Antioxidants Rescue Mitochondrial Transport in Differentiated Alzheimer’s Disease Trans-Mitochondrial Cybrid Cells

Human subjects and creation of cybrid cell lines

Both AD patients and non-AD controls were recruited from the University of Kansas Alzheimer’s Disease Center (KUADC). Subjects with AD met the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association criteria [20].Non-AD subjects were cognitively normal and age-matched to AD subjects. This study was approved by the University of Kansas Medical Center (KUMC) Institutional Review Board. All subjects provided written informed consent to participate in the study. The ages of AD and non-AD subject platelet donors were 73.6±2.96 and 75.8±5.04 years, respectively. Gender, age and disease status of donor patients are presented in Supplementary Table 1.

Cybrid cell lines were created on the human neuroblastoma cell (SH-SY5Y) nuclear background (by the KU ADC Mitochondrial Genomics and Metabolism Core) [21]. To create the cybrid cell lines used for this study, SH-SY5Y cells that were previously depleted of endogenous mtDNA (Rho0 cells) were fused with platelet cytoplasm from human subjects, and repopulated with mitochondria containing mtDNA from patients or controls as previously described [22]. Quantitative real-time polymerase chain reaction (PCR) showed that intact mtDNA copies were present in all cybrids without detectable large-scale deletion in the cells used in the present study (Supplementary Figure 1).

Cybrid growth and differentiation

AD and non-AD cybrids were grown in T75 tissue culture flasks in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (5.5 mM), 10% characterized fetal bovine serum (FBS; Gibco BRL, Logan, Utah), 100μg/ml pyruvate, 50μg/ml uridine, antibiotic-antimycotic, 100 Units/ml penicillin G, and 100μg/ml streptomycin [7]. Medium was changed every 2 days. Twelve mm plastic coverslips and cell culture plates were coated with 1.5 mg/ml poly-D-lysine (Sigma-Aldrich, St. Louis, Missouri, USA, dissolved in sterile H₂O) for 2 h at room temperature, and were rinsed twice with sterile H₂O before use. Harvest of proliferating cybrid cells was initiated using 0.1% trypsin (Invitrogen, Carlsbad, CA) in phosphate-buffered saline (PBS, Invitrogen, Carlsbad, CA) for 5 min at 37°C. Trypsin activity was inactivated by an equal volume of culture media containing serum. Cells were then harvested by centrifugation and re-suspended in culture media. Three thousand cells in 0.25 ml culture media were added to each well of a 24-well-plate with coverslips inside or 40,000 cells to each well of a 6-well-plate. One day later, culture media were changed to the differentiation media [neurobasal media supplemented with 1× B27 (Invitrogen, Carlsbad, CA) and 0.5 mM glutamine, and antibiotic-antimycotic] with 10 nM staurosporine (SAT, Sigma-Aldrich Corp, St Louis, Missouri, USA). We made 25μM stock SAT in dimethyl sulfoxide (DMSO) and stored it at –80°C). Approximately, half of the differentiation media was replaced (with 10 nM SAT) every day as previously described [23, 24]. Based on the measurement of length of neuronal processes, day-14 cybrid cells reached plateau after differentiation in day-35 (data not shown), indicating that differentiation was considered to complete on day 14 of SAT treatment. To assess the effects of antioxidant on AD cells, 10μM probucol (Sigma-Aldrich Corp, St Louis, Missouri, USA) or 200μM ascorbic acid (AA, Sigma-Aldrich Corp, St Louis, Missouri, USA) were added to the media along the differentiation.

Respiratory chain complexes enzyme activities and ATP levels measurement

Enzyme complex I (NADH-ubiquinone reductase), complex IV (cytochrome c oxidase, CcO) activity, and ATP levels were determined as described previously [7]. Briefly, cybrid cells were washed with ice-cold PBS, and then harvested, centrifuged, and suspended in 50μl of isolation buffer containing 250 mM sucrose, 20 mM HEPES, and 1 mM EDTA. Cell suspensions (containing ∼3-4 mg of protein/ml) were added to a cuvette containing 0.95 ml of 1× assay buffer (10 mM Tris-HCl, and 120 mM KCl), and the reaction volume was brought to 1.05 ml with the addition of 1× enzyme dilution buffer (10 mM Tris-HCl, pH 7.0). The reaction was then initiated by the addition of 50μl of ferrocytochrome substrate solution (0.22 mM). The change in absorbance of cytochrome c at 550 nm was measured using a Shimadzu (Kyoto, Japan) UV1200 spectrophotometer. Activity is expressed as micromoles of cytochrome oxidized per min^–1 mg^–1 protein using an extinction coefficient of 18.64 mM^–1 cm^–1. ATP levels were determined using an ATP Bioluminescence Assay Kit (Roche) following the manufacturer’s instruction [9, 25]. Briefly, cells were harvested, incubated on ice for 15 min, and centrifuged at 13,000 g for 10 min. ATP levels were measured using a Luminescence plate reader (Molecular Devices) with an integration time of 10 s.

Determination of ROS generation with electron paramagnetic resonance (EPR) analysis

The oxidation of the cyclic hydroxylamine 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) was used to measure the ROS levels as described in our previous study [26, 27]. Cybrid cells cultured on 6-well plates were washed with PBS and then incubated with 100μM CMH at 37°C for 30 min, followed by one wash of cold PBS. After that, cybrid cells were washed with ice-cold PBS, and then harvested with 100μl of PBS for each well.

EPR measurements were performed using a Bruker EMXplus X-band EPR spectrometer running Bruker Xenon acquisition/processing software and equipped with a dual mode cavity (ER4116DM) and Oxford ESR900 cryostat. For each sample ∼50μl was loaded into a 1.5 mm O.D. micropipette (Blaubrand intraMARK), sealed with Bruker X-Sealant and placed into a 4 mm O.D. EPR tube (Wilmad Labglass 707-SQ-250M). The spectra were recorded at room temperature with the following acquisition parameters: microwave frequency 9.64 GHz; center magnetic field, 3440 gauss; scan range, 100 gauss; microwave power, 2 mW; modulation frequency, 100 kHz; modulation amplitude, 1 gauss and time constant, 82 ms. Signal intensity was determined as the height of the central peak on the up-field side of the midpoint.

Determination of mitochondrial ROS generation with MitoSox Red and mitochondrial membrane potential (ΔrmPsi) with TMRM

Cybrid cells were seeded at low density onto Lab-Tek eight-well chamber slides (10000 cells/well). Mitochondrial ROS generation was determined using MitoSox Red (Invitrogen), a unique fluorogenic dye highly selective for detection of superoxide production in live cell mitochondria. Cells were incubated with fresh medium containing 2.5μM MitoSox for 30 min. To detect mitochondrial membrane potential, cells were co-stained with TMRM (100 nM; Invitrogen) for 30 min. Mitochondria were labeled with Mitotracker Red (MTRed, 100 nM, Invitrogen) or and MitoTracker Green without interference of mitochondrial membrane potential (MTGreen, 100nM, Invitrogen) for 30 min at 37°C before fixation to visualize mitochondrial morphology.

Fluorescence images were acquired on a Leica SP5 confocal microscope and analyzed using Leica LAS AF software (Leica Wetzlar). Excitation wavelengths were 543 nm for MitoSox, TMRM or MTRed, and 488 nm for MTGreen, respectively. Fluorescent signals were quantified using NIH Image J software. Post-acquisition processing was performed using MetaMorph (Molecular Devices) and NIH Image J software for quantification of fluorescent signals and measurement of mitochondrial density. Fluorescence intensity was quantified from the same sized mitochondrial area. Mitochondrial density and fluorescent intensity were quantified by an investigator blinded to experimental groups. More than 50 clearly identifiable mitochondria from randomly selected cells were measured and quantified.

Measurement of mitochondrial movement in AD and non-AD cybrid cells

To measure mitochondrial movement, both AD and non-AD cybrid cells were incubated with 50 nM MitoTrackerRed (MTRed; Molecular Probes, Invitrogen) for 10 min at 37°C. Time-lapse recordings of labeled mitochondrial movement were acquired on a Carl Zeiss (Axiovert 200) microscope with an incubation system (PeCon) to maintain cybrid cells at 37°C during image collection. Collection of image stacks and velocity measurements were made using Axiovision Software as previously described [8, 25]. For standard recordings, images of mitochondria in one axon per cybrid cell were collected every 3 s for 2 min.

Mitochondria in each frame of every video recording were individually tracked using AxioVision Software. The average velocity was calculated during the 2-min recording period. Then, the velocities of mitochondria in the process were averaged to obtain the average velocity for mitochondrial movement. In addition, the percentage of movable mitochondria, a total of mitochondrial traveling distance (total distance traveled regardless of direction during the recording period), and mitochondrial density in each process were determined according to the previous studies with modifications [7, 15]. Exposure periods (30–50 ms) were kept to a minimum to limit the phototoxicity. All data were obtained from five cell lines of either AD or non-AD cybrids.

Statistical analysis

Data are presented as mean±SEM. Statistical analysis was performed using Statview software (SAS Institute, Version 5.0.1). Differences between means were assessed by Student’s t-test or one-way analysis of variance (ANOVA) with Fisher post hoc test. p < 0.05 was considered significant.

Differentiation of AD and non-AD cybrid cells

We observed morphological changes in staurosporine (SAT)-treated non-AD (Fig. 1A II) and AD cybrid cells (Fig. 1A IV) under non-serum conditions. SAT serves as a strong inducer of differentiation in SH-SY5Y cells. SAT concentrations between 4 and 25 nM are not toxic [19, 28], so we treated cybrid cells with 10 nM SAT for 14 days. Differentiated cells began to appear 3 days after treatment and differentiation was complete on day 14. Over the course of a month, no cell detachment or apoptosis (based on stains to detect apoptosis) was observed. Typical differentiated morphology appeared as long and straight neuronal-like processes stretched out in non-AD cybrid cells (Fig. 1A II). In contrast, differentiated AD cells revealed much shorter neuronal like processes than differentiated non-AD cells (Fig. 1A IV). Neuronal process length showed a 3.6-fold increase in differentiated non-AD cybrid cells compared to non-differentiated non-AD cells, whereas we observed only a 1.4-fold increase in the processes in the differentiated AD cybrid cells (Fig. 1B). Process lengths between the undifferentiated non-AD and AD cybrid cells were comparable (p > 0.05). These results suggest that SH-SY5Y AD cybrid cells do not differentiate normally when induced bySAT.

Mitochondrial density in differentiated AD and non-AD cybrid cells

We compared mitochondrial morphology in differentiated AD and non-AD cybrid cells by staining with a mitochondrial marker. In non-AD cybrid cells, mitochondria were shorter and scattered along the differentiated neuronal processes (Fig. 1C I and II), whereas in AD cells the mitochondria were concentrated within the shortened processes (Fig. 1C III and IV). After 14 days of SAT treatment, the number of mitochondria within the processes was significantly lower in AD cybrid cells than it was in non-AD cybrid cells (Fig. 1D). Each of non-AD cybrid cells contained about 35 mitochondria within its differentiated processes, whereas each of the AD cells contained about 11 mitochondria in its less differentiated processes (Fig. 1D). To confirm that the increased numbers of mitochondria in processes of non-AD cells represented a higher mitochondrial density, and was not simply due to the longer length of the differentiated processes in non-AD cells, we also calculated the number of mitochondria per 100μm of process. Indeed, mitochondrial density was significantly higher in non-AD cells than AD cells (Fig. 1E), indicating that the content of mitochondria within AD cybrid processes was reduced.

Mitochondrial dysfunction in differentiated AD cybrid cells

We next evaluated mitochondrial function by measuring respiratory chain enzyme activities, mitochondrial membrane potential, and reactive oxygen species (ROS) generation in differentiated AD and non-AD cybrid cells. The enzymatic activities of complex I and IV were significantly reduced in differentiated AD cybrid cells compared to non-AD cybrids (Fig. 2A-B). Similarly, ATP levels in AD cybrid cells were lower than that in non-AD cells (Fig. 2C).

ROS levels was detected by EPR studies usingthe cyclic hydroxylamine 1-hydroxy-3-methoxy-carbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) as a spin trapping agent. Substantial ROS production was detected in AD cybrid cells compared to non-AD cybrid cells (Fig. 2D). The amount of the ROS level was quantified by measuring the height of the middle EPR peak from each spectrum (representative images are shown in Fig. 2E).

Given that mitochondria are a major source of ROS generation and that ROS accumulation affects mitochondrial function, we measured mitochondrial ROS generation. MitoSox staining intensity, which reflects mitochondrial ROS production, was increased in AD cybrid cells (Fig. 2F, G). Mitosox signal was enhanced and aggregated within the terminals of the differentiated AD neurons, suggesting that abnormal ROS accumulation in neuronal process terminals impair process differentiation in ADneurons.

We next assessed inner mitochondrial membrane potential in differentiated AD and non-AD cybrid cells using the fluorescent probe TMRM. Mitochondrial depolarization (disrupting or decreasing the membrane potential) reduces the mitochondrial staining intensity. Compared to non-AD mitochondria, the intensity of TMRM staining was significantly lower in AD cybrid cells (Fig. 2H, I). Decreased TMRM signal was observed in the axon-like neuronal processes of AD cybrid neurons (Fig. 2I). These results demonstrate impaired mitochondrial function and increased mitochondrial ROS generation/accumulation in differentiated cybrids that contain AD-derived mitochondria.

Impairment of mitochondrial motility in axons of AD cybrid neurons

After differentiation, both human AD and non-AD cybrid neuronal cells formed axon-like neuronal processes, which allowed us to directly evaluate the effects of AD subject-derived mitochondria on axonal mitochondrial transport and movement. We assessed mitochondrial motility by measuring the percentage of movable and stationary mitochondria, travel distance, and velocity. The percentage of moving mitochondria was significantly reduced in AD neuronal processes compared to non-AD processes (Fig. 3A). Conversely, increased numbers of stationary mitochondria were observed within AD neuronal processes (Fig. 3B), indicating defects of mitochondrial movement in AD cybrids in addition to impaired differentiation and lower mitochondrial process density of mitochondria. The velocity and total travel distance of mitochondria were also significantly decreased in AD neuronal processes (Fig. 3C-D). Quantification of mitochondrial movement and kymography revealed significantly reduced anterograde and retrograde mitochondrial movement in AD neuronal processes compared to non-AD cybrids (Fig. 3E-G). These data demonstrate mitochondrial transport and trafficking is impaired within AD-derived neuronal processes.

Effects of antioxidants on cell differentiation, mitochondrial function, and axonal transports

Given that elevated ROS levels (Fig. 2D-G) might contribute to defective differentiation (Fig. 1A, B) as well as impaired mitochondrial morphology (Fig. 1C-E) and function (Fig. 2A-C, H, I) in AD cybrid cells, we examined whether antioxidants could rescue AD cybrid differentiation defects. Treatment with two antioxidants, probucol and ascorbic acid (AA) significantly increased neuronal processes (Fig. 4A, B) and mitochondrial density (Fig. 4C-E) within processes in AD cybrid cells compared to vehicle-treated control cells under normal differentiation condition. Furthermore, in the presence of antioxidants, AD cybrid cells displayed an improvement in mitochondrial function as shown by increased complex I and IV activity, ATP levels (Fig. 5A-C), and mitochondrial membrane potential ((Fig. 5G, I). Treatment of probucol or AA completely abolished ROS production/accumulation in AD cybrid cells (Fig. 5D-F, H). These data suggest that antioxidant treatment reverses perturbed AD-specific mitochondrial morphology and function in AD cybrid neurons.

Probucol and AA also improved mitochondrial transport and motility (Fig. 6). There was an increase in the percentage of movable mitochondria (Fig. 6A), as well as a decrease in the percentage of stationary ones in AD cybrid cells (Fig. 6B). Mitochondrial movement velocity and total travel distance were also rescued (Fig. 6C, D). Quantification of mitochondrial movement and kymography revealed that the antioxidant treatments increased both anterograde and retrograde mitochondrial movement in AD neuronal processes (Fig. 6E-G).