Using genetically modified extracellular vesicles as a non-invasive strategy to evaluate brain-specific cargo
Rufino-Ramos, D. et al. (2022) Using genetically modified extracellular vesicles as a non-invasive strategy to evaluate brain-specific cargoBiomaterials 281, 121366. DOI: https://doi.org/10.1016/j.biomaterials.2022.121366
Extracellular vesicles (EVs) are small nanosized vesicles utilized for cell-to-cell communication by almost every cell within the brain. These vesicles contain lipids, proteins, and nucleic acid cargo vital for proper cellular homeostasis and maintenance of physiological condition. Despite unique cargo carried within these vesicles, it has proven challenging to track the release of EVs due to the large quantity within the nervous system, the relatively large distances they can travel, and their small size. Attempting to use fluorescent tags for proper identification has also proven challenging due to the high degree of autofluorescence these tags can generate. The aim of this study was to create a platform to improve identification of in vivo vesicle release to determine cells of origin more accurately in the nervous system.
This study utilized a transfusible construct tagging tetraspanin CD63, a hallmark vesicular membrane marker of EVs. This tag was equipped with both fluorescent and bioluminescent tags and was termed NoMi (NanoluciferaseoutsideandMCheryinside). NanoLuc was chosen as the bioluminescent tag for EV secretion due to its small size (19.1 kDa), lack of need for ATP and Mg2+, and extremely bright luminescence. Several other labels on the NoMi tag included copGFP, mCherry and FLAG, all to provide easier EV detection by immunofluorescence staining and pull-down assays. The Flag tag enabled isolation of the NoMi-EVs from the blood through affinity-tag capture.
Intracranial injection of the construct into the striatum of mice was paired with retro-orbital administration of the Nano-Glo® In Vivo substrate Fluorofurimazine (FFz) for bioluminescence expression within the EVs. The expression of the tag in the EVs was validated with immunofluorescence staining of coronal brain sections 13 days after injection. These NoMi-EVs appeared to spread throughout the brain after injection, as there was a gradient of NanoLuc luminescence up to 800µm away from the injection site. Increased RNA levels of the copGFP tag outside the brain compartment demonstrated that brain labeled EVs are released into the blood stream. To further demonstrate that EVs spread through the bloodstream, primary human neural progenitor cells (hNPCs) were transduced with NoMi before implantation into mouse brains. These hNPC transduced animals demonstrated a similar increase in copGFP-RNA levels in the periphery.
Results of this proof-of-concept study demonstrate the power of their NoMi construct in identifying release in vivo activity of EVs. This strategy shows the potential for combining a robust bioluminescent tag for EV detection with a Flag tag. However, further optimization is necessary before this approach can be generally applied to study EV activity within complicated neuronal research.
Keywords: bioluminescent imaging, luciferase, NanoLuc, Extracellular vesicle, EVs, Central Nervous System, human Neural Progenitor Cells
