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  1. Article ; Online: Cellular and Subcellular Localisation of Kv4-Associated KChIP Proteins in the Rat Cerebellum

    Rocío Alfaro-Ruíz / Carolina Aguado / Alejandro Martín-Belmonte / Ana Esther Moreno-Martínez / Rafael Luján

    International Journal of Molecular Sciences, Vol 21, Iss 6403, p

    2020  Volume 6403

    Abstract: The K + channel interacting proteins (KChIPs) are a family of cytosolic proteins that interact with Kv4 channels, leading to higher current density, modulation of channel inactivation and faster recovery from inactivation. Using immunohistochemical ... ...

    Abstract The K + channel interacting proteins (KChIPs) are a family of cytosolic proteins that interact with Kv4 channels, leading to higher current density, modulation of channel inactivation and faster recovery from inactivation. Using immunohistochemical techniques at the light and electron microscopic level combined with quantitative analysis, we investigated the cellular and subcellular localisation of KChIP3 and KChIP4 to compare their distribution patterns with those for Kv4.2 and Kv4.3 in the cerebellar cortex. Immunohistochemistry at the light microscopic level demonstrated that KChIP3, KChIP4, Kv4.2 and Kv4.3 proteins were widely expressed in the cerebellum, with mostly overlapping patterns. Immunoelectron microscopic techniques showed that KChIP3, KChIP4, Kv4.2 and Kv4.3 shared virtually the same somato-dendritic domains of Purkinje cells and granule cells. Application of quantitative approaches showed that KChIP3 and KChIP4 were mainly membrane-associated, but also present at cytoplasmic sites close to the plasma membrane, in dendritic spines and shafts of Purkinje cells (PCs) and dendrites of granule cells (GCs). Similarly, immunoparticles for Kv4.2 and Kv4.3 were observed along the plasma membrane and at intracellular sites in the same neuron populations. In addition to the preferential postsynaptic distribution, KChIPs and Kv4 were also distributed presynaptically in parallel fibres and mossy fibres. Immunoparticles for KChIP3, KChIP4 and Kv4.3 were detected in parallel fibres, and KChIP3, KChIP4, Kv4.2 and Kv4.3 were found in parallel fibres, indicating that composition of KChIP and Kv4 seems to be input-dependent. Together, our findings unravelled previously uncharacterised KChIP and Kv4 subcellular localisation patterns in neurons, revealed that KChIP have additional Kv4-unrelated functions in the cerebellum and support the formation of macromolecular complexes between KChIP3 and KChIP4 with heterotetrameric Kv4.2/Kv4.3 channels.
    Keywords cerebellum ; potassium channel ; KChIP proteins ; electron microscopy ; immunohistochemistry ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Subject code 571
    Language English
    Publishing date 2020-09-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: The Expression and Localisation of G-Protein-Coupled Inwardly Rectifying Potassium (GIRK) Channels Is Differentially Altered in the Hippocampus of Two Mouse Models of Alzheimer’s Disease

    Rocío Alfaro-Ruiz / Alejandro Martín-Belmonte / Carolina Aguado / Félix Hernández / Ana Esther Moreno-Martínez / Jesús Ávila / Rafael Luján

    International Journal of Molecular Sciences, Vol 22, Iss 11106, p

    2021  Volume 11106

    Abstract: G protein-gated inwardly rectifying K + (GIRK) channels are the main targets controlling excitability and synaptic plasticity on hippocampal neurons. Consequently, dysfunction of GIRK-mediated signalling has been implicated in the pathophysiology of ... ...

    Abstract G protein-gated inwardly rectifying K + (GIRK) channels are the main targets controlling excitability and synaptic plasticity on hippocampal neurons. Consequently, dysfunction of GIRK-mediated signalling has been implicated in the pathophysiology of Alzheimer´s disease (AD). Here, we provide a quantitative description on the expression and localisation patterns of GIRK2 in two transgenic mice models of AD (P301S and APP/PS1 mice), combining histoblots and immunoelectron microscopic approaches. The histoblot technique revealed differences in the expression of GIRK2 in the two transgenic mice models. The expression of GIRK2 was significantly reduced in the hippocampus of P301S mice in a laminar-specific manner at 10 months of age but was unaltered in APP/PS1 mice at 12 months compared to age-matched wild type mice. Ultrastructural approaches using the pre-embedding immunogold technique, demonstrated that the subcellular localisation of GIRK2 was significantly reduced along the neuronal surface of CA1 pyramidal cells, but increased in its frequency at cytoplasmic sites, in both P301S and APP/PS1 mice. We also found a decrease in plasma membrane GIRK2 channels in axon terminals contacting dendritic spines of CA1 pyramidal cells in P301S and APP/PS1 mice. These data demonstrate for the first time a redistribution of GIRK channels from the plasma membrane to intracellular sites in different compartments of CA1 pyramidal cells. Altogether, the pre- and post-synaptic reduction of GIRK2 channels suggest that GIRK-mediated alteration of the excitability in pyramidal cells could contribute to the cognitive dysfunctions as described in the two AD animal models.
    Keywords Alzheimer´s disease ; hippocampus ; GIRK channels ; immunohistochemistry ; electron microscopy ; histoblot ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Subject code 572
    Language English
    Publishing date 2021-10-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Alteration in the Synaptic and Extrasynaptic Organization of AMPA Receptors in the Hippocampus of P301S Tau Transgenic Mice

    Rocio Alfaro-Ruiz / Carolina Aguado / Alejandro Martín-Belmonte / Ana Esther Moreno-Martínez / Jesús Merchán-Rubira / Félix Hernández / Jesús Ávila / Yugo Fukazawa / Rafael Luján

    International Journal of Molecular Sciences, Vol 23, Iss 13527, p

    2022  Volume 13527

    Abstract: Tau pathology is a hallmark of Alzheimer’s disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR ... ...

    Abstract Tau pathology is a hallmark of Alzheimer’s disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the hippocampus in 10-month-old P301S mice. The labeling density for GluA1-4 in the excitatory synapses established on spines was significantly reduced in P301S mice, compared to age-matched wild-type mice, in the strata radiatum and lacunosum-moleculare but unaltered in the stratum oriens. The density of synaptic GluA1-4 established on interneuron dendrites was significantly reduced in P301S mice in the three strata. The labeling density for GluA1-4 at extrasynaptic sites was significantly reduced in several postsynaptic compartments of CA1 pyramidal cells and interneurons in the three dendritic layers in P301S mice. Our data demonstrate that the progressive accumulation of phospho-tau is associated with alteration of AMPARs on the surface of different neuron types, including synaptic and extrasynaptic membranes, leading to a decline in the trafficking and synaptic transmission, thereby likely contributing to the pathological events taking place in AD.
    Keywords Alzheimer’s disease ; hippocampus ; AMPA receptors ; immunohistochemistry ; electron microscopy ; freeze-fracture ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Subject code 572 ; 571
    Language English
    Publishing date 2022-11-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: The Density of Group I mGlu 5 Receptors Is Reduced along the Neuronal Surface of Hippocampal Cells in a Mouse Model of Alzheimer’s Disease

    Alejandro Martín-Belmonte / Carolina Aguado / Rocío Alfaro-Ruiz / José Luis Albasanz / Mairena Martín / Ana Esther Moreno-Martínez / Yugo Fukazawa / Rafael Luján

    International Journal of Molecular Sciences, Vol 22, Iss 5867, p

    2021  Volume 5867

    Abstract: Metabotropic glutamate receptor subtype 5 (mGlu 5 ) is implicated in the pathophysiology of Alzheimer´s disease (AD). However, its alteration at the subcellular level in neurons is still unexplored. Here, we provide a quantitative description on the ... ...

    Abstract Metabotropic glutamate receptor subtype 5 (mGlu 5 ) is implicated in the pathophysiology of Alzheimer´s disease (AD). However, its alteration at the subcellular level in neurons is still unexplored. Here, we provide a quantitative description on the expression and localisation patterns of mGlu 5 in the APP/PS1 model of AD at 12 months of age, combining immunoblots, histoblots and high-resolution immunoelectron microscopic approaches. Immunoblots revealed that the total amount of mGlu 5 protein in the hippocampus, in addition to downstream molecules, i.e., G q/11 and PLCβ 1 , was similar in both APP/PS1 mice and age-matched wild type mice. Histoblots revealed that mGlu 5 expression in the brain and its laminar expression in the hippocampus was also unaltered. However, the ultrastructural techniques of SDS-FRL and pre-embedding immunogold demonstrated that the subcellular localisation of mGlu 5 was significantly reduced along the neuronal surface of hippocampal principal cells, including CA1 pyramidal cells and DG granule cells, in APP/PS1 mice at 12 months of age. The decrease in the surface localisation of mGlu 5 was accompanied by an increase in its frequency at intracellular sites in the two neuronal populations. Together, these data demonstrate, for the first time, a loss of mGlu 5 at the plasma membrane and accumulation at intracellular sites in different principal cells of the hippocampus in APP/PS1 mice, suggesting an alteration of the excitability and synaptic transmission that could contribute to the cognitive dysfunctions in this AD animal model. Further studies are required to elucidate the specificity of mGlu 5 -associated molecules and downstream signalling pathways in the progression of the pathology.
    Keywords Alzheimer’s disease ; hippocampus ; mGlu receptors ; immunohistochemistry ; electron microscopy ; freeze-fracture ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Subject code 572
    Language English
    Publishing date 2021-05-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Density of GABA B Receptors Is Reduced in Granule Cells of the Hippocampus in a Mouse Model of Alzheimer’s Disease

    Alejandro Martín-Belmonte / Carolina Aguado / Rocío Alfaro-Ruíz / Ana Esther Moreno-Martínez / Luis de la Ossa / José Martínez-Hernández / Alain Buisson / Ryuichi Shigemoto / Yugo Fukazawa / Rafael Luján

    International Journal of Molecular Sciences, Vol 21, Iss 2459, p

    2020  Volume 2459

    Abstract: Metabotropic γ-aminobutyric acid (GABA B ) receptors contribute to the control of network activity and information processing in hippocampal circuits by regulating neuronal excitability and synaptic transmission. The dysfunction in the dentate gyrus (DG) ...

    Abstract Metabotropic γ-aminobutyric acid (GABA B ) receptors contribute to the control of network activity and information processing in hippocampal circuits by regulating neuronal excitability and synaptic transmission. The dysfunction in the dentate gyrus (DG) has been implicated in Alzheimer´s disease (AD). Given the involvement of GABA B receptors in AD, to determine their subcellular localisation and possible alteration in granule cells of the DG in a mouse model of AD at 12 months of age, we used high-resolution immunoelectron microscopic analysis. Immunohistochemistry at the light microscopic level showed that the regional and cellular expression pattern of GABA B1 was similar in an AD model mouse expressing mutated human amyloid precursor protein and presenilin1 (APP/PS1) and in age-matched wild type mice. High-resolution immunoelectron microscopy revealed a distance-dependent gradient of immunolabelling for GABA B receptors, increasing from proximal to distal dendrites in both wild type and APP/PS1 mice. However, the overall density of GABA B receptors at the neuronal surface of these postsynaptic compartments of granule cells was significantly reduced in APP/PS1 mice. Parallel to this reduction in surface receptors, we found a significant increase in GABA B1 at cytoplasmic sites. GABA B receptors were also detected at presynaptic sites in the molecular layer of the DG. We also found a decrease in plasma membrane GABA B receptors in axon terminals contacting dendritic spines of granule cells, which was more pronounced in the outer than in the inner molecular layer. Altogether, our data showing post- and presynaptic reduction in surface GABA B receptors in the DG suggest the alteration of the GABA B -mediated modulation of excitability and synaptic transmission in granule cells, which may contribute to the cognitive dysfunctions in the APP/PS1 model of AD.
    Keywords Alzheimer´s disease ; hippocampus ; GABA B receptors ; ion channels ; immunohistochemistry ; electron microscopy ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Subject code 572
    Language English
    Publishing date 2020-04-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: Membrane palmitoylated protein 2 is a synaptic scaffold protein required for synaptic SK2-containing channel function

    Gukhan Kim / Rafael Luján / Jochen Schwenk / Melissa H Kelley / Carolina Aguado / Masahiko Watanabe / Bernd Fakler / James Maylie / John P Adelman

    eLife, Vol

    2016  Volume 5

    Abstract: Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca2+ influx, the ... ...

    Abstract Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca2+ influx, the synaptic SK2-containing channels modulate excitatory post-synaptic responses and the induction of synaptic plasticity. In addition, their activity- and protein kinase A-dependent trafficking contributes to expression of long-term potentiation (LTP). We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels. MPP2 and SK2 co-immunopurified from mouse brain, and co-immunoprecipitated when they were co-expressed in HEK293 cells. MPP2 is highly expressed in the post-synaptic density of dendritic spines on CA1 pyramidal neurons. Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP. Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels.
    Keywords synaptic transmission ; synaptic plasticity ; synaptic scaffold ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Subject code 571 ; 572
    Language English
    Publishing date 2016-02-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  7. Article ; Online: Transcriptome analysis in tissue sectors with contrasting crocins accumulation provides novel insights into apocarotenoid biosynthesis and regulation during chromoplast biogenesis

    Oussama Ahrazem / Javier Argandoña / Alessia Fiore / Carolina Aguado / Rafael Luján / Ángela Rubio-Moraga / Mónica Marro / Cuauhtémoc Araujo-Andrade / Pablo Loza-Alvarez / Gianfranco Diretto / Lourdes Gómez-Gómez

    Scientific Reports, Vol 8, Iss 1, Pp 1-

    2018  Volume 17

    Abstract: Abstract Crocins, the red soluble apocarotenoids of saffron, accumulate in the flowers of Crocus species in a developmental and tissue-specific manner. In Crocus sieberi, crocins accumulate in stigmas but also in a distinct yellow tepal sector, which we ... ...

    Abstract Abstract Crocins, the red soluble apocarotenoids of saffron, accumulate in the flowers of Crocus species in a developmental and tissue-specific manner. In Crocus sieberi, crocins accumulate in stigmas but also in a distinct yellow tepal sector, which we demonstrate contains chromoplast converted from amyloplasts. Secondary metabolites were analysed by LC-DAD-HRMS, revealing the progressive accumulation of crocetin and crocins in the yellow sector, which were also localized in situ by Raman microspectroscopy. To understand the underlying mechanisms of crocin biosynthesis, we sequenced the C. sieberi tepal transcriptome of two differentially pigmented sectors (yellow and white) at two developmental stages (6 and 8) by Illumina sequencing. A total of 154 million high-quality reads were generated and assembled into 248,099 transcripts. Differentially expressed gene analysis resulted in the identification of several potential candidate genes involved in crocin metabolism and regulation. The results provide a first profile of the molecular events related to the dynamics of crocetin and crocin accumulation during tepal development, and present new information concerning apocarotenoid biosynthesis regulators and their accumulation in Crocus. Further, reveals genes that were previously unknown to affect crocin formation, which could be used to improve crocin accumulation in Crocus plants and the commercial quality of saffron spice.
    Keywords Medicine ; R ; Science ; Q
    Subject code 580
    Language English
    Publishing date 2018-02-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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