7 results on '"Pérez Siles G"'
Search Results
2. Advances and challenges in modeling inherited peripheral neuropathies using iPSCs.
- Author
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Van Lent J, Prior R, Pérez Siles G, Cutrupi AN, Kennerson ML, Vangansewinkel T, Wolfs E, Mukherjee-Clavin B, Nevin Z, Judge L, Conklin B, Tyynismaa H, Clark AJ, Bennett DL, Van Den Bosch L, Saporta M, and Timmerman V
- Subjects
- Humans, Animals, Peripheral Nervous System Diseases genetics, Peripheral Nervous System Diseases pathology, Peripheral Nervous System Diseases therapy, Organoids metabolism, Models, Biological, Induced Pluripotent Stem Cells metabolism, Induced Pluripotent Stem Cells cytology
- Abstract
Inherited peripheral neuropathies (IPNs) are a group of diseases associated with mutations in various genes with fundamental roles in the development and function of peripheral nerves. Over the past 10 years, significant advances in identifying molecular disease mechanisms underlying axonal and myelin degeneration, acquired from cellular biology studies and transgenic fly and rodent models, have facilitated the development of promising treatment strategies. However, no clinical treatment has emerged to date. This lack of treatment highlights the urgent need for more biologically and clinically relevant models recapitulating IPNs. For both neurodevelopmental and neurodegenerative diseases, patient-specific induced pluripotent stem cells (iPSCs) are a particularly powerful platform for disease modeling and preclinical studies. In this review, we provide an update on different in vitro human cellular IPN models, including traditional two-dimensional monoculture iPSC derivatives, and recent advances in more complex human iPSC-based systems using microfluidic chips, organoids, and assembloids., (© 2024. The Author(s).)
- Published
- 2024
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3. Structural Determinants of the Neuronal Glycine Transporter 2 for the Selective Inhibitors ALX1393 and ORG25543.
- Author
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Benito-Muñoz C, Perona A, Felipe R, Pérez-Siles G, Núñez E, Aragón C, and López-Corcuera B
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- Animals, Benzamides pharmacology, Neurons, Serine analogs & derivatives, Drosophila melanogaster, Glycine Plasma Membrane Transport Proteins genetics
- Abstract
The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission by controlling the extracellular concentration of synaptic glycine and the supply of neurotransmitter to the presynaptic terminal. Spinal cord glycinergic neurons present in the dorsal horn diminish their activity in pathological pain conditions and behave as gate keepers of the touch-pain circuitry. The pharmacological blockade of GlyT2 reduces the progression of the painful signal to rostral areas of the central nervous system by increasing glycine extracellular levels, so it has analgesic action. O -[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-l-serine (ALX1393) and N -[[1-(dimethylamino)cyclopentyl]methyl]-3,5-dimethoxy-4-(phenylmethoxy)benzamide (ORG25543) are two selective GlyT2 inhibitors with nanomolar affinity for the transporter and analgesic effects in pain animal models, although with deficiencies which preclude further clinical development. In this report, we performed a comparative ligand docking of ALX1393 and ORG25543 on a validated GlyT2 structural model including all ligand sites constructed by homology with the crystallized dopamine transporter from Drosophila melanogaster . Molecular dynamics simulations and energy analysis of the complex and functional analysis of a series of point mutants permitted to determine the structural determinants of ALX1393 and ORG25543 discrimination by GlyT2. The ligands establish simultaneous contacts with residues present in transmembrane domains 1, 3, 6, and 8 and block the transporter in outward-facing conformation and hence inhibit glycine transport. In addition, differential interactions of ALX1393 with the cation bound at Na1 site and ORG25543 with TM10 define the differential sites of the inhibitors and explain some of their individual features. Structural information about the interactions with GlyT2 may provide useful tools for new drug discovery.
- Published
- 2021
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4. A novel dominant hyperekplexia mutation Y705C alters trafficking and biochemical properties of the presynaptic glycine transporter GlyT2.
- Author
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Giménez C, Pérez-Siles G, Martínez-Villarreal J, Arribas-González E, Jiménez E, Núñez E, de Juan-Sanz J, Fernández-Sánchez E, García-Tardón N, Ibáñez I, Romanelli V, Nevado J, James VM, Topf M, Chung SK, Thomas RH, Desviat LR, Aragón C, Zafra F, Rees MI, Lapunzina P, Harvey RJ, and López-Corcuera B
- Subjects
- Amino Acid Substitution, Animals, Female, Glycine genetics, Glycine metabolism, Humans, Ion Transport genetics, Male, Presynaptic Terminals, Protein Transport genetics, Spain, United Kingdom, Genes, Dominant, Genetic Diseases, Inborn genetics, Genetic Diseases, Inborn metabolism, Glycine Plasma Membrane Transport Proteins genetics, Glycine Plasma Membrane Transport Proteins metabolism, Mutation, Missense, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Nervous System Diseases genetics, Nervous System Diseases metabolism
- Abstract
Hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, producing hypertonia and apnea episodes. Although rare, this orphan disorder can have serious consequences, including sudden infant death. Dominant and recessive mutations in the human glycine receptor (GlyR) α1 gene (GLRA1) are the major cause of this disorder. However, recessive mutations in the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 gene (SLC6A5) are rapidly emerging as a second major cause of startle disease. In this study, systematic DNA sequencing of SLC6A5 revealed a new dominant GlyT2 mutation: pY705C (c.2114A→G) in transmembrane domain 11, in eight individuals from Spain and the United Kingdom. Curiously, individuals harboring this mutation show significant variation in clinical presentation. In addition to classical hyperekplexia symptoms, some individuals had abnormal respiration, facial dysmorphism, delayed motor development, or intellectual disability. We functionally characterized this mutation using molecular modeling, electrophysiology, [(3)H]glycine transport, cell surface expression, and cysteine labeling assays. We found that the introduced cysteine interacts with the cysteine pair Cys-311-Cys-320 in the second external loop of GlyT2. This interaction impairs transporter maturation through the secretory pathway, reduces surface expression, and inhibits transport function. Additionally, Y705C presents altered H(+) and Zn(2+) dependence of glycine transport that may affect the function of glycinergic neurotransmission in vivo.
- Published
- 2012
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5. An aspartate residue in the external vestibule of GLYT2 (glycine transporter 2) controls cation access and transport coupling.
- Author
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Pérez-Siles G, Núñez E, Morreale A, Jiménez E, Leo-Macías A, Pita G, Cherubino F, Sangaletti R, Bossi E, Ortíz AR, Aragón C, and López-Corcuera B
- Subjects
- Amino Acid Substitution, Animals, Aspartic Acid chemistry, COS Cells, Chlorocebus aethiops, Conserved Sequence, Electrophysiological Phenomena, Female, Glycine Plasma Membrane Transport Proteins genetics, In Vitro Techniques, Ion Transport drug effects, Models, Molecular, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Mutant Proteins chemistry, Mutant Proteins genetics, Mutant Proteins metabolism, Oocytes metabolism, Rats, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Sodium metabolism, Spiro Compounds pharmacology, Xenopus laevis, Glycine Plasma Membrane Transport Proteins chemistry, Glycine Plasma Membrane Transport Proteins metabolism
- Abstract
Synaptic glycine levels are controlled by GLYTs (glycine transporters). GLYT1 is the main regulator of synaptic glycine concentrations and catalyses Na+-Cl--glycine co-transport with a 2:1:1 stoichiometry. In contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na+. Using molecular interaction potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na+ interactions. Replacement of Asp471 in this region reduced Na+ affinity and Na+ co-operativity of transport, an effect not produced in the homologous position (Asp295) in GLYT1. Unlike the GLYT1-Asp295 mutation, this Asp471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [3H]glycine accumulation. The homologous Asp471 and Asp295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na+ and Li+-induced conformational changes. Although these two cations had opposite effects on GLYT1, they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. On the basis of these findings, we propose a role for Asp471 in controlling cation access to GLYT2 Na+ sites, ion coupling during transport and the subsequent conformational changes.
- Published
- 2012
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6. Molecular basis of the differential interaction with lithium of glycine transporters GLYT1 and GLYT2.
- Author
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Pérez-Siles G, Morreale A, Leo-Macías A, Pita G, Ortíz AR, Aragón C, and López-Corcuera B
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, Glycine metabolism, Lithium metabolism, Protein Binding physiology, Protein Transport physiology, Glycine Plasma Membrane Transport Proteins metabolism, Lithium physiology
- Abstract
Glycine synaptic levels are controlled by glycine transporters (GLYTs) catalyzing Na(+)/Cl(-)/glycine cotransport. GLYT1 displays a 2:1 :1 stoichiometry and is the main regulator of extracellular glycine concentrations. The neuronal GLYT2, with higher sodium coupling (3:1 :1), supplies glycine to the pre-synaptic terminal to refill synaptic vesicles. In this work, using structural homology modelling and molecular dynamics simulations of GLYTs, we predict the conservation of the two sodium sites present in the template (leucine transporter from Aquifex aeolicus), and confirm its use by mutagenesis and functional analysis. GLYTs Na1 and Na2 sites show differential cation selectivity, as inferred from the action of lithium, a non-transport-supporting ion, on Na(+)-site mutants. GLYTs lithium responses were unchanged in Na1-site mutants, but abolished or inverted in mutants of Na2 site, which binds lithium in the presence of low sodium concentrations and therefore, controls lithium responses. Here, we report, for the first time, that lithium exerts opposite actions on GLYTs isoforms. Glycine transport by GLYT1 is inhibited by lithium whereas GLYT2 transport is stimulated, and this effect is more evident at increased glycine concentrations. In contrast to GLYT1, high and low affinity lithium-binding processes were detected in GLYT2., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)
- Published
- 2011
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7. Subcellular localization of the neuronal glycine transporter GLYT2 in brainstem.
- Author
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Núñez E, Pérez-Siles G, Rodenstein L, Alonso-Torres P, Zafra F, Jiménez E, Aragón C, and López-Corcuera B
- Subjects
- Animals, Biomarkers metabolism, Brain Stem cytology, Cell Membrane metabolism, Cell Membrane ultrastructure, Humans, Immunohistochemistry, Microscopy, Immunoelectron, Neurons ultrastructure, Rats, Rats, Wistar, rab GTP-Binding Proteins genetics, rab GTP-Binding Proteins metabolism, rab4 GTP-Binding Proteins genetics, rab4 GTP-Binding Proteins metabolism, Brain Stem metabolism, Glycine metabolism, Glycine Plasma Membrane Transport Proteins metabolism, Neurons metabolism
- Abstract
The neuronal glycine transporter GLYT2 belongs to the neurotransmitter:sodium:symporter (NSS) family and removes glycine from the synaptic cleft, thereby aiding the termination of the glycinergic signal and achieving the reloading of the presynaptic terminal. The task fulfilled by this transporter is fine tuned by regulating both transport activity and intracellular trafficking. Different stimuli such as neuronal activity or protein kinase C (PKC) activation can control GLYT2 surface levels although the intracellular compartments where GLYT2 resides are largely unknown. Here, by biochemical and immunological techniques in combination with electron and confocal microscopy, we have investigated the subcellular distribution of GLYT2 in rat brainstem tissue, and characterized the vesicles that contain the transporter. GLYT2 is shown to be present in small and larger vesicles that contain the synaptic vesicle protein synaptophysin, the recycling endosome small GTPase Rab11, and in the larger vesicle population, the vesicular inhibitory amino acid transporter VIAAT. Rab5A, the GABA transporter GAT1, synaptotagmin2 and synaptobrevin2 (VAMP2) were not present. Coexpression of a Rab11 dominant negative mutant with recombinant GLYT2 impaired transporter trafficking and glycine transport. Dual immunogold labeling of brainstem synaptosomes showed a very close proximity of GLYT2 and Rab11. Therefore, the intracellular GLYT2 resides in a subset of endosomal membranes and may traffic around several compartments, mainly Rab11-positive endosomes.
- Published
- 2009
- Full Text
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