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Page 30 Chakraborty et al. Extracell Vesicles Circ Nucleic Acids 2023;4:27-43 https://dx.doi.org/10.20517/evcna.2023.05

[46]
[45]
spreading of diseases. Viruses such as HIV-1 , SARS-CoV-2 or Herpes were shown to exploit TNTs as
[47]
a way to propagate while evading the immune system. TNTs were also proposed to be involved in the
spreading of various neurodegenerative pathologies because they are able to mediate the propagation of
[48]
aggregate-prone proteins accumulating in different NDs (reviewed by Soraya Victoria and Zurzolo ).
Finally, cancer cells also appear to use TNTs as a way to survive chemo- and radio-therapy and adapt to
their microenvironment [49-52] . In this review, we summarize the structural and functional characteristics of
TNTs and their role in the propagation of aggregate-prone proteins in different NDs.

MECHANISM OF TNT FORMATION
Molecular players of cytoskeletal regulation in TNT formation
Two main mechanisms of TNT formation have been proposed: cell dislodgement and protrusion-
elongation [Figure 1B]. So far, these formation mechanisms have not been correlated to any difference in
structure or functionality. As mature neurons are post-mitotic in nature and exhibit low migratory
phenotype past embryonic development, cell dislodgement does not appear to be a favored formation
mechanism for TNTs between neuronal cells, which is consistent with previous observations in
catecholaminergic-derived neuronal cell line (CAD cells) [53,54] .

Therefore, hereafter we focus on the protrusion-elongation mechanism.

Other Actin-based protrusions have been described and studied before the discovery of TNTs, such as
filopodia, microvilli, or stereocilia. Interestingly, common players have been identified in the formation
mechanism of these structures, hinting us towards the probable actors involved in TNT formation.
Supported by current literature , one of the models describing the critical steps involved in TNT formation
[53]
through protrusion-elongation begins with a signaling cascade leading to the activation of Rho GTPases.
This in turn leads to the activation and clustering of membrane-bending proteins to locally induce negative
membrane curvature, which is associated with the recruitment of Actin polymerizers and Actin bundlers to
create and elongate a bundle of Actin filament that will push the membrane and grow the protrusion . In
[55]
the past years, some actors shown to positively regulate TNT formation have strengthened this model, such
[56]
[53]
[57]
as the G-protein Rab35 , the I-BAR protein IRSp53 , or the Actin bundler Eps8 . Eventually, fusion
occurs at the tip of the TNT, probably through a process of activation and recruitment of adhesive proteins
associated with Actin polymerization to drive the force required to break the membrane tension, as
observed in myoblast fusion . Consistent with this hypothesis, very recent data support the role of the N-
[58]
Cadherin-α-Catenin complex, as well as of tetraspanins (CD9 and CD81) in the process of fusion with the
receiving cell [Figure 1B] [30,59,60] . Following the formation of the structure, motor proteins would mediate the
transport of cargoes [54,61] . The nature of cargoes transferred from a donor to an acceptor cell seems to be a
well-organized event, with the involvement of molecular motors, intracellular/extracellular
components, type and health of the connected cells, and potentially several other influential factors
that are yet to be discovered. The functional nature of TNTs will be discussed subsequently.

What regulates the formation of a TNT with an acceptor cell and whether this process is random or guided
remains largely unknown. In rat hippocampal neurons and astrocytes, p53 leads to caspase-3 activation,
subsequently leading to the cleavage of the calcium-binding protein S100A4 in TNT-initiating cells. This
consequently results in an extracellular gradient of S100A4 which was shown to direct TNT formation
towards other cell . It is yet the only known mechanism of guidance of TNTs via chemotactic cues, but it
[62]
leads us to think that the general directionality of TNT growth might be regulated through similar
processes.