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Biomedical subjects

E B Ziff

Publications and source records attributed to E B Ziff.

At least 19 recordsLinked to original sources
PubMed2001

PICK1 targets activated protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2.

The PICK1 protein interacts in neurons with the AMPA-type glutamate receptor subunit 2 (GluR2) and with several other membrane receptors via its single PDZ domain. We show that PICK1 also binds in neurons and in heterologous cells to protein kinase Calpha (PKCalpha) and that the interaction is highly dependent on the activation of the kinase. The formation of PICK1-PKCalpha complexes is strongly induced by TPA, and PICK1-PKCalpha complexes are cotargeted with PICK1-GluR2 complexes to spines, where GluR2 is found to be phosphorylated by PKC on serine 880. PICK1 also reduces the plasma membrane levels of the GluR2 subunit, consistent with a targeting function of PICK1 and a PKC-facilitated release of GluR2 from the synaptic anchoring proteins ABP and GRIP. This work indicates that PICK1 functions as a targeting and transport protein that directs the activated form of PKCalpha to GluR2 in spines, leading to the activity-dependent release of GluR2 from synaptic anchor proteins and the PICK1-dependent transport of GluR2 from the synaptic membrane.

3T3 Cells↗
PubMed2001

Differential cellular and subcellular localization of ampa receptor-binding protein and glutamate receptor-interacting protein.

Excitatory synaptic currents in the mammalian brain are typically mediated by the neurotransmitter glutamate, acting at AMPA receptors. We used immunocytochemistry to investigate the distribution of AMPA receptor-binding protein (ABP) in the cerebral neocortex. ABP was most prominent in pyramidal neurons, although it was also present (at lower levels) in interneurons. ABP and its putative binding partners, the GluR2/3 subunits of the AMPA receptor, exhibited prominent cellular colocalization. Under appropriate processing conditions, colocalization could also be documented in puncta, many of which could be recognized as dendritic spines. However, a sizable minority of GluR2/3-positive puncta were immunonegative for ABP. Because glutamate receptor-interacting protein (GRIP) may also anchor GluR2, we studied the relative distribution of ABP and GRIP. There was extensive colocalization of these two antigens at the cellular level, although GRIP, unlike ABP, was strongest in nonpyramidal neurons. Different parts of a single dendrite could stain selectively for ABP or GRIP. To further characterize this heterogeneity, we investigated punctate staining of neuropil using synaptophysin and the membrane tracer DiA to identify probable synapses. Some puncta were comparably positive for both ABP and GRIP, but the majority were strongly positive for one antigen and only weakly positive or immunonegative for the other. This heterogeneity could be seen even within adjacent spines of a single dendrite. These data suggest that ABP may act as a scaffold for AMPA receptors either in concert with or independently from GRIP.

Animals↗
PubMed2001

Transcription factors in melanocyte development: distinct roles for Pax-3 and Mitf.

A transgenic mouse model was used to examine the roles of the murine transcription factors Pax-3 and Mitf in melanocyte development. Transgenic mice expressing beta-galactosidase from the dopachrome tautomerase (Dct) promoter were generated and found to express the transgene in developing melanoblasts as early as embryonic day (E) 9.5. These mice express the transgene in a pattern characteristic of endogenous Dct expression. Transgenic mice were intercrossed with two murine coat color mutants, Splotch (Sp), containing a mutation in the murine Pax3 gene, and Mitf(mi), with a mutation in the basic-helix-loop-helix-leucine zipper gene Mitf. Transgenic heterozygous mutant animals were crossed to generate transgenic embryos for analysis. Examination of beta-galactosidase-expressing melanoblasts in mutant embryos reveals that Mitf is required in vivo for survival of melanoblasts up to the migration staging area in neural crest development. Examination of Mitf(mi)/+ embryos shows that there are diminished numbers of melanoblasts in the heterozygous state early in melanocyte development, consistent with a gene dosage-dependent effect upon cell survival. However, quantification and analysis of melanoblast growth during the migratory phase suggests that melanoblasts then increase in number more rapidly in the heterozygous embryo. In contrast to Mitf(mi)/Mitf(mi) embryos, Sp/Sp embryos exhibit melanoblasts that have migrated to characteristic locations along the melanoblast migratory pathway, but are greatly reduced in number compared to control littermates. Together, these results support a model for melanocyte development whereby Pax3 is required to expand a pool of committed melanoblasts or restricted progenitor cells early in development, whereas Mitf facilitates survival of the melanoblast in a gene dosage-dependent manner within and immediately after emigration from the dorsal neural tube, and may also directly or indirectly affect the rate at which melanoblast number increases during dorsolateral pathway migration.

Animals↗
PubMed2000

Synaptophysin regulates clathrin-independent endocytosis of synaptic vesicles.

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.

Animals↗
PubMed2000

Role of NMDA receptor functional domains in excitatory cell death.

The mechanisms by which the NMDA receptor (NMDAR) induces excitotoxicity were investigated using a novel assay. We quantitated the capacity of wild type and mutant receptors for cell killing in CHO cells and cultured cortical neurons by measuring the activity of a co-transfected firefly luciferase expression plasmid. NR1 subunit pore mutations that block Ca(2+) influx, and deletion of the NR1 cytoplasmic C-terminal domain, which functions in Ca(2+) regulation of receptor currents, decreased NMDAR mediated cell killing. We also transfected the NR1 pore mutants and C-terminal truncations in the presence of co-expressed exogenous wild type subunits. The pore and C-terminal truncation mutants acted in a dominant negative fashion and increased the survival of NMDAR-expressing CHO cells. Although physiological studies of similar NMDA receptor mutants have been carried out in heterologous cell lines, their functions in neurons remain relatively unknown. We show that expression of pore mutants and specific C terminal truncation mutants in cultured cortical neurons also exerts dominant negative function and protects these primary cells from endogenous receptor induced excitotoxic death. These results implicate positive actions of the selectivity filter and of the NR1 C-terminal domain in a Ca(2+)-dependent mechanism for NMDAR excitotoxicity. They also indicate that the mutant receptors which show diminished excitotoxicity and dominant negative action in heterologous cells can co-assemble with endogenous subunits in primary neurons and block NMDAR-dependent excitotoxic death.

Animals↗
PubMed2000

Mutagenesis reveals a role for ABP/GRIP binding to GluR2 in synaptic surface accumulation of the AMPA receptor.

We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Cell-density-dependent regulation of expression and glycosylation of dopachrome tautomerase/tyrosinase-related protein-2.

The expression of the dopachrome tautomerase gene (Dct) and its protein product, tyrosinase-related protein-2, was studied in the cultured, phorbol-ester-dependent murine melanocyte cell line melan-a. Increased cell density was found to stimulate Dct expression both in cells stably transfected with a Dct promoter-lacZ construct and endogenously in nontransfected cells. Increased Dct expression under these conditions corresponds to increased tyrosinase-related protein-2 production. Tyrosinase-related protein-2 was found to exist in two distinct glycoforms with different endoglycosidase sensitivities. Density-dependent expression of tyrosinase-related protein-2 was independent of time of cell growth, cell proliferation, and soluble factors, implying that cell-cell contact is the important determinant governing increased Dct expression under these conditions. Tyrp1 gene expression and tyrosinase-related protein-1 production were also induced under similar conditions. The results show that cell-cell contact between melanocytes induces a coordinated response at both transcriptional and nontranscriptional levels that induces production of the tyrosinase-related proteins that have a significant role in melanization.

Animals↗
PubMed2000

Consistent and selective expression of the discoidin domain receptor-1 tyrosine kinase in human brain tumors.

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.

Adolescent↗
PubMed1999

Recent excitement in the ionotropic glutamate receptor field.

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.

Animals↗
PubMed1999

ABP: a novel AMPA receptor binding protein.

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.

Animals↗
PubMed1999

Mxi1 is a repressor of the c-Myc promoter and reverses activation by USF.

The basic region/helix-loop-helix/leucine zipper (B-HLH-LZ) oncoprotein c-Myc is abundant in proliferating cells and forms heterodimers with Max protein that bind to E-box sites in DNA and stimulate genes required for proliferation. A second B-HLH-LZ protein, Mxi1, is induced during terminal differentiation, and forms heterodimers with Max that also bind E-boxes but tether the mSin3 transcriptional repressor protein along with histone deacetylase thereby antagonizing Myc-dependent activation. We show that Mxi1 also antagonizes Myc by a second pathway, repression of transcription from the major c-myc promoter, P2. Repression was independent of Mxi1 binding to mSin3 but dependent on the Mxi1 LZ and COOH-terminal sequences, including putative casein kinase II phosphorylation sites. Repression targeted elements of the myc P2 promoter core (-35/+10), where it reversed transactivation by the constitutive transcription factor, USF. We show that Zn2+ induction of a stably transfected, metallothionein promoter-regulated mxi1 gene blocked the ability of serum to induce transcription of the endogenous c-myc gene and cell entry into S phase. Thus, induction of Mxi1 in terminally differentiating cells may block Myc function by repressing the c-myc gene P2 promoter, as well as by antagonizing Myc-dependent transactivation through E-boxes.

3T3 Cells↗
PubMed1999

FGF-2 potentiates Ca(2+)-dependent inactivation of NMDA receptor currents in hippocampal 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.

Actinin↗
PubMed1998

AP-1, CREB and CBP transcription factors differentially regulate the tyrosine hydroxylase gene.

The tyrosine hydroxylase (TH) gene encodes the rate-limiting enzyme in the biosynthesis of catecholamines. We have investigated the roles of two elements of the TH promoter, the TH-'Fat Specific Element' (TH-FSE) which binds the Fos-Jun complex, and the cAMP Response Element (CRE), which binds CREB and the co-activator protein, CREB Binding Protein (CBP) in regulating TH gene transcription. In PC12 cells, the TH-FSE was required for induction by NGF while the CRE was required for induction by cAMP. We show that both elements can function independently and contribute strongly to TH promoter basal activity in PC12 cells. We employed transient expression in the F9 teratocarcinoma cell line to vary experimentally the levels of the nuclear regulators implicated in TH control by the PC12 studies. In F9 cells, the TH promoter was strongly activated by Fos and Jun, and by PKA-stimulated CREB protein. In F9 and NIH3T3 cells, CBP, a co-activator which targets Fos-Jun and PKA-stimulated CREB, also induced the TH promoter. Immunohistochemical studies in rat brain regions enriched in dopaminergic neurons, including the midbrain and olfactory bulb (OB), suggest that Fos-Jun and CREB make differential contributions to TH gene activity in different tissues. Whereas changes in Fos protein levels parallel decreases in TH protein upon olfactory deprivation, CBP levels remain unchanged. This suggests that CRE-associated factors, including CBP, are not major regulators in the OB. In contrast, the presence of CREB and the absence of Fos immunoreactivity in midbrain dopaminergic cells suggests that the CRE is the primary regulator in this region.

3T3 Cells↗
PubMed1998

The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and alpha- and beta-SNAPs.

In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.

Adenosine Triphosphate↗
PubMed1998

Novel anchorage of GluR2/3 to the postsynaptic density by the AMPA receptor-binding protein ABP.

We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors.

Amino Acid Sequence↗
PubMed1998

Genetic elements regulating HES-1 induction in Wnt-1-transformed PC12 cells.

PC12 cells differentiate in response to nerve growth factor from a chromaffin cell to a sympathetic neuronal phenotype. Wnt-1 is a secreted signaling factor required for development of mammalian midbrain and cerebellum. PC12 cells transformed by Wnt-1 fail to express several differentiation-specific genes in response to nerve growth factor. We have previously shown that HES-1, a negative regulator of neuronal differentiation, is increased in Wnt-1/PC12 cells (P. S. Issack and E. B. Ziff. Altered expression of helix-loop-helix transcriptional regulators and cyclin D1 in Wnt-1-transformed PC12 cells. Cell Growth & Differ., 9: 837-845). Here, we show that the HES-1 promoter is more active in Wnt-1/PC12 cells relative to PC12 and that the binding sites for the transcription factor RBP-J kappa contribute to this induction. We also identify two additional promoter elements required for elevated HES-1 expression. One element binds Wnt-1-induced protein complexes in a sequence-specific manner. Identification of Wnt-1 responsive elements in potential target genes may provide clues to nuclear pathways regulated by Wnt-1.

Animals↗
PubMed1998

Altered expression of helix-loop-helix transcriptional regulators and cyclin D1 in Wnt-1-transformed PC12 cells.

Nerve growth factor induces PC12 cells to differentiate from a chromaffin cell to a sympathetic neuronal phenotype. In contrast, PC12 cells, which stably express Wnt-1, a secreted signaling factor required for development of mammalian midbrain and cerebellum, fail to express differentiation-specific genes in response to nerve growth factor. Analysis of factors binding to E box-containing regulatory elements of the terminal differentiation gene encoding peripherin suggested a differentiation-specific control of expression of helix-loop-helix transcriptional regulators. Specifically, the MASH-1 (mammalian achaete-scute homologue) helix-loop-helix transcription factor, which plays a positive role in neuronal differentiation, is reduced in Wnt-1/PC12 cells, and HES-1, a negative regulator of MASH-1, is increased. These data suggest that the differentiation block may result from induction of HES-1. Wnt-1/PC12 cells also proliferate more rapidly and express increased levels of cyclin D1. Thus, Wnt-1 may block the differentiation and enhance the proliferation of PC12 cells by activating HES-1 and cyclin D1 and repressing MASH-1.

Animals↗