Novel autophagy inducers by accelerating lysosomal clustering against Parkinson’s disease

  1. Yuki Date
  2. Yukiko Sasazawa
  3. Mitsuhiro Kitagawa
  4. Kentaro Gejima
  5. Ayami Suzuki
  6. Hideyuki Saya
  7. Yasuyuki Kida
  8. Masaya Imoto
  9. Eisuke Itakura
  10. Nobutaka Hattori
  11. Shinji Saiki

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Abstract

The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including α-synuclein (αSyn) associated with the pathogenesis of Parkinson’s disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble αSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.

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  1. Version published to 10.7554/elife.98649 on eLife
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    Reply to the reviewers

    Manuscript number: RC-2023-02232

    Corresponding author(s): Shinji, Saiki and Nobutaka, Hattori

    1. General Statements [optional]

    Thank you for the review of our paper entitled “Identification of novel autophagy inducers by accelerating lysosomal clustering against Parkinson's disease” (RC-2023-02232). We have carefully read the critiques and planed experiments. Below we include point-by-point responses to the questions raised by the reviewers. We have also carried out some experiments and highlighted the revised sentences in the transferred manuscript in red. The numbers of pages and lines are indicated based on the MS Word transferred manuscript. We believe this revision plans appropriately addresses the issues raised by Reviewers. Finally, all the authors would like to thank again the Editor and Reviewers for improving our manuscript by providing their invaluable comments and suggestions.

    Point-by-point description of the revisions

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The manuscript by Date et al employed a cell model by stably expressing LGP120-mCherry and GFP-gamma-tubulin to carry out high-content screening in search of chemical compounds that enhance lysosomal clustering and autophagy. They found 6 clinically approved drugs categorized as topoisomerase II inhibitors and the benzimidazole class. They further validated these compounds by a set of well-designed experiments including autophagy flux assays and mTOR dependence. In the mechanistic study, they demonstrated the compounds induce lysosomal clustering in a JIP4-TRPML1-dependent manner. In a PD cell model, one of the compounds albendazole exhibited the effect on boosting the degradation of insoluble alpha-synuclein. The study is of interest, and the cell model and the approach generated by the authors would be transferable for future studies of other high-content imaging screening. Most of the data is clear and convincing.

    Major comment

    1. In addition to its role in facilitating a-syn turnover by autophagy, Is the chemical protective against a-syn toxicity?

    RESPONSE:

    As suggested by the Reviewer, we examined the cytotoxicity of aSyn aggregates in SH-SY5Y cells overexpressing aSyn-GFP by LDH assay. As shown in the revised version of Fig. 1, aSyn aggregates induced by introducing aSyn fibrils into SH-SY5Y cells overexpressing aSyn-GFP did not exhibit any cytotoxicity. In addition, we observed no significant change in cell death after 8 hours of treatment with albendazole compared with DMSO.

    Previous studies have reported that induced pluripotent stem cells (iPSCs) derived from patients with PD with a triplication of the human SNCA genomic locus exhibited reduced capacity for differentiation into dopaminergic or GABAergic neurons, decreased neurite outgrowth, and lower neuronal activity compared with control cultures, albeit without showing cytotoxicity (Cell Death and Disease 6: e1994, Oliveira et al., 2015). Given this context, we were thus unable to conduct the suggested assessment due to technical limitations. Therefore, we consider the evaluation of the recovery of aSyn toxicity by drug treatment challenging in this cellular model using fibril aSyn.

    __Revised Fig 1. __

    SH-SY5Y cells overexpressing aSyn-GFP were transfected with aSyn fibril for 48 h and treated with the indicated albendazole concentrations for 8 h. The cytotoxicity was measured by using Cytotoxicity LDH Assay Kit-WST kit.

    1. Please elaborate why albendazole does not change the levels of soluble a-syn, but those of insoluble, as shown Fig 8D.

    RESPONSE:

    The unchanged aSyn-GFP levels in the soluble fraction (Fig. 8D) are likely due to the abundance of soluble aSyn-GFP. To evaluate the autophagic degradation of aSyn monomers, we used SH-SY5Y cells stably expressing aSyn-Halo and measured aSyn degradation by quantifying cleaved Halo. As shown in the revised version of Fig. 2, albendazole treatment induced a higher cleavage rate of Halo than DMSO treatment for 8 h, suggesting that albendazole degrades both aSyn monomers and aSyn aggregates. We have added the data in Fig. S7A, and the description of these experiments in the Results section (page 10, lines 359 to 364).

    __Revised Fig. 2. __

    SH-SY5Y cells expressing aSyn-Halo were labeled for 20 min with 100 nM of tetramethylrhodamine-conjugated ligand in a nutrient-rich medium. After washing with phosphate-buffered saline and incubating in normal medium for 30 min, the cells were treated with 10 µM albendazole for 8 h. The experiments were performed in triplicate. Cell lysates were separated by electrophoresis and analyzed by in-gel fluorescence detection (left). The HaloTMR band intensity was normalized by the sum of the band intensities of HaloTMR-aSyn and HaloTMR. The vertical axis of the graph represents the intensity multiplied by 100. Mean values of data from five or three experiments are shown. The graph data are expressed as mean ± standard deviation. ****P

    1. Fig 6A shows that some of the compounds (Teniposide, Amsacrine) affect the levels of JIP4. Can albendazole also reduce JIP4 levels. It might be interesting to test this, as JIP4 is important for lysosomal clustering.

    RESPONSE:

    As the Reviewer pointed out, JIP4 is essential for lysosome accumulation. However, our data showed decreased JIP4 levels with the addition of lysosomal-clustering compounds. We hypothesized that this response was caused by the autophagy-induced degradation of JIP4. The decrease in JIP4 levels was detected by western blot after 4 h of treatment with 10 μM of teniposide. Moreover, the decrease in JIP4 levels induced by teniposide was suppressed by co-treatment with bafilomycin A1, indicating that JIP4 was degraded by teniposide-induced autophagy, as shown in the revised version of Fig. 3. We have added the data in Fig. S6 and the related description of these experiments in the Results section (page 8, lines 289 to 293).

    __Revised Fig 3. __

    SH-SY5Y cells were treated with 10 µM teniposide and with or without 30 nM bafilomycin A1 for 4 h. Cell lysates were immunoblotted with anti-JIP4 and actin antibodies.

    Minor comments: The writing is good generally. Please tide up the text in a few occasions to make the expressions more formal.

    RESPONSE: We have revised our manuscript to adopt a more formal tone.

    Reviewer #1 (Significance (Required)):

    Significance: The study generated a new approach for high-throughput screening of compounds to enhance lysosomal clustering. Audience: Basic and clinical research Expertise: Programmed cell death, neurodegenerative diseases

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this study, the authors focused on lysosome positioning and autophagy activity to search for novel agents effective against Parkinson's disease. As a result, several compounds were successively identified, including Topoisomerase inhibitors and Benzimidazole. Authors showed that these agents regulate lysosomal positioning through different pathways but commonly require JIP4 to regulate lysosomal positioning and subsequent autophagy. They also showed that albendazole treatment promoted the degradation of insoluble ubiquitinated proteins and αSyn in cultured cells.

    Major Comments.

    1. Two compounds, for instance teniposide and albendazole both requires JIP4 and/or TRPML1 to regulate lysosomal positioning and autophagy but their action seems different. What is the actual mechanism by which these compounds require JIP4/TRPML1. How inhibition of Topoisomerase leads to increase of JIP4 phosphorylation? Do teniposide and albendazole both affect calcium release from TRPML1?

    RESPONSE:

    We previously reported that acrolein/H2O2 accelerates lysosomal retrograde trafficking by TRPML1 and phosphorylated JIP4. Mechanistically, JIP4 was phosphorylated by CaMK2G activated by Ca2+ released from TRPML1 (EMBO J 41: e111476, Sasazawa et al., 2022). TRPML1 acts as a reactive oxygen species (ROS) sensor in lysosomes (Nat Commun 7: 12109, Zhang et al., 2016). We concluded that acrolein induces ROS production, which then activates TRPML1. (EMBO J 41: e111476, Sasazawa et al., 2022). Therefore, topoisomerase inhibitors (topo-i) may induce ROS and stimulate TRPML1. We examined intracellular ROS levels in response to topo-i. As shown in revised Fig. 4A, the topo-i teniposide, etoposide, and amsacrine significantly increased ROS levels. Moreover, N-acetyl-L-cysteine, an ROS scavenger, partially attenuated lysosomal clustering induced by topo-i (revised Fig. 4B). In addition, Ca2+ imaging showed that teniposide, but not albendazole, upregulates Ca2+ flux (revised Fig. 4C). Based on the activity of CaMK2G siRNA as shown in Fig. 5D, 5E, and S5, topo-i may activate TRPML1 in a ROS-dependent manner and increase PI(3,5)P2 binding with TRPML1 (Nat Commun 1, 38, Dong et al., 2010). Consecutive Ca2+ release via TRPML1 activated CaMK2G and is followed by enhanced lysosomal transport toward the MTOC via JIP4 phosphorylation.

    We have added the revised Fig.4A and 4B data in Fig. S8A and S8B, and the related description of these experiments in the Discussion section (page 11, lines 401 to 409). We have also added the data in revised Fig. 4C to Fig. S6 and the related description of these experiments in the Results section (page 7, lines 266 to 267).

    Conversely, we showed that benzimidazoles, including albendazole, induce lysosomal clustering mediated by JIP4, TRPML1, ALG2, and Rab7. Moreover, benzimidazoles showed lysosomal clustering activity within a narrow concentration range, as shown in Fig. S7D. Benzimidazoles inhibit tubulin polymerization (*Int J Paras *18:885–936. Lacey et al., 1988). We hypothesized that the effect of tubulin polymerization induced by benzimidazole plays a key role in the induction of lysosomal clustering as described in the Discussion section. To clarify this, we observed the behavior of tubulin filaments in response to various albendazole concentrations under confocal microscopy. As shown in revised Fig. 4D, conditions where albendazole was administered to induce lysosomal clustering, tubulin filaments were observed only near the MTOC, and the filaments in the cell periphery were disassembled. In contrast, when exposed to higher albendazole concentrations, tubulin filaments throughout the cell were disassembled, resulting in the inhibition of lysosomal clustering This would explain why benzimidazole exerts lysosomal clustering activity within a narrow concentration range. Under JIP4, TRPML1, ALG2 and Rab7 silencing, lysosomes may fail to interact with microtubules, resulting in the inhibition of lysosomal clustering. We postulated that albendazole-induced lysosomal clustering is not mediated by factors activated by specific stimuli in lysosomal transport but, rather, is induced by spatially constraining conventional lysosomal transport mediated by various adaptors (i.e., JIP4, TRPML1, ALG2, and Rab7) through tubulin disassembly. We have added the data in Fig. S9C and the related description of these experiments in the Discussion section (page 12, lines 428 to 436).

    A B

    C

    D

    __Revised Fig. 4. __

    1. SH-SY5Y cells were treated with the indicated compounds (10 µM) for 4 h. The amount of intracellular reactive oxygen species (ROS) is examined by ROS Assay Kit -Highly Sensitive DCFH-DA (Dojindo) and the normalized pixels above threshold as measured using an INCellAnalyzer 2200 and ImageJ.
    2. SH-SY5Y cell lines were pretreated with 0.1 mM N-acetyl-L-cysteine (NAC) for 24 h and then treated with the indicated compound (10 µM) for an additional 4 Cells were fixed and stained with anti-g-tubulin (green) and anti-LAMP2 (red) antibodies. Lysosomal distribution was examined using an INCellAnalyzer 2200 and quantified using ImageJ software.
    3. SH-SY5Y cells were treated with teniposide, amsacrine, etoposide, albendazole (1, 5, 10 µM), oxibendazole (0.1, 0.5, and 1 µM), or and mebendazole (0.5,1, and 5 µM) for 4 h, and stained with Fluo4-AM for 30 min. The fluorescence intensity was measured using a plate reader.
    4. SH-SY5Y cells were treated with albendazole (10 and 100 µM) or nocodazole (0.5 and 10 µM) for 4 h. Cells were fixed and stained with LAMP1 (red) and a-tubulin (green) antibodies.
    1. The authors should clarify the functional advantage of these drugs identified in this study as drugs for Parkinson's disease by comparing with known autophagy inducers such as Torin1 or rapamycin.

    RESPONSE:

    To evaluate the functional advantage of lysosome-clustering compounds over Torin1, we evaluated the degradation activity of insoluble aSyn induced by the addition of aSyn fibrils to aSyn-GFP cells. Torin1 induced the degradation of insoluble aSyn by autophagy, as shown in revised Fig. 5A. However, the degradation activity of albendazole was more vigorous, as shown in revised Fig. 5B. In contrast, we observed that Torin1 exhibited more autophagic induction activity than albendazole, as assessed using Halo-LC3. Similar results were obtained with teniposide (revised Fig. 5C). These results suggest that albendazole, with its ability to concentrate lysosomes around the degradation substrate, facilitates more effective degradation of insoluble aSyn than Torin1. This presents a significant advantage in the development of therapeutics for Parkinson's Disease. Moreover, Torin1 acts on the upstream signals of autophagy by inhibiting mTORC1, potentially impacting diverse cellular responses. Conversely, compounds that induce lysosomal clustering target the final step of autophagic degradation, which may have fewer side effects. We have added the description of these experiments in the Results section (page 10, lines 366 to 380) and the Discussion section (page 11 lines 410 to 412) and presented the data in Fig. S7B–S7E and Fig. 6D.

    A____ ____B

    C

    __Revised Fig. 5. __

    1. SH-SY5Y cells overexpressing aSyn-GFP were transfected with aSyn fibril (0.2 µg/mL) using Lipofectamine 3000. After 48 h, the transfection reagent was washed out, and the SH-SY5Y cells were treated with 100 nM Torin1 with or without 100 nM bafilomycin A1 for 8 h (B). Cell lysates were separated into Triton X-100–soluble (soluble) and pellet fractions (insoluble), then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with the indicated antibody(left). The amount of insoluble aSyn was quantified using Image J software (C).
    2. SH-SY5Y cells overexpressing aSyn-GFP were transfected with aSyn fibril (0.2 µg/mL) using Lipofectamine 3000. After 48 h, and washing out the transfection reagent, SH-SY5Y cells were treated with albendazole (10μM) with or without 100 nM Torin1 for 8 h. Cell lysates were separated into Triton X-100–soluble (soluble) and pellet fractions (insoluble), then subjected to SDS-PAGE and immunoblotting with the indicated antibody (left). The amount of insoluble aSyn was quantified using Image J software.
    3. SH-SY5Y cells stably expressing Halo-LC3 were labeled for 20 min with 100 nM TMR-conjugated ligand in a nutrient-rich medium. After washing with PBS and incubating the cells in normal medium for 30 min, cells were treated with DMSO, teniposide (10 μM), albendazole (10 µM), and/or Torin1 (100 nM) for 8 h. Cell lysates were immunoblotted with the indicated antibody and analyzed by in-gel fluorescence detection (left). The HaloTMR band intensity was normalized by the sum of the band intensities of HaloTMR-LC3B and HaloTMR (right).
    1. Related to the previous question, in Fig.6A and B additional data comparing novel compounds with established autophagy inducers, such as torin1 and rapamycin, should be included and discussed.

    RESPONSE:

    As indicated in a previous response, we evaluated the autophagic induction activity of Torin1, and the results have been added to Fig. 6D. In addition, co-treatment with Torin1 and teniposide or albendazole induced autophagy more effectively than Torin1 treatment alone, without affecting mTOR inhibition activity (revised Fig. 4C). These findings indicate that the induction of autophagy by lysosomal clustering compounds is not caused by autophagosome formation but by the formation of autolysosomes. We have added a description of these experiments in the Results section (page 9, lines 316 to 322) and have added the data in Fig. 6D.

    1. The authors should examined whether increased degradation of insoluble proteins and αSyn are dependent on JIP4.

    RESPONSE:

    As the Reviewer suggested, we have examined whether lysosomal accumulation through the JIP4-TRPML1 pathway is crucial for the degradation of aSyn aggregates. We evaluated the degradation activity of insoluble aSyn induced by the addition of aSyn fibrils to aSyn-GFP cells when JIP4, TMEM55B, or TRPML1 were knocked down. Interestingly, the insoluble fraction assay showed that JIP4 and TRPML1 knockdown regulated the decrease of aSyn-GFP and p-aSyn levels in the insoluble fraction for both DMSO and albendazole treatments. The results were particularly more pronounced with TRPML1 knockdown. However, the knockdown of TMEM55B did not produce such findings (revised Fig. 6). These data suggest that lysosomal clustering via the JIP4–TRPML1 pathway plays a significant role in aSyn degradation. We have added a relevant description in the Results section (page 10, lines 373 to 380) and have added the data in Fig. S7F and S7G.

    __Revised Fig. 6. __

    SH-SY5Y cells over-expressing aSyn-GFP were transfected with the indicated siRNAs for 24 h and then transfected with aSyn fibril (0.2 µg/mL) using Lipofectamine 3000 for 48 h. After washing out the transfection reagent, the SH-SY5Y cells were treated with dimethyl sulfoxide or albendazole (10 μM) for 8 h. Cell lysates were separated into Triton X-100–soluble (soluble) and pellet fractions (insoluble) and subjected to SDS-PAGE and immunoblotting with the indicated antibody. The bar graph presents the ratio of the insoluble aSyn-GFP to the soluble GAPDH or insoluble p-aSyn to the soluble GAPDH of the intensity of the data in panel F. Data are expressed as mean ± standard deviation.

    1. Authors only utilized. SH-SY5Y cells in this study. It is important to examine whether these compounds also regulate lysosomal positioning and autophagy in other cell lines.

    RESPONSE:

    As per the Reviewer’s suggestion, we evaluated the lysosomal-clustering activity induced by topo-i and benzimidazole in human adenocarcinoma HeLa cells. As shown in revised Fig. 7A and 7B compounds do not induce lysosomal clustering or autophagy in HeLa cells. Furthermore, in the case of benzimidazole, they transport lysosomes to the cell periphery. Previously, we found that oxidative stress accumulates lysosomes in a neuroblastoma-specific manner through the TRPML1–phosphoJIP4-dependent mechanism (EMBO J 41: e111476, Sasazawa et al., 2022). Since we have demonstrated that topo-i-mediated lysosomal trafficking is dependent on the TRPML1–phosphorylated JIP4 complex, we hypothesized that several molecules involved in lysosomal trafficking are absent in HeLa cells.

    In contrast, we showed that albendazole-induced lysosomal clustering is due to tubulin depolymerization. Therefore, we examined the relationships between tubulin depolymerization and lysosomal clustering induced by albendazole in HeLa cells and found that albendazole did not induce lysosomal clustering but rather inhibited it at higher concentrations (revised Fig. 7B). Interestingly, similar to SH-SY5Y cells, a low albendazole concentration (10 μM) induced tubulin depolymerization only at the cell periphery, whereas a high concentration (100 μM) depolymerized the entire cell (revised Fig. 7C). However, unlike SH-SY5Y cells, no characteristic accumulation of tubulin filaments was observed near the MTOC under low albendazole concentration (10 µM); instead, they were arranged around the nucleus. Concurrently, lysosomes were around these dispersed tubulin filaments. Therefore, the differences in the effects of benzimidazole in HeLa and SH-SY5Y cells lies in the dose-dependent effects on the state of tubulin filaments. We have added a relevant description in the Discussions section (page 12, lines 419 to 422, and 437 to 453).

    __ A__

    __ B__

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    __Revised Fig. 7. __

    HeLa cells were treated with teniposide (10 μM), amsacrine (10 μM), etoposide (10 μM), albendazole (10 μM), oxibendazole (1 μM), or mebendazole (5 μM). Images were captured using an INCellAnalyzer2200. INCellAnalyzer2200 images were analyzed using ImageJ for lysosomal clustering. The graph presents the lysosomal clustering values (n > 30). Data are expressed as mean ± standard deviation (SD). ****P HeLa cells were treated with teniposide (10 μM), amsacrine (10 μM), etoposide (10 μM), albendazole (10 μM), oxibendazole (1 μM), or mebendazole (5 μM) for 4 h. Cell lysates were immunoblotted with the indicated antibodies. The amount of LC3II was estimated using Image J software (bottom panel). HeLa cells were treated with albendazole at specified concentrations (in µM). After treatment, cells were fixed and stained with anti-g-tubulin (green) and anti-LAMP2 (red) antibodies, followed by imaging with an INCellAnalyzer2200. INCellAnalyzer2200 images were processed and analyzed using ImageJ for lysosomal clustering. The graph presents the lysosomal clustering values (n > 30). Data are expressed as mean ± SD. *P  HeLa cells were treated with albendazole (10 and 25 µM) for 4 h. Cells were fixed and stained with LAMP1 (red) and a-tubulin (green) antibodies.

    1. The authors conclude that the six compounds do not mediate mTOR signaling in Fig. 3, but should more carefully describe in the manuscript why they performed this experiment and what the results mean for.

    RESPONSE:

    As per the Reviewer’s advice, we have changed the description in the manuscript as follows:

    Previous studies have shown that lysosomal retrograde transport regulates autophagic flux by facilitating autophagosome formation by suppressing mTORC1 and expediting fusion between autophagosomes and lysosomes (Kimura et al, 2008; Korolchuk et al, 2011). Conversely, we recent found that acrolein/H2O2 induces lysosomal clustering in an mTOR-independent manner (Sasazawa et al., 2022). In this study, we aimed to identify pharmacologic agents that act downstream rather than upstream in the autophagy pathway, with the goal of minimizing side effects. Therefore, we evaluated the effects of the compounds on the mTOR pathway. As shown in Fig. 3, these compounds induced lysosomal clustering without affecting mTOR activity, indicating their potential as promising candidates for PD therapy. We have added the description of these experiments in the Results section (page 6, lines 202 to 208 and line 217).

    Minor comments.

    1. The name of the compound should be written in the red point of Fig.2A.

    RESPONSE:

    We have included the names of the six compounds identified and are listed in Fig. 2A.

    1. Regarding images of Fig.2B, the magnified images and quantitative data should be added.

    RESPONSE:

    We have included magnified images, as well as the quantitative results of lysosome clustering analysis using INCellAnalyzer2200 in Fig. 2B.

    1. The results of Fig.2C need to be explained more carefully. A quantitative data is missing.

    RESPONSE:

    We have included the quantitative results of western blot in Fig. 2B.

    1. Fig.S2, which compares autophagy activity with conventional agents, should be quantified and added to the Fig.3.

    RESPONSE:

    We have presented the results of RFP/GFP quantification performed by FACS analysis using SH-SY5Y cells stably expressing RFP-GFP-LC3 in Fig. S2, which is equivalent to the quantification of the data in the Fig. S2 image. These data are now presented as Fig. S2B. Since Fig. 3 focuses on mTOR signaling, we preferred to retain the figure number.

    1. In the statistical analysis of Fig.4B, the clustering value was increased by siRILP, which should be briefly described in the manuscript.

    RESPONSE:

    On the contrary, the enhancement of lysosomal retrograde transport in RILP knockdown cells in Fig. 4B suggests the potential involvement of RILP in anterograde transport. However, to the best of our knowledge, no reports have investigated this matter. We presume that negative feedback mechanisms may be present. We have added this description to the Results section (page 7 lines 238 to 241).

    1. In Fig.4A and B, it is possible that the knockdown efficiency of siRILP and siTMEM55B was not sufficient to observe the effect on lysosomes, and this concern should be described in the manuscript.

    RESPONSE:

    We established starvation conditions, which induce TMEM55B-dependent lysosomal retrograde transport, as a positive control and evaluated the lysosomal induction activity of compounds when TMEM55B was knocked down. As shown below, lysosome accumulation was suppressed only when subjected to starvation treatment, indicating sufficient knockdown efficiency of TMEM55B. These compounds induced lysosomal clustering independently of TMEM55B, unlike under starvation conditions. We have added a description of these experiments in the Results section and presented the data in Fig. S4A (page 7, lines 232 to 237).

    On the other hand, we were unable to establish a positive control for RILP knockdown experiments because conditions that regulate RILP-dependent lysosomal distribution dependent are not understood. While we cannot completely rule out the possibility of insufficient knockdown efficiency, considering that RILP knockdown appears to paradoxically enhance lysosomal induction, as mentioned above, it is reasonable to assume that the knockdown effect has occurred.

    __Revised Fig. 8. __

    SH-SY5Y cells were transfected with TMEM55B siRNA for 48 h and then treated with teniposide (10 μM), albendazole (10 μM), or starvation medium for 4 h. Cells were fixed and stained with anti-g-tubulin (green) and anti-LAMP2 (red) antibodies. Images were captured using an INCellAnalyzer2200. INCellAnalyzer2200 images were analyzed using ImageJ for lysosomal clustering. The graph presents the lysosomal clustering values (n > 30). Data are expressed as mean ± standard deviation (SD). ****P

    1. The authors should add the results of the WB experiment showing the amount of JIP4 protein in Fig.5G.

    RESPONSE:

    We have added western blot data that introduce flag-JIP4 into JIP4KO SH-SY5Y cells, which are presented in Fig. 5G.

    1. In Fig.5F, images of JIP4KO cells that do not express FLAG-JIP4 should be added as controls, and further quantification should be done on cells in all three conditions.

    RESPONSE:

    We have added immunofluorescence data that do not express flag-JIP4 in Fig. 5F, which had been obtained simultaneously during the acquisition of other images. Furthermore, we quantified lysosomal distribution, which is shown in Fig. 5E. Using ImageJ, we automatically delineated approximately 70% of the cell area toward the cell center and designated the region excluded from this area as the cellular peripheral region (revised Fig. 9A). Subsequently, we quantified the proportion of lysosomes contained within that region in cells expressing flag-JIP4 (revised Fig. 9B). We have added this experimental data in Fig. 5E.

    A B

    __Revised Fig. 9. __

    1. Approximately 70% of the cell area toward the cell center was automatically delineated using ImageJ, with the region excluded from this defined as the cellular peripheral region.
    2. JIP4 KO cells were transfected with flag-tagged JIP4 (wild-type and T217A) for 24 h and treated with teniposide (10 μM) for 4 h. Cell lysates underwent SDS-PAGE and were immunoblotted with anti-JIP4 and anti-actin The graph displays the percentage of cells with peripheral lysosomes. Data are expressed as mean ± standard deviation. *P 20).
    1. In Fig.6A, the total amount of JIP4 seems to change in some agent treatments, which needs to be explained.

    RESPONSE:

    As per our response to Reviewer 1, we evaluated the decrease in JIP4 expression by WB after 4 h of treatment with 10 μM teniposide. The teniposide-induced decrease of JIP4 was suppressed by bafilomycinA1 co-treatment, indicating that JIP4 was degraded by teniposide-induced autophagy (revised Fig. 3). We have added the data in Fig. S6, and the related description of these experiments have been added to the Results section (page 8, lines 289 to 293).

    1. In Fig.7C and D, the effect of drug treatment on the amount of ubiquitinated proteins should also be checked.

    RESPONSE:

    We have included ubiquitin protein blots in Fig. 7C and 7D.

    1. In Fig.8B, it is described that lysosomes are more localized in αSyn by drug treatment, but more convincing images and quantitative data are needed.

    RESPONSE:

    . The colocalization of LAMP2 and aSyn-GFP aggregates was assessed by measuring the fluorescence values of lysosomes in contact within the aSyn-GFP aggregation area using ImageJ. We have added this quantified data in Fig. 8D.

    Reviewer #2 (Significance (Required)): Although the reviewer appreciates the discovery of novel drugs to induce autophagy through regulating lysosomal positioning, the detailed action of these compounds and their superiority in the field are not clear.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)):____ __ In this manuscript, Date et al. sought to identify compounds that promote protein aggregates clearance - in particular those formed by mutant alpha synuclein. Briefly the authors screened a library of clinically approved compounds for inducers of lysosomal clustering followed by a secondary screen for autophagy inducers. By this two-step procedure, the authors identified three topoisomerase inhibitors and three anthelmintics as hits. Next, the authors unveiled that lysosomal clustering induced by these compounds is independent of mTORC1 but requires TRPML1 and JIP4. Moreover, the topoisomerase inhibitors hits involved phosphorylation of JIP4 while the anthelmintics additionally required Rab7 and ALG2. Intriguingly, the authors found that lysosomal clustering was prerequisite to autophagy induction. Focusing on the class of anthelmintics (i.e. albendazole) the authors showed that these induce autophagy to degrade aggregates formed upon proteasome inhibition. Lastly, the authors demonstrated that albendazole also led to increased degradation of αSyn aggregates through autophagy induction.

    Major points

    1. Most importantly, the authors need to tone down the significance of their findings throughout the manuscript. For examples, they should restrain from using "nullified" when it is really reduced only by 10-25 %.

    RESPONSE:

    We have changed the description in the manuscript according to the Reviewer’s suggestion.

    1. The authors claim that the topoisomerase inhibitors led to JIP4 phosphorylation while Figure 5C actually shows the opposite (partially reduced phosphorylation compared to DMSO treatment) and the Jak3 inhibitor has no obvious effect. The authors should quantify the phostag results.

    RESPONSE:

    We agree with the Reviewer that the Phos-tag PAGE results of JIP4 in Fig. 5C is complicated, and the bands were not clear. We have replaced these with more robust data (Fig. 5C).

    1. Figure 6A/B: Why do all compounds except Mebendazole affect the abundance of JIP4?

    RESPONSE:

    As per our response to Reviewer 1, we evaluated the decrease in JIP4 expression by WB after 4 h of treatment with 10 μM teniposide. The teniposide-induced decrease of JIP4 was suppressed by bafilomycinA1 co-treatment, indicating that JIP4 was degraded by teniposide-induced autophagy (revised Fig. 3). We have added the data in Fig. S6, and the related description of these experiments have been added to the Results section (page 8, lines 289 to 293).

    1. Figure 7C: The blot is not convincing. The authors should quantify this effect.

    RESPONSE:

    We evaluated and confirmed the degradation of p62 by albendazole, as shown in Fig. 7C.

    Reviewer #3 (Significance (Required)):

    Overall, the work of Date and colleague highlights the role of lysosomal clustering in clearing protein aggregates. Importantly, the identified classes of compounds might open new avenues for rationalizing treatment strategies for neurodegenerative diseases. However, several critical points remain.

  3. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, Date et al. sought to identify compounds that promote protein aggregates clearance - in particular those formed by mutant alpha synuclein. Briefly the authors screened a library of clinically approved compounds for inducers of lysosomal clustering followed by a secondary screen for autophagy inducers. By this two-step procedure, the authors identified three topoisomerase inhibitors and three anthelmintics as hits. Next, the authors unveiled that lysosomal clustering induced by these compounds is independent of mTORC1 but requires TRPML1 and JIP4. Moreover, the topoisomerase inhibitors hits involved phosphorylation of JIP4 while the anthelmintics additionally required Rab7 and ALG2. Intriguingly, the authors found that lysosomal clustering was prerequisite to autophagy induction. Focusing on the class of anthelmintics (i.e. albendazole) the authors showed that these induce autophagy to degrade aggregates formed upon proteasome inhibition. Lastly, the authors demonstrated that albendazole also led to increased degradation of αSyn aggregates through autophagy induction.

    Major points

    1. Most importantly, the authors need to tone down the significance of their findings throughout the manuscript. For examples, they should restrain from using "nullified" when it is really reduced only by 10-25 %.
    2. The authors claim that the topoisomerase inhibitors led to JIP4 phosphorylation while Figure 5C actually shows the opposite (partially reduced phosphorylation compared to DMSO treatment) and the Jak3 inhibitor has no obvious effect. The authors should quantify the phostag results.
    3. Figure 6A/B: Why do all compounds except Mebendazole affect the abundance of JIP4?
    4. Figure 7C: The blot is not convincing. The authors should quantify this effect.

    Significance

    Overall, the work of Date and colleague highlights the role of lysosomal clustering in clearing protein aggregates. Importantly, the identified classes of compounds might open new avenues for rationalizing treatment strategies for neurodegenerative diseases. However, several critical points remain.

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    Referee #2

    Evidence, reproducibility and clarity

    In this study, the authors focused on lysosome positioning and autophagy activity to search for novel agents effective against Parkinson's disease. As a result, several compounds were successively identified, including Topoisomerase inhibitors and Benzimidazole. Authors showed that these agents regulate lysosomal positioning through different pathways but commonly require JIP4 to regulate lysosomal positioning and subsequent autophagy. They also showed that albendazole treatment promoted the degradation of insoluble ubiquitinated proteins and αSyn in cultured cells.

    Major Comments.

    • Two compounds, for instance teniposide and albendazole both requires JIP4 and/or TRPML1 to regulate lysosomal positioning and autophagy but their action seems different. What is the actual mechanism by which these compounds require JIP4/TRPML1. How inhibition of Topoisomerase leads to increase of JIP4 phosphorylation? Do teniposide and albendazole both affect calcium release from TRPML1?
    • The authors should clarify the functional advantage of these drugs identified in this study as drugs for Parkinson's disease by comparing with known autophagy inducers such as Torin1 or rapamycin.
    • Related to the previous question, in Fig.6A and B additional data comparing novel compounds with established autophagy inducers, such as torin1 and rapamycin, should be included and discussed.
    • The authors should examined whether increased degradation of insoluble proteins and αSyn are dependent on JIP4.
    • Authors only utilized. SH-SY5Y cells in this study. It is important to examine whether these compounds also regulate lysosomal positioning and autophagy in other cell lines.
    • The authors conclude that the six compounds do not mediate mTOR signaling in Fig. 3, but should more carefully describe in the manuscript why they performed this experiment and what the results mean for.

    Minor comments.

    • The name of the compound should be written in the red point of Fig.2A.
    • Regarding images of Fig.2B, the magnified images and quantitative data should be added.
    • The results of Fig.2C need to be explained more carefully. A quantitative data is missing.
    • Fig.S2, which compares autophagy activity with conventional agents, should be quantified and added to the Fig.3.
    • In the statistical analysis of Fig.4B, the clustering value was increased by siRILP, which should be briefly described in the manuscript.
    • In Fig.4A and B, it is possible that the knockdown efficiency of siRILP and siTMEM55B was not sufficient to observe the effect on lysosomes, and this concern should be described in the manuscript.
    • The authors should add the results of the WB experiment showing the amount of JIP4 protein in Fig.5G.
    • In Fig.5F, images of JIP4KO cells that do not express FLAG-JIP4 should be added as controls, and further quantification should be done on cells in all three conditions.
    • In Fig.6A, the total amount of JIP4 seems to change in some agent treatments, which needs to be explained.
    • In Fig.7C and D, the effect of drug treatment on the amount of ubiquitinated proteins should also be checked.
    • In Fig.8B, it is described that lysosomes are more localized in αSyn by drug treatment, but more convincing images and quantitative data are needed.

    Significance

    Although the reviewer appreciates the discovery of novel drugs to induce autophagy through regulating lysosomal positioning, the detailed action of these compounds and their superiority in the field are not clear.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Date et al employed a cell model by stably expressing LGP120-mCherry and GFP-gamma-tubulin to carry out high-content screening in search of chemical compounds that enhance lysosomal clustering and autophagy. They found 6 clinically approved drugs categorized as topoisomerase II inhibitors and the benzimidazole class. They further validated these compounds by a set of well-designed experiments including autophagy flux assays and mTOR dependence. In the mechanistic study, they demonstrated the compounds induce lysosomal clustering in a JIP4-TRPML1-dependent manner. In a PD cell model, one of the compounds albendazole exhibited the effect on boosting the degradation of insoluble alpha-synuclein. The study is of interest, and the cell model and the approach generated by the authors would be transferable for future studies of other high-content imaging screening. Most of the data is clear and convincing.

    Major comments:

    1. In addition to its role in facilitating a-syn turnover by autophagy, Is the chemical protective against a-syn toxicity?
    2. Please elaborate why albendazole does not change the levels of soluble a-syn, but those of insoluble, as shown Fig 8D.
    3. Fig 6A shows that some of the compounds (Teniposide, Amsacrine) affect the levels of JIP4. Can albendazole also reduce JIP4 levels. It might be interesting to test this, as JIP4 is important for lysosomal clustering.

    Minor comments:

    The writing is good generally. Please tide up the text in a few occasions to make the expressions more formal.

    Significance

    Significance: The study generated a new approach for high-throughput screening of compounds to enhance lysosomal clustering.

    Audience: Basic and clinical research

    Expertise: Programmed cell death, neurodegenerative diseases

  6. Version published to 10.1101/2023.11.07.566147v1 on bioRxiv