Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles

Summary

Here, we present the methodology for concisely assessing autophagosome marker LC3-II levels in extracellular vesicles (EVs) by immunoblotting. Analysis for LC3-II levels in EVs, autolysosome formation, and omegasome formation suggests the new role of STX6 in the release of LC3-II-positive EVs when autophagosome-lysosome fusion is inhibited.

Abstract

(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf cytoplasmic contents, subsequently fusing with lysosomes for degradation. Beyond the canonical role, autophagy-related genes also modulate a secretory pathway involving the release of inflammatory molecules, tissue repair factors, and extracellular vesicles (EVs). Notably, the process of disseminating pathological proteins between cells, particularly in neurodegenerative diseases affecting the brain and spinal cord, underscores the significance of understanding this phenomenon. Recent research suggests that the transactive response DNA-binding protein 43 kDa (TDP-43), a key player in amyotrophic lateral sclerosis and frontotemporal lobar degeneration, is released in an autophagy-dependent manner via EVs enriched with the autophagosome marker microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II), especially when autophagosome-lysosome fusion is inhibited.

To elucidate the mechanism underlying the formation and release of LC3-II-positive EVs, it is imperative to establish an accessible and reproducible method for evaluating both intracellular and extracellular LC3-II-positive vesicles. This study presents a detailed protocol for assessing LC3-II levels via immunoblotting in cellular and EV fractions obtained through differential centrifugation. Bafilomycin A1 (Baf), an inhibitor of autophagosome-lysosome fusion, serves as a positive control to enhance the levels of intracellular and extracellular LC3-II-positive vesicles. Tumor susceptibility gene 101 (TSG101) is used as a marker for multivesicular bodies. Applying this protocol, it is demonstrated that siRNA-mediated knockdown of syntaxin-6 (STX6), a genetic risk factor for sporadic Creutzfeldt-Jakob disease, augments LC3-II levels in the EV fraction of cells treated with Baf while showing no significant effect on TSG101 levels. These findings suggest that STX6 may negatively regulate the extracellular release of LC3-II via EVs, particularly under conditions where autophagosome-lysosome fusion is impaired. Combined with established methods for evaluating autophagy, this protocol provides valuable insights into the role of specific molecules in the formation and release of LC3-II-positive EVs.

Introduction

Transactivation response DNA-binding protein 43 (TDP-43) is a widely expressed heterogeneous nuclear ribonucleoprotein involved in regulating exon splicing, gene transcription, and mRNA stability, all vital for cell survival^1,2. In neurodegenerative conditions like amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), a nuclear protein TDP-43 abnormally accumulates in the cytoplasm. This shift results in a loss of TDP-43 function in the nucleus and a toxic gain-of-function in the cytoplasm. Pathological accumulation of TDP-43 begins in specific regions of the brain and spinal cord, spreading through these areas in a prion-like fashion, a process closely associated with disease progression³. However, the exact mechanism by which TDP-43 pathology spreads through the brain and spinal cord remains unknown.

TDP-43 is secreted through extracellular vesicles (EVs), and elevated levels of TDP-43 are detected in the plasma and cerebrospinal fluid (CSF) of ALS and FTLD-TDP patients^4,5,6. CSF from patients diagnosed with ALS and FTLD induces intracellular mislocalization and aggregation of endogenous TDP-43 in human glioma cells⁷. Thus, TDP-43 released extracellularly via EVs may mediate the cell-to-cell spreading of TDP-43 pathology.

Autophagy is a well-preserved cellular degradation mechanism that involves the enclosure of unwanted substances within double-membrane vesicles, known as autophagosomes, which are marked by LC3. These autophagosomes fuse with lysosomal-associated membrane protein 1 (LAMP1)-positive lysosomes to form autolysosomes (LC3⁺/LAMP1⁺), leading to the degradation of their contents⁸. Histological analyses indicate that autophagosomes engulfing inclusions accumulate in the neurons of sporadic ALS patients⁹. Some causal genes of familial ALS and FTLD-TDP are linked to the regulation of autophagy^10,11,12. These findings suggest that autophagy is suppressed in ALS and FTLD-TDP patients.

The previous study indicated that TDP-43 is secreted via EVs positive for the autophagosome marker LC3-II when autophagosome-lysosome fusion is inhibited¹³. Dysregulation of the autophagy-lysosome pathway might cause not only the intracellular accumulation of TDP-43 but also the extracellular release of TDP-43 via EVs¹⁴. However, it remains unknown how LC3-II positive EVs are released and how significant this is in the spreading of TDP-43 pathology.

EVs are classified into large EVs (100 to 1000 nm in diameter), which are produced from cell-surface budding, and small EVs (50 to 150 nm in diameter), which are produced from the budding of endosomal membranes toward the interior of the endosome (known as exosomes) and the Golgi apparatus. To separately collect large and small EVs, we perform sequential centrifugation and collect pellets by centrifugation at 20,000 × g and 110,000 × g, respectively. The P1 EV fraction (20,000 × g pellets) is prepared to collect large EVs, and the P2 EV fraction (110,000 × g pellets) is prepared to collect small EVs¹⁵. The methodology to assess LC3-II levels in large and small EVs derived from sequential centrifugation by immunoblotting is stable and reproducible¹⁶. In addition to analyzing LC3-II levels in EVs, analyzing autolysosome and omegasome formation enhances understanding of how dysregulation of autophagy leads to the release of LC3-II positive EVs. Here, the present study shows the suppressive role of syntaxin 6 (STX6), a SNARE protein, which promotes the movement of transport vesicles to target membranes¹⁷, in the release of LC3-II positive EVs when autophagosome-lysosome fusion is inhibited.

Protocol

1. Preparation of the cell, P1, and P2 EV fraction from the cultured medium of HeLa cells

1. Preparation of the P1 and P2 EV fraction from the cultured medium of HeLa cells
   1. Day 1
      1. Count the cells using a cell counter and seed 1 × 10⁶ cells on a 6 cm plate containing a culture medium composed of DMEM High Glucose, 10% FBS, and 1% penicillin/streptomycin. Incubate the cells at 37 °C with 5% CO₂ and controlled humidity for 24 h.
   2. Day 2 (optional)
      1. Prepare 20 µM siRNA solution.
      2. Mix 100 µL of reduced serum medium and 3 µL of siRNA in a low-retention tube to prepare solution A.
      3. Mix 100 µL of reduced serum medium and 5 µL of transfection reagent in a low-retention tube to prepare solution B.
      4. Mix solution A and solution B, then incubate the mixture at room temperature (RT) for 5 min.
      5. Add the mixture of solutions A and B to the medium used for culturing HeLa cells. Incubate the cells at 37 °C with 5% CO₂ and controlled humidity for 24 h.
   3. Day 3
      1. Wash the cells with PBS and expose them to 500 µL of 0.25% trypsin and 1 mM EDTA solution at 37 °C with 5% CO₂ and controlled humidity for 5 min.
      2. Add 2.5 mL of the culture medium to the cells, and use a Pasteur transfer pipette with a 200 µL pipette tip attached to prepare a single-cell suspension.
      3. Count the cells using a cell counter, and seed 1 × 10⁶ cells on a 10 cm plate. Incubate the cells at 37 °C with 5% CO₂ and controlled humidity for 24 h.
         NOTE: It is necessary to prepare at least 1 × 10⁶ cells to collect EVs from the culture medium required for immunoblotting analysis.
   4. Day 4
      1. Prepare culture medium (see step 1.1.1.1) containing either vehicle (DMSO) or 100 nM Bafilomycin A1 (Baf).
      2. Expose the cells to the medium containing either vehicle or 100 nM Baf and incubate them at 37 °C with 5% CO₂ and controlled humidity for 24 h.
   5. Day 5
      1. Collect all the culture medium from the 10 cm plate, transfer it into a 15 mL tube, and centrifuge it at 3,000 × g for 10 min at 4 °C to remove cell debris.
         NOTE: The remaining cells in the 10 cm plate will be used in section 1.2.1.
      2. For the isolation of the P1 EV fraction, transfer the supernatant after centrifugation at 3,000 × g into a centrifugation tube, and then centrifuge it at 20,000 × g at 4 °C for 1 h.
      3. For the isolation of the P2 EV fraction, transfer the supernatant after centrifugation at 20,000 × g into an ultracentrifuge tube, and then centrifuge it at 110,000 × g at 4 °C for 1 h.
      4. After wiping off the excess moisture inside the tube with a laboratory wipe, resuspend the remaining pellets in the centrifugation tube in 50 µL of 2x sample buffer [2.5% SDS, 125mM Tris-HCl buffer (pH 6.8), 30% glycerol, 10% 2-mercaptoethanol, 0.4% Bromophenol Blue] to prepare the P1 EV fraction.
      5. Remove the supernatant by decanting, wipe off the excess moisture inside the tube with a laboratory wipe, and then resuspend the remaining pellets in the ultracentrifugation tube in 50 µL of 2x sample buffer to prepare the P2 EV fraction.
      6. Incubate the P1 and P2 EV fractions at 100 °C on a heat block for 10 min.
         NOTE: Do not shake the tubes after centrifugation at 20,000 × g and 110,000 × g to avoid accidentally discarding the remaining pellets. After tilting the tube to transfer the supernatant, maintain the tube's position to prevent the pellet from being immersed in the remaining supernatant again.
2. Preparation of the cell fraction from cultured HeLa cells
   1. Day 5
      1. After transfer of the culture medium from the 10 cm plate into a 15 mL tube, wash the cells in the 10 cm dish with PBS and expose them to 2 mL of 0.25% trypsin and 1 mM EDTA solution at 37 °C with 5% CO₂ and controlled humidity for 5 min.
      2. Add 8 mL of growth medium to the cells, then use a Pasteur transfer pipette with a 200 µL pipette tip to pipette the cells and prepare a single-cell suspension.
      3. Transfer the cell suspension into a 15 mL tube and centrifuge it at 1,000 × g for 10 min at 4 °C.
      4. Remove the supernatant by an aspirator, add PBS to the cell pellet, and then centrifuge at 1,000 × g for 5 min at 4 °C.
      5. Remove the PBS using an aspirator and sonicate the cell pellet in 1 mL of A68 buffer [10 mM Tris-HCl buffer, pH 7.5, 0.8 M NaCl, 1 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)] containing 1% sarkosyl.
      6. Measure the protein concentration of the cell lysate using a BCA protein assay kit.
      7. Mix 300 µL of the cell lysate with 100 µL of 4x sample buffer and incubate the mixture at 100 °C on a heat block for 10 min to prepare the cell fraction.

2. Immunoblotting analysis

1. Separate equivalent amounts of proteins in the samples by SDS-PAGE¹⁸.
2. Transfer the separated proteins onto polyvinylidene difluoride (PVDF) membranes.
3. After transfer, block the membranes with the blocking agent for 20 min.
4. Incubate the blocked membrane overnight with the indicated primary antibody in Tris buffer containing 10% (v/v) calf serum at RT.
5. Wash the membrane with Tris buffer for 5 s and then incubate it with a biotin-conjugated secondary antibody at RT for 2 h.
6. Wash the membrane with Tris buffer for 5 s and then incubate it with Tris buffer containing 0.4% (v/v) solutions A and solution B from the kit at RT for 1 h.
7. Wash the membrane with PBS, and then incubate it with PBS containing 0.04% (w/v) diaminobenzidine, 0.8% (w/v) NiCl₂·6H₂O, and 0.3% (v/v) H₂O₂ for colorimetric detection of bands.
   NOTE: For the detection of signals, a chemiluminescence method is also acceptable.
8. Digitize the image of the membrane by a scanner and subject it to densitometry analysis using Fiji.
   1. Open the digitized image using Fiji, and convert the RGB color image to an 8-bit image by clicking Image | Type | 8-bit.
   2. Click on the rectangle tool and adjust the size of the rectangle by dragging it to surround the bands. Identify the band to be analyzed by clicking Analyze | Gels | Select First lane.
   3. Position the rectangle on the second and the subsequent bands, and identify the bands for analysis by clicking Analyze | Gels | Select Next Lane.
   4. After recognizing the final band, measure the densitometry across the rectangle by clicking Analyze | Gels | Plot Lanes.
   5. Surround the signal area of the band with a straight line, click the wand tool, and click in the area to calculate the signal intensity of the band.

3. Analysis of the number of autophagosomes and autolysosomes

1. Day 1
   1. Count the number of HeLa cells using a cell counter, and seed 1 × 10⁵ HeLa cells expressing LAMP1-GFP and mCherry-LC3 on a 3.5 cm dish. Incubate the cells at 37 °C with 5% CO₂ and controlled humidity for 24 h.
2. Day 2 (optional)
   1. Perform the steps described in section 1.1.2 in 3.5 cm dishes.
3. Day 3
   1. Discard the culture medium from the 3.5 cm dishes.
   2. Wash cells with PBS, expose them to 400 µL of 0.25% trypsin and 1 mM EDTA solution, and incubate at 37 °C with 5% CO₂ and controlled humidity for 5 min.
   3. Add 1.6 mL of the growth medium to the cells, and pipette them with a Pasteur transfer pipette to prepare a single-cell suspension.
   4. Count the number of cells using the cell counter, and seed 1 × 10⁴ cells on an 8-chambered coverslip. Incubate the cells at 37 °C with 5% CO₂ and controlled humidity for 24 h.
      NOTE: Two wells of the chambered coverslip are prepared for control and STX6 knockdown cells, respectively, following DMSO or Baf treatment as described in section 3.4.2.
4. Day 4
   1. Prepare the growth medium without phenol red (DMEM without Phenol Red, 10% FBS, 1% penicillin/streptomycin), containing either vehicle (DMSO) or 100 nM Baf dissolved in DMSO.
   2. Replace the culture medium with the medium without phenol red, containing either vehicle or 100 nM Baf, and incubate for 4 h.
      NOTE: To assess the effect of Baf on autophagosome and autolysosome numbers, prepare vehicle-treated cells as controls.
   3. Stain the nuclei with 0.33 µg/mL Hoechst 33342 for 30 min before observation.
   4. Conduct Live cell imaging using a 60x objective lens on a confocal laser microscope and capture ten different frames.
      1. Open the RGB merged image in Fiji, and split it into the respective red, green, and blue image components by clicking on Image | Color | Split Channels.
      2. Set a constant threshold for the red and green signals in each image by clicking Image | Adjust | Threshold for each experiment.
      3. Count the number of areas positive for red or green signals by clicking Analyze | Analyze Particles. Check the Summarize box, then click OK. Record the values from the Count to identify autophagosome- and lysosome-associated vesicles.
      4. To overlay the green image on the red image, set the transfer mode to AND by clicking Edit | Paste Control. Copy the green image and then paste it onto the red image to merge them, highlighting areas that are positive for both red and green signals.
      5. To identify autolysosome numbers, count the number of areas positive for both red and green signals by clicking Analyze | Analyze Particles. Check the Summarize box, click OK, and record the values in the Count section to identify vesicles associated with autophagosomes and lysosomes.
   5. Divide the total number of autolysosomes and autophagosomes by the total number of cells to calculate the number of autolysosomes and autophagosomes per cell.
      NOTE: Count at least 35 cells in each of the three independent experiments.

4. Analysis for omegasome formation

1. Day 1
   1. Count the number of HeLa cells using a cell counter, and seed 1 × 10⁵ cells on a 3.5 cm dish. Incubate the cells at 37 °C with 5% CO₂ and controlled humidity for 24 h.
2. Day 2 (optional)
   1. Perform the steps described in section 1.1.2.
3. Day 3
   1. Prepare a single-cell suspension as described in steps 3.3.1-3.3.2.
   2. Count the cells using the cell counter, and seed 1 × 10⁵ cells on a 3.5 cm dish. Incubate the cells at 37 °C with 5% CO₂ and controlled humidity for 24 h.
4. Day 4
   1. Mix 1 µg of pEGFP-C1-hAtg13, 3 µL of the DNA transfection reagent, and 100 µL of reduced serum medium in a low retention tube. Incubate the mixture for 20 min, then add it to the cultured medium.
   2. Incubate the cells at 37 °C with 5% CO₂ and controlled humidity for 24 h.
5. Day 5
   1. Trypsinize the cells as described in steps 3.3.1-3.3.2.
   2. Add 2.5 mL of the growth medium to the cells. Pipette the mixture using a Pasteur transfer pipette to prepare a single-cell suspension.
   3. Count the cells using the cell counter, and seed 1 × 10⁴ cells on an 8-chambered coverslip. Incubate the cells at 37 °C with 5% CO₂ and controlled humidity for 24 h.
6. Day 6
   1. Follow steps 3.4.1-3.4.4 to conduct live cell imaging of the cells from step 4.5.3 using a confocal laser microscope. Capture ten different frames of images.
      1. Follow steps 3.4.4.1-3.4.4.3 to split the RGB merged images into the respective red, green, and blue image components, set a constant threshold for the green signals in each image, and determine GFP signal intensity. Record the values from the Total Area section to determine the signal intensity of GFP-ATG13.
      2. Divide the total signal intensity by the number of cells examined to calculate the signal intensity per cell.
         NOTE: Count at least 34 cells in each of the three independent experiments.

Results

As shown in previous studies, Baf treatment increased the levels of TSG101 (P1: P < 0.01, P2: P = 0.012, as determined by two-way ANOVA in EZR¹⁹) and LC3-II (P1: P < 0.01, P2: P < 0.01, as determined by two-way ANOVA in EZR¹⁹) in the EV-rich fraction. Notably, Baf treatment also increased the levels of STX6 (P1: P < 0.01, P2: P < 0.01, as determined by two-way ANOVA in EZR¹⁹) in the EV fraction (Figure 1A)...

Discussion

The immunoblotting study revealed LC3-II and TSG101 levels in the cellular fraction, the microvesicle-rich P1 EV fraction, and the exosome-rich P2 EV fraction. Live-cell imaging was used to examine autophagosomes, autolysosomes, and omegasomes, providing insight into whether STX6 knockdown influences autophagy. These combined results suggest that STX6 knockdown affects the release of LC3-II positive EVs, potentially linked to dysregulation of the autophagy pathway. A key advantage of this method is its ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25 % Trypsin EDTA	Fujifilm Wako	201-16945
10 cm Dish	Thermo Fisher Scientific	150464
15 mL Tube	Thermo Fisher Scientific	339650
200 μL Pipette Tip	Nippon Genetics	FG-301	pipetting
2-Mercaptoethanol	Nacalai Tesque	21417-52	a material for sample buffer solution
3,3'-Diaminobenzidine Tetrahydrochloride	Nacalai Tesque	11009-41	a material for DAB solution
3.5 cm Dish	Thermo Fisher Scientific	150460
6 cm Dish	TrueLine	TR4001
Aluminium Block Thermostatic Baths (dry thermobaths)	EYELA	273860
Aspirator	SANSYO	SAP-102	inhaling solution
Avanti JXN-30	Beckman Coulter	B34193
Bafilomycin A1	Adipogen	BVT-0252
Biotin-conjugated Goat Anti-rabbit IgG Antibody	Vector Laboratories	BA-1000	2nd antibody for immunoblotting
Biotin-conjugated Horse Antimouse IgG Antibody	Vector Laboratories	BA-2000	2nd antibody for immunoblotting
Blocking One	Nacalai Tesque	03953-95	a material for immunoblotting
Bromophenol Blue	Nacalai Tesque	05808-61	a material for sample buffer solution
Calf Serum	cytiva	SH30073.03
CanoScan LiDE 220	Canon	CSLIDE220	Scanner
Centrifuge 5702 R	eppendolf	5703000039
Counting Slides Dual Chamber	Bio-Rad	1450015J	cell counting
Digital Sonifier 450	BRANSON
Dimethyl Sulfoxide	nacalai tasque	09659-14	vehicle
DMEM High Glucose	Nacalai Tesque	08458-45	culture medium
DMEM without Phenol Red	Nacalai Tesque	08489-45	culture medium
EGTA	Dojindo	348-01311	a material for A68 solution
Excel	Microsoft	version 16.16.27	satistical analysis
EZR	Reference No. 24	version 1.68	satistical analysis
FBS	Sigma	173012	Culture medium
Fiji	NIH		Image analysis tool
Glycerol	Nacalai Tesque	09886-05	a material for sample buffer solution
Hoehst33342	Dojindo	H342
Hydrogen Peroxide	Fujifilm Wako	080-0186	a material for DAB solution
Kimwipe S-200	NIPPON PAPER CRECIA	62011	cleaning wipe
Low Retention Tube	Nippon Genetics	FG-MCT015CLB	siRNA and DNA transfection
LSM780 Confocal Laser Microscope	Carl Zeiss
Monoclonal Mouse Anti-LC3 Antibody	MBL	M186-3	1st antibody for immunoblotting
Nickel(II) Chloride Hexahydrate	Fujifilm Wako	149-01041	a material for DAB solution
N-Lauroylsarcosine Sodium Salt	Nacalai Tesque	20117-12
Optima XE-90 Ultracentrifuge	Beckman Coulter	A94471
Opti-MEM I Reduced Serum Medium	Thermo Fisher Scientific	31985-070	siRNA and DNA transfection
pEGFP-C1-hAtg13	Addgene	22875
Penicillin/Streptomycin	Nacalai Tesque	26253-84	Culture medium
Pierce BCA Protein Assay Kits	Thermo Fisher Scientific	23225
Polyclonal Rabbit Anti-PCNA Antibody	BioAcademia	70-080	1st antibody for immunoblotting
Polyclonal Rabbit Anti-syntaxin 6 Antibody	ProteinTech	10841-1-AP	1st antibody for immunoblotting
Polyclonal Rabbit Anti-TSG101 Antibody	ProteinTech	28283-1-AP	1st antibody for immunoblotting
Polyclonal Rabbit Anti-ULK1 Antibody	ProteinTech	20986-1-AP	1st antibody for immunoblotting
Polyvinylidene Difluoride Membrane	Milliore	IPVH00010	a material for immunoblotting
R	R development core team	version 4.4.1	satistical analysis
RNAiMAX	Thermo Fisher Scientific	13778	siRNA transfection reagent
siRNA STX6	Thermo Fisher Scientific	HSS115604	siRNA for transfection
Sodium Chloride	Nacalai Tesque	31320-05	a material for Tris buffer and A68 solution
Sodium Dodecyl Sulfate	Fujifilm Wako	192-13981	a material for sample buffer solution
SPARK Microplate Reader	TECAN
Stealth RNAi Negative Control Duplexes, Med GC	Thermo Fisher Scientific	12935300	siRNA transfection
Sucrose	Fujifilm Wako	193-00025	a material for A68 solution
TC20 Automated Cell Counter with Thermal Printer	Bio-Rad	1450109J1	cell counting
Thermobath	TOKYO RIKAKIKAI	MG-3100	incubation
TransIT-293	Mirus Bio	MIR 2700	DNA transfection reagent
TransIT-LT1	Mirus Bio	MIR2300	DNA transfection reagent
Tris(hydroxymethyl)aminomethane	Nacalai Tesque	35406-75	a material for Tris buffer, sample buffer and A68 solution
Trypan Blue Dye 0.40%	Bio-Rad	1450021	cell counting
Ultra-Clear Open-Top Tube, 16 x 96mm	Beckman Coulter	361706	collecting for the P1 EV fraction
Ultra-Clear Tube, 14 x 89mm	Beckman Coulter	344059	collecting for the P2 EV fraction
Vectastain ABC Standard Kit	Vector Laboratories	PK-4000	immunoblotting
Wash Bottle	As One	1-4640-02	washing membrane
μ-Slide 8 Well High	ibidi	80806