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Page 8 Graner. Extracell Vesicles Circ Nucleic Acids 2020;1:3-19 I http://dx.doi.org/10.20517/evcna.2020.08

membrane; “kiss-and-run” fusion at the ER; endosomal membrane fusion - aka, back fusion; and endosomal
rupture). They used an elegant system to dissect these mechanisms, starting with a cell line expressing a
GAL3 fluorescent reporter that is detectable upon binding beta-galactoside aggregates, which would be
present if endosomes ruptured. Upon exosome incubation, such rupture was not evident. They next used
exosomes from cells expressing intralumenal GFP-tagged CD63 that were loaded onto cells expressing a
labeled (mCherry) anti-GFP nanobody intracellularly. If exosomes fuse at the plasma membrane, the red
nanobody would accumulate there; if exosomes back fuse inside the endosome, the red nanobody would
accumulate intracellularly. There would be co-localization with GFP at each of these sites. Their experiments
revealed intracellular co-fluorescence, indicating endosomal release; and correlative light and electron
microscopy (CLEM) also showed lysosomal localization, suggesting movement through the endo-lysosomal
pathway. As these results suggested that protein transfer may occur via exosomes, they attempted treatment
of a Huntington’s Disease (polyQ, HTT-Q74) murine model. They used their exosome loading protocol to
place the HSP40 chaperone homolog DNAJB6 into exosomes. In intrathecally injected transgenic mice, they
showed reduction in poly-Q aggregates in mouse brains and spinal cords, indicating functional transfer of
the chaperone to reduce aggregates.

Continuing the theme of in vitro/in vivo work, Nicole Noren Hooten (NIH National Institute on Aging,
US) introduced the potential role of EVs in type 2 diabetes mellitus (T2DM). Previous studies found that
larger EVs (microparticles) derived from blood cells were elevated in patients with T2DM. Nicole’s work
examined smaller vesicles (exosome and microvesicle range) in a large longitudinal study was comprised of
euglycemic individuals, pre-diabetics, and T2DM patients (NIH HANDLS study). Precipitated plasma EV
concentrations were elevated in T2DM patients, and this increased as pre-diabetic patients progressed to full
disease. T2DM EVs had lower amounts of insulin signaling proteins and induced inflammatory responses
when incubated with monocytes and B cells, thus suggesting that those EVs may promote inflammation in
patients. Longitudinal and cross-sectional analysis of EV inflammatory proteins showed that levels of VEGFA
were associated with diabetes development in this expansive cohort. They followed this with differential
centrifugation of plasma EVs (10K × g and 120K × g fractions). Curiously, the 10K EV fraction from T2DM
patients increased endothelial cell migration vs. those EVs from euglycemic controls, and those cells showed
increased actin rearrangements and lamellipodia formation. This raises the possibility that plasma EVs in
T2DM patients may contribute to detrimental vascular changes that are related to the diabetic pathology.

Y. Peng Loh (NIH, National Institute of Child Health and Human Development, US) completed the EV
Function session with her group’s latest work on exosomal carboxypeptidase E (CPE) in tumor vesicles. CPE
mRNA overexpression is correlated with poorer overall survival in patients with hepatocellular carcinoma
(HCC), and is generally elevated in serum exosomes of cancer patients as well as in highly malignant cancer
cell lines. In vitro, exosomes from high metastatic HCC (more malignant) cell lines induce proliferation and
invasion in lower malignant HCC cells, and this can be blunted if the exosomes contain shRNA against
CPE. Loh et al., transfected CPE shRNA into HEK exosome producer cells to treat high metastatic HCC cells
with those exosomes, which reduced proliferation in those cells, perhaps by suppressing CCND1 and MYC.
This suggests that CPE is a good cancer biomarker, a good anti-cancer target, and that exosomal delivery of
shRNA may be a therapeutic avenue against cancer.

With increasing interest in the roles of EVs in microbiology, the next session featured talks on bacterial EVs,
hosted by Meta Kuehn (Duke University, US) and G Marcela Rodriguez (Rutgers University, US). Marcela
started the session with a talk on EV (membrane vesicle, MV) production in mycobacteria. Mycobacteria
MVs display phospho- and glycolipid content consistent with the plasma cell membrane, but the bacteria
is surrounded by a thick cell wall, which makes the MV release somewhat of a mystery. Mycobacterial
MV production is regulated by several genes; strains lacking the “vesiculogenesis and immune response
regulator” (viR) gene demonstrate enhanced vesicle production. Environmental iron availability also