Open Access
Amyloid-driven tau accumulation on mitochondria potentially leads to cognitive deterioration in Alzheimer’s disease
(MDPI, 2021) Cuadrado-Tejedor, Mar; Pérez-González, Marta; Alfaro-Ruiz, Rocío; Badesso, Sara; Sucunza, Diego; Espelosín, María; Ursúa, Susana; Lachén Montes, Mercedes; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Luján, Rafael; García-Osta, Ana; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua
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.
Open Access
Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis
(Elsevier, 2022) Colomé, Núria; Abian, Joaquín; Aloria, Kerman; Arizmendi, Jesús M.; Barceló-Batllori, Silvia; Braga-Lagache, Sophie; Burlet-Schiltz, Odile; Carrascal, Montse; Casal, Ignacio J.; Chicano-Gálvez, Eduard; Chiva, Cristina; Clemente, Luis F.; Elortza, Félix; Estanyol, Josep M.; Fernández Irigoyen, Joaquín; Fernández-Puente, Patricia; Fidalgo, María J.; Froment, Carine; Fuentes, Manuel; Fuentes-Almagro, Carlos; Gay, Marina; Hainard, Alexandre; Heller, Manfred; Hernández, María Luisa; Ibarrola, Nieves; Iloro, Ibon; Kieselbach, Thomas; Lario, Antonio; Locard-Paulet, Marie; Marina-Ramírez, Anabel; Martín, Luna; Morato-López, Esperanza; Muñoz, Javier; Navajas, Rosana; Odena, Antonia M.; Odriozola, Leticia; Oliveira, Eliandre de; Paradela, Alberto; Pasquarello, Carla; Rios, Vivian de los; Ruiz-Romero, Cristina; Sabidó, Eduard; Sánchez del Pino, Manuel; Sancho, Jaime; Santamaría Martínez, Enrique; Schaeffer-Reiss, Christine; Schneider, Justine; Torre, Carolina de la; Valero, Luz M.; Vilaseca, Marta; Wu, Shuai; Wu, Linfeng; Ximénez de Embún, Pilar; Canals, Francesc; Corrales, Fernando J.; ProteoRed-ISCIII; EuPA; Ciencias de la Salud; Osasun Zientziak
Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.
Open Access
New in vivo approach to broaden the thioredoxin family interactome in chloroplasts
(MDPI, 2022) Ancín Rípodas, María; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Larraya Reta, Luis María; Fernández San Millán, Alicia; Veramendi Charola, Jon; Farrán Blanch, Inmaculada; Ciencias de la Salud; Osasun Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMAB
Post-translational redox modifications provide an important mechanism for the control of major cellular processes. Thioredoxins (Trxs), which are key actors in this regulatory mechanism, are ubiquitous proteins that catalyse thiol-disulfide exchange reactions. In chloroplasts, Trx f, Trx m and NADPH-dependent Trx reductase C (NTRC) have been identified as transmitters of the redox signal by transferring electrons to downstream target enzymes. The number of characterised Trx targets has greatly increased in the last few years, but most of them were determined using in vitro procedures lacking isoform specificity. With this background, we have developed a new in vivo approach based on the overexpression of His-tagged single-cysteine mutants of Trx f, Trx m or NTRC into Nicotiana benthamiana plants. The over-expressed mutated Trxs, capable of forming a stable mixed disulfide bond with target proteins in plants, were immobilised on affinity columns packed with Ni-NTA agarose, and the covalently linked targets were eluted with dithiothreitol and identified by mass spectrometry-based proteomics. The in vivo approach allowed identification of 6, 9 and 42 new potential targets for Trx f, Trx m and NTRC, respectively, and an apparent specificity between NTRC and Trxs was achieved. Functional analysis showed that these targets are involved in several cellular processes.
Open Access
Maraviroc prevents hcc development by suppressing macrophages and the liver progenitor cell response in a murine chronic liver disease model
(MDPI, 2021) Passman, Adam M.; Strauss, Robyn P.; McSpadden, Sarah B.; Finch-Edmondson, Megan; Andrewartha, Neil; Woo, Ken H.; Diepeveen, Luke A.; Zhao, Weihao; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Medina-Ruiz, Laura; Szpakowska, Martyna; Chevigné, Andy; Park, Hyerin; Carlessi, Rodrigo; Tirnitz-Parker, Janina; Blanco, José R.; London, Roslyn; Callus, Bernard A.; Elsegood, Caryn L.; Baker, Murray V.; Martínez, Alfredo; Yeoh, George C.T.; Ochoa-Callejero, Laura; Ciencias de la Salud; Osasun Zientziak
Maraviroc (MVC), a CCR5 antagonist, reduces liver fibrosis, injury and tumour burden in mice fed a hepatocarcinogenic diet, suggesting it has potential as a cancer therapeutic. We investigated the effect of MVC on liver progenitor cells (LPCs) and macrophages as both have a role in hepatocarcinogenesis. Mice were fed the hepatocarcinogenic choline-deficient, ethionine-supple-mented diet (CDE) ± MVC, and immunohistochemistry, RNA and protein expression were used to determine LPC and macrophage abundance, migration and related molecular mechanisms. MVC reduced LPC numbers in CDE mice by 54%, with a smaller reduction seen in macrophages. Tran-script and protein abundance of LPC-associated markers correlated with this reduction. The CDE diet activated phosphorylation of AKT and STAT3 and was inhibited by MVC. LPCs did not express Ccr5 in our model; in contrast, macrophages expressed high levels of this receptor, suggesting the effect of MVC is mediated by targeting macrophages. MVC reduced CD45+ cells and macrophage migration in liver and blocked the CDE-induced transition of liver macrophages from an M1-to M2-tumour-associated macrophage (TAM) phenotype. These findings suggest MVC has potential as a re-purposed therapeutic agent for treating chronic liver diseases where M2-TAM and LPC numbers are increased, and the incidence of HCC is enhanced.
Open Access
Successful biocontrol of Pichia spp. strains against Botrytis cinerea infection in apple fruit: unraveling protection mechanisms from proteomic insights
(Elseiver, 2024-05-25) Fernández San Millán, Alicia; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Larraya Reta, Luis María; Ancín Rípodas, María; Farrán Blanch, Inmaculada; Veramendi Charola, Jon; Institute for Multidisciplinary Research in Applied Biology - IMAB; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Botrytis cinerea causes major crop losses, especially under postharvest conditions. We have found that Pichia fermentans Pf-31 and Pichia terricola Pt-36 are two promising yeast strains that are able to efficiently control B. cinerea infection in apples. This effect is most pronounced when the yeasts are applied as live cells, although dead cells or culture filtrates also show some degree of control. Both strains arrest spore germination, inhibit mycelial growth, strongly attach to hyphae and promote their own proliferation in the fruit when B. cinerea is present, probably due to preferential colonization of apple wounds. Indeed, this metabolism enhancement was corroborated by a proteomic analysis, which revealed the differentially accumulated yeast proteins that contribute towards this antagonistic behavior. Besides the boost in proteins involved in energetic metabolism, other changes in proteins related to cell envelope composition are implicated in the biocontrol abilities of both strains, and this might be to facilitate hyphal adhesion or biofilm formation. The results of this study are of great value because they promote a deep understanding of the proteins that undergo changes during yeast antagonistic interactions, but also because they provide new insights into the proteomes of non-Saccharomyces yeasts, which have not been previously described.
Open Access
Progressive modulation of the human olfactory bulb transcriptome during Alzheimer´s disease evolution: novel insights into the olfactory signaling across proteinopathies
(Impact Journals, 2017) Lachén Montes, Mercedes; Zelaya Huerta, María Victoria; Segura, Víctor; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua, PC025; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Alzheimer´s disease (AD) is characterized by progressive dementia, initially presenting olfactory dysfunction. Despite the olfactory bulb (OB) is the first central structure of the olfactory pathway, we lack a complete molecular characterization of the transcriptional events that occurs in this olfactory area during AD progression. To address this gap in knowledge, we have assessed the genome-wide expression in postmortem OBs from subjects with varying degree of AD pathology. A stagedependent deregulation of specific pathways was observed, revealing transmembrane transport, and neuroinflammation as part of the functional modules that are disrupted across AD grading. Potential drivers of neurodegeneration predicted by networkdriven transcriptomics were monitored across different types of dementia, including progressive supranuclear palsy (PSP), mixed dementia, and frontotemporal lobar degeneration (FTLD). Epidermal growth factor receptor (EGFR) expression was significantly increased in the OB of AD and mixed dementia subjects. Moreover, a significant increment in the activation of signal transducer and activator of transcription 3 (STAT3) was exclusively detected in advanced AD stages, whereas total STAT3 levels were specifically overexpressed in mixed dementia. Furthermore, transcription factors deregulated in the OB of mixed dementia subjects such as cAMP Responsive Element Binding Protein 1 (CREB1) and AP-1 Transcription Factor Subunit (c-Jun) were not differentially modulated at olfactory level across AD grading. On the other hand, olfactory expression of this signal transducer panel was unchanged in PSP and FTLD subjects. Taken together, this study unveils cross-disease similarities and differences for specific signal transducers, providing mechanistic clues to the intriguing divergence of AD pathology across proteinopathies.
Open Access
Striatal synaptic bioenergetic and autophagic decline in premotor experimental parkinsonism
(Oxford University Press, 2022) Merino Galán, Leyre; Jiménez Urbieta, Haritz; Zamarbide, Marta; Rodríguez Chinchilla, Tatiana; Belloso Iguerategui, Arantzazu; Santamaría Martínez, Enrique; Fernández Irigoyen, Joaquín; Aiastui, Ana; Doudnikoff, Evelyne.; Bézard, Erwan; Ouro, Alberto; Knafo, Shira; Gago, Belén; Quiroga Varela, Ana; Rodríguez Oroz, María Cruz; Ciencias de la Salud; Osasun Zientziak
Synaptic impairment might precede neuronal degeneration in Parkinson’s disease. However, the intimate mechanisms altering synaptic function by the accumulation of presynaptic α-synuclein in striatal dopaminergic terminals before dopaminergic death occurs, have not been elucidated. Our aim is to unravel the sequence of synaptic functional and structural changes preceding symptomatic dopaminergic cell death. As such, we evaluated the temporal sequence of functional and structural changes at striatal synapses before parkinsonian motor features appear in a rat model of progressive dopaminergic death induced by overexpression of the human mutated A53T α-synuclein in the substantia nigra pars compacta, a protein transported to these synapses. Sequential window acquisition of all theoretical mass spectra proteomics identified deregulated proteins involved first in energy metabolism and later, in vesicle cycling and autophagy. After protein deregulation and when α-synuclein accumulated at striatal synapses, alterations to mitochondrial bioenergetics were observed using a Seahorse XF96 analyser. Sustained dysfunctional mitochondrial bioenergetics was followed by a decrease in the number of dopaminergic terminals, morphological and ultrastructural alterations, and an abnormal accumulation of autophagic/endocytic vesicles inside the remaining dopaminergic fibres was evident by electron microscopy. The total mitochondrial population remained unchanged whereas the number of ultrastructurally damaged mitochondria increases as the pathological process evolved. We also observed ultrastructural signs of plasticity within glutamatergic synapses before the expression of motor abnormalities, such as a reduction in axospinous synapses and an increase in perforated postsynaptic densities. Overall, we found that a synaptic energetic failure and accumulation of dysfunctional organelles occur sequentially at the dopaminergic terminals as the earliest events preceding structural changes and cell death. We also identify key proteins involved in these earliest functional abnormalities that may be modulated and serve as therapeutic targets to counterbalance the degeneration of dopaminergic cells to delay or prevent the development of Parkinson’s disease.
Open Access
Ultrahigh sensitive detection of tau protein as Alzheimer's biomarker via microfluidics and nanofunctionalized optical fiber sensors
(Wiley, 2022) Chiavaioli, Francesco; Santano Rivero, Desiree; Del Villar, Ignacio; Socorro Leránoz, Abián Bentor; Zhang, Xuejun; Li, Kaiwei; Santamaría Martínez, Enrique; Fernández Irigoyen, Joaquín; Baldini, Francesco; Van den Hove, Daniel L. A.; Shi, Lei; Bi, Wei; Guo, Tuan; Giannetti, Ambra; Matías Maestro, Ignacio; Institute of Smart Cities - ISC; Ingeniería Eléctrica y Electrónica; Ingeniaritza Elektrikoa eta Elektronikoa; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Alzheimer’s disease (AD) is one of the most common neurodegenerative illnesses displaying the highest death rate in the elderly. However, the existing AD diagnostic system remains elusive due to lack of a technology that may ensure enough sensitivity and reproducibility, detection accuracy, and specificity. Herein, a straightforward approach is reported to realize lab-on-fiber (LoF) technology for AD biomarker detection based on a D-shaped single-mode fiber combined with nanometer-scale metal-oxide film. The proposed sensing system, which permits the generation of lossy-mode resonance (LMR), remarkably increases the evanescent field of light guided through the fiber, and hence the fiber-surrounding medium interaction. Moreover, such optical sensors are highly repeatable in results and can safely be embedded into a compact and stable microfluidic system. Herein, the specific detection of Tau protein (as one of the classical AD biomarkers that is highly correlated with AD progression) in a complex biofluid with a detection limit of 10 12 M and over a wide concentration range (10 3 –10 μg mL 1 ) is successfully demonstrated. The proposed LoF biosensor is an appealing solution for rapid, sub-microliter dose and highly sensitive detection of analytes at low concentrations, hereby having the potential toward early screening and personalized medicine in AD.
Open Access
Effect of immunology biomarkers associated with hip fracture and fracture risk in older adults
(BMC, 2023) Cedeño Veloz, Bernardo Abel; Lozano Vicario, Lucía; Zambom Ferraresi, Fabrício; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Rodríguez-García, Alba; Romero Ortuno, Román; Mondragón Rubio, Jaime; Ruiz-Ruiz, Javier; Ramírez Vélez, Robinson; Izquierdo Redín, Mikel; Martínez Velilla, Nicolás; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Osteoporosis is a skeletal disease that can increase the risk of fractures, leading to adverse health and socioeconomic consequences. However, current clinical methods have limitations in accurately estimating fracture risk, particularly in older adults. Thus, new technologies are necessary to improve the accuracy of fracture risk estimation. In this observational study, we aimed to explore the association between serum cytokines and hip fracture status in older adults, and their associations with fracture risk using the FRAX reference tool. We investigated the use of a proximity extension assay (PEA) with Olink. We compared the characteristics of the population, functional status and detailed body composition (determined using densitometry) between groups. We enrolled 40 participants, including 20 with hip fracture and 20 without fracture, and studied 46 cytokines in their serum. After conducting a score plot and two unpaired t-tests using the Benjamini-Hochberg method, we found that Interleukin 6 (IL-6), Lymphotoxin-alpha (LT-α), Fms-related tyrosine kinase 3 ligand (FLT3LG), Colony stimulating factor 1 (CSF1), and Chemokine (C-C motif) ligand 7 (CCL7) were significantly different between fracture and non-fracture patients (p < 0.05). IL-6 had a moderate correlation with FRAX (R2 = 0.409, p < 0.001), while CSF1 and CCL7 had weak correlations with FRAX. LT-α and FLT3LG exhibited a negative correlation with the risk of fracture. Our results suggest that targeted proteomic tools have the capability to identify differentially regulated proteins and may serve as potential markers for estimating fracture risk. However, longitudinal studies will be necessary to validate these results and determine the temporal patterns of changes in cytokine profiles.
Open Access
Amyotrophic lateral sclerosis is accompanied by protein derangements in the olfactory bulb-tract axis
(MDPI, 2020) Lachén Montes, Mercedes; Mendizuri, Naroa; Ausín, Karina; Andrés Benito, Pol; Ferrer, Isidro; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua, Ref. 0011-1411-2020-000028
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by progressive muscle paralysis due to the degeneration of upper and lower motor neurons. Recent studies point out an involvement of the non-motor axis during disease progression. Despite smell impairment being considered a potential non-motor finding in ALS, the pathobiochemistry at the olfactory level remains unknown. Here, we applied an olfactory quantitative proteotyping approach to analyze the magnitude of the olfactory bulb (OB) proteostatic imbalance in ALS subjects (n = 12) with respect to controls (n = 8). Around 3% of the quantified OB proteome was differentially expressed, pinpointing aberrant protein expression involved in vesicle-mediated transport, macroautophagy, axon development and gliogenesis in ALS subjects. The overproduction of olfactory marker protein (OMP) points out an imbalance in the olfactory signal transduction in ALS. Accompanying the specific overexpression of glial fibrillary acidic protein (GFAP) and Bcl-xL in the olfactory tract (OT), a tangled disruption of signaling routes was evidenced across the OB–OT axis in ALS. In particular, the OB survival signaling dynamics clearly differ between ALS and frontotemporal lobar degeneration (FTLD), two faces of TDP-43 proteinopathy. To the best of our knowledge, this is the first report on high-throughput molecular characterization of the olfactory proteostasis in ALS.