Alzheimer's 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 study, 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 and tau functional interactomes. To implement a computational repurposing of drug candidates with the capacity to reverse early AD‐related olfactory omics signatures (OMSs), we generated a consensual 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‐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, Insulin‐like growth factor 1 (IGF‐1), microtubules, and Polo‐like kinase (PLK) represented a family of drugs with detrimental potential to induce olfactory AD‐associated gene expression changes. To validate the potential therapeutic effects of the proposed drugs, in vitro assays were performed. These validation experiments 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 (Aβ)‐induced neurotoxicity. Taken together, our data pointed out that OMSs may be useful as substrates for drug repurposing to propose novel neuroprotective treatments against AD.

Due to the segmented functions and complexity of the human brain, the characterization of molecular profiles within specific areas such as brain structures and biofluids is essential to unveil the molecular basis for structure specialization as well as the molecular imbalance associated with neurodegenerative and psychiatric diseases. Much of our knowledge about brain functionality derives from neurophysiological, anatomical, and transcriptomic approaches. More recently, laser capture and imaging proteomics, technological and computational developments in LC-MS/MS, as well as antibody/aptamer-based platforms have allowed the generation of novel cellular, spatial, and post-translational dimensions as well as innovative facets in biomarker validation and druggable target identification. Proteomics is a powerful toolbox to functionally characterize, quantify and localize the extensive protein catalog of the human brain across physiological and pathological states. Brain function depends on multi-dimensional protein homeostasis and its elucidation will help us to characterize biological pathways that are essential to properly maintain cognitive functions. In addition, comprehensive human brain pathological proteomes may be the basis in computational drug-repositioning methods as a strategy for unveiling potential new therapies in neurodegenerative and psychiatric disorders.

Alzheimer’s disease (AD) is a neurodegenerative olfactory disorder affecting millions of people worldwide. Alterations in the hexosamine- or glucose-related pathways have been described through AD progression. Specifically, an alteration in glucosamine 6 phosphate isomerase 2 (GNPDA2) protein levels has been observed in olfactory areas of AD subjects. However, the biological role of GNPDA2 in neurodegeneration remains unknown. Using mass spectrometry, multiple GNPDA2 interactors were identified in human nasal epithelial cells (NECs) mainly involved in intraciliary transport. Moreover, GNPDA2 overexpression induced an increment in NEC proliferation rates, accompanied by transcriptomic alterations in Type II interferon signaling or cellular stress responses. In contrast, the presence of beta-amyloid or mutated Tau-P301L in GNPDA2-overexpressing NECs induced a slowdown in the proliferative capacity in parallel with a disruption in protein processing. The proteomic characterization of Tau-P301L transgenic zebrafish embryos demonstrated that GNPDA2 overexpression interfered with collagen biosynthesis and RNA/protein processing, without inducing additional changes in axonal outgrowth defects or neuronal cell death. In humans, a significant increase in serum GNPDA2 levels was observed across multiple neurological proteinopathies (AD, Lewy body dementia, progressive supranuclear palsy, mixed dementia and amyotrophic lateral sclerosis) (n = 215). These data shed new light on GNPDA2-dependent mechanisms associated with the neurodegenerative process beyond the hexosamine route.

Alzheimer’s 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 (Aβ)-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. STATEMENTS: Data availability statement: Mass-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 statement: This 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 disclosure: Authors declare that they have no conflicts of interest/financial disclosures. Ethics approval and patient consent statement: According 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).

Background: The amygdala is involved in the emotional expression, memory processing and managing stimulatory input. Although amygdala atrophy is early evidenced in Alzheimer’s 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. Methods: Label-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. Results: Amygdaloid 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 Aβ 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. Conclusions: Early 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 summary: The 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 Alzheimer’s 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.

Alzheimer's disease (AD) is a neurodegenerative olfactory disorder affecting millions of people worldwide. Alterations in the hexosamine or glucose-related pathways have been described through AD progression. Specifically, an alteration in glucosamine 6 phosphate isomerase 2 (GNPDA2) protein levels has been observed in olfactory areas from AD subjects. However, the biological role of GNPDA2 in neurodegeneration remains unknown. Using mass-spectrometry, multiple GNPDA2 interactors were identified in human nasal epithelial cells (NECs) mainly involved in intraciliary transport. Moreover, GNPDA2 overexpression induced an increment in NEC proliferation rates, accompanied by transcriptomic alterations in Type II interferon signalling or cellular stress responses. In contrast, the presence of beta-amyloid or mutated Tau-P301L in GNPDA2-overexpressing NECs induced a slow-down in the proliferative capacity in parallel with a disruption in protein processing. The proteomic characterization of Tau-P301L transgenic zebrafish embryos demonstrated that GNPDA2 overexpression interfered with collagen biosynthesis and RNA/protein processing, without inducing additional changes in axonal outgrowth defects nor neuronal cell death. In humans, a significant increase in serum GNPDA2 levels was observed across multiple neurological proteinopathies (AD, Lewy body dementia, progressive supranuclear palsy, mixed-dementia, and amyotrophic lateral sclerosis) (n=187). These data shed new light on GNPDA2-dependent mechanisms associated to the neurodegenerative process beyond the hexosamine route.