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Chakraborty et al. Extracell Vesicles Circ Nucleic Acids 2023;4:27-43 https://dx.doi.org/10.20517/evcna.2023.05 Page 37

microglia and human peripheral blood mononuclear cells-derived microglia form extensive intercellular
[119]
networks of TNTs that contribute to the movement of α-Syn fibrils between cells . Our recent work also
highlights the movement of such fibrils preferentially from neuronal to microglial cells via TNTs. The extent
of TNT-mediated fibril movement from neuronal to microglial cells was significantly higher than in the
other direction, implicating a significant role of TNTs in mediating neuron-glia communication . These
[96]
results altogether provide a significant resource for understanding PD pathology spread between different
cell types of the brain. However, a comprehensive understanding of the (patho)physiological significance of
such differences in transfer and its contribution to PD spread in human brains is currently lacking.

Huntington’s disease
HD manifestation occurs due to extensive “CAG” repeats in exon 1 of the Huntingtin gene, which generates
mutant protein. Although not a lot of attention has been paid to the extent of TNT-mediated transfer of
mHtt between cells, there exist several lines of evidence, in vitro and in vivo, that confirm the involvement
of TNTs in the spread of pathology. mHtt movement between CAD neuronal cells and primary cerebellar
granule neurons has been reported to occur via TNTs . Subsequent reports from the group of
[123]
Subramaniam have shown the involvement of Rhes protein in the regulation of TNT formation and transfer
of mHtt between striatal neuronal cell lines and primary neurons, which the authors referred to as “Rhes
tunnels” . In vivo, transfer of mHtt between medium spiny neurons of striatum, as well as from striatum
[124]
to cortex, is reported to be dependent on Rhes-mediated connections between cells . With the
[125]
understanding of Rhes-regulated mHtt transfer via TNTs, it would be interesting to assess the roles of glial
cells in mHtt pathology spread.

CONCLUSION AND FUTURE PERSPECTIVES
The discovery and consecutive studies on TNTs in the past decades have enlarged the picture of
intercellular communication mechanisms. However, in order to understand the challenges to overcome in
the future, it is important to recognize the limitations of the field. First, TNTs are fragile structures and do
not survive most fixation protocols. A recently published paper used microfluidics and AFM indentation to
[126]
display the elastic properties of TNTs in human embryonic kidney cells, enabling them to resist bending .
They revealed that TNTs formed between cells separating faster than 0.5 µm/min are highly unstable. The
frailty of these structures might have initially represented an important limitation for the field, but
nowadays, protocols for fixation, identification and characterization of TNTs have been well
documented . Another issue that has been extensively addressed is the lack of specific markers to
[127]
distinguish TNTs from other TNT-like structures. Their ability to transfer vesicles and organelles between
cells is unique. However, even though live imaging of such transfer is critical to demonstrate their
functionalities, the low frequency of these events does not allow for robust quantification. Currently,
thorough studies on TNTs rely on the combination of several parameters to distinguish them from filopodia
in fixed and live samples for quantification: they should hover over the substrate, have a length above 10 μ
m, contain Actin and have a diameter below 1 μm. The combination of these parameters serves the purpose
of decreasing the number of false positives when studying TNTs, as confusion between filopodia and TNTs
is the main bias to avoid. A major drawback of such a method is the increase in false negatives. This analysis
allows the identification of a specific subtype of long, non-adherent TNTs, yet does not consider structures
too close to the substrate or smaller than 10 μm [55-68] . Finally, the field of TNTs faces the same challenge as
studies on EVs, or even filopodia. The difficulty of purifying these structures and the complexity of the
phenomenon studied requires us to categorize them using distinct terminology. Therefore, terms such as
“exosome”, “ectosome”, “migrasomes”, “filopodia”, or “TNT”, are likely to encompass a multitude of
structures with important structural and functional differences, as suggested by numerous studies in the
field [13,128,129] . In other words, semantics could provide a biased perception of the actual processes undergoing
in living cells. The molecular similarities of the formation mechanisms of TNTs, filopodia and EVs, along