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The neocortex of P301S mice, used as a model of fronto-temporal lobar degeneration linked to tau mutation (FTLD-tau), and wild-type mice, both aged 9 months, were analyzed with conventional label-free phosphoproteomics 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 328 corresponding to 524 phosphorylation sites. The majority of dysregulated phosphoproteins, most of them hyperphosphorylated, were proteins of the membranes, synapses, membrane trafficking, membrane vesicles linked to endo- and exocytosis, cytoplasmic vesicles, and cytoskeleton. Another group was composed of kinases. In contrast, proteins linked to DNA, RNA metabolism, RNA splicing, and protein synthesis were hypophosphorylated. Other pathways modulating energy metabolism, cell signaling, Golgi apparatus, carbohydrates, and lipids are also targets of dysregulated protein phosphorylation in P301S mice. The present results, together with accompanying immunohistochemical and Western-blotting studies, show widespread abnormal phosphorylation of proteins, in addition to protein tau, in P301S mice. These observations point to dysregulated protein phosphorylation as a relevant contributory pathogenic component of tauopathies.

The neocortex of P301S mice, used as a model of frontotemporal degeneration linked to tau mutation, and wild-type mice, both aged 9 months, were analyzed with conventional label-free phosphoproteomics 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 328 corresponding to 524 phosphorylation sites. The majority of dysregulated phosphoproteins, most of them hyper-phosphorylated, were proteins of the membranes, synapses, membrane trafficking, membrane vesicles linked to endo- and exocytosis, cytoplasmic vesicles, and cytoskeleton. Another group was composed of kinases. In contrast, proteins linked to DNA, RNA metabolism, RNA splicing, and protein synthesis were hypo-phosphorylated. Other pathways modulating energy metabolism, cell signalling, Golgi apparatus, carbohydrates, and lipids are also targets of dysregulated protein phosphorylation in P301S mice. The present results, together with accompanying immunohistochemical and western blotting studies, show widespread abnormal phosphorylation of proteins, in addition to protein tau, in P301S mice. These observations point to dysregulated protein phosphorylation as a relevant contributory pathogenic component of tauopathies.

This study, which performs an extensive mass spectrometry-based analysis of 19 brain regions from both left and right hemispheres, presents the first draft of the human brain interhemispheric proteome. This high-resolution proteomics data provides comprehensive coverage of 3300 experimentally measured (nonhypothetical) proteins across multiple regions, allowing the characterization of protein-centric interhemispheric differences and synapse biology, and portrays the regional mapping of specific regions for brain disorder biomarkers. In the context of the Human Proteome Project (HPP), the interhemispheric proteome data reveal specific markers like chimerin 2 (CHN2) in the cerebellar vermis, olfactory marker protein (OMP) in the olfactory bulb, and ankyrin repeat domain 63 (ANKRD63) in basal ganglia, in line with regional brain transcriptomes mapped in the Human Protein Atlas (HPA). In addition, an in silico analysis pipeline was used to predict the structure and function of the uncharacterized uPE1 protein ANKRD63, and parallel reaction monitoring (PRM) was applied to validate its region-specific expression. Finally, we have built the Interhemispheric Brain Proteome Map (IBPM) Portal (www.brainprot.org) to stimulate the scientific community's interest in the brain molecular landscape and accelerate and support research in neuroproteomics. Data are available via ProteomeXchange with identifier PXD019936.

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 completion and annotation of the human proteome require the availability of information related to protein function. Currently, more than 1800 human genes constitute the "dark proteome," which include missing proteins, uncharacterized human genes validated at protein level, smORFs, proteins from lncRNAs, or any uncharacterized transcripts. During the last years, different experimental workflows based on multi-omics analyses, bioinformatics, and in vitro and in vivo studies have been promoted by the Human Proteome Project Consortium to enhance the annotation of dark proteins. In this chapter, we describe a method that utilizes recombinant proteins and antibody arrays to establish a straightforward methodology in order to rapidly characterize potential functional features of dark proteins associated to intracellular signaling dynamics and extracellular immune response in human cell cultures. Further validating the method, this workflow was applied to probe changes in the activation patterns of kinases and transcription factors as well as in cytokine production modulated by the dark C1orf128 (PITHD1) protein in human olfactory neuroepithelial cells.

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.

Monocytic and granulocytic myeloid-derived suppressor cells together with tumor-infiltrating macrophages constitute the main tumor-infiltrating immunosuppressive myeloid populations. Due to the phenotypic resemblance to conventional myeloid cells, their identification and purification from within the tumors is technically difficult and makes their study a challenge. We differentiated myeloid cells modeling the three main tumor-infiltrating types together with uncommitted macrophages, using ex vivo differentiation methods resembling the tumor microenvironment. The phenotype and proteome of these cells was compared to identify linage-dependent relationships and cancer-specific interactome expression modules. The relationships between monocytic MDSCs and TAMs, monocytic MDSCs and granulocytic MDSCs, and hierarchical relationships of expression networks and transcription factors due to lineage and cancer polarization were mapped. Highly purified immunosuppressive myeloid cell populations that model tumor-infiltrating counterparts were systematically analyzed by quantitative proteomics. Full functional interactome maps have been generated to characterize at high resolution the relationships between the three main myeloid tumor-infiltrating cell types. Our data highlights the biological processes related to each cell type, and uncover novel shared and differential molecular targets. Moreover, the high numbers and fidelity of ex vivo-generated subsets to their natu-ral tumor-shaped counterparts enable their use for validation of new treatments in high-throughput experiments. The Oncoimmunology Group is funded by the Spanish Association against Cancer (AECC, PROYE16001ESCO); Instituto de Salud Carlos III (ISCIII)-FEDER Project Grants FIS PI17/02119, FIS PI20/00010; COV20/00000, and TRANSPOCART ICI19/00069; Biomedicine Project Grant from the Department of Health of the Government of Navarre-FEDER funds (BMED 050-2019); strategic projects from the Department of Industry, Government of Navarre (AGATA, Ref. 0011-1411-2020-000013; LINTERNA, Ref. 0011-1411-2020-000033; DESCARTHES, 0011-1411-2019-000058); European Project Horizon 2020 Improved Vaccination for Older Adults (ISOLDA; ID: 848166).

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.

The completion and annotation of the human proteome require the availability of information related to protein function. Currently, more than 1800 human genes constitute the "dark proteome," which include missing proteins, uncharacterized human genes validated at protein level, smORFs, proteins from lncRNAs, or any uncharacterized transcripts. During the last years, different experimental workflows based on multi-omics analyses, bioinformatics, and in vitro and in vivo studies have been promoted by the Human Proteome Project Consortium to enhance the annotation of dark proteins. In this chapter, we describe a method that utilizes recombinant proteins and antibody arrays to establish a straightforward methodology in order to rapidly characterize potential functional features of dark proteins associated to intracellular signaling dynamics and extracellular immune response in human cell cultures. Further validating the method, this workflow was applied to probe changes in the activation patterns of kinases and transcription factors as well as in cytokine production modulated by the dark C1orf128 (PITHD1) protein in human olfactory neuroepithelial cells.

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. The mechanisms of adaptation to long-term silencing of AKT isoforms of pancreatic 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 up-regulation, 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 vivopharmacological co-inhibition of AKT and mitochondrial metabolism effectively controlled pancreatic adenocarcinoma growth in pre-clinical models.

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.

Incluye material complementario The Human Proteome Project (HPP) consortium aims to functionally characterize the dark proteome. On the basis of the relevance of olfaction in early neurodegeneration, we have analyzed the dark proteome using data mining in public resources and omics data sets derived from the human olfactory system. Multiple dark proteins localize at synaptic terminals and may be involved in amyloidopathies such as Alzheimer's disease (AD). We have characterized the dark PITH domain-containing protein 1 (PITHD1) in olfactory metabolism using bioinformatics, proteomics, in vitro and in vivo studies, and neuropathology. PITHD1-/- mice exhibit olfactory bulb (OB) proteome changes related to synaptic transmission, cognition, and memory. OB PITHD1 expression increases with age in wild-type (WT) mice and decreases in Tg2576 AD mice at late stages. The analysis across 6 neurological disorders reveals that olfactory tract (OT) PITHD1 is specifically upregulated in human AD. Stimulation of olfactory neuroepithelial (ON) cells with PITHD1 alters the ON phosphoproteome, modifies the proliferation rate, and induces a pro-inflammatory phenotype. This workflow applied by the Spanish C-HPP and Human Brain Proteome Project (HBPP) teams across the ON-OB-OT axis can be adapted as a guidance to decipher functional features of dark proteins. Data are available via ProteomeXchange with identifiers PXD018784 and PXD021634. This work was funded by grants from the Spanish Ministry of Science Innovation and Universities (ref. PID2019-110356RB-I00 to JFI and ES) and Department of Economic and Business Development from Government of Navarra (ref. 0011-1411-2020-000028) to ES and from MEXT-JSPS 17K08884 and Takeda Science Foundation to IO. The Proteomics Unit of Navarrabiomed is a member of Proteored (PRB3-ISCIII) and is supported by grant PT17/0019/009 to JFI, of the PE I+D+I 2013–2016 funded by ISCIII and FEDER. MLM was supported by a postdoctoral fellowship from the Public University of Navarra (UPNA).

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.

(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).

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.

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.

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.