Papers by Cezar m. Tigaret
Molecular Pharmacology 69 Pp 1251 1259, Apr 1, 2006
N-Methyl-D-aspartate (NMDA) receptor (NMDAR) activity regulates the net number of ␣-amino-3-hydro... more N-Methyl-D-aspartate (NMDA) receptor (NMDAR) activity regulates the net number of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR) at the cell surface by modulating the balance between AMPAR membrane insertion and endocytosis. In this study, we addressed the role of NMDAR subtypes and of NMDAR-mediated Ca 2ϩ influx in the NMDAR-induced endocytosis of GluR2-containing AMPARs in primary murine hippocampal neurons. We found that NMDAR activation enhanced the endocytosis of AMPARs containing the GluR2 splice variants with short, but not long, cytoplasmic tails. NMDA-induced GluR2 endocytosis was completely inhibited by pharmacological block of NR2B-containing NMDARs. In turn, preferential block of NR2A-containing □ S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material.
Subcellular localisation of recombinant a- and ?-synuclein
Mol Cell Neurosci, 2005
α-Synuclein, a protein implicated in neurodegenerative diseases and of elusive physiological func... more α-Synuclein, a protein implicated in neurodegenerative diseases and of elusive physiological function owes its name to an observed presence in presynaptic and nuclear compartments. However, its nuclear localisation has remained controversial. We expressed synuclein–eGFP fusion proteins in organotypic rat hippocampal slice cultures and murine hippocampal primary neurons using a Sindbis virus expression system. Recombinant full-length α-synuclein accumulated in presynaptic locations, mimicking its native distribution. Expression of deletion mutant α-synuclein revealed that presynaptic targeting depended on the presence of its N-terminal and core region. This domain also causes nuclear exclusion of the α-synuclein fusion protein. In contrast, the C-terminal domain of α-synuclein directs fusion proteins into the nuclear compartment. The related protein γ-synuclein contains a similar N-terminal and core domain as α-synuclein. However, γ-synuclein lacks a C-terminal domain that causes nuclear localisation of the fusion protein, suggesting that the two synucleins might have different roles relating to the cell nucleus.
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 25, 2015
Membrane trafficking of AMPA receptors (AMPARs) is critical for neuronal function and plasticity.... more Membrane trafficking of AMPA receptors (AMPARs) is critical for neuronal function and plasticity. Although rapid forms of AMPAR internalization during long-term depression (LTD) require clathrin and dynamin, the mechanisms governing constitutive AMPAR turnover and internalization of AMPARs during slow homeostatic forms of synaptic plasticity remain unexplored. Here, we show that, in contrast to LTD, constitutive AMPAR internalization and homeostatic AMPAR downscaling in rat neurons do not require dynamin or clathrin function. Instead, constitutive AMPAR trafficking is blocked by a Rac1 inhibitor and is regulated by a dynamic nonstructural pool of F-actin. Our findings reveal a novel role for neuronal clathrin-independent endocytosis controlled by actin dynamics and suggest that the interplay between different modes of receptor endocytosis provides for segregation between distinct modes of neuronal plasticity.
Subcellular localisation of recombinant alpha- and gamma-synuclein
Molecular and cellular neurosciences, 2005
alpha-Synuclein, a protein implicated in neurodegenerative diseases and of elusive physiological ... more alpha-Synuclein, a protein implicated in neurodegenerative diseases and of elusive physiological function owes its name to an observed presence in presynaptic and nuclear compartments. However, its nuclear localisation has remained controversial. We expressed synuclein-eGFP fusion proteins in organotypic rat hippocampal slice cultures and murine hippocampal primary neurons using a Sindbis virus expression system. Recombinant full-length alpha-synuclein accumulated in presynaptic locations, mimicking its native distribution. Expression of deletion mutant alpha-synuclein revealed that presynaptic targeting depended on the presence of its N-terminal and core region. This domain also causes nuclear exclusion of the alpha-synuclein fusion protein. In contrast, the C-terminal domain of alpha-synuclein directs fusion proteins into the nuclear compartment. The related protein gamma-synuclein contains a similar N-terminal and core domain as alpha-synuclein. However, gamma-synuclein lacks a C...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2003
Precise refinement of synaptic connectivity is the result of activity-dependent mechanisms in whi... more Precise refinement of synaptic connectivity is the result of activity-dependent mechanisms in which coincidence-dependent calcium signaling by NMDA receptors (NMDARs) under control of the voltage-dependent Mg2+ block might play a special role. In the developing rodent trigeminal system, the pattern of synaptic connections between whisker-specific inputs and their target cells in the brainstem is refined to form functionally and morphologically distinct units (barrelettes). To test the role of NMDA receptor signaling in this process, we introduced the N598R mutation into the native NR1 gene. This leads to the expression of functional NMDARs that are Mg2+ insensitive and Ca2+ impermeable. Newborn mice expressing exclusively NR1 N598R-containing NMDARs do not show any whisker-related patterning in the brainstem, whereas the topographic projection of trigeminal afferents and gross brain morphology appear normal. Furthermore, the NR1 N598R mutation does not affect expression levels of NM...
Molecular Pharmacology, 2006
N-Methyl-D-aspartate (NMDA) receptor (NMDAR) activity regulates the net number of ␣-amino-3-hydro... more N-Methyl-D-aspartate (NMDA) receptor (NMDAR) activity regulates the net number of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR) at the cell surface by modulating the balance between AMPAR membrane insertion and endocytosis. In this study, we addressed the role of NMDAR subtypes and of NMDAR-mediated Ca 2ϩ influx in the NMDAR-induced endocytosis of GluR2-containing AMPARs in primary murine hippocampal neurons. We found that NMDAR activation enhanced the endocytosis of AMPARs containing the GluR2 splice variants with short, but not long, cytoplasmic tails. NMDA-induced GluR2 endocytosis was completely inhibited by pharmacological block of NR2B-containing NMDARs. In turn, preferential block of NR2A-containing □ S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material.
Subcellular localisation of recombinant α- and γ-synuclein
Molecular and Cellular Neuroscience, 2005
alpha-Synuclein, a protein implicated in neurodegenerative diseases and of elusive physiological ... more alpha-Synuclein, a protein implicated in neurodegenerative diseases and of elusive physiological function owes its name to an observed presence in presynaptic and nuclear compartments. However, its nuclear localisation has remained controversial. We expressed synuclein-eGFP fusion proteins in organotypic rat hippocampal slice cultures and murine hippocampal primary neurons using a Sindbis virus expression system. Recombinant full-length alpha-synuclein accumulated in presynaptic locations, mimicking its native distribution. Expression of deletion mutant alpha-synuclein revealed that presynaptic targeting depended on the presence of its N-terminal and core region. This domain also causes nuclear exclusion of the alpha-synuclein fusion protein. In contrast, the C-terminal domain of alpha-synuclein directs fusion proteins into the nuclear compartment. The related protein gamma-synuclein contains a similar N-terminal and core domain as alpha-synuclein. However, gamma-synuclein lacks a C-terminal domain that causes nuclear localisation of the fusion protein, suggesting that the two synucleins might have different roles relating to the cell nucleus.
Learning & Memory, 2009
The NMDA receptor (NMDAR) subunit GluN1 is an obligatory component of NMDARs without a known func... more The NMDA receptor (NMDAR) subunit GluN1 is an obligatory component of NMDARs without a known functional homolog and is expressed in almost every neuronal cell type. The NMDAR system is a coincidence detector with critical roles in spatial learning and synaptic plasticity. Its coincidence detection property is crucial for the induction of hippocampal long-term potentiation (LTP). We have generated a mutant mouse model expressing a hypomorph of the Grin1 N598R allele, which leads to a minority (about 10%) of coincidence detection-impaired NMDARs. Surprisingly, these animals revealed specific functional changes in the dentate gyrus (DG) of the hippocampal formation. Early LTP was expressed normally in area CA1 in vivo, but was completely suppressed at perforant path-granule cell synapses in the DG. In addition, there was a pronounced reduction in the amplitude of the evoked population spike in the DG. These specific changes were accompanied by behavioral impairments in spatial recognition, spatial learning, reversal learning, and retention. Our data show that minor changes in GluN1-dependent NMDAR physiology can cause dramatic consequences in synaptic signaling in a subregion-specific fashion despite the nonredundant nature of the GluN1 gene and its global expression.
Measurement of Hippocampal Levels of Cellular Second Messengers Following In Situ Freezing
Journal of Neurochemistry, 1993
The in situ freezing technique has been widely used to fix labile metabolites and cellular second... more The in situ freezing technique has been widely used to fix labile metabolites and cellular second messengers in cerebral cortex. In this study, we isolated specific brain regions at 0 degree C from coronal sections of frozen heads following in situ brain freezing and measured regional concentrations of labile metabolites and cellular messengers. These levels in the cortex were compared with those in cortical punches obtained at freezing temperature (less than -40 degrees C) from the same in situ frozen brains and those of cortex dissected from decapitated animals. In both isoflurane- and pentobarbital-anesthetized animals, we observed that the levels of lactate, free fatty acids, inositol 1,4,5-trisphosphate, and diacylglycerol, as well as the proportion of protein kinase C associated with the membrane fraction, were similar in cortical punches taken at freezing temperature and those dissected at 0 degree C. However, with animals decapitated at room temperature, cortical and hippocampal levels of lactate, free fatty acids, and inositol 1,4,5-trisphosphate and the proportion of membrane protein kinase C were significantly higher than those of corresponding brain regions isolated at 0 degree C from in situ frozen brains (p < 0.05). These results indicate that dissection of cortex and hippocampus at 0 degree C following in situ freezing will eliminate decapitation-induced production of artifacts and changes in the levels of cellular second messengers such as inositol 1,4,5-trisphosphate, diacylglycerol, and protein kinase C. The present technique, used in conjunction with in situ freezing, will fix cellular second messengers and labile metabolites in several regions of brain and may facilitate accurate characterization of molecular and cellular mechanisms underlying CNS function.
European Journal of Pharmacology, 1987
The rationale of this study was to obtain a highly specific inhibitor of phospholipase C by raisi... more The rationale of this study was to obtain a highly specific inhibitor of phospholipase C by raising rabbit antibodies against the purified bacterial phospholipase C. The antibodies inhibited the enzyme activity in vitro and, as shown by immunofluorescence, cross-reacted with the membrane-bound phospholipase C of isolated guinea-pig smooth muscle cells. Incubation (0-4 h) of guinea-pig taenia coli and ileum with antibodies resulted in a progressive inhibition (up to 85%) of the contractile response evoked by 2 #M acetylcholine or 2 #M histamine but did not inhibit significantly the contraction produced by prostaglandin F2~ (0.1 #M). These inhibitory antibodies presumably represent the 'missing tool' needed to establish unequivocally if a given agonist acts via stimulation of the membrane-bound phospholipase C, and implicitly phosphoinositide hydrolysis.
European Journal of Neuroscience, 2008
In adult brain the majority of AMPA glutamate receptor (GluR) subunits contain GluR2. In knock-ou... more In adult brain the majority of AMPA glutamate receptor (GluR) subunits contain GluR2. In knock-out (KO) mice the absence of GluR2 results in consequences for synaptic plasticity including cognitive impairments. Here the morphology of dendritic spines and their synaptic contacts was analysed via three-dimensional reconstruction of serial electron micrographs from dentate gyrus (DG) of adult wild type (WT) and GluR2 KO mice. Pre-embedding immunocytochemical staining was used to examine the distribution and subcellular localization of AMPA receptor GluR1 and N-methyl-d-aspartate receptor NR1 subunits. There were no significant changes in synapse density in the DG of GluR2 KO compared with WT mice. However, in GluR2 KO mice there was a significant decrease in the percentage of synapses on mushroom spines but an increase in synapses on thin spines. There was also a large decrease in the proportion of synapses with complex perforated ⁄ segmented post-synaptic densities (PSDs) (25 vs. 78% in WT) but an increase in synapses with macular PSDs (75 vs. 22%). These data were coupled in GluR2 KO mice with significant decreases in volume and surface area of mushroom spines and their PSDs. In both GluR2 KO and WT mice, NR1 and GluR1 receptors were present in dendrites and spines but there was a significant reduction in NR1 labelling of spine membranes and cytoplasm in GluR2 KO mice, and a small decrease in GluR1 immunolabelling in membranes and cytoplasm of spines in GluR2 KO compared with WT mice. Our data demonstrate that the absence of GluR2 has a significant effect on both DG synapse and spine cytoarchitecture and the expression of NR1 receptors.
Phospholipase C is a transmembrane protein. Immunocytochemical evidence
Acta Histochemica et Cytochemica, 1988
... Biology, Faculty of Medicine, University of Jassy. POPESCU LAURENTZIU M. Division of Cell Bio... more ... Biology, Faculty of Medicine, University of Jassy. POPESCU LAURENTZIU M. Division of Cell Biology, Faculty of Medicine, University of Jassy. ... cell types (skeletal and smooth muscle cells, nerve cells, fibroblasts, leukocytes, erythrocytes) and species (frog, guinea-pig, rat, man). ...
At glutamatergic synapses, induction of associative synaptic plasticity requires time-correlated ... more At glutamatergic synapses, induction of associative synaptic plasticity requires time-correlated presynaptic and postsynaptic spikes to activate postsynaptic NMDA receptors (NMDARs). The magnitudes of the ensuing Ca2+ transients within dendritic spines are thought to determine the amplitude and direction of synaptic change. In contrast, we show that at mature hippocampal Schaffer collateral synapses the magnitudes of Ca2+ transients during plasticity induction do not match this rule. Indeed, LTP induced by time-correlated pre- and postsynaptic spikes instead requires the sequential activation of NMDARs followed by voltage-sensitive Ca2+ channels within dendritic spines. Furthermore, LTP requires inhibition of SK channels by mGluR1, which removes a negative feedback loop that constitutively regulates NMDARs. Therefore, rather than being controlled simply by the magnitude of the postsynaptic calcium 2+ rise, LTP induction requires the coordinated activation of distinct sources of Ca2+ and mGluR1-dependent facilitation of NMDAR function.
[Molecular mechanism of tolerance for vasodilator nitrates]
Revista de medicină internă, neurologe, psihiatrie, neurochirurgie, dermato-venerologie. Medicină internă
Decreased alpha 1-adrenergic receptors after experimental brain injury
Journal of Neurotrauma
The magnitude of neuronal damage in central nervous system (CNS) injury may be related, in part, ... more The magnitude of neuronal damage in central nervous system (CNS) injury may be related, in part, to alterations in the balance between excitatory and inhibitory neurotransmitters. Previous studies have implicated a role of central inhibitory noradrenergic mechanisms in the pathophysiologic sequelae of traumatic brain injury. In the present study, we examined alpha 1-adrenergic receptor binding after parasaggital lateral fluid percussion (FP) brain injury of moderate severity (2.3 atm) in the rat. At 30 min following injury, the specific binding of [3H]prazosin to membranes isolated from left cortex (injury site) was reduced by 37% in brain-injured animals when compared to sham-operated noninjured animals (p < 0.05). However, there were no significant differences in [3H]prazosin binding to membranes of either contralateral (right) cortex or left and right hippocampi between brain-injured and sham-operated animals. Conversely, at 24 h posttrauma, specific binding to membranes of le...
NEUROSCIENCE: More AMPAR Garnish
Science, 2009
ABSTRACT
Neuron, 2010
The Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is critically required for the synapti... more The Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is critically required for the synaptic recruitment of AMPA-type glutamate receptors (AMPARs) during both development and plasticity. However, the underlying mechanism is unknown. Using singleparticle tracking of AMPARs, we show that CaMKII activation and postsynaptic translocation induce the synaptic trapping of AMPARs diffusing in the membrane. AMPAR immobilization requires both phosphorylation of the auxiliary subunit Stargazin and its binding to PDZ domain scaffolds. It does not depend on the PDZ binding domain of GluA1 AMPAR subunit nor its phosphorylation at Ser831. Finally, CaMKII-dependent AMPAR immobilization regulates short-term plasticity. Thus, NMDA-dependent Ca 2+ influx in the post-synapse triggers a CaMKII-and Stargazin-dependent decrease in AMPAR diffusional exchange at synapses that controls synaptic function.
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Papers by Cezar m. Tigaret