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Loh et al. Extracell Vesicles Circ Nucleic Acids 2023;4:568-87 https://dx.doi.org/10.20517/evcna.2023.34 Page 582

released from internalized sEVs into the cytoplasm/nucleus of recipient cells. More studies in the future will
illuminate this possibility for other RSP proteins. Another advantage of transporting these RSP protein/
peptide molecules in sEVs to distant targets for intercellular communication may be to increase their half-
life in circulation since sEVs are stable and the cargo can be protected from degradation. This may possibly
expand the functions of such hormones or neurotrophic factors as they reach more distant targets. It is
likely, as more studies are done, that many other peptide hormones, neurotrophic factors, and regulated
secretory proteins could be found in EVs. This new concept is in its infancy and many questions remain
unanswered.

Most critical is how the hormones and neurotrophic factors, including their precursors, get packaged into
[133]
EVs/sEVs. Based on the discovery of Nobel Laurette Gunter Blobel , these hormones and neurotrophic
factors have an N-terminal signal peptide that directs them into the RER cisternae and into the secretory
vesicle pathway. One possibility is that RSP proteins may go into vesicles formed by budding from the TGN,
which then fuse with endosomes. Transmembrane cargo, such as the transmembrane form of CPE/NF-α1,
would then be retained on the endosome until it matured into a MVB, which would allow invagination and
ILV formation. Since transmembrane CPE/NF-α1 is a sorting receptor to which prohormones and
proBDNF bind tightly at an acidic pH, they may stay associated and ride with CPE/NF-α1 into ILVs.
Another possibility is that RSP proteins, after secretion, could be endocytosed and end up in endosomes
and then in ILVs. Alternatively, ILVs could be formed through the endoplasmic reticulum (ER)/Golgi
secretory pathway, packaging RSP proteins in them during the process. The ILVs are then taken up by
MVBs and released as sEV or extracellular vesicles [Figure 1]. A recent report studying the prostate-like
Secondary Cell (SC) of Drosophila male accessory gland demonstrated the presence of Rab6 positive-DCVs
and -ILVs within the same compartments derived from the TGN. Then Rab 6 in the membrane in these
organelles is transitioned to Rab11 for both Rab11 -exosome and DCV biogenesis, indicating that these two
[134]
processes may be interdependent . Indeed, Rab GTPases are known to control many aspects of vesicle
trafficking by acting as regulatable switches . While the SCs are a specialized secretory system, these
[135]
observations indicate the sharing of communication between the RSP and the endosomal systems for DCV
and EV biogenesis and hence the possible packaging of RSP cargo into sEVs. More studies on mammalian
secretory cells will determine if this communication between the RSP and endosomal system also occurs.

Another important question is, since these hormones and neurotrophic factors are packaged with other
specific components such as RNA and microRNA in EVs, how is that accomplished? Are they randomly or
selectively co-packaged? Finally, although it is clear that the hormones or neurotrophic factors in the sEVs
are taken up by recipient cells and exert specific physiological effects as reflected by changes in the cell’s
mRNA and microRNA profiles, the details of the mechanism of action remain elusive. Many more cell
biological studies in the future are necessary to shed light on these questions. It is hoped that our inclusion
of a review of the molecular players involved in the intracellular trafficking of hormones to the RSP will
inspire more cell biologists to study the molecular mechanisms in detail for the packaging of RSP proteins
into EVs/sEVs. Furthermore, the secretion, uptake, and internalization of these EVs and the physiological
function of the internalized hormones and neurotrophic factors in the recipient cells warrant exploration.

DECLARATIONS
Authors’ contributions
Wrote and edited the manuscript: Loh YP, Xiao L, Park JJ

Availability of data and materials
Not applicable.