Exosomal Aβ-Binding Proteins Identified by “In Silico” Analysis Represent Putative Blood-Derived Biomarker Candidates for Alzheimer´s Disease

Abstract

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 Aβ-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 Aβ-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.

Novel exosome biomarker candidates for Alzheimer´s disease unraveled through Mass Spectrometry analysis

“…In addition, it was shown that C4BPα limits the complement activation by Aβ and/or death cells in AD brains, possibly protecting the neuronal environment from immune activation [56]. Moreover, a previous bioinformatic analysis by our group [29] identi ed both AACT and C4BPα as Aβ-binding proteins in the exosomal proteome, constructed by the overlap of serum-, plasma-and CSF-exosomal proteomes available in databases namely EXOCARTA, Vesiclepedia or EVpedia. Taken together, we hypothesized that AACT and C4BPα could represent putative AD exosomal biomarker candidates, and thus both markers were tested in serum-derived exosomes from Control and AD cases.…”

“…For further analyses, exosomal proteomes obtained through MS (list of gene names from proteins identi ed) were overlapped with an 'in silico' serum-derived exosomal gene list. This list was collated using public databases, namely Vesiclepedia, EVpedia and Exocarta and complemented following a literature review on exosomal AD related proteins [11,29]. More than 70% of the exosomal proteins identi ed by MS and isolated either with ExoQ or ExoS were also present in the serum-derived exosomal gene list, reinforcing the exosomal nature of the samples (Fig.…”

“…EVs, with exosome-like characteristics, were isolated from serum samples as previously described [21,28,29]. Two distinct exosomes isolation methods were used: the precipitation-based ExoQuick Serum Exosome Precipitation Solution (System Biosciences) (ExoQ) and the column-based ExoSpin Blood Exosome Puri cation Kit (Cell guidance systems) (ExoS).…”

“…Proteomes obtained by MS (list of gene names from proteins identi ed) were initially overlapped through Venn diagrams with a serum exosomal list (Exo Serum list), obtained from databases and from literature search as described in [29], using the bioinformatics and evolutionary genomics website (http://bioinformatics.psb.ugent.be/webtools/Venn/; accessed on 3 rd February 2020), in order to determine the percentage of exosomal proteins present in EVs samples analysed. Only MS proteins for which gene name was available were included in the overlap.…”

Role of Exosomes in Brain Diseases

“…In recent years, exosomes have attracted considerable interest in the study of brain diseases, such as Alzheimer's disease, Parkinson's disease, stroke, and traumatic brain injury, due to their critical importance in the disease process and potential value for clinical application. The role and molecular mechanisms of exosomes carrying proteins related to the brain diseases [amyloid precursor protein (APP), α-synuclein (α-syn), mHtt, PrPsc] have been emphatically explored (Hartmann et al, 2017 ; Leblanc et al, 2017 ; Wang J. K. T. et al, 2017 ; Hill, 2019 ; Li B. et al, 2020 ; Pan et al, 2020 ; Perez-Gonzalez et al, 2020 ; Singh and Muqit, 2020 ; Tsunemi et al, 2020 , 2021 ; Ananbeh et al, 2021 ; Soares Martins et al, 2021a ). Notably, their ability to transport cargo is a key mechanism involved in the spread of disease.…”