-
[show abstract]
[hide abstract]
ABSTRACT: Neuronal development and synaptic plasticity are highly regulated processes in which protein kinases play a key role. Recently, increasing attention has been paid to a serine/threonine protein kinase called mammalian target of rapamycin (mTOR) that has well-known functions in cell proliferation and growth. In neuronal cells, mTOR is implicated in multiple processes, including transcription, ubiquitin-dependent proteolysis, and microtubule and actin dynamics, all of which are crucial for neuronal development and long-term modification of synaptic strength. The aim of this article is to present our current understanding of mTOR functions in axon guidance, dendritic tree development, formation of dendritic spines, and in several forms of long-term synaptic plasticity. We also aim to present explanation for the mTOR effects on neurons at the level of mTORregulated genes and proteins.
Molecular Neurobiology 01/2007; 34(3):205-19. · 5.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The syntheses and photophysical characterization of ZP9, 2-{2-chloro-6-hydroxy-3-oxo-5-[(2-{[pyridin-2-ylmethyl-(1H-pyrrol-2-ylmethyl)amino]methyl}phenylamino)methyl]-3H-xanthen-9-yl}benzoic acid, and ZP10, 2-{2-chloro-6-hydroxy-5-[(2-{[(1-methyl-1H-pyrrol-2-ylmethyl)pyridin-2-ylmethylamino]methyl}phenylamino)methyl]-3-oxo-3H-xanthen-9-yl}benzoic acid, two asymmetrically derivatized fluorescein-based dyes, are described. These sensors each contain an aniline-based ligand moiety functionalized with a pyridyl-amine-pyrrole group and have dissociation constants for Zn(II) in the sub-micromolar (ZP9) and low-micromolar (ZP10) range, which we define as "midrange". They give approximately 12- (ZP9) and approximately 7-fold (ZP10) fluorescence turn-on immediately following Zn(II) addition at neutral pH and exhibit improved selectivity for Zn(II) compared to the di-(2-picolyl)amine-based Zinpyr (ZP) sensors. Confocal microscopy studies indicate that such asymmetrical fluorescein-based probes are cell permeable and Zn(II) responsive in vivo.
Inorganic Chemistry 12/2006; 45(24):9748-57. · 4.60 Impact Factor
-
Dongmei Cheng,
Casper C Hoogenraad,
John Rush,
Elizabeth Ramm,
Max A Schlager,
Duc M Duong,
Ping Xu,
Sameera R Wijayawardana,
John Hanfelt,
Terunaga Nakagawa, Morgan Sheng,
Junmin Peng
[show abstract]
[hide abstract]
ABSTRACT: The postsynaptic density (PSD) of central excitatory synapses is essential for postsynaptic signaling, and its components are heterogeneous among different neuronal subtypes and brain structures. Here we report large scale relative and absolute quantification of proteins in PSDs purified from adult rat forebrain and cerebellum. PSD protein profiles were determined using the cleavable ICAT strategy and LC-MS/MS. A total of 296 proteins were identified and quantified with 43 proteins exhibiting statistically significant abundance change between forebrain and cerebellum, indicating marked molecular heterogeneity of PSDs between different brain regions. Moreover we utilized absolute quantification strategy, in which synthetic isotope-labeled peptides were used as internal standards, to measure the molar abundance of 32 key PSD proteins in forebrain and cerebellum. These data confirm the abundance of calcium/calmodulin-dependent protein kinase II and PSD-95 and reveal unexpected stoichiometric ratios between glutamate receptors, scaffold proteins, and signaling molecules in the PSD. Our data also demonstrate that the absolute quantification method is well suited for targeted quantitative proteomic analysis. Overall this study delineates a crucial molecular difference between forebrain and cerebellar PSDs and provides a quantitative framework for measuring the molecular stoichiometry of the PSD.
Molecular & Cellular Proteomics 07/2006; 5(6):1158-70. · 7.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In neuroscience, rats have several advantages over mice as a model organism. For instance, behavioral experiments are more advanced and the larger size of the brain is better suited for surgical manipulation and biochemistry. Furthermore, the vascular physiology of rats is considered closer to human, providing clinical relevance. Because transgenesis rates achieved by conventional pronuclear injection are extremely low (0.2-3.5%), the availability of transgenic rats in neuroscience is limited. Lentivirus infection is an efficient way to integrate exogenous genes into the genome of a one-cell embryo to generate transgenic animals. We report here the generation of synapsin I promoter driven GRIP1-transgenic rats using lentiviral transgenesis. GRIP1 was chosen as a transgene because it interacts with AMPA receptors and is involved in glutamate receptor signaling. From a single infection experiment, 45% of the offspring carried the transgene and 40% achieved germ-line transmission. The expression of GRIP1 was observed at low levels in brain, spinal cord and testis. Interestingly, one transgenic copy lacked a 147 bp fragment in the GRIP1 coding region most likely caused by alternative splicing of genomic lentiviral RNA. Co-immunoprecipitation from rat brains showed that transgenic GRIP1 is in complex with the endogenous GluR2 subunit of AMPA receptors. These results indicate that functional transgenic GRIP1 protein is expressed in rat brain using lentiviral vectors containing a human synapsin I promoter. Tissue specific lentiviral transgenic rats will be a powerful tool for various applications in modern neuroscience.
Journal of Neuroscience Methods 05/2006; 152(1-2):1-9. · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Excitatory synapses are formed on dendritic spines, postsynaptic structures that change during development and in response to synaptic activity. Once mature, however, spines can remain stable for many months. The molecular mechanisms that control the formation and elimination, motility and stability, and size and shape of dendritic spines are being revealed. Multiple signaling pathways, particularly those involving Rho and Ras family small GTPases, converge on the actin cytoskeleton to regulate spine morphology and dynamics bidirectionally. Numerous cell surface receptors, scaffold proteins and actin binding proteins are concentrated in spines and engaged in spine morphogenesis.
Current Opinion in Neurobiology 03/2006; 16(1):95-101. · 7.44 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Most excitatory synaptic transmissions in the central nervous system are mediated by the neurotransmitter glutamate. Binding of glutamate released from the presynaptic membrane causes glutamate receptors in the postsynaptic membrane to open, which results in a transient depolarization of the postsynaptic membrane. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) subtype of glutamate receptors is responsible for the majority of excitatory postsynaptic currents and is thought to play a central role in synaptic plasticity. Because modulation of glutamate receptors is believed to be involved in the basic mechanism underlying information storage in the brain, the molecular architecture of native AMPA receptors (AMPA-Rs) is of great interest. Previously, we have shown that AMPA-Rs purified from the brain are tightly associated with members of the stargazin/TARP (transmembrane AMPA receptor regulatory protein) family of membrane proteins [Nakagawa et al., Nature 433 (2005), pp. 545-549]. Here, we present a three-dimensional (3D) density map of the hetero-tetrameric AMPA-R without associated stargazin/TARP proteins as determined by cryo-negative stain single-particle electron microscopy. In the absence of stargazin/TARP proteins, the density representing the transmembrane region of the AMPA-R particles is substantially smaller, corroborating our previous analysis that was based solely on projection images.
Biological Chemistry 03/2006; 387(2):179-87. · 2.96 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Dendritic spines show rapid motility and plastic morphology, which may mediate information storage in the brain. It is presently believed that polymerization/depolymerization of actin is the primary determinant of spine motility and morphogenesis. Here, we show that myosin IIB, a molecular motor that binds and contracts actin filaments, is essential for normal spine morphology and dynamics and represents a distinct biophysical pathway to control spine size and shape. Myosin IIB is enriched in the postsynaptic density (PSD) of neurons. Pharmacologic or genetic inhibition of myosin IIB alters protrusive motility of spines, destabilizes their classical mushroom-head morphology, and impairs excitatory synaptic transmission. Thus, the structure and function of spines is regulated by an actin-based motor in addition to the polymerization state of actin.
Neuron 02/2006; 49(2):175-82. · 14.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The compound NTA-DCF consists of two components, a dichlorofluorescein (DCF) reporter and a nitrilotriacetic acid (NTA) functionality. The latter binds polyhistidine sequences selectively through a bridging metal ion. The NTA-DCF conjugate has photophysical properties similar to those of the parent DCF fluorophore both by itself and as its nickel(II) complex. The insensitivity of the emission to paramagnetic ions allows the probe to label His6-tagged proteins fluorescently on the extracellular surfaces of HEK 293-T and HeLa cells.
Journal of the American Chemical Society 02/2006; 128(2):418-9. · 9.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The molecular mechanisms that determine the size and complexity of the neuronal dendritic tree are unclear. Here, we show that the phosphoinositide-3' kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway promotes the growth and branching of dendrites in cultured hippocampal neurons. Constitutively active mutants of Ras, PI3K, and Akt, or RNA interference (RNAi) knockdown of lipid phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome Ten), induced growth and elaboration of dendrites that was blocked by mTOR inhibitor rapamycin and/or by overexpression of eIF-4E binding protein 1 (4E-BP1), which inhibits translation of 5' capped mRNAs. The effect of PI3K on dendrites was lost in more mature neurons (>14 d in vitro). Dendritic complexity was reduced by inhibition of PI3K and by RNAi knockdown of mTOR or p70 ribosomal S6 kinase (p70S6K, an effector of mTOR). A rapamycin-resistant mutant of mTOR "rescued" the morphogenetic effects of PI3K in the presence of rapamycin. By regulating global and/or local protein translation, and as a convergence point for multiple signaling pathways, mTOR could play a central role in the control of dendrite growth and branching during development and in response to activity.
Journal of Neuroscience 01/2006; 25(49):11300-12. · 7.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: QZ1, 2-[2-chloro-6-hydroxy-3-oxo-5-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, and QZ2, 2-[6-hydroxy-3-oxo-4,5-bis-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, two fluorescein-based dyes derivatized with 8-aminoquinoline, have been prepared and their photophysical, thermodynamic, and zinc-binding kinetic properties determined. Because of their low background fluorescence and highly emissive Zn(II) complexes, QZ1 and QZ2 have a large dynamic range, with approximately 42- and approximately 150-fold fluorescence enhancements upon Zn(II) coordination, respectively. These dyes have micromolar K(d) values for Zn(II) and are selective for Zn(II) over biologically relevant concentrations of the alkali and alkaline earth metals. The Zn(II) complexes also fluoresce brightly in the presence of excess Mn(II), Fe(II), Co(II), Cd(II), and Hg(II), offering improved specificity for Zn(II) over di(2-picolyl)amine-based Zn(II) sensors. Stopped-flow kinetic investigations indicate that QZ1 and QZ2 bind Zn(II) with k(on) values of (3-4) x 10(6) M(-1) s(-1), compared to (6-8) x 10(5) M(-1) s(-1) for select ZP (Zinpyr) dyes, at 4.3 degrees C. Dissociation of Zn(II) from QZ1 and QZ2 occurs with k(off) values of 150 and 160 s(-1), over 5 orders of magnitude larger than those for ZP probes, achieving reversibility on the biological (millisecond) time scale. Laser scanning confocal and two-photon microscopy studies reveal that QZ2 is cell-permeable and Zn(II)-responsive in vivo. Because of its weaker affinity for Zn(II), QZ2 responds to higher concentrations of intracellular Zn(II) than members of the ZP family, illustrating that binding affinity is an important parameter for Zn(II) detection in vivo.
Journal of the American Chemical Society 01/2006; 127(48):16812-23. · 9.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mitochondrial morphology within cells is controlled by precisely regulated rates of fusion and fission . During programmed cell death (PCD), mitochondria undergo extensive fragmentation and ultimately caspase-independent elimination through a process known as mitoptosis . Though this increased fragmentation is due to increased fission through the recruitment of the dynamin-like GTPase Drp1 to mitochondria , as well as to a block in mitochondrial fusion , cellular mechanisms underlying these processes remain unclear. Here, we describe a mechanism for the increased mitochondrial Drp1 levels and subsequent stimulation of mitochondrial fission seen during PCD. We observed Bax/Bak-mediated release of DDP/TIMM8a, a mitochondrial intermembrane space (IMS) protein , into the cytoplasm, where it binds to and promotes the mitochondrial redistribution of Drp1, a mediator of mitochondrial fission. Using both loss- and gain-of-function assays, we also demonstrate that the Drp1- and DDP/TIMM8a-dependent mitochondrial fragmentation observed during PCD is an important step in mitoptosis, which in turn is involved in caspase-independent cell death. Thus, following Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), IMS proteins released comprise not only apoptogenic factors such as cytochrome c involved in caspase activation but also DDP/TIMM8a, which activates Drp1-mediated fission to promote mitochondrial fragmentation and subsequently elimination during PCD.
Current Biology 01/2006; 15(23):2112-8. · 9.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The total molecular mass of individual postsynaptic densities (PSDs) isolated from rat forebrain was measured by scanning transmission EM. PSDs had a mean diameter of 360 nm and molecular mass of 1.10 +/- 0.36 GDa. Because the mass represents the sum of the molecular masses of all of the molecules comprising a PSD, it becomes possible to derive the number of copies of each protein, once its relative mass contribution is known. Mass contributions of PSD-95, synapse-associated protein (SAP)97, and alpha-Ca2+/calmodulin-dependent protein kinase II (CaMKII) were determined by quantitative gel electrophoresis of PSD fractions. The number of PSD-95 molecules per average PSD, contributing 2.3% of the mass of the PSD, was calculated to be 300, whereas the number of SAP97 molecules, contributing 0.9% of the mass of the PSD, was 90. The alpha-CaMKII holoenzymes, which contribute 6% of the mass when brains are homogenized within 2 min of interrupting blood flow, have 80 holoenzymes associated with a typical PSD. When blood flow is interrupted 15 min before homogenization, the average mass of PSDs increases by approximately 40%. The additional alpha-CaMKII associated with PSDs accounts for up to 20% of this mass increase, representing the addition of 100-200 alpha-CaMKII holoenzymes.
Proceedings of the National Academy of Sciences 09/2005; 102(32):11551-6. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Nature Neuroscience 08/2005; 8(7):906-15. · 15.53 Impact Factor
-
Yinghua Zhu,
Daniel Pak,
Yi Qin,
Stefanie G McCormack,
Myung J Kim,
Joel P Baumgart,
Vanisree Velamoor,
Yves P Auberson,
Pavel Osten,
Linda van Aelst, Morgan Sheng,
J Julius Zhu
[show abstract]
[hide abstract]
ABSTRACT: The related small GTPases Ras and Rap1 are important for signaling synaptic AMPA receptor (-R) trafficking during long-term potentiation (LTP) and long-term depression (LTD), respectively. Rap2, which shares 60% identity to Rap1, is present at excitatory synapses, but its functional role is unknown. Here, we report that Rap2 activity, stimulated by NR2A-containing NMDA-R activation, depresses AMPA-R-mediated synaptic transmission via activation of JNK rather than Erk1/2 or p38 MAPK. Moreover, Rap2 controls synaptic removal of AMPA-Rs with long cytoplasmic termini during depotentiation. Thus, Rap2-JNK pathway, which opposes the action of the NR2A-containing NMDA-R-stimulated Ras-ERK1/2 signaling and complements the NR2B-containing NMDA-R-stimulated Rap1-p38 MAPK signaling, channels the specific signaling for depotentiating central synapses.
Neuron 07/2005; 46(6):905-16. · 14.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: NMDA receptors (NMDARs) control bidirectional synaptic plasticity by regulating postsynaptic AMPA receptors (AMPARs). Here we show that NMDAR activation can have differential effects on AMPAR trafficking, depending on the subunit composition of NMDARs. In mature cultured neurons, NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1, primarily by regulating its surface insertion. In mature neurons, NR2B is coupled to inhibition rather than activation of the Ras-ERK pathway, which drives surface delivery of GluR1. Moreover, the synaptic Ras GTPase activating protein (GAP) SynGAP is selectively associated with NR2B-NMDARs in brain and is required for inhibition of NMDAR-dependent ERK activation. Preferential coupling of NR2B to SynGAP could explain the subtype-specific function of NR2B-NMDARs in inhibition of Ras-ERK, removal of synaptic AMPARs, and weakening of synaptic transmission.
Neuron 07/2005; 46(5):745-60. · 14.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Molecular and Cellular Neuroscience 05/2005; 28(4):650-60. · 3.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (LAR-RPTP) bind to liprin-alpha (SYD2) and are implicated in axon guidance. We report that LAR-RPTP is concentrated in mature synapses in cultured rat hippocampal neurons, and is important for the development and maintenance of excitatory synapses in hippocampal neurons. RNA interference (RNAi) knockdown of LAR or dominant-negative disruption of LAR function results in loss of excitatory synapses and dendritic spines, reduction of surface AMPA receptors, impairment of dendritic targeting of the cadherin-beta-catenin complex, and reduction in the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Cadherin, beta-catenin and GluR2/3 are tyrosine phosphoproteins that coimmunoprecipitate with liprin-alpha and GRIP from rat brain extracts. We propose that the cadherin-beta-catenin complex is cotransported with AMPA receptors to synapses and dendritic spines by a mechanism that involves binding of liprin-alpha to LAR-RPTP and tyrosine dephosphorylation by LAR-RPTP.
Nature Neuroscience 05/2005; 8(4):458-67. · 15.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Biological Chemistry 04/2005; 280(9):8172-9. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ionotropic glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system. Their modulation is believed to affect learning and memory, and their dysfunction has been implicated in the pathogenesis of neurological and psychiatric diseases. Despite a wealth of functional data, little is known about the intact, three-dimensional structure of these ligand-gated ion channels. Here, we present the structure of native AMPA receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; AMPA-Rs) purified from rat brain, as determined by single-particle electron microscopy. Unlike the homotetrameric recombinant GluR2 (ref. 3), the native heterotetrameric AMPA-R adopted various conformations, which reflect primarily a variable separation of the two dimeric extracellular amino-terminal domains. Members of the stargazin/TARP family of transmembrane proteins co-purified with AMPA-Rs and contributed to the density representing the transmembrane region of the complex. Glutamate and cyclothiazide markedly altered the conformational equilibrium of the channel complex, suggesting that desensitization is related to separation of the N-terminal domains. These data provide a glimpse of the conformational changes of an important ligand-gated ion channel of the brain.
Nature 03/2005; 433(7025):545-9. · 36.28 Impact Factor
-
Science 03/2005; 307(5713):1207-8. · 31.20 Impact Factor
