Open Access
Network-driven proteogenomics unveils an aging-related imbalance in the olfactory IκBα-NFκB p65 complex functionality in Tg2576 Alzheimer’s disease mouse model
(MDPI, 2017) Palomino Alonso, Maialen; Lachén Montes, Mercedes; González Morales, Andrea; Ausín, Karina; Pérez Mediavilla, Alberto; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua, PC023-24; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Olfaction is often deregulated in Alzheimer’s disease (AD) patients, and is also impaired in transgenic Tg2576 AD mice, which overexpress the Swedish mutated form of human amyloid precursor protein (APP). However, little is known about the molecular mechanisms that accompany the neurodegeneration of olfactory structures in aged Tg2576 mice. For that, we have applied proteome- and transcriptome-wide approaches to probe molecular disturbances in the olfactory bulb (OB) dissected from aged Tg2576 mice (18 months of age) as compared to those of age matched wild-type (WT) littermates. Some over-represented biological functions were directly relevant to neuronal homeostasis and processes of learning, cognition, and behavior. In addition to the modulation of CAMP responsive element binding protein 1 (CREB1) and APP interactomes, an imbalance in the functionality of the IκBα-NFκB p65 complex was observed during the aging process in the OB of Tg2576 mice. At two months of age, the phosphorylated isoforms of olfactory IκBα and NFκB p65 were inversely regulated in transgenic mice. However, both phosphorylated proteins were increased at 6 months of age, while a specific drop in IκBα levels was detected in 18-month-old Tg2576 mice, suggesting a transient activation of NFκB in the OB of Tg2576 mice. Taken together, our data provide a metabolic map of olfactory alterations in aged Tg2576 mice, reflecting the progressive effect of APP overproduction and β-amyloid (Aβ) accumulation on the OB homeostasis in aged stages.
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
Fiber-based early diagnosis of venous thromboembolic disease by label-free D-dimer detection
(Elsevier, 2019) Zubiate Orzanco, Pablo; Urrutia Azcona, Aitor; Ruiz Zamarreño, Carlos; Egea Urra, Josune; Fernández Irigoyen, Joaquín; Giannetti, Ambra; Baldini, Francesco; Díaz Lucas, Silvia; Matías Maestro, Ignacio; Arregui San Martín, Francisco Javier; Santamaría Martínez, Enrique; Chiavaioli, Francesco; Del Villar, Ignacio; Ingeniaritza Elektrikoa, Elektronikoaren eta Telekomunikazio Ingeniaritzaren; Institute of Smart Cities - ISC; Ingeniería Eléctrica, Electrónica y de Comunicación; Gobierno de Navarra / Nafarroako Gobernua
D-dimer is a useful diagnostic biomarker for deep vein thrombosis or pulmonary embolism, collectively referred to as venous thromboembolism (VTE). The ability to detect in real-time the amount of D-dimer with a fast and reliable method is a key step to anticipate the appearance of these diseases. Here, the results of a highly specific and sensitive biosensor for the detection of D-dimer based on lossy mode resonance in fiber optics are presented. The unique features of specialty fibers in light management integrated with microfluidics allow detecting D-dimer in human serum with a detection limit of 100 ng/mL, a value 5-fold below the clinical cutoff value. Comparison of the results achieved with mass-spectrometry-based proteomics, which allows for the identification of beta- and gamma-chains of fibrinogen, demonstrates the ability of our platform to specifically (>90%) recognize D-dimer. Therefore, this technology potentially represents a paradigm shift in the development of a simple, high-specificity and label-free biosensing platform, which can be applied to speed up diagnostic healthcare processes of venous thromboembolism toward an early diagnostic and personalized treatment system.
Open Access
The proteome of Medicago truncatula in response to ammonium and urea nutrition reveals the role of membrane proteins and enzymes of root lignification
(Elsevier, 2019) Royo Castillejo, Beatriz; Esteban Terradillos, Raquel; Buezo Bravo, Javier; Santamaría Martínez, Enrique; Fernández Irigoyen, Joaquín; Becker, Dirk; Morán Juez, José Fernando; Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMAB; Ciencias; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Plants differ widely in their growth and tolerance responses to ammonium and urea nutrition, while derived phenotypes seem markedly different from plants grown under nitrate supply. Plant responses to N sources are complex, and the traits involved remain unknown. This work reports a comprehensive and quantitative root proteomic study on the NH4+-tolerant legume Medicago truncatula grown under axenic conditions with either nitrate, NH4+ or urea supply as sole N source by using the iTRAQ method. Sixty-one different proteins among the three N sources were identified. Interestingly, among the proteomic responses, urea nutrition displayed greater similarity to nitrate than to ammonium nutrition. We found remarkable differences in membrane proteins that play roles in sensing the N form, and regulate the intracellular pH and the uptake of N. Also, several groups of proteins were differentially expressed in the C metabolism pathway involved in reorganizing N assimilation. In addition, enzymes related to phenylpropanoid metabolism, including the peroxidases POD2, POD6, POD7 and POD11, which were up-regulated under ammonium nutrition, contributed to the reinforcement of cell walls, as confirmed by specific staining of lignin. Thus, we identified cell wall lignification as an important tolerance mechanism of root cells associated with the stunted phenotype typical of plants grown under ammonium nutrition.
Open Access
Early-onset molecular derangements in the olfactory bulb of Tg2576 mice: novel insights into the stress-responsive olfactory kinase dynamics in Alzheimer’s disease
(Frontiers Media, 2019) Lachén Montes, Mercedes; González Morales, Andrea; Palomino Alonso, Maialen; Ausín, Karina; Gómez-Ochoa, Marta; Zelaya Huerta, María Victoria; Ferrer, Isidro; Pérez Mediavilla, Alberto; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The olfactory bulb (OB) is the first processing station in the olfactory pathway. Despite smell impairment, which is considered an early event in Alzheimer’s disease (AD), little is known about the initial molecular disturbances that accompany the AD development at olfactory level. We have interrogated the time-dependent OB molecular landscape in Tg2576 AD mice prior to the appearance of neuropathological amyloid plaques (2-, and 6-month-old), using combinatorial omics analysis. The metabolic modulation induced by overproduction of human mutated amyloid precursor protein (APP) clearly differs between both time points. Besides the progressive perturbation of the APP interactome, functional network analysis unveiled an inverse regulation of downstream extracellular signal-regulated kinase (ERK1/2), and p38 mitogen-activated protein kinase (MAPK) routes in 2-month-old Tg2576 mice with respect to wild-type (WT) mice. In contrast, Akt and MAPK kinase 4 (SEK1)/ stress-activated protein kinase (SAPK) axis were parallel activated in the OB of 6-months-old-Tg2576 mice. Furthermore, a survival kinome profiling performed during the aging process (2-, 6-, and 18-month-old) revealed that olfactory APP overexpression leads to changes in the activation dynamics of protein kinase A (PKA), and SEK1/MKK4-SAPK/JNK between 6 and 18 months of age, when memory deficits appear and AD pathology is well established in transgenic mice. Interestingly, both olfactory pathways were differentially activated in a stage-dependent manner in human sporadic AD subjects with different neuropathological grading. Taken together, our data reflect the early impact of mutated APP on the OB molecular homeostasis, highlighting the progressive modulation of specific signaling pathways during the olfactory amyloidogenic pathology.
Open Access
Neuroanatomical quantitative proteomics reveals common pathogenic biological routes between amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)
(MDPI, 2019) Iridoy Zulet, Marina; Zubiri, Irene; Zelaya Huerta, María Victoria; Martínez, Leire; Ausín, Karina; Lachén Montes, Mercedes; Santamaría Martínez, Enrique; Fernández Irigoyen, Joaquín; Jericó Pascual, Ivonne; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
(1) Background: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders with an overlap in clinical presentation and neuropathology. Common and differential mechanisms leading to protein expression changes and neurodegeneration in ALS and FTD were studied trough a deep neuroproteome mapping of the spinal cord. (2) Methods: A liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the spinal cord from ALS-TAR DNA-binding protein 43 (TDP-43) subjects, ubiquitin-positive frontotemporal lobar degeneration (FTLD-U) subjects and controls without neurodegenerative disease was performed. (3) Results: 281 differentially expressed proteins were detected among ALS versus controls, while 52 proteins were dysregulated among FTLD-U versus controls. Thirty-three differential proteins were shared between both syndromes. The resulting data was subjected to network-driven proteomics analysis, revealing mitochondrial dysfunction and metabolic impairment, both for ALS and FTLD-U that could be validated through the confirmation of expression levels changes of the Prohibitin (PHB) complex. (4) Conclusions: ALS-TDP-43 and FTLD-U share molecular and functional alterations, although part of the proteostatic impairment is region-and disease-specific. We have confirmed the involvement of specific proteins previously associated with ALS (Galectin 2 (LGALS3), Transthyretin (TTR), Protein S100-A6 (S100A6), and Protein S100-A11 (S100A11)) and have shown the involvement of proteins not previously described in the ALS context (Methanethiol oxidase (SELENBP1), Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN-1), Calcyclin-binding protein (CACYBP) and Rho-associated protein kinase 2 (ROCK2)). © 2018 by the authors. Licensee MDPI, Basel, Switzerland.
Open Access
Oncolytic adenovirus Delta-24-RGD induces a widespread glioma proteotype remodeling during autophagy
(Elsevier, 2018) González Morales, Andrea; Zabaleta, Aintzane; García Moure, Marc; Alonso Roldán, Marta; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Adenovirus Delta-24-RGD has shown a remarkable efficacy in a phase I clinical trial for glioblastoma. Delta-24-RGD induces autophagy in glioma cells, however, the molecular derangements associated with Delta-24-RGD infection remains poorly understood. Here, proteomics was applied to characterize the glioma metabolic disturbances soon after Delta-24-RGD internalization and late in infection. Minutes post-infection, a rapid survival reprogramming of glioma cells was evidenced by an early c-Jun activation and a time-dependent dephosphorylation of multiple survival kinases. At 48 h post-infection (hpi), a severe intracellular proteostasis impairment was characterized, detecting differentially expressed proteins related to mRNA splicing, cytoskeletal organization, oxidative response, and inflammation. Specific kinase-regulated protein interactomes for Delta-24-RGD-modulated proteome revealed interferences with the activation dynamics of protein kinases C and A (PKC, PKA), tyrosine-protein kinase Src (c-Src), glycogen synthase kinase-3 (GSK-3) as well as serine/threonine-protein phosphatases 1 and 2A (PP1, PP2A) at 48hpi, in parallel with adenoviral protein overproduction. Moreover, the late activation of the nuclear factor kappa B (NF-κB) correlates with the extracellular increment of specific cytokines involved in migration, and activation of different inflammatory cells. Taken together, our integrative analysis provides further insights into the effects triggered by Delta-24-RGD in the modulation of tumor suppression and immune response against glioma. Significance: The current study provides new insights regarding the molecular mechanisms governing the glioma metabolism during Delta-24-RGD oncolytic adenoviral therapy. The compilation and analysis of intracellular and extracellular proteomics have led us to characterize: i) the cell survival reprogramming during Delta-24-RGD internalization, ii) the proteostatic disarrangement induced by Delta-24-RGD during the autophagic stage, iii) the protein interactomes for Delta-24-RGD-modulated proteome, iv) the regulatory effects on kinase dynamics induced by Delta-24-RGD late in infection, and v) the overproduction of multitasking cytokines upon Delta-24-RGD treatment. We consider that the quantitative molecular maps generated in this study may establish the foundations for the development of complementary adenoviral based-vectors to increase the potency against glioma.
Open Access
Olfactory bulb neuroproteomics reveals a chronological perturbation of survival routes and a disruption of prohibitin complex during Alzheimer's disease progression
(Springer Nature, 2017) Lachén Montes, Mercedes; González Morales, Andrea; Zelaya Huerta, María Victoria; Pérez Valderrama, Estela; Ausín, Karina; Ferrer, Isidro; 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
Olfactory dysfunction is among the earliest features of Alzheimer’s disease (AD). Although neuropathological abnormalities have been detected in the olfactory bulb (OB), little is known about its dynamic biology. Here, OB- proteome analysis showed a stage-dependent synaptic proteostasis impairment during AD evolution. In addition to progressive modulation of tau and amyloid precursor protein (APP) interactomes, network-driven proteomics revealed an early disruption of upstream and downstream p38 MAPK pathway and a subsequent impairment of Phosphoinositide-dependent protein kinase 1 (PDK1)/Protein kinase C (PKC) signaling axis in the OB from AD subjects. Moreover, a mitochondrial imbalance was evidenced by a depletion of Prohibitin-2 (Phb2) levels and a specific decrease in the phosphorylated isoforms of Phb1 in intermediate and advanced AD stages. Interestingly, olfactory Phb subunits were also deregulated across different types of dementia. Phb2 showed a specific up-regulation in mixed dementia, while Phb1 isoforms were down-regulated in frontotemporal lobar degeneration (FTLD). However, no differences were observed in the olfactory expression of Phb subunits in progressive supranuclear palsy (PSP). To sum up, our data reflect, in part, the missing links in the biochemical understanding of olfactory dysfunction in AD, unveiling Phb complex as a differential driver of neurodegeneration at olfactory level.