FedExosome: profiling AD‐related mitochondrial DNA and microRNAs isolated from neuronal‐enriched peripheral blood packages

Abstract

AbstractBackgroundAlzheimer’s disease (AD) is a complex disease characterized by impaired memory, diminished cognition, and specific brain pathology (i.e., accumulation of amyloid beta plaques and tau tangles). AD progression is often difficult to detect with the earliest clinical symptoms appearing decades after key neuropathological changes have occurred. Accurate diagnosis early, between initial disease onset and clinical manifestation, could reveal critical aspects of pathogenesis and inform the development of effective therapeutic strategies to prevent neuronal death. Evidence suggests that early alterations in the AD brain can propagate to local and distal cells through the biological packages secreted by neurons. These neuronal‐enriched extracellular vesicles (NEEVs) can cross the blood‐brain barrier and are capable of mediating systemic inflammation in response to CNS‐sourced stress through their mitochondrial DNA (mtDNA) and microRNA (miRNA) cargo. Circulating NEEVs may therefore serve as an easily accessible, early indicator of the forthcoming neuropathological changes in the AD brain. Here, we develop a higher throughput workflow for profiling mtDNA and miRNA in plasma NEEVs and apply the workflow to clinical samples collected longitudinally from participants in the Texas Alzheimer’s Research and Care Consortium.MethodHuman plasma samples were processed using a two‐step method involving (1) precipitation of total exosomes with ExoQuick (SBI) and (2) immunoprecipitation‐based enrichment of NEEVs with a biotinylated antibody against a well‐established neuronal surface marker (CD171). Captured NEEVs were stained and analyzed using flow cytometry as well as nanoparticle tracking analysis (NTA). DNA and RNA were then extracted for mtDNA quantification and miRNA sequencing.ResultThe protocol modifications implemented in this project were compared based on particle size, distribution, and purity as well as absolute mtDNA load and spike‐in normalized miRNA read counts. These data not only demonstrate the feasibility of profiling the mtDNA and miRNA from 500µL of plasma NEEVs but have also resulted in a higher throughput workflow for processing TARCC patient samples.ConclusionThis work represents the first successful attempt to simultaneously quantify mtDNA and sequence miRNA in the relatively small subpopulation of plasma EVs that originate from neurons. Future studies will harness the protocol developed herein to assess the biomarker potential of NEEVs in the TARCC cohort.