α-Synuclein fibrils subvert lysosome structure and function for the propagation of protein misfolding between cells through tunneling nanotubes

Aysegul Dilsizoglu Senol, Maura Samarani, Sylvie Syan, Carlos M. Guardia, Takashi Nonaka, Nalan Liv, Patricia Latour-Lambert, Masato Hasegawa, Judith Klumperman, Juan S Bonifacino, et al.

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Aysegul Dilsizoglu Senol, Maura Samarani, Sylvie Syan, Carlos M. Guardia, Takashi Nonaka, et al. α-Synuclein fibrils subvert lysosome structure and function for the propagation of protein misfolding between cells through tunneling nanotubes. PLoS Biology, 2021, 19 (7), pp.e3001287. 10.1371/journal.pbio.3001287. pasteur-03274582

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α-Synuclein fibrils subvert lysosome structure and function for the
propagation of protein misfolding between cells through tunneling
nanotubes

4 Aysegul Dilsizoglu Senol¹§, Maura Samarani¹§, Sylvie Syan¹, Carlos M. Guardia², Takashi

5 Nonaka³, Nalan Liv⁴, Patricia Latour Lambert⁵, Masato Hasegawa³, Judith Klumperman⁴, Juan

6 S. Bonifacino², Chiara Zurzolo¹*

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1'Unité de Trafic Membranaire et Pathogénèse, Département de Biologie Cellulaire et de
l'Infection, Institut Pasteur, Paris, France.

9 ²Neurosciences and Cellular and Structural Division, *Eunice Kennedy Shriver* National
10 Institute of Child Health and Human Development, National Institutes of Health, Bethesda,
11 MD, USA.

12 ^3Dementia Research Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.

13 ^4Section Cell Biology, Center for Molecular Medicine, University Medical Center Utrecht,
14 Utrecht University, Utrecht, Netherlands.

15 ^5Dynamique des Interaction Hôte-Pathogène, Département de Biologie Cellulaire et de
16 l'Infection, Institut Pasteur, Paris, France.

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§These authors contributed equally

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*Corresponding Author

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E-mail: chiara.zurzolo@pasteur.fr (CZ)
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Abstract

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The accumulation of α-synuclein aggregates in specific brain regions is a hallmark of synucleinopathies including Parkinson's disease. α-Synuclein 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-synuclein aggregates is unclear. Here, by using super-resolution and electron microscopy, we find that α-synuclein fibrils affect the morphology of lysosomes and impair their function in neuronal cells.

In addition, we demonstrate that α-synuclein fibrils induce peripheral redistribution of lysosomes, likely mediated by TFEB, increasing the efficiency of α-synuclein fibrils' transfer to neighboring cells. We also show that lysosomal membrane permeabilization allows the seeding of soluble α-synuclein in cells that have taken up α-synuclein fibrils from the culture medium and, more importantly, in healthy cells in co-culture, 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 co-culture system, we determine the origin and nature of the lysosomes transferred between cells and we show that donor cells bearing α-synuclein 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-synuclein 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.
41 **Introduction**

The accumulation of amyloidogenic proteins in different regions of the brain is a hallmark of neurodegenerative diseases (NDs) leading to cellular dysfunction, loss of synaptic communication, and deficits in specific brain functions [1,2]. The misfolded α-synuclein (α-syn) protein accumulates in intraneuronal inclusions known as Lewy bodies and Lewy neurites in NDs known as synucleinopathies, including the second most common ND, Parkinson's Disease (PD) [3–5]. These inclusions are associated with loss of neuronal and glial cells in specific brain regions, leading to both motor (e.g., bradykinesia, tremor, rigidity) and non-motor (e.g., cognitive impairment, depression, anxiety) symptoms [6,7].

α-Syn is a small (140 amino acids) cytoplasmic protein enriched in the brain and localized to presynaptic terminals [8]. Although the precise function of α-syn remains poorly understood, it has been shown to promote membrane curvature, thus contributing to synaptic trafficking and vesicle budding [9–12], to modulate dopamine release through its association with presynaptic terminal SNARE complexes [13], and to be involved in synaptic attenuation [14]. A recent study has characterized *in vitro* a mechanism by which α-syn stabilizes, in a concentration-dependent manner, the docking of synaptic vesicles on the plasma membrane by establishing a dynamic link between the two membranes [15]. In PD brains, the spread of the pathology correlates with the presence of α-syn inclusions and follows a specific and predictable route through interconnected brain regions, mirroring the different stages of the disease [5,16]. These findings supported the hypothesis, later confirmed experimentally, that α-syn aggregates can be transferred from one cell to another both *in vitro* [17–22], and *in vivo* [23–27]. Furthermore, mounting evidence suggests that α-syn aggregates spread in a “prion-like” manner, as they are able to self-propagate by acting as “seeds” and inducing the misfolding of soluble native α-syn [19,27–35]. Thus, understanding how α-syn aggregates spread and how/where in the cell they induce misfolding of the soluble protein is crucial: i) to elucidate the pathogenesis of the disease, and ii) to devise a therapy to inhibit the progression of the pathology. Different mechanisms depending on either cell-to-cell contact or secretion have been proposed for the spread of α-syn fibrils [35]. We and others have found that, in *in vitro* and *ex vivo* cultures, tunneling nanotubes (TNTs) play a major role in this process [19–21,36].

TNTs are actin-based, thin cellular protrusions that connect remote cells [37]. They have been identified in various cell types *in vitro* [38–41] and *in vivo* [42–47]. TNTs are open-ended structures [48,49] that allow the exchange of various cargos, including entire organelles, such as mitochondria and lysosomes, between cells [19,37–40]. In addition to α-syn fibrils, other
amyloidogenic proteins such as prion protein [50–52], huntingtin [53], and tau [52,54,55] use TNTs as highways for transfer to naïve cells, suggesting that TNTs are common routes for the spread of pathogenic proteins between cells. Of particular interest, we showed that α-syn fibrils can be efficiently transferred between neuronal CAD cells, primary neurons, primary astrocytes, and human neural progenitor cells, and between organotypic hippocampal slices and astrocytes. We found that α-syn fibrils are mostly localized inside lysosomes in all of these cell types [19,21,56] and could be transferred inside lysosomes through TNTs from “infected” donor cells to naïve acceptor cells in co-cultures. Importantly, once arrived in the acceptor cells, α-syn fibrils were able to induce the aggregation of soluble α-syn [19]. How this occurs, however, is not clear.

Lysosomes are cytoplasmic, membrane-enclosed, acidic organelles that have long been considered as the “dustbin” and “recycling center” of the cells, as they are responsible for the degradation of both exogenous and intracellular cargos destined to lysosomes via endocytic, phagocytic [57,58], and autophagic pathways [59,60]. However, in recent years, mounting evidence revealed that lysosomes play a more complex role in the regulation of cellular homeostasis, as they are involved in crucial processes such as plasma membrane repair, transcriptional control, nutrient sensing, signaling, and energy metabolism [61–65]. It is therefore not surprising that lysosomal dysfunction is involved in a plethora of pathological conditions, including NDs [66]. Notably, the levels of lysosome-associated membrane proteins 1 and 2 (LAMP1 and LAMP2) and lysosomal enzymes Cathepsin B and D were found to be significantly reduced in PD patient brains, mouse and cell models [67–72]. Moreover, lysosomal membrane permeabilization (LMP) has been reported in cells treated with protein aggregates including α-syn [73,74].

In the present study, we focused on the role of lysosomes in the intercellular spread of α-syn fibrils in neuronal cells. Particularly, we studied how α-syn fibrils affect lysosomes and how these organelles contribute to the spread and propagation of α-syn aggregates through TNTs. We found that α-syn fibrils alter the morphology of lysosomes and compromise lysosomal function. We demonstrated that α-syn fibrils induce the peripheral distribution of the lysosomes exhibiting functional impairment, likely mediated by TFEB, and showed that this peripheral positioning enhances the efficiency of α-syn fibrils’ transfer in a cell-to-cell contact-dependent manner. In addition, we demonstrated that TFEB translocated to the nucleus in the presence of α-syn fibrils, probably contributing to the peripheral distribution of lysosomes. We also found that LMP, which is well described in cells following the uptake of α-syn fibrils from the medium, also occurs in acceptor cells following the transfer of lysosomes bearing α-syn fibrils
from neighboring donor cells. Thus, by facilitating the escape of α-syn fibrils from lysosomes after their transfer, LMP appears to be a relevant mechanism for the seeding of soluble α-syn and propagation of the pathology in neighboring cells. Importantly, our super-resolution microscopy data suggest that lysosomes create an optimal environment for seeding and are a hub for the conversion of soluble α-syn into aggregates. Finally, by using a heterotypic cell co-culture, we were able to track the origin and nature of the lysosomes transferred between cells. We found that cells overwhelmed by the presence of α-syn fibrils impairing lysosomal function transfer more damaged lysosomes to recipient cells and, in return, they receive healthy lysosomes from neighboring cells devoid of α-syn, suggesting a potential rescue mechanism in α-syn pathology.
118 **Results**

119 **α-Syn fibrils affect the morphology and function of lysosomes.**

120 We have previously reported that the majority of exogenous α-syn fibrils internalized by 121 neuronal catecholaminergic cell line CAD localize to lysosomes [19]. We confirmed these 122 findings by performing object-based 3D colocalization analysis (https://imaris.oxinst.com) in 123 CAD cells challenged with α-syn fibrils for 18 h prior to labelling for the lysosomal marker 124 LAMP1. We detected 74 ± 1% of α-syn fibrils colocalizing with lysosomes (LAMP1+ 125 organelles), confirming that the majority of α-syn fibrils localize to lysosomes following their 126 uptake (Fig 1A). In addition, we found 60 ± 1% of lysosomes colocalizing with α-syn fibrils 127 (Fig 1A). To further investigate the lysosomal localization of α-syn fibrils and the possible 128 morphological consequences of this localization, we performed structured illumination 129 microscopy (SIM). We found that α-syn fibrils localized either inside lysosomes (Fig 1B, 130 magenta arrow and enlarged images on the right) or at the lysosomal membrane (Fig 1B, green 131 arrow and enlarged images on the right). We then measured the diameter of lysosomes in 132 control cells and in α-syn fibril-treated cells (where we distinguished between lysosomes 133 containing or not containing α-syn fibrils). The average diameter of lysosomes in control cells 134 and of lysosomes not containing α-syn fibrils in treated cells was almost identical (0.43 ± 0.01 135 µm) whereas lysosomes containing α-syn fibrils increased their diameter by 50% (0.63 ± 0.01 136 µm; Fig 1B, graph), demonstrating that the enlargement induced by α-syn fibrils is restricted to 137 the lysosomes containing fibrils.

138 To further investigate the morphology of compartments containing α-syn fibrils, we used a 139 correlative light-electron microscopy (CLEM) approach to identify α-syn-positive and - 140 negative lysosomes by fluorescence microscopy (FM) and study their corresponding 141 morphology by electron microscopy (EM). First, we cultured CAD cells transiently transfected 142 with a plasmid encoding LAMP1-GFP and loaded with Alexa 568-tagged α-syn fibrils on 143 gridded coverslips, and imaged α-syn fibril-treated cells in FM while marking coordinates of 144 α-syn positive (Alexa568+/GFP+) and negative (Alexa568-/GFP+) LAMP1-GFP lysosomes. 145 Following FM imaging, the CAD cells were prepared for EM by keeping their orientation, and 146 serial sections were collected from the region containing the previously marked coordinates 147 (see also Material and Methods) [75,76]. Then, the FM signal was correlated to EM images. 148 Using this approach, we overcame the lack of suitable tools to identify α-syn fibrils directly by 149 EM and were able to compare the structural properties of lysosomes containing or not 150 containing α-syn fibrils. LAMP1-GFP-positive organelles colocalizing with α-syn fibrils in FM
were structurally identified as lysosomes by the presence of electron-dense heterogenous luminal content, degraded membranes, and occasional intraluminal vesicles as morphological criteria [77]. Lysosomes not containing α-syn fibrils presented a healthy lysosome morphology with spherical shape and smooth membranes [77,78] (Fig 1C, green square, number 1). Interestingly, most lysosomes containing α-syn fibrils were found to be quite heterogeneous in structure (Fig 1C, red squares numbers 2, 3, and 4). Some had an unusual luminal content and displayed curvy limiting membrane, suggesting that the presence of the fibrils could deform the organelles (Fig 1C, red squares numbers 3, and 4). The aberrant, curvy organelles were completely absent in control cells not treated with α-syn fibrils (S1A Fig). A subset of lysosomes containing α-syn fibrils, as defined by FM, had a healthy morphology (Fig 1C, red square, number 2), which could represent an early stage of fibrillar accumulation.

Next, we used another CLEM approach to analyse the morphology and size of α-syn-positive lysosomes with high-resolution. CAD cells loaded with Alexa 568-tagged α-syn fibrils were prepared for EM following the flat embedding Tokuyasu technique. This technique preserves the fluorescence signal of α-syn fibrils in ultrathin sections prepared for EM, and in addition allows to perform immunolabelling (both gold and fluorescent) of endogenous LAMP1 [79] (Fig 1D). Following a recently developed high-throughput on-section CLEM approach [80], we analysed 50 LAMP1-immunolabelled lysosomes in control cells (S1B Fig), 50 α-syn-positive, and 50 α-syn-negative lysosomes in treated cells. For each lysosome, we correlated EM ultrastructural details (surface area and perimeter) and the presence of LAMP1 to the FM images showing the presence or absence of α-syn fibrils. Using this approach, we confirmed that the lysosomes bearing α-syn fibrils were significantly larger (perimeter: 2.24 ± 0.14 µm and area: 0.16 ± 0.01 µm²) compared to lysosomes devoid of α-syn fibrils (perimeter: 1.16 ± 0.06 µm and area: 0.08 ± 0.01 µm²) and control lysosomes (perimeter: 1.16 ± 0.04 µm and area: 0.08 ± 0.01 µm²; Fig 1D, graphs).

**Fig 1. α-Syn fibrils affect the morphology of lysosomes. (A)** Representative confocal images of control and Alexa 568-tagged α-syn fibril-treated (18 h) CAD cells, immunolabelled for LAMP1 (green). Colocalization between LAMP1+ puncta and α-syn fibrils is indicated by arrows in the inset of a selected region delimited by a square. % of α-syn fibrils colocalizing with LAMP1+ puncta (74 ± 1%) and % of LAMP1+ puncta colocalizing with α-syn fibrils (60 ± 1 %) performed by object-based 3D colocalization method (Imaris software) is presented. Mean ± S.E.M, n=3 (30 cells per condition). Scale bar: 10 µm (for inset: 2 µm). **(B)** Super-
resolution (SR) images of control (left panel) and Alexa 568-tagged α-syn fibril-treated CAD cells for 18 h (right panel), immunolabelled for LAMP1 (far-red) antibody (pseudo colored in grey). Merge images of each condition are presented with additional HCS CellMask™ Blue staining. Magenta and green arrows indicate the selected lysosomes having α-syn fibrils inside the lysosomal lumen and on the lysosomal membrane, respectively, where higher magnifications of these lysosomes and 3D reconstructions are represented in magenta and green squares, respectively. Scale bar: 10 µm (for insets: 2 µm, for magenta and green insets: 0.5 µm).

Average diameter (µm) of lysosomes in control (0.43 ± 0.01) and in α-syn fibril-treated cells, the latter sub-grouped as: lysosomes without α-syn fibrils (0.43 ± 0.01) and lysosomes with α-syn fibrils (0.63 ± 0.01), is presented. Mean ± S.E.M, lysosomes' diameters were measured in SR images of eight α-syn fibril-treated cells and seven control cells (155 lysosomes per condition). Images were acquired by spinning-disk microscopy with SR module. ns=not significant, ****P<0.0001 by Kruskal-Wallis nonparametric ANOVA test followed by Dunn's multiple comparison tests. (C) FM image of LAMP1-GFP transfected and α-syn fibril-treated CAD cells for 18 h (left panel). Enlarged merged images of correlative resin EM and FM images of four selected lysosomes not containing (number 1, indicated by green square) and containing α-syn fibrils (numbers 2, 3, 4, indicated by red squares) are presented. Lysosomes number 1 and 2 are further magnified (blue squares) and presented within the same image. Lyso: lysosome, PM: plasma membrane, LD: lipid droplets. Scale bar: 10 µm (for red and green insets: 0.5 µm, for blue insets: 0.2 µm). (D) Overlay of on-section correlative EM images immuno-gold labelled for LAMP1¹⁰ and FM images of LAMP1-GFP transfected and α-syn fibril-treated CAD cells (18 h). Insets of three selected regions (indicated by red, purple, and black squares) are presented. Average perimeter (µm) of lysosomes in control (1.16 ± 0.04) and in α-syn fibril-treated cells: lysosomes without α-syn fibrils (1.16 ± 0.06) and lysosomes with α-syn fibrils (2.24 ± 0.14; upper graph) is presented. ns=not significant, ****P<0.0001 by one-way ANOVA followed by Tukey's multiple comparison tests. Average area (µm²) of lysosomes in control (0.08 ± 0.01) and in α-syn fibril-treated cells: lysosomes without α-syn fibrils (0.08 ± 0.01) and lysosomes with α-syn fibrils (0.16 ± 0.01; lower graph) is presented. Mean ± S.E.M. (50 cells per condition). ns=not significant, ****P<0.0001 by Kruskal-Wallis nonparametric ANOVA test followed by Dunn's multiple comparison tests. Scale bar: 10 µm (for insets: 0.5 µm). The data underlying this Figure may be found in S1 Data.
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In spite of these changes in lysosome morphology, the presence of α-syn fibrils had no effect on the abundance of lysosomes. Indeed, the average number of LAMP1-positive lysosomes per cell (control cells: 222 ± 6; α-syn fibril-treated cells: 218 ± 7; Fig 2A, left panel) and the level of LAMP1 (control: 0.9 ± 0.2 AU; α-syn: 0.7 ± 0.1 AU; Fig 2A, right panel) did not show any significant difference in the presence or absence of α-syn fibrils. Overall, these results indicate that, while the overall amount of lysosomes is not affected by α-syn fibrils, lysosomes containing α-syn exhibit changes in size and morphology.

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α-Syn fibrils have been shown to impair lysosomal function [67–72]. Thus, next we addressed their effect on lysosomes in our model system. We first assessed the acidity of the lysosomes in cells treated or not with α-syn fibrils by using a fluorogenic acidotropic dye, LysoTracker Deep Red (LysoTracker DR). The fluorescence intensity of LysoTracker DR was significantly decreased (~41 %) in α-syn fibril-treated cells compared to untreated control cells (Fig 2B), suggesting alkalinization of lysosomes in the presence of α-syn fibrils. Second, we used a DQ-BSA assay in which the fluorescent signal is detected only when the fluorogenic substrate is hydrolyzed by lysosomal proteases. We observed that the DQ-BSA fluorescence intensity was significantly decreased (~45 %) in the presence of α-syn fibrils compared to the control (Fig 2C), suggesting that the activity of the lysosomal proteases was compromised. Finally, we used a more specific approach to evaluate lysosomal function by quantifying the activity of the lysosomal enzyme Cathepsin B (CathB). The intracellular activity of CathB was detected as fluorescent puncta produced by the hydrolysis of the fluorogenic substrate Magic Red in live cells. We observed a significant decrease (~42 %) in CathB enzyme activity in cells challenged with α-syn fibrils compared to control cells (Fig 2D). Since the lysosomal pool was similar in control and α-syn fibril-treated cells, these data indicate that the decrease in the fluorescent signal of LysoTracker DR, DQ-BSA, and Magic Red is likely due to the presence of α-syn fibrils.

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Altogether, our data indicate that the majority of the α-syn fibrils localize to the lysosomes, where they induce lysosomal enlargement and structural changes, and impair lysosome function.

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**Fig 2. α-Syn fibrils affect the function of lysosomes. (A)** Average number of lysosomes (LAMP1+ puncta) per cell in control (222 ± 6) and in α-syn fibril-treated CAD cells for 18 h (218 ± 7) is presented. Mean ± S.E.M, *n*=5 (50 cells per condition). ns=not significant by Whitney-Mann *U*- test (left panel). Western blot showing LAMP1 and α-tubulin expression in

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control and α-syn fibril-treated cells. Integrated Density of LAMP1 protein expression levels normalized to α-tubulin in control (0.9 ± 0.2) and α-syn fibril-treated cells (0.7 ± 0.1) are presented in Arbitrary Units (AU). Mean ± S.E.M, n=3. ns=not significant by paired t-test. (B)
Representative confocal images of control and Alexa 568-tagged α-syn fibrils challenged CAD cells (18 h), labelled with LysoTracker DR (20 nM for 20 min; LysoTracker DR is pseudo colored in grey). Dashed lines represent the cell contour. Average Integrated Density of control cells (1.4 x 10⁷ ± 9 x 10⁵) and α-syn fibril-treated cells (8.3 x 10⁶ ± 5 x 10⁵) is presented. Mean ± S.E.M, n=3 (40 cells per condition). ****P<0.0001 by Mann-Whitney U-test. Scale bar: 10 µm. (C)
Representative confocal images of control and Alexa 568-tagged α-syn fibrils (red) challenged CAD cells for 18 h, treated with DQ-BSA (green) for 90 min. Dashed lines represent the cell contour. Average Integrated Density measured for control cells (3.8 x 10⁶ ± 1 x 10⁵) and α-syn fibril-treated cells (1.7 x 10⁶ ± 6 x 10⁴) is presented. Mean ± S.E.M, n=3 (25 cells per condition). ****P<0.0001 by Mann-Whitney U-test. Scale bar: 10 µm. (D)
Representative confocal images of control and Alexa 488-tagged α-syn fibrils (green) challenged CAD cells for 18 h treated with 1X Magic Red Cathepsin B (red). Dashed lines represent the cell contour. Average Integrated Density measured for control cells (1.3 x 10⁶ ± 6 x 10⁴) and α-syn fibril-treated cells (7.5 x 10⁵ ± 5 x 10⁴) is presented. Mean ± S.E.M, n=5 (100 cells per condition). ****P<0.0001 by Mann-Whitney U-test. Scale bar: 10 µm. The data underlying this Figure may be found in S1 Data.

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269 **Lysosomes containing α-syn fibrils undergo Lysosomal Membrane Permeabilization**
( **LMP** ).
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271 α-Syn [73,74], as well as other amyloidogenic aggregates such as tau [81,82] and amyloid beta
272 [83], induce LMP following their uptake from the culture medium in different cell types. This
273 allows escape of α-syn aggregates from the lysosome to act on the soluble α-syn in the cytosol.
274 However, the fate of these lysosomes has not been assessed. Furthermore, we previously
275 demonstrated that α-syn spread between cells occurs mainly by direct cell-to-cell transfer of
276 lysosomes bearing aggregates through tunneling nanotubes (TNTs), cytoplasmic protrusions
277 extending from the cell periphery and connecting distant cells [21,35,52] (S2 Fig). Therefore,
278 the question arises as to whether LMP also occurs after lysosomes bearing α-syn have been
279 transferred to healthy neighboring cells. To address this question, we assessed first whether α-
280 syn fibrils also induced LMP in our neuronal CAD cells. We used the Galectin-3 (Gal3) puncta
281 assay, based on the localization of Gal3 to damaged lysosomes. CAD cells were transiently
282 transfected with Gal3-GFP and challenged or not with α-syn fibrils for 18 h, prior to labelling
283 for LAMP1 (Figs 3A and S3A number 1). After image acquisition by confocal microscopy, we
284 quantified Gal3-GFP puncta formation in α-syn fibril-treated and non-treated cells. We detected
285 a significantly higher percentage of cells exhibiting Gal3-GFP puncta in the presence of α-syn
286 fibrils (46 ± 2%) compared to the control condition (7 ± 1%) (Fig 3B, left graph), in which most
287 cells displayed cytosolic Gal3-GFP [73,74]. However, in cells exhibiting Gal3-GFP puncta,
288 there was no significant difference in the average number of puncta between control and α-syn
289 fibril-treated cells (8 ± 1 and 12 ± 1 respectively; Fig 3B, right graph). In addition, we performed
290 object-based 3D colocalization analysis to evaluate the association of Gal3-GFP with both α-
291 syn fibrils and LAMP1+ lysosomes. We detected 38 ± 4% of Gal3-GFP puncta colocalizing
292 with α-syn fibrils, 47 ± 3% of Gal3-GFP puncta colocalizing with LAMP1+ lysosomes, and 35
293 ± 3% of Gal3-GFP puncta colocalizing with both α-syn fibrils and lysosomes (Fig 3A, magenta
294 arrowheads, and C). These findings demonstrate that more than one third of Gal3-GFP puncta
295 are recruited to the ruptured lysosomes containing α-syn fibrils. Furthermore, by using SIM,
296 we could clearly observe the recruitment of Gal3-GFP to the damaged lysosomes positive for
297 α-syn fibrils, which appeared to be either inside their lumen (Fig 3D) or at their membrane (Fig
298 3E).
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Since lysosomes under LMP can be targeted to the autophagy pathway in order to be degraded (a process called lysophagy) [84], next we investigated this possible scenario in CAD cells treated with α-syn fibrils in comparison with control cells. As a positive control we treated CAD cells with L-leucyl-L-leucine methyl ester (LLOMe), which is a well-known inducer of LMP. Cells were then labeled for ubiquitin, LC3, and LAMP1, and colocalization of these markers were analysed. As expected, in CAD cells treated with LLOMe, we could detect good colocalization between LC3, ubiquitin and LAMP1, whereas in control cells we could only detect a few LC3 and ubiquitin puncta and no colocalization between these puncta and lysosomes (S4 Fig). In three independent experiments, we could only detect one lysosome that colocalized with LC3 and ubiquitin in α-syn fibril-treated cells (S4 Fig); thus, lysophagy was an extremely rare event under these conditions and was not statistically different from the control condition.

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312 **Fig 3. α-Syn fibrils induce LMP. (A)** Representative confocal images of Gal3-GFP transfected
313 control (upper panel) and Alexa 568-tagged α-syn fibril-treated CAD cells for 18 h (lower
panel), labelled with LAMP1-Alexa 647 antibody (pseudo colored in grey). Higher magnifications of the selected region in α-syn fibril-treated cells (white squares) are presented on the upper-left part of each image. Arrowheads indicate a lysosome having α-syn fibrils colocalizing with Gal3 puncta. Scale bar: 10 µm (for insets: 5 µm). (B) % of control (7 ± 1%) and α-syn fibril-treated CAD cells for 18 h (46 ± 2%) under LMP (left graph), and average number of Gal3-GFP puncta for control (8 ± 1) and α-syn fibril-treated cells (12 ± 1) under LMP (right graph). Mean ± S.E.M, n=3 (50 cells per condition). ****P<0.0001 by Mann-Whitney U-test. (C) % of Gal3-GFP puncta colocalizing with α-syn fibrils (38 ± 4%), with LAMP1 (47 ± 3%), and with both α-syn fibrils and LAMP1 (35 ± 3%) calculated by object-based 3D colocalization (Imaris software). Mean ± S.E.M, n=3 (30 cells per condition). (D) SR image showing the recruitment of Gal3-GFP to a lysosome containing α-syn fibrils puncta in its lumen. (E) SR image showing the recruitment of Gal3-GFP to a lysosome having α-syn fibrils at its membrane. In D and E, insets of two selected regions (white squares) from different Z-planes of the same Gal3-GFP transfected and α-syn fibril-treated CAD cell, labelled for HCS CellMask™ Blue, DAPI, and LAMP1 (pseudo colored in grey) are presented. Images were acquired with SR SIM (Zeiss, LSM 780-Elyra PS.1). Scale bars: 10 µm (for insets: 1 µm). The data underlying this Figure may be found in S1 Data.

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332 α-Syn fibrils seed de novo formation of aggregates at the lysosomes.

333 LMP has been proposed to be a relevant mechanism for α-syn fibrils to escape from lysosomes and interact with soluble α-syn to induce seeding [19,85]. Therefore, we investigated the seeding activity of exogenous α-syn fibrils in CAD cells. To this aim, CAD cells were transiently transfected with a plasmid encoding α-syn-GFP, a soluble fluorescent reporter of α-syn, and the cells were challenged or not with Alexa 568-tagged α-syn fibrils for 18 h. The formation of α-syn-GFP aggregates detected as green puncta was then quantified (Figs 4A and S3A number 2). We found that 76 ± 5% of cells treated with α-syn fibrils produced newly formed α-syn-GFP aggregates, whereas this percentage was significantly lower (13 ± 2%) in control cells transfected with α-syn-GFP only (Fig 4B, left graph). In addition, the average number of newly formed α-syn-GFP puncta detected in the cells treated with the fibrils (27 ± 3) was significantly higher than the α-syn-GFP puncta detected in the control condition (12 ± 1; Fig 4B, right graph), suggesting that the exogenous α-syn fibrils induce efficient seeding of the soluble protein. These data were in line with previous findings [86–89]; however, the
question remains as to whether lysosomes participate to this seeding event and how/where in the cell it occurs. To investigate whether seeding is associated with the lysosomes, we performed super-resolution microscopy in CAD cells transfected with α-syn-GFP, loaded with α-syn fibrils and labelled for LAMP1. We observed three different scenarios: i) lysosomes with both α-syn fibrils and α-syn-GFP aggregates colocalizing at the lysosomal membrane (Fig 4C), ii) lysosomes with α-syn fibrils and α-syn-GFP aggregates colocalizing inside their lumen (Fig 4D), and iii) lysosomes containing both α-syn fibrils and α-syn-GFP aggregates inside their lumen, but not necessarily co-localizing (Fig 4E). In addition, we performed object-based 3D colocalization analysis between newly formed α-syn aggregates (α-syn-GFP puncta), α-syn fibrils and lysosomes. We observed that 69 ± 6 % of the newly formed aggregates colocalized with LAMP1+ lysosomes, 74 ± 7% colocalized with α-syn fibrils, and 63 ± 6 % of them were positive for both α-syn fibrils and LAMP1+ organelles (Fig 4F), indicating that in CAD cells the majority of the seeding takes place at lysosomal compartments following α-syn fibrils uptake.

To understand whether the localization of α-syn-GFP aggregates to lysosomes was consistent with the beginning of the seeding event and not a post seeding delivery of newly formed fibrils, we monitored the occurrence of seeding by live imaging microscopy. We first transfected CAD cells with soluble α-syn-GFP and then immediately after the addition of α-syn fibrils we started to monitor the seeding event by detecting the formation of green puncta (α-syn-GFP newly formed aggregates) in these cells compared to control cells by live spinning-disk microscopy.

We found that the seeding first became apparent about 2 h from the addition of the α-syn fibrils and increased with time (S1 Movie and S5A Fig). We did not observe any newly formed α-syn-GFP puncta in untreated control cells (S2 Movie and S5B Fig). Next, we performed super-resolution microscopy in order to investigate whether these newly formed aggregates were associated with the lysosomes by immunolabelling LAMP1 at different time points (1h, 2h, and 4h). Interestingly, at all these time points (even in the rare events found at 1 hour), we found that newly formed aggregates colocalized with α-syn fibrils and LAMP1 (Fig 4G-I). Therefore, newly formed α-syn-GFP aggregates following α-syn fibrils addition are associated with lysosomes at the very beginning of the seeding event, supporting our hypothesis that lysosomes function as hubs for the seeding of newly formed α-syn aggregates.

Overall, these results indicate that exogenous α-syn fibrils induce *de novo* aggregation of soluble α-syn in association with lysosomes.
**Fig 4. α-Syn fibrils seed de novo formation of aggregates associated with lysosomes. (A)** Representative confocal images of α-syn-GFP transfected control or Alexa 568-tagged α-syn fibril-treated CAD cells (18 h). Scale bar: 10 µm (for insets: 5 µm). (B) % of control (13 ± 2%) and α-syn fibril-treated CAD cells for 18 h (76 ± 5%) that formed α-syn-GFP aggregates (left graph). Mean ± S.E.M, *n*=3 (45 cells per condition). ****P<0.0001 by two tailed *t*-test. Average number of α-syn-GFP puncta per cell in control (12 ± 1) and α-syn fibril-treated cells (27 ± 3) (right graph). Mean ± S.E.M, *n*=3 (45 cells per condition). **P*=0.005 by Mann-Whitney *U*-test. (C) SR image showing α-syn fibrils puncta and α-syn-GFP+ aggregate colocalizing on the lysosomal membrane in CAD cells treated with α-syn fibrils for 18 h. (D) SR image showing colocalization between α-syn fibrils puncta and α-syn-GFP+ aggregate inside the lysosomal lumen, and α-syn fibrils on the lysosomal membrane in CAD cells treated with α-syn fibrils for 18 h. (E) SR image showing α-syn fibrils puncta and α-syn-GFP+ aggregate inside the lysosomal lumen in CAD cells treated with α-syn fibrils for 18 h. (F) Object-based 3D colocalization analysis (Imaris software) between α-syn-GFP aggregates and α-syn fibrils (74 ± 7%), α-syn-GFP aggregates and LAMP1+ lysosomes (69 ± 6%), and α-syn-GFP aggregates with both α-syn fibrils and LAMP1+ lysosomes (63 ± 6%). Mean ± S.E.M, *n*=2 (16 super-resolution images, ~2000 lysosomes were analysed). (G) SR image showing α-syn fibrils puncta and α-syn-GFP+ aggregate colocalizing with LAMP1 on the lysosomal membrane in CAD cells treated with α-syn fibrils for 1 h. (H) SR image demonstrating α-syn fibrils puncta and α-syn-GFP+ aggregate colocalizing with LAMP1 both on the lysosomal membrane and inside the lysosomal lumen in CAD cells treated with α-syn fibrils for 2 h. (I) SR image showing α-syn fibrils puncta and α-syn-GFP+ aggregate colocalizing with LAMP1 on the lysosomal membrane in CAD cells treated with α-syn fibrils for 4 h. In C, D, E, G, H, I insets of selected regions (white squares) from different α-syn-GFP transfected and α-syn fibril-treated CAD cells, labelled for DAPI and LAMP1 (pseudo colored in grey) are presented. Images were acquired with a spinning-disk microscope with SR module. Scale bars: 10 µm (for insets: 1 µm; 0.5 µm for inset in G). The data underlying this Figure may be found in S1 Data.

**α-Syn-loaded lysosomes localize to the cell periphery and are less functional.**

The results presented above highlight the role of lysosomes in the propagation of α-syn misfolding after uptake of the fibrils from the culture medium. However, they do not address i) how propagation of misfolding occurs following the transfer of lysosomes containing α-syn aggregates to healthy neighboring cells [19,21], and ii) whether/how lysosome damage induced
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by α-syn aggregates favors the spread of pathology [19,32]. Lysosomes are dynamic organelles
that move bidirectionally between the perinuclear and peripheral regions of the cell [90–92].

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As our results indicate that the α-syn fibrils interfere with lysosome morphology and function, we hypothesized that they could also have an impact on lysosome positioning. Therefore, we evaluated the distribution of lysosomes in CAD cells challenged with α-syn fibrils compared to control cells. Cells were labelled for LAMP1, HCS CellMaskTM Blue (cell outline stain), and DAPI (nuclear stain), and the number of lysosomes in the perinuclear and peripheral regions in each condition was quantified (see Materials and Methods; Fig 5A). In agreement with previous findings that lysosomes are mainly located in the perinuclear region in healthy non-polarized cells [93], we detected 57 ± 1% of the lysosomes at the perinuclear region in control CAD cells (Fig 5A, white arrows, and B). In contrast, in cells treated with α-syn fibrils, the situation was reversed, with the majority of lysosomes (56 ± 1%) found in the peripheral region (Fig 5A, green, red, yellow arrows, and B).

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Since our results indicate that α-syn fibrils affect lysosomal pH and function (Fig 2B-D), to understand whether there is a correlation between lysosome distribution and function, we next evaluated the distribution of lysosomes containing α-syn fibrils and quantified the perinuclear and peripheral distribution of only functional lysosomes positive for LysoTracker DR and Magic Red CathB. We found that α-syn fibril-containing LysoTracker DR positive and Magic Red CathB positive lysosomes were more perinuclear (54 ± 2% and 56 ± 1%, respectively; Fig 5C), suggesting that peripheral lysosomes containing α-syn fibrils are less functional.

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These results thus show that α-syn fibrils promote redistribution of lysosomes towards the cell periphery. Furthermore, this pool of peripheral lysosomes is less acidic and has less degradative capacity.

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**Peripheral α-syn-loaded lysosomes are preferentially transferred to neighboring cells.**

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Our next question was to understand whether the effect of α-syn on lysosome positioning could be relevant for the propagation of the pathology and in which way. Peripheral lysosomes could be more prone to exocytosis and therefore could increase the secretion of α-syn fibrils to the extracellular environment. Alternatively, peripheral lysosomes could be more apt for transfer to healthy neighboring cells through TNTs. To distinguish between these alternative scenarios, we first examined lysosomal exocytosis by comparing the amount of CathB released into the culture medium of control and α-syn fibril-treated CAD cells using an ELISA method. These experiments showed no significant difference in the amount of CathB released by control (70

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± 1 ng/mL) and α-syn fibril-treated cells (75 ± 7 ng/mL; Fig 5D). As an alternative approach, we measured the integrated fluorescence density of LAMP1 at the plasma membrane (PM) in cells treated or not treated with α-syn fibrils. To this end, we used an antibody recognizing a luminal epitope of LAMP1, which is displayed to the extracellular milieu after fusion of lysosomes with the PM in non-permeabilized cells (Fig 5E). We did not observe a difference in the average integrated density of PM-associated LAMP1 between control (9x10⁴ ± 2x10⁴ AU) and α-syn fibril-treated cells (9x10⁴ ± 3x10⁴ AU; Fig 5E, graph). These results indicate that, despite their different distributions, lysosomes are exocytosed at a similar rate in α-syn fibril-treated and control cells.

Next, to understand whether the peripheral positioning of lysosomes could affect their transfer to neighboring cells, we compared the efficiency of lysosome transfer by using a co-culture assay where donor cells were transiently transfected with LAMP1-GFP and either treated or not with α-syn fibrils for 18 h (α-syn co-culture and control co-culture, respectively), and acceptor cells were transiently transfected with H2B-mCherry (Figs 5F and S3B). We observed that the percentage of acceptor cells that received LAMP1-GFP puncta (lysosomes) from donor cells was significantly higher in the α-syn co-culture compared to the control co-culture (29 ± 3% and 18 ± 2%, respectively) (Fig 5G, left graph). Lysosome transfer through secretion was evaluated by a "secretion test" (S3I Fig) whereby acceptor cells treated with the conditioned medium of control or α-syn fibril-loaded donor cells were quantified for the presence of LAMP1-GFP puncta. We did not observe a significant difference between the two secretion tests (9 ± 2% and 7 ± 1% in control and α-syn secretion, respectively; Fig 5G, left graph). In addition, we did not observe any difference in the average number of LAMP1-GFP puncta detected per acceptor cell either in the co-culture conditions (2 ± 0.2 for both co-culture conditions) or in the secretion test conditions (1 ± 0.1 for both secretion conditions; Fig 5G, right graph). These results confirm our previous data that lysosome transfer from donor to acceptor cells is predominantly mediated by cell-to-cell contact, likely through TNTs rather than secretion [19,21,22]. Importantly, we showed that donor cells challenged with α-syn fibrils transfer lysosomes more efficiently to the acceptor cells compared to control donor cells. Moreover, we performed object-based 2D colocalization analysis in the acceptor cells that received both LAMP1-GFP+ lysosomes and α-syn fibrils in order to evaluate the number of lysosomes containing α-syn fibrils transferred to the acceptor cells. We found 51 ± 6% of α-syn fibrils colocalizing with LAMP1-GFP+ lysosomes and 51 ± 6% of lysosomes colocalizing
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with α-syn fibrils, suggesting that half of the lysosomes transferred from donor to acceptor cells
carried α-syn fibrils (Fig 5H).

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Taken together, our data indicate that lysosomes that are more peripherally located in the presence of α-syn fibrils are not secreted more compared to control cells, but instead are more efficiently transferred to acceptor cells by cell-to-cell contact.

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**Fig 5. α-Syn-loaded lysosomes localize to the cell periphery, are less functional, and are preferentially transferred to neighboring cells. (A)** Representative confocal images of control and Alexa 568-tagged α-syn fibril-treated CAD cells (18 h) labelled for LAMP1-Alexa 488 antibody, HCS CellMaskTM Blue, and DAPI. White arrows indicate the perinuclear lysosome clusters in control cells; green, red, and yellow arrows indicate peripheral lysosomes, α-syn fibrils, and lysosomes colocalizing with α-syn fibrils, respectively. Scale bar: 10 µm. **(B)** % of LAMP1+ lysosomes in perinuclear (purple dots) and peripheral regions (black dots) of the control cells and cells challenged with α-syn fibrils is presented. In control cells: % of perinuclear lysosomes: 57 ± 1%, and peripheral lysosomes: 43 ± 1%; in α-syn fibril-treated cells: % of perinuclear lysosomes: 44 ± 1%, and peripheral lysosomes: 56 ± 1%. Mean ± S.E.M, *n*=5 (40 cells per condition). ns=not significant, ****P<0.0001 by one-way ANOVA followed by Tukey's multiple comparison tests. **(C)** Graph representing the distribution of LysoTracker DR+ and Magic Red CathB+ lysosomes colocalizing with the α-syn fibrils in perinuclear (purple dots) and peripheral region (black dots). % of LysoTracker DR+ lysosomes colocalizing with α-syn fibrils in perinuclear region: 54 ± 2%, and in peripheral region: 46 ± 2%. % of Magic Red CathB+ lysosomes colocalizing with α-syn fibrils in perinuclear region: 56 ± 1%, and in peripheral region: 44 ± 1%. Mean ± S.E.M, *n*=4 (40 cells per condition). ns=not significant by one-way ANOVA followed by Tukey's multiple comparison tests. **(D)** Quantity of secreted CathB (ELISA) in the media of control (70 ± 1 ng/mL) and α-syn fibril-treated CAD cells (75 ± 7 ng/mL). Mean ± S.E.M, *n*=3. ns=not significant by Mann-Whitney *U*-test. **(E)** Representative confocal images of control and Alexa 568-tagged α-syn fibril-treated CAD cells for 18 h, immuno-labelled for LAMP1-Alexa 488 antibody in non-permeabilized conditions. Scale bar: 10 µm. Average Integrated Density of LAMP1 immunofluorescence at the cell surface in control (9x10⁴ ± 2x10⁴) and in α-syn fibril-treated cells (9x10⁴ ± 3x10⁴) is presented. Mean ± S.E.M, *n*=3 (20 cells per condition). ns=not significant by paired *t*-test. Scale bar: 10 µm. **(F)** Representative confocal images of donor and acceptor cells after 24 h of co-culture. Donor cells were transfected with LAMP1-GFP and treated with Alexa 568-tagged α-syn fibrils

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or not (referred to as “α-syn co-culture” and “control co-culture”, respectively). Acceptor cells were transfected with H2B-mCherry. Scale bar: 20 µm. (G) % of acceptor cells that received LAMP1+ puncta (lysosomes) from donor cells in control co-culture (18 ± 2%), in α-syn co-culture (29 ± 3%), in control secretion test (9 ± 2%), and in α-syn secretion test (7 ± 1%) (left graph). Mean ± S.E.M, n=4 (150 acceptor cells per condition). ns=not significant, ***P=0.0004 by one-way ANOVA followed by Tukey's multiple comparison tests. The average number of LAMP1-GFP+ puncta detected per acceptor cell in control co-culture (2 ± 0.2), in α-syn co-culture (2 ± 0.2), in control secretion (1 ± 0.1), and in α-syn secretion (1 ± 0.1) is presented (right graph). Mean ± S.E.M, n=4 (150 acceptor cells per condition). ns=not significant, ***P=0.0004 by Kruskal-Wallis nonparametric ANOVA test followed by Dunn's multiple comparison tests. (H) % of α-syn fibril puncta colocalizing with LAMP1-GFP+ puncta (51 ± 6%) and % of LAMP1-GFP+ puncta colocalizing with α-syn fibrils puncta (51 ± 6%) is presented. Mean ± S.E.M, n=4 (20 acceptor cells that received both α-syn fibrils and LAMP1-GFP puncta were analysed). The data underlying this Figure may be found in S1 Data.

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525 TFEB as a player in lysosome redistribution upon α-syn loading.

526 The Transcription Factor EB (TFEB) regulates lysosome biogenesis and function by 527 coordinating the expression of lysosomal components [94]. Furthermore, TFEB has also been 528 shown to influence lysosome positioning [95–97]. To investigate a possible role of TFEB in 529 the peripheral redistribution of lysosomes upon α-syn loading, we first asked whether the 530 lysosomal loading of α-syn fibrils triggers TFEB nuclear translocation. To address this 531 question, we expressed TFEB-WT-GFP in CAD cells challenged or not with α-syn fibrils for 532 18 h (S3A Fig number 3). We found a significant increase in the percentage of CAD cells 533 having nuclear TFEB upon α-syn loading compared to control cells (control: 18 ± 4%; α-syn: 534 57 ± 6%, Fig 6A).

535 Next, we assessed the effect of TFEB overexpression on lysosome distribution in CAD cells. 536 We transiently transfected CAD cells with plasmids encoding wild-type TFEB (TFEB-WT- 537 GFP) or constitutively active mutant TFEB (TFEB-mut-GFP) (i.e., nuclear TFEB), or with 538 empty plasmid CMV-GFP (control), and quantified the number of lysosomes in the perinuclear 539 and peripheral regions. We found that cells overexpressing the constitutively active form of 540 TFEB showed more peripheral lysosomes compared to both control cells and cells 541 overexpressing TFEB-WT-GFP (TFEB-mut-GFP: 59 ± 1%; TFEB-WT-GFP: 54 ± 2%; CMV-
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GFP: 52 ± 1%; Fig 6B). These results indicate that TFEB promotes the peripheral distribution
of lysosomes in CAD cells.

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To determine whether TFEB participates in peripheral redistribution of lysosomes upon α-syn
loading, we silenced the expression of TFEB in CAD cells by RNA interference (Fig 6C). CAD
cells transfected with scrambled siRNA (control) or siTFEB were challenged with α-syn fibrils
for 18 h. Then, we quantified the positioning of lysosomes containing α-syn fibrils (Fig 6D).
As expected, control cells showed more peripheral α-syn-loaded lysosomes (52.1 ± 1%). In
contrast, in siTFEB-treated CAD cells α-syn-loaded lysosomes were slightly but significantly
more perinuclear (50.7 ± 1%). These findings indicate that silencing of endogenous TFEB
reduced the peripheral redistribution of lysosomes upon α-syn loading, suggesting the
involvement of TFEB in this event.

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**Fig 6. The role of TFEB in lysosome redistribution upon α-syn loading. (A) % of cells**
overexpressing TFEB-WT-GFP having nuclear TFEB in control CAD cells (18 ± 4%) and α-syn fibril-treated CAD cells for 18 h (57 ± 6%). Mean ± S.E.M, *n*=3 (50 cells per condition).

****P<0.0001 by Unpaired t test. (B) % of LAMP1+ lysosome distribution in perinuclear (purple dots) and peripheral regions (black dots) of cells overexpressing CMV-GFP (control), TFEB-WT-GFP, or TFEB-mut-GFP. In control CMV-GFP cells: % of perinuclear lysosomes: 48 ± 1%, and peripheral lysosomes: 52 ± 1%; in TFEB-WT-GFP cells: % of perinuclear lysosomes: 46 ± 2%, and peripheral lysosomes: 54 ± 2%; in TFEB-mut-GFP cells: % of perinuclear lysosomes: 41 ± 1%, and peripheral lysosomes: 59 ± 1%. Mean ± S.E.M, *n*=3 (30 cells per condition). ***P<0.001 by one-way ANOVA followed by Tukey's multiple comparison tests. (C) Western blot prepared from cell extracts collected from control, scrambled siRNA (sicontrol)-, and siTFEB-pretreated CAD cells for 60 h, showing TFEB and GAPDH (loading control) expression. (D) Graph representing the distribution of lysosomes containing α-syn fibrils in perinuclear (purple dots) and peripheral region (black dots) of α-syn fibril-treated sicontrol and siTFEB cells. % of lysosomes containing α-syn fibrils of sicontrol cells in perinuclear region: 49 ± 1%, and in peripheral region: 52 ± 1%. % of lysosomes containing α-syn fibrils of siTFEB cells in perinuclear region: 51 ± 1%, and in peripheral region: 49 ± 1%. Mean ± S.E.M, *n*=4 (30 cells per condition). ns=not significant, *P*=0.0288 by one-way ANOVA followed by Tukey's multiple comparison tests. The data underlying this Figure may be found in S1 Data.

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To further investigate the effect of lysosome positioning on α-syn fibrils transfer, we set-up different co-culture assays where we monitored the amount of α-syn transfer using donor cells with more peripheral or more perinuclear lysosomes. To this end, we respectively overexpressed or silenced the Arf-like small GTPase Arl8b, which controls the movement of lysosomes towards the cell periphery [98,99]. To examine the effect of peripheral lysosome redistribution, Arl8b-GFP transfected or control donor CAD cells were loaded with α-syn fibrils and co-cultured with acceptor CAD cells transiently transfected with H2B-GFP to distinguish them from the donors (referred to as Arl8b-GFP co-culture and control co-culture, respectively; Figs 7A and S3C). We then quantified the presence of α-syn fibrils in the acceptor cells in each co-culture condition. We detected a significant increase in the percentage of acceptor cells that received α-syn fibrils in the Arl8b-GFP co-culture (65 ± 4%) compared to control co-culture (49 ± 4%; Fig 7B, left graph). As a control, we monitored the transfer of α-syn fibrils through the culture supernatant, thus checking a possible effect on transfer through a secretion mechanism. As previously found [19], the amount of α-syn transfer by secretion was very low compared with the transfer in co-culture, and did not significantly change in the two co-culture conditions (8 ± 2% and 10 ± 2%, in control and Arl8b-GFP secretion conditions, respectively; Fig 7B, left graph). Despite changes in the percentage of cells that took up α-syn fibrils in co-cultures, we did not find a significant difference in the average number of α-syn puncta per acceptor cell in any of these conditions (in average: 3 ± 0.4, 3.4 ± 0.4, 1.3 ± 0.4, 2 ± 0.3 α-syn fibrils puncta per acceptor cell in control co-culture, Arl8b-GFP co-culture, control secretion, and Arl8b-GFP secretion, respectively; Fig 7B, right graph).

To corroborate these observations in a different cell type, we performed the same experiment in HeLa cells (Figs S3D and S6) in which the effect of Arl8b on lysosome positioning was extensively characterized [90,91]. We observed that the percentage of acceptor cells that received α-syn fibrils was 28 ± 3% and 60 ± 5% in control and Arl8b-GFP co-cultures, respectively (S6A and B Fig, left graph). Again, we did not observe any significant increase in the transfer of α-syn fibrils through secretion from the donors overexpressing Arl8b-GFP in comparison to control donors (8 ± 3% and 1 ± 1% respectively; S6B Fig, left graph). As in CAD cells, the average number of α-syn puncta per acceptor cell was not significantly different in HeLa co-cultures and secretion conditions (3 ± 1 and 5 ± 1 in control and Arl8b-GFP co-cultures, respectively; 1 ± 0.0 and 2 ± 0.2 in control and Arl8b-GFP secretion tests, respectively; S6B Fig, right graph).
Furthermore, we silenced the expression of Arl8b in CAD cells by RNA interference (Fig 7C).

Briefly, we pretreated donor cells with siRNA targeting Arl8b (siArl8b) and scrambled siRNA (sicontrol) for 60 h prior to loading with α-syn fibrils for 18 h; in parallel, untreated control cells were challenged with α-syn fibrils. These donors were then co-cultured with acceptor cells transiently transfected with H2B-GFP (Figs 7D and S3E). In control co-culture and in sicontrol co-culture, the percentage of acceptor cells that received α-syn fibrils was 55 ± 4% and 60 ± 3%, respectively, whereas in siArl8b co-culture we observed a significant lower percentage of 28 ± 3% (Fig 7E, left graph). We did not detect any significant difference in α-syn transfer through secretion (6 ± 1%, 9 ± 1%, and 6 ± 1% in control, s, and siArl8b secretion conditions, respectively; Fig 7E, left graph). The average number of α-syn puncta detected in acceptor cells in co-cultures and secretion conditions was not significantly changed (1-2 puncta per cell in all the conditions; Fig 7E, right graph).

To further test the importance of lysosome positioning in α-syn fibrils transfer, we performed similar experiments in HeLa cells with knock out (KO) of components of the lysosome-positioning machinery. These components included the myrlysin (also known as BORCS5) subunit of BORC, which promotes recruitment of Arl8b to membranes [100], and the kinesins KIF1B and KIF5B [101–105], which drive lysosome movement towards the cell periphery [90]. KO of these proteins impairs lysosome movement towards the cell periphery [90,100]. These KO donor cells, in parallel to control donors (WT HeLa) were loaded with α-syn fibrils and co-cultured with WT HeLa cells expressing H2B-GFP (Figs S3F and S6C). Acceptor cells were then analysed for the presence of α-syn fibrils. The percentage of acceptor cells that received α-syn fibrils in control co-culture was 29 ± 2%, whereas in myrlysin-KO and KIF1B-KIF5B-double-KO (KIF1B-5B dKO) co-cultures this percentage was significantly decreased to 16 ± 2% and 12 ± 2%, respectively (S6D Fig, left graph). α-Syn fibrils’ transfer through secretion was similar in all the conditions (5 ± 2%, 6 ± 1%, 6 ± 1% in control, myrlysin-KO, and KIF1B-5B dKO secretion conditions, respectively; S6D Fig, left graph). We did not observe a significant difference in the average number of transferred α-syn puncta in any of the co-cultures/secretion conditions (1-2 puncta per cell in all the conditions; S6D Fig, right graph).

Overall, these data indicate that the contact-dependent transfer of α-syn is affected by the position of lysosomes, with more transfer when lysosomes are peripheral and less transfer when lysosomes are perinuclear.

As we have shown that lysosomes containing α-syn are transferred from acceptor cells inside TNTs [52] (S2 Fig), we wondered whether TNT formation was affected by the manipulations
that alter lysosome positioning. Thus, we performed two sets of TNT counting experiments in control, Arl8b-GFP-overexpressing or siArl8b/sicontrol-treated CAD cells in the presence or absence of α-syn fibrils. These cells were labelled with WGA and the percentage of TNT-connected cells was quantified for each condition (see Materials and Methods). We did not observe a significant difference in the percentage of TNT-connected cells between control (45 ± 3%) and Arl8b-GFP-overexpressing cells (46 ± 2%; Fig 7F, left graph). However, in the presence of α-syn fibrils the percentage of TNT-connected cells was significantly increased to 60 ± 5% in control and 64 ± 5% in Arl8b-GFP-overexpressing cells (Fig 7F, left graph). Similarly, the percentage of TNT-connected cells remained unchanged between control (44 ± 4%), sicontrol (45 ± 4%), and siArl8b (43 ± 4%) pretreated cells, whereas in the presence of α-syn fibrils the percentage of TNT-connected cells significantly increased to 62 ± 4% in control, 62 ± 5% in sicontrol, and 66 ± 3% in siArl8b-treated cells (Fig 7F, right graph). These data indicate that lysosome positioning has no effect on TNT formation per se, but it is the presence of α-syn fibrils that increases the number of TNT-connected cells, in line with previous findings [19].

Taken together, these results indicate that lysosome positioning affects the efficiency of α-syn fibrils' transfer. Moreover, α-syn fibrils increase the number of TNT-connected cells independent of lysosome positioning.

**Fig 7. Lysosome positioning affects the efficiency of α-syn fibrils' transfer. (A)** Representative confocal images of donor and acceptor cells after 24 h of co-culture. Donor cells were either loaded with Alexa 568-tagged α-syn fibrils only (control co-culture) or transfected with Arl8b-GFP prior to be treated with α-syn fibrils (Arl8b-GFP co-culture). Acceptor cells were transfected with H2B-GFP. Co-cultures were labelled with HCS CellMaskTM Blue and DAPI. Arrows indicate the α-syn fibrils received by acceptor cells and asterisks indicate the cells having α-syn fibrils in each co-culture condition. Scale bar: 10 µm. (B) % of acceptor cells received α-syn fibrils in control co-culture (49 ± 4%), in Arl8b-GFP co-culture (65 ± 4%), in control secretion (8 ± 2%), and in Arl8b-GFP secretion (10 ± 2%) (left graph). Mean ± S.E.M, n=3 (90 acceptor cells per condition). ns=not significant, **P=0.0038 by Kruskal-Wallis nonparametric ANOVA test followed by Dunn's multiple comparison tests. Average number of α-syn fibrils puncta in control co-culture (3 ± 0.4), in Arl8b-GFP co-culture (3.4 ± 0.4), in control secretion (1.3 ± 0.4), and in Arl8b-GFP secretion (2 ± 0.3) (right graph). Mean ± S.E.M, n=3 (90 acceptor cells per condition). ns=not significant, by one-way ANOVA followed by
Tukey's multiple comparison tests. (C) Western blot prepared from cell extracts collected from control, scrambled siRNA (sicontrol)-, and siArl8b-pretreated CAD cells for 60 h, showing Arl8b and α-tubulin (loading control) expression. Integrated Density of Arl8b protein expression levels normalized to α-tubulin in control (0.80 ± 0.03), sicontrol- (0.82 ± 0.02), and siArl8b-pretreated cells (0.19 ± 0.09) are presented in Arbitrary Units (AU). Mean ± S.E.M, *n*=3. ns= not significant, **P*=0.0004 by one-way ANOVA followed by Tukey's multiple comparison tests. (D) Representative confocal images of donor and acceptor cells after 24 h of co-culture. Donor CAD cells were pretreated with sicontrol or siArl8b for 60 h or left untreated prior to be challenged with Alexa 568-tagged α-syn fibrils (referred to as control, sicontrol, and siArl8b co-cultures, respectively). Acceptor cells were transfected with H2B-GFP. Co-cultures were labelled with HCS CellMask™ Blue and DAPI. Arrows indicate the α-syn fibrils received by acceptor cells and asterisks indicate the cells having α-syn fibrils in each co-culture condition. Scale bar: 10 µm. (E) % of acceptor cells that received α-syn fibrils in control co-culture (55 ± 4%), sicontrol co-culture (60 ± 3%), siArl8b co-culture (28 ± 3%), control secretion (6 ± 1%), sicontrol secretion (9 ± 1%), and siArl8b secretion (6 ± 1%) (left graph). Mean ± S.E.M, *n*=4 (80 acceptor cells per condition). ns= not significant, ****P<0.0001 by one-way ANOVA followed by Tukey's multiple comparison tests. Average number of α-syn puncta per acceptor cell in control co-culture (2 ± 0.2), sicontrol co-culture (2 ± 0.1), siArl8b co-culture (2 ± 0.3), control secretion (1 ± 0.2), sicontrol secretion (2 ± 0.2), and siArl8b secretion (2 ± 0.3) (right graph). Mean ± S.E.M, *n*=4 (80 acceptor cells per condition). ns= not significant by Kruskal-Wallis nonparametric ANOVA test followed by Dunn's multiple comparison tests. (F) Control, Arl8b-GFP transfected, and sicontrol- or siArl8b-pretreated CAD cells for 60 h were challenged or not with α-syn fibrils (18 h) and labelled with WGA for TNT counting experiments. % of TNT-connected cells in control (45 ± 3%), Arl8b-GFP (46 ± 2%), control-α-syn (60 ± 5%), and Arl8b-GFP-α-syn (64 ± 5%) conditions. Mean ± S.E.M, *n*=4 (115 cells per condition). ns= not significant (left graph). % of TNT-connected cells in control (44 ± 4%), sicontrol (45 ± 4%), siArl8b (43 ± 4%), control-α-syn (62 ± 4%), sicontrol-α-syn (62 ± 5%), and siArl8b-α-syn (66 ± 3%) is presented (right graph). Mean ± S.E.M, *n*=3 (90 cells per condition). ns= not significant, \*P<0.05, \*\*P<0.01 by one-way ANOVA followed by Tukey's multiple comparison tests. The data underlying this Figure may be found in S1 Data.

α-Syn fibrils induce LMP upon arrival in the acceptor cells.
Our data show that more than 50% of α-syn fibrils in acceptor cells colocalize with LAMP1, indicating that they are still associated with lysosomes after the transfer (Fig 5H). Moreover, we have previously reported that α-syn fibrils can induce seeding of soluble α-syn once they are transferred to acceptor cells [19]. Based on the results presented here one possible explanation for this observation would be that α-syn fibrils also trigger LMP in acceptor cells following their transfer, similar to what we have observed in donor cells (Fig 3A and B). To assess this hypothesis, we performed co-culture experiments where donor cells were either transiently transfected with H2B-mCherry (control co-culture) or treated with α-syn fibrils for 18 h (α-syn co-culture), and co-cultured with acceptor cells transiently transfected with Gal3-GFP (Figs 8A and S3G number 1). Acceptor cells were then evaluated for the presence of Gal3-GFP puncta. The percentage of acceptor cells exhibiting LMP (Fig 8A, white arrows) was significantly higher in the α-syn co-culture (33 ± 5%) compared to control co-culture (6 ± 2%) (Fig 8B, upper graph). We also quantified the average number of Gal3-GFP puncta per acceptor cell and we found a significant increase in α-syn co-culture (14 ± 1) compared to control co-culture where some LMP occurs spontaneously (8 ± 1; Fig 8B, lower graph). We also performed super-resolution microscopy in α-syn co-culture labelled for lysosomes with antibody to LAMP1 and observed Gal3-GFP and α-syn fibrils puncta both in close proximity inside the lysosomal lumen (Fig 8C) and colocalizing on the lysosomal membrane (Fig 8D). These results thus show that, following their transfer, α-syn-containing lysosomes undergo LMP in acceptor cells.

**Fig 8. α-Syn fibrils induce LMP upon arrival in the acceptor cells.** (A) Representative confocal images of donor and acceptor cells after 24 h of co-culture. Donor cells were transfected with H2B-mCherry (control co-culture) or treated with Alexa 568-tagged α-syn fibrils (α-syn co-culture). Acceptor cells were transfected with Gal3-GFP. Co-cultures were labelled with HCS CellMask™ Blue. Red arrows indicate the α-syn fibrils received by acceptor cells, white arrows indicate the Gal3-GFP puncta formation (LMP) in the acceptor cells that received α-syn fibrils. Scale bar: 10 µm. (B) % of acceptor cells under LMP in control (6 ± 2%) and α-syn (33 ± 5%) co-cultures (upper graph). Average number of Gal3-GFP puncta formation in acceptor cells under LMP in control (8 ± 1) and α-syn (14 ± 1) co-cultures (lower graph). Mean ± S.E.M, n=5 (50 acceptor cells per condition). *P=0.04, ****P<0.0001 by Mann-Whitney *U*-test. (C) SR image of a co-culture where donor cells were treated with α-syn fibrils for 18 h and acceptor cells were transfected with Gal3-GFP, labelled for LAMP1-Alexa 647
antibody (pseudo colored in grey). A selected region where Gal3-GFP puncta were observed (indicated by a white square in the acceptor cell of the co-culture image), was further magnified to observe one lysosome (indicated by the small white square) having α-syn fibrils puncta and recruited Gal3-GFP. Image was acquired with a Spinning Disk microscope with SR module. Scale bar: 10 µm (for the inset: 0.2 µm). (D) SR image of an acceptor cell demonstrating Gal3-GFP puncta recruited at the lysosomal membrane where α-syn fibrils were detected in close proximity. Selected lysosome is indicated by a white square. Image was acquired by SR SIM (Zeiss, LSM 780-Elyra PS.1). Scale bar: 5 µm (for the inset: 0.5 µm). In C, the signal was boosted to make the selected lysosome visible; in D, the acceptor cell was presented with HCS CellMask™ Blue labelling as this cell was poorly transfected but accumulation of Gal3-GFP puncta was clearly detectable. The data underlying this Figure may be found in S1 Data.

750

α-Syn fibrils induce seeding after their transfer into acceptor cells and this event is preferentially associated with lysosomes.

To further investigate the seeding mechanism in acceptor cells that received lysosomes containing α-syn fibrils, we used again our α-syn-GFP soluble fluorescent reporter expressed in acceptor cells co-cultured with donor cells, either challenged with α-syn fibrils or transfected with H2B-mCherry (α-syn co-culture and control co-culture respectively; Figs 9A and S3G number 2). We then quantified the presence of α-syn-GFP puncta (indicating aggregation of the soluble protein; Fig 9A, white arrows) in the acceptor cells that received α-syn fibrils (Fig 9A, red arrows). We found that 27 ± 4% of acceptor cells contained 7 ± 1 α-syn-GFP puncta in α-syn co-culture, whereas in control co-culture only 10 ± 4% of the acceptor cells had 2 ± 0.2 α-syn-GFP puncta (Fig 9B).

Next, to address whether the seeding in acceptor cells was associated with lysosomes, we performed super-resolution microscopy. After labelling the cells in co-culture with antibody to LAMP1, we examined the subcellular localization of α-syn-GFP aggregates in acceptor cells. We observed four different scenarios: i) lysosomes containing α-syn fibrils and colocalizing with α-syn-GFP puncta surrounding the lysosomal membrane (Fig 9C), ii) lysosomes filled with α-syn fibrils with discrete α-syn-GFP deposits on their membrane (Fig 9D), iii) lysosomes containing α-syn-GFP puncta with α-syn fibrils in close proximity (Fig 9E), and iv) lysosomes containing both α-syn fibrils and α-syn-GFP puncta partially colocalizing in their lumen (Fig 9F). In addition, we performed 3D object-based colocalization analysis between α-syn-GFP
puncta, α-syn fibrils, and LAMP1+ lysosomes. We observed that 60 ± 7% of the newly formed α-syn-GFP aggregates colocalized with LAMP1+ lysosomes (Fig 9G). Overall, these data indicate that the majority of the seeding of endogenously expressed soluble α-syn takes place at lysosomal compartments in acceptor cells after the transfer of α-syn fibrils.

**Fig 9. α-syn fibrils induce seeding after their transfer into acceptor cells and this event is frequently associated with lysosomes.** (A) Representative confocal images of donor and acceptor cells after 24 h of co-culture. Donor cells were transfected with H2B-mCherry (control co-culture) or treated with Alexa 568-tagged α-syn fibrils for 18 h (α-syn co-culture). Acceptor cells were transfected with α-syn-GFP. Inset of acceptor cells (indicated by white squares) is presented at the right top corner of acceptor cells' panel. White arrows indicate α-syn-GFP aggregate formation, red arrows indicate α-syn fibrils received by acceptor cells in α-syn co-culture. Scale bar: 10 µm (for the inset: 5 µm). (B) % of acceptor cells that formed α-syn-GFP aggregates in control (10 ± 4%) and in α-syn (27 ± 4%) co-cultures (left graph). Average number of α-syn-GFP puncta detected in acceptor cells in control (2 ± 0.2) and in α-syn (7 ± 1) co-cultures is presented (right graph). Mean ± S.E.M, *n*=3 (150 acceptor cells per condition). *P*=0.01 and **P*=0.003 for % of aggregates and average number of aggregates in control versus α-syn co-cultures, respectively, by Mann Whitney *U*-test. (C) SR image showing a lysosome containing α-syn fibrils puncta colocalizing with α-syn-GFP+ aggregate at the lysosomal membrane. (D) SR image showing a lysosome containing α-syn fibrils where α-syn-GFP+ aggregate is located mostly in a restricted portion of the lysosomal membrane. (E) SR image showing a lysosome having α-syn-GFP+ aggregate where α-syn fibrils puncta were found to be in close proximity of the lysosomes. (F) SR image showing a lysosome where both α-syn fibrils and α-syn-GFP aggregates are located to the lumen of the lysosome and partially colocalizing. (G) Object-based 3D analysis (Imaris software) of colocalization between α-syn-GFP and LAMP1+ lysosomes (60 ± 7%). Mean ± S.E.M, *n*=2 (15 super-resolution images, ~3000 lysosomes were analysed). Insets of images in C, D (acquired with Spinning-Disk microscope with SR module) and E, F (acquired with SR SIM, Zeiss, LSM 780-Elyra PS.1) from four selected regions (white squares) and from different acceptor cells transfected with α-syn-GFP and that received α-syn fibrils, labelled for LAMP1 Alexa 647 antibody (pseudo colored in grey). Scale bars: 10 µm (for the insets: 1 µm). The data underlying this Figure may be found in S1 Data.
**α-Syn-loaded cells transfer damaged lysosomes to healthy cells and receive healthy lysosomes in return.**

In our homotypic co-culture system between CAD cells, we could not address the nature of the transferred lysosomes (i.e., whether the lysosomes that are transferred have LMP or not) nor the directionality of lysosome transfer (from donor to acceptor and/or from acceptor to donor). To answer these fundamental questions, we designed a heterotypic co-culture assay using mouse CAD cells as donors and human HeLa cells as acceptors. Briefly, CAD cells were transiently transfected with Gal3-GFP, loaded or not with α-syn fibrils, and used as the donor cell population; on the other hand, HeLa cells stably expressing Gal3-Turquoise were used as the acceptor cell population. Donor and acceptor cells were co-cultured for 24 h prior to be fixed and immunolabelled with either a specific anti-mouse LAMP1 antibody (to detect the lysosomes originated from donor cells) or a specific anti-human LAMP1 antibody (to detect the lysosomes originated from acceptor cells; S3H Fig).

First, we tested this heterotypic system by evaluating: i) the efficiency of α-syn fibrils' transfer from donor CAD cells to acceptor HeLa cells, and ii) the occurrence of seeding in the acceptor cells. The percentage of acceptor HeLa cells that received α-syn fibrils from donors was 65 ± 3% with an average of 4 ± 0.2 α-syn puncta per acceptor cell (S7A-B Fig). As expected, also in this case, both the percentage of α-syn fibrils (7 ± 2%) and the average number of α-syn fibrils puncta per acceptor cell (2 ± 0.2) was significantly lower in the secretion condition (S7B Fig). When donor CAD cells loaded with α-syn fibrils were co-cultured with HeLa cells transiently transfected with soluble α-syn-GFP, we again observed the aggregation of soluble α-syn-GFP (S7C Fig, green arrows) in acceptor HeLa cells that received α-syn fibrils from donor CAD cells (S7C Fig, red arrows). Taken together, these results indicate that α-syn fibrils can be efficiently transferred in a cell-to-cell contact manner and can induce seeding in the acceptor cells following their transfer also in a heterotypic mouse-human co-culture assay.

As we could recognize the origin of lysosomes, we then used this system to better quantify the transfer of all lysosomes and of lysosomes under LMP from donor to acceptor cells. To this end, CAD cells were either loaded or not with α-syn fibrils and co-cultured with HeLa cells expressing Gal3-Turquoise. Cells in co-culture were then immunolabelled with the antibody to mouse LAMP1 to detect the transfer of lysosomes originated from donor cells (Fig 10A). The percentage of acceptor HeLa cells that received donors' lysosomes was significantly higher in α-syn co-culture (52 ± 4%) compared to control co-culture (32 ± 5%), whereas the average number of transferred lysosomes (LAMP1+ puncta) did not change (3 ± 0.3 in both co-cultures;
Fig 10B). Furthermore, we observed that in α-syn co-culture both lysosomes containing α-syn fibrils (Figs 10A, yellow arrows, and S7G, red arrow) or not (Figs 10A, white arrows, and S7G, white arrow) were transferred to acceptor cells.

Next, to evaluate the amount of lysosomes having LMP that were transferred from CAD cells to HeLa cells, we quantified the occurrence of LMP in CAD cells in our heterotypic co-culture condition. Thus, we counted the number of Gal3-GFP puncta in donor CAD cells transfected with Gal3-GFP, challenged or not with α-syn fibrils, and co-cultured with HeLa-Gal3-Turquoise cells (S7D Fig, green arrows). As expected, the percentage of donor CAD cells having LMP was significantly higher in α-syn loaded donor cells (11 ± 4%) compared to control donors (2 ± 2%), whereas the average number of Gal3-GFP puncta per cell did not show any statistically significant change (10 ± 2 and 14 ± 4 in control and α-syn donors, respectively; S7E Fig). These findings indicate that, in co-culture conditions, donor CAD cells challenged with α-syn fibrils undergo LMP similar to what we showed earlier (Fig 3A and B). Next, we evaluated the transfer of donors' lysosomes with LMP by quantifying the presence of Gal3-GFP puncta in acceptor HeLa-Gal3-Turquoise cells both in control and α-syn co-cultures (Fig 10C). We found a significantly higher percentage of HeLa cells with Gal3-GFP puncta in the α-syn co-culture (13 ± 2%; Fig 10C, green arrows, and D, left graph) compared to control co-culture (4 ± 1%; Fig 10D, left graph). We did not observe a significant difference between the average number of Gal3-GFP puncta per acceptor cell in the two co-cultures (2 ± 0.2 and 2 ± 0.3 in control and α-syn co-cultures, respectively; Fig 10D, right graph). These data indicate that CAD cells challenged with α-syn fibrils transfer more damaged lysosomes (i.e., undergoing LMP) to acceptor cells compared to control CAD cells. In addition, we were able to detect donors' lysosomes colocalizing both with α-syn fibrils and Gal3-GFP puncta in acceptor HeLa cells (Fig 10E), demonstrating that damaged lysosomes carrying α-syn fibrils are transferred into the acceptor cells.

By taking advantage of HeLa cells stably expressing Gal3-Turquoise as acceptor cells, we also evaluated the occurrence of LMP in acceptor cells by assessing the Gal3-Turquoise puncta (Fig 10F, turquoise arrows). We found that 18 ± 3% of acceptor cells undergo LMP with an average of 10 ± 1 Gal3-Turquoise puncta per cell in α-syn co-culture (Fig 10G). LMP in the acceptor cells of control co-culture was significantly lower (3 ± 1%) with an average of 9 ± 1 Gal3-Turquoise puncta per cell (Fig 10G). In addition, by using antibody to human LAMP1, we also detected colocalization with Gal3-Turquoise puncta in acceptor cells (S7F Fig, arrow). These results suggest that α-syn fibrils derived from CAD cells are able to induce LMP in the acceptor
HeLa cells following their transfer. Interestingly, in very few cases we could also detect colocalization between Gal3-Turquoise puncta and donors' lysosomes labelled with anti-mouse LAMP1 antibody in acceptor HeLa cells (S7G Fig, white arrow), suggesting that damaged donors' lysosomes can undergo LMP upon arrival to the acceptor cells.

Finally, to evaluate lysosome transfer from acceptor to donor cells, the CAD-HeLa co-culture was immunolabelled with anti-human LAMP1 antibody and donor CAD cells were evaluated for the presence of acceptors' lysosomes (Fig 10H). The percentage of donor cells that received acceptors' lysosomes in control co-culture was 14 ± 4% whereas in α-syn co-culture this percentage was significantly higher (34 ± 5%; Fig 10I, left graph). The average number of acceptors' lysosomes per donor cell in both co-culture conditions was similar (2 ± 0.3 and 2 ± 0.1 in control and α-syn co-cultures, respectively; Fig 10I, right graph). To understand whether lysosomes transferred from acceptor to donor cells were damaged, we quantified the presence of Gal3-Turquoise puncta in donor CAD cells in both in the α-syn and control co-cultures (S7H Fig). Only 4 ± 1% of donor cells resulted positive for Gal3-Turquoise puncta in α-syn co-culture, while we could barely (0.3 ± 0.3%) observe donor cells positive for Gal3-Turquoise puncta in control co-culture (S7I Fig). Taken together, these results demonstrate that donor cells challenged with α-syn fibrils receive healthy lysosomes from acceptor cells, while the transfer of damaged lysosomes from the acceptor cells is a rare event. This suggests the occurrence of a rescue mechanism towards donor cells overloaded with α-syn, similar to what was previously reported in the case of a lysosomal disease [106].

**Fig 10. Intercellular lysosome transfer is reciprocal. (A) Representative confocal image of CAD-HeLa co-culture where donor mouse CAD cells were treated with Alexa 568-tagged α-syn fibrils (18 h) and co-cultured with acceptor human HeLa stable Gal3-Turquoise cells for 24 h, labelled for anti-LAMP1 mouse antibody and DAPI. In acceptor HeLa Gal3-Turquoise cells, arrows indicate donors' lysosomes containing (yellow) or not (white) α-syn fibrils. Inset represents a selected region (white square) of one acceptor cell containing donors' lysosomes. Scale bar: 10 µm (for the inset: 2 µm). (B) % of acceptor HeLa cells that received lysosomes from donor CAD cells in control (32 ± 5%) and in α-syn (52 ± 4%) co-cultures (left graph).**

**P=0.006 by two-tailed t-test. Average number of lysosomes transferred from donor to acceptor cells in control (3 ± 0.3) and in α-syn (3 ± 0.3) co-cultures (right graph). Mean ± S.E.M. ns= not significant by Mann-Whitney U-test. (C) Representative confocal image of CAD-HeLa co-culture where Gal3-GFP transfected donor CAD cells were treated with Alexa
568-tagged α-syn fibrils for 18 h and co-cultured with HeLa Gal3-Turquoise for 24 h, labelled with DAPI. In acceptor HeLa Gal3-Turquoise cells, red arrows indicate α-syn fibrils, green arrows indicate Gal3-GFP puncta (lysosomes under LMP received from donor cells). Scale bar: 10 µm. (D) % of acceptor HeLa cells that received Gal3-GFP puncta from donor cells in control (4 ± 1%) and in α-syn (13 ± 2%) co-cultures (left graph); average number of Gal3-GFP puncta per acceptor HeLa cell in control (2 ± 0.3) and in α-syn (2 ± 0.2) co-cultures (right graph). Mean ± S.E.M. ns= not significant, *P=0.04 by Mann-Whitney U-test. (E) Acceptor HeLa Gal3-Turquoise cell that received a donor's lysosome containing α-syn fibrils and under LMP. Colocalization between LAMP1 (labelled with anti-LAMP1 mouse antibody), α-syn fibrils, and Gal3-GFP is presented in the inset of the selected area (white square). Scale bar: 10 µm (for the inset: 1 µm). (F) Representative confocal image of LMP in HeLa Gal3-Turquoise (pseudo colored in yellow) acceptor cell in CAD-HeLa co-culture. In acceptor HeLa Gal3-Turquoise cells, red arrows indicate the α-syn fibrils, turquoise arrows indicate the Gal3Turquoise puncta. Scale bar: 10 µm. (G) % of acceptor HeLa cells under LMP in control (3 ± 1%) and in α-syn (18 ± 3%) co-cultures is presented (left graph); average number of Gal3-Turquoise puncta per acceptor cell under LMP in control (9 ± 1) and in α-syn (10 ± 1) co-cultures is presented (right graph). Mean ± S.E.M. ns= not significant, *P=0.03 by Mann-Whitney U-test. (H) Representative confocal image of CAD-HeLa co-culture where donor CAD cells were treated with Alexa 568-tagged α-syn fibrils for 18 h and co-cultured with HeLa Gal3-Turquoise for 24 h, labelled for anti-LAMP1 human antibody and DAPI. In donor CAD cells, white arrows indicate acceptors' lysosomes. Inset of a selected region of a donor CAD cell (white square) showing acceptors' lysosomes in higher magnification. Scale bar: 10 µm (for the inset: 5 µm). (I) % of acceptors' lysosomes transferred from acceptor to donor cells in control (14 ± 4%) and in α-syn (34 ± 5%) co-cultures (left graph). **P=0.002 by two-tailed t-test. Average number of acceptors' lysosomes per donor cell in control (2 ± 0.3) and in α-syn (2 ± 0.1) co-cultures (right graph). Mean ± S.E.M. ns= not significant by Mann-Whitney U-test. In CAD-HeLa co-culture model, data was collected from seven independent experiments in which four and three of them were labelled with anti-LAMP1 mouse and human antibodies, respectively. According to the experiment, in average 100 acceptor or donor cells were analysed for each condition per experiment. The data underlying this Figure may be found in S1 Data.
933

**Discussion**

934

α-Syn plays a crucial role in the generation and progression of synucleinopathies including PD.

935

Indeed, the pathology correlates with the presence of α-syn inclusions spreading through interconnected brain regions in a “prion-like” manner [5, 16, 107]. By using a co-culture system,

936

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

937

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

938

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

939

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

940

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

941

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

942

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

943

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

944

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

945

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

946

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

947

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

948

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

949

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

950

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

951

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

952

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

953

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

954

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

955

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

956

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

957

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

958

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

959

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

960

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

961

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

962

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

963

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

964

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,

965

we previously demonstrated that α-syn fibrils can be transferred from donor to acceptor cells inside of lysosomes through TNTs and induce the seeding in the recipient cells [19]. However,
cells [74]. Specifically, LMP has been described in several cell types challenged with α-syn fibrils, such as the neuronal cell lines SH-SY5Y and N27, as well as human dopaminergic neurons derived from induced pluripotent stem cells [73,74]. Here, we showed that α-syn fibrils induce LMP also in neuronal CAD cells (Fig 3A and B). By using SIM, we observed the recruitment of Gal3 to the damaged lysosomes displaying α-syn fibrils in their lumen or limiting membrane (Fig 3D and E). Importantly, we demonstrated that α-syn fibrils trigger LMP also in the recipient cells following their intercellular transfer (Fig 8A and B). LMP in acceptor cells has been only reported in a co-culture system where donor N27 cells expressing α-syn were pretreated with the mitochondrial toxin MPP⁺ (1-methyl-4-phenylpyridine) to induce PD-like pathology and co-cultured with acceptor cells stably expressing Gal3 [73]. Our results indicate that, without inducing a PD-like pathology in donor cells, the transfer of α-syn fibrils ‘per se’ is sufficient to induce LMP in acceptor cells both in homotypic (Fig 8A and B) and heterotypic co-culture systems (Fig 10F and G). In addition, by performing super-resolution microscopy, we also detected the recruitment of Gal3 to damaged lysosomes bearing α-syn fibrils either in their lumen or at their membrane in the acceptor cells, similar to what we observed in donor cells (Fig 8C and D). Interestingly, our results show that lysosomes containing α-syn fibrils are not targeted for lysophagy [84] (S4 Fig), possibly suggesting a rescue mechanism such as the one orchestrated by the ESCRT machinery [111,112]. Considering the evidence supporting autophagy impairment in neurodegenerative diseases and more specifically following α-syn accumulation [55,113], and the recent findings that impaired autophagic flux contributes to the occurrence of LMP [114], it will be interesting to assess the contribution of autophagy to LMP, lysosomal dysfunction, and propagation of α-syn.

The underlying mechanism of LMP induction by α-syn fibrils is unclear. Several studies have reported the ability of α-syn protofibrils to permeabilize membranes [115-117]; thus, as suggested by Freeman et al., the luminal α-syn fibrils could induce membrane curvature causing the rupture of lysosomes [73]. Our correlative EM data show a wavy limiting membrane in lysosomes containing α-syn fibrils compared to lysosomes devoid of α-syn fibrils, supporting this hypothesis. Moreover, oxidative stress could also compromise lysosomal integrity [118]. Consistently, we have previously reported that α-syn fibrils increase ROS levels in CAD cells [19], and enhanced ROS levels have been found in PD mouse models and PD patients [68,119]. Since α-syn fibrils located to the lysosomes need to directly interact with the soluble cytosolic α-syn in order to induce the seeding, LMP has been proposed as a relevant mechanism to allow the escape of α-syn fibrils from the lysosomes [19,85]. Co-localization between Gal3 and aggregated α-syn has been reported in the basal forebrain of Diffused Lewy Body Disease
patients but not in normal, age-matched controls, supporting the role of lysosome rupture in α-syn pathology and progression [85]. In the current study, we detected efficient seeding of soluble α-syn both in donor cells treated with α-syn fibrils and in acceptor cells that received the fibrils inside lysosomes from donor cells (Figs 4A and B and 9A and B). By using super-resolution microscopy, we observed that 69 ± 6% and 60 ± 7% of the newly formed α-syn aggregates colocalize with LAMP1+ lysosomes in the donor and acceptor cells, respectively. These data together with live imaging show that the seeding event begins shortly after the internalization of α-syn fibrils and occurs preferentially at the lysosomes (Figs 4C-I, 9C-G, S5A, and S1 Movie). It has been shown that specific environments inside membranous and vesicular structures favor the aggregation of the α-syn protein [120,121]; thus, our results indicate that lysosomes can create a confined environment facilitating the conversion of soluble α-syn.

Do lysosomes contribute to the spread of the disease? Lysosomes are highly dynamic organelles that move bidirectionally along microtubules between the center and the periphery of the cell [122]. In non-polarized cells, lysosomes are mostly clustered at the perinuclear region, but a more dynamic pool is located at the cell periphery [90,92,100]. Here, we demonstrated that α-syn fibrils act on lysosome positioning by inducing their peripheral redistribution (Fig 5A and B). We also found that peripheral α-syn-containing lysosomes exhibit lysosomal dysfunction (i.e., disturbed pH and decreased lysosomal enzyme activities; Fig 5C). In addition, we showed that donor cells challenged with α-syn fibrils, thus having more peripheral lysosomes, transfer lysosomes more efficiently to the recipient cells compared to untreated donor cells (Fig 5F and G). Moreover, the peripheral distribution of lysosomes positively affects α-syn fibrils' transfer both in CAD and HeLa cells (Figs 7A and B and S6A and B).

The induction of LMP has been recently shown to promote nuclear translocation of TFEB [123], which influences lysosomal motility and exocytosis [95]. Furthermore, TFEB overexpression promotes α-syn clearance [124]. Thus, we investigated whether the loading of lysosomes with α-syn fibrils triggers TFEB nuclear translocation and whether this translocation participates in α-syn-induced distribution of lysosomes to the cell periphery. We found that α-syn fibrils induced TFEB translocation to the nucleus (Fig 6A). Moreover, expression of active TFEB correlates with more peripheral distribution of lysosomes (Fig 6B), while silencing of the endogenous TFEB prevents the peripheral redistribution of lysosomes upon α-syn loading (Fig 6D) suggesting the involvement of TFEB in this event. However, how this occurs is not yet clear. Previous reports suggested that TFEB-regulated transcription induced lysosomal docking to the plasma membrane, thus promoting the peripheral distribution of lysosomes in
1034 HeLa cells [95]. However, TFEB and the related transcription factor TFE3 have been reported
1035 to contribute to perinuclear positioning of lysosomes through transcriptional activation of the
1036 lysosomal protein TMEM55B under starvation conditions in HeLa cells [96]. It has also been
1037 reported that overexpression of TFEB promotes perinuclear positioning of the lysosomes in
1038 mouse fibroblast cells defective for the lysosomal transporter cystinosin [97]. Given these
1039 controversies in the literature, the exact role of TFEB in the regulation of lysosome positioning
1040 remains to be addressed in the future.

1041 To our knowledge, this is the first report showing that α-syn fibrils influence lysosome
1042 positioning, favoring α-syn fibrils transfer in a cell-to-cell contact-dependent manner via
1043 lysosomes. These findings suggest that lysosomes play an important role in the intercellular
1044 trafficking of α-syn fibrils. It is worth noting that α-syn fibrils do not affect lysosomal
1045 exocytosis (Fig 5D and E). In addition, in the secretion tests used in each co-culture experiment,
1046 we never detected any significant difference in the transfer of α-syn fibrils and/or lysosomes
1047 through the cell supernatant. Although this was not the aim of our study, these results further
1048 confirm that transfer of α-syn fibrils primarily relies on TNT- and/or cell-to-cell-contact-
1049 dependent mechanisms in accordance with our previous findings [19,20,22]. The mechanism
1050 by which α-syn fibrils enhance TNT formation is still largely unknown. However, we and others
1051 demonstrated that cells form more TNTs under stress, such as when they are treated with H₂O₂
1052 [125,126]. In line with these findings, we previously reported that CAD cells bearing α-syn
1053 fibrils exhibit sustained increase in ROS levels [19]. Therefore, α-syn fibrils can indeed trigger
1054 stress in the cells in order to induce TNT formation. In addition, it has been reported that α-syn
1055 could induce membrane remodeling, such as membrane expansion and formation of cylindrical
1056 tubes, and that the extent of membrane expansion correlated linearly with the density of α-syn
1057 [127]. Thus, further studies are essential to address how α-syn fibrils participate in the formation
1058 of TNTs.

1059 Lysosome transfer through TNTs has been shown to be bidirectional [106]. However, there is
1060 no study on the directionality/origin and the nature (whether they are healthy or damaged) of
1061 the lysosomes transferred between cells in the presence of the α-syn fibrils. Thus, in the current
1062 study, we studied the transfer of both healthy and damaged (i.e., under LMP) lysosomes
1063 between CAD (donor) and HeLa (acceptor) cells and found that the transfer of both types of
1064 lysosomes is bidirectional (Fig 10A, B, H, and I). We showed that α-syn fibril-treated donor
1065 cells transfer more lysosomes (both healthy and damaged) to acceptor cells compared to
1066 untreated control donor cells. Interestingly, we observed the recruitment of acceptor's Gal3 to
1067 the donor's lysosomes transferred in an acceptor cell (S7G Fig), suggesting that the rupture of
donors' lysosomes containing α-syn fibrils could occur after their transfer in the recipient cells.

Focusing on the transfer of lysosomes from acceptors to donors, we found that α-syn fibril-
treated donor cells received more healthy acceptors' lysosomes compared to control untreated
donor cells (Fig 10H and I). We also detected damaged acceptors' lysosomes in α-syn fibril-
treated donor cells although this was a very rare event (S7H and I Fig). Based on these data we
suggest that in the context of α-syn spreading, donor cells loaded with α-syn fibrils, try to get
rid of α-syn fibrils through the transfer of lysosomes, thus promoting the spreading of the fibrils.

On the other hand, donor cells receive healthy lysosomes from acceptor cells, suggesting a
potential rescue mechanism. A similar rescue mechanism has been previously reported in
cystinosin-deficient fibroblasts, which receive cystinosin-bearing lysosomes from healthy
macrophages through TNTs [106].

In light of these findings, we propose a working model on how lysosomes contribute to the
transfer and propagation of α-syn fibrils (Fig 11). After internalization, α-syn fibrils localize to
lysosomes (Fig 11, number 1), where they induce LMP and recruit Gal3 (Fig 11, number 2). α-
Syn fibrils escape from damaged lysosomes and seed soluble α-syn in donor cells, an event that
occurs mostly in association with the lysosomes (Fig 11, number 3). Following α-syn fibrils
internalization, TFEB translocates to the nucleus (Fig 11, number 4) and participates in the
redistribution of lysosomes containing α-syn fibrils, likely under LMP or functionally impaired,
towards the cell periphery (Fig 11, number 5). Here, lysosomes are not exocytosed (Fig 11,
number 6), but are more prone to be transferred to other cells through TNTs. Indeed, cells
loaded with α-syn fibrils transfer more lysosomes (both under LMP or not) to acceptor cells
compared to healthy cells through TNTs (Fig 11, number 7). Once lysosomes reach the acceptor
cells, α-syn fibrils escape from lysosomes already under LMP or from lysosomes that undergo
LMP when they arrive in the acceptor cells. This again allows α-syn fibrils to seed soluble α-
syn in the acceptor cells, mostly in association with the lysosomes (Fig 11, number 8) and
further triggering LMP (Fig 11, number 9). In an attempt to rescue α-syn-overloaded donor
cells, acceptor cells transfer healthy lysosomes to donor cells (Fig 11, number 10).

In conclusion, our data explain how lysosomes are involved in the transfer and propagation of
α-syn aggregates, and the crucial role that they play in this process. In agreement with these
findings, we have previously reported that α-syn fibrils can be efficiently transferred between
neurons, primary astrocytes, neurons and astrocytes, astrocytes and organotypic hippocampal
slices and between iPSCs and they were associated to lysosomes in these co-culture systems
[19,21,56]; however we have used here undifferentiated neuronal CAD cells line, for their easy
manipulation. Although our results contribute to a better understanding of the mechanism of
1102 development and progression of α-syn pathology, opening new avenues for possible therapeutic
1103 approaches against synucleinopathies, it will be necessary in the future to investigate the
1104 transfer of α-syn containing lysosomes in differentiated primary neurons and/or in ex-vivo/in-
1105 vivo systems to further validate our data and overcome the limitation of this study.
1106

1107 **Fig 11. Working Model.** 1) α-Syn fibrils localize to lysosomes following their uptake. 2) α-Syn fibrils induce LMP (i.e., recruit Gal3 to the ruptured lysosomes) in donor cells following their uptake, allowing their escape from lysosomes. 3) α-Syn fibrils interact with soluble α-syn and induce seeding in donor cells. 4) Following α-syn fibrils internalization, TFEB translocates to the nucleus. 5) Lysosomes (possibly under LMP, as their pH is higher and lysosomal activities is lower) having the α-syn fibrils move towards the cell periphery. 6) Peripheral lysosomes are not preferentially exocytosed. 7) Lysosomes having α-syn (both under LMP or not) are transferred to acceptor cells along with healthy lysosomes. 8) α-Syn fibrils induce LMP following their transfer to acceptor cells. 9) α-Syn fibrils induce further seeding following their transfer to acceptor cells. 10) Acceptor cells transfer healthy lysosomes to donors.
1117 **Materials and Methods**

1118

1119 **Cell culture, transfection, siRNA treatment**

1120 The mouse catecholaminergic neuronal cells (CAD cells) generated from a brain tumour of
1121 transgenic mice by targeted oncogenesis (kind gift of Hubert Laude; Institut National de la
1122 Recherche Agronomique, Jouy-en-Josas, France), were cultured in Opti-MEM<sup>R</sup> GlutaMAX<sup>TM</sup>
1123 (Gibco) including 10% foetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen-Strep).
1124 CAD cells express neuronal properties, but they lack neuronal morphology, thus stay
1125 undifferentiated in the presence of serum in their medium. This situation can be reversed by
1126 serum starvation and differentiation can be induced [128]. In this study, we used CAD cells in
1127 their undifferentiated state as this condition is optimal to study TNTs [19,125,129–131]. HeLa
1128 cells WT, HeLa cells myrlysin-KO and KIF1B-KIF5B double KO [100,132] and HeLa cells
1129 stably expressing Gal3-Turquoise were cultured in DMEM-GlutaMAX<sup>TM</sup> including 4.5 g/L D
1130 glutamine and pyruvate (Gibco) supplemented with 10% of FBS. CAD and HeLa cells were
1131 transiently transfected with Gal3-GFP, Arl8b-GFP, fluorescently tagged human histone H2B
1132 (H2B-GFP or H2B-mCherry) plasmids (final concentration: 1 µg for 150,000 cells/35mm Ibidi
1133 µ-dishes, 3 µg for 800.000 cells/T25 flasks) by Lipofectamine 2000 (Invitrogen) according to
1134 the manufacturer's instructions.

1135 To downregulate Arl8b and TFEB, 30 pmol of unique 27mer siRNA duplex against mouse
1136 Arl8b and TFEB (CliniSciences and OriGene respectively) was introduced to CAD cells in
1137 parallel to siRNA scramble (sicontrol) by using Lipofectamine RNAiMAX (Invitrogen)
1138 according to the manufacturer's instructions, and incubated 60h prior to be used in co-culture
1139 experiments and in Western blot analysis.

1140 To induce lysophagy, CAD cells were treated with 1 mM L-Leucyl-L-Leucine methyl ester
1141 (LLOMe) for 3h.

1142

1143 **Generation of stable HeLa mTurquoise-Galectin3 cell line**

1144 In order to generate stable HeLa cell line expressing a mTurquoise-Galectin3 fluorescent
1145 reporter, we used a well-established Sleeping Beauty (SB) transposable system that originates
1146 from an inactive transposon in salmonid fish and belongs to the Tc1/mariner-type transposon
1147 family, as previously described [133,134]. Briefly, the insert sfiI-mTurquoise-Galectin3-sfiI
1148 was amplified from pE mTurquoise-Galectin3 plasmid by polymerase chain reaction (PCR)
1149 where SfiI-mTurquoise-forward (AGG CCT CTG AGG CCG CCA CCA TGG TGA GCA
1150 AGG G) and Gal3-SfiI-reverse (AGG CCT GAC AGG CCT TAT CAT GGT ATA TGA AGC
1151 AC) primers (IDT-DNA Company) were used with Flash Phusion High Fidelity PCR Master
1152 Mix (ThermoFisher Scientific). Next, the insert was cut by sfiI enzyme and inserted into the
1153 constitutive vector pSBbi-PUR, a gift from Eric Kowarz, (Addgene Plasmid #60523) that was
cut by sfiI enzyme and dephosphorylated by Rapid DNA Dephos & Ligation Kit (Roche).
1154 Transformation was performed with on stBL3 competent bacteria following the ligation of sfiI-
pSBbi-PUR-sfiI vector and insert sfiI-mTurquoise-Galectin3-sfiI, and colonies were selected
1155 on LB broth agar plates containing amphicilin. Clones were then sequenced (Eurofins
1156 Genomic) in order to verify the existence of the insert and maxiprep for the pSBbi-PUR-
mTurquoise-Galectin3 plasmid was performed by using the Invitrogen™ PureLink™ HiPure
1157 Plasmid Maxiprep Kit. Next, HeLa cells seeded on 6 well plates (300.000 cells per well) were
1158 co-transfected with 9 µg of pSBbi-PUR-mTurquoise-Galectin3 and 100 ng SB transposase
1159 expression vector pCMV (CAT)T7-SB100, a gift from Eric Kowarz (Addgene plasmid #
1160 34879) by using XtremGene® transfection reagent (Roche). 24 h following the transfection,
1161 cells were subjected to puromycin (1 µg/mL). Selection was carried out for 2 to 10 days. Cells
1162 were then cultivated for four weeks without selection medium and monoclonal selection was
1163 performed on 96 well plates (1 cell/well). Clones exhibited expected mTurquoise fluorescent
1164 were further tested for classical behavior for HeLa cells (low Galectin-3 fluorescent in the
1165 whole cell and a brilliant signal upon vacuole rupture) and used in the experiments described
1166 in the current study.
1167
1168
1169

1170

1171 **Preparation and internalization of α-syn fibrils**

1172 Human wild-type α-syn in pRK172, a construct containing α-syn that lacks cysteine because of
mutagenesis of codon 136 (TAC to TAT) was transformed into *Escherichia coli* BL21 (DE3)
1173 as previously reported. Expression and purification were performed as described previously
1174 [86]. Briefly, the protein concentrations of monomeric α-syn were measured by RP-HPLC as
described previously [86,135]. Purified recombinant α-syn monomers (~5 mg/mL) containing
1175 30 mM Tris-HCl, pH 7.5, 10 mM DTT, and 0.1% sodium azide were incubated at 37°C with
shaking using a horizontal shaker (TAITEC) at 200 rpm. Following the incubation for 7 days,
1176 the samples were ultracentrifuged at 100.000 g for 20 min at room temperature (RT), and the
1177 ppt fraction was recovered as α-syn fibrils. They were re-suspended in saline prior to be
1178 ultracentrifuged again. The pellets were re-suspended in saline and sonicated using a cup horn
1179
1180
1181 ultracentrifuged again. The pellets were re-suspended in saline and sonicated using a cup horn
1182 sonicator (Sonifier SFX, Branson) at 35% power for 180 s (total 240 s, 30 s on, 10 s off). The
1183 fibrils were labelled with Alexa Fluor 488 or 568 Protein Labelling Kit (Invitrogen) following
1184 the manufacturer's instructions. After the incubation of fibrils with Alexa Fluor dyes, samples
1185 were ultracentrifuged again and the pellets were re-suspended in 30 mM Tris-HCl, pH 7.5 prior
1186 to be ultracentifuged one more time. Finally, labelled α-syn fibrils were resuspended in saline
1187 containing 0.1% sodium azide and the protein concentration of the fibrils was determined by
1188 RP-HPLC as described before [86,135]. Seeding activity of the fibrils were checked by adding
1189 the labelled fibrils (3 µg) to 100 µL of 1 mg/mL α-syn monomer solution in 30 µM Thioflavin
1190 T and 80 mM Hepes, pH 7.5. Amyloid-like fibril formation was continuously monitored by
1191 measuring the Thioflavin T fluorescence (excitation 442 nm, emission 485 nm) with a
1192 microplate reader (Infinite 200, TECAN).

1193 0.5 µM Alexa 488 or Alexa 568-tagged α-syn fibrils were diluted in the appropriate medium
1194 and sonicated for 5 min at 80% amplitude with a pulse cycle of 5 s on and 2 s off in an ultrasonic
1195 water bath Vibra-Cell 75,041 (BioBlocks Scientific). Fibrils were then introduced to cells
1196 immediately and incubated for 18 h. Before cells were processed for further procedures (e.g.,
1197 co-culture preparation, fixation, etc.), they were washed 3 times with diluted trypsin (1/3) in
1198 phosphate buffered saline (PBS). In parallel, control cells were subjected to the same treatments
1199 as described above without the addition of α-syn fibrils.

1200

1201 Immunocytochemistry for lysosomes

1202 CAD and/or HeLa cells and co-cultures (CAD-CAD and CAD-HeLa) were fixed with 4%
1203 paraformaldehyde (PFA) for 20 min at room temperature (RT). After 3 washes with PBS, cells
1204 were permeabilized with 0.1% Triton X-100 in PBS for 3 min at RT. Cells were then washed
1205 with PBS and nonspecific antibody binding sites were blocked by using 10% of goat serum
1206 (GS) in PBS for 30 min. Cells were then incubated with primary rat monoclonal anti-mouse
1207 LAMP1 (1D4B) or anti-human LAMP1 (H4A3) primary antibodies with the dilution of 1/200
1208 in blocking solution for 1 h. Antibodies were purchased from Developmental Studies
1209 Hybridoma Bank (DSHB), Iowa City, IA, USA. Cells were then washed 3 times with PBS and
1210 incubated for secondary antibodies (anti-rat Alexa-647 or anti-rat Alexa-488 purchased from
1211 Invitrogen, Carlsbad, CA) diluted 1:500 in blocking solution for 2 h at RT. After 3 washes with
1212 PBS, cells were further stained with HCS CellMaskTM Blue (Invitrogen) for 15 min at RT
1213 (diluted 1:5000 in PBS). Cells were then washed 3 times with PBS and further stained for DAPI
1214 (diluted 1:5000 in PBS) for 5 min. Finally, cells were washed and mounted with Aqua-
1215 Poly/Mount (Polysciences, Inc.). Image acquisitions were performed by confocal microscopy
1216 with identical settings.

1217

1218 **Immunocytochemistry for lysophagy**

1219 CAD cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature (RT).
1220 After 3 washes with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 3 min
1221 at RT. Cells were then washed with PBS and nonspecific antibody binding sites were blocked
1222 by using 2% of bovine serum albumin (BSA) in PBS for 30 min. Cells were then incubated
1223 with primary rabbit anti-mouse LC3 (1:500, MBL International), mouse anti-Ubiquitin (1:1000,
1224 FK2, Enzo), and rat monoclonal anti-mouse LAMP1 (1D4B) (1:100, DSHB) in blocking
1225 solution for 1 h. Cells were then washed 3 times with PBS and incubated for secondary
1226 antibodies (anti-rabbit Alexa 488, anti-mouse Alexa-647 from Invitrogen, Carlsbad, CA; and
1227 anti-rat CF™405M from Sigma) diluted 1:500 in blocking solution for 1 h at RT. Finally, cells
1228 were washed and mounted with Aqua-Poly/Mount (Polysciences, Inc.). Image acquisitions
1229 were performed by confocal microscopy with identical settings.

1230

1231 **LysoTracker Deep Red staining, DQ-BSA assay, and Cathepsin B activity assay**

1232 CAD cells were plated on 35 mm Ibidi µ-dishes (100.000 cells/dish). Alexa 568-tagged α-syn
1233 fibrils were introduced or not (control) to the cell culture medium 24 h after plating. Following
1234 18 h of α-syn fibril uptake, 20 nM LysoTracker Deep Red (Invitrogen) was prepared in culture
1235 medium and cells were incubated for 20 min at 37°C. Cells were then fixed with 4% PFA at
1236 RT and stained for HCS CellMask™ Blue and DAPI as described before. Image acquisitions
1237 were performed by confocal microscopy with identical settings.

1238 To detect Cathepsin B (CathB) activity, CAD cells were plated on 35 mm Ibidi µ-dishes
1239 (100.000 cells/dish). Alexa 488-tagged α-syn fibrils were introduced or not (control) to the cell
1240 culture medium 24 h after plating. Following 18 h of α-syn fibril uptake, cells were treated with
1241 1X Magic Red Cathepsin solution, provided by Magic Red Cathepsin Assay kit
1242 (ImmunoChemistry Technologies LLC) and prepared through the manufacturer's
1243 recommendation, for 15 min at 37°C. Cells were then immediately imaged by confocal
1244 microscopy in live condition. The intracellular activity of CathB was detected as red fluorescent
1245 puncta produced by the hydrolysis of the fluorogenic substrate introduced. All images were
1246 acquired with the identical settings. For both experiments integrated density of LysoTracker
1247 Deep Red and CathB signal were measured by using FiJi software after a region of interest
1248 (ROI) is manually created by the freehand selection tool and the measurements were expressed
1249 in arbitrary unit (AU).

1250 For DQ-BSA assay, CAD cells were plated on 35 mm Ibidi µ-dishes (130.000 cells/dish). Alexa
1251 568-tagged α-syn fibrils were introduced or not (control) to the cell culture medium 24 h after.

1252 Following 18 h of α-syn fibrils uptake, 10 µg/mL DQ Green BSA (Thermo Fisher Scientific)
1253 was prepared in culture medium and cells were incubated for 90 min at 37°C. Cells were then
1254 fixed with 4% PFA at RT. Image acquisitions were performed by confocal microscopy with
1255 identical settings. Integrated density of DQ-BSA signal were measured by using FiJi software
1256 after a region of interest (ROI) is manually created by the freehand selection tool and the
1257 measurements were expressed in arbitrary unit (AU).

1258

1259 **TNT labelling**

1260 To preserve the structure of TNTs, cells were fixed by a two-step fixation protocol as described
1261 previously [20,129]. Briefly, cells were first fixed with fixative solution-1 (2% PFA, 0.05%
1262 glutaraldehyde (GA) and 0.2 M HEPES in PBS) for 20 min at 37°C, then with fixative solution-
1263 2 (4% PFA and 0.2 M HEPES in PBS) for an additional 20 min at 37°C. After several washes,
1264 cells were labelled with 3.3 µg/µL Wheat Germ Agglutinin (WGA)-Alexa Fluor-488 nm
1265 conjugate solution (Life Technologies) for 20 min at RT for membrane detection. As there is
1266 no specific marker for TNTs, WGA labelling was used to detect TNTs as described in detail in
1267 the quantification section.

1268

1269 **Lysosomal exocytosis analysis**

1270 *Evaluation of released Cathepsin B by ELISA*

1271 CAD cells were plated in B12-wells (80.000 cells/well). Cells were then treated with Alexa
1272 568-tagged α-syn fibrils or not for 18 h. Soluble lysosomal Cathepsin B (CathB) released in
1273 culture medium was evaluated by using Human Cathepsin B ELISA Kit (Abcam) following the
1274 manufacturer's instructions. Briefly, aliquots of culture medium derived from control cells and
1275 cells challenged with α-syn fibrils were incubated in 96-well plates precoated with a CathB
1276 specific mouse monoclonal antibody for 90 min at 37°C. Biotinylated CathB-specific goat
1277 polyclonal antibody was then added on the cells for 60 min at 37°C. Cells were then washed
1278 with tris-buffered saline (TBS) prior to the addition of Avidin-Biotin-Peroxidase Complex for
1279 30 min at 37°C. Unbound conjugates were washed away with TBS buffer five times. 3,3',5,5'-
1280 Tetramethylbenzidine was then used to visualize the horse radish peroxidase (HRP) enzymatic
1281 reaction (O.D. absorbance: 450 nm). Released CathB content is expressed as nanograms of
1282 CathB/mL of culture medium.

1283

1284 *Evaluation of LAMP1 at the plasma membrane*

1285 CAD cells were plated in 35 mm Ibidi µ-dishes (120.000 cells/dish). Cells were then treated
1286 with Alexa 568-tagged α-syn fibrils or not for 18 h. Live cells were the incubated with the
1287 primary rat monoclonal anti-mouse Lamp1 (1D4B) antibody recognizing an epitope at the
1288 luminal region of the protein, diluted in complete medium, for 30 min at 4°C. After washing
1289 with PBS, cells were fixed in 2% PFA in PBS for 10 min at RT. Next, cells were incubated
1290 with the Alexa Fluor 488-conjugated goat anti-rat secondary antibody diluted in PBS for 30
1291 min at RT. Cells were then imaged by confocal microscopy.

1292

1293 **Detection of LMP and seeding**

1294 CAD cells were plated on 35 mm Ibidi µ-dishes (100.000 cells/dish) and transiently transfected
1295 with 1 µg of Gal3-GFP (for LMP) or 1 µg of α-syn-GFP plasmids (for seeding). 24 h later cells
1296 were either treated with Alexa 568-tagged α-syn fibrils for 18 h or left untreated. In co-culture
1297 experiments, cells transiently transfected with Gal3-GFP or α-syn-GFP plasmids were co-
1298 cultured for 24 h with donor cells previously treated for 18 h with Alexa 568-tagged α-syn
1299 fibrils or not. Cells were then fixed with 4% PFA for 20 min at RT and stained for DAPI.
1300 Following image acquisition with confocal microscopy, cells were quantified for the presence
1301 of Gal3-GFP or α-syn-GFP puncta by using "Spot detector" tool of the ICY software. Note that
1302 a threshold of five puncta is applied to all images analysed and cells bearing puncta above this
1303 threshold were considered as having LMP.

1304

1305 **Co-culture experiments**

1306 Donor cells were plated on 35 mm well plates (400.000/well) and transfected with suitable
1307 plasmids according to the experiment (LAMP1-GFP, Arl8b-GFP, Gal3-GFP, H2B-mCherry)
1308 or pre-treated with siRNA Arl8b or siRNA-scrambled for 60 h. Myrlysin-KO and KIF1B-5B
1309 dKO HeLa cells were directly used as donors. Donor cells (in parallel to untreated control cells)
1310 were then challenged with Alexa 568-tagged α-syn fibrils for 18 h (or not according to the
experiment). They were then washed once with PBS and 3 times with 1:3 diluted trypsin before co-culture preparation.

Acceptor cells were plated on T25 flasks (800.000/flask) and transfected with suitable plasmids according to the experiment (Gal3-GFP, H2B-mCherry, H2B-GFP, α-syn-GFP). HeLa Gal3-Turquoise cell line was used directly as acceptor cell population. Both donor and acceptor cells were then detached (mechanically and by trypsinization for CAD and HeLa cells respectively) and counted. Co-culture was prepared in a 1:1 ratio (100.000 donor and 100.000 acceptors on 35 mm Ibidi µ-dishes) for 24 h. Cells were then fixed with 4% PFA for 20 min at RT and stained for HCS CellMaskTM Blue and DAPI as described before and mounted. Schematic presentation of different co-culture preparations was presented in S3 Fig.

Of note, co-cultures were named with the predominant treatment of the donor cells: i) co-cultures having donor cells treated with α-syn fibrils or not were referred to as “α-syn co-culture” and “control co-culture” respectively; ii) in the cases where all donor populations were treated with α-syn fibrils, in order to emphasize the different treatments between donor cells, co-cultures were named by these treatments (e.g., Arl8b-GFP over-expressing or siArl8b pretreated donors were referred to as “Arl8b-GFP co-culture” and “siArl8b co-culture” respectively).

### Secretion test

In all co-culture conditions, donor cells and acceptor cells were also plated separately during the co-culture preparation (100.000 cells/35 mm Ibidi µ-dishes) for the total duration of co-culture incubation (24 h). Then the conditioned medium collected from donor cells were transferred onto acceptor cells. Acceptor cells were then quantified for the presence of specific cargos (e.g., lysosomes, α-syn fibrils) to assess the cargo transfer through secretion in each co-culture condition. Secretion tests for particular experiments were referred to as predominant treatment of the donor cells as described above (e.g., “control secretion”, “α-syn secretion”, “Arl8b-GFP secretion”).

### Confocal microscopy

After fixation and immunostaining, images were acquired with an inverted laser scanning confocal microscope LSM700 (Zeiss), with a 63X objective (zoom 1.0). Images were acquired using the ZEN acquisition software (Zeiss). In all experiments, we acquired Z-stacks covering
1343 the whole volume of cells. For the experiments where integrated density was measured in
1344 control and α-syn treated cells (LysoTracker Deep Red, Magic Red CathB, DQ-BSA assays,
1345 and evaluation of LAMP1 at the plasma membrane in non-permeabilized condition), all settings
1346 (including the laser power and exposure line time) were kept identical. In some experiments
1347 line averaging were also used in order to improve the signal to noise ratio.

1348

1349 **Super-Resolution (SR) Microscopy**

1350 *Sample preparation*

1351 24-well plates containing sterile cover slips (Deckglaser 12 mm) were coated with fibronectin
1352 (25 µg/mL) diluted in PBS and kept at 37°C for 1 h. Cover slips were then washed 3 times with
1353 PBS. CAD cells were then plated on cover slips (20.000 cells/coverslip). Gal3-GFP or α-syn-
1354 GFP transfected and α-syn treated cells or mixture of donor and acceptor cells (co-cultures
1355 prepared for LMP and seeding experiments) were then plated on coverslips. Cells were then
1356 fixed with 4% PFA for 20 min at RT and immunolabelled with primary rat monoclonal anti-
1357 mouse LAMP1 (1D4B) and secondary anti-rat Alexa 647 antibodies as described before. Cells
1358 were then stained with DAPI and HCS CellMask™ Blue and mounted with Fluoromount-G
1359 (Southern Biotech) mounting medium.

1360

1361 *Structured Illumination Microscopy (SIM)*

1362 SIM was performed on a Zeiss LSM780 Elyra PS1 microscope (Carl Zeiss, Germany) using
1363 100X/1.4 oil Plan Apo objective with a 1.518 refractive index oil (GE Healthcare Life Science)
1364 and an EMCCD Andor Ixon 887 1 K camera for the detection. SIM images were processed
1365 with ZEN software and then aligned with ZEN using 100-nm TetraSpeck microspheres
1366 (ThermoFisher Scientific) embedded in the same conditions as the sample.

1367

1368 *Spinning Disk- Live Super-Resolution (SR) Confocal Microscopy*

1369 Images were acquired with an inverted Eclipse Ti Nikon microscope equipped with a CSU- X1
1370 spinning disk confocal scanning unit (Yokogawa), with an EMCCD Camera (Evolve 512 Delta,
1371 Photometrics), and with Live SR super-resolution module (Gataca systems), using a x100 or
1372 x60 1.4 NA PL-APO VC oil objective lenses controlled by MetaMorph software. SR images
1373 were denoised by using Safir software [136].
1374

1375 **Live imaging microscopy**

1376 Time-lapse microscopy imaging was performed on an inverted Spinning Disk microscope
1377 (Elipse Ti microscope system, Nikon Instruments, Melville, NY, U.S.A.) using 60X 1.4 NA
1378 CSU oil immersion objective lens and Laser illumination 488 only or together with 561. For
1379 live cell imaging, the 37°C temperature was controlled with an Air Stream Stage Incubator,
1380 which also controlled humidity. Cells were plated in Ibidi µ-Dish 35 mm and incubated with
1381 5% CO₂ during image acquisition. Image processing and movies were realized using
1382 MetaMorph and ImageJ/Fiji software.

1383

1384 **Electron Microscopy**

1385 *Correlative Resin Electron Microscopy*

1386 For correlation of FM and resin-embedded EM of CAD cells, FM imaging was performed prior
1387 to sample preparation in EM. Cells were transiently transfected with LAMP1-GFP using
1388 Lipofectamine 2000 transfection reagent for 24 h and seeded on carbon-coated, gridded
1389 coverslips prepared as described before [76]. 24 h later, they were treated with α-syn- Alexa568
1390 fibrils for 18 h. Cells were then washed in 0.1 M phosphate buffer (PB) and fixed in 4%
1391 formaldehyde in 0.1 M PB buffer after which fluorescent Z-stacks of cells of interest were
1392 obtained for the GFP and Alexa 568 signal. The position of cells relative to the pattern etched
1393 in the coverslip was registered using polarized light. After FM imaging, the same specimens
1394 were prepared for EM by fixing with 2.5% glutaraldehyde + 2% formaldehyde in 0.1 M PB
1395 buffer for 2 h, and post fixation with osmium tetroxide and uranyl acetate, Fixed cells were
1396 dehydrated using a graded ethanol series, and embedded in Epon resin which was polymerized
1397 for 48 h at 65°C. After polymerization, the glass coverslip was removed from the Epon block
1398 by dissolving it in hydrogen fluoride. The exposed Epon surface was thoroughly cleaned with
1399 distilled water and left to harden overnight at 63°C. Areas of the resin block containing the cells
1400 previously imaged by FM were cut out using a clean razor blade, and glued to empty Epon
1401 sample stubs, with the basal side of the cells facing outwards [75,76]. From these blocks, 70
1402 nm sections were cut and collected on formvar and carbon coated copper 50 mesh support grids.
1403 Thin EPON sections were imaged in a Tecnai 12 TEM (Thermo Fischer Scientific, Eindhoven,
1404 The Netherlands) equipped with a Veleta 2k×2k CCD camera (EMSIS, Munster, Germany),
1405 operating at 80 kV. For correlation of FM and EM data, the TEM image of a ROI was overlayed
1406 with FM data using Photoshop. Multiple corresponding spots (e.g. nuclei) on images were
1407 manually selected, after which the correct scaling and transformation steps were performed and
1408 high-precision overlays of FM and EM data were generated. We used only linear transformation
1409 options to achieve the overlays shown in the Figures.

1410

1411 *On-section Correlative Microscopy of thawed cryosections*

1412 Similarly, CAD cells were grown on carbon-coated, gridded coverslips, incubated with α-syn
1413 Alexa 568-tagged fibrils for 18 h and fixed in 0.1 M PB (pH 7.4) containing 4% PFA that was
1414 added to an equal volume of medium. Fixed cells were flat embedded in 12% gelatin by keeping
1415 their orientation, cryoprotected in 2.3 M sucrose, and plunge frozen according to previous
1416 protocols [137,138]. 70 nm cryosections were picked up on formvar coated copper grids, after
1417 which they were immunolabelled for LAMP1 with a secondary Alexa 488-tagged rabbit
1418 antibody and 10 nm gold-conjugated Protein A (Cell Microscopy Core, UMC Utrecht, The
1419 Netherlands). Finally, sections were labelled using Hoechst 33342 to outline nuclei for
1420 correlation. The grids were washed with dH2O and sandwiched between a microscope slide
1421 and a no. 1 coverslip in 2% methylcellulose in dH2O. Sections were imaged in a Deltavision
1422 RT wide field fluorescence microscope (GE Healthcare, U.S.A.) equipped with a Cascade II
1423 EM-CCD camera (Photometrics, U.S.A.). Grids were first imaged at 40× magnification to form
1424 a map of the section, after which regions of interest (ROIs) were selected using 100×
1425 magnification. After imaging the grids were removed from the microscope slide, thoroughly
1426 rinsed with H2O and contrasted for EM and embedded in methylcellulose containing uranyl
1427 acetate. After drying, ROIs were imaged using a Tecnai 12 TEM (Thermo Fischer Scientific)
1428 operating at 80 kV, equipped with a Veleta 2k×2k CCD camera (EMSIS, Munster, Germany)
1429 camera, and running serialEM software. Registration of thin section FM and EM data was
1430 performed using Adobe Photoshop, similar to above.

1431

1432 **Western immunoblots**

1433 For the Western blot analysis, cells were rinsed with PBS and then scraped in RIPA buffer (50
1434 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1%
1435 SDS). The resulting soluble fractions were centrifuged at 18,000 g for 5 min at 4°C, and then,
1436 the supernatants were taken as the whole cell lysate, mixed with Laemmli sample buffer (1 M
1437 Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 5% β-mercaptoethanol, and 0.02% bromophenol
1438 blue), and boiled for 5 min. For Western blots, equal amounts of proteins (30 µg/lane) were
1439 separated on SDS-PAGE gels and transferred to nitrocellulose membranes (GE Healthcare Life
1440 sciences), following standard procedures. Membranes were then blocked in Tris-buffered saline
1441 containing 0.1% Tween-20 and 5% non-fat dried milk for 1 h and probed overnight at 4°C with
1442 primary antibodies. The membranes were then washed and exposed for 1h at RT to the anti-
1443 rabbit/anti-mouse peroxidase-conjugated antibodies (1:1000) The specific protein bands were
1444 visualized using the ECL-immunoblotting chemiluminescence system (GE Healthcare Life
1445 sciences) and the ImageQuant LAS 500TM camera (GE Healthcare Life sciences). Primary
1446 antibodies used in this study: rat anti-Lamp1 (1:100, DSHB), anti-Arl8b (1:100), rabbit anti-
1447 TFEB (1:1000, Bethyl Laboratories), mouse anti-tubulin (1:1000), rabbit anti-GAPDH (1:5000,
1448 Boster). To determine the apparent molecular weights of the protein bands, a PageRuler plus
1449 prestained protein ladder (Thermo Fisher Scientific) was used.
1450

1451

**Quantification analysis**

1452

*Quantification for lysosomal positioning*

1453 We performed 2D analysis with the Z-projection where we used a two-step detection protocol
1454 in ICY software in order to assess the lysosomal positioning. In the first step, we used a script
1455 to detect two ROIs: i) the limiting membrane of whole cell that was detected with HCS
1456 CellMask™ Blue staining; ii) the nuclear region that was detected by DAPI staining. Important
1457 to note that in the cases where perinuclear region of the cells including microtubule organizing
1458 center (MTOC) was not detected in Z-projection, this ROI was manually corrected. In the
1459 second step of the protocol another script was used where "Spot detector" and "Colocalization"
1460 modules were combined. Thus, we were able to detect the number of lysosomes, the number of
1461 α-syn fibrils puncta, and the number of lysosomes colocalizing with the α-syn fibrils. Number
1462 of peripheral lysosomes were calculated by simply subtracting the number of centrally located
1463 lysosomes from the whole number of lysosomes and the percentages central and peripheral
1464 lysosomes were calculated accordingly. Same protocols were used to quantify the LysoTracker
1465 Deep Red and MagicRed Cathepsin B positive lysosomes where HCS CellMask™ Blue and
1466 Cell Tracker Blue (Invitrogen) staining were used in addition to DAPI staining respectively.
1467

1468

TNT counting

1469 A semi-automatized “TNT counting” tool in ICY software was used for TNT counting
1470 experiments as previously described [20,129]. Briefly, total number of cells were counted in
1471 each image and the connection between each cell pair was evaluated by scanning through the
1472 Z-stacks. Cell pairs that are connected by a protrusion that fits to certain criteria that are used
1473 to discriminate TNTs from other cellular protrusions (such as having thin, straight, and
1474 uninterrupted connections that are not touching to the substratum) were counted as “TNTs” and
1475 connected by a line using a free-hand line tool. Total number of cells and number of TNT-
1476 connected cells were automatically counted by the tool. Thus, data were presented as percentage
1477 of TNT connected cells.

1478
1479 Lysosome size (on-section CLEM)

1480 FM and EM images from the same section were overlaid and correlated to each other. LAMP1
1481 immunogold labelled organelles (lysosomes) were selected blindly and their size was measured
1482 using the “lasso” tool in Fiji. Afterwards the lysosomes were clustered based on presence of α-
1483 syn fibrils, and their size distribution was plotted.

1484
1485 Lysosome size (SR microscopy)

1486 Diameter of lysosomes were measured by using free-hand straight line tool of FiJi software.
1487 For each condition (control lysosomes and lysosomes containing α-syn or not), SR images were
1488 zoomed, and diameter of randomly selected lysosomes was measured.

1489
1490 Lysosome number

1491 The average number of lysosomes in control cells and in cells treated with α-syn fibrils were
1492 counted by using the “Spot detector” tool in ICY software.

1493
1494 Object-based 3D colocalization analysis (Imaris software)

1495 ROI for each was created by using “Surface” tool in the Surpass mode in Imaris. For each cell
1496 puncta of interest (Gal3-GFP, α-syn-GFP, α-syn fibrils and LAMP1+ puncta) in different colors
1497 were detected by “Spots” tool. Then colocalization was performed first for each pair of interest,
1498 and then for triple presence of the puncta by using “Colocalization” tool.

1499
1500 Statistical analysis

1501 Data collected at least from 3 independent experiments was presented as Mean ± SEM. Exact
1502 number of experiments and number of cells analysed per condition for each experiment is
1503 presented in the Figure legends. All data were first subjected to a D'Agostino-Pearson omnibus
1504 normality test. Values having Gaussian distribution were further analysed by two-tailed t-test
and one-way ANOVA followed by Tukey's multiple comparison tests for paired and multiple comparisons respectively. Values having non-Gaussian distribution were analysed by Whitney-Mann *U*-test and Kruskal-Wallis nonparametric ANOVA test followed by Dunn's multiple comparison tests for paired and multiple comparisons respectively. Paired set of data were analysed by *paired-t* test.
1510 Acknowledgements

1511 The authors thank all the lab members for useful discussion. We are grateful to Institut Pasteur
1512 Image Analysis Hub (IAH) for their help in quantitative image analysis, Audrey Salles from
1513 Unit of Technology and Service Photonic BioImaging platform (UTechS PBI, Institut Pasteur)
1514 and Cécile Leduc from Cell Polarity, Migration and Cancer unit, Institut Pasteur, for their help
1515 in SIM acquisitions. UTechS PBI, part of the France-BioImaging infrastructure network (FBI)
1516 acknowledges Agence Nationale de la Recherche (ANR-10-INSB-04), ANR/FBI and the
1517 Région Ile-de-France Domaine d'Intérêt Majeur-Malinf program for the use of SIM. We thank
1518 Alexandre Dufour for writing the script for the lysosomal positioning analysis, Eric Kowarz for
1519 the pSBbi-PUR and pCMV (CAT) T7-SB100 plasmids, Andrea Ballabio (TIGEM) for TFEB-
1520 WT-GFP and TFEB-mut-GFP plasmids, and Jost Enninga (Institut Pasteur) and Kristine
1521 Schauer (Institut Curie) for discussion. We also acknowledge Cilia de Heus (UMCU) for her
1522 assistance in CLEM experiments.
1523 References

1524 1. Soto C, Pritzkow S. Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases. Nat Neurosci. 2018;21: 1332–1340. doi:10.1038/s41593-018-0235-9

1527 2. Gan L, Cookson MR, Petrucelli L, La Spada AR. Converging pathways in neurodegeneration, from genetics to mechanisms. Nat Neurosci. 2018;21: 1300–1309. doi:10.1038/s41593-018-0237-7

1530 3. Poewe W. Nature Reviews Disease Primers. Nat Rev Dis Primer. : 52.

1531 4. Volpicelli-Daley LA, Gamble KL, Schultheiss CE, Riddle DM, West AB, Lee VM-Y. Formation of α-synuclein Lewy neurite-like aggregates in axons impedes the transport of distinct endosomes. Martin TFJ, editor. Mol Biol Cell. 2014;25: 4010–4023. doi:10.1091/mbc.e14-02-0741

1535 5. Braak H, Tredici KD, Rüb U, de Vos RAI, Jansen Steur ENH, Braak E. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging. 2003;24: 197–211. doi:10.1016/S0197-4580(02)00065-9

1538 6. Takamatsu Y, Fujita M, Ho GJ, Wada R, Sugama S, Takenouchi T, et al. Motor and Nonmotor Symptoms of Parkinson's Disease: Antagonistic Pleiotropy Phenomena Derived from α-Synuclein Evolvability? Park Dis. 2018;2018: 5789424. doi:10.1155/2018/5789424

1542 7. McCann H, Stevens CH, Cartwright H, Halliday GM. α-Synucleinopathy phenotypes. Parkinsonism Relat Disord. 2014;20: 62–67. doi:10.1016/S1353-8020(13)70017-8

1544 8. Taguchi K, Watanabe Y, Tsujimura A, Tanaka M. Brain region-dependent differential expression of alpha-synuclein: α-Synuclein Differential Expression. J Comp Neurol. 2016;524: 1236–1258. doi:10.1002/cne.23901

1547 9. Chandra S, Chen X, Rizo J, Jahn R, Südhof TC. A Broken α-Helix in Folded α-Synuclein. J Biol Chem. 2003;278: 15313–15318. doi:10.1074/jbc.M213128200

1549 10. Varkey J, Isas JM, Mizuno N, Jensen MB, Bhatia VK, Jao CC, et al. Membrane Curvature Induction and Tubulation Are Common Features of Synucleins and Apolipoproteins. J Biol Chem. 2010;285: 32486–32493. doi:10.1074/jbc.M110.139576

1552 11. Meade RM, Fairlie DP, Mason JM. Alpha-synuclein structure and Parkinson's disease – lessons and emerging principles. Mol Neurodegener. 2019;14:29: 10.1186/s13024-019-0329-1. doi:10.1186/s13024-019-0329-1

1555 12. Huang M, Wang B, Li X, Fu C, Wang C, Kang X. α-Synuclein: A Multifunctional Player in Exocytosis, Endocytosis, and Vesicle Recycling. Front Neurosci. 2019;13: 28. doi:10.3389/fnins.2019.00028
1558 13. Burré J, Sharma M, Tsetsenis T, Buchman V, Südhof TC. α-Synuclein Promotes SNARE-Complex Assembly in vivo and in vitro. Science. 2011; 1663–1667.

1560 14. Sun J, Wang L, Bao H, Premi S, Das U, Chapman ER, et al. Functional cooperation of α-synuclein and VAMP2 in synaptic vesicle recycling. Proc Natl Acad Sci. 2019;116: 11113–11115. doi:10.1073/pnas.1903049116

1563 15. Man WK, Tahirbegi B, Vrettas MD, Preet S, Ying L, Vendruscolo M, et al. The docking of synaptic vesicles on the presynaptic membrane induced by α-synuclein is modulated by lipid composition. Nat Commun. 2021;12: 927. doi:10.1038/s41467-021-21027-4

1566 16. Braak H, Rub U, Gai WP, Del Tredici K. Idiopathic Parkinson's disease: possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen. J Neural Transm. 2003;110: 517–536. doi:10.1007/s00702-002-0808-2

1569 17. Bae E-J, Yang N-Y, Song M, Lee CS, Lee JS, Jung BC, et al. Glucocerebrosidase depletion enhances cell-to-cell transmission of α-synuclein. Nat Commun. 2014;5: doi:10.1038/ncomms5755. doi:10.1038/ncomms5755

1572 18. Domert J, Sackmann C, Severinsson E, Agholme L, Bergström J, Ingelsson M, et al. Aggregated Alpha-Synuclein Transfer Efficiently between Cultured Human Neuron-Like Cells and Localize to Lysosomes. Witt SN, editor. PLOS ONE. 2016;11: e0168700. doi:10.1371/journal.pone.0168700

1576 19. Abounit S, Bousset L, Loria F, Zhu S, Chaumont F, Pieri L, et al. Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes. EMBO J. 2016;35: 2120–2138. doi:10.15252/embj.201593411

1579 20. Dilsizoglu Senol A, Pepe A, Grudina C, Sassoon N, Reiko U, Bousset L, et al. Effect of tolytoxin on tunneling nanotube formation and function. Sci Rep. 2019;9: 5741. doi:10.1038/s41598-019-42161-6

1582 21. Loria F, Vargas JY, Bousset L, Syan S, Salles A, Melki R, et al. α-Synuclein transfer between neurons and astrocytes indicates that astrocytes play a role in degradation rather than in spreading. Acta Neuropathol (Berl). 2017;134: 789–808. doi:10.1007/s00401-017-1746-2

1586 22. Vargas JY, Loria F, Wu Y, Córdova G, Nonaka T, Bellow S, et al. The Wnt/Ca²⁺ pathway is involved in interneuronal communication mediated by tunneling nanotubes. EMBO J. 2019;38: e101230. doi:10.15252/embj.2018101230

1589 23. Desplats P, Lee H-J, Bae E-J, Patrick C, Rockenstein E, Crews L, et al. Inclusion formation and neuronal cell death through neuron-to-neuron transmission of -synuclein. Proc Natl Acad Sci. 2009;106: 13010–13015. doi:10.1073/pnas.0903691106

1592 24. Luk KC, Kehm V, Carroll J, Zhang B, O'Brien P, Trojanowski JQ, et al. Pathological -Synuclein Transmission Initiates Parkinson-like Neurodegeneration in Nontransgenic Mice. Science. 2012;338: 949–953. doi:10.1126/science.1227157
1595 25. Recasens A, Dehay B, Bové J, Carballo-Carbajal I, Dovero S, Pérez-Villalba A, et al. Lewy body extracts from Parkinson disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys: LB-Induced Pathology. Ann Neurol. 2014;75: 351–362. doi:10.1002/ana.24066

1599 26. Kordower JH, Chu Y, Hauser RA, Freeman TB, Olanow CW. Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease. Nat Med. 2008;14: 504–506. doi:10.1038/nm1747

1602 27. Masuda-Suzukake M, Nonaka T, Hosokawa M, Kubo M, Shimozawa A, Akiyama H, et al. Pathological alpha-synuclein propagates through neural networks. 2014;2: http://www.actaneurocomms.org/content/2/1/88.

1605 28. Ayers JI, Brooks MM, Rutherford NJ, Howard JK, Sorrentino ZA, Riffe CJ, et al. Robust Central Nervous System Pathology in Transgenic Mice following Peripheral Injection of α-Synuclein Fibrils. J Virol. 2017;91: e02095-16.

1608 29. Brundin P, Melki R, Kopito R. Prion-like transmission of protein aggregates in neurodegenerative diseases. Nat Rev Mol Cell Biol. 2010;11: 301–307. doi:10.1038/nrm2873

1611 30. Masuda-Suzukake M, Nonaka T, Hosokawa M, Oikawa T, Arai T, Akiyama H, et al. Prion-like spreading of pathological α-synuclein in brain. Brain. 2013;136: 1128–1138. doi:10.1093/brain/awt037

1614 31. Rey NL, Wesson DW, Brundin P. The olfactory bulb as the entry site for prion-like propagation in neurodegenerative diseases. Neurobiol Dis. 2018;109: 226–248. doi:10.1016/j.nbd.2016.12.013

1617 32. Victoria GS, Zurzolo C. The spread of prion-like proteins by lysosomes and tunneling nanotubes: Implications for neurodegenerative diseases. J Cell Biol. 2017;216: 2633–2644. doi:10.1083/jcb.201701047

1620 33. Angot E, Steiner JA, Lema Tomé CM, Ekström P, Mattsson B, Björklund A, et al. Alpha-Synuclein Cell-to-Cell Transfer and Seeding in Grafted Dopaminergic Neurons In Vivo. 1621 Mosley RL, editor. PLoS ONE. 2012;7: e39465. doi:10.1371/journal.pone.0039465

1623 34. Hansen C, Angot E, Bergström A-L, Steiner JA, Pieri L, Paul G, et al. α-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells. J Clin Invest. 2011;121: 715–725. doi:10.1172/JCI43366

1626 35. Vargas JY, Grudina C, Zurzolo C. The prion-like spreading of α-synuclein: From in vitro to in vivo models of Parkinson's disease. Ageing Res Rev. 2019;50: 89–101. doi:10.1016/j.arr.2019.01.012

1629 36. Dieriks BV, Park TI-H, Fourie C, Faull RLM, Dragunow M, Curtis MA. α-synuclein transfer through tunneling nanotubes occurs in SH-SY5Y cells and primary brain pericytes from Parkinson's disease patients. Sci Rep. 2017;7: 42984. doi:10.1038/srep42984
1632 37. Rustom A. Nanotubular Highways for Intercellular Organelle Transport. Science.
1633 2004;303: 1007-1010. doi:10.1126/science.1093133

1634 38. Abounit S, Zurzolo C. Wiring through tunneling nanotubes - from electrical signals to organelle transfer. J Cell Sci. 2012;125: 1089-1098. doi:10.1242/jcs.083279

1636 39. Marzo L, Gousset K, Zurzolo C. Multifaceted Roles of Tunneling Nanotubes in Intercellular Communication. Front Physiol. 2012;3: doi: 10.3389/fphys.2012.00072.
1637

1638 doi:10.3389/fphys.2012.00072
1639 40. Gerdes H-H, Bukoreshtliev NV, Barroso JFV. Tunneling nanotubes: A new route for the exchange of components between animal cells. FEBS Lett. 2007;581: 2194-2201.
1640

1641 doi:10.1016/j.febslet.2007.03.071
1642 41. Cordero Cervantes D, Zurzolo C. Peering into tunneling nanotubes—The path forward.
1643 EMBO J. 2021 [cited 29 Mar 2021]. doi:10.15252/embj.2020105789

1644 42. Chinnery HR, Pearlman E, McMenamin PG. Cutting Edge: Membrane Nanotubes In Vivo: A Feature of MHC Class II + Cells in the Mouse Cornea. J Immunol. 2008;180: 5779-5783. doi:10.4049/jimmunol.180.9.5779

1647 43. Lou E, Fujisawa S, Morozov A, Barlas A, Romin Y, Dogan Y, et al. Tunneling Nanotubes Provide a Unique Conduit for Intercellular Transfer of Cellular Contents in Human Malignant Pleural Mesothelioma. Yang P-C, editor. PLoS ONE. 2012;7: e33093.
1648
1649
1650 doi:10.1371/journal.pone.0033093

1651 44. Pasquier J, Guerrouahen BS, Al Thawadi H, Ghiabi P, Maleki M, Abu-Kaoud N, et al. Preferential transfer of mitochondria from endothelial to cancer cells through tunneling nanotubes modulates chemoresistance. J Transl Med. 2013;11: 94.
1652
1653
1654 doi:10.1186/1479-5876-11-94

1655 45. Osswald M, Jung E, Sahm F, Solecki G, Venkataramani V, Blaes J, et al. Brain tumour cells interconnect to a functional and resistant network. Nature. 2015;528: 93-98.
1656
1657 doi:10.1038/nature16071

1658 46. Seyed-Razavi Y, Hickey MJ, Kuffová L, McMenamin PG, Chinnery HR. Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction. Immunol Cell Biol. 2013;91: 89-95. doi:10.1038/icb.2012.52

1661 47. Alarcon-Martinez L, Villafranca-Baughman D, Quintero H, Kacerovsky JB, Dotigny F, Murai KK, et al. Interpericyte tunnelling nanotubes regulate neurovascular coupling. Nature. 2020;585: 91-95. doi:10.1038/s41586-020-2589-x

1664 48. Sartori-Rupp A, Cordero Cervantes D, Pepe A, Gousset K, Delage E, Corroyer-Dulmont S, et al. Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells. Nat Commun. 2019;10: https://doi.org/10.1038/s41467-018-08178-7.
1665
1666
1667 doi:10.1038/s41467-018-08178-7
1668 49. Zurzolo C. Tunneling nanotubes: Reshaping connectivity. Curr Opin Cell Biol. 2021;71: 139-147. doi:10.1016/j.ceb.2021.03.003

1670 50. Gousset K, Schiff E, Langevin C, Marijanovic Z, Caputo A, Browman DT, et al. Prions hijack tunnelling nanotubes for intercellular spread. Nat Cell Biol. 2009;11: 328-336. doi:10.1038/ncb1841

1673 51. Victoria GS, Arkhipenko A, Zhu S, Syan S, Zurzolo C. Astrocyte-to-neuron intercellular prion transfer is mediated by cell-cell contact. Sci Rep. 2016;6: 20762. doi:10.1038/srep20762

1676 52. Abounit S, Wu JW, Duff K, Victoria GS, Zurzolo C. Tunneling nanotubes: A possible highway in the spreading of tau and other prion-like proteins in neurodegenerative diseases. Prion. 2016;10: 344-351. doi:10.1080/19336896.2016.1223003

1679 53. Costanzo M, Abounit S, Marzo L, Danckaert A, Chamoun Z, Roux P, et al. Transfer of polyglutamine aggregates in neuronal cells occurs in tunneling nanotubes. J Cell Sci. 2013;126: 3678-3685. doi:10.1242/jcs.126086

1682 54. Tardivel M, Bégard S, Bousset L, Dujardin S, Coens A, Melki R, et al. Tunneling nanotube (TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies. Acta Neuropathol Commun. 2016;4: 117. doi:10.1186/s40478-016-0386-4

1685 55. Chastagner P, Loria F, Vargas JY, Tois J, I Diamond M, Okafo G, et al. Fate and propagation of endogenously formed Tau aggregates in neuronal cells. EMBO Mol Med. 2020;12. doi:10.15252/emmm.202012025

1688 56. Grudina C, Kouroupi G, Nonaka T, Hasegawa M, Matsas R, Zurzolo C. Human NPCs can degrade α-syn fibrils and transfer them preferentially in a cell contact-dependent manner possibly through TNT-like structures. Neurobiol Dis. 2019;132: 104609. doi:10.1016/j.nbd.2019.104609

1692 57. Luzio JP, Parkinson MDJ, Gray SR, Bright NA. The delivery of endocytosed cargo to lysosomes. Biochem Soc Trans. 2009;37: 1019-1021. doi:10.1042/BST0371019

1694 58. Luzio JP, Pryor PR, Bright NA. Lysosomes: fusion and function. Nat Rev Mol Cell Biol. 2007;8: 622-632. doi:10.1038/nrm2217

1696 59. Singh R, Cuervo AM. Autophagy in the Cellular Energetic Balance. Cell Metab. 2011;13: 495-504. doi:10.1016/j.cmet.2011.04.004

1698 60. Rabinowitz JD, White E. Autophagy and Metabolism. Science. 2010;330: 1344-1348. doi:10.1126/science.1193497

1700 61. Settembre C, Fraldi A, Medina DL, Ballabio A. Signals from the lysosome: a control centre for cellular clearance and energy metabolism. Nat Rev Mol Cell Biol. 2013;14: 283-296. doi:10.1038/nrm3565
1703 62. Settembre C, Ballabio A. Lysosomal Adaptation: How the Lysosome Responds to External Cues. Cold Spring Harb Perspect Biol. 2014;6: a016907-a016907.
doi:10.1101/cshperspect.a016907

1706 63. Lim C-Y, Zoncu R. The lysosome as a command-and-control center for cellular metabolism. J Cell Biol. 2016;214: 653-664. doi:10.1083/jcb.201607005

1708 64. Perera RM, Zoncu R. The Lysosome as a Regulatory Hub. Annu Rev Cell Dev Biol. 2016;32: 223-253. doi:10.1146/annurev-cellbio-111315-125125

1710 65. Shin HR, Zoncu R. The Lysosome at the Intersection of Cellular Growth and Destruction. Dev Cell. 2020;54: 226-238. doi:10.1016/j.devcel.2020.06.010

1712 66. Fraldi A, Klein AD, Medina DL, Settembre C. Brain Disorders Due to Lysosomal Dysfunction. Annu Rev Neurosci. 2016;39: 277-295. doi:10.1146/annurev-neuro-070815-014031

1715 67. Chu Y, Dodiya H, Aebischer P, Olanow CW, Kordower JH. Alterations in lysosomal and proteasomal markers in Parkinson's disease: Relationship to alpha-synuclein inclusions. Neurobiol Dis. 2009;35: 385-398. doi:10.1016/j.nbd.2009.05.023

1718 68. Dehay B, Bove J, Rodriguez-Muela N, Perier C, Recasens A, Boya P, et al. Pathogenic Lysosomal Depletion in Parkinson's Disease. J Neurosci. 2010;30: 12535-12544.
doi:10.1523/JNEUROSCI.1920-10.2010

1721 69. Mazzulli JR, Zunke F, Isacson O, Studer L, Krainc D. α-Synuclein-induced lysosomal dysfunction occurs through disruptions in protein trafficking in human midbrain synucleinopathy models. Proc Natl Acad Sci. 2016;113: 1931-1936.
doi:10.1073/pnas.1520335113

1725 70. Hoffmann A-C, Minakaki G, Menges S, Salvi R, Savitskiy S, Kazman A, et al. Extracellular aggregated alpha synuclein primarily triggers lysosomal dysfunction in neural cells prevented by trehalose. Sci Rep. 2019;9: 544. doi:10.1038/s41598-018-35811-8

1728 71. Mazzulli JR, Xu Y-H, Sun Y, Knight AL, McLean PJ, Caldwell GA, et al. Gaucher Disease Glucocerebrosidase and α-Synuclein Form a Bidirectional Pathogenic Loop in Synucleinopathies. Cell. 2011;146: 37-52. doi:10.1016/j.cell.2011.06.001

1731 72. Boya P. Lysosomal Function and Dysfunction: Mechanism and Disease. Antioxid Redox Signal. 2012;17: 766-774. doi:10.1089/ars.2011.4405

1733 73. Freeman D, Cedillos R, Choyke S, Lukic Z, McGuire K, Marvin S, et al. Alpha-Synuclein Induces Lysosomal Rupture and Cathepsin Dependent Reactive Oxygen Species Following Endocytosis. Kahle PJ, editor. PLoS ONE. 2013;8: e62143.
doi:10.1371/journal.pone.0062143

1737 74. Flavin WP, Bousset L, Green ZC, Chu Y, Skarpathiotis S, Chaney MJ, et al. Endocytic vesicle rupture is a conserved mechanism of cellular invasion by amyloid proteins. Acta Neuropathol (Berl). 2017;134: 629-653. doi:10.1007/s00401-017-1722-x
1740 75. Jongsma ML, Bakker J, Cabukusta B, Liv N, Elsland D, Fermie J, et al. SKIP - HOPS recruits
1741 TBC 1D15 for a Rab7-to-Arl8b identity switch to control late endosome transport.
1742 EMBO J. 2020;39. doi:10.15252/embj.2019102301

1743 76. Fermie J, Liv N, ten Brink C, van Donselaar EG, Müller WH, Schieber NL, et al. Single
1744 organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D
electron microscopy. Traffic. 2018;19: 354-369. doi:10.1111/tra.12557

1746 77. Meel E, Klumperman J. Imaging and imagination: understanding the endo-lysosomal
1747 system. Histochem Cell Biol. 2008;129: 253-266. doi:10.1007/s00418-008-0384-0

1748 78. Klumperman J, Raposo G. The Complex Ultrastructure of the Endolysosomal System.
1749 Cold Spring Harb Perspect Biol. 2014;6: a016857-a016857.
1750 doi:10.1101/cshperspect.a016857

1751 79. Mohammadian S, Fokkema J, Agronskaia AV, Liv N, de Heus C, van Donselaar E, et al.
1752 High accuracy, fiducial marker-based image registration of correlative microscopy
images. Sci Rep. 2019;9: 3211. doi:10.1038/s41598-019-40098-4

1754 80. Cortese K, Vicidomini G, Gagliani MC, Boccacci P, Diaspro A, Tacchetti C. High Data
1755 Output Method for 3-D Correlative Light-Electron Microscopy Using Ultrathin
1756 Cryosections. In: Sousa AA, Kruhlak MJ, editors. Nanoimaging. Totowa, NJ: Humana
1757 Press; 2013. pp. 417-437. doi:10.1007/978-1-62703-137-0_23

1758 81. Calafate S, Flavin W, Verstreken P, Moechars D. Loss of Bin1 Promotes the Propagation
1759 of Tau Pathology. Cell Rep. 2016;17: 931-940. doi:10.1016/j.celrep.2016.09.063

1760 82. Papadopoulos C, Kirchner P, Bug M, Grum D, Koerver L, Schulze N, et al. VCP /p97
1761 cooperates with YOD 1, UBXD 1 and PLAA to drive clearance of ruptured lysosomes by
autophagy. EMBO J. 2017;36: 135-150. doi:10.15252/embj.201695148

1763 83. Oku Y, Murakami K, Irie K, Hoseki J, Sakai Y. Synthesized Aβ42 Caused Intracellular
1764 Oxidative Damage, Leading to Cell Death, via Lysosome Rupture. Cell Struct Funct.
1765 2017;42: 71-79. doi:10.1247/csf.17006

1766 84. Maejima I, Takahashi A, Omori H, Kimura T, Takabatake Y, Saitoh T, et al. Autophagy
1767 sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury.
EMBO J. 2013;32: 2336-2347. doi:10.1038/emboj.2013.171

1769 85. Jiang P, Gan M, Yen S-H, McLean PJ, Dickson DW. Impaired endo-lysosomal membrane
1770 integrity accelerates the seeding progression of α-synuclein aggregates. Sci Rep.
1771 2017;7: 7690. doi:10.1038/s41598-017-08149-w

1772 86. Nonaka T, Watanabe ST, Iwatsubo T, Hasegawa M. Seeded Aggregation and Toxicity of
1773 α-Synuclein and Tau: CELLULAR MODELS OF NEURODEGENERATIVE DISEASES. J Biol
1774 Chem. 2010;285: 34885-34898. doi:10.1074/jbc.M110.148460
1775 87. Shrivastava AN, Bousset L, Renner M, Redeker V, Savistchenko J, Triller A, et al.
1776 Differential Membrane Binding and Seeding of Distinct α-Synuclein Fibrillar
1777 Polymorphs. Biophys J. 2020;118: 1301–1320. doi:10.1016/j.bpj.2020.01.022

1778 88. Yang X, Williams JK, Yan R, Mouradian MM, Baum J. Increased Dynamics of α-Synuclein
1779 Fibrils by β-Synuclein Leads to Reduced Seeding and Cytotoxicity. Sci Rep. 2019;9:
1780 17579. doi:10.1038/s41598-019-54063-8

1781 89. Sangwan S, Sahay S, Murray KA, Morgan S, Guenther EL, Jiang L, et al. Inhibition of
1782 synucleinopathic seeding by rationally designed inhibitors. eLife. 2020;9: e46775.
1783 doi:10.7554/eLife.46775

1784 90. Guardia CM, Farías GG, Jia R, Pu J, Bonifacino JS. BORC Functions Upstream of Kinesins
1785 1 and 3 to Coordinate Regional Movement of Lysosomes along Different Microtubule
1786 Tracks. Cell Rep. 2016;17: 1950–1961. doi:10.1016/j.celrep.2016.10.062

1787 91. Pu J, Guardia CM, Keren-Kaplan T, Bonifacino JS. Mechanisms and functions of
1788 lysosome positioning. J Cell Sci. 2016;129: 4329–4339. doi:10.1242/jcs.196287

1789 92. Cabukusta B, Neefjes J. Mechanisms of lysosomal positioning and movement. Traffic.
1790 2018;19: 761–769. doi:10.1111/tra.12587

1791 93. Jongsma MLM, Berlin I, Wijdeven RHM, Janssen L, Janssen GMC, Garstka MA, et al. An
1792 ER-Associated Pathway Defines Endosomal Architecture for Controlled Cargo
1793 Transport. Cell. 2016;166: 152–166. doi:10.1016/j.cell.2016.05.078

1794 94. Bajaj L, Lotfi P, Pal R, Ronza A di, Sharma J, Sardiello M. Lysosome biogenesis in health
1795 and disease. J Neurochem. 2019;148: 573–589. doi:10.1111/jnc.14564

1796 95. Medina DL, Fraldi A, Bouche V, Annunziata F, Mansueto G, Spampanato C, et al.
1797 Transcriptional Activation of Lysosomal Exocytosis Promotes Cellular Clearance. Dev
1798 Cell. 2011;21: 421–430. doi:10.1016/j.devcel.2011.07.016

1799 96. Willett R, Martina JA, Zewe JP, Wills R, Hammond GRV, Puertollano R. TFEB regulates
1800 lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to
1801 lysosomes. Nat Commun. 2017;8: 1580. doi:10.1038/s41467-017-01871-z

1802 97. Zhang J, Johnson JL, He J, Napolitano G, Ramadass M, Rocca C, et al. Cystinosin, the
1803 small GTPase Rab11, and the Rab7 effector RILP regulate intracellular trafficking of the
1804 chaperone-mediated autophagy receptor LAMP2A. J Biol Chem. 2017;292: 10328–
1805 10346. doi:10.1074/jbc.M116.764076

1806 98. Hofmann I, Munro S. An N-terminally acetylated Arf-like GTPase is localised to
1807 lysosomes and affects their motility. J Cell Sci. 2006;119: 1494–1503.
1808 doi:10.1242/jcs.02958

1809 99. Bagshaw RD, Callahan JW, Mahuran DJ. The Arf-family protein, Arl8b, is involved in the
1810 spatial distribution of lysosomes. Biochem Biophys Res Commun. 2006;344: 1186–
1811 1191. doi:10.1016/j.bbrc.2006.03.221
1812 100. Pu J, Schindler C, Jia R, Jarnik M, Backlund P, Bonifacino JS. BORC, a Multisubunit Complex that Regulates Lysosome Positioning. Dev Cell. 2015;33: 176-188. doi:10.1016/j.devcel.2015.02.011

1815 101. Nakata T, Hirokawa N. Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport. J Cell Biol. 1995;131: 1039-1053. doi:10.1083/jcb.131.4.1039

1818 102. Rosa-Ferreira C, Munro S. Arl8 and SKIP Act Together to Link Lysosomes to Kinesin-1. Dev Cell. 2011;21: 1171-1178. doi:10.1016/j.devcel.2011.10.007

1820 103. Korolchuk VI, Saiki S, Lichtenberg M, Siddiqi FH, Roberts EA, Imarisio S, et al. Lysosomal positioning coordinates cellular nutrient responses. Nat Cell Biol. 2011;13: 453-460. doi:10.1038/ncb2204

1823 104. Matsushita M, Tanaka S, Nakamura N, Inoue H, Kanazawa H. A Novel Kinesin-Like Protein, KIF1Bβ3 Is Involved in the Movement of Lysosomes to the Cell Periphery in Non-Neuronal Cells: Novel Kinesin Motor for Lysosomal Movement. Traffic. 2004;5: 140-151. doi:10.1111/j.1600-0854.2003.00165.x

1827 105. Bentley M, Decker H, Luisi J, Banker G. A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations. J Cell Biol. 2015;208: 273-281. doi:10.1083/jcb.201408056

1830 106. Naphade S, Sharma J, Gaide Chevronnay HP, Shook MA, Yeagy BA, Rocca CJ, et al. Brief Reports: Lysosomal Cross-Correction by Hematopoietic Stem Cell-Derived Macrophages Via Tunneling Nanotubes: Lysosomal Cross-Correction by Stem Cells Via TNTs. STEM CELLS. 2015;33: 301-309. doi:10.1002/stem.1835

1834 107. Del Tredici K, Braak H. Review: Sporadic Parkinson's disease: development and distribution of α-synuclein pathology. Neuropathol Appl Neurobiol. 2016;42: 33-50. doi:10.1111/nan.12298

1837 108. Usenovic M, Tresse E, Mazzulli JR, Taylor JP, Krainc D. Deficiency of ATP13A2 Leads to Lysosomal Dysfunction, -Synuclein Accumulation, and Neurotoxicity. J Neurosci. 2012;32: 4240-4246. doi:10.1523/JNEUROSCI.5575-11.2012

1840 109. Chen PM, Gombart ZJ, Chen JW. Chloroquine treatment of ARPE-19 cells leads to lysosome dilation and intracellular lipid accumulation: possible implications of lysosomal dysfunction in macular degeneration. Cell Biosci. 2011;1: 10. doi:10.1186/2045-3701-1-10

1844 110. Boya P, Kroemer G. Lysosomal membrane permeabilization in cell death. Oncogene. 2008;27: 6434-6451. doi:10.1038/onc.2008.310

1846 111. Radulovic M, Schink KO, Wenzel EM, Nähse V, Bongiovanni A, Lafont F, et al. ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival. EMBO J. 2018;37. doi:10.15252/embj.201899753
1849 112. Skowyra ML, Schlesinger PH, Naismith TV, Hanson PI. Triggered recruitment of ESCRT machinery promotes endolysosomal repair. Science. 2018;360: eaar5078. doi:10.1126/science.aar5078

1852 113. Lynch-Day MA, Mao K, Wang K, Zhao M, Klionsky DJ. The Role of Autophagy in Parkinson's Disease. Cold Spring Harb Perspect Med. 2012;2: a009357-a009357. doi:10.1101/cshperspect.a009357

1855 114. Cui L, Zhao L-P, Ye J-Y, Yang L, Huang Y, Jiang X-P, et al. The Lysosomal Membrane Protein Lamp2 Alleviates Lysosomal Cell Death by Promoting Autophagic Flux in Ischemic Cardiomyocytes. Front Cell Dev Biol. 2020;8: 31. doi:10.3389/fcell.2020.00031

1859 115. Lashuel HA, Petre BM, Wall J, Simon M, Nowak RJ, Walz T, et al. α-Synuclein, Especially the Parkinson's Disease-associated Mutants, Forms Pore-like Annular and Tubular Protofibrils. J Mol Biol. 2002;322: 1089-1102. doi:10.1016/S0022-2836(02)00735-0

1862 116. Tosatto L, Andrighetti AO, Plotegher N, Antonini V, Tessari I, Ricci L, et al. Alpha-synuclein pore forming activity upon membrane association. Biochim Biophys Acta BBA - Biomembr. 2012;1818: 2876-2883. doi:10.1016/j.bbamem.2012.07.007

1865 117. Volles MJ, Lansbury PT. Vesicle Permeabilization by Protofibrillar α-Synuclein Is Sensitive to Parkinson's Disease-Linked Mutations and Occurs by a Pore-like Mechanism†. Biochemistry. 2002;41: 4595-4602. doi:10.1021/bi0121353

1868 118. Butler D, Bahr BA. Oxidative Stress and Lysosomes: CNS-Related Consequences and Implications for Lysosomal Enhancement Strategies and Induction of Autophagy. Antioxid Redox Signal. 2006;8: 185-196. doi:10.1089/ars.2006.8.185

1871 119. Perier C, Tieu K, Guegan C, Caspersen C, Jackson-Lewis V, Carelli V, et al. Complex I deficiency primes Bax-dependent neuronal apoptosis through mitochondrial oxidative damage. Proc Natl Acad Sci. 2005;102: 19126-19131. doi:10.1073/pnas.0508215102

1874 120. Bourdenx M, Dehay B. What lysosomes actually tell us about Parkinson's disease? Ageing Res Rev. 2016;32: 140-149. doi:10.1016/j.arr.2016.02.008

1876 121. Trinkaus VA, Riera-Tur I, Martínez-Sánchez A, Guo Q, Arzberger T, Baumeister W, et al. In situ architecture of neuronal α-Synuclein inclusions. : 46.

1878 122. Matteoni R, Kreis TE. Translocation and Clustering of Endosomes and Lysosomes Depends on Micro bules. J Cell Biol. 1987;105: 1253-1265.

1880 123. Nakamura S, Shigeyama S, Minami S, Shima T, Akayama S, Matsuda T, et al. LC3 lipidation is essential for TFEB activation during the lysosomal damage response to kidney injury. Nat Cell Biol. 2020;22: 1252-1263. doi:10.1038/s41556-020-00583-9

1883 124. Decressac M, Mattsson B, Weikop P, Lundblad M, Jakobsson J, Bjorklund A. TFEB-mediated autophagy rescues midbrain dopamine neurons from -synuclein toxicity. Proc Natl Acad Sci. 2013;110: E1817-E1826. doi:10.1073/pnas.1305623110
1886 125. Gousset K, Marzo L, Commere P-H, Zurzolo C. Myo10 is a key regulator of TNT formation in neuronal cells. J Cell Sci. 2013;126: 4424-4435. doi:10.1242/jcs.129239

1888 126. Wang Y, Cui J, Sun X, Zhang Y. Tunneling-nanotube development in astrocytes depends on p53 activation. Cell Death Differ. 2011;18: 732-742. doi:10.1038/cdd.2010.147

1890 127. Shi Z, Sachs J, Rhoades E, Baumgart T. Biophysics of α-synuclein induced membrane remodelling. 2015; 19.

1892 128. Qi Y, Wang JKT, McMillian M, Chikaraishi DM. Characterization of a CNS Cell Line, CAD, in which Morphological Differentiation Is Initiated by Serum Deprivation. J Neurosci. 1997;17: 1217-1225. doi:10.1523/JNEUROSCI.17-04-01217.1997

1895 129. Abounit S, Delage E, Zurzolo C. Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking. Curr Protoc Cell Biol. 2015;67: doi: 10.1002/0471143030.cb1210s67. doi:10.1002/0471143030.cb1210s67

1898 130. Zhu S, Abounit S, Korth C, Zurzolo C. Transfer of disrupted-in-schizophrenia 1 aggregates between neuronal-like cells occurs in tunnelling nanotubes and is promoted by dopamine. Open Biol. 2017;7: 160328. doi:10.1098/rsob.160328

1901 131. Delage E, Cervantes DC, Pénard E, Schmitt C, Syan S, Disanza A, et al. Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes. Sci Rep. 2016;6: 39632. doi:10.1038/srep39632

1904 132. Jia R, Guardia CM, Pu J, Chen Y, Bonifacino JS. BORC coordinates encounter and fusion of lysosomes with autophagosomes. Autophagy. 2017;13: 1648-1663. doi:10.1080/15548627.2017.1343768

1907 133. Kowarz E, Löscher D, Marschalek R. Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines. Biotechnol J. 2015;10: 647-653. doi:10.1002/biot.201400821

1910 134. Mátés L, Chuah MKL, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, et al. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009;41: 753-761. doi:10.1038/ng.343

1913 135. Nonaka T, Iwatsubo T, Hasegawa M. Ubiquitination of α-Synuclein †. Biochemistry. 2005;44: 361-368. doi:10.1021/bi0485528

1915 136. Boulanger J, Kervrann C, Bouthemy P, Elbau P, Sibarita J-B, Salamero J. Patch-Based Nonlocal Functional for Denoising Fluorescence Microscopy Image Sequences. IEEE Trans Med Imaging. 2010;29: 442-454. doi:10.1109/TMI.2009.2033991

1918 137. Oorschot V, de Wit H, Annaert WG, Klumperman J. A Novel Flat-embedding Method to Prepare Ultrathin Cryosections from Cultured Cells in Their In Situ Orientation. J Histochem Cytochem. 2002;50: 1067-1080. doi:10.1177/002215540205000809

1921 138. Slot JW, Geuze HJ. Cryosectioning and immunolabeling. Nat Protoc. 2007;2: 2480-2491. doi:10.1038/nprot.2007.365
1923 **Supporting information**

1924 **S1 Fig.** EM images of control CAD cells. (A) Resin embedded EM images of control CAD cells. Three examples of control lysosomes (indicated by blue squares) selected from different regions (indicated by red squares) of three different cells are presented. Scale bar for the cells: 5 µm, for the selected regions: 1 µm, and for lysosomes: 0.2 µm. (B) On-section EM images of control CAD cells immunogold labelled with LAMP1¹⁰. Two examples of lysosomes were presented in insets selected from regions indicated by red and blue squares. Scale bar: 1 µm (for the insets: 0.2 µm).

1931

1932 **S2 Fig.** α-Syn fibrils are transferred from donor to acceptor cells inside of lysosomes through TNTs. Representative image of a TNT having a lysosome containing α-syn fibrils which is formed between α-syn loaded donor cell for 18 h (D) and H2B-GFP transfected acceptor cell (A) in 24 h of CAD-CAD co-culture. Z projection of donor and acceptor cells and lysosomes labelled with LAMP1 is presented (upper panels). Z projection of merged image with an additional staining of HCS CellMask™ Blue, a bottom section (section no:3) where TNT is not visible and an upper section (section no:8) where TNT is visible are presented (middle panels). Arrows in the orthogonal view of the upper section and in insets are indicating the lysosome containing α-syn fibrils inside of the TNT (lower panels). Scale bar: 10 µm (for insets: 5 µm).

1942

1943 **S3 Fig.** Schematic presentation of experimental designs. (A) CAD cells were transiently transfected with Gal3-GFP (1), α-syn-GFP (2), or TFEB-WT-GFP (3) and treated with Alexa 568-tagged α-syn fibrils for 18 h; cells were then analysed for the presence of Gal3-GFP puncta, α-syn-GFP puncta, or nuclear TFEB respectively. (B) Donor CAD cells were transiently transfected with LAMP1-GFP and either treated with Alexa 568-tagged α-syn fibrils for 18 h (co-culture prepared from these donors was referred to as “α-syn co-culture”) or left untreated (co-culture prepared from these donors was referred to as “control co-culture”). Donor cells were then co-cultured with acceptor CAD cells transiently transfected with H2B-mCherry for 24 h. Efficiency of the LAMP1-GFP+ lysosome transfer was measured in each condition. (C) Donor CAD cells were transiently transfected with Arl8b-GFP (co-culture prepared from these donors was referred to as “Arl8b co-culture”) or not (co-culture prepared from these donors was referred to as “control co-culture”) prior to be loaded with Alexa 568-
tagged α-syn fibrils for 18 h. Arl8b-GFP expressing donors (having more peripheral lysosomes) and control donors were then co-cultured with acceptor CAD cells that were transiently transfected with H2B-GFP for 24 h. Efficiency of the α-syn fibrils' transfer was measured in each condition. (D) Donor HeLa cells were transiently transfected with Arl8b-GFP (co-culture prepared from these donors was referred to as "Arl8b co-culture") or not transfected (co-culture prepared from these donors was referred to as "control co-culture") prior to be loaded with Alexa 568-tagged α-syn fibrils for 18 h. Arl8b-GFP expressing donors (having more peripheral lysosomes) and control donors were then co-cultured with acceptor HeLa cells that were transiently transfected with H2B-GFP for 24 h. Efficiency of the α-syn fibrils' transfer was measured in each condition. (E) Three donor CAD cell populations were prepared: untreated control cells, cells pretreated with siconrol (scramble) or with siArl8b. Co-cultures were referred to as "control", "siconrol", and "siArl8b" co-cultures to distinguish the treatments applied to the donor cells. Donors having perinuclear lysosomes (siArl8b pretreated) and control donors (siconrol pretreated or untreated) were loaded with Alexa 568-tagged α-syn fibrils for 18 h prior to be co-cultured with acceptor CAD cells transfected with H2B-GFP for 24 h. Efficiency of the α-syn fibrils' transfer was measured in each condition. (F) Donor control cells (WT HeLa) or myrlysin-KO HeLa cells or KIF1B-5B dKO HeLa cells were treated with Alexa 568-tagged α-syn fibrils for 18 h. Control donors (WT HeLa) and donors having perinuclear lysosomes (myrlysin-KO and KIF1B-5B dKO HeLa cells) were then co-cultured with the acceptor WT HeLa cells transiently transfected with H2B-GFP for 24 h. Co-cultures were referred to as "control", "myrlysin-KO", and "KIF1B-5B dKO" co-cultures to distinguish the treatments applied to the donor cells. Efficiency of the α-syn fibrils' transfer was measured in each condition. (G) Donor CAD cells were either transfected with H2B-mCherry (co-culture prepared from these donors was referred to as "control co-culture") or treated with Alexa 568-tagged α-syn fibrils for 18 h (co-culture prepared from these donors was referred to as "α-syn co-culture") were co-cultured with acceptor CAD cells were either transiently transfected with Gal3-GFP (1) or α-syn-GFP (2). Donor and acceptor cells were then co-cultured for 24 h. Acceptors cells were analysed for the presence of Gal3-GFP or α-syn-GFP puncta, respectively. (H) Donor CAD cells transiently transfected with Gal3-GFP and treated with Alexa 568-tagged α-syn fibrils for 18 h were co-cultured with acceptor HeLa cells stably expressing Gal3-Turquoise for 24 h. Co-cultures were either immunolabelled with LAMP1 (mouse) antibody or LAMP1 (human) antibody to detect lysosomes derived from donor and acceptor cells, respectively. (I) Secretion test: in parallel to co-culture preparations where donor and acceptor cells were mixed in 1:1 ratio, donors and acceptor cells were also plated separately and medium
1989 of acceptor cells were replaced by the conditioned medium collected from donor cells. Acceptor
1990 cells were then analysed for the presence of different cargos (e.g., α-syn fibrils, lysosomes) in
1991 order to evaluate cargo transfer from donor to acceptor cells through secretion.

1992
1993 **S4 Fig. α-Syn fibrils do not induce lysophagy.** Representative confocal images of CAD cells control, treated with 1 mM LLOMe for 3 h, or treated with Alexa 568-tagged α-syn fibrils for 18 h and immunolabelled with LC3-Alexa 488, Ubiquitin FK2-Alexa 647, and Lamp1-CF™405M (pseudo colored in grey) antibodies. Light blue arrows indicate lysosomes under lysophagy. n=3 (60 cells analysed per condition). Scale bar: 10 µm.
1994
1995
1996
1997

1998
1999 **S5 Fig. Timeframes of the S1 and S2 Movies of CAD cells overexpressing soluble α-syn-GFP treated or not with Alexa 568-tagged α-syn fibrils up to 4 h.** (A) Timeframes at 0/1/2/3/4 hours of the S1 Movie of CAD cells overexpressing soluble α-syn-GFP monitored after the administration of α-syn fibrils. (B) Timeframes at 0/1/2/3/4 hours of the S2 Movie of CAD cells overexpressing soluble α-syn-GFP.
2000
2001
2002
2003
2004

2005 **S6 Fig. Lysosome positioning affects the efficiency of α-syn fibrils' transfer in HeLa cells.**
2006 (A) Representative confocal images of donor and acceptor HeLa cells after 24 h of co-culture. Donor cells were either treated with Alexa 568-tagged α-syn fibrils for 18 h (referred as "control co-culture") or they were transfected with Arl8b-GFP prior to be treated with α-syn fibrils (referred as "Arl8b-GFP co-culture"). Acceptor cells were transfected with H2B-GFP. 24 h later, co-cultures were labelled with HCS CellMask™ Blue and DAPI. Arrows indicate the α-syn fibrils received by acceptor cells and asterisks indicate the cells having α-syn fibrils in each co-culture condition. Scale bar: 10 µm. (B) % of acceptor cells received α-syn fibrils in control co-culture (28 ± 3%), Arl8b-GFP co-culture (60 ± 5%), control secretion (1 ± 1%), and Arl8b-GFP secretion (8 ± 3%) is presented (left graph); average number of α-syn puncta in control co-culture (3 ± 1), Arl8b-GFP co-culture (5 ± 1), control secretion (1 ± 0.0), and in Arl8b-GFP secretion (2 ± 0.2) is presented (right graph). Mean ± S.E.M, n=3 (70 acceptor cells per condition). ns=not significant, ***P=0.0001 by Kruskal-Wallis nonparametric ANOVA test followed by Dunn's multiple comparison tests. (C) Representative confocal images of donor and acceptor HeLa cells after 24 h of co-culture. WT, myrlysin-KO, and KIF1B-5B dKO HeLa cells were loaded with Alexa 568-tagged α-syn fibrils (referred as control, myrlysin-KO, and KIF1B-5B dKO co-cultures, respectively) and co-cultured with acceptor cells transfected with
2022 H2B-GFP. Co-cultures were labelled with HCS CellMask™ Blue and DAPI. Arrows indicate the α-syn fibrils received by acceptor cells and asterisks indicate the cells having α-syn fibrils in each co-culture condition. Scale bar: 10 µm. (D) % of acceptor cells received α-syn fibrils in control co-culture (29 ± 2%), myrlysin-KO co-culture (16 ± 2%), KIF1B-5B dKO co-culture (12 ± 2%), control secretion (5 ± 2%), myrlysin-KO secretion (6 ± 1%), and KIF1B-5B dKO secretion (6 ± 1%) is presented (left graph); average number of α-syn in control co-culture (2 ± 0.3), myrlysin-KO co-culture (2 ± 0.3), KIF1B-5B dKO co-culture (2 ± 0.4), control secretion (1 ± 0.4), myrlysin-KO secretion (1 ± 0.2), and KIF1B-5B dKO secretion (1 ± 0.2) is presented (right graph). Mean ± S.E.M, n=3 (90 acceptor cells per condition). ns=not significant, **P=0.016, ****P<0.0001 by Kruskal-Wallis nonparametric ANOVA test followed by Dunn's multiple comparison tests. The data underlying this Figure may be found in S1 Data.

2033

**S7 Fig. Validation of CAD-HeLa co-culture system with additional information. (A)**
Representative confocal image of donor CAD cells loaded with Alexa 568-tagged α-syn fibrils (18 h) co-cultured with HeLa Gal3-Turquoise acceptor cells for 24 h. In acceptor HeLa cells, arrows indicate α-syn fibrils. Scale bar: 10 µm. (B) % of acceptor HeLa cells received α-syn fibrils in co-culture (65 ± 3%) and in secretion test (7 ± 2%) is presented (left graph). Mean ± S.E.M. ****P<0.0001 by two-tailed t-test. Average number of α-syn fibrils puncta per acceptor cell in co-culture (4 ± 0.2) and in secretion test (2 ± 0.2) is presented (right graph). ns=not significant by Mann-Whitney U-test. (C) Representative confocal image of donor CAD cells loaded with Alexa 568-tagged α-syn fibrils (18 h) co-cultured with acceptor WT HeLa cells transfected with α-syn-GFP for 24 h. Red arrows indicate α-syn fibrils, green arrows indicate α-syn-GFP puncta formation (seeding) in acceptor HeLa cells. Scale bar: 10 µm. (D) Representative confocal image of Gal3-GFP transfected and Alexa 568-tagged α-syn fibril loaded donor CAD cells (18 h) co-cultured with HeLa Gal3-Turquoise cells for 24 h. In donor CAD cell, green arrows indicate Gal3-GFP puncta formation. Scale bar: 10 µm. (E) % of donor CAD cells under LMP in control (2 ± 2%) and in α-syn (11 ± 4%) co-cultures is presented (left graph); average number of Gal3-GFP puncta in donor cells under LMP in control (10 ± 2) and in α-syn (14 ± 4) co-cultures is presented (right graph). Mean ± S.E.M. ns=not significant, by *P=0.04 Mann-Whitney U-test. (F) HeLa Gal3-Turquoise acceptor cell under LMP labelled for LAMP1 human Alexa 647 antibody (pseudo colored in grey). Colocalization between Gal3-Turquoise puncta and LAMP1 (indicated by arrows) is presented in a selected region indicated by the square. Scale bar: 10 µm (for the inset: 1 µm). (G) HeLa Gal3-Turquoise acceptor cell
2055 under LMP labelled with LAMP1 mouse Alexa 647 antibody (pseudo colored in grey).
2056 Colocalization between Gal3-Turquoise puncta and donor's lysosome (white arrows), and
2057 colocalization between α-syn fibrils puncta and donor's lysosome (red arrows) are presented in
2058 a selected region indicated by a square. Scale bar: 10 µm (for the inset: 1 µm). (H)
2059 Representative confocal image of Alexa 568-tagged α-syn fibril-treated donor CAD cells for
2060 18 h co-cultured with HeLa Gal3-Turquoise cells (pseudo colored in yellow) for 24 h. Higher
2061 magnification of a selected region (white square) in donor CAD cell is presented. Arrows
2062 indicate a Gal3-Turquoise puncta detected in a donor CAD cell. "Merge" image is saturated for
2063 the better visualization of the Gal3-Turquoise puncta. Scale bar: 10 µm (for the inset: 2 µm).
2064 (I) % of donor CAD cells received Gal3-Turquoise puncta in control (0.3 ± 0.3%) and in α-syn
2065 (4 ± 1%) co-cultures. Mean ± S.E.M. **P=0.003 by Mann-Whitney U-test. In CAD-HeLa co-
2066 culture model, data was collected from seven independent experiments in which four and three
2067 of them were labelled with anti-LAMP1 mouse and human antibodies, respectively. According
2068 to the experiment, in average 100 acceptor or donor cells were analysed for each condition per
2069 experiment. The data underlying this Figure may be found in S1 Data.

2070

2071 **S1 Movie.** Seeding of α-syn-GFP aggregates in CAD cells overexpressing soluble α-syn-GFP treated with α-syn fibrils up to 4 h. Timeframes of the max projection of 36 slides (step size: 0.20 µm) with a total physical thickness of 7.2 µm, with 5 minutes of interval time. Video was acquired using laser 488 in a Spinning Disk microscope.

2075

2076 **S2 Movie.** Monitoring of CAD cells overexpressing soluble α-syn-GFP up to 4 h.
2077 Timeframes of the max projection of 21 slides (step size: 0.48 µm) with a total physical
2078 thickness of 9.55 µm, with 15 minutes of interval time. Video was acquired using laser 488 in
2079 a Spinning Disk microscope.
<figure><img src="image_3.png" /><figcaption>Figure-1</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure-2</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure-3</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure-4</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Figure-5</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure-6</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Figure-7</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure-8</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Figure-9</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>Figure-10</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>Figure-11</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>Figure S1</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>Figure S2</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>Figure S3</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>Figure S4</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>Figure S5</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>Figure S6</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>Figure S7</figcaption></figure>
## Funding

Association pour la Recherche sur la Sclérose Latérale Amyotrophique et Autres Maladies du Motoneurone (ARSLA 2016), INNOV 39-2019 Flash Start (Institut Pasteur) supported ADS, Access to Correlative Light and Electron Microscopy Flagship Node was supported by Euro-BioImaging grant (EuBI_AYDI109) for ADS,

MS was supported by France Alzheimer (S-FB17026) and the Marie Skłodowska-Curie Action H2020-MSCA-IF-2019 EU project 897378

Agence Nationale de la Recherche (ANR-16-CE16-0019-01), Fondation pour la Recherche Médicale (FRM-2016-DEQ20160334896) and LECMA-Vaincre Alzheimer (2016 / FR-16020) and France Alzheimer (AAP SM 2017#1674) foundations financially supported CZ, Intramural Program of NICHD (project ZIA HD001607) supported JSB, Grant-in-Aids for Scientific Research (JP26117005), CREST, JST (JP18071300) and AMED Brain/MINDS (JP18dm0207019) supported MH, Brain Science Foundation supported TN, NL was supported by the Netherlands Organization for Scientific Research (NWO) through a ZonMW-TOP grant (91216006) to JK.

Agence Nationale de la Recherche (ANR-10-INSB-04), ANR/FBI and the Région Ile-de-France Domaine d'Intérêt Majeur-Malinf program supported the use of structured illumination microscopy (SIM) at the BioImagerie Photonic platform at Institut Pasteur