Biology subjects

Fernandez-Irigoyen, J.

Publications and source records attributed to Fernandez-Irigoyen, J..

10 recordsLinked to original sources

bioRxiv2026

A generalizable normalization framework to decouple protocol and instrument effects: Application to high-sensitivity proteomics multicentric study (PME13)

Multicenter studies are essential for benchmarking analytical workflows, yet their interpretation is often confounded by the combined effects of experimental protocols and instrumentation. To address this challenge, we introduce a simple normalization-based analytical framework, the recovery metric ({rho}), designed to decouple protocol driven effects from instrument dependent variability. We applied this framework to the 13th Proteomics Multicentric Experiment (PME13), a large multicentric proteomics dataset generated across 27 laboratories using high sensitivity workflows and varying sample preparation protocols. By leveraging a common digested reference sample, {rho} enables direct cross-comparison of all datasets on a unified scale, effectively minimizing instrument-related biases. Using this approach, we demonstrate that apparent instrument dependent trends are largely removed when evaluated through {rho}, revealing consistent protocol driven effects across laboratories. Statistical modeling identified key variables influencing {rho}, including sample input amount, reduction and alkylation, and the use of n-dodecyl-{beta}-D-maltoside (DDM). While DDM was associated with improved {rho}, reduction and alkylation and additional handling steps led to reduced performance, particularly at low input levels. We further highlight practical considerations for the application of ratio based normalization, including the occurrence of values exceeding theoretical bounds, which reflect deviations from underlying assumptions and require appropriate filtering. Overall, this work establishes a generalizable analytical strategy for disentangling confounding factors in multicentric datasets and provides practical guidelines for optimizing high sensitivity proteomics (HSP) workflows. The proposed framework is broadly applicable to other analytical fields where cross laboratory comparability is required.

bioinformatics↗

bioRxiv2026

Multi-omics characterization of SIRT3 metabolism and its adaptation to the presence of amyloid-beta oligomers in nasal epithelial cells

Sirtuins (SIRTs) are nicotinamide adenine dinucleotide (NAD{square})-dependent deacetylases that regulate cellular homeostasis in a multifactorial manner. Although alterations in SIRT signaling are evidenced in both olfactory dysfunction and Alzheimers disease (AD), the specific role of sirtuin 3 (SIRT3) in olfactory metabolism remains unknown. Here, we have evidenced a partial interdependency between SIRT3 and SIRT5 deacetylase members in human nasal epithelial cell cultures (hNECs). A multi-omic integrative approach applied to conditions of SIRT3 silencing or overexpression revealed that hNEC metabolism is markedly more sensitive to reduced SIRT3 levels, identifying specific transcripts and phosphorylation sites belonging to inflammatory and redox mediators that are tightly regulated by SIRT3 in hNECs. Following exposure to oligomeric A{beta} peptide, phosphoproteomic alterations promoted an activation trend of stress-induced senescence and apoptotic signaling in SIRT3-silenced hNECs, whereas induced activation of mitotic phase-related pathways, Hippo signaling, and glycogen metabolism were evidenced in SIRT3-overexpressing hNECs. From a translational point of view, a dissimilar sex-dependent profile in serum SIRT protein levels (SIRT1 and SIRT6) was observed across multiple neurological disorders including AD, mixed dementia, frontotemporal lobar degeneration and amyotrophic lateral sclerosis. These data shed new light on novel SIRT-dependent mechanisms associated with neurodegeneration, underscoring that the maintenance of optimal SIRT3 protein levels may partially counteract the detrimental effects induced by A{beta} oligomers in AD at olfactory level.

neuroscience↗

bioRxiv2025

Phospho-signalling deregulation of the mTOR pathway in fibroblasts from LRRK2 Parkinson s patients

Activating mutations at the leucine-rich repeat kinase 2 (LRRK2) gene are the most frequent genetic cause of Parkinsons Disease (PD). Using in-depth mass-spectrometry, here we analysed the impact of LRRK2 mutations on the proteome and phospho-proteome of skin fibroblasts from a large LRRK2 clinical cohort from Spain. The cohort encompassed G2019S LRRK2-associated PD (L2PD) (n=15), G2019S LRRK2 non-manifesting carriers (L2NMCs) (n=13), idiopathic PD (iPD) (n=12), and control subjects (n=14) (total n=54). The proteome analysis revealed 89 differential proteins in G2019S L2PD (58 down/ 31 up) and 168 in G2019S L2NMCs (127 down / 41 up) compared to controls. Most of these proteome changes involved deficits in proteins related to mitochondrial energy and ribosomal protein synthesis. At the phospho-proteome level, we identified 394 differential phospho-sites in G2019S L2PD (188 hyper / 206 hypo), 367 in G2019S L2NMCs (215 hyper / 152 hypo), and 428 in iPD (214 hyper / 214 hypo) compared with controls. These phospho-proteomic changes included hyper-phosphorylated proteins associated with the mTOR and Rho GTPase pathways. Notably, the top hyper-phosphorylated hit in G2019S carriers was pThr37/46 4E-BP1, a key effector of the mTOR pathway controlling protein synthesis, and this protein was also deregulated in iPD. In summary, our study uncovers peripheral deregulation of mTOR phospho-signalling associated with LRRK2 mutations, which is related to deficits in mitochondrial and ribosomal proteins, and affects converging pathways altered in iPD.

neuroscience↗

bioRxiv2025

Early, sex-dependent and progressive proteomic imbalance in the amygdala during Alzheimers disease progression

BackgroundThe amygdala is involved in the emotional expression, memory processing and managing stimulatory input. Although amygdala atrophy is early evidenced in Alzheimers Disease (AD), the molecular mechanisms disrupted in initial neuropathological stages are still unknown. In the present study, we investigated the proteomic impairment of the amygdaloid region from AD-Braak stage I-II and III-IV subjects to better understand the neuropathological processes occurred early in this area and to identify potential targets that may face AD from the beginning of the disease. MethodsLabel-free quantitative proteomics was applied using an Orbitrap Exploris 480 mass-spectrometer in 24 postmortem amygdala specimens derived from non-demented (n=3F/5M), AD-Braak stage I-II (n=4F/4M) and AD-Braak stage III-IV (n=4F/4M). Data analysis was performed using MaxQuant and Perseus software (two-way Student T-test; p<0.05). Metascape and Ingenuity Pathway Analysis softwares were considered for biological interpretation. Connectivity map platform was used for drug repurposing analyses. Transcriptomic/proteomic data of other brain regions were obtained from AlzData, Neuropro, and Agora repositories. ResultsAmygdaloid proteome of AD-Braak stage I-II and III-IV subjects compared to controls revealed a progressive proteomic impairment with a minimal overlap across Braak stages. Some of the amygdaloid DEPs were known interactors of human A{beta} plaques, APP, or Tau proteins or were previously identified at transcriptional or translational level in other brain regions affected by AD. Interestingly, amygdaloid proteome was more severely affected in women than in men with a particular protein expression profile associated to each AD stage. Comparing our sex-dependent differential proteome datasets with transcriptomic data of different brain regions, we identified potential sex-specific proteins related to cognitive decline and neurodegeneration. Finally, data-driven drug repositioning using amygdaloid omics profiles unveiled that most of the small molecule candidates were neuropathological stage and/or sex-specific. ConclusionsEarly and sex-specific amygdaloid proteome dysregulation in AD highlights the consideration of a deliberate stratification by sex in future research and clinical trials to develop effective therapeutic strategies in AD for both sexes. Plain English summaryThe amygdala is a brain region involved in the expression of emotions, memory processing and managing incoming stimulus. Atrophy of this area is evidenced at the first stages of Alzheimers Disease (AD), pointing out a potential involvement of amygdala in the pathology of this disease. However, the molecular changes occurred early in this area are not fully understood. To this end, we interrogated the proteome of amygdala postmortem samples came from subjects of early AD stages. By applying data and functional analyses, we observed a stage-dependent and progressive proteomic impairment in this area. We detected proteins differentially expressed that were already known to interact with well-stablished neuropathological proteins or were altered in other brain areas. Importantly, data stratification by sex revealed that protein expression changes of amygdala were more abundant in women than men across AD progression. After comparing our results with published data in different brain regions affected by AD, we identified sex-specific proteins that could be used as biomarkers of cognitive decline and neurodegeneration. Finally, a drug repositioning-based approach proposed candidates with the potential to reverse amygdaloid malignant AD signature more effectively in one sex than in other or just in one sex. These observations highlight the consideration to include sex differences in future research to develop more precise and effective treatments in AD. Highlights{middle dot} Amygdaloid proteome experiences an increasing impairment across early neuropathological stages of AD, with a minimal overlap between Braak I-II and Braak III-IV {middle dot} Some of the amygdaloid DEPs are known interactors of human neuropathological A{beta} plaques, APP, or Tau proteins, or are potentially connected with them direct or indirectly {middle dot} Protein expression changes in AD amygdala are more abundant in women than men across Braak staging, with a particular protein expression profile associated to each AD stage {middle dot} There are potential sex-specific proteins related to cognitive decline and neurodegeneration expressed in amygdala and other different brain regions {middle dot} Drug candidates that potentially reverse the neuropathological amygdaloid proteome are sex-specific, highlighting the need to consider sex stratification in future research to improve results translatability and progress on the field.

neuroscience↗

bioRxiv2025

Olfactory proteomics reveals the capacity of the HDAC1 inhibitor pyroxamide to halt the α-synuclein preformed fibrils-induced damage in nasal epithelial, microglial and dopaminergic neuronal cell lines

Parkinsons disease (PD) is the second most common neurodegenerative disorder mainly characterized by the degeneration of dopaminergic neurons originating in the substantia nigra (SN) pars compacta and projecting to other brain regions, giving rise to motor and non-motor symptoms. Despite significant progress in understanding the molecular and cellular disruptions associated with PD, there remains an unmet clinical need for effective therapies. In this study, proteomic analysis of the olfactory tract (OT) in controls with no known neurological history (n=17) and PD subjects (n=21) revealed Lewy body disease (LBD) stage-dependent proteostatic impairment, accompanied by progressive modulation of the alpha-synuclein (-syn) functional interactome. Differential OT omic profiles (OMS) were used in a computational drug repurposing approach, reveling the HDAC1 inhibitor pyroxamide as one of the top drug candidates with in silico potential to restore altered OMS. To explore the potential therapeutic effects of pyroxamide, in vitro assays were performed using -syn preformed fibrils (PFFs). Pyroxamide treatment reduced -syn PFFs-induced toxicity in olfactory epithelial, microglial and dopaminergic neuronal cell lines, producing a protective effect against hydrogen peroxide-induced damage exclusively in brain-derived cell types. These findings confirm the suitability of omics profiles in drug repurposing workflows against PD, offering valuable insights into the potential of HDAC1 inhibitors in the therapeutic pipeline of PD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/679944v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1e7a8e8org.highwire.dtl.DTLVardef@229c2org.highwire.dtl.DTLVardef@39f5corg.highwire.dtl.DTLVardef@1d35105_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Microglial SIRT2 deficiency aggravates cognitive decline and amyloid pathology in Alzheimer's disease

Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, has been implicated in aging and neurodegenerative diseases such as Alzheimers disease (AD). While global SIRT2 inhibition has shown promise in reducing amyloid-beta pathology and cognitive deficits in different mouse models of AD, peripheral SIRT2 inhibition has been associated with adverse effects, such as increased inflammation. This suggests that targeted inhibition of specific cellular populations within the brain may represent a more precise and effective approach for the treatment of AD. To explore this hypothesis, we generated a conditional microglial SIRT2 knockout mouse model in the context of AD. Our results reveal that microglial SIRT2 reduction does not confer protective effects in the APP/PS1 model; rather, it aggravates cognitive decline, accelerates amyloid plaque deposition, and increases levels of pro-inflammatory cytokines at early stages of AD pathology. Transcriptomic analysis further indicates that SIRT2-deficient microglia exhibit altered expression of genes associated with aging and synaptic dysfunction. This phenotype was accompanied by increased phagocytosis of synaptic elements and impaired long-term potentiation. These findings suggest that while SIRT2 inhibition in some contexts may be beneficial, targeted inhibition within microglia could accelerate AD progression, underscoring the need for cell-specific approaches when considering SIRT2 as a therapeutic target.

neuroscience↗

bioRxiv2024

A double hit affecting the IKZF1-IKZF2 tandem in immune cells of schizophrenic patients regulate specific symptoms

Schizophrenia is a complex multifactorial disorder and increasing evidence suggests the involvement of immune dysregulations in its pathogenesis. We observed that IKZF1 and IKZF2, classic immune-related transcription factors (TFs), were both downregulated in patients peripheral blood mononuclear cells (PBMCs) but not in their brain. We generated a new mutant mouse model with a reduction in Ikzf1 and Ikzf2 to study the impact of those changes. Such mice developed deficits in the three dimensions (positive-negative-cognitive) of schizophrenic-like phenotypes associated with alterations in structural synaptic plasticity. We then studied the secretomes of cultured PBMCs obtained from human patients and identified potentially secreted molecules, which depended on IKZF1 and IKZF2 levels, and that in turn have an impact on neural synchrony, structural synaptic plasticity and schizophrenic-like symptoms in in vivo and in vitro models. Our results point out that IKZF1-IKZF2-dependent immune signals negatively impact on essential neural circuits involved in schizophrenia.

neuroscience↗

bioRxiv2023

Neuropathological stage-dependent proteome mapping of the olfactory tract in Alzheimer's disease: From early olfactory-related omics signatures to computational repurposing of drug candidates

Alzheimers disease (AD) is the most common form of dementia, characterized by an early olfactory dysfunction, progressive memory loss and behavioral deterioration. Albeit substantial progress has been made in characterizing AD-associated molecular and cellular events, there is an unmet clinical need for new therapies. In this work, olfactory tract proteotyping performed in controls and AD subjects (n=17/group) showed a Braak stage-dependent proteostatic impairment accompanied by the progressive modulation of amyloid precursor protein (APP) and tau functional interactomes. To implement a computational repurposing of drug candidates with capacity to reverse early AD-related olfactory omics signatures, we generated a consensual olfactory omics signatures (OMSs) database compiling differential omics datasets obtained by mass-spectrometry or RNA-sequencing derived from initial AD across the olfactory axis. Using the Connectivity Map (CMAP)-based drug repurposing approach, PKC, EGFR, Aurora kinase, Glycogen synthase kinase and CDK inhibitors were the top pharmacologic classes capable to restore multiple OMSs, whereas compounds with targeted activity to inhibit PI3K, IGF-1, microtubules and PLK represented a family of drugs with detrimental potential to induce olfactory AD-associated gene expression changes. In-vitro validation assays revealed that pretreatment of human neuron-like SH-SY5Y cells with the EGFR inhibitor AG-1478 showed a neuroprotective effect against hydrogen peroxide-induced damage while the pretreatment with the Aurora kinase inhibitor Reversine reduced amyloid-beta (A{beta})-induced neurotoxicity. Taken together, our data pointed out that olfactory omics signatures may be useful as substrates for drug repurposing to propose novel neuroprotective treatments against AD. STATEMENTSO_ST_ABSData availability statementC_ST_ABSMass-spectrometry data and search results files were deposited in the Proteome Xchange Consortium via the JPOST partner repository (https://repository.jpostdb.org) with the identifier PXD038061 for ProteomeXchange and JPST001921 for jPOST (for reviewers: https://repository.jpostdb.org/preview/1400199357636bce4231af5 Access key: 8609). The data supporting the findings of this study are available in Supplementary Material. Raw data are available from the corresponding author, upon reasonable request. Funding statementThis work was funded by grants from the Spanish Ministry of Science, Innovation and Universities (Ref. PID2019-110356RB-I00/AEI/10.13039/501100011033) to J.F.-I. and E.S. and the Department of Economic and Business Development from Government of Navarra (Ref. 0011-1411-2023-000028 to E.S.). PC-C was supported by a predoctoral fellowship from the Public University of Navarra (UPNA). ML-M is supported by a postdoctoral fellowship from Miguel Servet Foundation-Navarrabiomed. EA-C is supported by "Programa MRR Investigo 2023" in the framework of the European Union recovery and resilience facility. Conflict of interest disclosureAuthors declare that they have no conflicts of interest/financial disclosures. Ethics approval and patient consent statementAccording to the Spanish Law 14/2007 of Biomedical Research, inform written consent from several Spanish Neurological Tissue Banks was obtained for research purposes from relatives of subjects included in this study. According to the Declaration of Helsinki, all assessments, post-mortem evaluations, and experimental procedures were previously approved by the Clinical Ethics Committee of Navarra Health Service (Study code: PI_2019/108).

neuroscience↗

bioRxiv2022

GLUT1 ablation in astrocytes paradoxically improves central and peripheral glucose metabolism via enhanced insulin-stimulated ATP release

Astrocytes are considered an essential source of blood-borne glucose or its metabolites to neurons. Nonetheless, the necessity of the main astrocyte glucose transporter, i.e. GLUT1, for brain glucose metabolism has not been defined. Unexpectedly, we found that brain glucose metabolism was paradoxically augmented in mice with astrocytic GLUT1 ablation (GLUT11'GFAP mice). These mice also exhibited improved peripheral glucose metabolism especially in obesity, rendering them metabolically healthier. Importantly, GLUT11'GFAP mice did not present cognitive alterations. Mechanistically, we observed that GLUT1-ablated astrocytes exhibited increased insulin receptor-dependent ATP release, and both astrocyte insulin signalling and brain purinergic signalling are essential for improved brain function and systemic glucose metabolism. Collectively, we demonstrate that astrocytic GLUT1 is central to the regulation of brain energetics, yet its ablation triggers a reprogramming of brain metabolism sufficient to sustain energy requirements, peripheral glucose homeostasis and cognitive function.

neuroscience↗

bioRxiv2022

Metabolic dyshomeostasis induced by SARS-CoV-2 structural proteins reveals immunological insights into viral olfactory interactions

One of the most common symptoms in COVID-19 is a sudden loss of smell. SARS-CoV-2 has been detected in the olfactory bulb (OB) from animal models and sporadically in COVID-19 patients. To decipher the specific role over the SARS-CoV-2 proteome at olfactory level, we characterized the in-depth molecular imbalance induced by the expression of GFP-tagged SARS-CoV-2 structural proteins (M, N, E, S) on mouse OB cells. Transcriptomic and proteomic trajectories uncovered a widespread metabolic remodeling commonly converging in extracellular matrix organization, lipid metabolism and signaling by receptor tyrosine kinases. The molecular singularities and specific interactome expression modules were also characterized for each viral structural factor. The intracellular molecular imbalance induced by each SARS-CoV-2 structural protein was accompanied by differential activation dynamics in survival and immunological routes in parallel with a differentiated secretion profile of chemokines in OB cells. Machine learning through a proteotranscriptomic data integration uncovered TGF-beta signaling as a confluent activation node by the SARS-CoV-2 structural proteome. Taken together, these data provide important avenues for understanding the multifunctional immunomodulatory properties of SARS-CoV-2 M, N, S and E proteins beyond their intrinsic role in virion formation, deciphering mechanistic clues to the olfactory inflammation observed in COVID-19 patients.

systems biology↗