Turchinovich, A. | Search bioRxiv

bioRxiv2020

Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles

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.

neuroscience↗

bioRxiv2019

Dissection of demethylation and toxicity induced gene expression changes after decitabine treatment.

The DNA methyltransferase inhibitor decitabine (DAC) is a widely used drug for both fundamental epigenetics studies and anti-cancer therapy. Besides DNA demethylation, DAC also induces cell toxicity associated with DNA damage. The dual-mode of DAC action on cells provides a significant hurdle to study genes which expression is regulated by CpG methylation. In this work, we performed the analysis of global DNA methylation levels in cultured cancer cells after treatment with increasing doses of DAC and have found the U-shaped curve of the de-methylation efficacy induced by the drug. Specifically, high doses of DAC induced significantly lower DNA hypomethylation as compared to hundred-fold less concentrated doses. At the same time, the impact of DAC on cell viability was dose-dependent. These findings allowed dissecting the demethylation and the cell toxicity impact of DAC on gene expression in subsequent mRNA-seq experiments. Surprisingly, the number of genes that were upregulated due to DNA hypomethylation was comparable to the number of genes induced by DAC toxicity. Furthermore, we show that high DAC concentrations induce downregulation of housekeeping genes which are most widely used for RT-qPCR normalization (including GAPDH, actin and tubulin). The latter suggests that genes unaffected by DAC treatment would manifest themselves as upregulated when their expression is normalized on a downregulated housekeeping reference. Finally, we show that expression of most human oncogenes and tumor-suppressor genes remains unaffected after DAC treatment, and only a few of them were upregulated due to DNA hypomethylation. Our work stresses the importance of closely studying the correlation of the degree of DNA demethylation induced by varying doses of DAC with changes in gene expression levels to avoid erroneous conclusions regarding epigenetic silencing of a gene.

molecular biology↗