Multiplexed mRNA analysis of brain-derived extracellular vesicles upon experimental stroke in mice reveals increased mRNA content with potential relevance to inflammation and recovery processes

Abstract

Extracellular vesicles (EVs) are lipid bilayer-enclosed structures that represent newly discovered means for cell-to-cell communication as well as promising disease biomarkers and therapeutic tools. Apart from proteins, lipids, and metabolites, EVs can deliver genetic information such as mRNA, eliciting a response in the recipient cells. In the present study, we have analyzed the mRNA content of brain-derived EVs (BDEVs) isolated 72 h after experimental stroke in mice and compared them to controls (shams) using nCounter® Nanostring panels, with or without prior RNA isolation. We found that both panels show similar results when comparing upregulated mRNAs in stroke. Notably, the highest upregulated mRNAs were related to processes of stress and immune system responses, but also to anatomical structure development, cell differentiation, and extracellular matrix organization, thus indicating that regenerative mechanisms already take place at this time-point. The five top overrepresented mRNAs in stroke mice were confirmed by RT-qPCR and, interestingly, found to be full-length. We could reveal that the majority of the mRNA cargo in BDEVs was of microglial origin and predominantly present in small BDEVs (≤ 200 nm in diameter). However, the EV population with the highest increase in the total BDEVs pool at 72 h after stroke was of oligodendrocytic origin. Our study shows that nCounter® panels are a good tool to study mRNA content in tissue-derived EVs as they can be carried out even without previous mRNA isolation, and that the mRNA cargo of BDEVs indicates a possible participation in inflammatory but also recovery processes after stroke.

Extracellular RNA profiles in non-small cell lung cancer plasma

“…In our study, we used RNA-seq to analyze the species of total RNAs in the plasma of NSCLC patients and NCs. According to previous studies, the amount of exRNA in 5–7 µL plasma is approximately 10 pg ( 12 ). Thus, there is <1 ng of RNA in 500 µL of plasma.…”

“…In this study, we used CIRI to identify 203 extracellular circRNAs, but the frequently predicted circRNAs were low in abundance, and the predicted circRNAs differed greatly between individuals. This is because exRNAs in plasma are low in abundance and fragmented ( 12 , 42 ), which makes it difficult to completely present an exRNA expression profile. Additionally, the source of exRNA in the plasma is complex, and the health status of individuals differ.…”

“…The RNAs from the cells of whole-body tissues, and to some extent, the exRNAs of plasma, reflect the health of the body ( 11 ). Thus, circulating exRNAs are useful biomarkers for many diseases, including central nervous system disease, renal disease, and cancer ( 10 , 12 , 13 ). Many studies have reported possible NSCLC biomarkers, including miRNAs (e.g., mir-21 and mir-126) ( 14 , 15 ), and circRNAs (e.g., hsa_circ_0033155 and hsa_circ_0014130) ( 16 , 17 ).…”

Efficient enzyme-free isolation of brain-derived extracellular vesicles

“…With the previous experiments, we demonstrated that the isolated BDEV populations with and without collagenase were similar in terms of protein composition. To investigate similarities/differences among the BDEV preparations further, we then employed the Nanostring nCounter® Neurodegeneration panel to assess mRNA composition using the same protocol as previously described (Bub et al, 2022). For the analysis, we pooled F1 and F2 human BDEVs in both instances, isolated with (0.5 mg/mL) and without collagenase.…”

Mechanisms and Biomarker Potential of Extracellular Vesicles in Stroke

“…These oligodendrocyte-derived EVs have been found to contain a vast number of proteins, including tetraspanins, heat shock proteins, myelin proteins PLP and CNP, Alix, and Tsg101, and RNA [ 141 ]. In a recent study, exploring the mRNA content of brain-derived EVs in mice 72 h post MCAO, although the majority of mRNA content was microglial in origin, the EV population with the greatest increase was actually derived from oligodendrocytes [ 142 ]. Taken together, these results reveal that oligodendrocytes are likely to be dynamic players in stroke outcome.…”