cDNAs and Viral Vectors
Full length and cytoplasmic (cyto) mitochondrial ferritin (FTMT) cDNAs were produced using standard techniques and oligonucleotide-mediated PCR mutagenesis. FTMT was cloned from human genomic DNA as previously described 
. The major portion of the mitochondrial localization leader (194 nucleic acids) was truncated to obtain the cytoFTMT with resulting cytoplasmic localization 
To express transgenes, we constructed replication deficient, type 5 adenovirus (AdV5, deleted for E1/E3) expression vectors, based on the BD Adeno-XTM Expression System 1 (BD Biosciences Clontech, Carlsbad, CA, USA). Briefly, the cDNAs were inserted into appropriate restriction sites in pShuttle-2 and then subcloned into pAd-X plasmid to generate the recombinant AdV.
We recovered, isolated and propagated viral stocks using human embryonic kidney cells (HEK-293, ATCC, Manassas, VA, USA, #CRL-1573). Working stocks of virus were produced using double CsCl density gradient purification. The final purified vector preparations were dialyzed using TNMG buffer (10 mM Tris, 150 mM NaCl, 1 mM MgCl2, 5% glycerol). The recombinant AdV5 viruses were titered on HEK293 cells using immunodetection for single infectious units. The titers, in units of pfu/mL, were as follows: cytoFTMT AdV 2.6×1011, FTMT 1.9×1011, lacZ AdV 3.7×1010, L*H AdV 2×1010, and FTH AdV 1.4×1010. The eGFP AdV used had a titer of 1×1011 pfu/mL and was purchased from the University of Pittsburgh Vector Core Facility.
Cell Line and Western Blot
Human osteosarcoma cells U2OS (ATCC, #HTB-96) were grown and maintained in complete medium (90% DMEM, 10% FBS) and transduced with cytoFTMT, FTMT and lacZ AdV using a multiplicity of infection (MOI) of 30. Cells expressing the different ferritins and control reporter (lacZ) were harvested at 48 h post-transduction and lysed in a detergent solution containing protease inhibitor cocktail and EDTA (Pierce, Rockford, IL, USA). Total protein content was measured with a bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL). Equivalent amounts of clarified samples were resolved on 4–20% polyacrylamide gradient gels (Pierce). Primary antibody used for the western blot analysis was mouse monoclonal to mitochondrial ferritin (ab55027, Abcam, Boston MA). We used chemiluminescence to expose the immunoreactive bands on film (1651454, Eastman Kodak, Rochester, NY). The gels were stripped and reprobed with mouse monoclonal for β-actin (sc-47778, Santa Cruz). Secondary antibodies were goat anti-rabbit horseradish peroxidase (HRP) conjugate (1858415, Pierce, Rockford, IL) and goat anti-mouse HRP conjugate (1858413, Pierce).
Inductively Coupled Plasma Emission Spectroscopy (ICP-ES)
We transduced U2OS cells with AdV coding for the different ferritins and lacZ control using MOI of 30. At 24 hours post-transduction, the culture media was supplemented with 1 mg/ml holotransferrin (T0665, Sigma-Aldrich, St. Louis, MO) and 1 mM ferric citrate (F3388, Sigma-Aldrich). At 48 hours post-transduction all culture media was aspirated and cells were washed, harvested and counted.
For cell digestion, the cells were spun down in Oak Ridge centrifuge tubes (3119-0010, Nalgene), resuspended in 70% nitric acid and heated in an autoclave with vacuum for moisture removal for 1 hour at 90°C. The dried and digested material was then resuspended in 5 ml of 1% nitric acid, and the tubes were agitated in an ultrasonic water bath for one hour to bring the cell pellets into solution. We used a Perkin Elmer Optima 3000DV ICP (Perkin Elmer, Waltham, MI) in axial mode for the analysis. The results are obtained as mg/L (ppm) in the solutions and then normalized by cell number. The detection limit for iron on this instrument is 0.003 ppm.
We used the Vybrant cytotoxicity assay (V-23111, Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. We measured the amount of released enzyme glucose 6-phosphate dehydrogenase (G6PD) of U2OS cells 48 hours after the AdV transduction with the different ferritin transgenes and lacZ control. The fluorescence was measured with a Tecan Safire 2 fluorescence plate reader (Tecan Group Ltd., Durham, NC) using excitation/emission ~530/590 nm. A background fluorescence value of the cell medium alone was subtracted from each value. The values were normalized to the fluorescence of lysed cells representing the total cellular G6PD. For positive control, we used 50 µM camptothecin (C9911, Sigma-Aldrich).
The significance of the ICP-ES results and the cytotoxicity assay was calculated using Origin 7.5 software (Northampton, MA). We used one-way analysis of variance (ANOVA) to look at differences of the group means, followed by Tukey’s method for pair-wise differences with a confidence interval of 0.95.
Immunocytochemistry and Subcellular Localization Study
U2OS cells were grown on 35 mm glass bottom cell culture dishes (WillCo 64-0758, Warner Instruments, Hamden, CT) and transduced with reporter transgenes. At 48 h post-transduction, cells were fixed using 4% paraformaldehyde (PFA). The cells were washed with phosphate buffer saline (PBS) and 0.2% Tween 20 (Bio-Rad, Hercules, CA) and then probed using antigen-specific antibodies followed by the secondary reagents as described below. We used mouse monoclonal to mitochondrial ferritin (ab55027, Abcam), MitoTarcker Orange (M7510, Invitrogen, Carlsbad, CA), mouse monoclonal to LAMP2, lysosome marker (ab25631, Abcam), and rabbit polyclonal to Golgi protein marker GOLH4 (ab28049, Abcam). Cell nuclei were counterstained with Hoechst 3342 (Calbiochem, La Jolla, CA).
In Vivo Studies
All animal experiments were approved by the Carnegie Mellon Institutional Animal Care and Use Committee (IACUC). Adult female (N
10) C57BL mice (Harlan, Indianapolis, IN), 5–7 weeks old, were anesthetized with an intraperitoneal cocktail of ketamine/xylazine. The mice were placed supine and a cannula was inserted into the nasal cavity approximately 7 mm deep. A viral vector solution (10 µl) was slowly administered intranasally over 5–10 minutes. One nasal passage received cytoFTMT AdV and the opposite nasal passage received the eGFP AdV control. The mice remained supine for at least 15 minutes post-administration to aid vector absorption to the olfactory epithelium. Mice were intubated prior to this procedure to maintain a clear airway. Approximately 48 h after the viral administration, iron supplement solution of 10 mM ferric citrate (Sigma, #F3388) and 2 mg/ml holotransferrin (#T0665, Sigma) was introduced to both nasal passages by the same intranasal instillation procedure. At 48 hours later, the mice were imaged. After imaging, animals were perfused transcardially with PBS and then with 4% PFA, and the brains were either embedded in paraffin or flash frozen in optimal cutting temperature (OCT) compound (EMS, Hatfield, PA) and stored at −80°C.
MRI was performed at 7 T using a Bruker Biospec imaging system (Bruker, Billerica, MA). The mice were anesthetized, intubated, placed on a mechanical ventilator, and maintained on 0.75% isoflurane in 70% O2
and 30% N2
O inhalation gas during the imaging sessions. For in vivo
imaging we used a quadrature receive mouse brain surface coil (Rapid Biomedical, Columbus, OH) and for excitation we used a 72 mm birdcage coil (Bruker). T2
*-weighted images were acquired using a 3D gradient-echo (GRE) sequence with TE/TR
7/100 ms, 20° flip angle, 4 averages, 128×128×128 matrix, field of view
1.5×1.5×1.5 cm and 117 µm isotropic resolution. The total in vivo
scan time was 1 hour 49 min. For the ex vivo
imaging, we used an 11.7 T Bruker microimaging system with a volume coil and a 3D GRE sequence with TE/TR
8/100 ms, 20° flip angle, 4 averages, 256×256×256 matrix, field of view
1.28×1.28×1.28 cm and 50 µm isotropic resolution. The total ex vivo
scan time was 7 h 16 min.
Immunohistochemistry and Histology
Frozen brain tissue was cryosectioned in 20 µm thick slices, and paraffin embedded tissue was sectioned into 10 µm thick slices. All tissue sections were mounted on glass slides. The slides were incubated for one hour at room temperature with primary antibodies in a humidified chamber, washed and incubated for 45 min with secondary antibodies. We used rabbit polyclonal antibody to Olfactory Marker Protein (OMP) (ab62609, Abcam) and mouse monoclonal antibody to mitochondrial ferritin (ab55027, Abcam), with secondary Alexa Fluor 594 F(ab’)2 fragment of goat anti-rabbit (A1172, Invitrogen), goat anti-rabbit Alexa Fluor Alexa 635 (A31576, Invitrogen) and Alexa Fluor 568 F(ab’)2 fragment of goat anti-mouse (A11019, Invitrogen). Nuclei were counterstained with Hoechst 3342 (Calbiochem). Control sections were incubated with the secondary antibodies without the presence of the primary antibody. For the Perls’ iron stain the sections were immersed in water with 2% potassium ferrocyanide and 2% concentrated hydrochloric acid for 30 min. Cell nuclei were counterstained with 0.5% nuclear fast red (H-3403, Vector Labs, Burlingame, CA). Paraffin embedding, sectioning and Hematoxylin & Eosin (H&E) staining was performed by AML Laboratories (Baltimore, MD).
Glass bottom cell culture dishes and glass cover slips were mounted after the final wash and were imaged using a Carl Zeiss LSM 510 Meta UV DuoScan inverted confocal laser-scanning microscope. RGB channels were collected sequentially with a matrix resolution of 1024×1024 and 4 averages.