��2005 . DO NO(MPT), T . [Autophagy 2:1, 39-46 January/February/March 2006] ��2006 Landes Bioscience Sara Rodriguez-Enriquez��� Insil Kim Robert T. Currin John J. Lemasters* Department of Cell and Developmental Biology University of North Carolina Chapel Hill, North Carolina USA ���Current Address: Instituto Nacional de Cardiolog��a Ignacio Ch��vez Department of Biochemistry Juan Badiano No. 1 Col. Seccion 16 Mexico *Correspondence to: John J. Lemasters Department of Cell Biology & Anatomy School of Medicine University of North Carolina at Chapel Hill CB#7090, 236 Taylor Hall Chapel Hill, North Carolina 27599-7090, USA Tel.: 919.966.5507 Fax: 919.966.1857 Email: lemaster@med.unc.edu Received 08/02/05 Accepted 10/10/05 Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=2229 KEY WORDS autophagy, fluorescence multiwell plate reader, LysoTracker Red, MitoTracker Green, mitochondrial permeability transition, mitophagy ABBREVIATIONS 3-MA 3-methyladenine CsA cyclosporin A JNK c-Jun N-terminal kinase LTR LysoTracker Red MTG MitoTracker Green MAPK mitogen-activated protein kinase MPT mitochondrial permeability transition PI3K phosphatidylinositol-3 kinase ACKNOWLEDGEMENTS We thank Dr. Lihua He and Jae-Sung Kim for helpful discussions and Ms. Sherry Grissom for expert technical assistance. This work was supported, in part, by Grants 1 P01 DK59340 and 5-R01 AG07218 from the National Institutes of Health.
Dr.
LANDES Dr. Rodriguez-Enriquez was supported by a fellowship from CONACyT-Mexico. Imaging facilities were supported, in part, by center grants 5-P30-DK34987 and 1-P50-AA11605 from the National Institutes of Health. Research Paper Tracker Dyes to Probe Mitochondrial Autophagy (Mitophagy) in Rat Hepatocytes ABSTRACT Mitochondria become targets for autophagic degradation after nutrient deprivation, a process also termed mitophagy. In this study, we used LysoTracker Red (LTR) and MitoTracker Green to characterize the kinetics of autophagosomal proliferation and mitophagy in cultured rat hepatocytes. Autophagy induced by nutrient deprivation plus glucagon increased LTR uptake assessed with a fluorescence plate reader and the number of LTR-labeled acidic organelles assessed with confocal microscopy in individual hepatocytes both by 4- to 6-fold. Serial imaging of hepatocytes coloaded with MitoTracker Green (MTG) revealed an average mitochondrial digestion time of 7.5
DISTRIBUTE min after autophagic induction. In the presence of protease inhibitors, digestion time more than doubled, and the total number of LTR-labeled organelles increased about 40%, but the proportion of the LTR-labeled acidic organelles containing MTG fluorescence remained constant at about 75%. Autophagy inhibitors, 3-methyladenine, wortmannin and LY204002, suppressed the increase of LTR uptake after nutrient deprivation by up to 85%, confirming that increased LTR uptake reflected autophagy induction. Cyclosporin A and NIM811, specific inhibitors of the mitochondrial permeability transition also decreased LTR uptake, whereas tacrolimus, an immunosuppressive reagent that does not inhibit the MPT, was without effect. In addition, the c-Jun N-terminal kinase (JNK) inhibitors, SCP25041 and SP600125, blocked LTR uptake by 47% and 61%, respectively, but ERK1, p38 and caspase inhibitors had no effect. The results show that mitochondria once selected for mitophagy are rapidly digested and support the concept that mitochondrial autophagy involves the MPT and signaling through PI3 kinase and possibly JNK. INTRODUCTION Autophagy is a process that digests injured, worn out and surplus organelles, such as mitochondria, and recovers their amino acids and other nutrients for other uses. Fasting is a potent inducer of autophagy in the liver, an effect mediated in part by glucagon, a hormone also stimulating hepatic glycogen breakdown.1,2 During nutritional stress, autophagy is the major mechanism for protein 3,4 By contrast after nutrient replenishment, insulin inhibits autophagy promotes hepatic glycogen formation.5 Autophagy has two important functions in cells: (1) removal of excess and damaged organelles and (2) liberation of free amino acids and other nutrients at times of nutrient depriv ation.
BIOSCIENCEdamagedimpaired,anddegradation.youngmitochondria 6 When autophagy is as in Atg7-deficient mice, deformed mito- chondria accumulate in the liver.7 As an organelle occupying 20% of the cytoplasmic volume of hepatocytes,8 mitochon- dria are a major target of autophagy. Even in well nourished animals, mitochondria turn over every 15 to 25 days by autophagy.9,10 Mitophagy may also help clear mitochondria with mutations of mitochondrial DNA (mtDNA).11-13 With aging, mitochondrial degra- dation through autophagy appears to become compromised to promote accumulation of mtDNA mutations and mitochondria. Lysosomes progressively accumulate lipofuscin to further impair mitochondrial degradation.12 Because appear to be selectively targeted for autophagy in some settings,14,15 the term mitophagy has been introduced for the specific process of mitochondrial autophagy.13 Although mechanisms targeting mitochondria for mitophagy remain poorly understood, a possible component of the targeting process is a signal from the mitochondria that will undergo autophagy. Previously, we proposed that the mitochondrial permeability transition (MPT) represents one such signal for mitophagy.14 The MPT results from opening of high conductance permeability transition (PT) pores in the mitochondrial inner membrane. Swelling after the MPT causes rupture of the outer membrane, which releases cytochrome c www.landesbioscience.com Autophagy 39

and other pro-apoptotic factors into the cytosol. Cyclosporin A (CsA) is an immunosuppressive undecapeptide that blocks the MPT and prevents MPT-dependent necrotic and apoptotic cell killing to hepatocytes and other cell types.16-19 Previously using a confocal fluorescence resonance energy transfer (FRET) technique to identify depolarizing mitochondria, CsA was shown to block mitochondrial depolarization after autophagic stimulation and the autophagosomal proliferation that followed. These observations supported the conclu- sion that the MPT initiates mitochondrial depolarization in mitophagy and promotes sequestration of depolarized mitochondria into autophagosomes.14 Methods to assess autophagy and mitophagy rely on techniques such as quantitative electron microscopy and release of radioactivity after labeling cellular proteins with radioisotopes.10,20,21 More recently, markers of acidic organelles like monodansylcadaverine or LysoTracker Red (LTR) have been used to study autophagy by fluorescence microscopy.14,22 A drawback of microscopy is that relatively few cells can be studied at a time and the inability to perform high throughput screening. Here, we evaluated LTR and MitoTracker Green (MTG) as probes of mitochondrial autophagy using correlative total LTR fluorescence measurements and confocal microscopy. Our results show that total LTR uptake increases as the lysosomal/ autophagosomal compartment expands after autophagic stimulation. This autophagy predominantly involves mitochondria, which undergo protease-dependent autophagic digestion within 10 min or less. 3-Methyladenine (3-MA), blockade of the MPT and inhibition of phosphatidylinositol-3 kinase (PI3K), which suppress autophagy, inhibited cellular LTR uptake. Inhibitors of c-Jun N-terminal kinase (JNK), but not inhibitors of other stress kinases or caspases, also block autophagy assessed by LTR uptake. MATERIALS AND METHODS Materials. LysoTracker Red and MitoTracker Green were obtained from Molecular Probes (Eugene, OR). CsA was obtained from Sigma Chemical (St. Louis, MO). SCP25041 was a gift of Celgene, Signal Research Division (San Diego, CA). SP600125 was obtained from A.G. Scientific (San Diego, CA). Wortmannin, LY294002, PD98059, SB203580, Z-VAD-fmk, DEVD-fmk, IETD-fmk, and LEHD-cho were purchased from Calbiochem- Novabiochem (La Jolla, CA). NIM-811 was the kind gift of Novartis (Basel, Switzerland). Tacrolimus was obtained from Fujisawa Healthcare (Deerfield, IL). All other reagents were of analytical grade from commercial sources. Hepatocyte isolation and culture. Primary rat hepatocytes were isolated from overnight fasted male Sprague-Dawley rats (200���250 g) by collagenase perfusion, as described previously.23 Cell viability routinely exceeded 90%, as assessed by trypan blue exclusion. Hepatocytes were plated on Type 1 collagen-coated 48-well microtiter plates (Falcon, Lincoln Park, NJ) at a density of 75,000 cells per well and cultured overnight in Waymouth���s MB-742/1 growth medium containing 27 mM NaHCO3, 2 mM L-glutamine, 10% fetal calf serum, 100 nM insulin and 10 nM dexamethasone, pH 7.4 at 37��C in 5% CO2/air. To induce autophagy, hepatocyte cultures were switched from serum-containing complete growth medium to serum-free Krebs-Ringer-HEPES buffer (KRH, in mM: 25 HEPES, 115 NaCl, 5 KCl, 1 KH2PO4, 1.2 MgSO4, and 2 CaCl2, pH 7.4 at 37��C in air) containing 1 ��M glucagon. In some experiments, 3-MA (10 mM), CsA (5 ��M), NIM811 (5 ��M), tacrolimus (5 ��M), wortmannin (0.5 ��M), 2-(4-mor- pholinyl)-8-phenylchromone (LY-294003, 10 ��M), PD98059 (100 ��M), SB203580 (100 ��M), SCP25041 (100 ��M), SP600125 (20 ��M), Z-VAD-fmk (100 ��M), DEVD-fmk (100 ��M), IETD-fmk (100 ��M), and LEHD-cho (100 ��M) were added 30 min before and then during autophagic induction. Loading of LysoTracker Red. After 70 min of nutrient deprivation with glucagon, LTR (25 to 500 nM) was added. After 20 min, each well was washed two times with fresh KRH and fixed with 2% paraformaldehyde in phosphate-buffered saline for 10 min at 4��C. The red fluorescence of LTR (590 nm) was measured immediately using a 544-nm (15-nm band pass) excitation filter and a 590-nm long pass emission filter with a FLUOstar multi-well fluorescence plate reader (BMG LabTechnologies, Offenburg, Germany). LTR fluorescence after various treatments was expressed as the percentage of LTR fluorescence of hepatocytes incubated in complete growth medium. Laser scanning confocal microscopy of LTR-labeled hepatocytes. Some hepatocytes were cultured overnight on Type I collagen-coated 40-mm round number 1.5 glass coverslips inside 60-mm Petri dishes at a density of 600,000 cells per dish.24 Until induction of autophagy, cells were kept in complete serum-containing Waymouth���s MB-742/1 growth medium. On the microscope stage, complete growth medium was switched to KRH plus 1 ��M glucagon. After 70 min, the hepatocytes were loaded with red-fluo- rescing LTR (200 nM) for 20 min and through-focus confocal images of LTR-loaded hepatocytes were taken at 2 ��m intervals with a Zeiss LSM 410 inverted laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) using a 63X oil/1.4 N.A. planapochromat objective lens. Excitation at 568 nm was provided by an argon/krypton laser, and fluorescence emission was measured through a 590-nm long pass barrier filter. Laser excitation energy was attenuated 100- to 1000-fold to minimize photobleaching and photodamage. Laser scanning confocal microscopy of hepatocytes coloaded with LTR and MTG. In other experiments, hepatocytes were cultured overnight on Type I collagen-coated 7-mm round number 1.5 coverslips fixed to the bottom of 35-mm Petri dishes (Corning, Ashland, MA) at a density of 300,000 cells per dish. Until induction of autophagy, cells were kept in complete Waymouth���s MB-742/1 growth medium. Hepatocytes were loaded with green-fluorescing MitoTracker Green (MTG, 0.5 ��M) for 60 min in humidified air at 37��C in Waymouth���s MB-742/1 growth medium containing 25 mM Na-HEPES. Afterwards, the MTG-loaded hepatocytes were washed 2 times with fresh complete growth medium and incubated with red-fluorescing LysoTracker Red (LTR, 0.5 ��M) for 20 min under identical conditions. After MTG and LTR had been loaded, one third of the initial concentration of LTR was kept in the medium for the duration of the experiment to maintain steady state loading. On the microscope stage, complete growth medium was switched to KRH plus 1 ��M glucagon in the presence or absence of proteases inhibitors (7.5 ��M pepstatin A or 10 ��M leupeptin) at 37��C. Time series of confocal images were collected every 1 to 2 min for up to 90 min after autophagic induction with a Zeiss LSM 510 inverted laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) using a 63X oil 1.4 N.A. planapochromat objective lens. Excitation of LTR at 543 nm was provided by a helium/neon laser, and fluorescence emission was measured through a 560-nm long pass barrier filter. Excitation of MTG at 488 nm was provided by an argon laser, and fluorescence emission was measured through a 500���550-nm band pass barrier filter. Laser excitation energy was attenuated 100- to 1000-fold to minimize photobleaching and photodamage, which was negligible in control experiments in complete growth medium. Measurement of the cellular content of lysosomes. The number of LTR-labeled organelles in confocal images was quantified from serial confocal images using the computer program Image Processing Kit 4.0.25 Briefly, a superimposing image was generated from two single slices through a difference of Gaussians. In each superimposing image, the threshold of each structure was adjusted and processed to get outlined structures. Each outlined structures were quantified using a mathematical function in Adobe Photoshop 7.0. Statistical analysis. Data are presented as means �� S.E.M. Differences between means were analyzed by the student���s t test using p 0.05 as the criterion of significance. RESULTS Increase of LTR uptake by cultured hepatocytes after nutrient depriva- tion plus glucagon. LTR accumulates inside endosomes, autophagosomes, lysosomes and autolysosomes (autophagosomes fused with lysosomes) by Mitochondrial Autophagy in Hepatocytes 40 Autophagy 2006 Vol. 2 Issue 1