RNA Transcripts in Salivary Extracellular Vesicle Cargo isolated from Aged Populations

Sicheng Wen¹Chang Yu²Maxfield Kelsey³Mandy Pereira¹Hannah Alaimo⁴Em Teixeira⁴Jenna Pracht⁴Lori A. Daiello⁴Jonathan Drake⁴John Sedivy³Zhijin Wu²Peter Quesenberry¹Jill Kreiling^3*

• ¹Department of Medicine, Division of Hematology/Oncology, Brown University, Rhode Island Hospital, Providence, United States
• ²Department of Biostatistics, Brown University School of Public Health, Providence, United States
• ³Brown Center on the Biology of Aging, Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, United States
• ⁴Department of Neurology, Warren Alpert Medical School of Brown University, Rhode Island Hospital Alzheimer's Disease and Memory Disorders Center, Providence, United States

We characterized the RNA cargo of extracellular vesicles (EVs) isolated from saliva (salEVs) from individuals over the age of 65 with normal cognition. Human saliva contains numerous factors, including DNA, RNA, and protein, that may reflect the health status of the individual. Many of these factors are contained within EVs. The contents of EVs are thought to mirror the cytoplasm of the cell of origin, providing insight into the health of the cell. The salEV RNA content was analyzed by RNA-seq and NanoString miRNA analysis. We found approximately 48.4% of the reads mapped to the human genome, with the remainder mapping to prokaryotic genomes. The transcripts included protein-coding RNA, long non-coding RNA, retrotransposons, and miRNAs. A significant number of the protein-coding transcripts were associated with pathways involved in neurodegenerative conditions. In addition, there was an enrichment of transcripts containing AP-2ε, HEYL, HES4, and TCFL5 transcription factor binding sites. We found that the lncRNA content was similar between samples, with PCBP1-AS1, TEX41, and PVT1 being the top represented transcripts. There were 286 miRNAs found in the salEV samples. The pathways predicted to be affected by the top represented miRNAs include Hippo signaling, TGF-β signaling, Wnt signaling, FoxO signaling, ErbB signaling, axon guidance, and mTOR signaling. We could detect retrotransposon transcripts from LINE, SINE, and LTR elements in salEVs. Retrotransposons are known to be derepressed with age and the onset of neurodegenerative diseases. When compared to blood-derived EVs, salEVs showed greater representation of transcripts associated with neurodegenerative pathways. The presence of these transcripts and retrotransposons in salEVs suggest that saliva may be used to screen for biomarkers of neurodegenerative diseases.