Exosomal Aß-Binding Proteins Identified by "In Silico" Analysis Represent Putative Blood-Derived Biomarker Candidates for Alzheimer´s Disease.

The potential of exosomes as biomarker resources for diagnostics and even for therapeutics has intensified research in the field, including in the context of Alzheimer´s disease (AD). The search for disease biomarkers in peripheral biofluids is advancing mainly due to the easy access it offers. In the study presented here, emphasis was given to the bioinformatic identification of putative exosomal candidates for AD. The exosomal proteomes of cerebrospinal fluid (CSF), serum and plasma, were obtained from three databases (ExoCarta, EVpedia and Vesiclepedia), and complemented with additional exosomal proteins already associated with AD but not found in the databases. The final biofluids' proteomes were submitted to gene ontology (GO) enrichment analysis and the exosomal Aß-binding proteins that can constitute putative candidates were identified. Among these candidates, gelsolin, a protein known to be involved in inhibiting Abeta fibril formation, was identified, and it was tested in human samples. The levels of this Aß-binding protein, with anti-amyloidogenic properties, were assessed in serum-derived exosomes isolated from controls and individuals with dementia, including AD cases, and revealed altered expression patterns. Identification of potential peripheral biomarker candidates for AD may be useful, not only for early disease diagnosis but also in drug trials and to monitor disease progression, allowing for a timely therapeutic intervention, which will positively impact the patient's quality of life.

Monitoring plasma protein aggregation during aging using conformation-specific antibodies and FTIR spectroscopy.

The loss of proteostasis during aging has been well described using different models, however little is known with respect to protein aggregation levels in biofluids with aging. Therefore, the aim of this study was to assess the pattern of age-related protein aggregation in human plasma using two distinct approaches: analysis with conformation-specific antibodies and FTIR spectroscopy. The latter has been widely used in biomedical research to study protein conformational changes in health and disease. Samples from a primary care based-cohort from the Aveiro region, Portugal, were used for slot-blot analyses followed by immunodetection with conformation-specific antibodies and for the acquisition of FTIR spectra. Immunoblot analyses revealed an age-dependent evolution of the protein conformational profile in human plasma, towards a decrease in prefibrillar oligomers and an increase in fibrillar structures. This finding was also supported by PLS-R multivariate analysis of FTIR data, where a positive correlation between the age of the donors and secondary structure of plasma proteins could be observed. Samples from younger donors are characterized by antiparallel ß-sheet-containing structures while intermolecular ß-sheets characterized older samples. Exclusion of age-associated co-morbidities improved the correlation between protein conformational profiles and aging. The results reveal structural changes in human plasma proteins from middle to old age, confirming the age-associated changes in protein aggregation, and support the applicability of FTIR as a reliable approach to study proteostasis during aging.

Exosome isolation from distinct biofluids using precipitation and column-based approaches.

The potential of exosomes as biomarker resources for diagnostics, prognostics and even for therapeutics is an area of intense research. Despite the various approaches available, there is no consensus with respect to the best methodology for isolating exosomes and to provide substantial yields with reliable quality. Differential centrifugation is the most commonly used method but it is time-consuming and requires large sample volumes, thus alternative methods are urgently needed. In this study two precipitation-based methods and one column-based approach were compared for exosome isolation from distinct biofluids (serum, plasma and cerebrospinal fluid). Exosome characterization included morphological analyses, determination of particle concentration, stability and exosome preparations' purity, using different complementary approaches such as Nanoparticle Tracking Analysis, Electrophoretic Light Scattering, Transmission Electron Microscopy, EXOCET colorimetric assay, protein quantification methods and western blotting. The three commercial kits tested successfully isolated exosomes from the biofluids under study, although ExoS showed the best performance in terms of exosome yield and purity. Data shows that methods other than differential centrifugation can be applied to quickly and efficiently isolate exosomes from reduced biofluid volumes. The possibility to use small volumes is fundamental in the context of translational and clinical research, thus the results here presented contribute significantly in this respect.