Rosa Gomez-villafuertes | Universidad Complutense de Madrid (original) (raw)
Papers by Rosa Gomez-villafuertes
Frontiers in Pharmacology, 2020
Monografías de la Real …, 2009
... En ratones deficientes de PKC-γ se observa una clara alteración de la transmisión sináptica e... more ... En ratones deficientes de PKC-γ se observa una clara alteración de la transmisión sináptica en el hipocampo y una inervación aberrante de las neu-ronas de Purkinje procedente de las fibras trepadoras de la corteza cerebelo-sa (22, 23). ...
Purinergic signalling, 2018
The pathogenesis of glaucoma involves numerous intracellular mechanisms including the purinergic ... more The pathogenesis of glaucoma involves numerous intracellular mechanisms including the purinergic system contribution. Furthermore, the presence and release of nucleotides and dinucleotides during the glaucomatous damage and the maintenance of degradation machinery through ecto-nucleotidase activity are participating in the modulation of the suitable extracellular complex balance. The aim of this study was to investigate the levels of diadenosine tetraphosphate (ApA) and the pattern of ecto-nucleotidase activity expression in glaucomatous retinas during the progress the pathology. ApA levels were analyzed by HPLC in glaucomatous retinas from the DBA/2J mice at 3, 9, 15, and 23 months of age. For that, retinas were dissected as flattened whole-mounts and stimulated in Ringer buffer with or without 59 mM KCl. NPP1 expression was analyzed by RT-PCR and western blot and its distribution was assessed by immunohistochemistry studies examined under confocal microscopy. Glaucomatous mice exh...
Frontiers in Cell and Developmental Biology
P2X7 receptor (P2RX7) is expressed strongly by most human cancers, including neuroblastoma, where... more P2X7 receptor (P2RX7) is expressed strongly by most human cancers, including neuroblastoma, where high levels of P2RX7 are correlated with a poor prognosis for patients. Tonic activation of P2X7 receptor favors cell metabolism and angiogenesis, thereby promoting cancer cell proliferation, immunosuppression, and metastasis. Although understanding the mechanisms that control P2X7 receptor levels in neuroblastoma cells could be biologically and clinically relevant, the intracellular signaling pathways involved in this regulation remain poorly understood. Here we show that (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), an allosteric inhibitor of dual specificity phosphatases (DUSP) 1 and 6, enhances the expression of P2X7 receptor in N2a neuroblastoma cells. We found that exposure to BCI induces the phosphorylation of mitogen-activated protein kinases p38 and JNK, while it prevents the phosphorylation of ERK1/2. BCI enhanced dual specificity phosphatase 1 expres...
Methods in molecular biology, 2020
Calcium is one of the most important intracellular messengers, triggering a wide range of cellula... more Calcium is one of the most important intracellular messengers, triggering a wide range of cellular responses. Changes in intracellular free calcium concentration can be measured using calcium sensitive fluorescent dyes, which are either EGTA- or BAPTA-based organic molecules that change their spectral properties in response to Ca2+ binding. One of the most common calcium indicators is the ratiometric dye Fura-2. The main advantage of using ratiometric dyes is that the ratio signal is independent of the illumination intensity, dye concentration, photobleaching, and focus changes among others, allowing for the concentration of intracellular calcium to be determined independently of these artifacts. In this protocol, we describe the use of Fura-2 to measure intracellular calcium elevations in single cultured cells after purinoceptor activation using a video-microscopy equipment. This method, usually known as calcium imaging, allows for real-time quantification of intracellular calcium ...
Brain Structure and Function, 2021
The purinergic system is one of the oldest cell-to-cell communication mechanisms and exhibits rel... more The purinergic system is one of the oldest cell-to-cell communication mechanisms and exhibits relevant functions in the regulation of the central nervous system (CNS) development. Amongst the components of the purinergic system, the ionotropic P2X7 receptor (P2X7R) stands out as a potential regulator of brain pathology and physiology. Thus, P2X7R is known to regulate crucial aspects of neuronal cell biology, including axonal elongation, path-finding, synapse formation and neuroprotection. Moreover, P2X7R modulates neuroinflammation and is posed as a therapeutic target in inflammatory, oncogenic and degenerative disorders. However, the lack of reliable technical and pharmacological approaches to detect this receptor represents a major hurdle in its study. Here, we took advantage of theP2rx7-EGFP reporter mouse, which expresses enhanced green fluorescence protein (EGFP) immediately downstream of theP2rx7proximal promoter, to conduct a detailed study of its distribution. We performed a...
Frontiers in pharmacology, 2017
Glaucoma is a neuropathology, often accompanied by an elevated intraocular pressure (IOP), which ... more Glaucoma is a neuropathology, often accompanied by an elevated intraocular pressure (IOP), which can lead to blindness. Since DBA/2J mice develop glaucoma, several studies of the physiopathology of glaucoma have been reported in this animal model. It is also known that purinergic receptors are involved in the pathology of glaucoma by controlling aqueous humor production and drainage and therefore controlling IOP. There are no studies on purinergic receptors in the DBA/2J model of glaucoma and their relation to the development of the pathology, so the aim of this study was to make an approach to the purinergic mechanisms involved in glaucoma. All the experiments were performed using DBA/2J and C57BL/6J mice and investigating P2Y1, P2Y2, and P2Y6 receptors. IOP measurements were made with a non-invasive rebound tonometer, and animals were instilled with diadenosine tetraphosphate (Ap4A) and the corresponding purinergic antagonists in order to see their effects on IOP. The expression o...
Frontiers in pharmacology, 2017
Adenosine triphosphate (ATP) is an important extracellular neurotransmitter that participates in ... more Adenosine triphosphate (ATP) is an important extracellular neurotransmitter that participates in several critical processes like cell differentiation, neuroprotection or axon guidance. Prior to its exocytosis, ATP must be stored in secretory vesicles, a process that is mediated by the Vesicular Nucleotide Transporter (VNUT). This transporter has been identified as the product of the SLC17A9 gene and it is prominently expressed in discrete brain areas, including the cerebellum. The main population of cerebellar neurons, the glutamatergic granule neurons, depends on purinergic signaling to trigger neuroprotective responses. However, while nucleotide receptors like P2X7 and P2Y13 are known to be involved in neuroprotection, the mechanisms that regulate ATP release in relation to such events are less clearly understood. In this work, we demonstrate that cerebellar granule cells express a functional VNUT that is involved in the regulation of ATP exocytosis. Numerous vesicles loaded with ...
Advances in experimental medicine and biology, Jan 17, 2017
The distribution of nucleotide P2Y receptors across different tissues suggests that they fulfil k... more The distribution of nucleotide P2Y receptors across different tissues suggests that they fulfil key roles in a number of physiological and pathological conditions. P2Y13 is one of the latest P2Y receptors identified, a novel member of the Gi-coupled P2Y receptor subfamily that responds to ADP, together with P2Y12 and P2Y14. Pharmacological studies drew attention to this new ADP receptor, with a pharmacology that overlaps that of P2Y12 receptors but with unique features and roles. The P2RY12-14 genes all reside on human chromosome 3 at 3q25.1 and their strong sequence homology supports their evolutionary origin through gene duplication. Polymorphisms of P2Y13 receptors have been reported in different human populations, yet their consequences remain unknown. The P2Y13 receptor is versatile in its signalling, extending beyond the canonical signalling of a Gi-coupled receptor. Not only can it couple to different G proteins (Gs/Gq) but the P2Y13 receptor can also trigger several intracel...
Epilepsia, Sep 21, 2017
ATP is released into the extracellular space during pathologic processes including increased neur... more ATP is released into the extracellular space during pathologic processes including increased neuronal firing. Once released, ATP acts on P2 receptors including ionotropic P2X and metabotropic P2Y receptors, resulting in changes to glial function and neuronal network excitability. Evidence suggests an involvement of P2Y receptors in the pathogenesis of epilepsy, but there has been no systematic effort to characterize the expression and function of the P2Y receptor family during seizures and in experimental and human epilepsy. Status epilepticus was induced using either intra-amygdala kainic acid or pilocarpine to characterize the acute- and long-term changes in hippocampal P2Y expression. P2Y expression was also investigated in brain tissue from patients with temporal lobe epilepsy. Finally, we analyzed the effects of two specific P2Y agonists, ADP and UTP, on seizure severity and seizure-induced cell death. Both intra-amygdala kainic acid and pilocarpine-induced status epilepticus i...
Neuropharmacology, Jan 7, 2015
Brain injury generates the release of a multitude of factors including extracellular nucleotides,... more Brain injury generates the release of a multitude of factors including extracellular nucleotides, which exhibit bi-functional properties and contribute to both detrimental actions in the acute phase and also protective and reparative actions in the later recovery phase to allow neuroregeneration. A promising strategy toward restoration of neuronal function is based on activation of endogenous adult neural stem/progenitor cells. The implication of purinergic signaling in stem cell biology, including regulation of proliferation, differentiation, and cell death has become evident in the last decade. In this regard, current strategies of acute transplantation of ependymal stem/progenitor cells after spinal cord injury restore altered expression of P2X4 and P2X7 receptors and improve functional locomotor recovery. The expression of both receptors is transcriptionally regulated by Sp1 factor, which plays a key role in the startup of the transcription machinery to induce regeneration-assoc...
Computational and Structural Biotechnology Journal, 2015
Neurobiology of Aging, 2012
The FASEB Journal, 2009
The precise mechanism by which mutant huntingtin elicits its toxicity remains unknown. However, s... more The precise mechanism by which mutant huntingtin elicits its toxicity remains unknown. However, synaptic alterations and increased susceptibility to neuronal death are known contributors to Huntington's disease (HD) symptomatology. While decreased metabolism has long been associated with HD, recent findings have surprisingly demonstrated reduced neuronal apoptosis in Caenorhabditis elegans and Drosophila models of HD by drugs that diminish ATP production. Interestingly, extracellular ATP has been recently reported to elicit neuronal death through stimulation of P2X7 receptors. These are ATP-gated cation channels known to modulate neurotransmitter release from neuronal presynaptic terminals and to regulate cytokine production and release from microglia. We hypothesized that alteration in P2X7-mediated calcium permeability may contribute to HD synaptic dysfunction and increased neuronal apoptosis. Using mouse and cellular models of HD, we demonstrate increased P2X7-receptor level and altered P2X7-mediated calcium permeability in somata and terminals of HD neurons. Furthermore, cultured neurons expressing mutant huntingtin showed increased susceptibility to apoptosis triggered by P2X7-receptor stimulation. Finally, in vivo administration of the P2X7-antagonist Brilliant Blue-G (BBG) to HD mice prevented neuronal apoptosis and attenuated body weight loss and motor-coordination deficits. These in vivo data strongly suggest that altered P2X7-receptor level and function contribute to HD pathogenesis and highlight the therapeutic potential of P2X7 receptor antagonists.
Neurochemistry International, 2009
![Research paper thumbnail of Adenosine triphosphate and diadenosine pentaphosphate induce [Ca2+]iincrease in rat basal ganglia aminergic terminals](https://attachments.academia-assets.com/106016114/thumbnails/1.jpg)
](Journal of Neuroscience Research, 2001
Journal of visualized experiments : JoVE, Dec 16, 2017
Understanding the mechanisms that control critical biological events of neural cell populations, ... more Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with sin...
Frontiers in Pharmacology, 2020
Monografías de la Real …, 2009
... En ratones deficientes de PKC-γ se observa una clara alteración de la transmisión sináptica e... more ... En ratones deficientes de PKC-γ se observa una clara alteración de la transmisión sináptica en el hipocampo y una inervación aberrante de las neu-ronas de Purkinje procedente de las fibras trepadoras de la corteza cerebelo-sa (22, 23). ...
Purinergic signalling, 2018
The pathogenesis of glaucoma involves numerous intracellular mechanisms including the purinergic ... more The pathogenesis of glaucoma involves numerous intracellular mechanisms including the purinergic system contribution. Furthermore, the presence and release of nucleotides and dinucleotides during the glaucomatous damage and the maintenance of degradation machinery through ecto-nucleotidase activity are participating in the modulation of the suitable extracellular complex balance. The aim of this study was to investigate the levels of diadenosine tetraphosphate (ApA) and the pattern of ecto-nucleotidase activity expression in glaucomatous retinas during the progress the pathology. ApA levels were analyzed by HPLC in glaucomatous retinas from the DBA/2J mice at 3, 9, 15, and 23 months of age. For that, retinas were dissected as flattened whole-mounts and stimulated in Ringer buffer with or without 59 mM KCl. NPP1 expression was analyzed by RT-PCR and western blot and its distribution was assessed by immunohistochemistry studies examined under confocal microscopy. Glaucomatous mice exh...
Frontiers in Cell and Developmental Biology
P2X7 receptor (P2RX7) is expressed strongly by most human cancers, including neuroblastoma, where... more P2X7 receptor (P2RX7) is expressed strongly by most human cancers, including neuroblastoma, where high levels of P2RX7 are correlated with a poor prognosis for patients. Tonic activation of P2X7 receptor favors cell metabolism and angiogenesis, thereby promoting cancer cell proliferation, immunosuppression, and metastasis. Although understanding the mechanisms that control P2X7 receptor levels in neuroblastoma cells could be biologically and clinically relevant, the intracellular signaling pathways involved in this regulation remain poorly understood. Here we show that (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), an allosteric inhibitor of dual specificity phosphatases (DUSP) 1 and 6, enhances the expression of P2X7 receptor in N2a neuroblastoma cells. We found that exposure to BCI induces the phosphorylation of mitogen-activated protein kinases p38 and JNK, while it prevents the phosphorylation of ERK1/2. BCI enhanced dual specificity phosphatase 1 expres...
Methods in molecular biology, 2020
Calcium is one of the most important intracellular messengers, triggering a wide range of cellula... more Calcium is one of the most important intracellular messengers, triggering a wide range of cellular responses. Changes in intracellular free calcium concentration can be measured using calcium sensitive fluorescent dyes, which are either EGTA- or BAPTA-based organic molecules that change their spectral properties in response to Ca2+ binding. One of the most common calcium indicators is the ratiometric dye Fura-2. The main advantage of using ratiometric dyes is that the ratio signal is independent of the illumination intensity, dye concentration, photobleaching, and focus changes among others, allowing for the concentration of intracellular calcium to be determined independently of these artifacts. In this protocol, we describe the use of Fura-2 to measure intracellular calcium elevations in single cultured cells after purinoceptor activation using a video-microscopy equipment. This method, usually known as calcium imaging, allows for real-time quantification of intracellular calcium ...
Brain Structure and Function, 2021
The purinergic system is one of the oldest cell-to-cell communication mechanisms and exhibits rel... more The purinergic system is one of the oldest cell-to-cell communication mechanisms and exhibits relevant functions in the regulation of the central nervous system (CNS) development. Amongst the components of the purinergic system, the ionotropic P2X7 receptor (P2X7R) stands out as a potential regulator of brain pathology and physiology. Thus, P2X7R is known to regulate crucial aspects of neuronal cell biology, including axonal elongation, path-finding, synapse formation and neuroprotection. Moreover, P2X7R modulates neuroinflammation and is posed as a therapeutic target in inflammatory, oncogenic and degenerative disorders. However, the lack of reliable technical and pharmacological approaches to detect this receptor represents a major hurdle in its study. Here, we took advantage of theP2rx7-EGFP reporter mouse, which expresses enhanced green fluorescence protein (EGFP) immediately downstream of theP2rx7proximal promoter, to conduct a detailed study of its distribution. We performed a...
Frontiers in pharmacology, 2017
Glaucoma is a neuropathology, often accompanied by an elevated intraocular pressure (IOP), which ... more Glaucoma is a neuropathology, often accompanied by an elevated intraocular pressure (IOP), which can lead to blindness. Since DBA/2J mice develop glaucoma, several studies of the physiopathology of glaucoma have been reported in this animal model. It is also known that purinergic receptors are involved in the pathology of glaucoma by controlling aqueous humor production and drainage and therefore controlling IOP. There are no studies on purinergic receptors in the DBA/2J model of glaucoma and their relation to the development of the pathology, so the aim of this study was to make an approach to the purinergic mechanisms involved in glaucoma. All the experiments were performed using DBA/2J and C57BL/6J mice and investigating P2Y1, P2Y2, and P2Y6 receptors. IOP measurements were made with a non-invasive rebound tonometer, and animals were instilled with diadenosine tetraphosphate (Ap4A) and the corresponding purinergic antagonists in order to see their effects on IOP. The expression o...
Frontiers in pharmacology, 2017
Adenosine triphosphate (ATP) is an important extracellular neurotransmitter that participates in ... more Adenosine triphosphate (ATP) is an important extracellular neurotransmitter that participates in several critical processes like cell differentiation, neuroprotection or axon guidance. Prior to its exocytosis, ATP must be stored in secretory vesicles, a process that is mediated by the Vesicular Nucleotide Transporter (VNUT). This transporter has been identified as the product of the SLC17A9 gene and it is prominently expressed in discrete brain areas, including the cerebellum. The main population of cerebellar neurons, the glutamatergic granule neurons, depends on purinergic signaling to trigger neuroprotective responses. However, while nucleotide receptors like P2X7 and P2Y13 are known to be involved in neuroprotection, the mechanisms that regulate ATP release in relation to such events are less clearly understood. In this work, we demonstrate that cerebellar granule cells express a functional VNUT that is involved in the regulation of ATP exocytosis. Numerous vesicles loaded with ...
Advances in experimental medicine and biology, Jan 17, 2017
The distribution of nucleotide P2Y receptors across different tissues suggests that they fulfil k... more The distribution of nucleotide P2Y receptors across different tissues suggests that they fulfil key roles in a number of physiological and pathological conditions. P2Y13 is one of the latest P2Y receptors identified, a novel member of the Gi-coupled P2Y receptor subfamily that responds to ADP, together with P2Y12 and P2Y14. Pharmacological studies drew attention to this new ADP receptor, with a pharmacology that overlaps that of P2Y12 receptors but with unique features and roles. The P2RY12-14 genes all reside on human chromosome 3 at 3q25.1 and their strong sequence homology supports their evolutionary origin through gene duplication. Polymorphisms of P2Y13 receptors have been reported in different human populations, yet their consequences remain unknown. The P2Y13 receptor is versatile in its signalling, extending beyond the canonical signalling of a Gi-coupled receptor. Not only can it couple to different G proteins (Gs/Gq) but the P2Y13 receptor can also trigger several intracel...
Epilepsia, Sep 21, 2017
ATP is released into the extracellular space during pathologic processes including increased neur... more ATP is released into the extracellular space during pathologic processes including increased neuronal firing. Once released, ATP acts on P2 receptors including ionotropic P2X and metabotropic P2Y receptors, resulting in changes to glial function and neuronal network excitability. Evidence suggests an involvement of P2Y receptors in the pathogenesis of epilepsy, but there has been no systematic effort to characterize the expression and function of the P2Y receptor family during seizures and in experimental and human epilepsy. Status epilepticus was induced using either intra-amygdala kainic acid or pilocarpine to characterize the acute- and long-term changes in hippocampal P2Y expression. P2Y expression was also investigated in brain tissue from patients with temporal lobe epilepsy. Finally, we analyzed the effects of two specific P2Y agonists, ADP and UTP, on seizure severity and seizure-induced cell death. Both intra-amygdala kainic acid and pilocarpine-induced status epilepticus i...
Neuropharmacology, Jan 7, 2015
Brain injury generates the release of a multitude of factors including extracellular nucleotides,... more Brain injury generates the release of a multitude of factors including extracellular nucleotides, which exhibit bi-functional properties and contribute to both detrimental actions in the acute phase and also protective and reparative actions in the later recovery phase to allow neuroregeneration. A promising strategy toward restoration of neuronal function is based on activation of endogenous adult neural stem/progenitor cells. The implication of purinergic signaling in stem cell biology, including regulation of proliferation, differentiation, and cell death has become evident in the last decade. In this regard, current strategies of acute transplantation of ependymal stem/progenitor cells after spinal cord injury restore altered expression of P2X4 and P2X7 receptors and improve functional locomotor recovery. The expression of both receptors is transcriptionally regulated by Sp1 factor, which plays a key role in the startup of the transcription machinery to induce regeneration-assoc...
Computational and Structural Biotechnology Journal, 2015
Neurobiology of Aging, 2012
The FASEB Journal, 2009
The precise mechanism by which mutant huntingtin elicits its toxicity remains unknown. However, s... more The precise mechanism by which mutant huntingtin elicits its toxicity remains unknown. However, synaptic alterations and increased susceptibility to neuronal death are known contributors to Huntington's disease (HD) symptomatology. While decreased metabolism has long been associated with HD, recent findings have surprisingly demonstrated reduced neuronal apoptosis in Caenorhabditis elegans and Drosophila models of HD by drugs that diminish ATP production. Interestingly, extracellular ATP has been recently reported to elicit neuronal death through stimulation of P2X7 receptors. These are ATP-gated cation channels known to modulate neurotransmitter release from neuronal presynaptic terminals and to regulate cytokine production and release from microglia. We hypothesized that alteration in P2X7-mediated calcium permeability may contribute to HD synaptic dysfunction and increased neuronal apoptosis. Using mouse and cellular models of HD, we demonstrate increased P2X7-receptor level and altered P2X7-mediated calcium permeability in somata and terminals of HD neurons. Furthermore, cultured neurons expressing mutant huntingtin showed increased susceptibility to apoptosis triggered by P2X7-receptor stimulation. Finally, in vivo administration of the P2X7-antagonist Brilliant Blue-G (BBG) to HD mice prevented neuronal apoptosis and attenuated body weight loss and motor-coordination deficits. These in vivo data strongly suggest that altered P2X7-receptor level and function contribute to HD pathogenesis and highlight the therapeutic potential of P2X7 receptor antagonists.
Neurochemistry International, 2009
Journal of Neuroscience Research, 2001
Journal of visualized experiments : JoVE, Dec 16, 2017
Understanding the mechanisms that control critical biological events of neural cell populations, ... more Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with sin...