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Rapamycin activates mammalian microautophagy
Journal of Pharmacological Sciences 140 (2019) 201e204
Contents lists available at ScienceDirect
Journal of Pharmacological Sciences journal homepage: www.elsevier.com/locate/jphs
Short Communication
Rapamycin activates mammalian microautophagy Masahiro Sato a, Takahiro Seki a, *, Ayumu Konno b, Hirokazu Hirai b, Yuki Kurauchi a, Akinori Hisatsune a, c, d, Hiroshi Katsuki a a
Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan Department of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Japan c Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto, Japan d Program for Leading Graduate Schools “HIGO (Health Life Science: Interdisciplinary and Glocal Oriented) Program”, Kumamoto University, Kumamoto, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 April 2019 Received in revised form 7 May 2019 Accepted 14 May 2019 Available online 24 May 2019
Autophagy-lysosome proteolysis is classified into macroautophagy (MA), microautophagy (mA) and chaperone-mediated autophagy (CMA). In contrast to MA and CMA, mA have been mainly studied in yeast. In 2011, mammalian mA was identified as a pathway to deliver cytosolic proteins into multivesicular bodies. However, its molecular mechanism is quite different from yeast mA. Using a cell-based method to evaluate mA and CMA, we revealed that rapamycin, an activator of yeast mA, significantly activated mammalian mA. Although rapamycin activates MA, mA was also activated by rapamycin in MAdeficient cells. These findings suggest that rapamycin is a first-identified activator of mammalian mA. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Microautophagy Chaperone-mediated autophagy Rapamycin
Removal of misfolded proteins by protein degradation systems is important for various neuronal functions and survival.1 There are two major protein degradation systems: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system.1 The latter is further classified into macroautophagy (MA), microautophagy (mA) and chaperone-mediated autophagy (CMA).2 Regarding UPS and MA, there are many reports about their involvements in physiological functions and disease pathogenesis.1 Recently, CMA has been focused on the relationship with Parkinson's disease and other neurodegenerative diseases.3 In contrast, most studies about mA have been conducted in yeast.4 In yeast mA, substrate are incorporated into vacuoles via the invagination of vacuolar membrane.4 However, the molecular mechanism of mammalian mA had remained unknown for a long period. In 2011, Sahu R et al identified a novel pathway to deliver cytosolic soluble proteins into late endosome/multivesicular bodies (MVBs) in an Hsc70-dependent manner and postulated this pathway as mammalian mA.5 Since mammalian mA is associated with endosomal sorting complex
* Corresponding author. Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 OeHonmachi, Chuo-ku, Kumamoto 862-0973, Japan. Fax: þ81 96 371 4182. E-mail address: [email protected] (T. Seki). Peer review under responsibility of Japanese Pharmacological Society.
required for transport (ESCRT) proteins that are involved in multivesicular formation,6 it is postulated that mA in yeast and mammalian cells are regulated in different manners. Recently, we have established a novel method to separately assess mA and CMA activities in mammalian cells.7 We used GAPDH fused with HaloTag (GAPDH-HT) as a marker of CMA and mA activities because GAPDH is recognized by Hsc70, which is commonly involved in both CMA and mA.3,5 This is the first method to evaluate mammalian mA in a single cell. Using this method, we revealed that prolonged serum deprivation activates CMA but does not affect mA in mammalian cells.7 Prolonged starvation is known to activate mA in yeast.4 These findings support the hypothesis that mA is divergently regulated in yeast and mammalian cells. It remains unknown how mA is regulated in mammalian cells and what stimuli activate mammalian mA. Rapamycin is an inhibitor of mammalian target of rapamycin complex 1 (mTORC1) and is widely used as a potent activator of MA.8 In addition, rapamycin is also known to activate mA in yeast.4 In the present study, we investigated the effect of rapamycin on mammalian mA using our method. LAMP2A (sense: 50 -GGCAGGAGUACUUAUUCUAGU-30 , antisense: 50 -UAGAAUAAGUACUCCUGCCAA-30 ) and TSG101 (sense: 50 CUAGUUCAAUGACUAUUAATT-30 , antisense: 50 -UUAAUAGUCAUUGAACUAGTT-30 ) siRNAs and MISSION siRNA Universal Negative Control were obtained from SigmaeAldrich (St. Louis, MO, USA).
https://doi.org/10.1016/j.jphs.2019.05.007 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Rapamycin was from AdipoGen Life Science (Liestal, Switzerland). The Nerve-Cell Culture System was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Penicillin/streptomycin solution was obtained from Nacalai Tesque (Kyoto, Japan). The HaloTag system (HaloTag vector and HaloTag ligand) was obtained from Promega (Madison, WI, USA). Flp-In system, neurobasal medium, B-27 serum free supplement, Lipofectamine RNAiMAX, and Lipofectamine 3000 were obtained from Invitrogen (Carlsbad, CA, USA). AD293 cells were obtained from Agilent Technologies (Santa Clara, CA, USA). Glass-bottomed culture dishes (35-mm diameter) were obtained from MatTek (Ashland, MA, USA). Mouse embryonic fibroblast (MEF) cells from Atg5-knockout (KO) mice and plasmid
to express LC3 constructs were kindly donated by Noboru Mizushima (University of Tokyo, Japan). AD293 and MEF cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 units/mL of penicillin and 100 mg/mL of streptomycin in a humidified atmosphere containing 5% CO2 at 37 C. Primary cultures of rat cortical neurons were prepared from E16 embryos of Wister/ ST rats as described previously.7 Dissociated neurons (3 105 cells/ dish) were cultured in the neurobasal medium supplemented with 2% B27 and 25 mM L-glutamate for 7 days in vitro (DIV). Transfection of siRNA and plasmid to express GFP-LC3 was conducted using Lipofectamine RNAiMAX and Lipofectamine 3000,
Fig. 1. Rapamycin activates mammalian mA in AD293 cells and primary cultured cortical neurons. (A) Punctate accumulation of GAPDH-HT in AD293 cells transfected with control, LAMP2A, or TSG101 siRNA (50 pmol). Cells were treated with vehicle (Veh, 0.1% DMSO) or rapamycin (500 nM) for 18 h after TMR-HT ligand labeling. Scale bars are 20 mm. (B) Quantitative analyses of GAPDH-HT puncta shown in A. * < p 0.05, ***p < 0.001 vs control siRNA-transfected cells treated with vehicle, #p < 0.05 (n ¼ 51e57). (C) Representative immunoblots of siRNA-mediated knockdown of LAMP2A and TSG101 in AD293/GAPDH-HT cells. Arrow indicates the specific band of TSG101. The amount of b-actin was evaluated as a loading control. (D) Punctate accumulation of GAPDH-HT in primary cultured cortical neurons transfected with control, LAMP2A, or TSG101 siRNA (50 pmol). Cells were treated with vehicle or rapamycin (500 nM) for 24 h after TMR-HT ligand labeling. Scale bars are 10 mm. (E) Quantitative analyses of GAPDH-HT puncta shown in C. **p < 0.01 vs control siRNA-transfected cells treated with vehicle, ##p < 0.01 (n ¼ 39e43). (F) Representative immunoblots of siRNA-mediated knockdown of LAMP2A and TSG101 in primary cultured cortical neurons. Arrow indicates the specific band of TSG101. The amount of b-actin was evaluated as a loading control.
M. Sato et al. / Journal of Pharmacological Sciences 140 (2019) 201e204
as described previously.7 To transiently express GAPDH-HT in primary cultured cortex neurons, we used adeno-associated virus serotype 9 (AAV9) vectors. The viral vectors expressed GAPDH-HT specifically in neurons under the control of the neuron-specific, modified synapsin I promoter.7 AAV9 vectors (2 1010 vg) were infected to cortical neurons on the glass bottom dishes on DIV1. Labeling with HT ligands fused with fluorescent dyes was conducted as described previously.7 Briefly, AD293/GAPDH-HT and Atg5-KO MEF cells on glass bottom dishes were labeled with 100 nM HT ligand fused with tetramethylrhodamine (TMR) for 10 min 2 days after transfection of siRNA or plasmids, cultured with vehicle (0.1% DMSO) or 500 nM rapamycin for 18 h and fixed with 4% paraformaldehyde (PFA). In this experiment, cells were treated with vehicle (0.1% DMSO) or 500 nM rapamycin. In primary cultured cortical neurons, cells were labeled with TMR-HT ligand on DIV6, cultured with vehicle or rapamycin for 24 h and fixed with 4% PFA. Fluorescent images of fixed cells were obtained using a confocal microscope, TCS SP5 (Leica, Wetzlar, Germany). We quantitatively analyzed the activities of CMA and mA by counting GAPDH-TH puncta labeled with TMR. All quantitative data are represented as mean ± standard error of the mean. Statistical differences were determined by one-way analysis of variance followed by post hoc test with Tukey's method. The difference was considered to be statistically significant, if p-value was less than 0.05. We have previously demonstrated that punctate accumulation of fluorescence-labeled GAPDH-HT under the knockdown of LAMP2A, a CMA-related protein, and TSG101, a mA-related protein, represents the activities of mA and CMA, respectively.7 In the present study, we investigated whether rapamycin affect mA/CMA activity in AD293 cells stably expressing GAPDH-HT.7 siRNAmediated knockdown of LAMP2A and TSG101 was conducted in the same method with our previous study,7 and these proteins were efficiently knocked down in the present study (Figs. 1C, F and 3C). Rapamycin significantly increased punctate accumulation of
203
GAPDH-HT in cells transfected with negative control siRNA and LAMP2A siRNA but did not affect it in cells transfected with TSG101 siRNA (Fig. 1A,B). Similar findings were observed in primary cultured cortical neurons, in which GAPDH-HT was transfected using adeno-associated viral vectors (Fig. 1D,E). An increase in GAPDH-HT puncta by rapamycin was more potent in neurons transfected with LAMP2A than in control neurons (Fig. 1D,E). This might be reflected by the excessive activation of mA for the compensation of impaired CMA. It is possible that this compensation is selectively observed in neurons. These findings suggest that rapamycin activates mammalian mA but does not activate CMA. Since rapamycin is a potent activator of MA, it is possible that an increase in GAPDH-HT puncta is reflected by MA activation. To exclude this possibility, AD293/GAPDH-HT cells were transiently transfected with GFP-LC3, which labels autophagosome. We rarely found the colocalization of GFP-LC3 puncta and GAPDH-HT puncta in the presence or absence of rapamycin (Fig. 2), suggesting that GAPDH-HT is not accumulated into autophagosomes even in the presence of rapamycin. In addition, we transiently transfected GAPDH-HT to mouse embryonic fibroblast (MEF) cells from MAdeficient Atg5 knockout (KO) mice. GAPDH-HT puncta were increased by rapamycin in Atg5-KO MEF cells transfected with control siRNA and LAMP2A siRNA but were not increased in cells transfected with TSG101 siRNA (Fig. 3A,B), similarly to AD293 cells and cortical neurons. These findings suggest that rapamycin activates mammalian mA, independently of MA. In the present study, we first identified rapamycin as an activator of mammalian mA. Then, how does rapamycin activate mammalian mA? Rapamycin is known to potently activate MA through the activation of ULK1, a MA initiator.8 In addition, rapamycin triggers the nuclear localization and activation of transcription factor EB (TFEB),9 which increases the expression of various genes related to MA and lysosome biogenesis.10 Namely, activation of MA by rapamycin is also mediated by the activation of TFEB. Because TFEB increases the lysosome biogenesis, rapamycin
Fig. 2. GAPDH-HT puncta are rarely colocalized with GFP-LC3-positive autophagosomes. Representative GAPDH-HT (left), GFP-LC3 (center) and merged (right) images of AD293 cells treated with vehicle (upper panels) and rapamycin (lower panels) for 18 h. Scale bars are 20 mm.
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Fig. 3. Rapamycin activates mammalian microautophagy in MA-deficient mouse embryonic fibroblast cells. (A) Punctate accumulation of GAPDH-HT in Atg5-knockout mouse embryonic fibroblast (MEF) cells transfected with control, LAMP2A, or TSG101 siRNA (50 pmol). Cells were treated with vehicle or rapamycin (500 nM) for 18 h after TMR-HT ligand labeling. Scale bars are 20 mm. (B) Quantitative analyses of GAPDH-HT puncta shown in A. *p < 0.05, ***p < 0.001 vs control siRNA-transfected cells treated with vehicle, ###p < 0.001 (n ¼ 45e47). (C) Representative immunoblots of siRNA-mediated knockdown of LAMP2A and TSG101 in Atg5-knockout MEF cells. The amount of b-actin was evaluated as a loading control.
could activate all pathways of autophagy-lysosome proteolysis. However, our present findings indicate that rapamycin fails to activate CMA. Therefore, an increase in lysosomes is not the main cause of the activation of mA by rapamycin. Homotypic fusion and protein sorting (HOPS) complex is involved in the fusion of MVBs and lysosomes.11 Vps11 and Vps18, the components of HOPS complex, are the target genes of TFEB.10 In addition, Rab7, which is reported to be involved in mammalian mA,12 is one of the target genes of TFEB.10 It is possible that TFEB-mediated increase in these proteins contributes to the activation of mammalian mA by rapamycin. According to this hypothesis, other TFEB inducers, including Torin-19, could be also activators of mammalian mA. Furthermore, HOPS complex is negatively regulated by mTORC1 through the phosphorylation of UV radiation resistance-associated gene product (UVRAG).13 Therefore, dephosphorylation of UVRAG by rapamycin might also be related to the activation of mammalian mA. Although further studies are necessary, our present findings provide the cue to reveal the detail mechanism how mA is regulated in mammalian cells. Recently, it has been reported that the decrease of CMA activity is involved in the onset and progression of various diseases, including neurodegenerative diseases.3 Indeed, an increase in specific miRNAs reduces the expression of Hsc70 and LAMP2A in the brains of Parkinson's disease patients.14 In addition, knockdown of LAMP2A in the midbrain causes Parkinson's disease-like symptoms in rats.15 Since Hsc70 is also involved in mammalian mA,5 it is considered that mammalian mA would be associated with various diseases. Taken together, in addition to CMA, mammalian mA could be a novel therapeutic target of various diseases. Conflicts of interests The authors declare no conflicts of interests. Acknowledgement This work is financially supported by Japan Society for the Promotion of Science KAKENHI [grant number 16K08276]. We
thank Prof. Noboru Mizushima (University of Tokyo, Japan) for giving us LC3 plasmids and Atg5-knockout MEF cells for this study.
References 1. Tanaka K, Matsuda N. Proteostasis and neurodegeneration: the roles of proteasomal degradation and autophagy. Biochim Biophys Acta Mol Cell Res. 2014;1843(1):197e204. https://doi.org/10.1016/j.bbamcr.2013.03.012. 2. Haspel JA, Choi AMK. Autophagy: a core cellular process with emerging links to pulmonary disease. Am J Respir Crit Care Med. 2011;184(11):1237e1246. https://doi.org/10.1164/rccm.201106-0966CI. 3. Li W, Nie T, Xu H, Yang J, Yang Q, Mao Z. Chaperone-mediated autophagy: advances from bench to bedside. Neurobiol Dis. 2019;122:41e48. https:// doi.org/10.1016/J.NBD.2018.05.010. 4. Li W, Li J, Bao J. Microautophagy: lesser-known self-eating. Cell Mol Life Sci. 2012;69(7):1125e1136. https://doi.org/10.1007/s00018-011-0865-5. 5. Sahu R, Kaushik S, Clement CC, et al. Microautophagy of cytosolic proteins by late endosomes. Dev Cell. 2011;20(1):131e139. https://doi.org/10.1016/ j.devcel.2010.12.003. 6. Hurley JH. ESCRT complexes and the biogenesis of multivesicular bodies. Curr Opin Cell Biol. 2008;20(1):4e11. https://doi.org/10.1016/j.ceb.2007.12.002. 7. Sato M, Seki T, Konno A, et al. Fluorescent-based evaluation of chaperonemediated autophagy and microautophagy activities in cultured cells. Genes Cells. 2016;21(8). https://doi.org/10.1111/gtc.12390. 8. Rabanal-Ruiz Y, Otten EG, Korolchuk VI. mTORC1 as the main gateway to autophagy. Essays Biochem. 2017;61(6):565e584. https://doi.org/10.1042/ EBC20170027. 9. Martina JA, Chen Y, Gucek M, Puertollano R. MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB. Autophagy. 2012;8(6):903e914. https://doi.org/10.4161/auto.19653. 10. Palmieri M, Impey S, Kang H, et al. Characterization of the CLEAR network reveals an integrated control of cellular clearance pathways. Hum Mol Genet. 2011;20(19):3852e3866. https://doi.org/10.1093/hmg/ddr306. 11. Balderhaar HJ, Ungermann C. CORVET and HOPS tethering complexes - coordinators of endosome and lysosome fusion. J Cell Sci. 2013;126(Pt 6): 1307e1316. https://doi.org/10.1242/jcs.107805. 12. Kawamura N, Sun-Wada G-H, Aoyama M, et al. Delivery of endosomes to lysosomes via microautophagy in the visceral endoderm of mouse embryos. Nat Commun. 2012;3(1):1071. https://doi.org/10.1038/ncomms2069. 13. Kim Y-M, Jung CH, Seo M, et al. mTORC1 phosphorylates UVRAG to negatively regulate autophagosome and endosome maturation. Mol Cell. 2015;57(2): 207e218. https://doi.org/10.1016/J.MOLCEL.2014.11.013. 14. Alvarez-Erviti L, Seow Y, Schapira AHV, Rodriguez-Oroz MC, Obeso JA, Cooper JM. Influence of microRNA deregulation on chaperone-mediated autophagy and a-synuclein pathology in Parkinson's disease. Cell Death Dis. 2013;4(3):e545ee548. https://doi.org/10.1038/cddis.2013.73. 15. Harada K, Hide I, Seki T, Tanaka S, Nakata Y, Sakai N. Extracellular ATP differentially modulates Toll-like receptor 4-mediated cell survival and death of microglia. J Neurochem. 2011;116(6). https://doi.org/10.1111/j.14714159.2011.07170.x.
Contents lists available at ScienceDirect
Journal of Pharmacological Sciences journal homepage: www.elsevier.com/locate/jphs
Short Communication
Rapamycin activates mammalian microautophagy Masahiro Sato a, Takahiro Seki a, *, Ayumu Konno b, Hirokazu Hirai b, Yuki Kurauchi a, Akinori Hisatsune a, c, d, Hiroshi Katsuki a a
Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan Department of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Japan c Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto, Japan d Program for Leading Graduate Schools “HIGO (Health Life Science: Interdisciplinary and Glocal Oriented) Program”, Kumamoto University, Kumamoto, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 April 2019 Received in revised form 7 May 2019 Accepted 14 May 2019 Available online 24 May 2019
Autophagy-lysosome proteolysis is classified into macroautophagy (MA), microautophagy (mA) and chaperone-mediated autophagy (CMA). In contrast to MA and CMA, mA have been mainly studied in yeast. In 2011, mammalian mA was identified as a pathway to deliver cytosolic proteins into multivesicular bodies. However, its molecular mechanism is quite different from yeast mA. Using a cell-based method to evaluate mA and CMA, we revealed that rapamycin, an activator of yeast mA, significantly activated mammalian mA. Although rapamycin activates MA, mA was also activated by rapamycin in MAdeficient cells. These findings suggest that rapamycin is a first-identified activator of mammalian mA. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Microautophagy Chaperone-mediated autophagy Rapamycin
Removal of misfolded proteins by protein degradation systems is important for various neuronal functions and survival.1 There are two major protein degradation systems: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system.1 The latter is further classified into macroautophagy (MA), microautophagy (mA) and chaperone-mediated autophagy (CMA).2 Regarding UPS and MA, there are many reports about their involvements in physiological functions and disease pathogenesis.1 Recently, CMA has been focused on the relationship with Parkinson's disease and other neurodegenerative diseases.3 In contrast, most studies about mA have been conducted in yeast.4 In yeast mA, substrate are incorporated into vacuoles via the invagination of vacuolar membrane.4 However, the molecular mechanism of mammalian mA had remained unknown for a long period. In 2011, Sahu R et al identified a novel pathway to deliver cytosolic soluble proteins into late endosome/multivesicular bodies (MVBs) in an Hsc70-dependent manner and postulated this pathway as mammalian mA.5 Since mammalian mA is associated with endosomal sorting complex
* Corresponding author. Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 OeHonmachi, Chuo-ku, Kumamoto 862-0973, Japan. Fax: þ81 96 371 4182. E-mail address: [email protected] (T. Seki). Peer review under responsibility of Japanese Pharmacological Society.
required for transport (ESCRT) proteins that are involved in multivesicular formation,6 it is postulated that mA in yeast and mammalian cells are regulated in different manners. Recently, we have established a novel method to separately assess mA and CMA activities in mammalian cells.7 We used GAPDH fused with HaloTag (GAPDH-HT) as a marker of CMA and mA activities because GAPDH is recognized by Hsc70, which is commonly involved in both CMA and mA.3,5 This is the first method to evaluate mammalian mA in a single cell. Using this method, we revealed that prolonged serum deprivation activates CMA but does not affect mA in mammalian cells.7 Prolonged starvation is known to activate mA in yeast.4 These findings support the hypothesis that mA is divergently regulated in yeast and mammalian cells. It remains unknown how mA is regulated in mammalian cells and what stimuli activate mammalian mA. Rapamycin is an inhibitor of mammalian target of rapamycin complex 1 (mTORC1) and is widely used as a potent activator of MA.8 In addition, rapamycin is also known to activate mA in yeast.4 In the present study, we investigated the effect of rapamycin on mammalian mA using our method. LAMP2A (sense: 50 -GGCAGGAGUACUUAUUCUAGU-30 , antisense: 50 -UAGAAUAAGUACUCCUGCCAA-30 ) and TSG101 (sense: 50 CUAGUUCAAUGACUAUUAATT-30 , antisense: 50 -UUAAUAGUCAUUGAACUAGTT-30 ) siRNAs and MISSION siRNA Universal Negative Control were obtained from SigmaeAldrich (St. Louis, MO, USA).
https://doi.org/10.1016/j.jphs.2019.05.007 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Rapamycin was from AdipoGen Life Science (Liestal, Switzerland). The Nerve-Cell Culture System was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Penicillin/streptomycin solution was obtained from Nacalai Tesque (Kyoto, Japan). The HaloTag system (HaloTag vector and HaloTag ligand) was obtained from Promega (Madison, WI, USA). Flp-In system, neurobasal medium, B-27 serum free supplement, Lipofectamine RNAiMAX, and Lipofectamine 3000 were obtained from Invitrogen (Carlsbad, CA, USA). AD293 cells were obtained from Agilent Technologies (Santa Clara, CA, USA). Glass-bottomed culture dishes (35-mm diameter) were obtained from MatTek (Ashland, MA, USA). Mouse embryonic fibroblast (MEF) cells from Atg5-knockout (KO) mice and plasmid
to express LC3 constructs were kindly donated by Noboru Mizushima (University of Tokyo, Japan). AD293 and MEF cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 units/mL of penicillin and 100 mg/mL of streptomycin in a humidified atmosphere containing 5% CO2 at 37 C. Primary cultures of rat cortical neurons were prepared from E16 embryos of Wister/ ST rats as described previously.7 Dissociated neurons (3 105 cells/ dish) were cultured in the neurobasal medium supplemented with 2% B27 and 25 mM L-glutamate for 7 days in vitro (DIV). Transfection of siRNA and plasmid to express GFP-LC3 was conducted using Lipofectamine RNAiMAX and Lipofectamine 3000,
Fig. 1. Rapamycin activates mammalian mA in AD293 cells and primary cultured cortical neurons. (A) Punctate accumulation of GAPDH-HT in AD293 cells transfected with control, LAMP2A, or TSG101 siRNA (50 pmol). Cells were treated with vehicle (Veh, 0.1% DMSO) or rapamycin (500 nM) for 18 h after TMR-HT ligand labeling. Scale bars are 20 mm. (B) Quantitative analyses of GAPDH-HT puncta shown in A. * < p 0.05, ***p < 0.001 vs control siRNA-transfected cells treated with vehicle, #p < 0.05 (n ¼ 51e57). (C) Representative immunoblots of siRNA-mediated knockdown of LAMP2A and TSG101 in AD293/GAPDH-HT cells. Arrow indicates the specific band of TSG101. The amount of b-actin was evaluated as a loading control. (D) Punctate accumulation of GAPDH-HT in primary cultured cortical neurons transfected with control, LAMP2A, or TSG101 siRNA (50 pmol). Cells were treated with vehicle or rapamycin (500 nM) for 24 h after TMR-HT ligand labeling. Scale bars are 10 mm. (E) Quantitative analyses of GAPDH-HT puncta shown in C. **p < 0.01 vs control siRNA-transfected cells treated with vehicle, ##p < 0.01 (n ¼ 39e43). (F) Representative immunoblots of siRNA-mediated knockdown of LAMP2A and TSG101 in primary cultured cortical neurons. Arrow indicates the specific band of TSG101. The amount of b-actin was evaluated as a loading control.
M. Sato et al. / Journal of Pharmacological Sciences 140 (2019) 201e204
as described previously.7 To transiently express GAPDH-HT in primary cultured cortex neurons, we used adeno-associated virus serotype 9 (AAV9) vectors. The viral vectors expressed GAPDH-HT specifically in neurons under the control of the neuron-specific, modified synapsin I promoter.7 AAV9 vectors (2 1010 vg) were infected to cortical neurons on the glass bottom dishes on DIV1. Labeling with HT ligands fused with fluorescent dyes was conducted as described previously.7 Briefly, AD293/GAPDH-HT and Atg5-KO MEF cells on glass bottom dishes were labeled with 100 nM HT ligand fused with tetramethylrhodamine (TMR) for 10 min 2 days after transfection of siRNA or plasmids, cultured with vehicle (0.1% DMSO) or 500 nM rapamycin for 18 h and fixed with 4% paraformaldehyde (PFA). In this experiment, cells were treated with vehicle (0.1% DMSO) or 500 nM rapamycin. In primary cultured cortical neurons, cells were labeled with TMR-HT ligand on DIV6, cultured with vehicle or rapamycin for 24 h and fixed with 4% PFA. Fluorescent images of fixed cells were obtained using a confocal microscope, TCS SP5 (Leica, Wetzlar, Germany). We quantitatively analyzed the activities of CMA and mA by counting GAPDH-TH puncta labeled with TMR. All quantitative data are represented as mean ± standard error of the mean. Statistical differences were determined by one-way analysis of variance followed by post hoc test with Tukey's method. The difference was considered to be statistically significant, if p-value was less than 0.05. We have previously demonstrated that punctate accumulation of fluorescence-labeled GAPDH-HT under the knockdown of LAMP2A, a CMA-related protein, and TSG101, a mA-related protein, represents the activities of mA and CMA, respectively.7 In the present study, we investigated whether rapamycin affect mA/CMA activity in AD293 cells stably expressing GAPDH-HT.7 siRNAmediated knockdown of LAMP2A and TSG101 was conducted in the same method with our previous study,7 and these proteins were efficiently knocked down in the present study (Figs. 1C, F and 3C). Rapamycin significantly increased punctate accumulation of
203
GAPDH-HT in cells transfected with negative control siRNA and LAMP2A siRNA but did not affect it in cells transfected with TSG101 siRNA (Fig. 1A,B). Similar findings were observed in primary cultured cortical neurons, in which GAPDH-HT was transfected using adeno-associated viral vectors (Fig. 1D,E). An increase in GAPDH-HT puncta by rapamycin was more potent in neurons transfected with LAMP2A than in control neurons (Fig. 1D,E). This might be reflected by the excessive activation of mA for the compensation of impaired CMA. It is possible that this compensation is selectively observed in neurons. These findings suggest that rapamycin activates mammalian mA but does not activate CMA. Since rapamycin is a potent activator of MA, it is possible that an increase in GAPDH-HT puncta is reflected by MA activation. To exclude this possibility, AD293/GAPDH-HT cells were transiently transfected with GFP-LC3, which labels autophagosome. We rarely found the colocalization of GFP-LC3 puncta and GAPDH-HT puncta in the presence or absence of rapamycin (Fig. 2), suggesting that GAPDH-HT is not accumulated into autophagosomes even in the presence of rapamycin. In addition, we transiently transfected GAPDH-HT to mouse embryonic fibroblast (MEF) cells from MAdeficient Atg5 knockout (KO) mice. GAPDH-HT puncta were increased by rapamycin in Atg5-KO MEF cells transfected with control siRNA and LAMP2A siRNA but were not increased in cells transfected with TSG101 siRNA (Fig. 3A,B), similarly to AD293 cells and cortical neurons. These findings suggest that rapamycin activates mammalian mA, independently of MA. In the present study, we first identified rapamycin as an activator of mammalian mA. Then, how does rapamycin activate mammalian mA? Rapamycin is known to potently activate MA through the activation of ULK1, a MA initiator.8 In addition, rapamycin triggers the nuclear localization and activation of transcription factor EB (TFEB),9 which increases the expression of various genes related to MA and lysosome biogenesis.10 Namely, activation of MA by rapamycin is also mediated by the activation of TFEB. Because TFEB increases the lysosome biogenesis, rapamycin
Fig. 2. GAPDH-HT puncta are rarely colocalized with GFP-LC3-positive autophagosomes. Representative GAPDH-HT (left), GFP-LC3 (center) and merged (right) images of AD293 cells treated with vehicle (upper panels) and rapamycin (lower panels) for 18 h. Scale bars are 20 mm.
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M. Sato et al. / Journal of Pharmacological Sciences 140 (2019) 201e204
Fig. 3. Rapamycin activates mammalian microautophagy in MA-deficient mouse embryonic fibroblast cells. (A) Punctate accumulation of GAPDH-HT in Atg5-knockout mouse embryonic fibroblast (MEF) cells transfected with control, LAMP2A, or TSG101 siRNA (50 pmol). Cells were treated with vehicle or rapamycin (500 nM) for 18 h after TMR-HT ligand labeling. Scale bars are 20 mm. (B) Quantitative analyses of GAPDH-HT puncta shown in A. *p < 0.05, ***p < 0.001 vs control siRNA-transfected cells treated with vehicle, ###p < 0.001 (n ¼ 45e47). (C) Representative immunoblots of siRNA-mediated knockdown of LAMP2A and TSG101 in Atg5-knockout MEF cells. The amount of b-actin was evaluated as a loading control.
could activate all pathways of autophagy-lysosome proteolysis. However, our present findings indicate that rapamycin fails to activate CMA. Therefore, an increase in lysosomes is not the main cause of the activation of mA by rapamycin. Homotypic fusion and protein sorting (HOPS) complex is involved in the fusion of MVBs and lysosomes.11 Vps11 and Vps18, the components of HOPS complex, are the target genes of TFEB.10 In addition, Rab7, which is reported to be involved in mammalian mA,12 is one of the target genes of TFEB.10 It is possible that TFEB-mediated increase in these proteins contributes to the activation of mammalian mA by rapamycin. According to this hypothesis, other TFEB inducers, including Torin-19, could be also activators of mammalian mA. Furthermore, HOPS complex is negatively regulated by mTORC1 through the phosphorylation of UV radiation resistance-associated gene product (UVRAG).13 Therefore, dephosphorylation of UVRAG by rapamycin might also be related to the activation of mammalian mA. Although further studies are necessary, our present findings provide the cue to reveal the detail mechanism how mA is regulated in mammalian cells. Recently, it has been reported that the decrease of CMA activity is involved in the onset and progression of various diseases, including neurodegenerative diseases.3 Indeed, an increase in specific miRNAs reduces the expression of Hsc70 and LAMP2A in the brains of Parkinson's disease patients.14 In addition, knockdown of LAMP2A in the midbrain causes Parkinson's disease-like symptoms in rats.15 Since Hsc70 is also involved in mammalian mA,5 it is considered that mammalian mA would be associated with various diseases. Taken together, in addition to CMA, mammalian mA could be a novel therapeutic target of various diseases. Conflicts of interests The authors declare no conflicts of interests. Acknowledgement This work is financially supported by Japan Society for the Promotion of Science KAKENHI [grant number 16K08276]. We
thank Prof. Noboru Mizushima (University of Tokyo, Japan) for giving us LC3 plasmids and Atg5-knockout MEF cells for this study.
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