α-Syn fibrils affect the morphology and function of lysosomes

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

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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 hours) CAD cells, immunolabeled 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 ± SEM, n = 3 (30 cells per condition). Scale bar: 10 μm (for inset: 2 μm). (B) SR images of control (left panel) and Alexa 568–tagged α-syn fibril-treated CAD cells for 18 hours (right panel), immunolabeled for LAMP1 (far-red) antibody (pseudo colored in gray). 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 subgrouped as the following: Lysosomes without α-syn fibrils (0.43 ± 0.01) and lysosomes with α-syn fibrils (0.63 ± 0.01) are presented. Mean ± SEM, lysosomes’ diameters were measured in SR images of 8 α-syn fibril-treated cells and 7 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 multiple comparison tests. (C) FM image of LAMP1-GFP transfected and α-syn fibril-treated CAD cells for 18 hours (left panel). Enlarged merged images of correlative resin EM and FM images of 4 selected lysosomes not containing (number 1, indicated by green square) and containing α-syn fibrils (numbers 2, 3, and 4, indicated by red squares) are presented. Lysosomes number 1 and 2 are further magnified (blue squares) and presented within the same image. 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 immunogold labeled for LAMP1¹⁰ and FM images of LAMP1-GFP transfected and α-syn fibril-treated CAD cells (18 hours). Insets of 3 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) are presented. ns = not significant, ****P < 0.0001 by 1-way ANOVA followed by Tukey 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) are presented. Mean ± SEM (50 cells per condition). ns = not significant, ****P < 0.0001 by Kruskal–Wallis nonparametric ANOVA test followed by Dunn multiple comparison tests. Scale bar: 10 μm (for insets: 0.5 μm). The data underlying this figure may be found in S1 Data. α-syn, α-synuclein; CAD, Cath.a-differentiated; EM, electron microscopy; FM, fluorescence microscopy; LAMP, lysosome-associated membrane protein; LD, lipid droplet; Lyso and lys, lysosome; PM, plasma membrane; SR, super-resolution; TEM, transmission electron microscopy.

https://doi.org/10.1371/journal.pbio.3001287.g001

To further investigate the morphology of compartments containing α-syn fibrils, we used a correlative light-electron microscopy (CLEM) approach to identify α-syn–positive and α-syn–negative lysosomes by fluorescence microscopy (FM) and study their corresponding morphology by electron microscopy (EM). First, we cultured CAD cells transiently transfected with a plasmid encoding LAMP1-GFP and loaded with Alexa 568–tagged α-syn fibrils on gridded coverslips and imaged α-syn fibril-treated cells in FM while marking coordinates of α-syn positive (Alexa568+/GFP+) and negative (Alexa568−/GFP+) LAMP1-GFP lysosomes. Following FM imaging, the CAD cells were prepared for EM by keeping their orientation, and serial sections were collected from the region containing the previously marked coordinates (see also Materials and methods) [75,76]. Then, the FM signal was correlated with EM images. Using this approach, we overcame the lack of suitable tools to identify α-syn fibrils directly by EM and were able to compare the structural properties of lysosomes containing or not 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 analyze 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 immunolabeling (both gold and fluorescent) of endogenous LAMP1 [79] (Fig 1D). Following a recently developed high-throughput on-section CLEM approach [80], we analyzed 50 LAMP1-immunolabeled lysosomes in control cells (S1B Fig) and 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).

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 arbitrary unit [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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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 hours (218 ± 7) is presented. Mean ± SEM, 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 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 AU. Mean ± SEM, 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 hours), labeled with LysoTracker DR (20 nM for 20 minutes; LysoTracker DR is pseudo colored in gray). Dashed lines represent the cell contour. Average integrated density of control cells (1.4 × 10⁷ ± 9 × 10⁵) and α-syn fibril-treated cells (8.3 × 10⁶ ± 5 × 10⁵) is presented. Mean ± SEM, 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 hours, treated with DQ-BSA (green) for 90 minutes. Dashed lines represent the cell contour. Average integrated density measured for control cells (3.8 × 10⁶ ± 1 × 10⁵) and α-syn fibril-treated cells (1.7 × 10⁶ ± 6 × 10⁴) is presented. Mean ± SEM, 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 hours treated with 1X Magic Red CathB (red). Dashed lines represent the cell contour. Average integrated density measured for control cells (1.3 × 10⁶ ± 6 × 10⁴) and α-syn fibril-treated cells (7.5 × 10⁵ ± 5 × 10⁴) is presented. Mean ± SEM, 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. α-syn, α-synuclein; AU, arbitrary unit; CAD, Cath.a-differentiated; CathB, Cathepsin B; LAMP, lysosome-associated membrane protein; LysoTracker DR, LysoTracker Deep Red.

https://doi.org/10.1371/journal.pbio.3001287.g002

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 (approximately 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 (approximately 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 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 (approximately 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.

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.

Lysosomes containing α-syn fibrils undergo lysosomal membrane permeabilization

α-Syn [73,74], as well as other amyloidogenic aggregates such as tau [81,82] and amyloid beta [83], induce LMP following their uptake from the culture medium in different cell types. This allows escape of α-syn aggregates from the lysosome to act on the soluble α-syn in the cytosol. However, the fate of these lysosomes has not been assessed. Furthermore, we previously demonstrated that α-syn spread between cells occurs mainly by direct cell-to-cell transfer of lysosomes bearing aggregates through TNTs, cytoplasmic protrusions extending from the cell periphery and connecting distant cells [21,35,52] (S2 Fig). Therefore, the question arises as to whether LMP also occurs after lysosomes bearing α-syn have been transferred to healthy neighboring cells. To address this question, we assessed first whether α-syn fibrils also induced LMP in our neuronal CAD cells. We used the Galectin-3 (Gal3) puncta assay based on the localization of Gal3 to damaged lysosomes. CAD cells were transiently transfected with Gal3-GFP and challenged or not with α-syn fibrils for 18 hours, prior to labeling for LAMP1 (Fig 3A, S3A Fig, number 1). After image acquisition by confocal microscopy, we quantified Gal3-GFP puncta formation in α-syn fibril-treated and untreated cells. We detected a significantly higher percentage of cells exhibiting Gal3-GFP puncta in the presence of α-syn fibrils (46 ± 2%) compared to the control condition (7 ± 1%) (Fig 3B, left graph), in which most cells displayed cytosolic Gal3-GFP [73,74]. However, in cells exhibiting Gal3-GFP puncta, there was no significant difference in the average number of puncta between control and α-syn fibril-treated cells (8 ± 1 and 12 ± 1, respectively; Fig 3B, right graph). In addition, we performed object-based 3D colocalization analysis to evaluate the association of Gal3-GFP with both α-syn fibrils and LAMP1+ lysosomes. We detected 38 ± 4% of Gal3-GFP puncta colocalizing with α-syn fibrils, 47 ± 3% of Gal3-GFP puncta colocalizing with LAMP1+ lysosomes, and 35 ± 3% of Gal3-GFP puncta colocalizing with both α-syn fibrils and lysosomes (Fig 3A, magenta arrowheads, and 3C). These findings demonstrate that more than one-third of Gal3-GFP puncta are recruited to the ruptured lysosomes containing α-syn fibrils. Furthermore, by using SIM, we could clearly observe the recruitment of Gal3-GFP to the damaged lysosomes positive for α-syn fibrils, which appeared to be either inside their lumen (Fig 3D) or at their membrane (Fig 3E).

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Fig 3. α-Syn fibrils induce LMP.

(A) Representative confocal images of Gal3-GFP transfected control (upper panel) and Alexa 568–tagged α-syn fibril-treated CAD cells for 18 hours (lower panel), labeled with LAMP1-Alexa 647 antibody (pseudo colored in gray). 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 hours (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 ± SEM, 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 ± SEM, 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 2 selected regions (white squares) from different Z-planes of the same Gal3-GFP transfected and α-syn fibril-treated CAD cell, labeled for HCS CellMask Blue, DAPI, and LAMP1 (pseudo colored in gray) 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. α-syn, α-synuclein; Gal3, Galectin-3; CAD, Cath.a-differentiated; LAMP, lysosome-associated membrane protein; LMP, lysosomal membrane permeabilization; SIM, structured illumination microscopy; SR, super-resolution.

https://doi.org/10.1371/journal.pbio.3001287.g003

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, microtubule-associated protein 1A/1B-light chain 3 (LC3), and LAMP1, and colocalization of these markers were analyzed. 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 3 independent experiments, we could only detect 1 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.

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

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 hours. The formation of α-syn-GFP aggregates detected as green puncta was then quantified (Fig 4A, S3A Fig, 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 SR microscopy in CAD cells transfected with α-syn-GFP, loaded with α-syn fibrils and labeled for LAMP1. We observed 3 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 colocalizing (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.

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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 hours). Scale bar: 10 μm (for insets: 5 μm). (B) % of control (13 ± 2%) and α-syn fibril-treated CAD cells for 18 hours (76 ± 5%) that formed α-syn-GFP aggregates (left graph). Mean ± SEM, n = 3 (45 cells per condition). ****P < 0.0001 by 2-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 ± SEM, 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 hours. (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 hours. (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 hours. (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 ± SEM, n = 2 (16 SR images, approximately 2,000 lysosomes were analyzed). (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 hour. (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 hours. (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 hours. In C, D, E, G, H, and I insets of selected regions (white squares) from different α-syn-GFP transfected and α-syn fibril-treated CAD cells, labeled for DAPI and LAMP1 (pseudo colored in gray) 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, α-synuclein; CAD, Cath.a-differentiated; LAMP, lysosome-associated membrane protein; SR, super-resolution.

https://doi.org/10.1371/journal.pbio.3001287.g004

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 hours from the addition of the α-syn fibrils and increased with time (S1 Movie, S5A Fig). We did not observe any newly formed α-syn-GFP puncta in untreated control cells (S2 Movie, S5B Fig). Next, we performed SR microscopy in order to investigate whether these newly formed aggregates were associated with the lysosomes by immunolabeling LAMP1 at different time points (1 hour, 2 hours, and 4 hours). 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–4I). 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.

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 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]. 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 labeled for LAMP1, HCS CellMask 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 nonpolarized cells [93], we detected 57 ± 1% of the lysosomes at the perinuclear region in control CAD cells (Fig 5A, white arrows, and 5B). 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 5B).

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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 hours) labeled for LAMP1-Alexa 488 antibody, HCS CellMask 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 ± SEM, n = 5 (40 cells per condition). ns = not significant, ****P < 0.0001 by 1-way ANOVA followed by Tukey 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 ± SEM, n = 4 (40 cells per condition). ns = not significant by 1-way ANOVA followed by Tukey 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 ± SEM, 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 hours, immuno-labeled 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 (9 × 10⁴ ± 2 × 10⁴) and in α-syn fibril-treated cells (9 × 10⁴ ± 3 × 10⁴) is presented. Mean ± SEM, 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 hours of coculture. Donor cells were transfected with LAMP1-GFP and treated with Alexa 568–tagged α-syn fibrils or not (referred to as “α-syn coculture” and “control coculture,” 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 coculture (18 ± 2%), in α-syn coculture (29 ± 3%), in control secretion test (9 ± 2%), and in α-syn secretion test (7 ± 1%) (left graph). Mean ± SEM, n = 4 (150 acceptor cells per condition). ns = not significant, ***P = 0.0004 by 1-way ANOVA followed by Tukey multiple comparison tests. The average number of LAMP1-GFP+ puncta detected per acceptor cell in control coculture (2 ± 0.2), in α-syn coculture (2 ± 0.2), in control secretion (1 ± 0.1), and in α-syn secretion (1 ± 0.1) is presented (right graph). Mean ± SEM, n = 4 (150 acceptor cells per condition). ns = not significant, ***P = 0.0004 by Kruskal–Wallis nonparametric ANOVA test followed by Dunn 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 ± SEM, n = 4 (20 acceptor cells that received both α-syn fibrils and LAMP1-GFP puncta were analyzed). The data underlying this figure may be found in S1 Data. α-syn, α-synuclein; AU, arbitrary unit; CAD, Cath.a-differentiated; CathB, Cathepsin B; LAMP, lysosome-associated membrane protein; LysoTracker DR, LysoTracker Deep Red.

https://doi.org/10.1371/journal.pbio.3001287.g005

Since our results indicate that α-syn fibrils affect lysosomal pH and function (Fig 2B–2D), 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.

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

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

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 ± 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 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 (9 × 10⁴ ± 2 × 10⁴ AU) and α-syn fibril-treated cells (9 × 10⁴ ± 3 × 10⁴ 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 coculture assay where donor cells were transiently transfected with LAMP1-GFP and either treated or not with α-syn fibrils for 18 hours (α-syn coculture and control coculture, respectively), and acceptor cells were transiently transfected with H2B-mCherry (Fig 5F, S3B Fig). We observed that the percentage of acceptor cells that received LAMP1-GFP puncta (lysosomes) from donor cells was significantly higher in the α-syn coculture compared to the control coculture (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 2 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 coculture conditions (2 ± 0.2 for both coculture 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 with α-syn fibrils, suggesting that half of the lysosomes transferred from donor to acceptor cells carried α-syn fibrils (Fig 5H).

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.

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

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

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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 hours (57 ± 6%). Mean ± SEM, 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 ± SEM, n = 3 (30 cells per condition). ***P < 0.001 by 1-way ANOVA followed by Tukey multiple comparison tests. (C) Western blot prepared from cell extracts collected from control, scrambled siRNA (sicontrol)-, and siTFEB-pretreated CAD cells for 60 hours, 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 ± SEM, n = 4 (30 cells per condition). ns = not significant, *P = 0.0288 by 1-way ANOVA followed by Tukey multiple comparison tests. The data underlying this figure may be found in S1 Data. α-syn, α-synuclein; CAD, Cath.a-differentiated; LAMP, lysosome-associated membrane protein; siRNA, small interfering RNA; siTFEB, siRNA targeting TFEB; sicontrol, scrambled siRNA; TFEB, transcription factor EB; WT, wild-type.

https://doi.org/10.1371/journal.pbio.3001287.g006

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

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 small interfering RNA (siRNA; control) or siRNA targeting TFEB (siTFEB) were challenged with α-syn fibrils for 18 hours. 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.

Lysosome positioning affects the efficiency of α-syn fibrils’ transfer

To further investigate the effect of lysosome positioning on α-syn fibrils transfer, we set up different coculture 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 toward 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 cocultured with acceptor CAD cells transiently transfected with H2B-GFP to distinguish them from the donors (referred to as Arl8b-GFP coculture and control coculture, respectively; Fig 7A, S3C Fig). We then quantified the presence of α-syn fibrils in the acceptor cells in each coculture condition. We detected a significant increase in the percentage of acceptor cells that received α-syn fibrils in the Arl8b-GFP coculture (65 ± 4%) compared to control coculture (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 coculture and did not significantly change in the 2 coculture 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 cocultures, 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, and 2 ± 0.3 α-syn fibrils puncta per acceptor cell in control coculture, Arl8b-GFP coculture, control secretion, and Arl8b-GFP secretion, respectively; Fig 7B, right graph).

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Fig 7. Lysosome positioning affects the efficiency of α-syn fibrils’ transfer.

(A) Representative confocal images of donor and acceptor cells after 24 hours of coculture. Donor cells were either loaded with Alexa 568–tagged α-syn fibrils only (control coculture) or transfected with Arl8b-GFP prior to be treated with α-syn fibrils (Arl8b-GFP coculture). Acceptor cells were transfected with H2B-GFP. Cocultures were labeled 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 coculture condition. Scale bar: 10 μm. (B) % of acceptor cells received α-syn fibrils in control coculture (49 ± 4%), in Arl8b-GFP coculture (65 ± 4%), in control secretion (8 ± 2%), and in Arl8b-GFP secretion (10 ± 2%) (left graph). Mean ± SEM, n = 3 (90 acceptor cells per condition). ns = not significant, **P = 0.0038 by Kruskal–Wallis nonparametric ANOVA test followed by Dunn multiple comparison tests. Average number of α-syn fibrils puncta in control coculture (3 ± 0.4), in Arl8b-GFP coculture (3.4 ± 0.4), in control secretion (1.3 ± 0.4), and in Arl8b-GFP secretion (2 ± 0.3) (right graph). Mean ± SEM, n = 3 (90 acceptor cells per condition). ns = not significant, by 1-way ANOVA followed by Tukey multiple comparison tests. (C) Western blot prepared from cell extracts collected from control, scrambled siRNA (sicontrol)-, and siArl8b-pretreated CAD cells for 60 hours, 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 AU. Mean ± SEM, n = 3. ns = not significant, ***P = 0.0004 by 1-way ANOVA followed by Tukey multiple comparison tests. (D) Representative confocal images of donor and acceptor cells after 24 hours of coculture. Donor CAD cells were pretreated with sicontrol or siArl8b for 60 hours or left untreated prior to be challenged with Alexa 568–tagged α-syn fibrils (referred to as control, sicontrol, and siArl8b cocultures, respectively). Acceptor cells were transfected with H2B-GFP. Cocultures were labeled 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 coculture condition. Scale bar: 10 μm. (E) % of acceptor cells that received α-syn fibrils in control coculture (55 ± 4%), sicontrol coculture (60 ± 3%), siArl8b coculture (28 ± 3%), control secretion (6 ± 1%), sicontrol secretion (9 ± 1%), and siArl8b secretion (6 ± 1%) (left graph). Mean ± SEM, n = 4 (80 acceptor cells per condition). ns = not significant, ****P < 0.0001 by 1-way ANOVA followed by Tukey multiple comparison tests. Average number of α-syn puncta per acceptor cell in control coculture (2 ± 0.2), sicontrol coculture (2 ± 0.1), siArl8b coculture (2 ± 0.3), control secretion (1 ± 0.2), sicontrol secretion (2 ± 0.2), and siArl8b secretion (2 ± 0.3) (right graph). Mean ± SEM, n = 4 (80 acceptor cells per condition). ns = not significant by Kruskal–Wallis nonparametric ANOVA test followed by Dunn multiple comparison tests. (F) Control, Arl8b-GFP transfected, and sicontrol- or siArl8b-pretreated CAD cells for 60 hours were challenged or not with α-syn fibrils (18 hours) and labeled 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 ± SEM, 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 ± SEM, n = 3 (90 cells per condition). ns = not significant, *P < 0.05, **P < 0.01 by 1-way ANOVA followed by Tukey multiple comparison tests. The data underlying this figure may be found in S1 Data. α-syn, α-synuclein; AU, arbitrary unit; CAD, Cath.a-differentiated; siRNA, small interfering RNA; siArl8b, siRNA targeting Arl8b; sicontrol, scrambled siRNA; TNT, tunneling nanotube; WGA, wheat germ agglutinin.

https://doi.org/10.1371/journal.pbio.3001287.g007

To corroborate these observations in a different cell type, we performed the same experiment in HeLa cells (S3D and S6 Figs) 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 cocultures, respectively (S6A and S6B 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 cocultures and secretion conditions (3 ± 1 and 5 ± 1 in control and Arl8b-GFP cocultures, 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 hours prior to loading with α-syn fibrils for 18 hours; in parallel, untreated control cells were challenged with α-syn fibrils. These donors were then cocultured with acceptor cells transiently transfected with H2B-GFP (Fig 7D, S3E Fig). In control coculture and in sicontrol coculture, the percentage of acceptor cells that received α-syn fibrils was 55 ± 4% and 60 ± 3%, respectively, whereas in siArl8b coculture 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, sicontrol, and siArl8b secretion conditions, respectively; Fig 7E, left graph). The average number of α-syn puncta detected in acceptor cells in cocultures and secretion conditions was not significantly changed (1 to 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 knockout (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 toward the cell periphery [90]. KO of these proteins impairs lysosome movement toward the cell periphery [90,100]. These KO donor cells, in parallel to control donors (WT HeLa), were loaded with α-syn fibrils and cocultured with WT HeLa cells expressing H2B-GFP (S3F and S6C Figs). Acceptor cells were then analyzed for the presence of α-syn fibrils. The percentage of acceptor cells that received α-syn fibrils in control coculture was 29 ± 2%, whereas in myrlysin-KO and KIF1B-KIF5B-double-KO (KIF1B-5B dKO) cocultures, 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%, and 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 cocultures/secretion conditions (1 to 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 2 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 labeled with wheat germ agglutinin (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.

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 3B). To assess this hypothesis, we performed coculture experiments where donor cells were either transiently transfected with H2B-mCherry (control coculture) or treated with α-syn fibrils for 18 hours (α-syn coculture) and cocultured with acceptor cells transiently transfected with Gal3-GFP (Fig 8A, S3G Fig, 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 coculture (33 ± 5%) compared to control coculture (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 coculture (14 ± 1) compared to control coculture where some LMP occurs spontaneously (8 ± 1; Fig 8B, lower graph). We also performed SR microscopy in α-syn coculture labeled 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.

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Fig 8. α-Syn fibrils induce LMP upon arrival in the acceptor cells.

(A) Representative confocal images of donor and acceptor cells after 24 hours of coculture. Donor cells were transfected with H2B-mCherry (control coculture) or treated with Alexa 568–tagged α-syn fibrils (α-syn coculture). Acceptor cells were transfected with Gal3-GFP. Cocultures were labeled 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%) cocultures (upper graph). Average number of Gal3-GFP puncta formation in acceptor cells under LMP in control (8 ± 1) and α-syn (14 ± 1) cocultures (lower graph). Mean ± SEM, n = 5 (50 acceptor cells per condition). *P = 0.04, ****P < 0.0001 by Mann–Whitney U test. (C) SR image of a coculture where donor cells were treated with α-syn fibrils for 18 hours and acceptor cells were transfected with Gal3-GFP, labeled for LAMP1-Alexa 647 antibody (pseudo colored in gray). A selected region where Gal3-GFP puncta were observed (indicated by a white square in the acceptor cell of the coculture 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 labeling 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. α-syn, α-synuclein; Gal3, Galectin-3; LAMP, lysosome-associated membrane protein; LMP, lysosomal membrane permeabilization; SIM, structured illumination microscopy; SR, super-resolution.

https://doi.org/10.1371/journal.pbio.3001287.g008

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 cocultured with donor cells, either challenged with α-syn fibrils or transfected with H2B-mCherry (α-syn coculture and control coculture, respectively; Fig 9A, S3G Fig, 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 coculture, whereas in control coculture, only 10 ± 4% of the acceptor cells had 2 ± 0.2 α-syn-GFP puncta (Fig 9B).

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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 hours of coculture. Donor cells were transfected with H2B-mCherry (control coculture) or treated with Alexa 568–tagged α-syn fibrils for 18 hours (α-syn coculture). 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, and red arrows indicate α-syn fibrils received by acceptor cells in α-syn coculture. 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%) cocultures (left graph). Average number of α-syn-GFP puncta detected in acceptor cells in control (2 ± 0.2) and in α-syn (7 ± 1) cocultures is presented (right graph). Mean ± SEM, 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 cocultures, 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 ± SEM, n = 2 (15 SR images, approximately 3,000 lysosomes were analyzed). Insets of images in C and D (acquired with spinning disk microscope with SR module) and E and F (acquired with SR SIM, Zeiss, LSM 780-Elyra PS.1) from 4 selected regions (white squares) and from different acceptor cells transfected with α-syn-GFP and that received α-syn fibrils, labeled for LAMP1 Alexa 647 antibody (pseudo colored in gray). Scale bars: 10 μm (for the insets: 1 μm). The data underlying this figure may be found in S1 Data. α-syn, α-synuclein; LAMP, lysosome-associated membrane protein; SIM, structured illumination microscopy; SR, super-resolution.

https://doi.org/10.1371/journal.pbio.3001287.g009

Next, to address whether the seeding in acceptor cells was associated with lysosomes, we performed SR microscopy. After labeling the cells in coculture with antibody to LAMP1, we examined the subcellular localization of α-syn-GFP aggregates in acceptor cells. We observed 4 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.

α-Syn–loaded cells transfer damaged lysosomes to healthy cells and receive healthy lysosomes in return

In our homotypic coculture 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 coculture 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 cocultured for 24 hours prior to be fixed and immunolabeled 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 and S7B 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 cocultured 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 coculture 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 cocultured with HeLa cells expressing Gal3-Turquoise. Cells in coculture were then immunolabeled 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 coculture (52 ± 4%) compared to control coculture (32 ± 5%), whereas the average number of transferred lysosomes (LAMP1+ puncta) did not change (3 ± 0.3 in both cocultures; Fig 10B). Furthermore, we observed that in α-syn coculture, both lysosomes containing α-syn fibrils (Fig 10A, yellow arrows; S7G Fig, red arrow) or not (Fig 10A, white arrows; S7G Fig, white arrow) were transferred to acceptor cells.

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Fig 10. Intercellular lysosome transfer is reciprocal.

(A) Representative confocal image of CAD-HeLa coculture where donor mouse CAD cells were treated with Alexa 568–tagged α-syn fibrils (18 hours) and cocultured with acceptor human HeLa stable Gal3-Turquoise cells for 24 hours, labeled 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%) cocultures (left graph). **P = 0.006 by 2-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) cocultures (right graph). Mean ± SEM. ns = not significant by Mann–Whitney U test. (C) Representative confocal image of CAD-HeLa coculture where Gal3-GFP transfected donor CAD cells were treated with Alexa 568–tagged α-syn fibrils for 18 hours and cocultured with HeLa Gal3-Turquoise for 24 hours, labeled with DAPI. In acceptor HeLa Gal3-Turquoise cells, red arrows indicate α-syn fibrils, and 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%) cocultures (left graph); average number of Gal3-GFP puncta per acceptor HeLa cell in control (2 ± 0.3) and in α-syn (2 ± 0.2) cocultures (right graph). Mean ± SEM. 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 (labeled 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 coculture. In acceptor HeLa Gal3-Turquoise cells, red arrows indicate the α-syn fibrils, and 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%) cocultures 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) cocultures is presented (right graph). Mean ± SEM. ns = not significant, *P = 0.03 by Mann–Whitney U test. (H) Representative confocal image of CAD-HeLa coculture where donor CAD cells were treated with Alexa 568–tagged α-syn fibrils for 18 hours and cocultured with HeLa Gal3-Turquoise for 24 hours, labeled 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%) cocultures (left graph). **P = 0.002 by 2-tailed t test. Average number of acceptors’ lysosomes per donor cell in control (2 ± 0.3) and in α-syn (2 ± 0.1) cocultures (right graph). Mean ± SEM. ns = not significant by Mann–Whitney U test. In CAD-HeLa coculture model, data were collected from 7 independent experiments in which 4 and 3 of them were labeled with anti-LAMP1 mouse and human antibodies, respectively. According to the experiment, in average, 100 acceptor or donor cells were analyzed for each condition per experiment. The data underlying this figure may be found in S1 Data. α-syn, α-synuclein; CAD, Cath.a-differentiated; Gal3, Galectin-3; LAMP, lysosome-associated membrane protein; LMP, lysosomal membrane permeabilization.

https://doi.org/10.1371/journal.pbio.3001287.g010

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 coculture 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 cocultured 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 coculture conditions, donor CAD cells challenged with α-syn fibrils undergo LMP similar to what we showed earlier (Fig 3A and 3B). 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 cocultures (Fig 10C). We found a significantly higher percentage of HeLa cells with Gal3-GFP puncta in the α-syn coculture (13 ± 2%; Fig 10C, green arrows, and D, left graph) compared to control coculture (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 2 cocultures (2 ± 0.2 and 2 ± 0.3 in control and α-syn cocultures, 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 coculture (Fig 10G). LMP in the acceptor cells of control coculture 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 labeled 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 coculture was immunolabeled 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 coculture was 14 ± 4%, whereas in α-syn coculture, this percentage was significantly higher (34 ± 5%; Fig 10I, left graph). The average number of acceptors’ lysosomes per donor cell in both coculture conditions was similar (2 ± 0.3 and 2 ± 0.1 in control and α-syn cocultures, 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 cocultures (S7H Fig). Only 4 ± 1% of donor cells resulted positive for Gal3-Turquoise puncta in α-syn coculture, while we could barely (0.3 ± 0.3%) observe donor cells positive for Gal3-Turquoise puncta in control coculture (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 toward donor cells overloaded with α-syn, similar to what was previously reported in the case of a lysosomal disease [106].

Cell culture, transfection, and siRNA treatment

The mouse catecholaminergic neuronal cells (CAD cells), generated from a brain tumor of transgenic mice by targeted oncogenesis (kind gift of Hubert Laude; Institut National de la Recherche Agronomique, Jouy-en-Josas, France), were cultured in Opti-MEM GlutaMAX (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA) including 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen-Strep). CAD cells express neuronal properties, but they lack neuronal morphology, thus stay undifferentiated in the presence of serum in their medium. This situation can be reversed by serum starvation, and differentiation can be induced [128]. In this study, we used CAD cells in their undifferentiated state as this condition is optimal to study TNTs [19,125,129–131]. HeLa cells WT, HeLa cells myrlysin-KO and KIF1B-KIF5B double KO [100,132], and HeLa cells stably expressing Gal3-Turquoise were cultured in DMEM-GlutaMAX including 4.5 g/L D glutamine and pyruvate (Gibco) supplemented with 10% of FBS. CAD and HeLa cells were transiently transfected with Gal3-GFP, Arl8b-GFP, fluorescently tagged human histone H2B (H2B-GFP or H2B-mCherry) plasmids (final concentration: 1 μg for 150,000 cells/35mm Ibidi μ-dishes, 3 μg for 800,000 cells/T25 flasks) by Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

To down-regulate Arl8b and TFEB, 30 pmol of unique 27mer siRNA duplex against mouse Arl8b and TFEB (CliniSciences, Nanterre, France and OriGene, Rockville, Maryland, USA, respectively) was introduced to CAD cells in parallel to siRNA scramble (sicontrol) by using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions and incubated 60 hours prior to be used in coculture experiments and in western blot analysis.

To induce lysophagy, CAD cells were treated with 1 mM LLOMe for 3 hours.

Generation of stable HeLa mTurquoise-Galectin3 cell line

In order to generate stable HeLa cell line expressing a mTurquoise-Galectin3 fluorescent reporter, we used a well-established Sleeping Beauty (SB) transposable system that originates from an inactive transposon in salmonid fish and belongs to the Tc1/mariner-type transposon family, as previously described [133,134]. Briefly, the insert sfiI-mTurquoise-Galectin3-sfiI was amplified from pE mTurquoise-Galectin3 plasmid by polymerase chain reaction (PCR) where SfiI-mTurquoise-forward (AGG CCT CTG AGG CCG CCA CCA TGG TGA GCA AGG G) and Gal3-SfiI-reverse (AGG CCT GAC AGG CCT TAT CAT GGT ATA TGA AGC AC) primers (Integrated DNA Technologies, Coralville, Iowa, USA) were used with Flash Phusion High Fidelity PCR Master Mix (Thermo Fisher Scientific, Eindhoven, the Netherlands). Next, the insert was cut by SfiI enzyme and inserted into the constitutive vector pSBbi-PUR, a gift from Eric Kowarz (Addgene Plasmid #60523), which was cut by sfiI enzyme and dephosphorylated by Rapid DNA Dephos & Ligation Kit (Roche, Basel, Switzerland). 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 on LB broth agar plates containing amphicilin. Clones were then sequenced (Eurofins Genomics, Luxembourg) 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 Plasmid Maxiprep Kit. Next, HeLa cells seeded on 6-well plates (300,000 cells per well) were co-transfected with 9 μg of pSBbi-PUR-mTurquoise-Galectin3 and 100 ng SB transposase expression vector pCMV (CAT)T7-SB100, a gift from Eric Kowarz (Addgene plasmid # 34879) by using XtremGene transfection reagent (Roche). Moreover, 24 hours following the transfection, cells were subjected to puromycin (1 μg/mL). Selection was carried out for 2 to 10 days. Cells were then cultivated for 4 weeks without selection medium, and monoclonal selection was performed on 96-well plates (1 cell/well). Clones exhibited expected mTurquoise fluorescent were further tested for classical behavior for HeLa cells (low Gal3 fluorescent in the whole cell and a brilliant signal upon vacuole rupture) and used in the experiments described in the current study.

Preparation and internalization of α-syn fibrils

Human WT 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) as previously reported. Expression and purification were performed as described previously [86]. Briefly, the protein concentrations of monomeric α-syn were measured by RP-HPLC as described previously [86,135]. Purified recombinant α-syn monomers (approximately 5 mg/mL) containing 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, the samples were ultracentrifuged at 100,000 g for 20 minutes at room temperature (RT), and the ppt fraction was recovered as α-syn fibrils. They were resuspended in saline prior to be ultracentrifuged again. The pellets were resuspended in saline and sonicated using a cup horn sonicator (Sonifier SFX, Branson, Brookfield, Connecticut, USA) at 35% power for 180 seconds (total 240 seconds, 30 seconds on, 10 seconds off). The fibrils were labeled with Alexa Fluor 488 or 568 Protein Labelling Kit (Invitrogen) following the manufacturer’s instructions. After the incubation of fibrils with Alexa Fluor dyes, samples were ultracentrifuged again, and the pellets were resuspended in 30 mM Tris-HCl, pH 7.5 prior to be ultracentifuged one more time. Finally, labeled α-syn fibrils were resuspended in saline containing 0.1% sodium azide, and the protein concentration of the fibrils was determined by RP-HPLC as described before [86,135]. Seeding activity of the fibrils were checked by adding the labeled fibrils (3 μg) to 100 μL of 1 mg/mL α-syn monomer solution in 30 μM Thioflavin T and 80 mM Hepes, pH 7.5. Amyloid-like fibril formation was continuously monitored by measuring the Thioflavin T fluorescence (excitation 442 nm, emission 485 nm) with a microplate reader (Infinite 200, TECAN).

A total of 0.5 μM Alexa 488 or Alexa 568–tagged α-syn fibrils were diluted in the appropriate medium and sonicated for 5 minutes at 80% amplitude with a pulse cycle of 5 seconds on and 2 seconds off in an ultrasonic water bath Vibra-Cell 75,041 (BioBlock Scientific, Strasbourg, France). Fibrils were then introduced to cells immediately and incubated for 18 hours. Before cells were processed for further procedures (e.g., coculture preparation, fixation, etc.), they were washed 3 times with diluted trypsin (1/3) in Phosphate-buffered saline (PBS). In parallel, control cells were subjected to the same treatments as described above without the addition of α-syn fibrils.

Immunocytochemistry for lysosomes

CAD and/or HeLa cells and cocultures (CAD-CAD and CAD-HeLa) were fixed with 4% paraformaldehyde (PFA) for 20 minutes at RT. After 3 washes with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 3 minutes at RT. Cells were then washed with PBS, and nonspecific antibody binding sites were blocked by using 10% of goat serum (GS) in PBS for 30 minutes. Cells were then incubated with primary rat monoclonal anti-mouse LAMP1 (1D4B) or anti-human LAMP1 (H4A3) primary antibodies with the dilution of 1/200 in blocking solution for 1 hour. Antibodies were purchased from Developmental Studies Hybridoma Bank (DSHB, Iowa City, Iowa, USA). Cells were then washed 3 times with PBS and incubated for secondary antibodies (anti-rat Alexa-647 or anti-rat Alexa-488 purchased from Invitrogen) diluted 1:500 in blocking solution for 2 hours at RT. After 3 washes with PBS, cells were further stained with HCS CellMask Blue (Invitrogen) for 15 minutes at RT (diluted 1:5,000 in PBS). Cells were then washed 3 times with PBS and further stained for DAPI (diluted 1:5,000 in PBS) for 5 minutes. Finally, cells were washed and mounted with Aqua-Poly/Mount (Polysciences, Warrington, Pennsylvania, USA). Image acquisitions were performed by confocal microscopy with identical settings.

Immunocytochemistry for lysophagy

CAD cells were fixed with 4% PFA for 20 minutes at RT. After 3 washes with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 3 minutes at RT. Cells were then washed with PBS, and nonspecific antibody binding sites were blocked by using 2% of bovine serum albumin (BSA) in PBS for 30 minutes. Cells were then incubated with primary rabbit anti-mouse LC3 (1:500, MBL International, Woburn, Massachusetts, USA), mouse anti-Ubiquitin (1:1,000, FK2, Enzo, Farmingdale, New York, USA), and rat monoclonal anti-mouse LAMP1 (1D4B) (1:100, DSHB) in blocking solution for 1 hour. Cells were then washed 3 times with PBS and incubated for secondary antibodies (anti-rabbit Alexa 488, anti-mouse Alexa-647 from Invitrogen; and anti-rat CF405M from Sigma, Saint Louis, Missouri, USA) diluted 1:500 in blocking solution for 1 hour at RT. Finally, cells were washed and mounted with Aqua-Poly/Mount (Polysciences). Image acquisitions were performed by confocal microscopy with identical settings.

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

CAD cells were plated on 35-mm Ibidi μ-dishes (100,000 cells/dish). Alexa 568–tagged α-syn fibrils were introduced or not (control) to the cell culture medium 24 hours after plating. Following 18 hours of α-syn fibril uptake, 20 nM LysoTracker DR (Invitrogen) was prepared in culture medium, and cells were incubated for 20 minutes at 37°C. Cells were then fixed with 4% PFA at RT and stained for HCS CellMask Blue and DAPI as described before. Image acquisitions were performed by confocal microscopy with identical settings.

To detect CathB activity, CAD cells were plated on 35-mm Ibidi μ-dishes (100,000 cells/dish). Alexa 488-tagged α-syn fibrils were introduced or not (control) to the cell culture medium 24 hours after plating. Following 18 hours of α-syn fibril uptake, cells were treated with 1X Magic Red Cathepsin solution, provided by Magic Red Cathepsin Assay kit (ImmunoChemistry Technologies, Bloomington, Minnesota, USA) and prepared through the manufacturer’s recommendation, for 15 minutes at 37°C. Cells were then immediately imaged by confocal microscopy in live condition. The intracellular activity of CathB was detected as red fluorescent puncta produced by the hydrolysis of the fluorogenic substrate introduced. All images were acquired with the identical settings. For both experiments, integrated density of LysoTracker DR and CathB signal was measured by using Fiji software after a region of interest (ROI) is manually created by the freehand selection tool and the measurements were expressed in AU.

For DQ-BSA assay, CAD cells were plated on 35-mm Ibidi μ-dishes (130,000 cells/dish). Alexa 568–tagged α-syn fibrils were introduced or not (control) to the cell culture medium 24 hours after. Following 18 hours of α-syn fibrils uptake, 10 μg/mL DQ Green BSA (Thermo Fisher Scientific) was prepared in culture medium, and cells were incubated for 90 minutes at 37°C. Cells were then fixed with 4% PFA at RT. Image acquisitions were performed by confocal microscopy with identical settings. Integrated density of DQ-BSA signal was measured by using Fiji software after a ROI is manually created by the freehand selection tool, and the measurements were expressed in AU.

TNT labeling

To preserve the structure of TNTs, cells were fixed by a 2-step fixation protocol as described previously [20,129]. Briefly, cells were first fixed with fixative solution-1 (2% PFA, 0.05% glutaraldehyde (GA) and 0.2 M HEPES in PBS) for 20 minutes at 37°C, then with fixative solution-2 (4% PFA and 0.2 M HEPES in PBS) for an additional 20 minutes at 37°C. After several washes, cells were labeled with 3.3 μg/μL WGA-Alexa Fluor-488 nm conjugate solution (Life Technologies, Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 20 minutes at RT for membrane detection. As there is no specific marker for TNTs, WGA labeling was used to detect TNTs as described in detail in the quantification section.

Lysosomal exocytosis analysis

Detection of LMP and seeding

CAD cells were plated on 35-mm Ibidi μ-dishes (100,000 cells/dish) and transiently transfected with 1 μg of Gal3-GFP (for LMP) or 1 μg of α-syn-GFP plasmids (for seeding). Moreover, 24 hours later, cells were either treated with Alexa 568–tagged α-syn fibrils for 18 hours or left untreated. In coculture experiments, cells transiently transfected with Gal3-GFP or α-syn-GFP plasmids were cocultured for 24 hours with donor cells previously treated for 18 hours with Alexa 568–tagged α-syn fibrils or not. Cells were then fixed with 4% PFA for 20 minutes at RT and stained for DAPI. Following image acquisition with confocal microscopy, cells were quantified for the presence of Gal3-GFP or α-syn-GFP puncta by using “Spot detector” tool of the ICY software. Note that a threshold of 5 puncta is applied to all images analyzed, and cells bearing puncta above this threshold were considered as having LMP.

Coculture experiments

Donor cells were plated on 35-mm well plates (400,000/well) and transfected with suitable plasmids according to the experiment (LAMP1-GFP, Arl8b-GFP, Gal3-GFP, and H2B-mCherry) or pretreated with siRNA Arl8b or siRNA-scrambled for 60 hours. Myrlysin-KO and KIF1B-5B dKO HeLa cells were directly used as donors. Donor cells (in parallel to untreated control cells) were then challenged with Alexa 568–tagged α-syn fibrils for 18 hours (or not according to the experiment). They were then washed once with PBS and 3 times with 1:3 diluted trypsin before coculture 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, and 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. Coculture was prepared in a 1:1 ratio (100,000 donor and 100,000 acceptors on 35-mm Ibidi μ-dishes) for 24 hours. Cells were then fixed with 4% PFA for 20 minutes at RT and stained for HCS CellMask Blue and DAPI as described before and mounted. Schematic presentation of different coculture preparations was presented in S3 Fig.

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

Confocal microscopy

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

Super-resolution microscopy

Live imaging microscopy

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

Electron microscopy

Western immunoblots

For the western blot analysis, cells were rinsed with PBS and then scraped in RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS). The resulting soluble fractions were centrifuged at 18,000 g for 5 minutes at 4°C, and then, the supernatants were taken as the whole cell lysate, mixed with Laemmli sample buffer (1 M Tris–HCl, pH 6.8, 20% glycerol, 4% SDS, 5% ß-mercaptoethanol, and 0.02% bromophenol blue), and boiled for 5 minutes. For western blots, equal amounts of proteins (30 μg/lane) were separated on SDS-PAGE gels and transferred to nitrocellulose membranes (GE Healthcare Life Sciences), following standard procedures. Membranes were then blocked in TBS containing 0.1% Tween-20 and 5% nonfat dried milk for 1 hour and probed overnight at 4°C with primary antibodies. The membranes were then washed and exposed for 1 hour at RT to the anti-rabbit/anti-mouse peroxidase-conjugated antibodies (1:1,000). The specific protein bands were visualized using the ECL immunoblotting chemiluminescence system (GE Healthcare Life Sciences) and the ImageQuant LAS 500TM camera (GE Healthcare Life Sciences). Primary antibodies used in this study are the following: rat anti-Lamp1 (1:100, DSHB), anti-Arl8b (1:100), rabbit anti-TFEB (1:1,000, Bethyl Laboratories, Montgomery, Texas, USA), mouse anti-tubulin (1:1,000), and rabbit anti-GAPDH (1:5,000, Boster, Pleasanton, California, USA). To determine the apparent molecular weights of the protein bands, a PageRuler plus prestained protein ladder (Thermo Fisher Scientific) was used.

Quantification analysis

Statistical analysis

Data collected at least from 3 independent experiments were presented as mean ± SEM. Exact number of experiments and number of cells analyzed per condition for each experiment is presented in the figure legends. All data were first subjected to a D’Agostino–Pearson omnibus normality test. Values having Gaussian distribution were further analyzed by 2-tailed t test and 1-way ANOVA followed by Tukey multiple comparison tests for paired and multiple comparisons, respectively. Values having non-Gaussian distribution were analyzed by Whitney–Mann U test and Kruskal–Wallis nonparametric ANOVA test followed by Dunn multiple comparison tests for paired and multiple comparisons, respectively. Paired set of data were analyzed by paired t test.