The accumulation of α-synuclein (α-syn) aggregates in specific brain regions is a hallmark of synucleinopathies including Parkinson disease (PD). α-Syn aggregates propagate in a “prion-like” manner and can be transferred inside lysosomes to recipient cells through tunneling nanotubes (TNTs). However, how lysosomes participate in the spreading of α-syn aggregates is unclear. Here, by using super-resolution (SR) and electron microscopy (EM), we find that α-syn fibrils affect the morphology of lysosomes and impair their function in neuronal cells. In addition, we demonstrate that α-syn fibrils induce peripheral redistribution of lysosomes, likely mediated by transcription factor EB (TFEB), increasing the efficiency of α-syn fibrils’ transfer to neighboring cells. We also show that lysosomal membrane permeabilization (LMP) allows the seeding of soluble α-syn in cells that have taken up α-syn fibrils from the culture medium, and, more importantly, in healthy cells in coculture, following lysosome-mediated transfer of the fibrils. Moreover, we demonstrate that seeding occurs mainly at lysosomes in both donor and acceptor cells, after uptake of α-syn fibrils from the medium and following their transfer, respectively. Finally, by using a heterotypic coculture system, we determine the origin and nature of the lysosomes transferred between cells, and we show that donor cells bearing α-syn fibrils transfer damaged lysosomes to acceptor cells, while also receiving healthy lysosomes from them. These findings thus contribute to the elucidation of the mechanism by which α-syn fibrils spread through TNTs, while also revealing the crucial role of lysosomes, working as a Trojan horse for both seeding and propagation of disease pathology.

Tunneling nanotubes (TNTs) are actin-containing membrane protrusions that play an essential role in long-range intercellular communication. They are involved in development of various diseases by allowing transfer of pathogens or protein aggregates as well as organelles such as mitochondria. Increase in TNT formation has been linked to many pathological conditions. Here we show that nM concentrations of tolytoxin, a cyanobacterial macrolide that targets actin by inhibition of its polymerization, significantly decrease the number of TNT-connected cells, as well as transfer of mitochondria and α-synuclein fibrils in two different cell lines of neuronal (SH-SY5Y) and epithelial (SW13) origin. As the cytoskeleton of the tested cell remain preserved, this macrolide could serve as a valuable tool for future therapies against diseases propagated by TNTs.

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