Biomedical subjects

Morgan Sheng

Publications and source records attributed to Morgan Sheng.

At least 37 records · Page 2Linked to original sources

PubMed2005

Polo-like kinases in the nervous system.

Polo like kinases (Plks) are key regulators of the cell cycle, but little is known about their functions in postmitotic cells such as neurons. Recent findings indicate that Plk2 and Plk3 are dynamically regulated in neurons by synaptic activity at the mRNA and protein levels. In COS cells, Plk2 and Plk3 interact with spine-associated Rap guanosine triphosphatase-activating protein (SPAR), a regulator of actin dynamics and dendritic spine morphology, leading to its degradation through the ubiquitin-proteasome system. Induction of Plk2 in hippocampal neurons eliminates SPAR protein, depletes a core postsynaptic scaffolding molecule (PSD-95), and causes loss of mature dendritic spines and synapses. These findings implicate neuronal Plks as mediators of activity-dependent change in molecular composition and morphology of synapses. Induction of Plks might provide a homeostatic mechanism for global dampening of synaptic strength following heightened neuronal activity ('synaptic scaling'). Synapse-specific actions of induced Plks are also possible, particularly in light of the discovery of phosphoserine/threonine peptide motifs as binding targets of the polo box domain, which could allow for 'priming' phosphorylation by upstream kinases that could 'tag' Plk substrates only in specific synapses.

Animals↗

PubMed2005

NSF interaction is important for direct insertion of GluR2 at synaptic sites.

Here, we use a cell surface thrombin cleavage assay to investigate directly the role of NSF in the surface delivery and synaptic accumulation of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors. In cultured hippocampal neurons, the GluR2 subunit (which specifically interacts with NSF) inserts rapidly into the plasma membrane from intracellular compartments and accumulates in synaptic sites. In contrast, surface accumulation of GluR3 (a subunit that does not interact with NSF) or a GluR2 mutant defective in NSF binding (DeltaA849-Q853) occurs initially at extrasynaptic sites and is kinetically slower than wild-type GluR2. Introducing a binding site for NSF into GluR3 (GluR3NSF) generates a subunit that behaves like GluR2 in terms of kinetics and site of surface insertion. These data suggest that the NSF interaction is necessary for rapid incorporation of AMPA receptor subunits into synapses and is sufficient to confer this property on GluR3.

Animals↗

PubMed2005

GRIP1 controls dendrite morphogenesis by regulating EphB receptor trafficking.

The function of the multi-PDZ domain scaffold protein GRIP1 (glutamate receptor interacting protein 1) in neurons is unclear. To explore the function of GRIP1 in hippocampal neurons, we used RNA interference (RNAi) to knock down the expression of GRIP1. Knockdown of GRIP1 by small interfering RNA (siRNA) in cultured hippocampal neurons caused a loss of dendrites, associated with mislocalization of the GRIP-interacting proteins GIuR2 (AMPA receptor subunit), EphB2 (receptor tyrosine kinase) and KIF5 (also known as kinesin 1; microtubule motor). The loss of dendrites by GRIP1-siRNA was rescued by overexpression of the extracellular domain of EphB2, and was phenocopied by overexpression of the intracellular domain of EphB2 and extracellular application of ephrinB-Fc fusion proteins. Neurons from EphB1-EphB2-EphB3 triple knockout mice showed abnormal dendrite morphogenesis. Disruption of the KIF5-GRIP1 interaction inhibited EphB2 trafficking and strongly impaired dendritic growth. These results indicate an important role for GRIP1 in dendrite morphogenesis by serving as an adaptor protein for kinesin-dependent transport of EphB receptors to dendrites.

Animals↗

PubMed2004

The 8-kDa dynein light chain binds to p53-binding protein 1 and mediates DNA damage-induced p53 nuclear accumulation.

The tumor suppressor protein p53 is known to undergo cytoplasmic dynein-dependent nuclear translocation in response to DNA damage. However, the molecular link between p53 and the minus end-directed microtubule motor dynein complex has not been described. We report here that the 8-kDa light chain (LC8) of dynein binds to p53-binding protein 1 (53BP1). The LC8-binding domain was mapped to a short peptide segment immediately N-terminal to the kinetochore localization region of 53BP1. The LC8-binding domain is completely separated from the p53-binding domain in 53BP1. Therefore, 53BP1 can potentially act as an adaptor to assemble p53 to the dynein complex. Unlike other known LC8-binding proteins, 53BP1 contains two distinct LC8-binding motifs that are arranged in tandem. We further showed that 53BP1 can directly associate with the dynein complex. Disruption of the interaction between LC8 and 53BP1 in vivo prevented DNA damage-induced nuclear accumulation of p53. These data illustrate that LC8 is able to function as a versatile acceptor to link a wide spectrum of molecular cargoes to the dynein motor.

Active Transport, Cell Nucleus↗

PubMed2004

The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses.

The proper intracellular distribution of mitochondria is assumed to be critical for normal physiology of neuronal cells, but direct evidence for this idea is lacking. Extension or movement of mitochondria into dendritic protrusions correlates with the development and morphological plasticity of spines. Molecular manipulations of dynamin-like GTPases Drp1 and OPA1 that reduce dendritic mitochondria content lead to loss of synapses and dendritic spines, whereas increasing dendritic mitochondrial content or mitochondrial activity enhances the number and plasticity of spines and synapses. Thus, the dendritic distribution of mitochondria is essential and limiting for the support of synapses. Reciprocally, synaptic activity modulates the motility and fusion/fission balance of mitochondria and controls mitochondrial distribution in dendrites.

Animals↗

PubMed2004

Quaternary structure, protein dynamics, and synaptic function of SAP97 controlled by L27 domain interactions.

Single-particle electron microscopy (EM) combined with biochemical measurements revealed the molecular shape of SAP97 and a monomer-dimer transition that depended on the N-terminal L27 domain. Overexpression of SAP97 drove GluR1 to synapses, potentiated AMPA receptor (AMPAR) excitatory postsynaptic currents (EPSCs), and occluded LTP. Synaptic potentiation and GluR1 delivery were dissociable by L27 domain mutants that inhibit multimerization of SAP97. Loss of potentiation was correlated with faster turnover of monomeric SAP97 mutants in dendritic spines. We propose that L27-mediated interactions of SAP97 with itself or other proteins regulate the synaptic delivery of AMPARs. RNAi knockdown of endogenous PSD-95 depleted surface GluR1 and impaired AMPA EPSCs. In contrast, RNAi knockdown of endogenous SAP97 reduced surface expression of both GluR1 and GluR2 and inhibited both AMPA and NMDA EPSCs. Thus SAP97 has a broader role than its close relative, PSD-95, in the maintenance of synaptic function.

Adaptor Proteins, Signal Transducing↗

PubMed2004

ZP8, a neuronal zinc sensor with improved dynamic range; imaging zinc in hippocampal slices with two-photon microscopy.

The synthesis of a difluorofluorescein monocarboxaldehyde platform and its use for preparing ZP8, a new member of the Zinpyr family of neuronal Zn(2+) sensors, are described. By combining an aniline photoinduced electron transfer (PET) switch and an electron-withdrawing fluorescein scaffold, ZP8 displays reduced background fluorescence and improved dynamic range compared to previous ZP probes. The bright sensor undergoes an 11-fold increase in fluorescence intensity upon Zn(2+) complexation (Phi = 0.03-0.35) with high selectivity over cellular concentrations of Ca(2+) and Mg(2+). In addition, sensors in the ZP family have been utilized for optical imaging in biological samples using two-photon microscopy (TPM). The cell-permeable ZP3 probe is capable of identifying natural pools of labile Zn(2+) within the mossy fiber synapses of live hippocampal slices using TPM, establishing the application of this technique for monitoring endogenous Zn(2+) stores.

Animals↗

PubMed2004

Actin/alpha-actinin-dependent transport of AMPA receptors in dendritic spines: role of the PDZ-LIM protein RIL.

The efficacy of excitatory transmission in the brain depends to a large extent on synaptic AMPA receptors, hence the importance of understanding the delivery and recycling of the receptors at the synaptic sites. Here we report a novel regulation of the AMPA receptor transport by a PDZ (postsynaptic density-95/Drosophila disc large tumor suppressor zona occludens 1) and LIM (Lin11/rat Isl-1/Mec3) domain-containing protein, RIL (reversion-induced LIM protein). We show that RIL binds to the AMPA glutamate receptor subunit GluR-A C-terminal peptide via its LIM domain and to alpha-actinin via its PDZ domain. RIL is enriched in the postsynaptic density fraction isolated from rat forebrain, strongly localizes to dendritic spines in cultured neurons, and coprecipitates, together with alpha-actinin, in a protein complex isolated by immunoprecipitation of AMPA receptors from forebrain synaptosomes. Functionally, in heterologous cells, RIL links AMPA receptors to the alpha-actinin/actin cytoskeleton, an effect that appears to apply selectively to the endosomal surface-internalized population of the receptors. In cultured neurons, an overexpression of recombinant RIL increases the accumulation of AMPA receptors in dendritic spines, both at the total level, as assessed by immunodetection of endogenous GluR-A-containing receptors, and at the synaptic surface, as assessed by recording of miniature EPSCs. Our results thus indicate that RIL directs the transport of GluR-A-containing AMPA receptors to and/or within dendritic spines, in an alpha-actinin/actin-dependent manner, and that such trafficking function promotes the synaptic accumulation of the receptors.

Actinin↗

PubMed2004

Subunit rules governing the sorting of internalized AMPA receptors in hippocampal neurons.

Removal of synaptic AMPA receptors is important for synaptic depression. Here, we characterize the roles of individual subunits in the inducible redistribution of AMPA receptors from the cell surface to intracellular compartments in cultured hippocampal neurons. The intracellular accumulation of GluR2 and GluR3 but not GluR1 is enhanced by AMPA, NMDA, or synaptic activity. After AMPA-induced internalization, homomeric GluR2 enters the recycling pathway, but following NMDA, GluR2 is diverted to late endosomes/lysosomes. In contrast, GluR1 remains in the recycling pathway, and GluR3 is targeted to lysosomes regardless of NMDA receptor activation. Interaction with NSF plays a role in regulated lysosomal targeting of GluR2. GluR1/GluR2 heteromeric receptors behave like GluR2 homomers, and endogenous AMPA receptors show differential activity-dependent sorting similar to homomeric GluR2. Thus, GluR2 is a key subunit that controls recycling and degradation of AMPA receptors after internalization.

Animals↗

PubMed2004

Intra- and intermolecular domain interactions of the C-terminal GTPase effector domain of the multimeric dynamin-like GTPase Drp1.

Mammalian Drp1 is a dynamin-like GTPase required for mitochondrial fission. Although it exists primarily as a cytosolic homo-tetramer in vivo, it can also self-assemble into higher order structures on the mitochondrial outer membrane, where it is required for proper mitochondrial division. Functional studies and sequence comparisons have revealed four different structural domains in Drp1, comprising N-terminal GTP-binding, middle, insert B, and C-terminal GTPase effector (GED) domains. Here we describe an intramolecular interaction within Drp1 between the GED and the N-terminal GTP-binding and middle domains. A point mutation (K679A) within the C-terminal GED domain inhibits this intramolecular association, without affecting the formation of Drp1 tetramers or the intermolecular associations among isolated C-terminal domains. Mutant Drp1 K679A exhibits impaired GTPase activity, and when overexpressed in mammalian cells it decreases mitochondrial division. Sedimentation experiments indicate that the K679A mutation either increases Drp1 complex formation or, more likely, decreases complex disassembly as compared with wild-type Drp1. Taken together, these data suggest that the C-terminal GED domain is important for stimulation of GTPase activity, formation and stability of higher order complexes, and efficient mitochondrial division.

Amino Acid Sequence↗

PubMed2004

Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity.

Activation of N-methyl-d-aspartate subtype glutamate receptors (NMDARs) is required for long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic transmission at hippocampal CA1 synapses, the proposed cellular substrates of learning and memory. However, little is known about how activation of NMDARs leads to these two opposing forms of synaptic plasticity. Using hippocampal slice preparations, we showed that selectively blocking NMDARs that contain the NR2B subunit abolishes the induction of LTD but not LTP. In contrast, preferential inhibition of NR2A-containing NMDARs prevents the induction of LTP without affecting LTD production. These results demonstrate that distinct NMDAR subunits are critical factors that determine the polarity of synaptic plasticity.

2-Amino-5-phosphonovalerate↗

PubMed2004

Transcriptional modification by a CASK-interacting nucleosome assembly protein.

CASK acts as a coactivator for Tbr-1, an essential transcription factor in cerebral cortex development. Presently, the molecular mechanism of the CASK coactivation effect is unclear. Here, we report that CASK binds to another nuclear protein, CINAP, which binds histones and facilitates nucleosome assembly. CINAP, via its interaction with CASK, forms a complex with Tbr-1, regulating expression of the genes controlled by Tbr-1 and CASK, such as NR2b and reelin. A knockdown of endogenous CINAP in hippocampal neurons reduces the promoter activity of NR2b. Moreover, NMDA stimulation results in a reduction in the level of CINAP protein, via a proteasomal degradation pathway, correlating with a decrease in NR2b expression in neurons. This study suggests that reduction of the CINAP protein level by synaptic stimulation contributes to regulation of the transcriptional activity of the Tbr-1/CASK/CINAP protein complex and thus modifies expression of the NR2b gene.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Semiquantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry.

The postsynaptic density (PSD) of central excitatory synapses plays a key role in postsynaptic signal transduction and contains a high concentration of glutamate receptors and associated scaffold and signaling proteins. We report here a comprehensive analysis of purified PSD fractions by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We identified 374 different proteins that copurified with the PSD structure and discovered thirteen phosphorylated sites from eight proteins. These proteins were classified into numerous functional groups, implying that the signaling pathways in the PSD are complex and diverse. Furthermore, using quantitative mass spectrometry, we measured the molar concentration and relative stoichiometries of a number of glutamate receptor subunits and scaffold proteins in the postsynaptic density. Thus this proteomic study reveals crucial information about molecular abundance as well as molecular diversity in the PSD, and provides a basis for further studies on the molecular mechanisms of synaptic function and plasticity.

Amino Acid Sequence↗

PubMed2004

Tyrosine phosphorylation of GluR2 is required for insulin-stimulated AMPA receptor endocytosis and LTD.

The alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors is subject to functionally distinct constitutive and regulated clathrin-dependent endocytosis, contributing to various forms of synaptic plasticity. In HEK293 cells transiently expressing GluR1 or GluR2 mutants containing domain deletions or point mutations in their intracellular carboxyl termini (CT), we found that deletion of the first 10 amino acids (834-843) selectively reduced the rate of constitutive AMPA receptor endocytosis, whereas truncation of the last 15 amino acids of the GluR2 CT, or point mutation of the tyrosine residues in this region, only eliminated the regulated (insulin-stimulated) endocytosis. Moreover, in hippocampal slices, both insulin treatment and low-frequency stimulation (LFS) specifically stimulated tyrosine phosphorylation of the GluR2 subunits of native AMPA receptors, and the enhanced phosphorylation appears necessary for both insulin- and LFS-induced long-term depression of AMPA receptor-mediated excitatory postsynaptic currents. Thus, our results demonstrate that constitutive and regulated AMPA receptor endocytosis requires different sequences within GluR CTs and tyrosine phosphorylation of GluR2 CT is required for the regulated AMPA receptor endocytosis and hence the expression of certain forms of synaptic plasticity.

Aging↗

PubMed2004

Cyclin-dependent kinase 5 phosphorylates the N-terminal domain of the postsynaptic density protein PSD-95 in neurons.

PSD-95 (postsynaptic density 95) is a postsynaptic scaffolding protein that links NMDA receptors to the cytoskeleton and signaling molecules. The N-terminal domain of PSD-95 is involved in the synaptic targeting and clustering of PSD-95 and in the clustering of NMDA receptors at synapses. The N-terminal domain of PSD-95 contains three consensus phosphorylation sites for cyclin-dependent kinase 5 (cdk5), a proline-directed serine-threonine kinase essential for brain development and implicated in synaptic plasticity, dopamine signaling, cocaine addiction, and neurodegenerative disorders. We report that PSD-95 is phosphorylated in the N-terminal domain by cdk5 in vitro and in vivo, and that this phosphorylation is not detectable in brain lysates of cdk5-/- mice. N-terminal phosphorylated PSD-95 is found in PSD fractions together with cdk5 and its activator, p35, suggesting a role for phosphorylated PSD-95 at synapses. In heterologous cells, coexpression of active cdk5 reduces the ability of PSD-95 to multimerize and to cluster neuronal ion channels, two functions attributed to the N-terminal domain of PSD-95. Consistent with these observations, the lack of cdk5 activity in cultured neurons results in larger clusters of PSD-95. In cdk5-/- cortical neurons, more prominent PSD-95 immunostained clusters are observed than in wild-type neurons. In hippocampal neurons, the expression of DNcdk5 (inactive form of cdk5) or of the triple alanine mutant (T19A, S25A, S35A) full-length PSD-95 results in increased PSD-95 cluster size. These results identify cdk5-dependent phosphorylation of the N-terminal domain of PSD-95 as a novel mechanism for regulating the clustering of PSD-95. Moreover, these observations support the possibility that cdk5-dependent phosphorylation of PSD-95 dynamically regulates the clustering of PSD-95/NMDA receptors at synapses, thus providing a possible mechanism for rapid changes in density and/or number of receptor at synapses.

Animals↗

PubMed2004

A tautomeric zinc sensor for ratiometric fluorescence imaging: application to nitric oxide-induced release of intracellular zinc.

Zinc is an essential metal ion for human growth and development, the disruption of cellular Zn(2+) homeostasis being implicated in several major disorders including Alzheimer's disease, diabetes, and cancer. The molecular mechanisms of Zn(2+) physiology and pathology are insufficiently understood, however, owing in part to the lack of tools for measuring changes in intracellular Zn(2+) concentrations with high spatial and temporal fidelity. To address this critical need, we have synthesized, characterized, and applied an intracellular fluorescent probe for the ratiometric imaging of Zn(2+) based on a tautomeric seminaphthofluorescein platform. Zin-naphthopyr 1 (ZNP1) affords single-excitation, dual-emission ratiometric detection of intracellular Zn(2+) through Zn(2+)-controlled switching between fluorescein and naphthofluorescein tautomeric forms. The probe features visible excitation and emission profiles, excellent selectivity responses for Zn(2+) over competing Ca(2+) and Mg(2+) ions at intracellular concentrations, a dissociation constant (K(d)) for Zn(2+) of <1 nM, and an 18-fold increase in fluorescence emission intensity ratio (lambda(624)/lambda(528)) upon zinc binding. We demonstrate the value of the ZNP1 platform for biological applications by imaging changes in intracellular [Zn(2+)] in living mammalian cells. Included is the ratiometric detection of endogenous pools of intracellular Zn(2+) after NO-induced release of Zn(2+) from cellular metalloproteins. We anticipate that ZNP1 and related probes should find utility for interrogating the biology of Zn(2+).

Animals↗

PubMed2004

Bright fluorescent chemosensor platforms for imaging endogenous pools of neuronal zinc.

A series of new fluorescent Zinpyr (ZP) chemosensors based on the fluorescein platform have been prepared and evaluated for imaging neuronal Zn(2+). A systematic synthetic survey of electronegative substitution patterns on a homologous ZP scaffold provides a basis for tuning the fluorescence responses of "off-on" photoinduced electron transfer (PET) probes by controlling fluorophore pK(a) values and attendant proton-induced interfering fluorescence of the metal-free (apo) probes at physiological pH. We further establish the value of these improved optical tools for interrogating the metalloneurochemistry of Zn(2+); the novel ZP3 fluorophore images endogenous stores of Zn(2+) in live hippocampal neurons and slices, including the first fluorescence detection of Zn(2+) in isolated dentate gyrus cultures. Our findings reveal that careful control of fluorophore pK(a) can minimize proton-induced fluorescence of the apo probes and that electronegative substitution offers a general strategy for tuning PET chemosensors for cellular studies. In addition to providing improved optical tools for Zn(2+) in the neurosciences, these results afford a rational starting point for creating superior fluorescent probes for biological applications.

Biosensing Techniques↗

PubMed2004

The dynamic turnover and functional roles of alpha-actinin in dendritic spines.

Excitatory synapses are located on actin-rich protrusions known as dendritic spines. alpha-Actinin is an actin binding protein enriched in the postsynaptic density (PSD) of excitatory synapses. Because it also binds to NMDA receptors and other PSD components, alpha-actinin has been proposed to link NMDA receptors and the PSD to the underlying actin cytoskeleton of the dendritic spine. Although alpha-actinin has been implicated in modulation of NMDA receptor activity, the cell biological function of alpha-actinin in neurons is unknown. We report here that alpha-actinin is concentrated in spines. Both the actin binding domain and the spectrin repeat region (which interacts with NMDA receptors) of alpha-actinin2 are required for spine targeting. In live imaging experiments, Venus-tagged alpha-actinin2 in dendritic spines showed faster turnover than PSD-95, as determined by fluorescent recovery after photobleaching (FRAP), and individual spines often showed marked fluctuations in alpha-actinin content over a time-scale of minutes. Overexpression of alpha-actinin2 increased the length and density of dendritic protrusions in cultured hippocampal neurons, an effect that requires the actin binding domain and the spectrin repeats of alpha-actinin. These results suggest that alpha-actinin regulates spine morphology and density.

Actinin↗