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Title: Profiling Small RNA from Brain Extracellular Vesicles in Individuals with Depression [NovaSeq]

ABSTRACT: Major depressive disorder (MDD) is a leading cause of disability with significant mortality risk. Despite progress in our understanding of the etiology of MDD, the underlying molecular changes in the brain remain poorly understood. Extracellular vesicles (EVs) are lipid-bound particles that can reflect the molecular signatures of the tissue of origin. We aimed to optimize a streamlined EV isolation protocol from post-mortem brain tissue and to determine whether EV RNA cargo, particularly microRNAs (miRNAs) have an MDD-specific profile. EVs were isolated from post-mortem human brain tissue. Quality was assessed using western blots, transmission electron microscopy, and microfluidic resistive pulse sensing. EV RNA was extracted and sequenced on Illumina platforms. Functional follow up was performed in silico. Quality assessment showed an enrichment of EV markers, as well as a size distribution of 30-200 nm in diameter, and no contamination with cellular debris. Small RNA profiling indicated the presence of several RNA biotypes, with miRNAs and transfer RNAs (tRNAs) being the most prominent. Exploring miRNA levels between groups revealed decreased expression of miR-92a-3p and miR-129-5p, which was validated by qPCR and was specific to EVs and not seen in bulk tissue. Finally, in silico functional analyses indicate potential roles for these two miRNAs in neurotransmission and synaptic plasticity. We provide a streamlined isolation protocol that yields EVs of high quality that are suitable for molecular follow up. Our findings warrant future investigations into brain EV miRNA dysregulation in MDD.

ORGANISM(S): Homo sapiens

PROVIDER: GSE255480 | GEO | 2024/03/20

REPOSITORIES: GEO

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Project description:Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque. Effects of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with macaque, suggesting opportunities for optimization. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

Dataset Information

Title: Profiling Small RNA from Brain Extracellular Vesicles in Individuals with Depression [NovaSeq]

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