Viviana Torres | Universidad de Concepción (original) (raw)
Papers by Viviana Torres
Cells
Neurons release neurotransmitters at a specialized region of the presynaptic membrane, the active... more Neurons release neurotransmitters at a specialized region of the presynaptic membrane, the active zone (AZ), where a complex meshwork of proteins organizes the release apparatus. The formation of this proteinaceous cytomatrix at the AZ (CAZ) depends on precise homo- and hetero-oligomerizations of distinct CAZ proteins. The CAZ protein CAST1/ERC2 contains four coiled-coil (CC) domains that interact with other CAZ proteins, but also promote self-assembly, which is an essential step for its integration during AZ formation. The self-assembly and synaptic recruitment of the Drosophila protein Bruchpilot (BRP), a partial homolog of CAST1/ERC2, is modulated by the serine-arginine protein kinase (SRPK79D). Here, we demonstrate that overexpression of the vertebrate SRPK2 regulates the self-assembly of CAST1/ERC2 in HEK293T, SH-SY5Y and HT-22 cells and the CC1 and CC4 domains are involved in this process. Moreover, the isoform SRPK2 forms a complex with CAST1/ERC2 when co-expressed in HEK293T...
Journal of neurochemistry, Jan 9, 2018
Dysregulated Wnt signaling is linked to major neurodegenerative diseases, including Alzheimer dis... more Dysregulated Wnt signaling is linked to major neurodegenerative diseases, including Alzheimer disease (AD). In mouse models of AD, activation of the canonical Wnt signaling pathway improves learning/memory, but the mechanism for this remains unclear. The decline in brain function in AD patients correlates with reduced glucose utilization by neurons. Here, we test whether improvements in glucose metabolism mediate the neuroprotective effects of Wnt in AD mouse model. APPswe/PS1dE9 transgenic mice were used to model AD, Andrographolide or Lithium was used to activate Wnt signaling, and cytochalasin B was used to block glucose uptake. Cognitive function was assessed by novel object recognition and memory flexibility tests. Glucose uptake and the glycolytic rate were determined using radiotracer glucose. The activities of key enzymes of glycolysis such as hexokinase (HK) and phosphofructokinase (PFK), Adenosine triphosphate (ATP)/ Adenosine diphosphate (ADP) levels and the pentose phosp...
Molecular neurobiology, Jan 29, 2018
Neurons are highly polarized cells displaying an elaborate architectural morphology. The design o... more Neurons are highly polarized cells displaying an elaborate architectural morphology. The design of their dendritic arborization and the distribution of their synapses contribute importantly to information processing in the brain. The growth and complexity of dendritic arbors are driven by the formation of synapses along their lengths. Synaptogenesis is augmented by the secretion of factors, like BDNF, Reelin, BMPs, or Wnts. Exo70 is a component of the exocyst complex, a protein complex that guides membrane addition and polarized exocytosis. While it has been linked to cytokinesis and the establishment of cell polarity, its role in synaptogenesis is poorly understood. In this report, we show that Exo70 plays a role in the arborization of dendrites and the development of synaptic connections between cultured hippocampal neurons. Specifically, while the overexpression of Exo70 increases dendritic arborization, synapse number, and spine density, the inhibition of Exo70 expression reduce...
Molecular Neurobiology
Among all the biological systems in vertebrates, the central nervous system (CNS) is the most com... more Among all the biological systems in vertebrates, the central nervous system (CNS) is the most complex, and its function depends on specialized contacts among neurons called synapses. The assembly and organization of synapses must be exquisitely regulated for a normal brain function and network activity. There has been a tremendous effort in recent decades to understand the molecular and cellular mechanisms participating in the formation of new synapses and their organization, maintenance, and regulation. At the vertebrate presynapses, proteins such as Piccolo, Bassoon, RIM, RIM-BPs, CAST/ELKS, liprin-α, and Munc13 are constant residents and participate in multiple and dynamic interactions with other regulatory proteins, which define network activity and normal brain function. Here, we review the function of these active zone (AZ) proteins and diverse factors involved in AZ assembly and maintenance, with an emphasis on axonal trafficking of precursor vesicles, protein homo- and hetero-oligomeric interactions as a mechanism of AZ trapping and stabilization, and the role of F-actin in presynaptic assembly and its modulation by Wnt signaling.
Neural plasticity, 2017
Synapses are complex structures that allow communication between neurons in the central nervous s... more Synapses are complex structures that allow communication between neurons in the central nervous system. Studies conducted in vertebrate and invertebrate models have contributed to the knowledge of the function of synaptic proteins. The functional synapse requires numerous protein complexes with specialized functions that are regulated in space and time to allow synaptic plasticity. However, their interplay during neuronal development, learning, and memory is poorly understood. Accumulating evidence links synapse proteins to neurodevelopmental, neuropsychiatric, and neurodegenerative diseases. In this review, we describe the way in which several proteins that participate in cell adhesion, scaffolding, exocytosis, and neurotransmitter reception from presynaptic and postsynaptic compartments, mainly from excitatory synapses, have been associated with several synaptopathies, and we relate their functions to the disease phenotype.
PloS one, 2016
Synaptic vesicles (SVs) fuse with the plasma membrane at a precise location called the presynapti... more Synaptic vesicles (SVs) fuse with the plasma membrane at a precise location called the presynaptic active zone (AZ). This fusion is coordinated by proteins embedded within a cytoskeletal matrix assembled at the AZ (CAZ). In the present study, we have identified a novel binding partner for the CAZ proteins Piccolo and Bassoon. This interacting protein, Trio, is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) known to regulate the dynamic assembly of actin and growth factor dependent axon guidance and synaptic growth. Trio was found to interact with the C-terminal PBH 9/10 domains of Piccolo and Bassoon via its own N-terminal Spectrin repeats, a domain that is also critical for its localization to the CAZ. Moreover, our data suggest that regions within the C-terminus of Trio negatively regulate its interactions with Piccolo/Bassoon. These findings provide a mechanism for the presynaptic targeting of Trio and support a model in which Piccolo and Bassoon play a ...
Journal of Prosthodontics, 2016
To assess the effects of terpenic denture cleanser on denture biofilm removal using scanning elec... more To assess the effects of terpenic denture cleanser on denture biofilm removal using scanning electron microscopy (SEM). The internal surface biofilm of four maxillary dentures was elucidated with Caristop-revelador Dual Tone, and 40 blue-stained specimens (0.6 cm × 0.4 cm × 2 mm) were obtained. These specimens were randomly assigned to one of the following four groups of 10 specimens each: control, Eci Clean, Fitty Dent, and terpenic denture cleanser. The period of immersion in each solution was 12 hours. Biofilm removal was evaluated using SEM, and morphologically varying areas of the SEM images were quantified with Imaris software. The data were analyzed using Kolmogorov-Smirnov, t-tests, ANOVA, and Tamhane's tests (p = 0.05). Data revealed that terpenic denture cleanser removed significantly more biofilm than any other treatment examined in this study. The t-tests revealed significant differences in the clean area that resulted from the use of the terpenic cleanser compared with the clean area that resulted from the use of Eci Clean (p = 0.013). Fitty Dent was the least effective and left dirty acrylic resin. The average areas with few removed layers were 59.3%, 43.3%, and 9.5% in Fitty Dent, Eci Clean, and terpenic cleanser groups, respectively. Tamhane's tests indicated that the Eci Clean and Fitty Dent groups were significantly different from the 0.5% terpenic cleanser group (p = 0.008). The terpenic denture cleanser was effective in removing denture biofilm.
Advances in Experimental Medicine and Biology, 1994
To identify a role for a Na:H (NHE) exchanger subtype in volume regulation in bovine corneal epit... more To identify a role for a Na:H (NHE) exchanger subtype in volume regulation in bovine corneal epithelium, we determined: 1. its sensitivity to inhibition by amiloride analogues 2. the effects of either Cl removal or hypertonicity on the intracellular pH. Our results suggest that volume regulatory responses elicited by stimulation of NHE-2 may help preserve epithelial barrier function in the face of increases in tear film osmolarity.
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1991
Very few mitochondria, undetectable levels of succinate dehydrogenase and glutamate dehydrogenase... more Very few mitochondria, undetectable levels of succinate dehydrogenase and glutamate dehydrogenase and significant activities of glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and malate dehydrogenase were observed in the deeper foot muscle tissue of Concholepas concholepas. 2. Malate dehydrogenase activity was resolved into two molecular forms, which differ in K~ values for oxaloacetate and malate, and sensitivity to inhibition by ~-ketoglutarate. 3. In addition to reoxidation of NADH generated from glycolysis, the function of malate dehydrogenase is discussed in connection with octopine synthesis and oxidation in the foot muscle of C. concholepas. 4. The results of initial rate and product inhibition studies of both forms of malate dehydrogenase are consistent with an Ordered Bi Bi kinetic mechanism, with NADH adding first and NAD ÷ leaving last. Oxaloacetate substrate inhibition is suggested to result from formation of an enzyme-NAD+-oxaloacetate dead end complex.
Neuron, Jan 24, 2003
Recent studies indicate that active zones (AZs)-sites of neurotransmitter release-may be assemble... more Recent studies indicate that active zones (AZs)-sites of neurotransmitter release-may be assembled from preassembled AZ precursor vesicles inserted into the presynaptic plasma membrane. Here we report that one putative AZ precursor vesicle of CNS synapses-the Piccolo-Bassoon transport vesicle (PTV)-carries a comprehensive set of AZ proteins genetically and functionally coupled to synaptic vesicle exocytosis. Time-lapse imaging reveals that PTVs are highly mobile, consistent with a role in intracellular transport. Quantitative analysis reveals that the Bassoon, Piccolo, and RIM content of individual PTVs is, on average, half of that of individual presynaptic boutons and shows that the synaptic content of these molecules can be quantitatively accounted for by incorporation of integer numbers (typically two to three) of PTVs into presynaptic membranes. These findings suggest that AZs are assembled from unitary amounts of AZ material carried on PTVs.
Journal of Neuroscience, 2012
Vesicular trafficking of presynaptic and postsynaptic components is emerging as a general cellula... more Vesicular trafficking of presynaptic and postsynaptic components is emerging as a general cellular mechanism for the delivery of scaffold proteins, ion channels, and receptors to nascent and mature synapses. However, the molecular mechanisms leading to the selection of cargos and their differential transport to subneuronal compartments are not well understood, in part because of the mixing of cargos at the plasma membrane and/or within endosomal compartments. In the present study, we have explored the cellular mechanisms of active zone precursor vesicle assembly at the Golgi in dissociated hippocampal neurons of Rattus norvegicus. Our studies show that Piccolo, Bassoon, and ELKS2/CAST exit the trans-Golgi network on a common vesicle that requires Piccolo and Bassoon for its proper assembly. In contrast, Munc13 and synaptic vesicle proteins use distinct sets of Golgi-derived transport vesicles, while RIM1␣ associates with vesicular membranes in a post-Golgi compartment. Furthermore, Piccolo and Bassoon are necessary for ELKS2/CAST to leave the Golgi in association with vesicles, and a core domain of Bassoon is sufficient to facilitate formation of these vesicles. While these findings support emerging principles regarding active zone differentiation, the cellular and molecular analyses reported here also indicate that the Piccolo-Bassoon transport vesicles leaving the Golgi may undergo further changes in protein composition before arriving at synaptic sites.
PLoS ONE, 2012
Arsenic main inorganic compound is arsenic trioxide (ATO) presented in solution mainly as arsenit... more Arsenic main inorganic compound is arsenic trioxide (ATO) presented in solution mainly as arsenite. ATO increases intracellular pH (pHi), cell proliferation and tumor growth. Sodium-proton exchangers (NHEs) modulate the pHi, with NHE1 playing significant roles. Whether ATO-increased cell proliferation results from altered NHEs expression and activity is unknown. We hypothesize that ATO increases cell proliferation by altering pHi due to increased NHEs-like transport activity. Madin-Darby canine kidney (MDCK) cells grown in 5 mmol/L D-glucose-containing DMEM were exposed to ATO (0.05, 0.5 or 5 mmol/L, 0-48 hours) in the absence or presence of 5-N,N-hexamethylene amiloride (HMA, 5-100 mmol/L, NHEs inhibitor), PD-98059 (30 mmol/L, MAPK1/2 inhibitor), Gö 6976 (10 mmol/L, PKCa, bI and m inhibitor), or Schering 28080 (10 mmol/L, H + / K + ATPase inhibitor) plus concanamycin (0.1 mmol/L, V type ATPases inhibitor). Incorporation of [ 3 H]thymidine was used to estimate cell proliferation, and counting cells with a hemocytometer to determine the cell number. The pHi was measured by fluorometry in 2,7-bicarboxyethyl-5,6-carboxyfluorescein loaded cells. The Na +-dependent HMA-sensitive NHEs-like mediated proton transport kinetics, NHE1 protein abundance in the total, cytoplasm and plasma membrane protein fractions, and phosphorylated and total p42/44 mitogen-activated protein kinases (p42/44 mapk) were also determined. Lowest ATO (0.05 mmol/L, ,0.01 ppm) used in this study increased cell proliferation, pHi, NHEs-like transport and plasma membrane NHE1 protein abundance, effects blocked by HMA, PD-98059 or Gö 6976. Cell-buffering capacity did not change by ATO. The results show that a low ATO concentration increases MDCK cells proliferation by NHEs (probably NHE1)-like transport dependent-increased pHi requiring p42/44 mapk and PKCa, bI and/or m activity. This finding could be crucial in diseases where uncontrolled cell growth occurs, such as tumor growth, and in circumstances where ATO, likely arsenite, is available at the drinking-water at these levels.
Journal of Neurochemistry, 2002
The influence of aurintricarboxylic acid (ATA), a neuroprotective compound, on the serotonin tran... more The influence of aurintricarboxylic acid (ATA), a neuroprotective compound, on the serotonin transporter expressed in JAR human placental choriocarcinoma cells was investigated. Treatment of the cells with ATA for 16 h led to a significant stimulation of the serotonm transporter activity. This effect was not observed, however, when the treatment was done for 1-2 h. The stimulatory effect was associated with an increase in the maximal velocity of the transport process with no significant change in the Michaelis-Menten constant. Northern blot hybridization revealed that ATA treatment caused a marked increase in the steady-state levels of serotonin transporter-specific transcripts. Treatment of the cells with ATA was found to increase tyrosine phosphorylation of a 180-kDa protein. The phosphotyrosine content of a protein of a similar molecular size increased dramatically when the cells were exposed to epidermal growth factor (EGF), suggesting that this protein may be the EGF receptor. Treatment of the cells with EGF for 24 h could reproduce the stimulatory effects of ATA on the serotonin transporter activity, the maximal velocity of the transport process, and the steady-state levels of the transporterspecific mRNAs. Genistein, a tyrosine kinase inhibitor, was able to block the stimulatory effect of ATA and EGF. It is concluded that EGF increases the serotonin transporter expression in JAR cells and that the neuroprotective compound ATA produces similar effects on the transporter most likely by activating the EGF receptor through tyrosine phosphorylation.
Journal of Biotechnology, 2010
Classic IRES sequences are notorious for exerting biased expression in favor of upstream coding r... more Classic IRES sequences are notorious for exerting biased expression in favor of upstream coding regions when placed into polycistronic vectors. Here, we report the development of a bicistronic lentiviral system based on the 1D/2A sequence from the foot-and-mouth disease virus that is able to maintain tightly balanced control of upstream and downstream protein expression for several days at a stoichiometry very closely approaching 1.0. Our results suggest that the 1D/2A sequence can be optimized in an FUGW lentiviral setting to coordinate expression of multiple polypeptides, presenting a potentially valuable tool to signaling network researchers and to the gene therapy community.
Journal of Biological Chemistry, 2002
Mutagenesis of recombinant 1 ␥-aminobutyric acid (GABA) receptors has previously identified five ... more Mutagenesis of recombinant 1 ␥-aminobutyric acid (GABA) receptors has previously identified five residues in the amino terminal extracellular domain that play an important role in GABA binding. Here, we present evidence that the tyrosine at position 102 of the 1 receptor is also associated with the agonist binding site. Wildtype and mutant 1 receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp. When Tyr-102 was mutated to cysteine, serine, tryptophan, or glycine the EC 50 increased 31-, 214-, 664-, and 8752-fold, respectively. An increase in the IC 50 was also observed for the competitive antagonist 3-APMPA, but not for the non-competitive antagonist picrotoxin. Y102C was accessible to modification by methanethiosulfonate, and this modification was prevented by both GABA and 3-APMPA. An interesting characteristic of the Y102S mutant receptor was that, in the absence of GABA, there was an unusually high oocyte resting conductance that was blocked by both 3-APMPA and picrotoxin, indicating spontaneously opening GABA receptors. It appears that mutation of Tyr-102 perturbs the binding site and gates the pore. We conclude that Tyr-102 is a component of the GABA binding domain and speculate that Tyr-102 might be important for coupling agonist binding to channel opening.
Journal of Biological Chemistry, 2005
Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of t... more Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.
Journal of Biological Chemistry, 1996
We have isolated a cDNA from a human placental choriocarcinoma cell cDNA library which, when expr... more We have isolated a cDNA from a human placental choriocarcinoma cell cDNA library which, when expressed in HeLa cells, induces a Na ؉-dependent amino acid transport system with preference for zwitterionic amino acids. Anionic amino acids, cationic amino acids, imino acids, and N-methylated amino acids are excluded by this system. These characteristics are identical to those described for the amino acid transporter B o. When expressed in Xenopus laevis oocytes that do not have detectable endogenous activity of the amino acid transporter B o , the cloned transporter increases alanine transport in the oocytes severalfold and induces alanine-evoked inward currents in the presence of Na ؉. The cDNA codes for a polypeptide containing 541 amino acids with 10 putative transmembrane domains. Amino acid sequence homology predicts this transporter (hATB o) to be a member of a superfamily consisting of the glutamate transporters, the neutral amino acid transport system ASCT, and the insulin-activable neutral/anionic amino acid transporter. Chromosomal assignment studies with somatic cell hybrid analysis and fluorescent in situ hybridization have located the ATB o gene to human chromosome 19q13.3.
Experimental Eye Research, 1998
The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) o... more The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) of Na : H exchange (NHE) and vacuolar H +-ATPase (i.e. V-type) activity to the regulation of intracellular pH (pH i). Furthermore, we characterized the effects on pH i regulation of exposure to 1 µ ET-1 under control and acid loaded conditions. With the pH sensitive dye, 2h,7h Bis (carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), the control pH i was 7n1 in NaCl (nominally HCO $-free) Ringers. Inhibition of NHE with 100 µ dimethylamiloride (DMA) rapidly decreased pH i by 0n37 units. Similarly, selective inhibition of V-type H +-ATPase with 10 µ bafilomycin A " decreased pH i by 0n22 units. Following acid loading in NaCl Ringers with a 20 m NH % Cl prepulse, pH i recovery was partially inhibited by exposure to either Na-free (NMGCl) Ringers, 100 µ DMA or 20 µ bafilomycin A ". Based on decreases in H + efflux resulting from selective inhibition of NHE and V-type H + pump activity, NHE activity accounts for 76 % of the pH i recovery following acid loading. Under control conditions, ET-1 (1 µ) had no effect on pH i whereas ET-1 completely suppressed pH i recovery following acid loading in NaCl or NMGCl Ringers. This inhibitory effect was largely due to stimulation of ET A because in the presence of BQ-123 (10 µ), a selective ET A receptor antagonist, pH i recovery was completely restored. Suppression of pH i recovery also occurred following stimulation of protein kinase C (PKC) with 10 −( phorbol myristate (PMA) whereas 10 −( 4 α phorbol 12,13 didecanoate (PDD) had no effect. ET-1 failed to suppress pH i recovery after inhibition of PKC with 0n5 µ calphostin C suggesting that the inhibition of pH i recovery by ET-1 is a consequence of PKC stimulation. Similarly, inhibition of Ca# +-dependent calmodulin stimulated CaM II kinase with KN-62 (10 µ) reversed the suppression of pH i recovery by ET-1. Preinhibition of either protein phosphatase (PP), PP-1, PP-2A or PP-2B activity with 1 µ phenylarsine oxide, 10 n okadaic acid, 10 µ cyclosporin A " or 20 µ BAPTA, also obviated the suppression of pH i recovery by ET-1. Therefore ET A receptor mediated inhibition of pH i regulation following acid loading could be a consequence of either PKC or CaMII kinase stimulation. Each one of these kinases may in turn phosphorylate and thereby stimulate the activities of PP-1, PP-2A or PP-2B. An increase in the activity of any one of these protein phosphatases could lead to dephosphorylation of the NHE and V-type H + pump. This alteration may prevent them from becoming adequately stimulated to elicit pH i recovery in response to acid loading.
Cells
Neurons release neurotransmitters at a specialized region of the presynaptic membrane, the active... more Neurons release neurotransmitters at a specialized region of the presynaptic membrane, the active zone (AZ), where a complex meshwork of proteins organizes the release apparatus. The formation of this proteinaceous cytomatrix at the AZ (CAZ) depends on precise homo- and hetero-oligomerizations of distinct CAZ proteins. The CAZ protein CAST1/ERC2 contains four coiled-coil (CC) domains that interact with other CAZ proteins, but also promote self-assembly, which is an essential step for its integration during AZ formation. The self-assembly and synaptic recruitment of the Drosophila protein Bruchpilot (BRP), a partial homolog of CAST1/ERC2, is modulated by the serine-arginine protein kinase (SRPK79D). Here, we demonstrate that overexpression of the vertebrate SRPK2 regulates the self-assembly of CAST1/ERC2 in HEK293T, SH-SY5Y and HT-22 cells and the CC1 and CC4 domains are involved in this process. Moreover, the isoform SRPK2 forms a complex with CAST1/ERC2 when co-expressed in HEK293T...
Journal of neurochemistry, Jan 9, 2018
Dysregulated Wnt signaling is linked to major neurodegenerative diseases, including Alzheimer dis... more Dysregulated Wnt signaling is linked to major neurodegenerative diseases, including Alzheimer disease (AD). In mouse models of AD, activation of the canonical Wnt signaling pathway improves learning/memory, but the mechanism for this remains unclear. The decline in brain function in AD patients correlates with reduced glucose utilization by neurons. Here, we test whether improvements in glucose metabolism mediate the neuroprotective effects of Wnt in AD mouse model. APPswe/PS1dE9 transgenic mice were used to model AD, Andrographolide or Lithium was used to activate Wnt signaling, and cytochalasin B was used to block glucose uptake. Cognitive function was assessed by novel object recognition and memory flexibility tests. Glucose uptake and the glycolytic rate were determined using radiotracer glucose. The activities of key enzymes of glycolysis such as hexokinase (HK) and phosphofructokinase (PFK), Adenosine triphosphate (ATP)/ Adenosine diphosphate (ADP) levels and the pentose phosp...
Molecular neurobiology, Jan 29, 2018
Neurons are highly polarized cells displaying an elaborate architectural morphology. The design o... more Neurons are highly polarized cells displaying an elaborate architectural morphology. The design of their dendritic arborization and the distribution of their synapses contribute importantly to information processing in the brain. The growth and complexity of dendritic arbors are driven by the formation of synapses along their lengths. Synaptogenesis is augmented by the secretion of factors, like BDNF, Reelin, BMPs, or Wnts. Exo70 is a component of the exocyst complex, a protein complex that guides membrane addition and polarized exocytosis. While it has been linked to cytokinesis and the establishment of cell polarity, its role in synaptogenesis is poorly understood. In this report, we show that Exo70 plays a role in the arborization of dendrites and the development of synaptic connections between cultured hippocampal neurons. Specifically, while the overexpression of Exo70 increases dendritic arborization, synapse number, and spine density, the inhibition of Exo70 expression reduce...
Molecular Neurobiology
Among all the biological systems in vertebrates, the central nervous system (CNS) is the most com... more Among all the biological systems in vertebrates, the central nervous system (CNS) is the most complex, and its function depends on specialized contacts among neurons called synapses. The assembly and organization of synapses must be exquisitely regulated for a normal brain function and network activity. There has been a tremendous effort in recent decades to understand the molecular and cellular mechanisms participating in the formation of new synapses and their organization, maintenance, and regulation. At the vertebrate presynapses, proteins such as Piccolo, Bassoon, RIM, RIM-BPs, CAST/ELKS, liprin-α, and Munc13 are constant residents and participate in multiple and dynamic interactions with other regulatory proteins, which define network activity and normal brain function. Here, we review the function of these active zone (AZ) proteins and diverse factors involved in AZ assembly and maintenance, with an emphasis on axonal trafficking of precursor vesicles, protein homo- and hetero-oligomeric interactions as a mechanism of AZ trapping and stabilization, and the role of F-actin in presynaptic assembly and its modulation by Wnt signaling.
Neural plasticity, 2017
Synapses are complex structures that allow communication between neurons in the central nervous s... more Synapses are complex structures that allow communication between neurons in the central nervous system. Studies conducted in vertebrate and invertebrate models have contributed to the knowledge of the function of synaptic proteins. The functional synapse requires numerous protein complexes with specialized functions that are regulated in space and time to allow synaptic plasticity. However, their interplay during neuronal development, learning, and memory is poorly understood. Accumulating evidence links synapse proteins to neurodevelopmental, neuropsychiatric, and neurodegenerative diseases. In this review, we describe the way in which several proteins that participate in cell adhesion, scaffolding, exocytosis, and neurotransmitter reception from presynaptic and postsynaptic compartments, mainly from excitatory synapses, have been associated with several synaptopathies, and we relate their functions to the disease phenotype.
PloS one, 2016
Synaptic vesicles (SVs) fuse with the plasma membrane at a precise location called the presynapti... more Synaptic vesicles (SVs) fuse with the plasma membrane at a precise location called the presynaptic active zone (AZ). This fusion is coordinated by proteins embedded within a cytoskeletal matrix assembled at the AZ (CAZ). In the present study, we have identified a novel binding partner for the CAZ proteins Piccolo and Bassoon. This interacting protein, Trio, is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) known to regulate the dynamic assembly of actin and growth factor dependent axon guidance and synaptic growth. Trio was found to interact with the C-terminal PBH 9/10 domains of Piccolo and Bassoon via its own N-terminal Spectrin repeats, a domain that is also critical for its localization to the CAZ. Moreover, our data suggest that regions within the C-terminus of Trio negatively regulate its interactions with Piccolo/Bassoon. These findings provide a mechanism for the presynaptic targeting of Trio and support a model in which Piccolo and Bassoon play a ...
Journal of Prosthodontics, 2016
To assess the effects of terpenic denture cleanser on denture biofilm removal using scanning elec... more To assess the effects of terpenic denture cleanser on denture biofilm removal using scanning electron microscopy (SEM). The internal surface biofilm of four maxillary dentures was elucidated with Caristop-revelador Dual Tone, and 40 blue-stained specimens (0.6 cm × 0.4 cm × 2 mm) were obtained. These specimens were randomly assigned to one of the following four groups of 10 specimens each: control, Eci Clean, Fitty Dent, and terpenic denture cleanser. The period of immersion in each solution was 12 hours. Biofilm removal was evaluated using SEM, and morphologically varying areas of the SEM images were quantified with Imaris software. The data were analyzed using Kolmogorov-Smirnov, t-tests, ANOVA, and Tamhane's tests (p = 0.05). Data revealed that terpenic denture cleanser removed significantly more biofilm than any other treatment examined in this study. The t-tests revealed significant differences in the clean area that resulted from the use of the terpenic cleanser compared with the clean area that resulted from the use of Eci Clean (p = 0.013). Fitty Dent was the least effective and left dirty acrylic resin. The average areas with few removed layers were 59.3%, 43.3%, and 9.5% in Fitty Dent, Eci Clean, and terpenic cleanser groups, respectively. Tamhane's tests indicated that the Eci Clean and Fitty Dent groups were significantly different from the 0.5% terpenic cleanser group (p = 0.008). The terpenic denture cleanser was effective in removing denture biofilm.
Advances in Experimental Medicine and Biology, 1994
To identify a role for a Na:H (NHE) exchanger subtype in volume regulation in bovine corneal epit... more To identify a role for a Na:H (NHE) exchanger subtype in volume regulation in bovine corneal epithelium, we determined: 1. its sensitivity to inhibition by amiloride analogues 2. the effects of either Cl removal or hypertonicity on the intracellular pH. Our results suggest that volume regulatory responses elicited by stimulation of NHE-2 may help preserve epithelial barrier function in the face of increases in tear film osmolarity.
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1991
Very few mitochondria, undetectable levels of succinate dehydrogenase and glutamate dehydrogenase... more Very few mitochondria, undetectable levels of succinate dehydrogenase and glutamate dehydrogenase and significant activities of glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and malate dehydrogenase were observed in the deeper foot muscle tissue of Concholepas concholepas. 2. Malate dehydrogenase activity was resolved into two molecular forms, which differ in K~ values for oxaloacetate and malate, and sensitivity to inhibition by ~-ketoglutarate. 3. In addition to reoxidation of NADH generated from glycolysis, the function of malate dehydrogenase is discussed in connection with octopine synthesis and oxidation in the foot muscle of C. concholepas. 4. The results of initial rate and product inhibition studies of both forms of malate dehydrogenase are consistent with an Ordered Bi Bi kinetic mechanism, with NADH adding first and NAD ÷ leaving last. Oxaloacetate substrate inhibition is suggested to result from formation of an enzyme-NAD+-oxaloacetate dead end complex.
Neuron, Jan 24, 2003
Recent studies indicate that active zones (AZs)-sites of neurotransmitter release-may be assemble... more Recent studies indicate that active zones (AZs)-sites of neurotransmitter release-may be assembled from preassembled AZ precursor vesicles inserted into the presynaptic plasma membrane. Here we report that one putative AZ precursor vesicle of CNS synapses-the Piccolo-Bassoon transport vesicle (PTV)-carries a comprehensive set of AZ proteins genetically and functionally coupled to synaptic vesicle exocytosis. Time-lapse imaging reveals that PTVs are highly mobile, consistent with a role in intracellular transport. Quantitative analysis reveals that the Bassoon, Piccolo, and RIM content of individual PTVs is, on average, half of that of individual presynaptic boutons and shows that the synaptic content of these molecules can be quantitatively accounted for by incorporation of integer numbers (typically two to three) of PTVs into presynaptic membranes. These findings suggest that AZs are assembled from unitary amounts of AZ material carried on PTVs.
Journal of Neuroscience, 2012
Vesicular trafficking of presynaptic and postsynaptic components is emerging as a general cellula... more Vesicular trafficking of presynaptic and postsynaptic components is emerging as a general cellular mechanism for the delivery of scaffold proteins, ion channels, and receptors to nascent and mature synapses. However, the molecular mechanisms leading to the selection of cargos and their differential transport to subneuronal compartments are not well understood, in part because of the mixing of cargos at the plasma membrane and/or within endosomal compartments. In the present study, we have explored the cellular mechanisms of active zone precursor vesicle assembly at the Golgi in dissociated hippocampal neurons of Rattus norvegicus. Our studies show that Piccolo, Bassoon, and ELKS2/CAST exit the trans-Golgi network on a common vesicle that requires Piccolo and Bassoon for its proper assembly. In contrast, Munc13 and synaptic vesicle proteins use distinct sets of Golgi-derived transport vesicles, while RIM1␣ associates with vesicular membranes in a post-Golgi compartment. Furthermore, Piccolo and Bassoon are necessary for ELKS2/CAST to leave the Golgi in association with vesicles, and a core domain of Bassoon is sufficient to facilitate formation of these vesicles. While these findings support emerging principles regarding active zone differentiation, the cellular and molecular analyses reported here also indicate that the Piccolo-Bassoon transport vesicles leaving the Golgi may undergo further changes in protein composition before arriving at synaptic sites.
PLoS ONE, 2012
Arsenic main inorganic compound is arsenic trioxide (ATO) presented in solution mainly as arsenit... more Arsenic main inorganic compound is arsenic trioxide (ATO) presented in solution mainly as arsenite. ATO increases intracellular pH (pHi), cell proliferation and tumor growth. Sodium-proton exchangers (NHEs) modulate the pHi, with NHE1 playing significant roles. Whether ATO-increased cell proliferation results from altered NHEs expression and activity is unknown. We hypothesize that ATO increases cell proliferation by altering pHi due to increased NHEs-like transport activity. Madin-Darby canine kidney (MDCK) cells grown in 5 mmol/L D-glucose-containing DMEM were exposed to ATO (0.05, 0.5 or 5 mmol/L, 0-48 hours) in the absence or presence of 5-N,N-hexamethylene amiloride (HMA, 5-100 mmol/L, NHEs inhibitor), PD-98059 (30 mmol/L, MAPK1/2 inhibitor), Gö 6976 (10 mmol/L, PKCa, bI and m inhibitor), or Schering 28080 (10 mmol/L, H + / K + ATPase inhibitor) plus concanamycin (0.1 mmol/L, V type ATPases inhibitor). Incorporation of [ 3 H]thymidine was used to estimate cell proliferation, and counting cells with a hemocytometer to determine the cell number. The pHi was measured by fluorometry in 2,7-bicarboxyethyl-5,6-carboxyfluorescein loaded cells. The Na +-dependent HMA-sensitive NHEs-like mediated proton transport kinetics, NHE1 protein abundance in the total, cytoplasm and plasma membrane protein fractions, and phosphorylated and total p42/44 mitogen-activated protein kinases (p42/44 mapk) were also determined. Lowest ATO (0.05 mmol/L, ,0.01 ppm) used in this study increased cell proliferation, pHi, NHEs-like transport and plasma membrane NHE1 protein abundance, effects blocked by HMA, PD-98059 or Gö 6976. Cell-buffering capacity did not change by ATO. The results show that a low ATO concentration increases MDCK cells proliferation by NHEs (probably NHE1)-like transport dependent-increased pHi requiring p42/44 mapk and PKCa, bI and/or m activity. This finding could be crucial in diseases where uncontrolled cell growth occurs, such as tumor growth, and in circumstances where ATO, likely arsenite, is available at the drinking-water at these levels.
Journal of Neurochemistry, 2002
The influence of aurintricarboxylic acid (ATA), a neuroprotective compound, on the serotonin tran... more The influence of aurintricarboxylic acid (ATA), a neuroprotective compound, on the serotonin transporter expressed in JAR human placental choriocarcinoma cells was investigated. Treatment of the cells with ATA for 16 h led to a significant stimulation of the serotonm transporter activity. This effect was not observed, however, when the treatment was done for 1-2 h. The stimulatory effect was associated with an increase in the maximal velocity of the transport process with no significant change in the Michaelis-Menten constant. Northern blot hybridization revealed that ATA treatment caused a marked increase in the steady-state levels of serotonin transporter-specific transcripts. Treatment of the cells with ATA was found to increase tyrosine phosphorylation of a 180-kDa protein. The phosphotyrosine content of a protein of a similar molecular size increased dramatically when the cells were exposed to epidermal growth factor (EGF), suggesting that this protein may be the EGF receptor. Treatment of the cells with EGF for 24 h could reproduce the stimulatory effects of ATA on the serotonin transporter activity, the maximal velocity of the transport process, and the steady-state levels of the transporterspecific mRNAs. Genistein, a tyrosine kinase inhibitor, was able to block the stimulatory effect of ATA and EGF. It is concluded that EGF increases the serotonin transporter expression in JAR cells and that the neuroprotective compound ATA produces similar effects on the transporter most likely by activating the EGF receptor through tyrosine phosphorylation.
Journal of Biotechnology, 2010
Classic IRES sequences are notorious for exerting biased expression in favor of upstream coding r... more Classic IRES sequences are notorious for exerting biased expression in favor of upstream coding regions when placed into polycistronic vectors. Here, we report the development of a bicistronic lentiviral system based on the 1D/2A sequence from the foot-and-mouth disease virus that is able to maintain tightly balanced control of upstream and downstream protein expression for several days at a stoichiometry very closely approaching 1.0. Our results suggest that the 1D/2A sequence can be optimized in an FUGW lentiviral setting to coordinate expression of multiple polypeptides, presenting a potentially valuable tool to signaling network researchers and to the gene therapy community.
Journal of Biological Chemistry, 2002
Mutagenesis of recombinant 1 ␥-aminobutyric acid (GABA) receptors has previously identified five ... more Mutagenesis of recombinant 1 ␥-aminobutyric acid (GABA) receptors has previously identified five residues in the amino terminal extracellular domain that play an important role in GABA binding. Here, we present evidence that the tyrosine at position 102 of the 1 receptor is also associated with the agonist binding site. Wildtype and mutant 1 receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp. When Tyr-102 was mutated to cysteine, serine, tryptophan, or glycine the EC 50 increased 31-, 214-, 664-, and 8752-fold, respectively. An increase in the IC 50 was also observed for the competitive antagonist 3-APMPA, but not for the non-competitive antagonist picrotoxin. Y102C was accessible to modification by methanethiosulfonate, and this modification was prevented by both GABA and 3-APMPA. An interesting characteristic of the Y102S mutant receptor was that, in the absence of GABA, there was an unusually high oocyte resting conductance that was blocked by both 3-APMPA and picrotoxin, indicating spontaneously opening GABA receptors. It appears that mutation of Tyr-102 perturbs the binding site and gates the pore. We conclude that Tyr-102 is a component of the GABA binding domain and speculate that Tyr-102 might be important for coupling agonist binding to channel opening.
Journal of Biological Chemistry, 2005
Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of t... more Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.
Journal of Biological Chemistry, 1996
We have isolated a cDNA from a human placental choriocarcinoma cell cDNA library which, when expr... more We have isolated a cDNA from a human placental choriocarcinoma cell cDNA library which, when expressed in HeLa cells, induces a Na ؉-dependent amino acid transport system with preference for zwitterionic amino acids. Anionic amino acids, cationic amino acids, imino acids, and N-methylated amino acids are excluded by this system. These characteristics are identical to those described for the amino acid transporter B o. When expressed in Xenopus laevis oocytes that do not have detectable endogenous activity of the amino acid transporter B o , the cloned transporter increases alanine transport in the oocytes severalfold and induces alanine-evoked inward currents in the presence of Na ؉. The cDNA codes for a polypeptide containing 541 amino acids with 10 putative transmembrane domains. Amino acid sequence homology predicts this transporter (hATB o) to be a member of a superfamily consisting of the glutamate transporters, the neutral amino acid transport system ASCT, and the insulin-activable neutral/anionic amino acid transporter. Chromosomal assignment studies with somatic cell hybrid analysis and fluorescent in situ hybridization have located the ATB o gene to human chromosome 19q13.3.
Experimental Eye Research, 1998
The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) o... more The contributions were determined in primary cultures of bovine corneal epithelial cells (BCEC) of Na : H exchange (NHE) and vacuolar H +-ATPase (i.e. V-type) activity to the regulation of intracellular pH (pH i). Furthermore, we characterized the effects on pH i regulation of exposure to 1 µ ET-1 under control and acid loaded conditions. With the pH sensitive dye, 2h,7h Bis (carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), the control pH i was 7n1 in NaCl (nominally HCO $-free) Ringers. Inhibition of NHE with 100 µ dimethylamiloride (DMA) rapidly decreased pH i by 0n37 units. Similarly, selective inhibition of V-type H +-ATPase with 10 µ bafilomycin A " decreased pH i by 0n22 units. Following acid loading in NaCl Ringers with a 20 m NH % Cl prepulse, pH i recovery was partially inhibited by exposure to either Na-free (NMGCl) Ringers, 100 µ DMA or 20 µ bafilomycin A ". Based on decreases in H + efflux resulting from selective inhibition of NHE and V-type H + pump activity, NHE activity accounts for 76 % of the pH i recovery following acid loading. Under control conditions, ET-1 (1 µ) had no effect on pH i whereas ET-1 completely suppressed pH i recovery following acid loading in NaCl or NMGCl Ringers. This inhibitory effect was largely due to stimulation of ET A because in the presence of BQ-123 (10 µ), a selective ET A receptor antagonist, pH i recovery was completely restored. Suppression of pH i recovery also occurred following stimulation of protein kinase C (PKC) with 10 −( phorbol myristate (PMA) whereas 10 −( 4 α phorbol 12,13 didecanoate (PDD) had no effect. ET-1 failed to suppress pH i recovery after inhibition of PKC with 0n5 µ calphostin C suggesting that the inhibition of pH i recovery by ET-1 is a consequence of PKC stimulation. Similarly, inhibition of Ca# +-dependent calmodulin stimulated CaM II kinase with KN-62 (10 µ) reversed the suppression of pH i recovery by ET-1. Preinhibition of either protein phosphatase (PP), PP-1, PP-2A or PP-2B activity with 1 µ phenylarsine oxide, 10 n okadaic acid, 10 µ cyclosporin A " or 20 µ BAPTA, also obviated the suppression of pH i recovery by ET-1. Therefore ET A receptor mediated inhibition of pH i regulation following acid loading could be a consequence of either PKC or CaMII kinase stimulation. Each one of these kinases may in turn phosphorylate and thereby stimulate the activities of PP-1, PP-2A or PP-2B. An increase in the activity of any one of these protein phosphatases could lead to dephosphorylation of the NHE and V-type H + pump. This alteration may prevent them from becoming adequately stimulated to elicit pH i recovery in response to acid loading.