Distributed System for Cognitive Stimulation Over Interactive TV.

Descripción y evaluación de sistema de estimulación cognitiva a través de la TDT orientada a personas con enfermedad de Parkinson, con supervisión por parte de sus terapeutas de forma remota. Abstract: This paper details the full design, implementation, and validation of an e-health service in order to improve the community health care services for patients with cognitive disorders. Specifically, the new service allows Parkinson’s disease patients benefit from the possibility of doing cognitive stimulation therapy (CST) at home by using a familiar device such as a TV set. Its use instead of a PC could be a major advantage for some patients whose lack of familiarity with the use of a PC means that they can do therapy only in the presence of a therapist. For these patients this solution could bring about a great improvement in their autonomy. At the same time, this service provides therapists with the ability to conduct follow-up of therapy sessions via the web,benefiting from greater and easier control of the therapy exercises performed by patients and allowing them to customize new exercises in accordance with the particular needs of each patient. As a result, this kind of CST is considered to be a complement of other therapies oriented to the Parkinson patients. Furthermore, with small changes, the system could be useful for patients with a different cognitive disease such as Alzheimer’s or mild cognitive impairment

Histoblot: A sensitive method to quantify the expression of proteins in normal and pathological conditions

The histoblot (in situ immunoblotting) technique is a simple, reproducible, and sensitive method for protein detection that allows both protein quantitation and analysis of tissue distribution. This easy and fast method allows the direct transfer of native proteins from unfixed frozen tissue sections by mechanical pressure to an immobilizing matrix. Proteins are directly blotted onto nitrocellulose membranes that are then immunolabelled similar to a western blot, but the result is an immunohistochemical imprint of the section retaining all proteins. The histoblot combines advantages of western blot and immunohistochemical methods and yields optimal accessibility of proteins blotted on membranes whilst also preserving anatomical resolution. In addition, it avoids chemical modifications, crosslinking, or semi-denaturation of proteins, which can alter the access of antibody to epitopes, as introduced by conventional immunohistochemistry. Therefore, the histoblot often enables the use of antibodies that do not recognise the target protein in fixed tissue samples. This method has become a trusted alternative to reveal and compare the regional distribution and expression profile of different proteins in the brain in physiological and pathological conditions. In addition, the technique exhibits a high subregional resolution, although is not suitable to unravel protein distribution at the cellular and subcellular levels. In this review, we introduce the histoblot procedure used in our laboratory on brain sections for the identification of quantitative changes of neurotransmitter receptors, ion channels and other signalling molecules in the brain. We also discuss the potentialities, limitations, and fundamental principles of this technique

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

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 model

Density of GABAB receptors is reduced in granule cells of the hippocampus in a mouse model of Alzheimer's disease

Metabotropic γ-aminobutyric acid (GABAB) 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 GABAB 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 GABAB1 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 GABAB receptors, increasing from proximal to distal dendrites in both wild type and APP/PS1 mice. However, the overall density of GABAB 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 GABAB1 at cytoplasmic sites. GABAB receptors were also detected at presynaptic sites in the molecular layer of the DG. We also found a decrease in plasma membrane GABAB 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 GABAB receptors in the DG suggest the alteration of the GABAB-mediated modulation of excitability and synaptic transmission in granule cells, which may contribute to the cognitive dysfunctions in the APP/PS1 model of A

Alteration in the Synaptic and Extrasynaptic Organization of AMPA Receptors in the Hippocampus of P301S Tau Transgenic Mice

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

Biohybrids for spinal cord injury repair

This is the peer reviewed version of the following article: Martínez-Ramos, C, Doblado, LR, Mocholi, EL, et al. Biohybrids for spinal cord injury repair. J Tissue Eng Regen Med. 2019; 13: 509-521, which has been published in final form at https://doi.org/10.1002/term.2816. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] Spinal cord injuries (SCIs) result in the loss of sensory and motor function with massive cell death and axon degeneration. We have previously shown that transplantation of spinal cord-derived ependymal progenitor cells (epSPC) significantly improves functional recovery after acute and chronic SCI in experimental models, via neuronal differentiation and trophic glial cell support. Here, we propose an improved procedure based on transplantation of epSPC in a tubular conduit of hyaluronic acid containing poly (lactic acid) fibres creating a biohybrid scaffold. In vitro analysis showed that the poly (lactic acid) fibres included in the conduit induce a preferential neuronal fate of the epSPC rather than glial differentiation, favouring elongation of cellular processes. The safety and efficacy of the biohybrid implantation was evaluated in a complete SCI rat model. The conduits allowed efficient epSPC transfer into the spinal cord, improving the preservation of the neuronal tissue by increasing the presence of neuronal fibres at the injury site and by reducing cavities and cyst formation. The biohybrid-implanted animals presented diminished astrocytic reactivity surrounding the scar area, an increased number of preserved neuronal fibres with a horizontal directional pattern, and enhanced coexpression of the growth cone marker GAP43. The biohybrids offer an improved method for cell transplantation with potential capabilities for neuronal tissue regeneration, opening a promising avenue for cell therapies and SCI treatment.Secretaria de Estado de Investigacion, Desarrollo e Innovacion, Grant/Award Number: MAT2015-66666-C3-1-R MINECO/FEDER MAT2015-66666-C3-2-R MINECO/FEDER; Spanish Ministry of Education, Culture and Sports through Laura Rodriguez Doblado, Grant/Award Number: FPU15/04975Martínez-Ramos, C.; Rodriguez Doblado, L.; López Mocholi, E.; Alastrue-Agudo, A.; Sánchez Petidier, M.; Giraldo-Reboloso, E.; Monleón Pradas, M.... (2019). Biohybrids for spinal cord injury repair. Journal of Tissue Engineering and Regenerative Medicine. 13(3):509-521. https://doi.org/10.1002/term.2816S509521133Ahuja, C. S., & Fehlings, M. (2016). Concise Review: Bridging the Gap: Novel Neuroregenerative and Neuroprotective Strategies in Spinal Cord Injury. 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Different modes of synaptic and extrasynaptic NMDA receptor alteration in the hippocampus of P301S tau transgenic mice

N-methyl-d-aspartate receptors (NMDARs) are pivotal players in the synaptic transmission and synaptic plasticity underlying learning and memory. Accordingly, dysfunction of NMDARs has been implicated in the pathophysiology of Alzheimer disease (AD). Here, we used histoblot and sodium dodecylsulphate-digested freeze-fracture replica labelling (SDS-FRL) techniques to investigate the expression and subcellular localisation of GluN1, the obligatory subunit of NMDARs, in the hippocampus of P301S mice. Histoblots showed that GluN1 expression was significantly reduced in the hippocampus of P301S mice in a laminar-specific manner at 10 months of age but was unaltered at 3 months. Using the SDS-FRL technique, excitatory synapses and extrasynaptic sites on spines of pyramidal cells and interneuron dendrites were analysed throughout all dendritic layers in the CA1 field. Our ultrastructural approach revealed a high density of GluN1 in synaptic sites and a substantially lower density at extrasynaptic sites. Labelling density for GluN1 in excitatory synapses established on spines was significantly reduced in P301S mice, compared with age-matched wild-type mice, in the stratum oriens (so), stratum radiatum (sr) and stratum lacunosum-moleculare (slm). Density for synaptic GluN1 on interneuron dendrites was significantly reduced in P301S mice in the so and sr but unaltered in the slm. Labelling density for GluN1 at extrasynaptic sites showed no significant differences in pyramidal cells, and only increased density in the interneuron dendrites of the sr. This differential alteration of synaptic versus extrasynaptic NMDARs supports the notion that the progressive accumulation of phospho-tau is associated with changes in NMDARs, in the absence of amyloid-β pathology, and may be involved in the mechanisms causing abnormal network activity of the hippocampal circui

Reduction in the neuronal surface of post and presynaptic GABA>B< receptors in the hippocampus in a mouse model of Alzheimer's disease

The hippocampus plays key roles in learning and memory and is a main target of Alzheimer's disease (AD), which causes progressive memory impairments. Despite numerous investigations about the processes required for the normal hippocampal functions, the neurotransmitter receptors involved in the synaptic deficits by which AD disables the hippocampus are not yet characterized. By combining histoblots, western blots, immunohistochemistry and high‐resolution immunoelectron microscopic methods for GABAB receptors, this study provides a quantitative description of the expression and the subcellular localization of GABAB1 in the hippocampus in a mouse model of AD at 1, 6 and 12 months of age. Western blots and histoblots showed that the total amount of protein and the laminar expression pattern of GABAB1 were similar in APP/PS1 mice and in age‐matched wild‐type mice. In contrast, immunoelectron microscopic techniques showed that the subcellular localization of GABAB1 subunit did not change significantly in APP/PS1 mice at 1 month of age, was significantly reduced in the stratum lacunosum‐moleculare of CA1 pyramidal cells at 6 months of age and significantly reduced at the membrane surface of CA1 pyramidal cells at 12 months of age. This reduction of plasma membrane GABAB1 was paralleled by a significant increase of the subunit at the intracellular sites. We further observed a decrease of membrane‐targeted GABAB receptors in axon terminals contacting CA1 pyramidal cells. Our data demonstrate compartment‐ and age‐dependent reduction of plasma membrane‐targeted GABAB receptors in the CA1 region of the hippocampus, suggesting that this decrease might be enough to alter the GABAB‐mediated synaptic transmission taking place in AD

High Biofilm Formation of Non-Smooth Candida parapsilosis Correlates with Increased Incorporation of GPI-Modified Wall Adhesins

Candida parapsilosis is among the most frequent causes of candidiasis. Clinical isolates of this species show large variations in colony morphotype, ranging from round and smooth to a variety of non-smooth irregular colony shapes. A non-smooth appearance is related to increased formation of pseudohyphae, higher capacity to form biofilms on abiotic surfaces, and invading agar. Here, we present a comprehensive study of the cell wall proteome of C. parapsilosis reference strain CDC317 and seven clinical isolates under planktonic and sessile conditions. This analysis resulted in the identification of 40 wall proteins, most of them homologs of known Candida albicans cell wall proteins, such as Gas, Crh, Bgl2, Cht2, Ecm33, Sap, Sod, Plb, Pir, Pga30, Pga59, and adhesin family members. Comparative analysis of exponentially growing and stationary phase planktonic cultures of CDC317 at 30 °C and 37 °C revealed only minor variations. However, comparison of smooth isolates to non-smooth isolates with high biofilm formation capacity showed an increase in abundance and diversity of putative wall adhesins from Als, Iff/Hyr, and Hwp families in the latter. This difference depended more strongly on strain phenotype than on the growth conditions, as it was observed in planktonic as well as biofilm cells. Thus, in the set of isolates analyzed, the high biofilm formation capacity of non-smooth C. parapsilosis isolates with elongated cellular phenotypes correlates with the increased surface expression of putative wall adhesins in accordance with their proposed cellular function.This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2013-47570-P and SAF2017-86188-P) and the regional government of Castilla-La Mancha (JCCM) (SBPLY/19/180501/000114), all co-financed by the EU (FEDER), to P.W.J.d.G. and E.E., the Consejería de Educación, Universidades e Investigación (GIC15/78 IT-990-16) of Gobierno Vasco-Eusko Jaurlaritza to E.E., and the FP7-PEOPLE-2013-ITN—Marie-Curie Action: “Initial Training Networks”: Molecular Mechanisms of Human Fungal Pathogen Host Interaction, ImResFun, MC-ITN-606786, to O.B