Fluorescent-based evaluation of chaperone-mediated autophagy and microautophagy activities in cultured cells.
- 1Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
- 2Department of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Japan.
- 3Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto, Japan.
- 4Program for Leading Graduate Schools “HIGO (Health life science: Interdisciplinary and Glocal Oriented) Program”, Kumamoto University, Kumamoto, Japan.
The autophagy-lysosome protein degradation is further classified into macroautophagy (MA), microautophagy (mA), and chaperone-mediated autophagy (CMA). While MA is involved in various functions and disease pathogenesis, little is known about CMA and mA because of the absence of easy methods to assess their activities. We have recently established a method to assess CMA activity using glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a CMA substrate, and HaloTag (HT) system. Another group has recently identified a mammalian mA pathway, in which substrates are delivered to late endosomes in an heat shock cognate protein (Hsc)70-dependent manner. Because Hsc70 is also involved in CMA, our method would detect both CMA and mA activities. In this study, we attempted to assess CMA and mA activities separately through the siRNA-mediated knockdown of CMA- and mA-related proteins. Knockdown of LAMP2A, a CMA-related protein, and TSG101, an mA-related protein, significantly but only partially decreased the punctate accumulation of GAPDH-HT in AD293 cells and primary cultured rat cortical neurons. Compounds that activate CMA significantly increased GAPDH-HT puncta in TSG101-knockdown cells, but not in LAMP2A-knockdown cells, suggesting that punctate accumulation of GAPDH-HT under LAMP2A- and TSG101-knockdown represents mA and CMA activities, respectively. We succeeded in establishing the method to separately evaluate CMA and mA activities by fluorescence observation.
- PMID: 27377049; DOI: 10.1111/gtc.12390
Intracellular protein degradation has an essential role to maintain protein homeostasis by degrading unfolded or misfolded proteins, especially in neurons (1). There are two major protein degradation systems, ubiquitin-proteasome system (UPS) and autophagy-lysosome system. The latter is further classified into three pathways, macroautophagy (MA), microautophagy (mA) and chaperone-mediated autophagy (CMA), by the routes of substrate delivery to lysosomes (2). Among these, UPS and MA have been widely studied about the physiological roles and the pathogenesis of various diseases (3-5).
On the contrary, there have been fewer studies concerning mA and CMA. About CMA, the substrate proteins are recognized by Hsc70, a molecular chaperone, and delivered to lysosome via LAMP2A, a lysosomal membrane protein (6). However, it remains unclear how CMA is regulated and how CMA affects the physiological functions and disease pathogenesis because of the absence of specific activity markers and easy method to assess the activity of CMA (6). In contrast, studies about MA have been explosively progressed after the findings of LC3-II, a specific marker of MA activity. To solve this problem, we established a novel method to assess CMA activity by observing the translocation of fluorescent-labeled CMA substrate, GAPDH fused with HaloTag (GAPDH-HT), from cytosol to lysosomes in a single cultured cell (7).
Recently, Sahu R et al demonstrated that cytosolic soluble proteins are incorporated into late endosome/multivesicular bodies (MVBs) in an Hsc70-dependent manner (8). This process is predicted to be the mammalian mA. Our novel method to monitor CMA activity utilizes GAPDH as a CMA substrate, which is recognized by Hsc70 and could be incorporated into late endosome by mA. Therefore, we assumed that we can detect mA as well as CMA using our novel method. In the present study, we report that CMA and mA activities can be separately assessed by siRNA-mediated knockdown of mA- and CMA-related proteins, respectively.
We confirmed that GAPDH-HT translocated from cytoplasm to late endosomes and MBVs by the immunostaining with anti-Rab7 and CD63 antibodies, markers of late endosomes and MVBs, respectively, in AD293 cells stably expressing GAPDH-HT (AD293/GAPDH-HT cells) (Fig. 1). This finding suggests that GAPDH-HT is delivered to late endosomes, probably via mA pathway.
Figure 1 Accumulation of GAPDH-HT to late endosomes. Localization of GAPDH-HT puncta to late endosomes/multivesicular bodies (MVBs). AD293/GAPDH-HT cells were fixed 18 h after TMR HT ligand labeling and immunostained with anti-Rab7 antibody (B) and anti-CD63 antibody (C). Rab7 and CD63 is a marker of late endosomes and MVBs, respectively. Scale bars are 10 μm.
Next, we attempted to detect CMA and mA activities separately by siRNA-mediated knockdown of mA- and CMA-related proteins. Transfection of LAMP2A, a CMA-related protein (9), and TSG101, a mA-related protein(8), siRNAs significantly, but partly, decreased the number of GAPDH-HT puncta in AD293/GAPDH-HT cells (Fig. 2A,B). Double knockdown of LAMP2A and TSG101 further decreased these puncta, although there was no significant difference in the number of its puncta between single and double knockdown (Fig. 2A,B). It is reported that retinoic acid receptor α (RARα) antagonist and mycophenolic acid (MPA) activates CMA via upregulation or stabilization of LAMP2A(10,11). LE540, a nonselective RAR antagonist, and MPA increased GAPDH-HT puncta in cells transfected with TSG101 siRNA, while the effects of these compounds were not observed in cells transfected with LAMP2A siRNA (Fig. 2C). These findings suggest that CMA and mA can be separately detected using GAPDH-HT by the knockdown of mA- and CMA-related proteins, respectively.
Figure 2 Separate assessment of CMA and mA activities by siRNA-mediate knockdown in AD293/GAPDH-HT cells. (A) Punctate accumulation of GAPDH-HT in siRNA-transfected AD293/GAPDH-HT cells. Cells were fixed 18 h after HT ligand labeling. Scale bars are 20 μm. (B) Quantitative analyses of GAPDH-HT puncta shown in A. * p < 0.05, ** p < 0.01 vs control siRNA, n = 44-53. (C) Quantitative analyses of GAPDH-HT puncta in AD293 cells transfected with control, LAMP2A or TSG101 siRNA (50 pmol) and treated with vehicle (Veh, 0.1 % DMSO), LE540 (LE, 10 μM) or mycophenolic acid (MPA, 5 μM) for 18 h after TMR-HT ligand labeling. ** p < 0.01 vs control siRNA-transfected cells treated with vehicle, # p < 0.05, ## p < 0.01, n = 37-45.
Next, we attempted to apply this method in primary cultured neurons. We transfected GAPDH-HT to primary cultured rat cortical neurons using adeno-associated virus serotype 9 (AAV9) vectors. GAPDH-HT accumulated to punctate structures 24 h incubation after the labeling of GAPDH-HT with TMR-HT ligands. This accumulation was significantly, but partly, decreased by siRNA-mediated knockdown of LAMP2A and TSG101, respectively. LE540 significantly increased GAPDH-HT puncta in neurons transfected with TSG101 siRNA, but did not affect the number of the puncta in cells transfected with LAMP2A siRNA. These findings suggest that CMA and mA activities can be separately assessed in primary cultured neuronal cells.
Importance of the study: We established a fluorescent-based method to assess CMA and mA activities, separately. Especially, this is the first report about the method to monitor mA activity in a single cell level. This method will contribute to the elucidation of physiological functions and involvement in disease pathogenesis of CMA and mA, which have not been fully elucidated.
Figure 3 Separate assessment of CMA and mA activities by siRNA-mediate knockdown n primary cultured cortex neurons. (A) Punctate accumulation of GAPDH-HT in siRNA-transfected cortex neurons. Cells were fixed 24 h after TMR HT ligand labeling. Scale bars are 20 μm. (B) Quantitative analyses of GAPDH-HT puncta shown in A. *** p < 0.001 vs control siRNA, n = 36-47.< 0.05, ## p < 0.01, n = 37-45. (C) Quantitative analyses of GAPDH-HT puncta in cortex neurons transfected with control, LAMP2A or TSG101 siRNA (30 pmol) and treated with vehicle (Veh, 0.1 % DMSO) or LE540 (LE, 1 μM) for 24 h after TMR-HT ligand labeling. ** p < 0.01, *** p < 0.001 vs control siRNA-transfected cells treated with vehicle, # p < 0.05, n = 18-20.
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