Novel autophagy inducers by accelerating lysosomal clustering against Parkinson’s disease
- Department of Biology, Graduate School of Science and Engineering, Chiba University, Inage-ku, Japan
- Department of Neurology, Juntendo University Faculty of Medicine, Japan
- Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Japan
- Division for Development of Autophagy Modulating Drugs, Juntendo University Faculty of Medicine, Japan
- Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Japan
- Division of Gene Regulation, Cancer Center, Fujita Health University, Japan
- Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Japan
- Department of Biology, Graduate School of Science, Chiba University, Inage-ku, Japan
- Neurodegenerative Disorders Collaborative Laboratory, RIKEN Center for Brain Science, Japan
- Department of Neurology, Institute of Medicine, University of Tsukuba, Japan
Figures
Figure 1
Method for screening lysosomal-clustering compounds.
(A) Scheme for the process of screening for lysosomal-clustering compounds. (B) Strategies for screening lysosomal-clustering compounds. If lysosomes accumulate around the microtubule-organizing …
-
Figure 1—source data 1
Data table of lysosomal clustering chemical screening.
- https://cdn.elifesciences.org/articles/98649/elife-98649-fig1-data1-v2.zip
Figure 2 with 2 supplements
Identification of autophagy inducers via lysosomal clustering.
(A) SH-SY5Y cells expressing red fluorescent protein (RFP)-green fluorescent protein (GFP) tandem fluorescent-tagged LC3 (R–G–LC3) were analyzed by flow cytometer after treatment with 63 …
-
Figure 2—source data 1
Uncropped blot images of Figure 2C.
- https://cdn.elifesciences.org/articles/98649/elife-98649-fig2-data1-v2.zip
Figure 2—figure supplement 1
Flow cytometer analysis of red fluorescent protein (RFP)-green fluorescent protein (GFP) tandem fluorescent-tagged LC3 (R–G–LC3) during second screening.
(A) SH-SY5Y tetracycline-on (Tet-on) cells expressing R-G-LC3 were cultured in the presence of doxycycline (Dox). After Dox removal, the cells were treated with either normal medium or subjected to …
Figure 2—figure supplement 2
Confocal images of SH-SY5Y expressing red fluorescent protein (RFP)-green fluorescent protein (GFP) tandem fluorescent-tagged LC3 (RFP-GFP-LC3).
(A) SH-SY5Y cells tetracycline-on (Tet-on) expressing RFP-GFP-LC3 were treated with dimethyl sulfoxide, starvation medium, Torin1 (1 μM), teniposide (10 μM), or albendazole (10 μM). Post-treatment, …
Figure 3
Lysosomal-clustering compounds are not dependent on mTORC1 activity.
(A) SH-SY5Y cells were treated with starvation medium, Torin1 (1 μM), teniposide (10 μM), amsacrine (10 μM), etoposide (10 μM), albendazole (10 μM), oxibendazole (1 μM), or mebendazole (5 μM) for 4 …
-
Figure 3—source data 1
Uncropped blot images of Figure 3A.
- https://cdn.elifesciences.org/articles/98649/elife-98649-fig3-data1-v2.zip
Figure 4 with 2 supplements
Lysosome-clustering compounds accumulate lysosomes in microtubule-organizing center (MTOC) in a JIP4-dependent manner.
(A) SH-SY5Y cells were transfected with the indicated siRNAs for 48 hr and then treated with teniposide (10 μM) and albendazole (10 μM) for 4 hr. Cells were fixed and stained with anti-γ-tubulin …
Figure 4—figure supplement 1
siRNAs and knockdown efficiency of each lysosomal factor.
(A, B) SH-SY5Y cells were treated with the specified siRNAs, and the knockdown efficiency of each siRNA was validated via reverse-transcription quantitative polymerase chain reaction (n=3 technical …
Figure 4—figure supplement 2
Lysosomal-clustering analysis of other lysosomal-clustering compounds using JIP4KO cells.
(A) SH-SY5Y cells were transfected with TMEM55B siRNA for 48 hr and then treated with teniposide (10 μM), albendazole (10 μM), or starvation medium for 4 hr. Cells were fixed and stained with …
Figure 5 with 1 supplement
Lysosomal clustering induced by topoisomerase inhibitor requires JIP4 phosphorylation.
(A) SH-SY5Y cells were treated with dimethyl sulfoxide (DMSO), teniposide (10 μM), amsacrine (10 μM), etoposide (10 μM), albendazole (10 μM), oxibendazole (1 μM), or mebendazole (5 μM) with or …
-
Figure 5—source data 1
Uncropped blot images of Figure 5C and 5G.
- https://cdn.elifesciences.org/articles/98649/elife-98649-fig5-data1-v2.zip
Figure 5—figure supplement 1
Calcium flux analysis of lysosomal clustering chemicals.
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 hr, and stained with …
Figure 6 with 1 supplement
Lysosomal clustering dependent on JIP4 is slightly involved in autophagy activity.
(A) SH-SY5Y cells stably expressing Halo-LC3 were labeled for 20 min with 100 nM tetramethylrhodamine (TMR)-conjugated ligand in a nutrient-rich medium. After washing with phosphate-buffered saline …
-
Figure 6—source data 1
Uncropped gel fluorescence images of Figure 6A and 6D.
- https://cdn.elifesciences.org/articles/98649/elife-98649-fig6-data1-v2.zip
Figure 6—figure supplement 1
Western blot analysis of JIP4 expression.
SH-SY5Y cells were treated with 10 µM teniposide and with or without 30 nM bafilomycin A1 for 4 hr. Cell lysates were immunoblotted with anti-JIP4 and actin antibodies.
Figure 7
JIP4-dependent lysosomal clustering is involved in aggresome clearance by MG132.
(A) SH-SY5Y cells were treated with MG132 (1 μM) for 16 hr to induce aggresome formation. After washing out MG132 with normal medium at intervals of 4, 8, or 12 hr, cell lysates were separated into …
-
Figure 7—source data 1
Uncropped blot images of Figure 7.
- https://cdn.elifesciences.org/articles/98649/elife-98649-fig7-data1-v2.zip
Figure 8 with 1 supplement
Albendazole reduces α-synuclein (αSyn)-green fluorescent protein (GFP) aggregates in a lysosome-dependent manner.
(A) SH-SY5Y cells overexpressing αSyn-GFP were transfected with αSyn fibril (0.2 µg/mL) using Lipofectamine 3000. After 48 hr, the cells were fixed and stained with an anti-LAMP2 antibody (red) and …
-
Figure 8—source data 1
Uncropped blot images of Figure 8D.
- https://cdn.elifesciences.org/articles/98649/elife-98649-fig8-data1-v2.zip
Figure 8—figure supplement 1
Analysis of the degradation of α-synuclein (αSyn) aggregates.
(A) SH-SY5Y cells expressing αSyn-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 …
Figure 9
Analysis of lysosomal clustering mechanism of action for topoisomerase inhibitors.
(A) SH-SY5Y cells were treated with the indicated compounds (10 µM) for 4 hr. The amount of intracellular reactive oxygen species (ROS) is examined by ROS Assay Kit -Highly Sensitive DCFH-DA …
-
Figure 9—source data 1
Uncropped blot images of Figure 9D.
- https://cdn.elifesciences.org/articles/98649/elife-98649-fig9-data1-v2.zip
Figure 10
Analysis of lysosomal clustering mechanism of action for benzimidazole mechanism.
(A) 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 …
-
Figure 10—source data 1
Uncropped blot images of Figure 10B.
- https://cdn.elifesciences.org/articles/98649/elife-98649-fig10-data1-v2.zip
