Loss of iron triggers PINK1/Parkin-independent mitophagy

Loss of iron triggers PINK1/Parkin-independent mitophagy A novel mitophagy assay uncovers a new PINK1/Parkin-independent mitophagy pathway induced by a decrease in iron levels. This pathway is active in fibroblasts of Parkinson patients with Parkin mutations and could be exploited as a potential therapy.


RNA interference
The shRNA construct against human BNIP3 was cloned into the pSuperRetro.Puro vector according to manufacturer's instructions (Oligoengine, Seattle, USA) and cells transduced with retroviral particles as described for the tandem tagged mitochondrial construct. The BNIP3 RNAi sequence used was GGAAAGAAGTTGAAAGCAT. Custom siRNAs against ATG5 and Beclin1 were purchased from Thermo Scientific, ATG5: GGAAUAUCCUGCAGAAGAAUU and Beclin1: GCUCAGUAUCAGAGAAAUUU. siRNAs were transfected using Lipofectamine RNAiMAX (Life Technologies) in accordance with manufacturer's instructions. Validated siRNA against PINK1 was purchased from Sigma-Aldrich (Product No. SASI_Hs01_00022856) and transfected using Transfectin (Bio-Rad) in accordance with manufacturers instructions. Non-targeting control siRNA was purchased from Thermo Scientific (Product No. D-001810-10-20).

Immunofluorescence and Fixed Cell Mitotracker Staining
Cells were grown on glass coverslips and cultured/treated as described above. For mitotracker staining cells were incubated in culture medium containing 200nM mitotracker Red FM for 30min and washed twice with PBS prior to fixation. Cells were fixed with 3.7% formaldehyde, 50mM HEPES pH7.4 for 20min. Formaldehyde was quenched with DMEM, 10mM HEPES pH7.4. Cells were permeablised with 0.2% NP-40 in PBS for 3min, blocked for 15min with 1% BSA in PBS (blocking buffer), incubated with primary antibody in blocking buffer for 1h at +37˚C, washed 3 x 10min in blocking buffer, incubated with Alexafluor coupled secondary antibodies (Life Technologies) in blocking buffer for 30min and washed 3 x 10min in blocking buffer. Cells were mounted using prolong gold mounting solution with dapi (Life Technologies) and visualised with a Nikon Eclipse Ti-S fluorescence microscope (Nikon, Kingston Upon Thames, UK). Primary antibodies were ATP synthase subunit beta (Abcam, ab13740, 1:500), LC3 (MBL, M152, 1:500), COXIV (Cell Signalling Technology, 4850, 1:500) and PINK1 (Novus Biologicals, BC100-494, 1:500). Images were quantified by manual counting of three fields of view using Nikon NIS-Elements software and images processed using Adobe Photoshop CS5.1 (Adobe, San Jose, USA).

Live Cell Lysotracker and Mitotracker Staining
Cells were grown on glass bottomed dishes and cultured/treated as described above. For lysotracker staining cells were incubated in culture medium containing 50nM LysoTracker Deep Red for 1 hour and for mitotracker staining cells were incubated in culture medium containing 200nM MitoTracker Deep Red FM for 30min. Cells were washed once with PBS and then incubated at +37˚C in DMEM, 10mM HEPES, without phenol red and images taken of live cells using a Nikon Eclipse TE200 microscope and Applied Precision softWoRx 5.5 software (GE Healthcare). Images were processed using Adobe Photoshop CS5.1.

Electron Microscopy
Cells were washed twice with PBS and subsequently lifted with 0.48mM EDTA in PBS. Cells were collected by centrifugation at 100xg for 2min. Cells were fixed with 2.5% glutaraldehyde, 2% formaldehyde in 0.1M sodium cacodylate. Fixed cells were dehydrated in graded ethanol and propylene oxide and embedded in durcupan resin (Sigma-Aldrich). Sections were cut on a Leica UCT ultramicrotome (Leica Microsystems, Milton Keynes, UK), collected on Pioloform coated 100 mesh copper grids and stained with uranyl acetate and lead citrate. Sections were examined on a Jeol 1200 EX electron microscope (Jeol, Welwyn Garden City, UK) and images taken on Fuji digital imaging plates and processed in a Ditabis plate scanner (Ditabis, Pforzheim, Germany). Images were quantified by manual counting of single or double membrane structures containing mitochondria. 50 cell sections (cross sections through 50 cells) were counted per condition for each independent experiment.

Oxygen Consumption
Oxygen consumption was measured using a Seahorse XF24 Extracellular Flux analyser (Seahorse Bioscience, North Billerica, USA). Cells were cultured in 24 well Seahorse plates at a density of 8x10 4 cells/well and incubated with DFP for the indicated length of time. Cell culture medium was replaced with unbuffered DMEM containing 200mM GlutaMax-1, 100mM Na pyruvate, 25mM glucose, 32mM NaCl and 40µM Phenol Red. Oxygen consumption was measured and respiration rate analysed with injections of 1µM oligomycin, 1µM FCCP and 10µM antimycin A as previously described for SH-SY5Y cells [4]. Results were normalised to total protein determined using the BCA assay (Thermo Scientific)

ATP/ADP Ratios
Cells were washed once in ice cold PBS, lysed in 300µL 5% percholric acid, vortexed and centrifuged at 17700xg for 3min. Supernatent was extracted with 3 washes of a 1:1 mixture of tri-n-octylamine and 1,1,2-trichlorotriflouroethane. The aqueous phase was separated by capillary electrophoresis and nucleotides detected by UV absorbance at 260nm as previously described [5].

Galactose Incubation
All cells were washed twice with glucose-free DMEM with 10mM galactose + 10% FBS. Cells grown under glucose were then incubated in DMEM containing 25mM glucose + 10% FBS and cells grown under galactose were incubated in glucose-free DMEM containing 10mM galactose + 10% FBS for 48 hours prior and during treatment with 1mM DFP as described above.

Reactive Oxygen Species Detection
Cells were cultured in 96 well plates at a density of 2.5x10 4 cells/well and treated with DFP as indicated. 5mM N-acetylcysteine was added at the same time as DFP or vehicle control. Following incubations cells were washed once with PBS and incubated for 30min at +37˚C in DMEM with 1% FBS containing 100µM 2',7'-dichlorofluorescin diacetate (DCFDA). Cells were washed once with Krebs-Ringer-HEPES (KRH) buffer and then 200µL KRH buffer was added to each well. 0.1% H 2 O 2 was added where indicated and fluorescence measured at excitation wavelength 485nm and emission wavelength 538nm using a SpectraMAX Gemini EM plate reader (Molecular Devices). Results were normalised to the amount of cell material which was measured using the sulforhodamine B assay performed as previously described [6].