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

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

Amyloid-driven tau accumulation on mitochondria potentially leads to cognitive deterioration in alzheimer’s disease

Despite the well-accepted role of the two main neuropathological markers (β-amyloid and tau) in the progression of Alzheimer’s disease, the interaction and specific contribution of each of them is not fully elucidated. To address this question, in the present study, an adeno-associated virus (AAV9) carrying the mutant P301L form of human tau, was injected into the dorsal hippocampi of APP/PS1 transgenic mice or wild type mice (WT). Three months after injections, memory tasks, biochemical and immunohistochemical analysis were performed. We found that the overexpression of hTauP301L accelerates memory deficits in APP/PS1 mice, but it did not affect memory function of WT mice. Likewise, biochemical assays showed that only in the case of APP/PS1-hTauP301L injected mice, an important accumulation of tau was observed in the insoluble urea fraction. Similarly, electron microscopy images revealed that numerous clusters of tau immunoparticles appear at the dendrites of APP/PS1 injected mice and not in WT animals, suggesting that the presence of amyloid is necessary to induce tau aggregation. Interestingly, these tau immunoparticles accumulate in dendritic mitochondria in the APP/PS1 mice, whereas most of mitochondria in WT injected mice remain free of tau immunoparticles. Taken together, it seems that amyloid induces tau aggregation and accumulation in the dendritic mitochondria and subsequently may alter synapse function, thus, contributing to accelerate cognitive decline in APP/PS1 mice.We gratefully acknowledge grant funding support from Ministry of Science and Innovation (MINECO) with exp. PID2019-104921RB-I00/MCI/AEI/10.13039/501100011033 as well as to the Foundation for Applied Medical Research, the University of Navarra (Pamplona, Spain) for financial support and the Asociación de Amigos of the University of Navarra for the grant to M.P.-G. and S.B. We also gratefully acknowledge grant funding support from Spanish Ministerio de Economía y Competitividad (RTI2018-095812-B-I00 MCIN/ AEI/10.13039/501100011033) y por FEDER una manera de hacer Europa and Junta de Comunidades de Castilla-La Mancha (SBPLY/17/180501/000229) to RL, from the Spanish Ministry of Science Innovation and Universities (Ref. PID2019-110356RB-I00/AEI/10.13039/501100011033) to J.F.-I. and E.S and from the Department of Economic and Business Development from Government of Navarra (INNOLFACT project; Ref. 0011-1411-2020-000028)

Nanoscale alterations in GABAB receptors and GIRK channel organization on the hippocampus of APP/PS1 mice

Alzheimer's disease (AD) is characterized by a reorganization of brain activity determining network hyperexcitability and loss of synaptic plasticity. Precisely, a dysfunction in metabotropic GABA(B) receptor signalling through G protein-gated inwardly rectifying K+ (GIRK or Kir3) channels on the hippocampus has been postulated. Thus, we determined the impact of amyloid-beta (A beta) pathology in GIRK channel density, subcellular distribution, and its association with GABA(B) receptors in hippocampal CA1 pyramidal neurons from the APP/PS1 mouse model using quantitative SDS-digested freeze-fracture replica labelling (SDS-FRL) and proximity ligation in situ assay (P-LISA). In wild type mice, single SDS-FRL detection revealed a similar dendritic gradient for GIRK1 and GIRK2 in CA1 pyramidal cells, with higher densities in spines, and GIRK3 showed a lower and uniform distribution. Double SDS-FRL showed a co-clustering of GIRK2 and GIRK1 in post- and presynaptic compartments, but not for GIRK2 and GIRK3. Likewise, double GABA(B1) and GIRK2 SDS-FRL detection displayed a high degree of co-clustering in nanodomains (40-50 nm) mostly in spines and axon terminals. In APP/PS1 mice, the density of GIRK2 and GIRK1, but not for GIRK3, was significantly reduced along the neuronal surface of CA1 pyramidal cells and in axon terminals contacting them. Importantly, GABA(B1) and GIRK2 co-clustering was not present in APP/PS1 mice. Similarly, P-LISA experiments revealed a significant reduction in GABA(B1) and GIRK2 interaction on the hippocampus of this animal model. Overall, our results provide compelling evidence showing a significant reduction on the cell surface density of pre- and postsynaptic GIRK1 and GIRK2, but not GIRK3, and a decline in GABA(B) receptors and GIRK2 channels co-clustering in hippocampal pyramidal neurons from APP/PS1 mice, thus suggesting that a disruption in the GABA(B) receptor-GIRK channel membrane assembly causes dysregulation in the GABA(B) signalling via GIRK channels in this AD animal model

Cisplatin resistance involves a metabolic reprogramming through ROS and PGC-1α in NSCLC which can be overcome by OXPHOS inhibition

Background: Platinum-based chemotherapy remains the standard of care for most lung cancer cases. However chemoresistance is often developed during the treatment, limiting clinical utility of this drug. Recently, the ability of tumor cells to adapt their metabolism has been associated to resistance to therapies. In this study, we first described the metabolic reprogramming of Non-Small Cell Lung Cancer (NSCLC) in response to cisplatin treatment. Methods: Cisplatin-resistant versions of the A549, H1299, and H460 cell lines were generated by continuous drug exposure. The long-term metabolic changes, as well as, the early response to cisplatin treatment were analyzed in both, parental and cisplatin-resistant cell lines. In addition, four Patient-derived xenograft models treated with cisplatin along with paired pre- and post-treatment biopsies from patients were studied. Furthermore, metabolic targeting of these changes in cell lines was performed downregulating PGC-1α expression through siRNA or using OXPHOS inhibitors (metformin and rotenone). Results: Two out of three cisplatin-resistant cell lines showed a stable increase in mitochondrial function, PGC1-α and mitochondrial mass with reduced glycolisis, that did not affect the cell cycle. This phenomenon was confirmed in vivo. Post-treatment NSCLC tumors showed an increase in mitochondrial mass, PGC-1α and a decrease in the GAPDH/MT-CO1 ratio. In addition, we demonstrated how a ROS-mediated metabolism reprogramming, involving PGC-1α and increased mitochondrial mass, is induced during short-time cisplatin exposure. Moreover, we tested how cells with increased PGC-1a induced by ZLN005 treatment, showed reduced cisplatin-driven apoptosis. Remarkably, the long-term metabolic changes, as well as the metabolic reprogramming during short-time cisplatin exposure can be exploited as an Achilles’ heel of NSCLC cells, as demonstrated by the increased sensitivity to PGC-1α interference or OXPHOS inhibition using metformin or rotenone. Conclusion: These results describe a new cisplatin resistance mechanism in NSCLC based on a metabolic reprogramming that is therapeutically exploitable through PGC-1α downregulation or OXPHOS inhibitors.Work in the authors’ laboratories is supported by ‘‘Instituto de Salud Carlos III’’ PI13/01806 and PIE14/0064 to M.P. A.C-B, received a Spanish Lung Cancer Group fellowship. R.L-B, is supported by Comunidad Autónoma de Madrid “Garantía juvenil” contrac

SK2 Channels Associate With mGlu1α Receptors and CaV2.1 Channels in Purkinje Cells

The small-conductance, Ca2+-activated K+ (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca2+ transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cerebellar PCs using SDS-digested freeze-fracture replica labeling (SDS-FRL) of high sensitivity combined with quantitative analyses. Immunogold particles for SK2 were observed on post- and pre-synaptic compartments showing both scattered and clustered distribution patterns. We found an axo-somato-dendritic gradient of the SK2 particle density increasing 12-fold from soma to dendritic spines. Using two different immunogold approaches, we also found that SK2 immunoparticles were frequently adjacent to, but never overlap with, the postsynaptic density of excitatory synapses in PC spines. Co-immunoprecipitation analysis demonstrated that SK2 channels form macromolecular complexes with two types of proteins that mobilize Ca2+: CaV2.1 channels and mGlu1α receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for CaV2.1 channels and mGlu1α receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to CaV2.1 channels, though they are not significantly co-clustered. These data demonstrate that SK2 channels located in different neuronal compartments can associate with distinct proteins mobilizing Ca2+, and suggest that the ultrastructural association of SK2 with CaV2.1 and mGlu1α provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca2+ transients in PCs

Guía de práctica clínica para el diagnóstico y manejo inicial de artritis reumatoide en el Seguro Social del Perú (EsSalud)

Background: Rheumatoid Arthritis (RA) has adverse health consequences, its early diagnosis and optimal management require recommendations based on high quality evidence adapted to each health system. Objective: To provide evidence-based clinical recommendations for the diagnosis and initial management of RA. Material and Methods: A Guideline Development Group (GDG) was established, including medical specialists and methodologists. The GDG formulated 10 clinical questions to be answered by this CPG. Systematic searches of published evidence in PubMed and CENTRAL were performed during December 2017 and July 2019 (including systematic reviews and -when it was considered pertinent- primary studies) were conducted. The evidence of the highest quality for answering each of the posed clinical questions was selected. The quality of the evidence was evaluated using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) methodology which was used in periodic work meetings by the GDG to review the evidence and formulate the recommendations, points of good clinical practice, and flowcharts. Finally, the CPG was approved with Resolution N° 132 – IETSI – ESSALUD – 2019. Results: This CPG addressed 10 clinical questions, divided into two topics: diagnosis and management. Based on these questions, 16 recommendations (5 strong and 11 weak), 28 points of good clinical practice, and 3 flowcharts were formulated. Conclusion: This article summarizes the methodology and evidence-based conclusions from the CPG for the diagnosis and initial management of RA in EsSalud.Introducción: La Artritis Reumatoide (AR) tiene adversas consecuencias en la salud su diagnóstico temprano y manejo óptimo requiere recomendaciones basadas en evidencia de alta calidad adaptadas a cada sistema de salud. Objetivo: Proveer recomendaciones clínicas basadas en evidencia para el diagnóstico y manejo inicial de la AR. Material y Métodos: Se conformó un grupo elaborador de la guía (GEG) que incluyó médicos especialistas y metodólogos. El GEG formuló 10 preguntas clínicas a ser respondidas por la presente GPC. Se realizaron búsquedas sistemáticas de la evidencia publicada en PubMed y CENTRAL entre diciembre 2017 a julio 2019 (revisiones sistemáticas y –cuando fue considerado pertinente– estudios primarios). Se seleccionó la evidencia de mayor calidad para responder cada una de las preguntas clínicas planteadas. La certeza de la evidencia fue evaluada usando la metodología Grading of Recommendations Assessment, Development, and Evaluation (GRADE) y en reuniones de trabajo periódicas, el GEG usó dicha metodología para revisar la evidencia y formular las recomendaciones, los puntos de buena práctica clínica y los flujogramas. Finalmente, la GPC fue aprobada con Resolución N° 132 – IETSI – ESSALUD – 2019. Resultados: La presente GPC abordó 10 preguntas clínicas, divididas en dos temas: diagnóstico y manejo. En base a dichas preguntas se formularon 16 recomendaciones (5 fuertes y 11 condicionales), 45 puntos de buena práctica clínica, y 3 flujogramas. Conclusión: El presente artículo resume la metodología y las conclusiones basadas en evidencias de la GPC para el diagnóstico y manejo inicial de la AR en EsSalud

Long daytime napping is associated with increased adiposity and type 2 diabetes in an elderly population with metabolic syndrome

Research examining associations between objectively-measured napping time and type 2 diabetes (T2D) is lacking. This study aimed to evaluate daytime napping in relation to T2D and adiposity measures in elderly individuals from the Mediterranean region. A cross-sectional analysis of baseline data from 2190 elderly participants with overweight/obesity and metabolic syndrome, in the PREDIMED-Plus trial, was carried out. Accelerometer-derived napping was measured. Prevalence ratios (PR) and 95% confidence intervals (CI) for T2D were obtained using multivariable-adjusted Cox regression with constant time. Linear regression models were fitted to examine associations of napping with body mass index (BMI) and waist circumference (WC). Participants napping ≥90 min had a higher prevalence of T2D (PR 1.37 (1.06, 1.78)) compared with those napping 5 to <30 min per day. Significant positive associations with BMI and WC were found in those participants napping ≥30 min as compared to those napping 5 to <30 min per day. The findings of this study suggest that longer daytime napping is associated with higher T2D prevalence and greater adiposity measures in an elderly Spanish population at high cardiovascular risk

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality