Phosphoproteome Microarray Analysis of Extracellular Particles as a Tool to Explore Novel Biomarker Candidates for Alzheimer's Disease.

Phosphorylation plays a key role in Alzheimer's disease (AD) pathogenesis, impacting distinct processes such as amyloid-beta (Aß) peptide production and tau phosphorylation. Impaired phosphorylation events contribute to senile plaques and neurofibrillary tangles' formation, two major histopathological hallmarks of AD. Blood-derived extracellular particles (bdEP) can represent a disease-related source of phosphobiomarker candidates, and hence, in this pilot study, bdEP of Control and AD cases were analyzed by a targeted phosphoproteomics approach using a high-density microarray that featured at least 1145 pan-specific and 913 phosphosite-specific antibodies. This approach, innovatively applied to bdEP, allowed the identification of 150 proteins whose expression levels and/or phosphorylation patterns were significantly altered across AD cases. Gene Ontology enrichment and Reactome pathway analysis unraveled potentially relevant molecular targets and disease-associated pathways, and protein-protein interaction networks were constructed to highlight key targets. The discriminatory value of both the total proteome and the phosphoproteome was evaluated by univariate and multivariate approaches. This pilot experiment supports that bdEP are enriched in phosphotargets relevant in an AD context, holding value as peripheral biomarker candidates for disease diagnosis.

Quantitative proteome analysis of LAP1-deficient human fibroblasts: A pilot approach for predicting the signaling pathways deregulated in LAP1-associated diseases.

Lamina-associated polypeptide 1 (LAP1), a ubiquitously expressed nuclear envelope protein, appears to be essential for the maintenance of cell homeostasis. Although rare, mutations in the human LAP1-encoding TOR1AIP1 gene cause severe diseases and can culminate in the premature death of affected individuals. Despite there is increasing evidence of the pathogenicity of TOR1AIP1 mutations, the current knowledge on LAP1's physiological roles in humans is limited; hence, investigation is required to elucidate the critical functions of this protein, which can be achieved by uncovering the molecular consequences of LAP1 depletion, a topic that remains largely unexplored. In this work, the proteome of patient-derived LAP1-deficient fibroblasts carrying a pathological TOR1AIP1 mutation (LAP1 E482A) was quantitatively analyzed to identify global changes in protein abundance levels relatively to control fibroblasts. An in silico functional enrichment analysis of the mass spectrometry-identified differentially expressed proteins was also performed, along with additional in vitro functional assays, to unveil the biological processes that are potentially dysfunctional in LAP1 E482A fibroblasts. Collectively, our findings suggest that LAP1 deficiency may induce significant alterations in various cellular activities, including DNA repair, messenger RNA degradation/translation, proteostasis and glutathione metabolism/antioxidant response. This study sheds light on possible new functions of human LAP1 and could set the basis for subsequent in-depth mechanistic investigations. Moreover, by identifying deregulated signaling pathways in LAP1-deficient cells, our work may offer valuable molecular targets for future disease-modifying therapies for TOR1AIP1-associated nuclear envelopathies.

FTIR Spectroscopy and Blood-Derived Extracellular Vesicles Duo in Alzheimer's Disease.

Background: Alzheimer's disease (AD) diagnosis is difficult, and new accurate tools based on peripheral biofluids are urgently needed. Extracellular vesicles (EVs) emerged as a valuable source of biomarker profiles for AD, since their cargo is disease-specific and these can be easily isolated from easily accessible biofluids, as blood. Fourier Transform Infrared (FTIR) spectroscopy can be employed to analyze EVs and obtain the spectroscopic profiles from different regions of the spectra, simultaneously characterizing carbohydrates, nucleic acids, proteins, and lipids. Objective: The aim of this study was to identify blood-derived EVs (bdEVs) spectroscopic signatures with AD discriminatory potential. Methods: Herein, FTIR spectra of bdEVs from two biofluids (serum and plasma) and distinct sets of Controls and AD cases were acquired, and EVs' spectra analyzed. Results: Analysis of bdEVs second derivative peaks area revealed differences between Controls and AD cases in distinct spectra regions, assigned to carbohydrates and nucleic acids, amides, and lipids. Conclusions: EVs' spectroscopic profiles presented AD discriminatory value, supporting the use of bdEVs combined with FTIR as a screening or complementary tool for AD diagnosis.

BIN1 rs744373 SNP and APOE alleles specifically associate to common diseases.

APOE ε4 and BIN1 are the two main genetic risk factors for sporadic Alzheimer's Disease (AD). Among several BIN1 variants, the rs744373 is frequently associated with AD risk by contributing to tau pathology and poor cognitive performance. This study addressed the association of APOE and BIN1 rs744373 to specific characteristics in a Portuguese primary care-based study group, denoted pcb-Cohort. The study included 590 participants from five primary care health centers in the Aveiro district of Portugal. Individuals were evaluated and scored for cognitive and clinical characteristics, and blood samples were collected from the volunteers meeting the inclusion and exclusion criteria (N = 505). APOE and BIN1 genotypes were determined, and their association with cognitive characteristics and other diseases that might contribute to cognitive deficits, namely depression, hypertension, type 2 diabetes, dyslipidemia, osteoarticular diseases, gastrointestinal diseases, cardiovascular and respiratory diseases, was assessed. The diseases attributed to the study group were those previously diagnosed and confirmed by specialists. The results generated through multivariate analysis show that APOE ε4 carriers significantly associated with poorer cognitive performance (OR = 2.527; p = 0.031). Additionally, there was a significant risk of dyslipidemia for APOE ε4 carriers (OR = 1.804; p = 0.036), whereas BIN1 rs744373 risk-allele carriers were at a significantly lower risk of having dyslipidemia (OR = 0.558; p = 0.006). Correlations were evident for respiratory diseases in which APOE ε4 showed a protective tendency (OR = 0.515; p = 0.088), and BIN1 had a significative protective profile (OR = 0.556; p = 0.026). Not of statistical significance, APOE ε2 showed a trend to protect against type 2 diabetes (OR = 0.342; p = 0.093), in contrast BIN1 rs744373 risk-allele carriers were more likely to exhibit the disease (OR = 1.491; p = 0.099). The data here presented clearly show, for the first time, that the two top genetic risk factors for sporadic AD impact a similar group of common diseases, namely dyslipidemia, respiratory diseases, and type 2 diabetes.