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OBJECTIVE The HEPAFIT study was aimed at examining the impact of a 6-month physical education intervention, considering various levels of exercise intensity, on hepatic fat and cardiometabolic health outcomes in adolescents with excess adiposity. METHODS Adolescents (n = 120), 11-17 years with excess adiposity by body fat >30%, were randomly assigned to one of the following 4 groups for 6 months: (1) standard physical education lessons, control (CTRL); (2) high-intensity physical education (HIPE); (3) low-to-moderate intensity physical education (LIPE) and (4) combined HIPE and LIPE (PLUS). The primary outcome was hepatic fat content measured by vibration-controlled transient elastography (controlled attenuation parameter [CAP]). Secondary outcomes were traditional cardiovascular health markers (body composition, serum lipids, aminotransferases and health-related physical fitness components). RESULTS Adjusted mixed effects linear models revealed a significant decrease in CAP levels in HIPE (-20.02 dB/m, p

Olfactory dysfunction is considered an early prodromal marker of many neurodegenerative diseases. Neuropathological changes and aberrant protein aggregates occur in the olfactory bulb (OB), triggering a tangled cascade of molecular events that is not completely understood across neurological disorders. This study aims to analyze commonalities and differences in the olfactory protein homeostasis across neurological backgrounds with different spectrums of smell dysfunction. For that, an integrative analysis was performed using OB proteomics datasets derived from subjects with Alzheimer’s disease (AD), Parkinson´s disease (PD), mixed dementia (mixD), dementia with Lewy bodies (DLB), frontotemporal lobar degeneration (FTLD-TDP43), progressive supranuclear palsy (PSP) and amyotrophic lateral sclerosis (ALS) with respect to OB proteome data from neurologically intact controls. A total of 80% of the differential expressed protein products were potentially disease-specific whereas the remaining 20% were commonly altered across two, three or four neurological phenotypes. A multi-level bioinformatic characterization revealed a subset of potential disease-specific transcription factors responsible for the downstream effects detected at the proteome level as well as specific densely connected protein complexes targeted by several neurological phenotypes. Interestingly, common or unique pathways and biofunctions were also identified, providing novel mechanistic clues about each neurological disease at olfactory level. The analysis of olfactory epithelium, olfactory tract and primary olfactory cortical proteotypes in a multi-disease format will functionally complement the OB dyshomeostasis, increasing our knowledge about the neurodegenerative process across the olfactory axis. This work was funded by grants from the Spanish Ministry of Science, Innovation and Universities (Ref. PID2019-110356RB-I00/ AEI / 10.13039/501100011033 to JF-I and ES) and the Department of Economic and Business Development of the Government of Navarra (Ref. 0011-14112020-000028 to ES).The Proteomics Platform of Navarrabiomed, member of Proteored (PRB3ISCIII), was supported by grant PT17/0019/009, of the PE I+D+I 2013-2016 funded by ISCIII and FEDER to JF. The Clinical Neuroproteomics Unit of Navarrabiomed is member of the Spanish Olfactory Network (ROE) (supported by grant RED2018-102662-T funded by Spanish Ministry of Science and Innovation) and the Global Consortium for Chemosensory Research (GCCR).

Myocardial fibrosis is a main contributor to the development of heart failure (HF). CT-1 (cardiotrophin-1) and Gal-3 (galectin-3) are increased in HF and associated with myocardial fibrosis. The aim of this study is to analyze whether CT-1 regulates Gal-3. Proteomic analysis revealed that Gal-3 was upregulated by CT-1 in human cardiac fibroblasts in parallel with other profibrotic and proinflammatory markers. CT-1 upregulation of Gal-3 was mediated by ERK (extracellular signal-regulated kinase) 1/2 and Stat-3 (signal transducer and activator of transcription 3) pathways. Male Wistar rats and B6CBAF1 mice treated with CT-1 (20 µg/kg per day) presented higher cardiac Gal-3 levels and myocardial fibrosis. In CT-1–treated rats, direct correlations were found between cardiac CT-1 and Gal-3 levels, as well as between Gal-3 and perivascular fibrosis. Gal-3 genetic disruption in human cardiac fibroblasts and pharmacological Gal-3 inhibition in mice prevented the profibrotic and proinflammatory effects of CT-1. Dahl salt-sensitive hypertensive rats with diastolic dysfunction showed increased cardiac CT-1 and Gal-3 expression together with cardiac fibrosis and inflammation. CT-1 and Gal-3 directly correlated with myocardial fibrosis. In HF patients, myocardial and plasma CT-1 and Gal-3 were increased and directly correlated. In addition, HF patients with high CT-1 and Gal-3 plasma levels presented an increased risk of cardiovascular death. Our data suggest that CT-1 upregulates Gal-3 which, in turn, mediates the proinflammatory and profibrotic myocardial effects of CT-1. The elevation of both molecules in HF patients identifies a subgroup of patients with a higher risk of cardiovascular mortality. The CT-1/Gal-3 axis emerges as a candidate therapeutic target and a potential prognostic biomarker in HF.

Tau hyperphosphorylation is the first step of neurofibrillary tangle (NFT) formation. In the present study, samples of the entorhinal cortex (EC) and frontal cortex area 8 (FC) of cases with NFT pathology classified as stages I–II, III–IV, and V–VI without comorbidities, and of middle‐aged (MA) individuals with no NFT pathology, were analyzed by conventional label‐free and SWATH‐MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) to assess the (phospho)proteomes. The total number of identified dysregulated phosphoproteins was 214 in the EC, 65 of which were dysregulated at the first stages (I–II) of NFT pathology; 167 phosphoproteins were dysregulated in the FC, 81 of them at stages I–II of NFT pathology. A large percentage of dysregulated phosphoproteins were identified in the two regions and at different stages of NFT progression. The main group of dysregulated phosphoproteins was made up of components of the membranes, cytoskeleton, synapses, proteins linked to membrane transport and ion channels, and kinases. The present results show abnormal phosphorylation of proteins at the first stages of NFT pathology in the elderly (in individuals clinically considered representative of normal aging) and sporadic Alzheimer's disease (sAD). Dysregulated protein phosphorylation in the FC precedes the formation of NFTs and SPs. The most active period of dysregulated phosphorylation is at stages III–IV when a subpopulation of individuals might be clinically categorized as suffering from mild cognitive impairment which is a preceding determinant stage in the progression to dementia. Altered phosphorylation of selected proteins, carried out by activation of several kinases, may alter membrane and cytoskeletal functions, among them synaptic transmission and membrane/cytoskeleton signaling. Besides their implications in sAD, the present observations suggest a molecular substrate for “benign” cognitive deterioration in “normal” brain aging., Dysregulated brain protein phosphorylation (DBPP) occurs at the first stages of neurofibrillary tangle (NFT) pathology (stages I‐II of Braak) in the frontal cortex (FC) and entorhinal cortex (EC). It progresses at the middle (stages III–IV), and advanced stages (V and VI) linked to cognitive impairment and dementia, respectively, in Alzheimer's disease.DBPP principally affects proteins of the cell membranes, cytoskeleton, synapses, protein, and energy metabolism, and it occurs in parallel with abnormal activation of multiple kinases. Many dysregulated phosphoproteins are shared with the FC and EC, and they are found at different stages of NFT pathology.DBPP conforms to a continuum between “normal” brain aging and Alzheimer's diseaseSince protein phosphorylation is crucial in protein signaling, DBPP implies severe dysfunction of critical molecular pathways.DBPP may contribute to progressive cell degeneration and disease progression in sAD. Since DBPP already occurs at the first stages of NFT pathology, which affects about 85% of individuals at the age of 65 years, we suggest that DBPP may contribute to “benign cognitive decline” in “normal” aging.

The most common form of mixed dementia (MixD) is constituted by abnormal protein deposits associated with Alzheimer's disease (AD) that coexist with vascular disease. Although olfactory dysfunction is considered a clinical sign of AD-related dementias, little is known about the impact of this sensorial impairment in MixD at the molecular level. To address this gap in knowledge, we assessed olfactory bulb (OB) proteome-wide expression in MixD subjects (n = 6) respect to neurologically intact controls (n = 7). Around 9% of the quantified proteins were differentially expressed, pinpointing aberrant proteostasis involved in synaptic transmission, nucleoside monophosphate and carbohydrate metabolism, and neuron projection regeneration. In addition, network-driven proteomics revealed a modulation in cell-survival related pathways such as ERK, AKT, and the PDK1-PKC axis. Part of the differential OB protein set was not specific of MixD, also being deregulated across different tauopathies, synucleinopathies, and tardopathies. However, the comparative functional analysis of OB proteome data between MixD and pure AD pathologies deciphered commonalities and differences between both related phenotypes. Finally, olfactory pro-teomics allowed to propose serum Prolow-density lipoprotein receptor-related protein 1 (LRP1) as a candidate marker to differentiate AD from MixD phenotypes. This work was funded by grants from the Spanish Ministry of Science, Innovation and Universities (Ref. PID2019-110356RB-I00/AEI/10.13039/501100011033) and the Department of Economic and Business Development from Government of Navarra (Ref. 0011-1411-2020-000028 to E.S.).

Altres ajuts: This work was supported by a grant from Department of Economic and Business Development from Government of Navarra (ref. 0011-1411-2020-000028) to ES. A close epidemiological link has been reported between cannabis use and schizophrenia (SCZ). However, biochemical markers in living humans related to the impact of cannabis in this disease are still missing. Olfactory neuroepithelium (ON) cells express neural features and offer a unique advantage to study biomarkers of psychiatric diseases. The aim of our study was to find exclusively deregulated proteins in ON cells of SCZ patients with and without a history of cannabis use. Thus, we compared the proteomic profiles of SCZ non-cannabis users (SCZ/nc) and SCZ cannabis users (SCZ/c) with control subjects non-cannabis users (C/nc) and control cannabis users (C/c). The results revealed that the main cascades affected in SCZ/nc were cell cycle, DNA replication, signal transduction and protein localization. Conversely, cannabis use in SCZ patients induced specific alterations in metabolism of RNA and metabolism of proteins. The levels of targeted proteins in each population were then correlated with cognitive performance and clinical scores. In SCZ/c, the expression levels of 2 proteins involved in the metabolism of RNA (MTREX and ZNF326) correlated with several cognitive markers and clinical signs. Moreover, use duration of cannabis negatively correlated with ZNF326 expression. These findings indicate that RNA-related proteins might be relevant to understand the influence of cannabis use on SCZ.

Simple Summary The standard of care for patients with newly diagnosed glioblastoma (GBM) comprises surgery followed by radio- and chemotherapy. In addition, dexamethasone is used to manage the development of inflammation within the brain in general, and particularly during treatment. The effects of dexamethasone on patient survival however remain controversial because several clinical studies suggest that dexamethasone could potentially restrict effective radiotherapy. With the idea to improve GBM therapy, we set out to identify small molecule inhibitors that could improve the killing of GBM cells when applied together with radiotherapy. We have identified a novel dexamethasone-induced mechanism that can directly protect GBM cells from radiotherapy and thus may contribute to the adverse effects observed in the clinic. Strikingly, this mechanism also sensitises GBM cells to tyrosine kinase inhibitors, thus encouraging the revision of the use of these inhibitors for the treatment of GBM, potentially in an adjuvant setting. Abstract (1) Background: Despite the indisputable effectiveness of dexamethasone (DEXA) to reduce inflammation in glioblastoma (GBM) patients, its influence on tumour progression and radiotherapy response remains controversial. (2) Methods: We analysed patient data and used expression and cell biological analyses to assess effects of DEXA on GBM cells. We tested the efficacy of tyrosine kinase inhibitors in vitro and in vivo. (3) Results: We confirm in our patient cohort that administration of DEXA correlates with worse overall survival and shorter time to relapse. In GBM cells and glioma stem-like cells (GSCs) DEXA down-regulates genes controlling G2/M and mitotic-spindle checkpoints, and it enables cells to override the spindle assembly checkpoint (SAC). Concurrently, DEXA up-regulates Platelet Derived Growth Factor Receptor (PDGFR) signalling, which stimulates expression of anti-apoptotic regulators BCL2L1 and MCL1, required for survival during extended mitosis. Importantly, the protective potential of DEXA is dependent on intact tyrosine kinase signalling and ponatinib, sunitinib and dasatinib, all effectively overcome the radio-protective and pro-proliferative activity of DEXA. Moreover, we discovered that DEXA-induced signalling creates a therapeutic vulnerability for sunitinib in GSCs and GBM cells in vitro and in vivo. (4) Conclusions: Our results reveal a novel DEXA-induced mechanism in GBM cells and provide a rationale for revisiting the use of tyrosine kinase inhibitors for the treatment of GBM.

Prohibitins are ubiquitous proteins involved in multiple biological functions depending on the cell type and their cellular localization. Prohibitins exert an essential role in the maintenance of mitochondrial structure and functionality. Besides alterations observed in metabolic and oncogenic processes, an imbalance in prohibitin system triggers mitochondrial defects leading to lifespan reduction in yeast and Caenorhabditis elegans, and impairments in mitochondrial homeostasis in neurological disorders such as Parkinson disease. In general, the aging and neurodegenerative processes are accompanied by a dysfunction of the sense of smell. Recent advances in olfactory proteomics have allowed to characterize a disruption of prohibitin subunits at the level of the olfactory bulb, unveiling prohibitin complex as a differential driver of neurodegeneration across different proteinopathies at olfactory level.

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-supplemented 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. Transcript 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.

Sporadic amyotrophic lateral sclerosis (sALS) is a fatal progressive neurodegenerative disease affecting upper and lower motor neurons. Biomarkers are useful to facilitate the diagnosis and/or prognosis of patients and to reveal possible mechanistic clues about the disease. This study aimed to identify and validate selected putative biomarkers in the cerebrospinal fluid (CSF) of sALS patients at early disease stages compared with age-matched controls and with other neurodegenerative diseases including Alzheimer disease (AD), spinal muscular atrophy type III (SMA), frontotemporal dementia behavioral variant (FTD), and multiple sclerosis (MS). SWATH acquisition on liquid chromatography-tandem mass spectrometry (LC&ndash, MS/MS) for protein quantitation, and ELISA for validation, were used in CSF samples of sALS cases at early stages of the disease. Analysis of mRNA and protein expression was carried out in the anterior horn of the lumbar spinal cord in post-mortem tissue of sALS cases (terminal stage) and controls using RTq-PCR, and Western blotting, and immunohistochemistry, respectively. SWATH acquisition on liquid chromatography-tandem mass spectrometry (LC&ndash, MS/MS) revealed 51 differentially expressed proteins in the CSF in sALS. Receiver operating characteristic (ROC) curves showed CXCL12 to be the most valuable candidate biomarker. We validated the values of CXCL12 in CSF with ELISA in two different cohorts. Besides sALS, increased CXCL12 levels were found in MS but were not altered in AD, SMA, and FTD. Therefore, increased CXCL12 levels in the CSF can be useful in the diagnoses of MS and sALS in the context of the clinical settings. CXCL12 immunoreactivity was localized in motor neurons in control and sALS, and in a few glial cells in sALS at the terminal stage, CXCR4 was in a subset of oligodendroglial-like cells and axonal ballooning of motor neurons in sALS, and CXCR7 in motor neurons in control and sALS, and reactive astrocytes in the pyramidal tracts in terminal sALS. CXCL12/CXCR4/CXCR7 axis in the spinal cord probably plays a complex role in inflammation, oligodendroglial and astrocyte signaling, and neuronal and axonal preservation in sALS.

Olfactory dysfunction is one of the prodromal symptoms in dementia with Lewy bodies (DLB). However, the molecular pathogenesis associated with decreased smell function remains largely undeciphered. We generated quantitative proteome maps to detect molecular alterations in olfactory bulbs (OB) derived from DLB subjects compared to neurologically intact controls. A total of 3214 olfactory proteins were quantified, and 99 proteins showed significant alterations in DLB cases. Protein interaction networks disrupted in DLB indicated an imbalance in translation and the synaptic vesicle cycle. These alterations were accompanied by alterations in AKT/MAPK/SEK1/p38 MAPK signaling pathways that showed a distinct expression profile across the OB&ndash, olfactory tract (OT) axis. Taken together, our data partially reflect the missing links in the biochemical understanding of olfactory dysfunction in DLB.

Alpha-synuclein (aSyn) protein levels are sufficient to drive Parkinson's disease (PD) and other synucleinopathies. Despite the biomedical/therapeutic potential of aSyn protein regulation, little is known about mechanisms that limit/control aSyn levels. Here, we investigate the role of a post-translational modification, N-terminal acetylation, in aSyn neurotoxicity. N-terminal acetylation occurs in all aSyn molecules and has been proposed to determine its lipid binding and aggregation capacities; however, its effect in aSyn stability/neurotoxicity has not been evaluated. We generated N-terminal mutants that alter or block physiological aSyn N-terminal acetylation in wild-type or pathological mutant E46K aSyn versions and confirmed N-terminal acetylation status by mass spectrometry. By optical pulse-labeling in living primary neurons we documented a reduced half-life and accumulation of aSyn N-terminal mutants. To analyze the effect of N-terminal acetylation mutants in neuronal toxicity we took advantage of a neuronal model where aSyn toxicity was scored by longitudinal survival analysis. Salient features of aSyn neurotoxicity were previously investigated with this approach. aSyn-dependent neuronal death was recapitulated either by higher aSyn protein levels in the case of WT aSyn, or by the combined effect of protein levels and enhanced neurotoxicity conveyed by the E46K mutation. aSyn N-terminal mutations decreased E46K aSyn-dependent neuronal death both by reducing protein levels and, importantly, by reducing the intrinsic E46K aSyn toxicity, being the D2P mutant the least toxic. Together, our results illustrate that the N-terminus determines, most likely through its acetylation, aSyn protein levels and toxicity, identifying this modification as a potential therapeutic target.

Galectin-3 (Gal-3) is increased in heart failure (HF) and promotes cardiac fibrosis and inflammation. We investigated whether Gal-3 modulates oxidative stress in human cardiac fibroblasts, in experimental animal models and in human aortic stenosis (AS). Using proteomics and immunodetection approaches, we have identified that Gal-3 down-regulated the antioxidant peroxiredoxin-4 (Prx-4) in cardiac fibroblasts. In parallel, Gal-3 increased peroxide, nitrotyrosine, malondialdehyde, and N-carboxymethyl-lysine levels and decreased total antioxidant capacity. Gal-3 decreased prohibitin-2 expression without modifying other mitochondrial proteins. Prx-4 silencing increased oxidative stress markers. In Gal-3-silenced cells and in heart from Gal-3 knockout mice, Prx-4 was increased and oxidative stress markers were decreased. Pharmacological inhibition of Gal-3 with modified citrus pectin restored cardiac Prx-4 as well as prohibitin-2 levels and improved oxidative status in spontaneously hypertensive rats. In serum from 87 patients with AS, Gal-3 negatively correlated with total antioxidant capacity and positively correlated with peroxide. In myocardial biopsies from 26 AS patients, Gal-3 up-regulation paralleled a decrease in Prx-4 and in prohibitin-2. Cardiac Gal-3 inversely correlated with Prx-4 levels in myocardial biopsies. These data suggest that Gal-3 decreased Prx-4 antioxidant system in cardiac fibroblasts, increasing oxidative stress. In pathological models presenting enhanced cardiac Gal-3, the decrease in Prx-4 expression paralleled increased oxidative stress. Gal-3 blockade restored Prx-4 expression and improved oxidative stress status. In AS, circulating levels of Gal-3 could reflect oxidative stress. The alteration of the balance between antioxidant systems and reactive oxygen species production could be a new pathogenic mechanism by which Gal-3 induces cardiac damage in HF.

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&ndash, 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.

The tumor-suppressor protein p16 is paradoxically overexpressed in cervical cancer (CC). Despite its potential as a biomarker, its clinical value and the reasons for its failure in tumor suppression remain unclear. Our purpose was to determine p16 clinical and biological significance in CC. p16 expression pattern was examined by immunohistochemistry in 78 CC cases (high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix &ndash, SCCCs). CC cell proliferation and invasion were monitored by real-time cell analysis and Transwell&reg, invasion assay, respectively. Cytoplasmic p16 interactors were identified from immunoprecipitated extracts by liquid chromatography-tandem mass spectrometry, and colocalization was confirmed by double-immunofluorescence. We observed that SCCCs showed significantly more cytoplasmic than nuclear p16 expression than HSILs. Importantly, nuclear p16 absence significantly predicted poor outcome in SCCC patients irrespective of other clinical parameters. Moreover, we demonstrated that cytoplasmic p16 interacted with CDK4 and other unreported proteins, such as BANF1, AKAP8 and AGTRAP, which could sequester p16 to avoid nuclear translocation, and then, impair its anti-tumor function. Our results suggest that the absence of nuclear p16 could be a diagnostic biomarker between HSIL and SCCC, and an independent prognostic biomarker in SCCC, and explain why p16 overexpression fails to stop CC growth.

Globular glial tauopathy (GGT) is a progressive neurodegenerative disease involving the grey matter and white matter (WM) and characterized by neuronal deposition of hyper-phosphorylated, abnormally conformed, truncated, oligomeric 4Rtau in neurons and in glial cells forming typical globular astrocyte and oligodendrocyte inclusions (GAIs and GOIs, respectively) and coiled bodies. Present studies centre on four genetic GGT cases from two unrelated families bearing the P301T mutation in MAPT and one case of sporadic GGT (sGGT) and one case of GGT linked to MAPT K317M mutation, for comparative purposes. Clinical and neuropathological manifestations and biochemical profiles of phospho-tau are subjected to individual variations in patients carrying the same mutation, even in carriers of the same family, independently of the age of onset, gender, and duration of the disease. Immunohistochemistry, western blotting, transcriptomic, proteomics and phosphoproteomics, and intra-cerebral inoculation of brain homogenates to wild-type (WT) mice were the methods employed. In GGT cases linked to MAPT P301T mutation, astrocyte markers GFAP, ALDH1L1, YKL40 mRNA and protein, GJA1 mRNA, and AQ4 protein are significantly increased; glutamate transporter GLT1 (EAAT2) and glucose transporter (SLC2A1) decreased; mitochondrial pyruvate carrier 1 (MPC1) increased, and mitochondrial uncoupling protein 5 (UCP5) almost absent in GAIs in frontal cortex (FC). Expression of oligodendrocyte markers OLIG1 and OLIG2mRNA, and myelin-related genes MBP, PLP1, CNP, MAG, MAL, MOG, and MOBP are significantly decreased in WM; CNPase, PLP1, and MBP antibodies reveal reduction and disruption of myelinated fibres; and SMI31 antibodies mark axonal damage in the WM. Altered expression of AQ4, GLUC-t, and GLT-1 is also observed in sGGT and in GGT linked to MAPT K317M mutation. These alterations point to primary astrogliopathy and oligodendrogliopathy in GGT. In addition, GGT linked to MAPT P301T mutation proteotypes unveil a proteostatic imbalance due to widespread (phospho)proteomic dearrangement in the FC and WM, triggering a disruption of neuron projection morphogenesis and synaptic transmission. Identification of hyper-phosphorylation of variegated proteins calls into question the concept of phospho-tau-only alteration in the pathogenesis of GGT. Finally, unilateral inoculation of sarkosyl-insoluble fractions of GGT homogenates from GGT linked to MAPT P301T, sGGT, and GGT linked to MAPT K317M mutation in the hippocampus, corpus callosum, or caudate/putamen in wild-type mice produces seeding, and time- and region-dependent spreading of phosphorylated, non-oligomeric, and non-truncated 4Rtau and 3Rtau, without GAIs and GOIs but only of coiled bodies. These experiments prove that host tau strains are important in the modulation of cellular vulnerability and phenotypes of phospho-tau aggregates. The project leading to these results received funding from 'La Caixa' Foundation under the agreement LCF/PR/HR19/52160007. The study was also supported by the Ministry of Economy and Competiveness (cofunded by European Regional Development Fund, ERDF, a way to build Europe): FIS PI17/000809 and IFI15/00035 fellowship to PA-B; and co-financed by ERDF under the program Interreg Poctefa: RedPrion 148/16; the Intra-CIBERNED 2019 collaborative project. ML-M was supported by a predoctoral fellowship from the Public University of Navarra (UPNA). The Proteomics Unit of Navarrabiomed is a member of Proteored, PRB3-ISCIII, and is supported by Grant PT17/0019/009, of the PE I + D + I 2013–2016 funded by ISCIII and FEDER.

Cancer cells acquire resistance to cytotoxic therapies targeting major survival pathways by adapting their metabolism. The AKT pathway is a major regulator of human pancreatic adenocarcinoma progression and a key pharmacological target. The mechanisms of adaptation to long-term silencing of AKT isoforms of human and mouse pancreatic adenocarcinoma cancer cells were studied. Following silencing, cancer cells remained quiescent for long periods of time, after which they recovered proliferative capacities. Adaptation caused profound proteomic changes largely affecting mitochondrial biogenesis, energy metabolism and acquisition of a number of distinct cancer stem cell (CSC) characteristics depending on the AKT isoform that was silenced. The adaptation to AKT1 silencing drove most de-differentiation and acquisition of stemness through C-MYC down-modulation and NANOG upregulation, which were required for survival of adapted CSCs. The changes associated to adaptation sensitized cancer cells to inhibitors targeting regulators of oxidative respiration and mitochondrial biogenesis. In vivo pharmacological co-inhibition of AKT and mitochondrial metabolism effectively controlled pancreatic adenocarcinoma growth in pre-clinical models. This research was supported by: Asociación Española Contra el Cáncer (AECC, PROYE16001ESCO); Instituto de Salud Carlos III, Spain (FIS project grant PI17/02119); 'Precipita' Crowdfunding grant (FECYT); Crowdfunding grant from Sociedad Española de Inmunología (SEI); and Government of Navarre project grant in biomedicine (BMED 050-2019). D.E. is funded by a Miguel Servet Fellowship (ISC III, CP12/03114, Spain); H.A. is supported by the Clinico Junior 2019 scholarship from AECC; M.Z. is supported by a scholarship from Universidad Pública de Navarra; M.G. is supported by a scholarship from the Government of Navarre; and L.C.D is supported by a DESCARTHES project grant (Industry department, Government of Navarre).

Background Frontotemporal lobar degeneration with TDP-43 immunoreactive inclusions (FTLD-TDP) may appear as sporadic (sFTLD-TDP) or linked to mutations in various genes including expansions of the non-coding region of C9ORF72 (c9FTLD). Objective Analysis of differential mRNA and protein expression in the frontal cortex in c9FLTD and evaluation with previous observations in frontal cortex in sFTLD-TDP and amyotrophic lateral sclerosis with TDP-43 inclusions. Methods Microarray hybridization and mass spectrometry-based quantitative proteomics followed by RT-qPCR, gel electrophoresis, and western blotting in frontal cortex area 8 in 19 c9FTLD cases and 14 age- and gender-matched controls. Results Microarray hybridization distinguish altered gene transcription related to DNA recombination, RNA splicing regulation, RNA polymerase transcription, myelin synthesis, calcium regulation, and ubiquitin-proteasome system in c9FTLD; proteomics performed in the same tissue samples pinpoints abnormal protein expression involving apoptosis, inflammation, metabolism of amino acids, metabolism of carbohydrates, metabolism of membrane lipid derivatives, microtubule dynamics, morphology of mitochondria, neuritogenesis, neurotransmission, phagocytosis, receptor-mediated endocytosis, synthesis of reactive oxygen species, and calcium signaling in c9FTLD. Conclusion Transcriptomics and proteomics, as well as bioinformatics processing of derived data, reveal similarly altered pathways in the frontal cortex in c9FTLD, but different RNAs and proteins are identified by these methods. Combined non-targeted '-omics' is a valuable approach to deciphering altered molecular pathways in FTLD provided that observations are approached with caution when assessing human postmortem brain samples.

Mild olfactory dysfunction has been observed in frontotemporal dementias (FTD). However, the underlying molecular mechanisms associated to this deficit are poorly understood. We applied quantitative proteomics to analyze pathological effects on the olfactory bulb (OB) from progressive supranuclear palsy (PSP) and frontotemporal lobar degeneration (FTLD-TDP43) subjects respect to elderly non-FTD group. Our data revealed: i) a mitochondrial and calcium homeostasis impairment in PSP and ii) a disruption of protein synthesis and vesicle trafficking in FTLD-TDP43. Although differential OB proteomes clearly differ between both FTD phenotypes, functional analyses pointed out an imbalance in survival signaling in both pathologies. A common alteration of olfactory mitogen-activated protein kinases (MAPKs), calcium/calmodulin dependent protein kinase II (CAMKII), and protein kinase C (PKC) signaling pathways was observed in PSP and FTLD subjects. In contrast, a specific shut off in mitogen-activated protein kinase kinase 4 (SEK1/MKK4)/stress-activated protein kinase (SAPK) axis was exclusively observed in PSP, whereas a specific phosphoinositide-dependent protein kinase 1 (PDK1) inactivation was observed in FTLD-TDP43. In summary, our data contribute to a better understanding of the molecular mechanisms that are modulated in PSP and FTLD-TDP43 at olfactory level, highlighting cross-disease similarities and differences in the regulation of survival pathways across FTD spectrum. SIGNIFICANCE: This work reflects differential olfactory molecular disarrangements in PSP and FTLD-TDP43, two clinically similar FTD disorders, but with different neuropathological signature. Besides FTDs present mild olfactory dysfunction, our data provide basic information for understanding the implication of the OB in the pathophysiology of FTDs.

Nowadays, diagnosis of neurodegenerative disorders is mainly based on neuroimaging and clinical symptoms, although postmortem neuropathological confirmation remains the gold standard diagnostic technique. Therefore, cerebrospinal fluid (CSF) proteome is considered a valuable molecular repository for diagnosing and targeting the neurodegenerative process. It is well known that olfactory dysfunction is among the earliest features of synucleinopathies such as Parkinson's disease (PD). Consequently, we consider that the application of tissue proteomics in primary olfactory structures is an ideal approach to explore early pathophysiological changes, detecting olfactory proteins that might be tested in CSF as potential biomarkers. Data mining of mass spectrometry-generated datasets has revealed that 30% of the olfactory bulb (OB) proteome is also localized in CSF. In this chapter, we describe a method that utilizes label-free quantitative proteomics and computational analysis to characterize human OB proteomes and potential cerebrospinal fluid (CSF) biomarkers associated with neurodegenerative syndromes. For that, we applied peptide fractionation methods, followed by tandem mass spectrometry (nanoLC-MS/MS), in silico analysis, and semi-quantitative orthogonal techniques in OB derived from PD subjects. After obtaining the differential OB proteome across Lewy-type alpha-synucleinopathy (LTS) stages and further validating the method, this workflow was applied to probe changes in NEGR1 (neuronal growth regulator 1) and GNPDA2 (glucosamine-6-phosphate deaminase 2) protein levels in CSF derived from parkinsonian subjects with respect to controls, observing an inverse correlation between both proteins and α-synuclein, the principal component analysis of Lewy pathology.

The brain is the most complex organ of the human body, and the study of the different diseases and injuries that affect it is far behind the ones that affect other organs. Some of these pathologies such as neurodegenerative diseases, physical injuries, and cancer present an important alteration in its inflammatory component, which affects their outcome in a positive or negative way. For this reason, it is important to characterize the joint expression of the cytokines and growth factors (GF) that are part of this inflammatory component. The cerebrospinal fluid (CSF) is in direct contact with the brain and spinal cord, being the best biofluid to study the cytokine and GF secretion patterns of these conditions. Currently, the proteomic workflows based on mass spectrometry (MS) are unable to easily detect these proteins in CSF. In this chapter, we describe a method based on cytokine membrane arrays to characterize, in a straightforward way, the secretion profile of different cytokines and GF at once in CSF.

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. This work is funded by grants from the Spanish Agencia Estatal de Investigación (AEI) and European Regional Development Fund (FEDER) (TEC2016-78047-R) and the Department of Economic Development from Government of Navarra (REF. PC023-24-25 , PC081-082 , and PI031). The Proteomics Unit of Navarrabiomed is a member of Proteored, PRB3-ISCIII, and is supported by grant PT17/0019/009, of the PE I+D+I 2013–2016 funded by ISCIII and FEDER. This work is partly funded by the European Commission, Directorate-General Communications Networks, Content and Technology (DG CONNECT) under the ERA-NET Cofund scheme - Horizon 2020 'Horizon 2020 – the Framework Programme for Research and Innovation ( 2014–2020 )' for the project OPTIMO and by National Research Council of Italy (CNR) within the Short Term Mobility program (STM 2017 ).

(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. This research is funded by the Navarre Association of ALS’s patients and relatives (ADELA-Navarra) and the Navarrabiomed-Miguel Servet Foundation, through “Ice Bucket Challenge” campaign donations. M.L.-M. was supported by a pre-doctoral fellowship from the Public University of Navarra (UPNA).

Aldosterone (Aldo) contributes to mitochondrial dysfunction and cardiac oxidative stress. Using a proteomic approach, A-kinase anchor protein (AKAP)-12 has been identified as a down-regulated protein by Aldo in human cardiac fibroblasts. We aim to characterize whether AKAP-12 down-regulation could be a deleterious mechanism which induces mitochondrial dysfunction and oxidative stress in cardiac cells. Aldo down-regulated AKAP-12 via its mineralocorticoid receptor, increased oxidative stress and induced mitochondrial dysfunction characterized by decreased mitochondrial-DNA and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expressions in human cardiac fibroblasts. CRISPR/Cas9-mediated knock-down of AKAP-12 produced similar deleterious effects in human cardiac fibroblasts. CRISPR/Cas9-mediated activation of AKAP-12 blunted Aldo effects on mitochondrial dysfunction and oxidative stress in human cardiac fibroblasts. In Aldo-salt-treated rats, cardiac AKAP-12, mitochondrial-DNA and PGC-1α expressions were decreased and paralleled increased oxidative stress. In myocardial biopsies from patients with aortic stenosis (AS, n = 26), AKAP-12, mitochondrial-DNA and PGC-1α expressions were decreased as compared to Controls (n = 13). Circulating Aldo levels inversely correlated with cardiac AKAP-12. PGC-1α positively associated with AKAP-12 and with mitochondrial-DNA. Aldo decreased AKAP-12 expression, impairing mitochondrial biogenesis and increasing cardiac oxidative stress. AKAP-12 down-regulation triggered by Aldo may represent an important event in the development of mitochondrial dysfunction and cardiac oxidative stress. This work was supported by: Miguel Servet contract CP13/00221 from the 'Instituto de Salud Carlos III-FEDER', Fondo de Investigaciones Sanitarias [PI15/02160], FIBROTARGETS project (FP7 #602904), ANR-MR focus (15-CE14-0032), Plan Estatal I+D+I 2013-2016 and Ministry of Economy and Competitiveness (SAF2016-79151-R). The Proteomics Unit of Navarrabiomed is a member of Proteored, PRB2-ISCIII, and is supported by grant PT13/0001, of the PE I+D+I 2013–2016 funded by ISCIII and FEDER.

Olfactory dysfunction is present in up to 90% of Alzheimer's disease (AD) patients. Although deposition of hyperphosphorylated tau and β-amyloid substrates are present in olfactory areas, the molecular mechanisms associated with decreased smell function are not completely understood. We have applied mass spectrometry-based quantitative proteomics to probe additional molecular disturbances in postmortem olfactory bulbs (OB) dissected from AD cases respect to neurologically intact controls (n=20, mean age 82.1 years). Relative proteome abundance measurements have revealed protein interaction networks progressively disturbed across AD stages suggesting an early imbalance in splicing factors, subsequent interrupted cycling of neurotransmitters, alteration in toxic and protective mechanisms of β-amyloid, and finally, a mitochondrial dysfunction together with disturbance in neuron-neuron adhesion. We also present novel molecular findings in the OB in an autopsy cohort composed by Lewy body disease (LBD), frontotemporal lobar degeneration (FTLD), mixed dementia, and progressive supranuclear palsy (PSP) cases (n = 41, mean age 79.7 years). Olfactory mediators deregulated during the progression of AD such as Visinin-like protein 1, RUFY3 protein, and Copine 6 were also differentially modulated in the OB in LBD, FTLD, and mixed dementia. Only Dipeptidyl aminopeptidase-like protein 6 showed a specific down-regulation in AD. However, no differences were observed in the olfactory expression of this protein panel in PSP subjects. This study demonstrates an olfactory progressive proteome modulation in AD, unveiling cross-disease similarities and differences especially for specific proteins involved in dendritic and axonic distributions that occur in the OB during the neurodegenerative process.

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. This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (Ref. SAF2014-59340-R To ES), the Department of Economic Development from Government of Navarra (Ref. PC023-PC024, PC025, PC081-82 and PI059 to ES and PI031 to JFI), the Instituto de Salud Carlos III and Fondos Feder Europeos (PI16/00066 to MMA), the Spanish Ministry of Science and Innovation (IEDI-2015-00638 to MMA), the Department of Health of the Government of Navarra (to MMA), the Basque Foundation for Health Research (BIOEF, BIO13/CI/005 to MMA), Asociación Pablo Ugarte-Fuerza, Julen (to MMA). AGM was supported by PEJ-2014-A-61949 (MINECO), and a pre-doctoral fellowship from Public University of Navarra (UPNA). The Proteomics Unit of Navarrabiomed is a member of Proteored, PRB3-ISCIII, and is supported by grant PT17/0019/0009, of the PE I+D+I 2013-2016 funded by ISCIII and FEDER.

// Andrea Gonzalez-Morales 1, 2, 3 , Aintzane Zabaleta 2, 4 , Elizabeth Guruceaga 2, 5 , Marta M. Alonso 2, 6, 7 , Marc Garcia-Moure 2, 6, 7 , Joaquin Fernandez-Irigoyen 1, 2, 3, * , Enrique Santamaria 1, 2, 3, * 1 Clinical Neuroproteomics Group, Navarrabiomed, Complejo Hospitalario de Navarra (CHN), Universidad Publica de Navarra (UPNA), Irunlarrea, Pamplona, Spain 2 IDISNA, Navarra Institute for Health Research, Pamplona, Spain 3 Proteored-ISCIII, Proteomics Unit, Navarrabiomed, Complejo Hospitalario de Navarra (CHN), Universidad Publica de Navarra (UPNA), Irunlarrea, Pamplona, Spain 4 Oncohematology Area, University Hospital of Navarra, Center for Applied Medical Research, CIBERONC, Pamplona, Spain 5 Bioinformatics Unit, Center for Applied Medical Research, University of Navarra, Pamplona, Spain 6 Program in Solid Tumors and Biomarkers, Foundation for the Applied Medical Research, Pamplona, Spain 7 Department of Pediatrics, University Hospital of Navarra, Pamplona, Spain * These authors share senior authorship Correspondence to: Enrique Santamaria, email: esantamma@navarra.es Keywords: adenovirus; Delta-24RGD infection; proteomics; glioma Received: February 06, 2018 Accepted: June 22, 2018 Published: July 24, 2018 ABSTRACT Glioblastoma multiforme (GBM) is the most common and aggressive type of malignant glioma. Oncolytic adenoviruses are being modified to exploit the aberrant expression of proteins in tumor cells to increase the antiglioma efficacy. E1A mutant adenovirus Delta-24-RGD (DNX-2401) has shown a favorable toxicity profile and remarkable efficacy in a first-in-human phase I clinical trial. However, the comprehensive modulation of glioma metabolism in response to Delta-24-RGD infection is poorly understood. Integrating mass spectrometry based-quantitative proteomics, physical and functional interaction data, and biochemical approaches, we conducted a cell-wide study of cytosolic, nuclear, and secreted glioma proteomes throughout the early time course of Delta-24-RGD infection. In addition to the severe proteostasis impairment detected during the first hours post-infection (hpi), Delta-24-RGD induces a transient inhibition of signal transducer and activator of transcription 3 (STAT3), and transcription factor AP-1 (c-JUN) between 3 and 10hpi, increasing the nuclear factor kappa B (NF-κB) activity at 6hpi. Furthermore, Delta-24-RGD specifically modulates the activation dynamics of protein kinase C (PKC), extracellular signal–regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) pathways early in infection. At extracellular level, Delta-24-RGD triggers a time –dependent dynamic production of multitasking cytokines, and chemotactic factors, suggesting potential pleiotropic effects on the immune system reactivation. Taken together, these data help us to understand the mechanisms used by Delta-24-RGD to exploit glioma proteome organization. Further mining of this proteomic resource may enable design and engineering complementary adenoviral based-vectors to increase the specificity and potency against glioma.

Thrombotic material retrieved from acute ischemic stroke (AIS) patients represents a valuable source of biological information. In this study, we have developed a clinical proteomics workflow to characterize the protein cargo of thrombi derived from AIS patients. To analyze the thrombus proteome in a large-scale format, we developed a workflow that combines the isolation of thrombus by endovascular thrombectomy and peptide chromatographic fractionation coupled to mass-spectrometry. Using this workflow, we have characterized a specific proteomic expression profile derived from four AIS patients included in this study. Around 1600 protein species were unambiguously identified in the analyzed material. Functional bioinformatics analyses were performed, emphasizing a clustering of proteins with immunological functions as well as cardiopathy-related proteins with blood-cell dependent functions and peripheral vascular processes. In addition, we established a reference proteomic fingerprint of 341 proteins commonly detected in all patients. Protein interactome network of this subproteome revealed protein clusters involved in the interaction of fibronectin with 14-3-3 proteins, TGFβ signaling, and TCP complex network. Taken together, our data contributes to the repertoire of the human thrombus proteome, serving as a reference library to increase our knowledge about the molecular basis of thrombus derived from AIS patients, paving the way toward the establishment of a quantitative approach necessary to detect and characterize potential novel biomarkers in the stroke field. This work was supported by Navarre Government Funding (Industry and Health department) through ADITECH (PT030) and Department of Economic Development from Government of Navarra (Ref. PC025), and Obra Social la Caixa to ES. KA are supported by PEJ-2014-A-61949 (MINECO). The Proteomics Unit of Navarrabiomed is a member of Proteored, PRB3-ISCIII, and is supported by grant PT17/0019/009 to JF, of the PE I+D+I 2013–2016 funded by ISCIII and FEDER

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. This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (Ref. SAF2014-59340-R), Department of Economic Development from Government of Navarra (Ref. PC023-24) and Obra Social la Caixa to Enrique Santamaría. Andrea González-Morales and Karina Ausín were supported by PEJ-2014-A-61949 and PEJ-2014-A-72151 (MINECO). Mercedes Lachén-Montes is supported by a predoctoral fellowship from the Public University of Navarra (UPNA). The Proteomics Unit of Navarrabiomed is a member of Proteored, PRB2-ISCIII, and is supported by grant PT13/0001, of the PE I + D + I 2013–2016 funded by ISCIII and FEDER.

Aims Galectin-3 (Gal-3), a β-galactoside-binding lectin involved in cardiac inflammation and fibrosis, could regulate oxidative stress, although the mechanisms have not been elucidated. We herein investigated the changes in oxidative stress-related mediators induced by Gal-3 in human cardiac fibroblasts and in pathological animal and human models of cardiac diseases. Results Using quantitative proteomics and immunodetection approaches, we have identified that Gal-3 down-regulated fumarate hydratase (FH) in human cardiac fibroblasts. In parallel, Gal-3 increased fumarate production in a time-dependent manner. Gal-3 treatment enhanced carbonylated proteins detected through OxyBlot technique. Interestingly, treatment of cells with fumarate induced oxidative stress, enhanced fibroblast activation markers and increased collagen and interleukin-6 secretion. In Gal-3-silenced cells and in heart from Gal-3 knock-out mice, FH was increased and fumarate was decreased. In myocardial biopsies from patients with aortic stenosis (AS, n =26), FH levels were decreased as compared to Controls ( n =13). Cardiac Gal-3 inversely correlated with FH levels in myocardial biopsies. In an experimental model of AS rats, pharmacological inhibition of Gal-3 restored cardiac FH, decreased fumarate concentration and improved oxidative status. Conclusion In human cardiac fibroblasts, Gal-3 decreased FH expression increasing fumarate concentration and promoting oxidative stress. In human AS, cardiac levels of Gal-3 inversely associated with FH. Gal-3 blockade restored FH and improved fumarate and oxidative stress status in AS rats. FH is therefore a key molecule mediating Gal-3-induced oxidative stress in cardiac cells.

Cardiac fibrosis is characterized by an excessive accumulation of extracellular matrix components, including collagens. Galectin-3 (Gal-3) and Cardiotrophin-1 (CT-1) are two profibrotic molecules that mediate Aldosterone (Aldo)-induced cardiac fibrosis. However the underlying mechanisms are not well defined. Our aim is to characterize changes in the proteome of human cardiac fibroblasts treated with Aldo, Gal-3 or CT-1 to identify new common proteins that might be new therapeutic targets in cardiac fibrosis. Using a quantitative proteomic approach in human cardiac fibroblasts, our results show that Aldo, Gal-3 and CT-1 modified the expression of 30, 17 and 89 proteins respectively, being common the reticulocalbin (RCN) family members. RCN-3 down-regulation triggered by Aldo, Gal-3 and CT-1 was verified. Treatment with recombinant RCN-3 decreased collagens expression in human cardiac fibroblasts through Akt phosphorylation. Interestingly, CRISPR/Cas9-mediated activation of RCN-3 decreased collagen production in human cardiac fibroblasts. In addition, recombinant RCN-3 blocked the profibrotic effects of Aldo, Gal-3 and CT-1. Interestingly, RCN-3 blunted the increase in collagens expression induced by other profibrotic stimuli, angiotensin II, in human cardiac fibroblasts. Our results suggest that RCN-3 emerges as a new potential negative regulator of collagen production and could represent a therapeutic target in the context of cardiac fibrosis.

Aldosterone (Aldo) could induce cardiac fibrosis, a hallmark of heart disease. Aldo direct effects on collagen production in cardiac fibroblasts remain controversial. Our aim is to characterize changes in the proteome of adult human cardiac fibroblasts treated with Aldo to identify new proteins altered that might be new therapeutic targets in cardiovascular diseases. Aldo increased collagens expressions in human cardiac fibroblasts. Complementary, using a quantitative proteomic approach, 30 proteins were found differentially expressed between control and Aldo-treated cardiac fibroblasts. Among these proteins, 7 were up-regulated and 23 were down-regulated by Aldo. From the up-regulated proteins, collagen type I, collagen type III, collagen type VI and S100-A11 were verified by Western blot. Moreover, protein interaction networks revealed a functional link between a third of Aldo-modulated proteome and specific survival routes. S100-A11 was identified as a possible link between Aldo and collagen. Interestingly, CRISPR/Cas9-mediated knock-down of S100-A11 blocked Aldo-induced collagen production in human cardiac fibroblasts. In adult human cardiac fibroblasts treated with Aldo, proteomic analyses revealed an increase in collagen production. S100-A11 was identified as a new regulator of Aldo-induced collagen production in human cardiac fibroblasts. These data could identify new candidate proteins for the treatment of cardiac fibrosis in cardiovascular diseases. Significance S100-A11 is identified by a proteomic approach as a novel regulator of Aldosterone-induced collagen production in human cardiac fibroblasts. Our data could identify new candidate proteins of interest for the treatment of cardiac fibrosis in cardiovascular diseases.

Pancreatic ductal adenocarcinoma, which represents 80% of pancreatic cancers, is mainly diagnosed when treatment with curative intent is not possible. Consequently, the overall five-year survival rate is extremely dismal—around 5% to 7%. In addition, pancreatic cancer is expected to become the second leading cause of cancer-related death by 2030. Therefore, advances in screening, prevention and treatment are urgently needed. Fortunately, a wide range of approaches could help shed light in this area. Beyond the use of cytological or histological samples focusing in diagnosis, a plethora of new approaches are currently being used for a deeper characterization of pancreatic ductal adenocarcinoma, including genetic, epigenetic, and/or proteo-transcriptomic techniques. Accordingly, the development of new analytical technologies using body fluids (blood, bile, urine, etc.) to analyze tumor derived molecules has become a priority in pancreatic ductal adenocarcinoma due to the hard accessibility to tumor samples. These types of technologies will lead us to improve the outcome of pancreatic ductal adenocarcinoma patients. This work was funded by grants from Department of Health from Government of Navarra (Ref. 008-2018), REFBIO II Pyrenees Biomedical Network from Programa INTERREG V-A España-Francia-Andora (Ref. BMK_PANC) and Sociedad Española de Oncología Médica (SEOM) to A.V. I.G.-B. is supported by a predoctoral fellowship from Department of Economic Development Government of Navarre 'Ayudas para la contratación de doctorandos y doctorandas por empresas y organismos de investigación y difusión de conocimientos: doctorados industriales 2018–2020'.

// Mercedes Lachen-Montes 1, 2 , Maria Victoria Zelaya 1, 2, 3 , Victor Segura 2, 4 , Joaquin Fernandez-Irigoyen 1, 2, 5, * and Enrique Santamaria 1, 2, 5, * 1 Clinical Neuroproteomics Group, Navarrabiomed, Departamento de Salud, Universidad Publica de Navarra, Pamplona, Spain 2 IDISNA, Navarra Institute for Health Research, Pamplona, Spain 3 Pathological Anatomy Department, Navarra Hospital Complex, Pamplona, Spain 4 Bioinformatics Unit, Center for Applied Medical Research, University of Navarra, Pamplona, Spain 5 Proteored-ISCIII, Proteomics Unit, Navarrabiomed, Departamento de Salud, Universidad Publica de Navarra, Pamplona, Spain * These authors share senior authorship Correspondence to: Enrique Santamaria, email: esantamma@navarra.es Keywords: Alzheimer, neurodegeneration, dementia, olfactory bulb, transcriptomics Received: January 21, 2017 Accepted: May 07, 2017 Published: May 23, 2017 ABSTRACT 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 stage-dependent 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 network-driven 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.

Olfactory dysfunction is an early event of Alzheimer's disease (AD). However, the mechanisms associated to AD neurodegeneration in olfactory areas are unknown. Here we used double-transgenic amyloid precursor protein/presenilin 1 (APPswe/PS1dE9) mice and label-free quantitative proteomics to analyze early pathological effects on the olfactory bulb (OB) during AD progression. Prior to β-amyloid plaque formation, 9 modulated proteins were detected on 3-month-old APP/PS1 mice while 16 differential expressed proteins were detected at 6 months, when β-amyloid plaques appear, indicating a moderate imbalance in cytoskeletal rearrangement, and synaptic plasticity in APP/PS1 OBs. Moreover, β-amyloid induced an inactivation of focal adhesion kinase (FAK) together with a transient activation of MEK1/2, leading to inactivation of ERK1/2 in 6-months APP/PS1 OBs. In contrast, the analysis of human OBs revealed a late activation of FAK in advanced AD stages, whereas ERK1/2 activation was enhanced across AD staging respect to controls. This survival potential was accompanied by the inhibition of the proapototic factor BAD in the OB across AD phenotypes. Our data contribute to a better understanding of the early molecular mechanisms that are modulated in AD neurodegeneration, highlighting significant differences in the regulation of survival pathways between APP/PS1 mice and sporadic human AD. Significance Loss of smell is involved in early stages of Alzheimer's disease (AD), usually preceding classic disease symptoms. However, the mechanisms governing this dysfunction are still poorly understood, losing its potential as a useful tool for clinical diagnosis. Our study characterizes potential AD-associated molecular changes in APP/PS1 mice olfactory bulb (OB) using MS-quantitative proteomics, revealing early cytoskeletal disruption and synaptic plasticity impairment. Moreover, an opposite pattern was found when comparing the activation status of specific survival pathways between APP/PS1 OBs and OBs derived from sAD subjects with different neuropathological grading. Our data reflect, in part, the progressive effect of APP overproduction and Aβ accumulation on the OB proteome during AD progression.

Primary liver cancer is the fifth most frequent neoplasm and the third most common cause of cancer-related death, with more than 500,000 new cases diagnosed yearly. The outcome for hepatocellular carcinoma (HCC) patients still remains dismal, partly because of our limited knowledge of its molecular pathogenesis and the difficulty in detecting the disease at its early stages. Therefore, studies aimed at the definition of the mechanisms associated with HCC progression and the identification of new biomarkers leading to early diagnosis and more effective therapeutic interventions are urgently needed. Proteomics is a rapidly expanding discipline that is expected to change the way in which diseases will be diagnosed, treated, and monitored in the near future. In the last few years, HCC has been extensively investigated using different proteomic approaches on HCC cell lines, animal models, and human tumor tissues. In this review, state-of-the-art technology on proteomics is overviewed, and recent advances in liver cancer proteomics and their clinical projections are discussed.

Methionine adenosyltransferases (MAT's) are central enzymes in living organisms that have been conserved with a high degree of homology among species. In the liver, MAT I and III, tetrameric and dimeric isoforms of the same catalytic subunit encoded by the gene MAT1A, account for the predominant portion of total body synthesis of S-adenosylmethionine (SAM), a versatile sulfonium ion-containing molecule involved in a variety of vital metabolic reactions and in the control of hepatocyte proliferation and differentiation. During the past 15 years 28 MAT1A mutations have been described in patients with elevated plasma methionines, total homocysteines at most only moderately elevated, and normal levels of tyrosine and other aminoacids. In this study we describe functional analyses that determine the MAT and tripolyphosphatase (PPPase) activities of 18 MAT1A variants, six of them novel, and none of them previously assayed for activity. With the exception of G69S and Y92H, all recombinant proteins showed impairment (usually severe) of MAT activity. Tripolyphosphate (PPPi) hydrolysis was decreased only in some mutant proteins but, when it was decreased MAT activity was always also impaired.

S-adenosylmethionine arises as a central molecule in the preservation of liver homeostasis as a chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. In the present work, we have attempted a comprehensive analysis of proteins associated with hepatocarcinogenesis in MAT1A knock out mice using a combination of two-dimensional electrophoresis and mass spectrometry, to then apply the resulting information to identify hallmarks of human HCC. Our results suggest the existence of individual-specific factors that might condition the development of preneoplastic lesions. Proteomic analysis allowed the identification of 151 differential proteins in MAT1A-/- mice tumors. Among all differential proteins, 27 changed in at least 50% of the analyzed tumors, and some of these alterations were already detected months before the development of HCC in the KO liver. The expression level of genes coding for 13 of these proteins was markedly decreased in human HCC. Interestingly, seven of these genes were also found to be down-regulated in a pretumoral condition such as cirrhosis, while depletion of only one marker was assessed in less severe liver disorders.

e16782 Background: New BKs are urgently needed in order to improve PDAC patient’s outcome. In this sense, the main aim of this multicenter and prospective project is to find predictive and/or prognostic BKs based on many cytokines that are produced in cancer microenvironment and can nowadays be accurately analyzed. Methods: Patients diagnosed of potentially resectable PDAC with poor prognostic factors and treated with neoadjuvant treatment are being included. Serial analyses (basal, with TC, with surgery and time of relapse/or after 1y of follow-up) of serum levels of 80 cytokines are being done. Results: With a median follow-up of 11,25 months (m) (4,67-38,28) 22 patients (pt) with median of age of 62,5 y-0, most of them men (63.6%) have been included, with radical surgery performed in 14 of them (2 pts with bypass). Seven operated pt received adjuvant treatment and 12 operated pt developed progression of disease. 18 pt have received nab-paclitaxel and gemcitabine as perioperative treatment and 4 received Folfirinox. Perineural and vascular invasion were confirmed after surgery in 45,5% and 27,3% of cases, respectively. Positive nodes were found in 40,9% of them, being the median of positive and resected nodes of 1 (0-14) and 26 respectively. Surgical margins were negative (R0) in 64,3% of cases (35,7% R1). Pathological response was described as partial in 13,6%, with no changes in 31,8% and progression in 13,6%. Globally, PFS and OS were 13,50 m (CI95%: 6,44-20,55) and 17,31 m (CI 95% 8,86-25,76). Significant differences (p = 0,002) were observed in cases in which radical surgery had been performed with PFS of 13,5 m (9,34-17,66) and 3.97 m (0-8,23) for unresectable patients. Regarding the cytokine array, we observed different PFS outcome based on basal MCP3 levels (low: 16,32 m CI 95%: 5,02-27,63; high: 7,52 m CI95%: 6,23-8.81, p = 0,023), basal Oncostatin (high: 7,12 m CI95%: 3,09-11,15, low: not reached; p = 0,007) or basal Eotaxin (low: 7,12 m CI 95%: 5,75-8,50; high: not reached, p = 0,029). A cytokine score was created based on these results, maintained significant differences for PFS (16,32 m CI95%: 5,07-27,57 vs 6,53 m CI95%: 2,75-10,32, p = 0.003). Our cytokine score showed a significant role as independent prognosis factor in multivariate analysis (HR: 0,196 CI95%: 0,042-0,918; p = 0.039). Conclusions: In potentially resectable/borderline PDAC our basal cytokine score seems to be able to discriminate as independent prognosis factor between those patients that will get significant benefit from neoadjuvant treatment from those that will not

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. This work was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (Ref. SAF2014-59340-R), Department of Economic Development from Government of Navarra (Ref. PC025), and Obra Social la Caixa to ES. AGM and KA are supported by PEJ-2014-A-61949 and PEJ-2014-A-72151 (MINECO). EPV is supported by PTA-2013/8711/I (MINECO). MLM is supported by a predoctoral fellowship from the Public University of Navarra (UPNA). The Proteomics Unit of Navarrabiomed is a member of Proteored, PRB2-ISCIII, and is supported by grant PT13/0001, of the PE I + D + I 2013-2016 funded by ISCIII and FEDER.

Olfactory dysfunction is one of the earliest features in Lewy-type alpha-synucleinopathies (LTSs) such as Parkinson's disease (PD). However, the underlying molecular mechanisms associated to smell impairment are poorly understood. Applying mass spectrometry–based quantitative proteomics in postmortem olfactory bulbs across limbic, early-neocortical, and neocortical LTS stages of parkinsonian patients, a proteostasis impairment, was observed, identifying 268 differentially expressed proteins between controls and PD phenotypes. In addition, network-driven proteomics revealed a modulation in ERK1/2, MKK3/6, and PDK1/PKC signaling axes. Moreover, a cross-disease study of selected olfactory molecules in sporadic Alzheimer's disease (AD) cases revealed different protein derangements in the modulation of secretagogin (SCGN), calcyclin-binding protein (CACYBP), and glucosamine 6 phosphate isomerase 2 (GNPDA2) between PD and AD. An inverse correlation between GNPDA2 and α-synuclein protein levels was also reflected in PD cerebrospinal fluid. Interestingly, PD patients exhibited significantly lower serum GNPDA2 levels than controls (n = 82/group). Our study provides important avenues for understanding the olfactory bulb proteostasis imbalance in PD, deciphering mechanistic clues to the equivalent smell deficits observed in AD and PD pathologies.