Quote: Rajasekaran S, Witt SN (2021) Trojan horses and tunnel nanotubes enable the spread of α-synuclein pathology in Parkinson’s disease. PLoS Biol 19 (7): e3001331. https://doi.org/10.1371/journal.pbio.3001331
Released: July 20, 2021
Copyright ©: © 2021 Rajasekaran, Witt. This is an open access article distributed under the terms of the Creative Commons Attribution License, which allows unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged.
Financing: The authors did not receive any specific funding for this work.
Competing interests: The authors have stated that there are no competing interests.
α-syn, alpha-synuclein; LB, Lewy body; LN, Lewy neurite; NAC, non-Aβ component of Alzheimer’s plaques; PD, Parkinson’s Disease; PFF, preformed fibril; siRNA, small interfering RNA; TNT, tunnel nanotube
Parkinson’s disease (PD) is due to the progressive degeneration of dopaminergic neurons in the midbrain, and the affected neurons contain inclusions called Lewy bodies (LBs) and Lewy neurites (LNs) that are linked with aggregates and amyloid fibers of the presynaptic protein alpha Synuclein (α-syn)  . In a classic study, Braak showed that LB / LN pathology develops in the enteric nervous system of Parkinson’s patients and hypothesized that “a putative environmental pathogen that can pass through the gastric epithelial lining is misfolding and aggregating in certain cell types α-syn could trigger the submucosal plexus and reach the brain via a successive series of projection neurons [2,3] . ”Li later found that neurons transplanted in 2 PD patients contained LBs 11-16 years later when the patients died, suggesting that the LBs had spread from host to graft  . These 3 studies and others [5,6] led the field to the current paradigm, ie an intestinal insult triggers α-syn to form cytotoxic, prion-like conformers, which then propagate along the intestinal-brain axis. How α-syn spreads is a mystery that needs to be solved. In this issue, Dilsizoglu Senol and colleagues report  focus on the role of lysosomes and tunnel nanotubes (TNTs) in the dissemination of pathogenic conformations of α-syn.
The α-syn protein consists of 3 domains: an amphipathic domain (residues 1 to 60); the hydrophobic NAC domain (non-Aβ component of Alzheimer’s disease plaques, residues 61 to 95); and an acid tail (residues 96 to 140). The intrinsically unfolded monomer tends to aggregate and fibrillate (Fig. 1A). In vitro, amyloid fibers of α-syn are produced by incubating α-syn monomers for several days with shaking. Sonication breaks the long fibers into short soluble amyloid seeds called preformed fibrils (PFFs) (Figure 1A). PFFs can be injected into mice or added to cells in culture.
Fig. 1. Cell-to-cell transmission via TNTs from dysfunctional lysosomes infected with pathogenic α-syn.
(A) The innumerable states of α-syn. Depending on the environment, monomeric α-syn itself associates to form soluble oligomers and ultimately to form insoluble amyloid fibers. PFFs are made by first generating amyloid fibers (shaking at 37 ° C for 7 days), washing, resuspending, and sonicating. PFFs are characterized in terms of size and concentration and are typically 50 nm in length. The addition of a cysteine residue to the carboxyl terminus of α-syn facilitates the labeling of PFFs with a fluorophore. PFFs are toxic to cells. (B) Dysfunctional lysosomes are transferred from cell to cell. The donor cells were undifferentiated mouse catecholaminergic neuronal (CAD) cells. CAD cells take up fluorescently labeled PFFs from the cell culture medium, and these particles enter the lysosomes. PFFs damage the lysosomes: Infected lysosomes double their size, have a higher pH value, have a reduced activity of lysosomal proteases and are leaky compared to uninfected lysosomes (left pictures). The infected lysosomes spread to the cell periphery, increasing the likelihood that they will tunnel out of the cells through nanotubes. A unique surface reaction takes place in a naive acceptor cell: PFFs embedded in the membrane of the infected lysosome catalyze the conversion of endogenous α-syn monomers into prion-like particles. α-syn, alpha-synuclein; CAD, Kath.a – differentiated; L, lysosome; N, core; PFF, preformed fibril; TNT, tunnel nanotube; λ, α-syn monomer; ■, PFF.
In previous work, the Abounit group showed that lysosomes filled with α-syn-PFFs spread intercellularly through TNTs  . TNTs consist of F-actin, have a diameter of 50 to 800 nm and lengths of up to several cell diameters and enable the selective transfer of organelles [9,10] . In their study in this issue  , Dilsizoglu Senol and colleagues analyzed the effects of fluorescently labeled PFFs on lysosome morphology, function, leakage, and spatial distribution of the recipient cell using high resolution microscopy, transmission electron microscopy, and several other techniques. The imaging showed that PFFs interact with lysosomes in different ways: some are luminal while others are surface-bound. In cells that were incubated with PFFs, there was a significant increase in both the size and the leakage of the lysosomes “infected” with PFFs, but no change in the number of lysosomes compared to untreated control cells. A strange change also occurred in the distribution of the lysosomes: cells relocated their leaky, dysfunctional α-syn-loaded lysosomes, compared to untreated control cells, preferentially from the perinuclear region to the periphery (Fig. Lysosomal exocytosis. Small interfering RNA (siRNA) -mediated Knockdown fromTFEB, the transcription factor that controls lysosomal biogenesis, eliminated this shift. Moving infected lysosomes to the cell periphery increases the likelihood of stumbling into a nanotube tunnel (or perhaps being escorted) and tunneling out of the donor into the naive acceptor cell, and this is exactly what has been captured using various techniques.
An important finding is that leaky infected lysosomes (aptly referred to as “Trojan horses”) are a platform for prions to proliferate because their membranes are embedded with PFFs. Regardless of whether the infected lysosomes are in donor or acceptor cells, α-syn monomers collide with and adhere to the surface of the infected lysosomes; consequently, the surface-embedded PFFs induce the conversion of surface-bound α-syn molecules into toxic, prion-like molecules (Figure 1B). These newly formed prion-like molecules then penetrate into infected or uninfected lysosomes, which ultimately reach healthy neighboring cells through TNTs for seminal pathology.
In addition to nanotubes, α-syn can be released from cells by secretion or cleavage of exosomes and uptake via pinocytosis or receptor-mediated endocytosis. All of these mechanisms are explored and even a cell surface receptor that binds to α-syn fibrils is identified  . One challenge is to show that nanotubes form in neurons along the gut-brain axis, but it is difficult to imagine that neurons lack such connections. If the mechanistic details of lysosome transfer by TNTs can be deciphered, one strategy is to look for molecules that inhibit such transfer. Inhibiting the formation of TNTs is another option, although doing so could have unintended consequences. The pathological spread could also be thwarted by the identification of drugs that bind tightly to α-syn prions. Such drugs would reverse the proliferation of amyloid in cells. Ultimately, reliable biomarkers for the disease must be found so that the disease can be treated long before the disease spreads to the brain. Drugs that inhibit cell-to-cell transfer or inhibit prion proliferation would be an excellent way to stop the disease from progressing.