Title: Profiling Small RNA from Brain Extracellular Vesicles in Individuals with Depression
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. Overall design: EVs were isolated from post-mortem human brain tissue of individuals who had MDD and died by suicide as well as psychiatrically healthy individuals who died naturally or by accident. RNA was isolated from EVs and small RNA libraries were constructed and sequenced. Isolations took place in 2 batches, the first batch was sequenced on Illumina HiSEQ, the second on Illumina NOVASEQ.