Abstract
BackgroundExosomes are a subset of extracellular vesicles 30–200 nm in diameter secreted from cells, which contain functional mRNAs and microRNAs. Cerebrospinal fluid (CSF) is the primary source for liquid biopsy to examine diseases in central nervous system. To date, there is no available method to analyze exosomal mRNAs comprehensively in human CSF.MethodsThe main purpose of this study is to established the methodology of comprehensive analysis of exosomal mRNAs in CSF by a highly sensitive next-generation sequencing. The signatures of CSF exosomal mRNAs were then compared between four normal healthy donors and four sporadic amyotrophic lateral sclerosis patients to identify disease-related biomarkers. Differentially expressed genes were identified by DESeq2.ResultsRNA sequencing from CSF exosomes was successfully performed, that was demonstrated by the high pearson’s product-moment correlation coefficient (r = 0.993) in the technical replicates. Also, position coverage analysis revealed that most detected mRNAs retained their integrity throughout their full-length in CSF exosomes. In CSF exosomes from normal healthy donors, an average of 14,807 genes were detected, of which 4580 genes were commonly detected among four individuals, including neuron-enriched genes such as TUBB3 and CAMK2A. In comparison with exosomal mRNAs in CSF from four patients with amyotrophic lateral sclerosis, 543 genes were significantly changed, as represented by CUEDC2. Gene Ontology analysis and pathway analysis with these genes revealed functional enrichment of ubiquitin-proteasome pathway, oxidative stress response, and unfolded protein response. These pathways are related to pathomechanisms of amyotrophic lateral sclerosis.ConclusionWe successfully established the methodology of comprehensive analysis of exosomal mRNAs in human CSF. It was shown to be useful to identify disease biomarkers for central nervous system. Several genes, such as CUEDC2, in CSF exosomes were suggested to be candidate disease biomarkers for amyotrophic lateral sclerosis.
Highlights
Exosomes are a subset of extracellular vesicles 30–200 nm in diameter secreted from cells, which contain functional mRNAs and microRNAs
We demonstrated that Extracellular vesicles (EVs) isolated from human Cerebrospinal fluid (CSF) were characterized as exosomes
We reported that more than 4500 genes were commonly detected in exosomes in CSF from normal healthy (NH) donors, and showed that housekeeping genes such as ACTB and GAPDH in the exosomes were abundant and consistent among four individuals
Summary
Exosomes are a subset of extracellular vesicles 30–200 nm in diameter secreted from cells, which contain functional mRNAs and microRNAs. Lötvall’s group reported the presence of mRNAs and microRNAs in exosomes secreted from mast cell lines. These exosomal RNAs were transferrable and functional in recipient cells [7]. Exosomal mRNAs were stable against RNase and freeze-thaw process [8, 9] These findings led to the hypothesis that exosomes play some important physiological roles. Perturbation in exosomal cargos in any disease state can be biomarkers for diagnosis, prognosis, and patient stratification. Their elucidation will lead to better understanding of disease mechanisms. Many studies have reported specific signatures of microRNAs in plasma or serum as candidate diagnostic biomarkers [10]
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