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Background: Extracellular vesicles (EVs) and non-coding RNAs (ncRNAs) are emerging contributors to Alzheimer's disease (AD) pathophysiology. Differential abundance of ncRNAs carried by EVs may provide valuable insights into underlying disease mechanisms. Brain tissue-derived EVs (bdEVs) are particularly relevant, as they may offer valuable insights about the tissue of origin. However, there is limited research on diverse ncRNA species in bdEVs in AD. Objective: This study explored whether the non-coding RNA composition of EVs isolated from post-mortem brain tissue is related to AD pathogenesis. Methods: bdEVs from age-matched late-stage AD patients (n = 23) and controls (n = 10) that had been separated and characterized in our previous study were used for RNA extraction, small RNA sequencing, and qPCR verification. Results: Significant differences of non-coding RNAs between AD and controls were found, especially for miRNAs and tRNAs. AD pathology-related miRNA and tRNA differences of bdEVs partially matched expression differences in source brain tissues. AD pathology had a more prominent association than biological sex with bdEV miRNA and tRNA components in late-stage AD brains. Conclusions: Our study provides further evidence that EV non-coding RNAs from human brain tissue, including but not limited to miRNAs, may be altered and contribute to AD pathogenesis. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is a progressive neurodegenerative disorder, with the strongest disease-associated changes observed at clinical or end-stage disease. Transcriptomic deregulation of miRNA expression can spread via "horizontal" RNA transfer through extracellular vesicles (EVs) to act in conjunction with proteins, leading to changes in mRNA, which can provide early signals to indicate forthcoming neuropathological changes in the brain. Here, we analysed the small RNA content, in particular, miRNA, contained in brain-derived EVs isolated from the frontal cortex of Alzheimer's subjects (n = 8) and neurological control subjects (n = 9). Brain-derived EVs were found to contain an upregulation of disease-associated miRNA. RNA species from brain-derived EVs were correlated with miRNA profiles obtained from matching total brain homogenate. These results provide a blueprint into the biological pathways potentially effected during disease that may be assisted by brain-derived EV RNA horizontal transfer.We also correlated the miRNA changes in the brain with those detected in peripheral EVs collected from serum of Alzheimer's disease patients (n = 23, and healthy controls, n = 23) and revealed a panel of miRNA that could be used as a liquid brain biopsy. Overall, our study provides the first interrogation of the small RNA contents in brain-derived EVs and how they could be used to understand the early pathological changes in Alzheimer's disease which will benefit the development of an early diagnostic blood test. [ABSTRACT FROM AUTHOR]

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, and mouse. Effects of tissue perfusion and a model 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 those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. 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. Finally, 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. [ABSTRACT FROM AUTHOR]

Background: Peripheral blood microRNAs (miRNA) have been identified as potential biomarkers for Alzheimer's disease (AD). Study results have generally been inconsistent and limited by sample heterogeneity. The aim of this study is to establish candidate blood miRNA biomarkers for AD by comparing differences in miRNA expression between participants with brain amyloid imaging-defined AD and normal cognition. Methods: Blood RNA was extracted from a subset of participants from the Australian Imaging Biomarkers Lifestyle Study of Ageing cohort (AIBL) with brain amyloid imaging results. MiRNA profiling was performed using small RNA sequencing on 71 participants, comprising 40 AD with high brain amyloid burden on imaging (amyloid positive) and 31 cognitively normal controls with low brain amyloid burden (amyloid negative). Cross-sectional comparisons were made between groups to examine differential miRNA expression levels using Fisher's exact tests. Replication of results was undertaken using a publicly available dataset of blood miRNA data of AD and controls. In silico analysis of downstream messenger RNA targets of candidate miRNAs was performed to elucidate potential biological function. Results: After quality control, 816 miRNAs were available for analysis. There were 71 significantly differentially expressed miRNAs between the AD and control groups (p < 0.05). Two of these miRNAs, miR-146b-5p and miR-15b-5p, were also significant in the replication cohort. Pathways analysis showed these miRNAs to be involved in innate immune system and regulation of the cell cycle, respectively, both of which have relevance to AD pathogenesis. Conclusion: Blood miR-146b-5p and miR15b-5p showed consistent differential expression in AD compared to controls. Further replication and translational studies in strictly phenotyped cohorts are needed to establish their role as biomarkers for AD to have clinical utility. [ABSTRACT FROM AUTHOR]

In rheumatoid arthritis (RA), extracellular vesicles (EVs) are associated with both the propagation and attenuation of joint inflammation and destruction. However, the specific EV content responsible for these processes is largely unknown. Investigations into identifying EV content are confounded by the challenges in obtaining high-quality EV preparations from synovial fluid. Implementing a size exclusion chromatography-based method of EV isolation, coupled with small RNA sequencing, we accurately characterised EV miRNAs in synovial fluid obtained from RA patients and investigated the differences between joints with high- and low-grade inflammation. Synovial fluid was obtained from the joints of 12 RA patients and, based on leukocyte counts, classified as either high (n = 7)- or low (n = 5)-grade inflammation. Using size exclusion chromatography, EVs were purified and small RNA was extracted and sequenced on a NextSeq 500. Sequencing reads were aligned to miRBase v21, and differences in miRNA profiles between RA patients with high- and low-grade joint inflammation were analysed. In total, 1972 distinct miRNAs were identified from RA synovial fluid EVs. miRNAs with less than five reads in fewer than five patients were filtered out, leaving 318 miRNAs for analysis. Analysis of the most abundant miRNAs suggested that they negatively regulate multiple genes relevant to inflammation, including signal transducer and activator of transcription 3 (STAT3), which lies downstream of IL-6 and has a pro-inflammatory role in RA. Synovial fluid from joints with high-grade inflammation contained 3.5-fold more EV miRNA per mL of synovial fluid (p = 0.0017). Seventy-eight EV miRNAs were differentially expressed between RA joints with high- and low-grade inflammation, and pathway analysis revealed that their target genes were commonly involved a variety of processes, including cellular apoptosis, proliferation and migration. Of the 49 miRNAs that were elevated in joints with high-grade inflammation, pathway analysis revealed that genes involved in cytokine-mediated signalling pathways were significantly enriched targets. In contrast, genes associated with reactive oxygen species signalling were significantly enriched as targets of the 29 miRNAs elevated in joints with low-grade inflammation. Our study identified an abundance of EV miRNAs from the synovial fluid of RA patients with the potential to modulate inflammation. In doing so, we defined potential mechanisms by which synovial fluid EVs may contribute to RA pathophysiology. [ABSTRACT FROM AUTHOR]