Gephyrin is a multifunctional scaffold protein essential for accumulation of inhibitory glycine a... more Gephyrin is a multifunctional scaffold protein essential for accumulation of inhibitory glycine and GABAA receptors at post-synaptic sites. The molecular events involved in gephyrin-dependent GABAA receptor clustering are still unclear. Evidence has been recently provided that gephyrin phosphorylation plays a key role in these processes. Gephyrin post-translational modifications have been shown to influence the structural remodeling of GABAergic synapses and synaptic plasticity by acting on post-synaptic scaffolding properties as well as stability. In addition, gephyrin phosphorylation and the subsequent phosphorylation-dependent recruitment of the chaperone molecule Pin1 provide a mechanism for the regulation of GABAergic signaling. Extensively characterized as pivotal enzyme controlling cell proliferation and differentiation, the prolyl-isomerase activity of Pin1 has been shown to regulate protein synthesis necessary to sustain the late phase of long-term potentiation at excitator...
Activation of p73 upon genotoxic treatment triggers apoptosis of tumor cells lacking functional p... more Activation of p73 upon genotoxic treatment triggers apoptosis of tumor cells lacking functional p53 and involves the activities of c-Abl and p300. Here, we demonstrate that conformational changes of p73 catalyzed by the prolyl isomerase Pin1 are crucial in this pathway. Lack of Pin1 reduces p73 stability, hampering its accumulation upon genotoxic stress. Indeed, we show that upon treatment with chemotherapeutic drugs c-Abl enhances the phosphorylation-dependent interaction between Pin1 and p73, and this in turn promotes p73 acetylation by p300. Consistently, the ability of c-Abl and p300 to increase p73 stability and transcriptional activity requires Pin1. As a consequence, Pin1 appears to be essential for activation of the apoptotic response by endogenous p73.
The tumour suppressor p53 is important in the cell decision to either arrest cell cycle progressi... more The tumour suppressor p53 is important in the cell decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 post-translational modifications and association with other proteins have been implicated in the regulation of its stability and transcriptional activities. Here we report that, on DNA damage, p53 interacts with Pin1, a peptidyl-prolyl isomerase, which regulates the function of many proteins involved in cell cycle control and apoptosis. The interaction is strictly dependent on p53 phosphorylation, and requires Ser 33, Thr 81 and Ser 315. On binding, Pin1 generates conformational changes in p53, enhancing its transactivation activity. Stabilization of p53 is impaired in UV-treated Pin1(-/-) cells owing to its inability to efficiently dissociate from Mdm2. As a consequence, a reduced p53-dependent response was detected in Pin1(-/-) cells, and this correlates with a diminished transcriptional activation of some p53-regulated genes. Our results suggest that, following stress-induced phosphorylation, p53 needs to form a complex with Pin1 and to undergo a conformational change to fulfil its biological roles.
Early in development, network activity in the hippocampus is characterized by recurrent synchrono... more Early in development, network activity in the hippocampus is characterized by recurrent synchronous bursts, whose cellular correlates are giant depolarizing potentials (GDPs). The propensity for generating GDPs is attributed to GABAergic synaptic transmission being depolarizing and excitatory in neonatal neurons. However, developmental regulation of intrinsic conductances may also influence GDPs generation. A likely candidate is the non-inactivating, low-threshold, muscarinic-sensitive K + current (M current; I m ), which down-regulates intrinsic bursting activity in adult hippocampal pyramidal neurons. Western blot analysis of homogenates of the CA3 hippocampal region showed that expression of the Kv7.2 subunit, one of the constituents of neuronal M channels, is weak in neonatal neurons, and markedly increases after the first postnatal week. Likewise, the density of I m was very low in neonatal CA3 pyramidal cells and increased later on. Spontaneously occurring intrinsic bursts in neonatal neurons were longer and more robust, and recurred more regularly, than in juvenile neurons. The I m blocker linopirdine only mildly affected intrinsic bursting in neonatal neurons, but strongly facilitated and regularized it in juvenile neurons. We conclude that the low expression of Kv7/M channels and the depolarizing action of GABA early after birth enhance intrinsic bursting and neuronal synchronization leading to generation of GDPs within the hippocampal network.
Covalent modi®cation of the promyelocytic leukaemia protein (PML) by SUMO-1 is a prerequisite for... more Covalent modi®cation of the promyelocytic leukaemia protein (PML) by SUMO-1 is a prerequisite for the assembly of nuclear bodies (NBs), subnuclear structures disrupted in various human diseases and linked to transcriptional and growth control. Here we demonstrate that p53 is recruited into NBs by a speci®c PML isoform (PML3) or by coexpression of SUMO-1 and hUbc9. NB targeting depends on the direct association of p53, through its core domain, with a C-terminal region of PML3. The relocalization of p53 into NBs enhances p53 transactivation in a promoter-speci®c manner and affects cell survival. Our results indicate the existence of a cross-talk between PML-and p53dependent growth suppression pathways, implying an important role for NBs and their resident proteins as modulators of p53 functions.
The microtubule-binding protein gephyrin is known to play a pivotal role in targeting and cluster... more The microtubule-binding protein gephyrin is known to play a pivotal role in targeting and clustering postsynaptic inhibitory receptors. Here, the Intracellular Antibodies Capture Technology (IATC) was used to select two single-chain antibody fragments or intrabodies, which, fused to nuclear localization signals (NLS), were able to efficiently and selectively remove gephyrin from glycine receptor (GlyR) clusters. Co-transfection of NLS-tagged individual intrabodies with gephyrin-enhanced green fluorescent protein (EGFP) in HEK 293 cells revealed a partial relocalization of gephyrin aggregates onto the nucleus or in the perinuclear area. When expressed in cultured neurons, these intrabodies caused a significant reduction in the number of immunoreactive GlyR clusters, which was associated with a decrease in the peak amplitude of glycine-evoked whole cell currents as assessed with electrophysiological experiments. Hampering protein function at a posttranslational level may represent an attractive alternative for interfering with gephyrin function in a more spatially localized manner.
Speed and reliability of synaptic transmission are essential for information coding in neuronal n... more Speed and reliability of synaptic transmission are essential for information coding in neuronal networks and require the presence of clustered neurotransmitter receptors at the plasma membrane in precise apposition to presynaptic terminals. Receptor clusterization is the result of highly regulated processes involving functional and structural proteins. Among the structural elements, microtubules are known to play a crucial role in anchoring of ␥-aminobutyric acid, type A (GABA A ) receptors. Here we show that microtubule depolymerization with nocodazole induces the declusterization of GABA A receptors and modifies the kinetic properties of GABAergic currents in cultured hippocampal neurons. In particular, this drug, applied either in the bath or via the patch pipette, induced the acceleration of the onset kinetics of miniature inhibitory postsynaptic currents (mIPSCs) without significantly affecting their frequency, thus suggesting a main postsynaptic site of action. After nocodazole treatment, current responses to ultrafast applications of GABA exhibited a faster rise time and an accelerated onset of desensitization. A quantitative analysis of GABA-evoked currents and model simulations suggest that declusterization affects the gating properties of GABA A receptors. In particular, a faster entry into the desensitized state of declustered GABA A receptors may account for the changes in the kinetic properties of mIPSCs after nocodazole treatment. Hence it appears that the clustered condition of GABA A receptors contributes in shaping GABAergic currents.
Gephyrin is a scaffold protein essential for stabilizing glycine and GABA A receptors at inhibito... more Gephyrin is a scaffold protein essential for stabilizing glycine and GABA A receptors at inhibitory synapses. Here, recombinant intrabodies against gephyrin (scFv-gephyrin) were used to assess whether this protein exerts a transynaptic action on GABA and glutamate release. Pair recordings from interconnected hippocampal cells in culture revealed a reduced probability of GABA release in scFv-gephyrin-transfected neurons compared with controls. This effect was associated with a significant decrease in VGAT, the vesicular GABA transporter, and in neuroligin 2 (NLG2), a protein that, interacting with neurexins, ensures the cross-talk between the post-and presynaptic sites. Interestingly, hampering gephyrin function also produced a significant reduction in VGLUT, the vesicular glutamate transporter, an effect accompanied by a significant decrease in frequency of miniature excitatory postsynaptic currents. Overexpressing NLG2 in gephyrin-deprived neurons rescued GABAergic but not glutamatergic innervation, suggesting that the observed changes in the latter were not due to a homeostatic compensatory mechanism. Pulldown experiments demonstrated that gephyrin interacts not only with NLG2 but also with NLG1, the isoform enriched at excitatory synapses. These results suggest a key role of gephyrin in regulating transynaptic signaling at both inhibitory and excitatory synapses.
Tonic inhibition plays a crucial role in regulating neuronal excitability because it sets the thr... more Tonic inhibition plays a crucial role in regulating neuronal excitability because it sets the threshold for action potential generation and integrates excitatory signals. Tonic currents are known to be largely mediated by extrasynaptic ␥-aminobutyric acid type A (GABA A ) receptors that are persistently activated by submicromolar concentrations of ambient GABA. We recently reported that, in cultured hippocampal neurons, the clustering of synaptic GABA A receptors significantly affects synaptic transmission (Petrini, E. M., Zacchi, P., Barberis, A., Mozrzymas, J. W., and Cherubini, E. (2003) J. Biol. Chem. 278, 16271-16279). In this work, we demonstrated that the clustering of extrasynaptic GABA A receptors modulated tonic inhibition. Depolymerization of the cytoskeleton with nocodazole promoted the disassembly of extrasynaptic clusters of ␦ and ␥ 2 subunitcontaining GABA A receptors. This effect was associated with a reduction in the amplitude of tonic currents and diminished shunting inhibition. Moreover, diffuse GABA A receptors were less sensitive to the GAT-1 inhibitor NO-711 and to flurazepam. Quantitative analysis of GABA-evoked currents after prolonged exposure to submicromolar concentrations of GABA and model simulations suggest that clustering affects the gating properties of extrasynaptic GABA A receptors. In particular, a larger occupancy of the singly and doubly bound desensitized states can account for the modulation of tonic inhibition recorded after nocodazole treatment. Moreover, comparison of tonic currents recorded during spontaneous activity and those elicited by exogenously applied low agonist concentrations allows estimation of the concentration of ambient GABA. In conclusion, receptor clustering appears to be an additional regulating factor for tonic inhibition.
We describe a method to select DNA encoding functional open reading frames (ORFs) from noncoding ... more We describe a method to select DNA encoding functional open reading frames (ORFs) from noncoding DNA within the context of a specific vector. Phage display has been used as an example, but any system requiring DNA encoding protein fragments, for example, the yeast two-hybrid system, could be used. By cloning DNA fragments upstream of a fusion gene, consisting of the -lactamase gene flanked by lox recombination sites, which is, in turn, upstream of gene 3 from fd phage, only those clones containing DNA fragments encoding ORFs confer ampicillin resistance and survive. After selection, the -lactamase gene can be removed by Cre recombinase, leaving a standard phage display vector with ORFs fused to gene 3. This vector has been tested on a plasmid containing tissue transglutaminase. All surviving clones analyzed by sequencing were found to contain ORFs, of which 83% were localized to known genes, and at least 80% produced immunologically detectable polypeptides. Use of a specific anti-tTG monoclonal antibody allowed the identification of clones containing the correct epitope. This approach could be applicable to the efficient selection of random ORFs representing the coding potential of whole organisms, and their subsequent downstream use in a number of different systems. 4 These authors contributed equally to this work. 5 Corresponding author. E-MAIL [email protected]; FAX (505) 667-2891. Article and publication are at
Proceedings of The National Academy of Sciences, 2007
At early developmental stages, correlated neuronal activity is thought to exert a critical contro... more At early developmental stages, correlated neuronal activity is thought to exert a critical control on functional and structural refinement of synaptic connections. In the hippocampus, between postnatal day 2 (P2) and P6, network-driven giant depolarizing potentials (GDPs) are generated by the synergistic action of glutamate and GABA, which is depolarizing and excitatory. Here the rising phase of GDPs was used to trigger Schaffer collateral stimulation in such a way that synchronized network activity was coincident with presynaptic activation of afferent input. This procedure produced a persistent increase in spontaneous and evoked ␣-amino-3-hydroxy-5-methyl-4-isoxadepropionic acid-mediated glutamatergic currents, an effect that required calcium influx through postsynaptic L-type calcium channels.
Gephyrin is a multifunctional scaffold protein essential for accumulation of inhibitory glycine a... more Gephyrin is a multifunctional scaffold protein essential for accumulation of inhibitory glycine and GABAA receptors at post-synaptic sites. The molecular events involved in gephyrin-dependent GABAA receptor clustering are still unclear. Evidence has been recently provided that gephyrin phosphorylation plays a key role in these processes. Gephyrin post-translational modifications have been shown to influence the structural remodeling of GABAergic synapses and synaptic plasticity by acting on post-synaptic scaffolding properties as well as stability. In addition, gephyrin phosphorylation and the subsequent phosphorylation-dependent recruitment of the chaperone molecule Pin1 provide a mechanism for the regulation of GABAergic signaling. Extensively characterized as pivotal enzyme controlling cell proliferation and differentiation, the prolyl-isomerase activity of Pin1 has been shown to regulate protein synthesis necessary to sustain the late phase of long-term potentiation at excitator...
Activation of p73 upon genotoxic treatment triggers apoptosis of tumor cells lacking functional p... more Activation of p73 upon genotoxic treatment triggers apoptosis of tumor cells lacking functional p53 and involves the activities of c-Abl and p300. Here, we demonstrate that conformational changes of p73 catalyzed by the prolyl isomerase Pin1 are crucial in this pathway. Lack of Pin1 reduces p73 stability, hampering its accumulation upon genotoxic stress. Indeed, we show that upon treatment with chemotherapeutic drugs c-Abl enhances the phosphorylation-dependent interaction between Pin1 and p73, and this in turn promotes p73 acetylation by p300. Consistently, the ability of c-Abl and p300 to increase p73 stability and transcriptional activity requires Pin1. As a consequence, Pin1 appears to be essential for activation of the apoptotic response by endogenous p73.
The tumour suppressor p53 is important in the cell decision to either arrest cell cycle progressi... more The tumour suppressor p53 is important in the cell decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 post-translational modifications and association with other proteins have been implicated in the regulation of its stability and transcriptional activities. Here we report that, on DNA damage, p53 interacts with Pin1, a peptidyl-prolyl isomerase, which regulates the function of many proteins involved in cell cycle control and apoptosis. The interaction is strictly dependent on p53 phosphorylation, and requires Ser 33, Thr 81 and Ser 315. On binding, Pin1 generates conformational changes in p53, enhancing its transactivation activity. Stabilization of p53 is impaired in UV-treated Pin1(-/-) cells owing to its inability to efficiently dissociate from Mdm2. As a consequence, a reduced p53-dependent response was detected in Pin1(-/-) cells, and this correlates with a diminished transcriptional activation of some p53-regulated genes. Our results suggest that, following stress-induced phosphorylation, p53 needs to form a complex with Pin1 and to undergo a conformational change to fulfil its biological roles.
Early in development, network activity in the hippocampus is characterized by recurrent synchrono... more Early in development, network activity in the hippocampus is characterized by recurrent synchronous bursts, whose cellular correlates are giant depolarizing potentials (GDPs). The propensity for generating GDPs is attributed to GABAergic synaptic transmission being depolarizing and excitatory in neonatal neurons. However, developmental regulation of intrinsic conductances may also influence GDPs generation. A likely candidate is the non-inactivating, low-threshold, muscarinic-sensitive K + current (M current; I m ), which down-regulates intrinsic bursting activity in adult hippocampal pyramidal neurons. Western blot analysis of homogenates of the CA3 hippocampal region showed that expression of the Kv7.2 subunit, one of the constituents of neuronal M channels, is weak in neonatal neurons, and markedly increases after the first postnatal week. Likewise, the density of I m was very low in neonatal CA3 pyramidal cells and increased later on. Spontaneously occurring intrinsic bursts in neonatal neurons were longer and more robust, and recurred more regularly, than in juvenile neurons. The I m blocker linopirdine only mildly affected intrinsic bursting in neonatal neurons, but strongly facilitated and regularized it in juvenile neurons. We conclude that the low expression of Kv7/M channels and the depolarizing action of GABA early after birth enhance intrinsic bursting and neuronal synchronization leading to generation of GDPs within the hippocampal network.
Covalent modi®cation of the promyelocytic leukaemia protein (PML) by SUMO-1 is a prerequisite for... more Covalent modi®cation of the promyelocytic leukaemia protein (PML) by SUMO-1 is a prerequisite for the assembly of nuclear bodies (NBs), subnuclear structures disrupted in various human diseases and linked to transcriptional and growth control. Here we demonstrate that p53 is recruited into NBs by a speci®c PML isoform (PML3) or by coexpression of SUMO-1 and hUbc9. NB targeting depends on the direct association of p53, through its core domain, with a C-terminal region of PML3. The relocalization of p53 into NBs enhances p53 transactivation in a promoter-speci®c manner and affects cell survival. Our results indicate the existence of a cross-talk between PML-and p53dependent growth suppression pathways, implying an important role for NBs and their resident proteins as modulators of p53 functions.
The microtubule-binding protein gephyrin is known to play a pivotal role in targeting and cluster... more The microtubule-binding protein gephyrin is known to play a pivotal role in targeting and clustering postsynaptic inhibitory receptors. Here, the Intracellular Antibodies Capture Technology (IATC) was used to select two single-chain antibody fragments or intrabodies, which, fused to nuclear localization signals (NLS), were able to efficiently and selectively remove gephyrin from glycine receptor (GlyR) clusters. Co-transfection of NLS-tagged individual intrabodies with gephyrin-enhanced green fluorescent protein (EGFP) in HEK 293 cells revealed a partial relocalization of gephyrin aggregates onto the nucleus or in the perinuclear area. When expressed in cultured neurons, these intrabodies caused a significant reduction in the number of immunoreactive GlyR clusters, which was associated with a decrease in the peak amplitude of glycine-evoked whole cell currents as assessed with electrophysiological experiments. Hampering protein function at a posttranslational level may represent an attractive alternative for interfering with gephyrin function in a more spatially localized manner.
Speed and reliability of synaptic transmission are essential for information coding in neuronal n... more Speed and reliability of synaptic transmission are essential for information coding in neuronal networks and require the presence of clustered neurotransmitter receptors at the plasma membrane in precise apposition to presynaptic terminals. Receptor clusterization is the result of highly regulated processes involving functional and structural proteins. Among the structural elements, microtubules are known to play a crucial role in anchoring of ␥-aminobutyric acid, type A (GABA A ) receptors. Here we show that microtubule depolymerization with nocodazole induces the declusterization of GABA A receptors and modifies the kinetic properties of GABAergic currents in cultured hippocampal neurons. In particular, this drug, applied either in the bath or via the patch pipette, induced the acceleration of the onset kinetics of miniature inhibitory postsynaptic currents (mIPSCs) without significantly affecting their frequency, thus suggesting a main postsynaptic site of action. After nocodazole treatment, current responses to ultrafast applications of GABA exhibited a faster rise time and an accelerated onset of desensitization. A quantitative analysis of GABA-evoked currents and model simulations suggest that declusterization affects the gating properties of GABA A receptors. In particular, a faster entry into the desensitized state of declustered GABA A receptors may account for the changes in the kinetic properties of mIPSCs after nocodazole treatment. Hence it appears that the clustered condition of GABA A receptors contributes in shaping GABAergic currents.
Gephyrin is a scaffold protein essential for stabilizing glycine and GABA A receptors at inhibito... more Gephyrin is a scaffold protein essential for stabilizing glycine and GABA A receptors at inhibitory synapses. Here, recombinant intrabodies against gephyrin (scFv-gephyrin) were used to assess whether this protein exerts a transynaptic action on GABA and glutamate release. Pair recordings from interconnected hippocampal cells in culture revealed a reduced probability of GABA release in scFv-gephyrin-transfected neurons compared with controls. This effect was associated with a significant decrease in VGAT, the vesicular GABA transporter, and in neuroligin 2 (NLG2), a protein that, interacting with neurexins, ensures the cross-talk between the post-and presynaptic sites. Interestingly, hampering gephyrin function also produced a significant reduction in VGLUT, the vesicular glutamate transporter, an effect accompanied by a significant decrease in frequency of miniature excitatory postsynaptic currents. Overexpressing NLG2 in gephyrin-deprived neurons rescued GABAergic but not glutamatergic innervation, suggesting that the observed changes in the latter were not due to a homeostatic compensatory mechanism. Pulldown experiments demonstrated that gephyrin interacts not only with NLG2 but also with NLG1, the isoform enriched at excitatory synapses. These results suggest a key role of gephyrin in regulating transynaptic signaling at both inhibitory and excitatory synapses.
Tonic inhibition plays a crucial role in regulating neuronal excitability because it sets the thr... more Tonic inhibition plays a crucial role in regulating neuronal excitability because it sets the threshold for action potential generation and integrates excitatory signals. Tonic currents are known to be largely mediated by extrasynaptic ␥-aminobutyric acid type A (GABA A ) receptors that are persistently activated by submicromolar concentrations of ambient GABA. We recently reported that, in cultured hippocampal neurons, the clustering of synaptic GABA A receptors significantly affects synaptic transmission (Petrini, E. M., Zacchi, P., Barberis, A., Mozrzymas, J. W., and Cherubini, E. (2003) J. Biol. Chem. 278, 16271-16279). In this work, we demonstrated that the clustering of extrasynaptic GABA A receptors modulated tonic inhibition. Depolymerization of the cytoskeleton with nocodazole promoted the disassembly of extrasynaptic clusters of ␦ and ␥ 2 subunitcontaining GABA A receptors. This effect was associated with a reduction in the amplitude of tonic currents and diminished shunting inhibition. Moreover, diffuse GABA A receptors were less sensitive to the GAT-1 inhibitor NO-711 and to flurazepam. Quantitative analysis of GABA-evoked currents after prolonged exposure to submicromolar concentrations of GABA and model simulations suggest that clustering affects the gating properties of extrasynaptic GABA A receptors. In particular, a larger occupancy of the singly and doubly bound desensitized states can account for the modulation of tonic inhibition recorded after nocodazole treatment. Moreover, comparison of tonic currents recorded during spontaneous activity and those elicited by exogenously applied low agonist concentrations allows estimation of the concentration of ambient GABA. In conclusion, receptor clustering appears to be an additional regulating factor for tonic inhibition.
We describe a method to select DNA encoding functional open reading frames (ORFs) from noncoding ... more We describe a method to select DNA encoding functional open reading frames (ORFs) from noncoding DNA within the context of a specific vector. Phage display has been used as an example, but any system requiring DNA encoding protein fragments, for example, the yeast two-hybrid system, could be used. By cloning DNA fragments upstream of a fusion gene, consisting of the -lactamase gene flanked by lox recombination sites, which is, in turn, upstream of gene 3 from fd phage, only those clones containing DNA fragments encoding ORFs confer ampicillin resistance and survive. After selection, the -lactamase gene can be removed by Cre recombinase, leaving a standard phage display vector with ORFs fused to gene 3. This vector has been tested on a plasmid containing tissue transglutaminase. All surviving clones analyzed by sequencing were found to contain ORFs, of which 83% were localized to known genes, and at least 80% produced immunologically detectable polypeptides. Use of a specific anti-tTG monoclonal antibody allowed the identification of clones containing the correct epitope. This approach could be applicable to the efficient selection of random ORFs representing the coding potential of whole organisms, and their subsequent downstream use in a number of different systems. 4 These authors contributed equally to this work. 5 Corresponding author. E-MAIL [email protected]; FAX (505) 667-2891. Article and publication are at
Proceedings of The National Academy of Sciences, 2007
At early developmental stages, correlated neuronal activity is thought to exert a critical contro... more At early developmental stages, correlated neuronal activity is thought to exert a critical control on functional and structural refinement of synaptic connections. In the hippocampus, between postnatal day 2 (P2) and P6, network-driven giant depolarizing potentials (GDPs) are generated by the synergistic action of glutamate and GABA, which is depolarizing and excitatory. Here the rising phase of GDPs was used to trigger Schaffer collateral stimulation in such a way that synchronized network activity was coincident with presynaptic activation of afferent input. This procedure produced a persistent increase in spontaneous and evoked ␣-amino-3-hydroxy-5-methyl-4-isoxadepropionic acid-mediated glutamatergic currents, an effect that required calcium influx through postsynaptic L-type calcium channels.
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Papers by P. Zacchi