Creation and Characterization of Mitochondrial DNA-Depleted Cell Lines with “Neuronal-Like” Properties

Journal ofNeurochemistryLippincott—Raven Publishers, Philadelphia© 1996 International Society for Neurochemistry

Creation and Characterization of MitochondrialDNA-Depleted Cell Lines with “Neuronal-Like” Properties

Scott W. Miller, *~patrjcjaA. Trimmer, *tW. Davis Parker, Jr., and Robert E. Davis

MitoKor, San Diego, California; and Departments of *Neurology, tNeuroscience, and ~Pediatrics,University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A.

Abstract: Mitochondrial dysfunction and attendant bi-oenergetic defects are increasingly recognized as playingan important role in neurodegenerative disorders. The in-creased attention on mitochondrial involvement points tothe need for developing cell lines that have neuron-likecharacteristics for the genetic analysis and modeling ofthese diseases. We describe the creation of respiratory-deficient SH-SY5Y neuroblastoma cell lines (p°6415)byselectively depleting mitochondrial DNA through pro-longed exposure to ethidium bromide. Oxygen consump-tion in these cells and activities of the electron transportchain enzyme complexes I and IV that contain subunitsencoded by the mitochondrial genome are eliminated. Incontrast, the function of complex II, a nuclear-encodedelectron transport chain component, is largely intact inthese cells. The p°64/5cells retain the ability to differenti-ate into cells with neuron-like phenotypes following treat-ment with phorbol ester or retinoic acid. Normal respi-ratory function is recovered by repopulation of p°64/5cells with exogenous human platelet mitochondria. Thep°6415cell line serves as a valuable model for the studyof neurologic diseases suspected of involving mitochon-drial dysfunction. Key Words: Mitochondria—Cyto-plasmic hybrid—p cell—SH-SY5Y cells—Electron trans-port chain—Reactive oxygen species.J. Neurochem. 67, 1897—1907 (1996).

In examining ETC defects, it is often unclearwhether the defect originates in the nuclear genome orthe mitochondrial genome or arises from secondarytoxic insults. An experimental approach to the studyof this problem has been to prepare cell lines that havebeen treated withethidium bromide (EtBr), a cationic,lipophilic agent that inhibits mtDNA replication andtranscription (Zybler et al., 1969; Nass, 1970). Aftermany replications in the presence of sublethal EtBrconcentrations, the resulting cells, termed p° (rhozero) cells, are devoid of mtDNA and ETC activity andare auxotrophic with respect to uridine and pyruvate(Desjardins et al., 1985; King and Attardi, 1989b).The p°cells can be repopulated with exogenous mito-chondria through fusion with cytoplasts or plateletsor microinjection of mitochondria (King and Attardi,1989a,b; Chomyn et al., 1994; Dunbar et al., 1995).In this manner, a defect encoded on the mitochondrialgenome can be transferred from an affected individualinto a host cell with a normal nuclear environment forstudy of the resulting phenotype.

Several studies have modeled mitochondrial dys-function in vitroby transforming p°cells with diseasedmitochondria. The metabolic defects arising from themitochondrial encephalomyopathy, lactic acidosis, andstroke-like episodes (MELAS) (Goto et al., 1990)tRNALeu(~R) gene mutation, and the myoclonic epi-

The electron transport chain (ETC) is located withinthe mitochondria and is uniquely composed of productsfrom two distinct genetic systems. Although the vastmajority of the gene products making up the ETC areencoded by the nuclear genome, 13 proteins, a set oftRNAs, and RNAs are encoded by mitochondrial DNA(mtDNA). Inheritance of mtDNA is maternal and cy-toplasmic rather than nuclear and follows non-Men-delian genetics (Anderson et al., 1981; Brown andWallace, 1994). Genetic lesions in mtDNA are knownto cause mitochondrial dysfunction resulting in defi-cient respiratory capacity and impaired cellular func-tion. Mitochondrial diseases involving ETC defectspredominantly affect postmitotic tissues with high en-ergy demands (Wallace et al., 1994; Beal, 1995).

Received March 15, 1996; revised manuscript received July 4,1996; accepted July 8, 1996.

Address correspondence and reprint requests to Dr. S. W. Millerat Department of Biochemistry, MitoKor, 11494 Sorrento ValleyRoad, San Diego, CA 92121, U.S.A.

Abbreviations used: cybrid, cytoplasmic hybrid; DCF, dichloro-fluorescein; DCF-DA, 2 ‘,7 ‘-dichlorohydrofiuorescein diacetate;DMEM, Dulbecco’s modified Eagle’s medium; EtBr, ethidium bro-mide; ETC, electron transport chain; FBS, fetal bovine serum; HBSS,Hanks’ balanced salt solution; MELAS, mitochondrial encephalo-myopathy, lactic acidosis, and stroke-like episodes; MERRF, myo-clonic epilepsy and ragged red fibers; mtDNA, mitochondrial DNA;NAO, lO-nonylacridine orange bromide; PEG, polyethylene glycol;RA, all-trans-retinoic acid; p°,rho zero (mitochondrial DNA defi-cient); ROS, reactive oxygen species; SDS, sodium dodecyl sulfate;SSC, saline—sodium citrate; TPA, 12-O-tetradecanoylphorbol 13-acetate.

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1898 S. W. MILLER ET AL.

lepsy and ragged red fibers (MERRF) encephalomyo-pathy 8344 and 8356 mutations (Shoffner et al., 1990;Silvestri et al., 1992; Zeviani et al., 1993) have beenexpressed in p°cells repopulated withdonor mitochon-dna (Yoneda et al., 1992; Chomyn et al., 1994; Dunbaret al., 1995). These studies have advanced our under-standing of the etiologic role of mitochondria dys-regulation in several diseases and have used rapidlydividing, mitotically activecells as recipients for mito-chondria. Replicating cells, however, may not be ap-propriate cellular models of neurodegenerative disor-ders that involve terminally differentiated cells.

We describe the preparation and characterization ofimmortalized,mtDNA-depleted lines that can be termi-nally differentiated into cells with a neuron-like pheno-type (p°33/5 and p°64/5). These cell lines can berepopulated withexogenous mitochondria from humandonors, creating stable cytoplasmic hybrid (cybrid)cell lines with the metabolic phenotype derived fromthe mitochondria donor. The technique described hereis suitable for studying energetic defects arising frommitochondnial genetic lesions within a cell with a neu-ron-like phenotype.

EXPERIMENTAL PROCEDURES

Tissue cultureReagents for tissue culture were purchased from Gibco-

BRL (Gaithersburg, MD, U.S.A.). lO-Nonylacridine orangebromide (NAO) was purchased from Molecular Bioprobes(Eugene, OR, U.S.A.). The short-chain ubiquinone ana-logue, coenzyme Qi, was a gift from Eisai Pharmaceuticals(Tokyo, Japan). All other reagents were from Sigma Chemi-cal Co. (St. Louis, MO, U.S.A.). SH-SY5Y neuroblastomacells (Biedleret al., 1973, 1978)were grown in tissue cultureflasks in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10% (wt/vol) heat-inactivated fetal bo-vine serum (FBS), penicillin (100 IU/ml), streptomycin(50 ~zg/ml), glucose (4,500 mg/L), 25 mM HEPES, andglutamate (584 mg/L) at 37°Cin 5%CO2 (growth medium).FBS was thawed overnight at 4°C,warmed to 37°C,andthen heated to 56°Cfor 30 mm for heat inactivation. Produc-tion and culturing of most p°cells require the presence ofuridine (50 ~g/ml) and pyruvate (100 ~sg/ml) to supportgrowth (Desjardins et al., 1985; King andAttardi, 1989a,b).The p°cells were produced by culturing SH-SY5Y cells inthe presence of EtBr at various concentrations and times asindicated. Cells were replated approximately once per weekand with medium changes every 2—3 days. Induction ofcell differentiation was a 3-week process. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) at 16 nM or all-trans-retinoic acid (RA) at 1 jiM to low-density cultures ofcells (1.75 >< l0~cells/cm

2) for 1 week was used to inducedifferentiation of the cells. During the second week, 1 jig!ml aphidicolin was added in addition to TPA and RA toeliminate proliferating cells. The final week of treatment waswith TPA or RA alone. Cell medium was changed everyother day.

Oxygen consumptionCells were removed from 75-cm2 culture flasks by treat-

ment with trypsin, rinsed one time with Hanks’ balanced

salt solution (HBSS; GibcoBRL), resuspended at 2.0 x l0~cells/ml in HBSS, and maintained at 37°C.A sample (80jil) of the cell suspension was introduced into a stirred po-larographic microchamber (Haas and Stumpf, 1984) in afinal volume of 330 jil in HBSS. Oxygen consumption wasmeasured by a Yellow Springs (Yellow Springs, OH,U.S.A.) Clark oxygen electrode (model 5531) and monitor(model 5300) at 37°C.Oxygen utilization was calculated asdescribed by Estabrook (1967).

Enzymatic assays and protein contentdeterminations

Citrate synthase activity was determined from 2 X l0~cells incubated at 30°Cin a cuvette containing 980 ,ul of0.04% (wt/vol) Triton X-l00, 0.1 mM 5,5’-dithio-bis(2-nitrobenzoic acid), and 100 mM Tris (pH 8.0) for 3 mmbefore assay. To initiate the reaction, JO-p1 aliquots each ofacetyl-CoA and oxaloacetic acid were addedto final concen-trations of 50 and500 pM, respectively. The contents of thecuvette were mixed by inversion, and the linear increase inabsorbance at 412 nm was recorded spectrophotometricallyfor 2—3 mm (Shepherd and Garland, 1969).

Complex I (NADH:ubiquinone oxidoreductase) activitywas determined essentially as described earlier using coen-zyme Q~(Parker et al., 1990, 1994). Membranes were lysedby incubation of cells at 2 x iO~cells/ml with 0.005% (wt/vol) digitonin in HBSS plus 5 mM EDTA (HBSS/EDTA)for 20 s at 23°C.The solubilization was stopped by additionof 50 volumes of cold HBSS/EDTA. The lysed cells werecentrifuged at 14,000 g for 10 mm at 4°C.The pellet wasdiluted to —~1mg/ml protein in HBSS/EDTA containing 1pM leupeptin, 1 pMpepstatin, and 100 ?uM phenylmethylsul-fonyl fluoride. Immediately before performing complex Iassays, a 200-jil aliquot of protein suspension in a 0.5-mlEppendorf tube was sonicated for 6 mm in an ice-packedcup horn sonicator (Sonifier 450; Branson, Danbury, CT,U.S.A.) at 50% (wt/vol) duty cycle at 50% power. ThecomplexIassay reaction wasperformed by sequentially add-ing 10 p1 of 4.2 mM coenzyme Q

1 in HPLC-grade methanol,10 p1 of 10 mM NADH, and 30—100 pg of protein to acuvette containing assay buffer ~25 mM potassium phos-phate (pH 8.0), 0.25 mM EDTA, and 1.5 mM potassiumcyanide] that had been preincubated at 30°Cfor 2 mm. Thetotal reaction volume was 1 ml. The change in absorbanceat 340 nm was measured for 120 s. This was immediatelyfollowedby addition of 5 p1 of 500 jiM rotenone (in HPLC-grade methanol), and the absorbance change was measuredfor another 120 s to determine the rotenone-sensitive activity.The complex I activity was calculated by subtracting therotenone-sensitive rate from the total rate along the linearportion of the curve and using 6.81 (mM cm)’ as the com-bined NADH-decylubiquinone extinction coefficient at 340nm. Complex II (succinic dehydrogenase) and complex IV(cytochrome c oxidase) activities were determined essen-tially as described earlier (Parker et al., 1990, 1994). Allenzymatic activities were normalized to total cellular proteinas determined by the BCA Protein Assay (Pierce, Rockford,IL, U.S.A.).

NAO uptakeCells were plated in 96-well microplates at 4—50 X i0

3cells per well overnight. Medium wasdecanted, and the cellswere rinsed once with HBSS. The cells were incubated withNAO (1 pg/ml) for 60 mm at 37°C,without CO

2, in a 100-pl volume of HBSS. The medium was decanted, and the

i. Neurochem., Vol. 67, No. 5, 1996

“NEURONAL-LIKE” p° CELLS 1899

cells were rinsed three times with 200 p1 of HBSS andleft inlOOpl of HBSS. Dye uptake was measured using a Millipore(Bedford, MA, U.S.A.) Cytofluor model 2350 fluorescencemeasurement system. The filter set for NAO was 485 nm(excitation) and530 nm (emission). Dye uptake by the cellswas optmmized for mncubatmon time, concentration, and cellnumber and was shown to be linear with respect to cellnumber under the conditions chosen (data not shown).

Slot blot analysis of mtDNATotal DNA from p°cells treated with various concentra-

tions of EtBr for 33 or 64 days was isolated by a Qiagen(Chatsworth, CA, U.S.A.) DNA Purification Kit and quantm-fled by absorbance at 260 nm and agarose gel electrophore-sis. Typically, the procedure yielded —~1 pg of cellular DNAfrom 1 x 106 cells. Ten micrograms of cellular DNA wasdenatured by treatmentwith 0.2 M NaOH in a 100-pu volumeat 65°Cfor 30 mm. The sample was neutralized with 100p1 of 2 M ammonium acetate. The DNA was vacuum-blottedonto a Zeta probe membrane (Bio-Rad, Richmond, CA,U.S.A.) prewetted with lOx saline—sodium citrate (SSC;1.5 mM NaCl and 150 mM sodium citrate, pH 7.0). Themembrane was exposed to UV light (254 nm, 125 mJ) andincubated with blocking buffer [0.2% (wt/vol) I-Block(Tropix, Bedford), O.5x SSC, and 0.1% (wt/vol) Tween-20] for 30 mm at ambient temperature. The membrane waswashed with hybridization buffer [5x SSC, 1% (wt!vol)sodium dodecyl sulfate (SDS), and 0.5% (wt/vol) bovineserum albumin] in an open small volume plastic dish. Alka-line phosphatase—oligonucleotide conjugate probe havingthe COXI subunit mtDNA sequence 5 ‘-CGTTTGGTATTG-GGTTATGGC-3’ was prepared as described by Ghosh etal. (1990). The membrane was incubated with 10 ml ofhybridization buffercontaining 2 pmol!ml alkaline phospha-tase—oligonucleotide conjugate for 60 mm at 42°C.Themembrane was washed three times with buffer I [lx SSCand 0.1% (wt!vol) SDS for 5 mm at room temperature],once with buffer 2 [0.5x SSC and 0.1% (wt!vol) SDS, for3 mm at 50°C],once with buffer 3 [lx SSC and 1% (wt!vol) Triton X-l00 for 3 mm at room temperature], oncewith buffer 4 (lx SSC for 10 mm at room temperature),and finally once briefly with development buffer (50 mMNaHCO, and 1 mM MgCl2, pH 9.5). The membrane wasdeveloped with Lumi-Phos 530 (Boehringer Mannheim, In-dianapolis, IN, U.S.A.) as per the manufacturer’s protocol.The mtDNA was quantified by comparison of the hybridiza-tion signal with signals obtained from known quantities ofplasmid (ranging from 1 x 10_Is to 1 x l0’~mol) con-taining the cytochrome c oxidase subunit I gene insert. Thelimit of detection of the alkaline phosphatase-oligonucleo-tide conjugate probes in the slot blot assay was determinedto be I x 10_17 mol of target DNA.

Platelet transformation of p°cellsThe p°cells were transformed by a modification of the

method of Chomyn et al. (1994). Platelets were isolatedfrom 10 ml of fresh blood drawn into a Becton Dickinson(Rutherford, NJ, U.S.A.) Vacutainer containing anticoagu-lant (acidcitrate dextrose). Acid citrate dextrose is superiorto EDTA as an anticoagulant for this application. The bloodwas kept on ice and used within 8 h. The blood was layeredover Histopaque (Sigma) in a 12-ml Accuspin tube andcentrifuged for 10 mm at 1,000 g at room temperature. Theplatelet-rich fraction was resuspended in 5 volumes of phos-phate-buffered saline, centrifuged at 1,700 g for 10 mm,

decanted, and resuspended in Ca2~-free minimal essential

medium (fusion medium). The p°cells were removed fromculture flasks by treatment with trypsin, rinsed two times,and finally resuspended in fusion medium before fusion. Thep°64/5 cells (4 x 10’) were combined with i0~or 108platelets in 2 ml of fusion medium and incubated for 10 mmat 23°C.Preparations of p°cells without added platelets andpreparations of platelets without added p°cells served asnegative controls and to monitor reversion of the p°cellphenotype. The cell mixtures were centrifuged at 300 g for10 mm and resuspended in 57 p1 of fusion medium. Cellularfusion was assisted by adding polyethylene glycol [PEG1000 (J. T. Baker), 70% (wt!vol)I in fusion medium tothe cells to achieve a final volume of 200 p1 [final PEGconcentration, 50% (wt/vol)]. Cells were incubated for 1.5mm at room temperature and then diluted to a final volumeof 10 ml with normal p°cell medium (SH-SY5Y mediumsupplemented with 100 pg!ml pyruvate and 50 pg!ml un-dine). The cell mixture containing fused cells was plated in75-cm2 flasks. The medium was changed on the followingday. The cells were allowed to recover for 1 week in p°medium with medium changes every 2 days. Cybrids repopu-lated with exogenous platelet mitochondria were selectedby culturing in medium lacking uridine and pyruvate andsupplemented with 10% (wt/vol) dialyzed heat-inactivatedFBS. These conditions were designed so that only aerobi-cally competent cells, i.e., cells successfully fused with exog-enous mitochondria, could survive. Dense colonies ( 3 mmin diameter) interspersed with a few small loose colonies(<3 mm) andindividual cells formed 3—5 weeks postfusion.The fusion efficiency based on the large dense colonies was0.025%. The reversion rates were calculated by plating 2x 106 p°cells in aflask, growing them in selection medium,and scoring for any surviving cells after a 4-week period.Based on the reversion rate for clone p°64!5( 10_6; seeTable 1), the fusion efficiency, and 4 x 10’ p°cells usedin the fusion procedure, fused cells will greatly outnumberany spontaneously reverted cell in each flask.

Cybrids were evaluated for ETC activity and differentia-tion 4—8 weeks after fusion. The cybrids were isolated as aheterogeneous population rather than as individual clones tominimize thepossibility of introducing a clonal bias. Karyo-typing was performed as described earlier (Rooney andCzepulkowski, 1986; Freshney, 1994). Cells were grownon coverslips in appropriate medium for Giemsa banding.Twenty cells from five cybrid cell lines were scored fordiploidy. No hyperdiploidy was observed in anyof thecybnidlines.

Quantification of reactive oxygen species (ROS)Cells were platedat 75,000 cells per well in 96-well plates

and cultured for 24 h in growth medium. The medium wasdecanted, and the cells were rinsed with 200 p1 of warmHBSS. These cells were then incubated with 30 pM 2 ‘,7 ‘-

dichlorodihydrofluorescein diacetate (DCF-DA; MolecularProbes) for 2 h in the cell culture incubator. The cells werewashed twice with 200 p1 of HBSS at 37°C.In someexperi-ments, sodium azide was added for 30 mm in 100 pl ofwarm HBSS. The fluorescence was read on a Cytofluor(model 2350; Millipore) with the excitation set at 485 nmand emission at 530 nm.

Confocal microscopyThe p°cells were grown in polylysine-coated chamber

slides (Lab-Tek; Nunc, Naperville, IL, U.S.A.) in medium

J. Neurochem., Vol. 67, No. 5, 1996


as described above. These cells were labeled with 5 pMNAO for 15 mm at 20°C.Platelets were collected as de-scribed above and labeled with MitoTracker CMXRos-H2(Molecular Probes) according to themanufacturer’s instruc-tions.

Both cell populations were washed with Ca2~-free mini-mal essential medium after labeling was complete. The sus-pension of labeled platelets (100 p1) was added to labeledp°cells growing in chamber slides. PEG solution (100 p1of 5 g of PEG 1000 in 2.5 ml of Ca2~-freeminimal essentialmedium) was added to each chamber for 1.5 mm at 37°C.The fusion was stopped by addition of 0.4 ml of Ca2~-freeminimal essential medium to each chamber and incubatingfor 10 mm at 37°C.The fusion medium was removed andreplaced with p° cell medium. In control fusions, Ca2~-free minimal essential medium was substituted for the PEGsolution. At intervals of 2 h to 7 days, fused and controlcultures were fixed with 3% (wt!vol) glutaraldehyde in 0.1M sodium phosphate buffer(pH 7.1) for 30 mm. The cham-bers and gaskets were removed, and slides were coveredwith Vectashield coverslips (Vector Laboratories, Burlin-game, CA, U.S.A.). Images of each cell were generated bymaking a composite of multiple images taken from focalplanes throughout the thickness of each cell (extended depthof focus photography) using a Zeiss Inverted Laser ScanConfocal Microscope (model LSM4IO).

DNA sequencingThe platelet-rich fraction from 3 ml of blood was isolated

by the Accuspin procedure as described above. The platelet-rich fraction was transferred to a centrifuge tube containing1 ml of 10 mM Tris-HC1 (pH 7.5) and 1 mM EDTA (Tris-EDTAbuffer), and leukocytes were collected by centrifuga-tion at 2,500 rpm for 10 mm. The leukocyte pellet wasresuspended in 5 ml of Tris-EDTA buffer, and 0.2 ml of20% (wt!vol) SDS and 0.1 ml of proteinase K at 20 mg/ml were added. After incubation at 37°Cfor 4 h with shakingthe lysate was extracted twice with phenol and twice withchloroformlisoamyl alcohol (24:1, vol/vol). DNA was pre-cmpitated by addition of a 10% volume of 3.0 M sodiumacetate (pH 5.0) and 2 volumes of ethanol. Following incu-bation at —20°Covernight, the precipitated DNA was col-lected by centrifugation, washed with 70% ethanol, brieflydried, and resuspended in 0.1—0.2 ml of Tris-EDTA buffer.The DNA concentration was determined by UV absorptionat 260 nm.

DNA was extracted from cultured cybrid cells followinglysis in 10 ml of lysis buffer [9 ml of Tris-EDTA buffer,800 p1 of 10% (wt/vol) SDS, and 200 p1 of proteinase Kat 20 mg/ml]. The resulting cell suspension was placed in50-ml conical centrifuge tubes andincubated for 4 h at 37°C.The lysate was extracted with phenol/chloroform, and theDNA was precipitated as described above.

Primers for PCR and DNA sequencing were chemicallysynthesized using an ABI 394 DNA/RNA Synthesizer (ABIDivision, Perkin Elmer, Foster City, CA, U.S.A.) using stan-dard /3-cyanoethylphosphoramidite chemistry. Tnitylatedprimers were deprotected with ammonium hydroxide andpurified using Oligonucleotide Purmficatmon Cartridges (ABIDmvmsmon, Perkin Elmer). Primer concentration was deter-mined by UV absorption at 260 nm.

A 297-hp fragment of the complex IV subunit I gene(mitochondrial nucleotides 6,914—7,210) was amplified byPCR using primers designed from the mitochondrial DNA

sequence published by Anderson et al. (1981). The PCRamplification product waspurified by agarose gel electropho-resis. PCR fragments were ligated with the TA-Cloning vec-tor pCRII (Invitrogen, San Diego, CA, U.S.A.), and thevector-ligated PCR fragments were transformed into compe-tent Escherichia coli cells of the strain XL1-Blue MRF’(Stratagene, San Diego). Transformed cells were spreadonto LB-agar plates containing ampicillin (50 pg!ml), kana-mycin (50pg/ml), isopropyl ~J-D-thiogalactopyranoside (20pg/ml), and ~9-galactosidase (100 pg/ml). The blue/whitecolor selection mechanism of the cloning vector allows fordetectionof white clones containing thedesired insert. Multi-ple white colonies were selected and propagated in culture.

Plasmid DNA containing the complex IV gene inserts wasisolated from 5 ml of bacterial cultures using the QIAwell96 Ultra Plasmid purification protocol (Qiagen). The iso-lated DNA was resuspended in 20 p1 of Tnis-EDTA buffer,and DNA concentrations were determined from A

26, ab-sorbance values.

Sequencingreactions using double-strandedplasmid DNAwere performed according to themanufacturer’s instructionsusing the Prism Ready Reaction DyeDeoxy Terminator Cy-cle Sequencing Kit (ABI Division, Perkin Elmer). TheDNAstrands were detected by fluorescence using the ABI 373AAutomated DNA Sequencer (ABI Division, Perkin Elmer).Electrophoresis and sequence analysis were performed usingthe ABI 373A Data Collection and Analysis Software andthe Sequence Navigator Software (ABI Division, Perkin El-mer). Ten different clones from each sample were sequencedto determine the mtDNA allele frequencies. The resultingsequences were aligned and compared with the publishedsequence (Anderson et al., 1981).

Western blottingCell extracts for western blotting were prepared by tryp-

sinizing cells, diluting with 10 volumes of DMEM, countingthe cells, and spinning for 10 mm at 400 g. The supernatantwas aspirated, and the cells were resuspended in 1 ml ofcold phosphate-buffered saline and centrifuged for 5 mm at400 g. The pellet was resuspended in 50 p1 of lysis buffer[1% (wt/vol) Triton X-100, 10 mMTnis (pH 7.5), 150mMNaC1, 2 mM phenylmethylsulfonyl fluoride, and 2 pg/mlaprotinin] per 1 x 106 cells and incubated on ice for 10mm. The cell lysate was centrifuged at 10,000 g for 10 mm.The Triton-soluble supernatant was decanted, and an aliquotwas withdrawn to determine total protein by theBCA ProteinAssay (Pierce). Twenty micrograms of protein was mixedwith 2 volumes of sample buffer [4% (wt/vol) SDS, 125mM Trms-HC1 (pH 6.8), 20% (wt!vol) glycerol, 5% (wt/vol) /3-mercaptoethanol, and 0.1% (wt/vol) bromophenolblue] and boiled for 2 mm before loading onto a 4—20%reducing Laemmli Tris-glycine, SDS-polyacrylamide gelfrom Novex (San Diego). After SDS-polyacrylamide gelelectrophoresis, the proteins were electrophoretically blottedonto nitrocellulose. Neuron-specific enolase (y homodimer)was detected using mouse anti-human neuron-specific eno-lase (y subunit specific; DAKO Corp., Carpenteria, CA,U.S.A.) as the primary antibody followed by treatment withan alkaline phosphatase-conjugated anti-mouse secondaryantibody and chemical development using NBT-BCIP/Sta-ble Mix (GibcoBRL). The antibody also detects a nonspe-cific protein at 50 kDa that is found in other nonneuronalcells.

J. Neurochem., Vol. 67. No. 5, 1996


FIG. 1. KCN-sensitive 02 consumption and slot blotting follow-ing EtBr treatment. 02 utilization was determined polarographi-cally in cells treated for either 33 (.) or 64 (0) days with varyingconcentrations of EtBr. Nonspecific 02 consumption was deter-mined in the presence of 1 mM KCN and was subtracted frommeasured total rates. Data are mean ±SEM (bars) values for atleast two independent experiments. Inset: SH-SY5Y cells weretreated for either 33 or 64 days with EtBr at 0, 0.1, 0.5, 1.0,and 5.0 ~ig/ml.Ten micrograms of total DNA was blotted ontonitrocellulose and hybridized with a DNA probe that is specificfor the mtDNA-encoded cytochrome c oxidase subunit I gene(see Experimental Procedures).

RESULTS

Production of p°SH-SY5Y cellsThe response of cells to mtDNA replication inhibi-

tor, EtBr, is cell line dependent (Desjardins et al.,1985; King and Attardi, 1989b). Therefore, SH-SY5Ycells were grown in culture under various differentconditions to optimize for EtBrconcentration and dura-tion of exposure. The EtBr concentrations testedranged from 0.1 to 5 jig/ml, with 5 j.tg/ml being maxi-mally effective. Higher EtBr concentrations resultedin cell death after 2—3 weeks. Cell lines for furtherstudy were exposed to the various EtBr concentrationsfor either 33 or 64 days. Cell lines treated for 33 or64 days with 5.0 jig/ml EtBr were designated p°33/5or p ‘64/5, respectively.

Production of respiration-deficient mutants wasmonitored by measuring KCN-inhibitable 02 utiliza-tion. Oxygen utilization decreased as a function of EtBrconcentration in both sets of cells and was undetectablein p°64/5(Fig. 1). Similarly, there was no detectableoxygen utilization sensmtive to either complex I inhibi-tion (rotenone) or complex III inhibition (antimycin)in these treated cells. In contrast, control cells wereextremely sensitive to both of these inhibitors, particu-larly rotenone (Fig. 2).

Once the p°64/5cells were shown to be respirationdeficient, the effects of long-term EtBr treatment onmtDNA content of the p°cells was examined. TotalDNA was extracted from 1 x 10’ parental SH-SY5Yand p064/5 neuroblastoma cells, and mtDNA was ana-lyzed by slot blot hybridization using an alkaline phos-phatase-conjugate oligonucleotide probe designed todetect a fragment of the mitochondria-encoded corn-

plex IV subunit 1 gene. Based on the 10 amol thresholdof detection determined for the alkaline phosphatase—oligonucleotide conjugate, the slot blot analysis dem-onstrated that the mtDNA content is below the lim-its of detection by this method (Fig. 1, inset). Thus,p°64/5cells have the p°phenotype as demonstratedby loss of 02 consumption and depleted mtDNA.

Mitochondrial enzyme assays of p°cellsEstablishment of a p°phenotype was accompanied

by loss of ETC enzymatic activity in those complexescontaining subunits encoded by the mitochondrial ge-nome (Table 1). Thus, both the p°64/5 cells andp033/5 cells exhibited no detectable complex I or IVactivity. In contrast, the activity ofthe nuclear-encodedETC enzyme complex, complex II (succinate dehydro-genase), was less perturbed following prolonged EtBrtreatment, suggesting relatively unimpaired processingof nuclear-encoded mitochondrial enzyme complexes.Activity of a mitochondnial matrix enzyme, citrate syn-thase, was comparable to control values in p 033/5 cellsand was decreased by ~-~50%in p°64/5cells. A selec-tive elimination of complex I and complex IV but notcomplex II activities in p°64/5 cells is consistent withloss of rntDNA in these cells.

Growth rates of p°64/5cellsThe p°cells described in the literature are dependent

on glycolysis, requiring both pyruvate and uridine forsurvival (Desjardins et al., 1985; King and Attardi,1989a,b). The growth rates of p‘64/5 cells with andwithout both uridine (50 pg/ml) and pyruvate (100pg/mi) were compared with that of the parental SH-SY5Y cells (Fig. 3). Pyruvate alone supported growthof the p°64/5 cells. However, p°64/5 cells grewslower than parental SH-SY5Y cells in medium con-taining pyruvate. Both these cell lines reached similarfinal cell densities. Cell death was noted after 3 days

FIG. 2. Responses of EtBr-treated cells to electron transportchain inhibitors. Cells were treated for 64 days with EtBr at vari-ous concentrations. Oxygen consumption was measured in thepresence of specific inhibitors of complex I (1.5 jiM rotenone),complex III (6 jig/mI antimycin), and complex IV (2 mM cya-nide). Data are mean ±SEM (bars) values for at least two inde-pendent experiments.



TABLE 1. Respiratory and biochemical activities in untreated parental and EtBr-treatedand reverted EtBr-treated p°cells

Cell02 consumption(nmol/min/mg)

Activity (mm mg~’)Reversion

rateComplex IV Complex I Complex II Citrate synthase

SH-SY5Y 3.25 ±0.57 2.092 ±0.222 23.2 ±3.1 28.49 174.4p°33/5 0.21 ±0.26 0.008 ±0.003 ND 30.69 158.2 <l0~’p°33/5revertant 3.72 ±0.77 5.490 ±0.281 ND 29.58 167.4p°64/5 0.00 ±0.03 0.010 ±0.030 0.0 ±0.05 7.20 83.2 <10_6Cybrid #01 3.31 ±0.64 2.130 ±0.352 27.7 ±4.7 ND 95.1Cybrid #02 4.12 ±0.48 1.930 ±0.113 30.5 ±2.0 ND 124.3

All activities are normalized to milligram of total cellular protein. ND, not determined. Data are mean ±SD values where indicated for atleast six independent experiments.

of growth in medium lacking pyruvate. The p°64/5cells did not appear to be auxotrophic for uridine asuridine alone did not support growth of p°64/5cells.The lack of uridine auxotrophy in the p°64/5 cellscontrasts withprevious studies(Desjardins et al., 1985;King and Attardi, 1989b) and may indicate functionaldihydroorotate dehydrogenase in the SH-SY5Y cellline or residual uridine in the dialyzedFBS. The depen-dency on pyruvate of the p°64/5cells for growth pro-vides a selectable marker for cybrid production follow-ing repopulation of p°cells with exogenous functionalmtDNA.

Aerobic competence of the p°6415cellularpopulation

Because all cells within a population may not haveachieved a p°phenotype during EtBr treatment, a fewaerobically competent cells could survive in the p°64/5cellularpopulation. Existence of aerobically competentcells in a populationcould contaminate the p°genotypeover several generations in culture. Presence of aerobi-cally competent cells in the p°64/5cellularpopulationwas determined by growing p°64/5cells in medium

FIG. 3. Dependence of p°cells on pyruvate for growth. Thep°64/5cells were propagated in the presence of 100 jig/mIpyruvate (U), 50 jig/mI uridine (C), both pyruvate and uridine(0), or no addition (A). Parental SH-SY5Y cells.) were grownas a positive control with no addition. Data are average cellnumbers per well of triplicate wells in a 24-well plate.

lacking uridine and pyruvate. Most cells died within2—3 weeks without the addition of pyruvate. One inevery 10’ p°33/5 and one in every i0~p°64/5 cellssurvived removal of pyruvate and uridine (Table 1).These few remaining aerobically competent cells wereexpanded and assayed for mitochondrial function. Theactivities of complex II, complex IV, citrate synthase,and 02 returned to control (or greater) levels in theseaerobically competent cells. This demonstrates that afew aerobically competent cells can be present in thep°64/5cellularpopulation. However, these cells repre-sent only a very small proportion of the total cellularpool. The p°cells have a low reversion rate but mustbe monitored carefully to ensure reversion does notoccur. We have since isolated p°cells treated for 118days at 5 pg/ml EtBr that do not survive long-termculture in pyruvate-free medium and do not revert toan aerobic phenotype.

Uptake of NAOThe p°cells lack mtDNA but still have mitochon-

dna. We evaluated the state of the mitochondria in p°neuroblastoma cells. The fluorescent dye, NAO, bindsselectively to cardiolipin, a molecule that is selectivelylocalized in the inner mitochondrial membrane. NAOuptake correlates with both number and size of mito-chondria (Leprat et al., 1990; Maftah et al., 1990).Binding of NAO was increased in p°64/5 cells (Fig.4). Mitochondria were stained with NAO and thenvisualized in confocal images of p°64/5cells (see Fig.7A). These rnitochondria are enlarged as demonstratedby confocal microscopy. These findings corroboratethe observations of Morais et al. (1988), who notedenlarged, misshapen mitochondria in electron micros-copy of p°cells.

p°6415cells differentiate following treatment withTPA and RA

Previous work suggested that p°cells could not beterminally differentiated into cells with stable pheno-types (Herzberg et al., 1993). However, p°64/5cellscould be differentiated into neuron-like cells in a man-ner similar to parental SH-SY5Y cells by treatmentwith either phorbol ester TPA (16 nM) or RA (1 jiM)

.1. Neurochem., Vol. 67, No. 5, 1996


cells expressed neuron-specific enolase, as is typicalof the differentiated SH-SY5Y parental cell line (Fig.6). Thus, p°neuroblastoma cells can be differentiatedinto neuronal-like cells based on morphological andbiochemical markers.

FIG. 4. Uptake of NAO by SH-SY5Y cells exposed to EtBr for64 days. Assay was performed in 96-well microplates. SH-SY5Ycells treated with various concentrations of EtBr for 64 days wereplated at 2 x io~cells per well 24 h before staining with 1 jig/ml NAO. Measurements were made as described in ExperimentalProcedures. Data are mean ±SD (bars) values of eight experi-ments.

(Pahiman et al., 1984). After 3 weeks of treatmentwith TPA or RA, the p°cells expressed long neuriteswith secretory granules typical of differentiating neu-roblastoma cells (Fig. 5). RA-differentiated p°64/5

FIG. 5. Differentiated SH-SY5Y andp°cells. SH-SY5Y (A—c) or p°64/5(D—F) cells were exposed to 0.1%ethanol as carrier control (A and D),16 nM TPA(B and E), or 1 jiM RA(Cand F) as described in ExperimentalProcedures.

Transfer of human platelet mitochondria to p°cells

The mitochondrial respiratory phenotype was re-stored to the p°64/5 neuroblastoma cells by fusionwith human platelets. Platelets lack nuclei but containmtDNA and are ideal sources for mitochondrial trans-fer experiments (Chomyn et al., 1994). Following fu-sion, the p°cells repopulated with exogenous mito-chondnia were selected by growth in medium lackingpyruvate and unidine. All aerobically incompetent cellsdie under these conditions. Karyotyping of the cybridcells confirmed that cybrids rather than polynuclearhybrids were produced by the fusion procedure (seeExperimental Procedures for details). On successfulmitochondrial transfer, the donor cells should expressnormal complex I and IV activities. These activitiesare absent in p°cells. ETC function for cybrid #01and cybrid #02 was similar to the activity of theseenzyme complexes in parental SH-SY5Y cells (Table


1904 S. W. MILLER ET AL

FIG. 6. Expression of neuron-specific enolase in differentiatedp°cells. Twenty micrograms of Triton-soluble protein derivedfrom SH-SY5Y cells and p ‘64/5 cells untreated (lanes A and D,respectively) or differentiated with either 1 jiM RA (lanes B andE, respectively) or 16 nM phorbol ester (lanes C and F, respec-tively) were fractionated by SDS-polyacrylamide gel electropho-resis (4—20% gels) and transferred to nitrocellulose, as de-scribed in Experimental Procedures. Neuron-specific enolase(arrow; y subunit) was detected using anti-human neuron-spe-cific enolase monoclonal antibody.

1). ETC function was assessed 4—6 weeks after fusionto permit mitochondrial repopulation. Assaying ETCactivity at early time points yielded low estimates ofETC activity for both complex I and complex IV.

The mtDNA is highly polymorphic and containsmultiple homoplasmic mutations that do not result inamino acid changes (silent mutations). This featurewas exploited to track the transfer of mtDNA fromhuman platelets into cybrids. A polymorphic, homo-plasmic silent mutation was found at nucleotide 7,028(C to T substitution) of the gene encoding for complexIV subunit I in a human blood donor. Following plate-let fusion with p°cells and selection in culture (~-~4—6 weeks), DNA was extracted and cloned from theparental SH-SY5Y and cybrid cell lines. Sequenceanalysis of the multiple clones revealed that cybrid butnot the parental cell line contained the homoplasmic(found in 10 of 10 DNA clones) silent 7,028 mutation,thus confirming successful transfer and repopulationof donor mitochondria in the cybrid. The absence ofwild-type clones provides further evidence that the re-cipient p°64/5neuroblastoma cells do not contain en-dogenous mtDNA and that cybrids are not reverted p°cells.

In addition, mitochondrial transfer into p°cells wasvisually confirmed by a fluorescent dye staining study.The p°cells were labeled withNAO, and mitochondniain donor platelets were labeled with MitoTrackerCMXRos-H2 (Fig. 7A and B). These two cellularpop-ulations with prelabeled mitochondria were fused toform transmitochondrial cybrids using PEG. In addi-tion to the endogenous NAO-labeled mitochondria, cy-brid cells contained clusters of red-stained mitochon-dria derived from donor platelets at 24 h (Fig. 7C)and 7 days (Fig. 7D) postfusion. Regions of overlap(yellow) between the two populations of labeled mito-chondria were easily visualized. Even after 7 days post-fusion, the two distinct labeled populations of mito-chondria were still evident in cybrid cells. Both mito-

chondrial populations assumed a typical, perinucleardistribution. Cells produced from control fusions (noPEG present) contained only endogenous (green) mi-tochondria (data not shown).

As a consequence of cellular respiration, ROS, i.e.,superoxide, hydrogen peroxide, and hydroxyl radicals,are produced. The p°64/5neuroblastoma cells shouldproduce relatively low levels of ROS. In contrast, res-cue of an aerobic phenotype should be followed by anincrease in ROS levels and recovery of the sensitivityto ETC-specific toxins. The ROS-sensitive dye DCF-DA was used to determine basal ROS production inparental SH-SY5Y, p°64/5, and cybrid cells. This dyeis readily incorporated into cells, and following hydro-lysis, the nonfluorescent substrate is released (Royalland Ischiropoulos, 1993). The substrate is oxidized bysuperoxide anions, peroxides, and other ROS into thefluorescent molecule dichlorofluorescein (DCF),which is retained inside living cells. Parental SH-SY5Y and cybrids exhibited similar levels of DCFfluorescence (Fig. 8). In contrast, p ‘64/5 cells showedreduced DCF fluorescence, in accordance with a lackof ETC function in these cells. Inhibition of complexIV activity with sodium azide increases production ofreactive hydroxyl radicals and other ROS in rodentbrain (Partridge et al., 1994). Parental SH-SY5Y andcybrids respond to increasing concentrations of sodiumazide with increased production of ROS, whereasp‘64/5 cells havea muted response. These results indi-cate that basal and sodium azide-induced DCF fluo-rescence is dependent on a functional ETC and confirmthat mitochondria and ETC function are recovered incybrids.

DISCUSSION

We report the creation of p°cells from an SH-SY5Yhuman neuroblastoma cell line. The p°status of theseSH-SY5Y-derived cells was demonstrated by the lackof 02 utilization, reduced production of ROS, and de-creased sensitivity to ETC toxins. Activities of ETCenzyme complexes containing subunits encoded by themitochondrial genome, i.e., complex I and IV, wereeliminated, whereas activity of complex II (encodedentirely by the nuclear genome) was maintained. Thecells were devoid of mtDNA and required exogenouspyruvate for growth. The concentration of EtBr re-quired to achieve the p°state was 10—100-fold higherthan that needed to produce p°fibroblasts (Desjardinset al., 1985) and p°osteosarcoma cells (King and At-tardi, 1989a,b), respectively. This suggests that sensi-tivity to EtBr treatment may be cell type specific andtherefore must be titrated for each cell type.

The mitochondrial genotype is difficult to alterusingstandard molecular techniques, but it can be manipu-lated by repopulating p°cells with exogenous mito-chondria taken from affected individuals. Osteosar-coma-derived and other rapidly replicating ~ cellshave been used successfully for transient expression

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FIG. 7. Confocal images of mitochondrial populations in p°cells, platelets, and cybrids. A: Distribution of mtDNA-depleted mitochondriain two p°cells after labeling with NAO (green). B: Mitochondria in platelets after being labeled with MitoTracker CMXRos-H2 (red).C and D: Appearance of cybrid cells after 24 h (C) and 7 days (D) postfusion. In addition to mtDNA-depleted (endogenous, green)mitochondria, there were clusters of platelet mitochondria (red) in cybrid cells. The yellow color indicates regions of overlap betweenthe two populations of mitochondria. Staining was performed as described in Experimental Procedures.

of exogenous mitochondrial genotypes (Yoneda et al.,1992; Chomyn eta!., 1994;Dunbar et al., 1995). How-ever, these p°cells continue to proliferate and cannotbe terminally differentiated into cells with neuron-likephenotypes. In addition, the mitochondrion is uniquein that it harbors mixtures of wild-type and mutantmtDNA (heteroplasmy). A mitochondrial diseasemanifests once the mutant allele exceeds a criticalthreshold, resulting in impaired bioenergetics (Brownand Wallace, 1994; Chomyn et al., 1994). We there-fore considered developing cellular models of mito-chondrial-based diseases using cells that can be termi-nally differentiated and growth arrested in culture. The

key feature of such cellular systems is elimination ofany potential propagative advantage inherent in aerobi-cally competent cells that possess a wild-type mito-chondrial genome as compared with respiratory-im-paired cells with mutated mitochondrial genes (Kingand Attardi, 1989a,b; Bourgeron et al., 1993). Oversuccessive generations a propagative advantage occurswhen cells expressing wild-type mitochondrial geno-types overgrow cells expressing mutant mitochondrialgenotypes. The result is dilution of the mutant pheno-type and its eventual disappearance. Several models ofrapidly dividing cybrids maintaining heteroplasmyover a long period have been produced (Yoneda et al.,

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FIG. 8. ROS species produced by SH-SY5Y cells, p°cells, andcybrids. SH-SY5Y (.), p°64/5(0), control cybrid #01 (0), andcontrol cybrid #02 (U) were preloaded with DCF-DA for 2 h andthen assayed as described in Experimental Procedures. The cellswere then exposed to sodium azide for 30 mm before determina-tion of the relative mean fluorescence (RMF). All values are nor-malized to the cell number in each well. Data are averages offour wells.

1992; Dunbar et al., 1995). Yet within these samestudies examples of rapid fluctuations in both wild-type and mutant mtDNA were noted in different clonesand different nuclear backgrounds.

The production of p°cells (p°64/5)from an SH-SY5Y neuroblastoma cell line described above over-comes many of these difficulties. These cells can bemaintained in a p°state and be terminally differenti-ated into cells with neuron-like phenotypes. On treat-ment with phorbol esters and retinoic acid, p°64/5cells flatten, extend neurites, and begin to express neu-ronal-specific markers, e.g., neuronal-specific enolase.In the p°state, these cells may be useful for identifyingprimary sites of action of mitochondrial toxins anddelineating mechanisms of cellular toxicity that aresuspected of involving mitochondrial dysfunction. Inessence these cells are “ETC knockouts.”

The aerobic phenotype of p°64/5cells can be res-cued by fusion with exogenous mitochondria from hu-man platelets. The resulting cybrid cell line is no longerdependent on pyruvate for survival, regains normaloxidative function, including production of ROS, andis again sensitive to mitochondrial toxins. Chromo-some counts reveal that these cells are cybrids andare not multinucleated hybrids. These cybrids derivedfrom p°64/5 cells uniquely possess the nuclear ge-nome of parental p°cells and the mitochondrial ge-nome of the human donor, as demonstrated by thetransfer of an exogenous polymorphic mutation inmtDNA from donor to p°cell.

In recent years, controversial findings have been re-ported implicating specific ETC defects in complexI and complex IV catalytic activity with Parkinson’sdisease and Alzheimer’ s disease, respectively (Parkeret al., 1989b, 1990). However, the precise origin ofthe ETC defect, whether mtDNA- or nuclear DNA-encoded, has not yet been elucidated. It would be ad-

vantageous to transfer dysfunctional mitochondriafrom such patients into cells possessing a normal, i.e.,non—disease-affected, nuclear genetic environment tosegregate mtDNA-encoded effects from nuclear DNA-encoded effects. It is important that the recipient cellsshould exhibit phenotypic characteristics that closelyresemble cells of affected tissues. This technique hasbeen useful in delineating the mitochondrial contribu-tion to several other metabolic diseases, i.e., MELAS(Goto et al., 1990), MERRF (Shoffner et al., 1990;Silvestri et al., 1992; Zeviani et al., 1993), and Leber’shereditary optic neuropathy (Wallace et al., 1988; Par-ker et al., 1989a). The p°64/5 cells described in thisstudy should be useful for the further study of thebiochemical, cellular, and tissue manifestations of neu-rologic diseases suspected of involving mitochondrialdysfunction and particularly valuable in isolating mito-chondrial contributions to excitotoxicity and apopto-sis in a neuronal environment. Preliminary reportsapplying this technology to the studyof mitochondrial-encoded complex IV and complex I defects in Alzhei-mer’s disease and Parkinson’s disease, respectively,havebeen recently reported (Glasco et al., 1995; Lakiset al., 1995; Swerdlow et al., 1996).

Acknowledgment: The authors thank Dr. Wendy Goldenfor performingchromosome counts, Drs.Eoin Fahy and Sou-mitra Ghosh for the oligonucleotide conjugates, and Dr. Co-rinna Herrnstadt for the cybrid DNA sequencing. SusanGlasco, Jan Parks, and James Lakis contributed substantiallyin the cell biology and enzymology studies. The authorswould like to thank Soumitra Ghosh for his rigorous criticalcommentary of the manuscript. W.D.P. was supported inpart by grants AG 10446 and AG094 17 from the NationalInstitute on Aging.

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Documents

Creation and Characterization of Mitochondrial DNA-Depleted Cell Lines with “Neuronal-Like” Properties