DOI: 10.1002/alz.082816 ISSN: 1552-5260

Blood‐derived extracellular vesicles phosphoproteome as a resource of biomarker candidates for Alzheimer’s disease

Tânia Soares Martins, Ilka Martins Rosa, Odete da Cruz e Silva, Ana Gabriela Henriques
  • Psychiatry and Mental health
  • Cellular and Molecular Neuroscience
  • Geriatrics and Gerontology
  • Neurology (clinical)
  • Developmental Neuroscience
  • Health Policy
  • Epidemiology

Abstract

Background

Protein phosphorylation plays a crucial role in cell communication, signaling and by extension is relevant to Alzheimer´s disease (AD) pathogenesis. Extracellular vesicles (EVs) are recognized as important mediators in intercellular communication and can be isolated from peripheral biofluids, where the content reflects the cellular origin. EVs’ role in AD pathophysiology and their value as biomarker resources is being unraveled. However, little is known regarding EVs’ phosphoproteome in AD and it has still to be determined if blood‐derived EVs are suitable for phosphoproteome profiling. Hence, this work aimed to characterize plasma‐derived EVs (pdEVs) and plasma supernatant EVs free (plasma‐depleted EVs) phosphoproteomes, to potential unravel disease mechanisms and/or new biomarker candidates for AD.

Method

A pool of human plasma was used for pdEVs isolation using a precipitation‐based methodology. Both pdEVs and plasma‐depleted EVs samples were analyzed using an antibody‐based phosphospecific microarray. Characterization of the phosphoproteomes obtained was achieved by Gene Ontology enrichment analysis and the most relevant Reactome pathways associated, using ClueGo and CluePedia plugins from Cytoscape v3.9.1. In addition, protein‐protein interaction network of the pdEVs phosphoproteome was retrieved from STRING database and analyzed using Cytoscape “Network Analyzer”, employing the betweenness centrality option to highlight central nodes. Further, the pdEVs phosphoproteome (corresponding gene names) was overlapped with a list of genes associated with AD, collated from DisGenet database.

Results

More than 70 proteins were enriched ≥40% in bdEVs when compared to plasma‐depleted. Top biological processes were mainly related to cell proliferation/division, apoptosis and phosphorylation. Reactome pathways analysis revealed terms associated to toll like receptor cascade and MAPK signaling. In the network constructed for the pdEVs enriched proteins, PTEN appeared as a central node. Additionally, overlap with AD gene list from DisGenet revealed that 60% of the pdEVs phosphoproteome is AD‐related. Among these, several kinases already described in senile plaques and/or neurofibrillary tangles were identified.

Conclusion

In sum, bdEVs phosphoproteome is enriched in AD‐related targets, exhibiting potential as a biomarker candidate resource for AD diagnosis.

This work was funded by the Alzheimer’s Association under Grant 2019‐AARG‐644347, supported by iBiMED‐UIDB/04501/2020,iBiMED‐UIDB/04501/2020,ADPro‐SAICT‐45‐2021‐02 and TSM individual PhD grant SFRH/BD/145979/2019. Health Ethics Committee ARS Centro(No.012804‐04.04.2012) and CNPD(No.369/2012).

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