein) and Shank/ProSAP family proteins, are also present at neuronal cholinergic synapses. To assess the function of this protein complex at neuronal cholinergic synapses in vivo, we examined ganglia in mice that lack PSD93. We find that neuronal cholinergic synapses form properly in PSD93 null mice. After denervation, however, synaptic clusters of nAChRs disassemble much faster in mice lacking PSD93 than those in wild-type mice. These results demonstrate that PSD93 is a key component of the postsynaptic scaffold at neuronal cholinergic synapses and plays an important role in synaptic stability. In addition, these results suggest that the mechanism of postsynaptic scaffolding is conserved between neuronal cholinergic and glutamatergic synapses.

Alternative Splicing↗
PubMed2004

Early events in glutamate receptor trafficking.

Glutamate receptors are the primary mediators of excitatory synaptic transmission in the mammalian central nervous system. Activity-dependent changes in the number of postsynaptic glutamate receptors underlie aspects of synaptic plasticity and provide a mechanism for information storage in the brain. Recent work shows that receptor exit from the endoplasmic reticulum represents a critical regulatory step in glutamate receptor trafficking to the neuronal cell surface.

Animals↗
PubMed2004

Targeted disruption of PSD-93 gene reduces platelet-activating factor-induced neurotoxicity in cultured cortical neurons.

PSD-93, a molecular adaptive protein, binds to and clusters the N-methyl-D-aspartate (NMDA) receptor and assembles a specific set of signaling proteins (for example neuronal nitric oxide synthase, nNOS) around the NMDA receptor at synapses in the central nervous system. This suggests that PSD-93 might mediate many NMDA receptor-dependent physiological and pathophysiological functions. We report here that PSD-93 colocalizes and interacts with the NMDA receptor and neuronal nitric oxide synthase in cultured cortical neurons. Targeted disruption of PSD-93 gene significantly prevented NMDA receptor-nitric oxide signaling-dependent neurotoxicity triggered via platelet-activating factor (PAF) receptor activation. In addition, the deficiency of PSD-93 markedly attenuated platelet-activating factor-induced increase in cyclic guanosine 3',5'-monophosphate (cGMP) and prevented platelet-activating factor-promoted formation of NMDA receptor-neuronal nitric oxide synthase complex. These findings indicate that PSD-93 is involved in the NMDA receptor--nitric oxide-mediated pathological processing of neuronal damage triggered via platelet--activating factor receptor activation. Since platelet-activating factor is a potent neuronal injury mediator during the development of brain trauma, seizures, and ischemia, the present work suggests that PSD-93 might contribute to molecular mechanisms of neuronal damage in these brain disorders.

Animals↗
PubMed2003

AMPA receptor trafficking at excitatory synapses.

Excitatory synapses in the CNS release glutamate, which acts primarily on two sides of ionotropic receptors: AMPA receptors and NMDA receptors. AMPA receptors mediate the postsynaptic depolarization that initiates neuronal firing, whereas NMDA receptors initiate synaptic plasticity. Recent studies have emphasized that distinct mechanisms control synaptic expression of these two receptor classes. Whereas NMDA receptor proteins are relatively fixed, AMPA receptors cycle synaptic membranes on and off. A large family of interacting proteins regulates AMPA receptor turnover at synapses and thereby influences synaptic strength. Furthermore, neuronal activity controls synaptic AMPA receptor trafficking, and this dynamic process plays a key role in the synaptic plasticity that is thought to underlie aspects of learning and memory.

Amino Acid Sequence↗
PubMed2003

Organization of postsynaptic density proteins and glutamate receptors in axodendritic and dendrodendritic synapses of the rat olfactory bulb.

Glutamate neurotransmission in the olfactory bulb involves both axodendritic synapses and dendrodendritic reciprocal synapses and possibly also extrasynaptic receptors. By using a sensitive immunogold procedure, we have investigated the organization of two synaptic scaffolding molecules, PSD-95 and PSD-93, as well as N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors, at these heterogeneous glutamate signaling sites. Immunolabeling for PSD-95 and PSD-93 was present in all major types of putative glutamatergic synapse, suggesting that these proteins are essential components of the synaptic signaling apparatus. The linear density and the subsynaptic distribution of PSD-95/PSD-93 gold particles did not differ significantly between axodendritic and dendrodendritic synapses. Antibodies recognizing NMDA and AMPA receptor subunits also labeled asymmetric synapses throughout the olfactory bulb. Immunolabeling for the AMPA receptor subunits GluR2/3 was similar in all types of synapse. In contrast, immunogold signals for the NR1 subunit of NMDA receptors varied significantly among different synapse populations, with olfactory nerve synapses in the glomerular layer showing the lowest labeling intensity. Although the lateral dendrites of mitral and tufted cells have been reported to respond to glutamate, they did not display significant plasma membrane labeling for the NR1 subunit or for PSD-95, suggesting that the physiological effects of glutamate at these sites are mediated by NMDA autoreceptors that are not clustered and occur only at a low density on the dendritic surface. Our quantitative analysis of olfactory bulb synapses indicates that the density of NMDA receptors is not determined by the complement of PSD-95/PSD-93. The latter molecules appear to be expressed in an all-or-none fashion and may form a standard lattice common to different types of glutamatergic synapse.

Animals↗
PubMed2003

Lipid- and protein-mediated multimerization of PSD-95: implications for receptor clustering and assembly of synaptic protein networks.

Postsynaptic density protein 95 (PSD-95/SAP-90) is a palmitoylated membrane-associated guanylate kinase that oligomerizes and clusters ion channels and associated signaling machinery at excitatory synapses in brain. However, the mechanism for PSD-95 oligomerization and its relationship to ion channel clustering remain uncertain. Here, we find that multimerization of PSD-95 is determined by only its first 13 amino acids, which also have a remarkable capacity to oligomerize heterologous proteins. Multimerization does not involve a covalent linkage but rather palmitoylation of two cysteine residues in the 13 amino acid motif. This lipid-mediated oligomerization is a specific property of the PSD-95 motif, because it is not observed with other palmitoylated domains. Clustering K+ channel Kv1.4 requires interaction of palmitoylated PSD-95 with tetrameric K+ channel subunits but, surprisingly, does not require multimerization of PSD-95. Finally, disrupting palmitoylation with 2-bromopalmitate disperses PSD-95/K+-channel clusters. These data suggest new models for K+ channel clustering by PSD-95 - a reversible process regulated by protein palmitoylation.

Animals↗
PubMed2003

Impaired NMDA receptor-mediated postsynaptic function and blunted NMDA receptor-dependent persistent pain in mice lacking postsynaptic density-93 protein.

Modification of synaptic NMDA receptor (NMDAR) expression influences NMDAR-mediated synaptic function and associated persistent pain. NMDARs directly bind to a family of membrane-associated guanylate kinases (MAGUKs) that regulate surface and synaptic NMDAR trafficking in the CNS. We report here that postsynaptic density-93 protein (PSD-93), a postsynaptic neuronal MAGUK, is expressed abundantly in spinal dorsal horn and forebrain, where it colocalizes and interacts with NMDAR subunits NR2A and NR2B. Targeted disruption of the PSD-93 gene reduces not only surface NR2A and NR2B expression but also NMDAR-mediated excitatory postsynaptic currents and potentials, without affecting surface AMPA receptor expression or its synaptic function, in the regions mentioned above. Furthermore, mice lacking PSD-93 exhibit blunted NMDAR-dependent persistent pain induced by peripheral nerve injury or injection of Complete Freund's Adjuvant, although they display intact nociceptive responsiveness to acute pain. PSD-93 appears to be important for NMDAR synaptic targeting and function and to be a potential biochemical target for the treatment of persistent pain.

Animals↗
PubMed2003

Postsynaptic density-95 mimics and occludes hippocampal long-term potentiation and enhances long-term depression.

Previous studies have shown that overexpression of the protein PSD-95 (postsynaptic density-95) selectively enhances AMPA receptor-mediated synaptic responses in hippocampal pyramidal cells. To determine whether this effect is related to synaptic plasticity at these synapses, we examined whether PSD-95 expression mimics long-term potentiation (LTP), and also whether it influences LTP and long-term depression (LTD) in hippocampal slice cultures. Using simultaneous recording from transfected or infected cells and control pyramidal cells, we found that PSD-95, similar to LTP, increases the amplitude and frequency of miniature EPSCs. It also converts silent synapses to functional synapses, as does LTP. In addition, LTP is completely occluded in cells expressing PSD-95, whereas LTD is greatly enhanced. These results suggest that common mechanisms are involved in controlling synaptic AMPA receptors by PSD-95 and synaptic plasticity.

Animals↗
PubMed2003

Functional studies and distribution define a family of transmembrane AMPA receptor regulatory proteins.

Functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in cerebellar granule cells requires stargazin, a member of a large family of four-pass transmembrane proteins. Here, we define a family of transmembrane AMPA receptor regulatory proteins (TARPs), which comprise stargazin, gamma-3, gamma-4, and gamma-8, but not related proteins, that mediate surface expression of AMPA receptors. TARPs exhibit discrete and complementary patterns of expression in both neurons and glia in the developing and mature central nervous system. In brain regions that express multiple isoforms, such as cerebral cortex, TARP-AMPA receptor complexes are strictly segregated, suggesting distinct roles for TARP isoforms. TARPs interact with AMPA receptors at the postsynaptic density, and surface expression of mature AMPA receptors requires a TARP. These studies indicate a general role for TARPs in controlling synaptic AMPA receptors throughout the central nervous system.

Animals↗
PubMed2003

Nitric oxide signaling specificity--the heart of the problem.

Nitric oxide (NO) is a gaseous free radical that functions as an endogenous mediator in numerous tissues. Because NO is both reactive and highly diffusible, its formation must be tightly regulated to control its synthesis and to specify its signaling. Indeed, molecular studies of the NO synthase (NOS) family of enzymes have elaborated a variety of mechanisms, including protein interactions, lipid modifications and protein phosphorylation cascades that spatially and temporally control NO biosynthesis. These mechanisms determine both the upstream cellular signals that stimulate NO formation and the downstream molecular targets for NO. Understanding these cellular pathways that control NOS will help us to elucidate the functional roles of NO and provide novel strategies to treat diseases associated with NO abnormalities.

Animals↗
PubMed2003

Effect of knock down of spinal cord PSD-93/chapsin-110 on persistent pain induced by complete Freund's adjuvant and peripheral nerve injury.

PSD-93/chapsin-110 is a neuronal PDZ domain-containing protein that binds to and clusters the N-methyl-D-aspartate receptor (NMDAR) at synapses in the central nervous system. It also assembles a specific set of signaling proteins around the NMDAR and mediates downstream signaling by the NMDAR. Thus, PSD-93/chapsin-110 might be involved in many physiological and pathophysiological actions triggered via the activation of the NMDAR. In the current study, we report that abundant PSD-93/chapsin-110 protein was detected in rat spinal cord, particularly in the superficial dorsal horn. The rats injected intrathecally with PSD-93/chapsin-110 antisense oligodeoxynucleotide every 24 h for 4 days displayed not only a remarkable decrease in spinal cord PSD-93/chapsin-110 expression but also a significant reduction in the paw withdrawal responses to thermal and mechanical stimuli during complete Freund's adjuvant-induced inflammatory pain and peripheral nerve injury-induced neuropathic pain. In contrast, the rats injected intrathecally with PSD-93/chapsin-110 missense oligodeoxynucleotide did not exhibit these changes. We also found that pretreatment with PSD-93/chapsin-110 antisense oligodeoxynucleotide did not change the locomotor activity or the responses to acute noxious thermal and mechanical stimuli in intact rats. The present results indicate that the deficiency of spinal cord PSD-93/chapsin-110 protein significantly attenuates thermal and mechanical hyperalgesia in complete Freund's adjuvant- or peripheral nerve injury-induced chronic pain. This suggests that spinal cord PSD-93/chapsin-110 might be involved in the central mechanism of chronic pain. Our work might provide a new target for the therapy of chronic pain.

Acute Disease↗