Establishment of a Novel Fluorescence-Based Method to Evaluate Chaperone-Mediated Autophagy in a Single Neuron

Background: Chaperone-mediated autophagy (CMA) is a selective autophagy-lysosome protein degradation pathway. The role of CMA in normal neuronal functions and in neural disease pathogenesis remains unclear, in part because there is no available method to monitor CMA activity at the single-cell level. Methodology/Principal Findings: We sought to establish a single-cell monitoring method by visualizing translocation of CMA substrates from the cytosol to lysosomes using the HaloTag (HT) system. GAPDH, a CMA substrate, was fused to HT (GAPDH-HT); this protein accumulated in the lysosomes of HeLa cells and cultured cerebellar Purkinje cells (PCs) after labeling with fluorescent dye-conjugated HT ligand. Lysosomal accumulation was enhanced by treatments that activate CMA and prevented by siRNA-mediated knockdown of LAMP2A, a lysosomal receptor for CMA, and by treatments that inactivate CMA. These results suggest that lysosomal accumulation of GAPDH-HT reflects CMA activity. Using this method, we revealed that mutant cPKC, which causes spinocerebellar ataxia type 14, decreased CMA activity in cultured PCs. Conclusion/Significance: In the present study, we established a novel fluorescent-based method to evaluate CMA activity in a single neuron. This novel method should be useful and valuable for evaluating the role of CMA in various neurona

Lysosomal translocation of GAPDH-HT in primary cultured PCs.

<p>(<i>A</i>) Representative fluorescent images of TMR-labeled GAPDH-HT immediately after (0 h, left) and 24 h after labeling (24 h, right) with TMR-HT ligand in primary cultured PCs. Images were projected from a Z-stack of images obtained by confocal laser microscopy. Bar = 20 µm. (<i>B</i>) Representative GAPDH-HT fluorescence (left), LAMP2 immunostaining (center) and merged (right) images of PC somata 21 h after labeling with TMR-HT ligand. Images were taken from the center of the Z-stack. Bar = 5 µm. (<i>C</i>) Representative GAPDH-HT fluorescence (left), LysoTracker red fluorescence (center) and merged (right) images of PC somata 21 h after labeling with TMR-OG ligand. Bar = 5 µm. (<i>D</i>) Representative fluorescent images of GAPDH-HT in PC somata treated with vehicle (0.1% DMSO, 0.1% methanol, left upper), H₂O₂ (100 µM, right upper), mycophenolic acid (MPA; 10 µM, left lower) and 6-aminonicotinamide (6-AN; 1 mM, right lower). Bar = 5 µm. (<i>E</i>) Quantitative analyses of lysosomal translocation of GAPDH-HT in PC somata treated with CMA activators. Dots of GAPDH-HT in each PC soma were counted in the center image from the Z-stack. Numbers of GAPDH-HT dots were significantly increased in the presence of CMA activators (H₂O₂, MPA and 6-AN). * p<0.01 vs PCs treated with vehicle (unpaired <i>t</i>-test, n = 60 for cells treated with vehicle, n = 30 for cells treated with CMA activators).</p

Lysosomal translocation of GAPDH-HT reflects CMA activity in HeLa cells.

<p>(<i>A</i>) Representative fluorescent images of GAPDH-HT 21 h after labeling with TMR-HT ligand in HeLa cells transfected with nontargeting-siRNA (left) or LAMP2A-siRNA (right). Bar = 20 µm. (<i>B,C</i>) Quantitative analyses of GAPDH-HT lysosomal translocation in cells transfected with nontargeting (Non)- and LAMP2A-siRNA. (<i>B</i>) Cells having more than 5 dots of GAPDH-HT were classified as GAPDH-HT dot-positive cells. We counted the number of GAPDH-HT dot-positive cells in 50–70 GAPDH-HT-expressing cells. (<i>C</i>) We assessed the number of GAPDH-HT dots per cell. The percentage of GAPDH-HT dot-positive cells and the number of GADPH-HT dots per cell were significantly decreased by siRNA-mediated LAMP2A-knockdown. ** p<0.001 vs cells treated with nontargeting-siRNA (unpaired <i>t</i>-test, n = 16 in <i>B</i>, n = 57 for nontargeting-siRNA and n = 74 for LAMP2A-siRNA in <i>C</i>). (<i>D</i>) Representative fluorescent images of GAPDH-HT taken 21 h after labeling with the TMR-HT ligand in HeLa cells treated with vehicle (0.1% DMSO, 0.1% methanol, left upper), serum free medium (0.1% DMSO, 0.1% methanol, center upper), H₂O₂ (100 µM, right upper), mycophenolic acid (MPA; 10 µM, left lower), SB202190 (20 µM, left center) or cycloheximide (CHX; 20 µg/ml, right lower). Bar = 20 µm. (<i>E,F</i>) Quantitative analyses of GAPDH-HT lysosomal translocation in cells treated with CMA activators or inhibitors the percentages of GAPDH-HT dot-positive cells (<i>E</i>) and the number of GAPDH-HT dots per cell (<i>F</i>). Percentages of GAPDH-HT dot-positive cells and the numbers of GAPDH-HT dots per cell were significantly increased by CMA activators (serum free medium, H₂O₂ and MPA), while they were significantly decreased by CMA inhibitors (SB202190 and CHX). * p<0.01, ** p<0.001 vs cells treated with vehicle (unpaired <i>t</i>-test, n = 12 for cells treated with vehicle, n = 8 for cells treated with CMA activators and inhibitors in <i>E</i>, n = 96 for vehicle, n = 61 for serum free, n = 45 for H₂O₂, n = 58 for MPA, n = 52 for SM202190 and n = 41 for CHX in <i>F</i>).</p

Visualization of CMA substrate translocation from cytosol to lysosomes using the HaloTag (HT) system.

<p>(<i>A</i>) Schematic illustration of CMA substrate-HT labeling with the HT ligand fused to fluorescent dye (gray circle). CMA substrate-HT in cytosol can be labeled with fluorescent dye since the HT reaction occurs at neutral pH. In contrast, CMA substrate-HT in lysosomes cannot be labeled since the HT reaction does not occur at acidic pH. (<i>B</i>) Schematic illustration of CMA substrate-HT translocation from cytosol to lysosome. Because the fluorescent dyes used in this study (TMR and OG) are not quenched at acidic pH, labeled CMA substrates can be visualized after translocation to lysosomes. (<i>C</i>) Representative fluorescent images of TMR-labeled GAPDH-HT immediately after (0 h, upper) and 21 h after labeling (21 h, lower) with TMR-HT ligand in HeLa cells. Arrows on the lower image indicate the cells with obvious GAPDH-HT dots. Bar = 20 µm. (<i>D</i>) Representative GAPDH-HT fluorescence (left), LAMP2 immunostaining (center) and merged (right) images of HeLa cells immediately after (0 h, upper panels) and 21 h after labeling (21 h, lower panels) with TMR-HT ligand. Bar = 5 µm. (<i>E</i>) Representative GAPDH-HT fluorescence (left), LysoTracker red fluorescence (center) and merged (right) images of HeLa cells 21 h after labeling with OG-HT ligand. Bar = 5 µm. (<i>F</i>) Representative GAPDH-HT fluorescence (left), LAMP2A immunostaining (center) and merged (right) images of HeLa cells 21 h after labeling with TMR-HT ligand. Bar = 5 µm.</p