Human Molecular Genetics, 2002, Vol. 11, No. 4 431-438
© 2002 Oxford University Press
Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells
Department of Molecular Biosciences, One Shields Avenue, School of Veterinary Medicine, University of California, Davis, CA 95616, USA, 1Section of Medical Genetics, Department of Genetics and Pathology, Rudbeck Laboratories, Dag Hammarsjölds väg 20, 751 85, Uppsala, Sweden, 2Rowe Program in Human Genetics, Departments of Biological Chemistry and Medicine, One Shields Avenue, School of Medicine, University of California, Davis, CA 95616, USA, 3Layton Biosciences, 709 East Evelyn Avenue, Sunnyvale, CA 94086, USA and 4Departments of Medical Genetics and Biology, University of Turku, FIN-20500 Turku, Finland
Received October 18, 2001; Revised and Accepted December 5, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Inheritance of one of three primary mutations at positions 11778, 3460 or 14484 of the mitochondrial genome in subunits of Complex I causes Leber’s Hereditary Optic Neuropathy (LHON), a specific degeneration of the optic nerve, resulting in bilateral blindness. It has been unclear why inheritance of a systemic mitochondrial mutation would result in a specific neurodegeneration. To address the neuron-specific degenerative phenotype of the LHON genotype, we have created cybrids using a neuronal precursor cell line, Ntera 2/D1 (NT2), containing mitochondria from patient lymphoblasts bearing the most common LHON mutation (11778) and the most severe LHON mutation (3460). The undifferentiated LHON-NT2 mutant cells were not significantly different from the parental cell control in terms of mtDNA/nDNA ratio, mitochondrial membrane potential, reactive oxygen species (ROS) production or the ability to reduce Alamar Blue. Differentiation of NT2s resulted in a neuronal morphology and neuron-specific pattern of gene expression, and a 3-fold reduction in mtDNA/nDNA ratio in both mutant and control cells; however, the differentiation protocol yielded significantly less LHON cells than controls, by 30%, indicating either a decreased proliferative potential or increased cell death of the LHON-NT2 cells. Differentiation of the cells to the neuronal form also resulted in significant increases in ROS production in the LHON-NT2 neurons versus controls, which is abolished by rotenone, a specific inhibitor of Complex I. We infer that the LHON genotype requires a differentiated neuronal environment in order to induce increased mitochondrial ROS, which may be the cause of the reduced NT2 yield; and suggest that the LHON degenerative phenotype may be the result of an increase in mitochondrial superoxide which is caused by the LHON mutations, possibly mediated through neuron-specific alterations in Complex I structure.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Leber’s Hereditary Optic Neuropathy (LHON) is caused by inheritance of mutations at positions 11778, 3460 or 14484 of the mitochondrial genome, each of which are components of Complex I (1–4). The clinical phenotype of LHON is the degeneration of retinal ganglion cells (RGCs), and a progressive degeneration of the optic nerve. One puzzling feature of LHON is the optic nerve specificity of the disease. Although the LHON mutations are usually homoplasmic (i.e. occur systemically), in most patients the only symptom is vision loss, the result of degeneration of the optic nerve. Studies in cybrids have demonstrated that the LHON mutations cause a decrease in molecular oxygen consumption in whole mitochondria supplied with Complex I substrates, but not Complex II substrates (5,6). However, in studies of Complex I activity in submitochondrial particles, estimates of the severity of an enzymatic defect in Complex I vary considerably (7–10). Thus, the defect in respiration observed in whole mitochondria does not appear completely attributable to a simple enzymatic defect of Complex I activity, and other hypotheses for LHON pathophysiology have been suggested, including an apoptotic hypothesis (11), and a reactive oxygen species (ROS) hypothesis, which are non-exclusive of each other (8,10,12).
One possible explanation for the optic neuron-specific phenotype of the LHON mitochondrial genotype is that the LHON mutations only express their pathogenicity in the context of a neuronal cell, perhaps because of differences in mitochondrial structure or function in neuronal cells. In order to address this possibility, we fused LHON mitochondria with the neural Ntera-2/D1 (NT2) human teratocarincoma cell line, which had been depleted of their mitochondria. The NT2 cell line is perhaps the most ‘physiological’ human neuronal cell line available. Precursor cells undergo differentiation into neuronal cells following retinoic acid (RA) treatment. Upon differentiation, the cells assume a neuronal morphology, have a neuronal pattern of gene expression, become responsive to neuron-specific stimuli and are electrophysiologically active (13–15). We have fused mitochondria from patient lymphoblasts bearing the most common LHON 11778 or 3460 mutations with the NT2 cell line, to determine if the introduction of LHON mutations into a neuronal environment causes cell-type specific effects or differentiation-specific effects.
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RESULTS |
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Construction of LHON-NT2 cybrids
LHON-NT2 cybrids were created as described in the schema (Fig. 1A) and Materials and Methods. DNA from all clones was subjected to PCR of the relevant mtDNA region and restriction fragment length polymorphism (RFLP) analysis was performed (Fig. 1B). Restriction enzymes were used to cleave the 499 bp PCR fragment (Fig. 1B, lane 1). The patterns of the fragments confirm the presence of the LHON mutations in each respective cell line, thus these LHON-NT2 cells contain pure (homoplasmic) LHON mtDNA.
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Verification of NT2 nuclear background in LHON-NT2 cybrids
The LHON-NT2 cells which contain homoplasmic LHON mtDNA also contain an NT2 nuclear genotype, as determined by several criteria. The LHON-NT2 clones were selected on the basis of geneticin (G418) resistance, which is conferred by the NT2 nucleus that was transfected with a vector encoding G418 resistance (M.McGrogan, unpublished data). Also, under light microscopy the 11778-1, 11778-2 and 3460-1 cell lines had an indistinguishable morphology from the NT2 control cells, i.e. cuboidal and attached (Fig. 2, left), which was different from the lymphoblastoid and unattached morphology of the LHON lymphoblast donor cells (not shown). To further confirm the nuclear genotype of the LHON-NT2 cybrids, 70 microsatellite markers representing chromosomes 1–20, 22 and X of the Marshfield screening set 8A (http://research.marshfieldclinic.org/genetics/) were amplified by PCR from total DNA. Of the 70 markers, 65 and 59 were variable (i.e. informative) between NT2 and L-910555 DNA (lymphoblasts with 11778 mutation) and NT2 and L-910615 DNA (lymphoblasts with 3460 mutation), respectively. An example of one marker, DXS6807, is shown in Figure 1C. In any particular run, any alleles of the 70 that did not amplify were excluded from the analysis. Cell lines 11778-1, 11778-2 and 3460-1 shared 65, 63 and 59 informative microsatellite alleles with the NT2 parent, respectively, and none with the L-910555 or L-910615 parent. Since the amplified nuclear microsatellite alleles were 100% identical to that of the parental NT2 DNA, the morphology of the undifferentiated cells is consistent with an NT2 nucleus, and the cells are geneticin resistant, there is no support for nuclear contamination from the LHON lymphoblast nuclei in the LHON-NT2 cybrids.
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Differentiation of NT2 neural cell precursors to neuronal form induces a 3-fold decrease in mtDNA/nDNA ratio
In order to determine whether the LHON-NT2 cells had a normal mtDNA/nDNA ratio, quantitative PCR analysis was performed. Differentiation of mutant and control precursor cells to neuronal form caused an
3-fold reduction in the mtDNA/nDNA ratio (Fig. 3). However, there was no significant difference in the mtDNA/nDNA ratio between mutants and controls in either the undifferentiated or differentiated form. This is the first report to our knowledge demonstrating that differentiation of a neuronal precursor cell reduces the mtDNA/nDNA ratio; a differentiation-dependent decrease in the number of mitochondria could be a contributor to the general observation that mitochondrial defects preferentially affect the neuronal cell type.
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Differentiation of LHON and parental lines induces neuronal genes
Differentiation of the NT2-LHON neuronal precursors into mature neurons was carried out (16); differentiated LHON-NT2 cells had a distinctly neuronal morphology that was indistinguishable from the differentiated parental NT2 cells (Fig. 2). Although we did not observe differences in neuronal morphology between differentiated LHON-NT2 and NT2 cells, we also assayed the expression of several genes in the undifferentiated and differentiated NT2 cells, to address whether the presence of the LHON mutations affected the qualitative gene expression of these markers of differentiation. Glia, undifferentiated neuronal cells, and differentiated neurons have a pattern of gene expression which is cell-specific (14–17). We observed a completely normal pattern of gene expression in the mutants versus controls (Fig. 4). For example, both undifferentiated and differentiated cells express vimentin, but do not express glial fibrillary acidic protein (GFAP), a marker of glial lineage. Also, differentiated neurons produced tau mRNA, which is not found in undifferentiated cells (17). In addition, both undifferentiated and differentiated cells express the intermediate neurofilament NF-M, the levels of which increase over time with RA treatment (17). Thus, the occurrence of the 11778 and 3460 mutations (not shown) exerted no observable effect on the qualitative pattern of gene expression relative to controls.
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No difference in mitochondrial membrane potential nor reduction of Alamar blue in mutants relative to controls
Mitochondrial membrane potential was measured in undifferentiated cells. No significant difference between LHON-NT2 cells and controls was observed (Fig. 5A). As a control, dinitrophenol (DNP) was added to depolarize the membrane potential, and a decrease in log fluorescence was observed for all cells treated with DNP, an example is shown in Figure 5A.
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Alamar blue is a dye whose reduction is correlated with mitochondrial electron transport chain activity. No significant differences in Alamar blue reduction were observed between LHON-NT2 and control cells in the undifferentiated or differentiated state (Fig. 5B). Thus, we did not observe any significant effects of the LHON mutations on membrane potential or the ability to reduce Alamar Blue.
Decreased yield of LHON-NT2 cells during differentiation
Although LHON-NT2 cells differentiate into neurons, we observed a subtle difference in yield of total cells (i.e. differentiated + undifferentiated) at 4 weeks from the start of differentiation. Figure 6 shows the total number of cells after differentiation in RA for 4 weeks. 2.5 x 107 cells were started for each cell line and, after 4 weeks, the cell population for NT2 cells increased 50-fold, while the LHON-NT2 cell population increased only 36-fold. Note that this yield includes both the differentiated overlying neurons (NT2 neurons) and the underlying supportive cells of glial lineage. This difference in cell number could be explained either by a decreased proliferation of the LHON-NT2 cells during differentiation or an increased cell death, or both.
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Increased ROS production in LHON-NT2 neurons relative to parental controls
One possible pathophysiological mechanism in mitochondrial disease (18,19) and specifically in LHON (8,10,12), is increased production of ROS in mutant cells. Superoxide and peroxides are two examples of ROS produced by mitochondria which can be assayed by the mitochondria-specific dye dihydroethidium (DHE) and the cell-general dye dichlorofluorescein diacetate (DCFDA) assays (20–24). No differences in ROS production were observed between undifferentiated NT2 and LHON-NT2 cell lines (Fig. 7), nor between osteosarcoma cell lines with and without these LHON mutations (data not shown). In contrast, after differentiation to the neuronal form, the LHON-NT2n cells produced about 2.5-fold more cellular hydroperoxide, as determined by the DCFDA assay, (P < 0.005, Fig. 7A). To identify the origin of increased ROS in LHON neurons, we used an assay specific for mitochondrial superoxide, the DHE assay (22,23,25). An increased production of mitochondrial superoxide was observed in differentiated LHON neurons relative to controls, and neurons bearing the more severe 3460 mutation produced more superoxide (Fig. 7B). We investigated the specificity of the assay by short treatments of the mutant and control neurons with specific mitochondrial inhibitors, antimycin A and rotenone (Fig. 7C). As expected, antimycin A, a specific inhibitor of Complex III, increased DHE fluorescence in all three groups of cells, demonstrating specificity of the assay for mitochondrial superoxide. In contrast, a short treatment of the cells with the Complex I-specific inhibitor rotenone, specifically inhibited the excess superoxide produced in the mutant neurons, but only to a small and not statistically significant extent in the controls. The simplest interpretation of these data is that the 11778 and 3460 mutations specifically cause an increase in mitochondrial superoxide in the differentiated neuronal environment which is related to the severity of the disease, and that the mutation-specific increase in superoxide is the direct result of electron flow through Complex I.
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DISCUSSION |
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In contrast to the pleiotropic phenotypes observed in other mitochondrial diseases, in patients affected with LHON the systemic inheritance of mtDNA mutation causes a strikingly specific and singular phenotype, i.e. the degeneration of the RGCs and optic nerve. The reason(s) for this specificity of neurodegeneration have not been clear, and we have addressed this problem by introducing LHON mutations into neural cells.
It was demonstrated that the 11778 and 3460 mutations from lymphoblastoid mitochondria were introduced into NT2 cybrids by PCR and restriction enzyme analysis (Fig. 1). There was no evidence of lymphoblastoid nuclear contamination of the cybrids, by selection with geneticin, by cellular morphology, and by microsatellite analysis.
LHON-NT2 cells differentiated into neurons, as demonstrated by a neuronal morphology and a neuron-specific pattern of gene expression (Figs 2 and 4). The differentiation process itself caused a 3-fold reduction in mtDNA/nDNA ratio in both mutant and control NT2 neurons (Fig. 3). This is to our knowledge the first demonstration that neurons have a significantly lower mtDNA/nDNA ratio than lymphoblasts, NT2 precursors and other reference cells such as fibroblasts that have about 1000 mtDNAs/nDNA or more. The decreased mitochondrial content of differentiated neurons could underlie the increased sensitivity of neurons to mitochondrial defects.
We observed a subtle defect in cellular yield during the differentiation of LHON-NT2 neurons versus controls, i.e. 9 x 108 cells were produced from the LHON-NT2s, versus 1.3 x 109 in the parental NT2s (Fig. 6). This defect could either be the result of decreased proliferative capacity or increased cell death during the differentiation process of the mutant cells, and we are currently investigating these possibilities. The analysis is complicated by the fact that the differentiation takes 4 weeks and is a mixture of cells destined to be neurons and glia.
Measurements of mitochondrial function could address the differences in cellular yield that were observed during differentiation. There was no strong support for a mitochondrial bioenergetic defect conferred by LHON mutations in NT2 or NT2 neurons, by the Alamar blue and TMRM assays (Fig. 5). It has previously been suggested that perhaps in optic neurons Complex I activity is limiting to the respiratory chain, and thus a small defect in the enzymatic activity of Complex I limits bioenergetic function, resulting in a specific bioenergetic problem in these cells (9). However, at the level of sensitivity of these assays, we do not observe a difference in bioenergetic function.
An alternative to the bioenergetic hypothesis is that the LHON mitochondria produce more ROS (26,27), and either the optic neurons are more sensitive to ROS, or that the LHON mitochondria produce more ROS specifically in the neuronal environment (this work). We only observed increases in mutants versus control cells in the differentiated neuronal state, and not in the undfferentiated NT2 state, nor in comparison of mutant and control osteosarcoma cybrids. We have observed an increase in ROS measured with DCFDA, which is a general assay of cellular peroxides, and also with DHE, which is a more specific assay of mitochondrial superoxide (Fig. 7). We observed that on average differentiated NT2 neuron controls produced less peroxide and superoxide than the undifferentiated NT2 neurons, but that the mutant differentiated neurons produced more peroxide and mitochondrial superoxide than the parental differentiated line. The fact that the LHON-NT2 neurons produced more superoxide than the undifferentiated controls supports the notion that the LHON mutations are causing a differentiation-dependent pathophysiological change, rather than, the simple inhibition of differentiation, and this is also consistent with the identical neuronal morphology of LHON-NT2s (Fig. 2), and identical pattern of neuron-specific gene expression between mutants and controls observed in Figure 4.
The observation that antimycin A produced a large increase in superoxide in the neurons supports the specificity of the DHE assay, as antimycin A is known to increase mitochondrial superoxide production. The inhibitability of ROS production specifically by the Complex I inhibitor rotenone suggests that the excess mitochondrial superoxide observed in mutants is derived from Complex I. The simplest inference from these data is that LHON mitochondria cause increased ROS production through alterations of the function of Complex I, specifically in a differentiated neuronal environment.
The observation of increased mitochondrial ROS in differentiated LHON cybrids suggests two pathophysiological mechanistic possibilities, but does not prove either one. The increased ROS are presumably the result of the LHON mutations, and may directly be the cause of increased cell death in the LHON disease, or not. Conversely, the increased ROS may be the consequence of some other process which is downstream of the LHON mutations. The yield defect in differentiating LHON-NT2 cells may allow the dissection of whether this is a proliferative versus cell death problem, and may provide an experimental means by which to test whether ROS are on the pathway to the cell yield defect, for example with antioxidants. To date, we have not observed significant differences in LHON-NT2 versus control neuronal death provoked by bolus addition of exogenous hydrogen peroxide, indicating that there is not direct additivity of the mitochondrial superoxide we observe with external H2O2 to produce neuronal death. This could either be explained by frank ROS toxicity not being the proximal cause of neuronal death in LHON, or by the non-physiological way (i.e. bolus addition of exogenous hydrogen peroxide) in which these neurons were exposed to ROS, and we are exploring other schemes for exposure to oxidative stress.
The reason for the neuron-specific increase in ROS is not known at this point, but two possibilities include: (i) that there is a neuron-specific alteration of Complex I structure that leads to increased ROS in the context of LHON mutations or (ii) that the LHON mutations lead to other pathophysiological changes, which then cause increased mitochondrial ROS. The first hypothesis is more direct, and is thus more simply tested.
These results, to our knowledge, provide the first experimental support to explain the cell specificity of the LHON degenerative phenotype, and also the first experimental support for the ‘oxidative stress hypothesis for LHON’, and could be relevant to LHON therapy, for example, with antioxidants. Another appealing feature of an oxidative stress hypothesis for LHON is that it has the potential to explain the 3-fold excess of LHON in males versus females if, for example, there is increased production of mitochondrial ROS in males versus female humans, as has been observed by others in laboratory animals (J.Vina, personal communication).
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MATERIALS AND METHODS |
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Cell culture
NT2 cells transfected with the geneticin (G418) resistance gene were maintained in DMEM supplemented with 10% FBS, 50 µg/ml uridine, 1 µM sodium pyruvate, penicillin and streptomycin, and 0.2 mg/ml G418. For differentiation, 2.5 x 106 cells were seeded in 75 cm2 flasks and treated with 10 µM RA in freshly prepared media twice a week for 5 weeks. The cells were then replated at 50 x 106 cells/T75 cm2 flasks, and following 1 day in culture without RA, the media was saved and new media supplemented with mitotic inhibitors (1 µM cytosine arabinoside and 10 µM fluorodeoxyuridine) was given for 1 week. Neurons were harvested by gentle trypsinization and plated onto poly-D-lysine-Matrigel or -astrocyte coated plates or coverslips, and given media consisting of 50% NT2 and 50% media saved from the 1 day in culture without RA. Differentiated neuronal cell lines are indicated with a ‘n’ after the name, i.e. NT2n.
Astrocyte preparation
Rat astrocytes were generously provided by Dr Patty Wong-Yim (University of California Davis), and were isolated according to the procedures described by Goslin and Banker (28). Frozen astrocytes were thawed and plated at a density of 12 000 cells/cm2 in MEM supplemented with 10% horse serum and 20% glucose. Astrocytes reached confluency within 10–14 days of initial plating.
Production of LHON-NT2 cybrids
A modified version of cybrid production described by King and Attardi (29) and Trounce and Wallace (30) was used to produce the LHON-NT2 cell lines (Fig. 1). For enucleation of donor cells, 107 lymphoblasts were resuspended in 3 ml 12.5% Ficoll containing 10 µg/ml cytochalasin B, and this was layered onto a discontinuous Ficoll gradient layer containing 10 µg/ml cytochalasin B. The gradient was centrifuged at 28 000 r.p.m. for 1 h at 37°C in a SW41 swinging bucket ultracentrifuge (Beckman Institute, Inc.), after which, the cytoplasts were removed and washed. The recipient cells, NT2, were treated 3 days prior to fusion with 1 µg/ml rhodamine-6-G (R6G) and harvested for fusion. The recipient and donor cells were mixed together and resuspended in 40–50% PEG for 1 min. Media was added and cells were plated overnight, and the next day, diluted into 100 mm plates. Colonies were picked 3–4 weeks later and screened for the presence of the 11778 or 3460 mutation by PCR. Approximately 10–20 colonies were screened for each fusion, two were homozygous for the 11778 mutation, 11778-1 and 11778-2, and one was homozygous for the 3460 mutation, 3460-1.
LHON mutation detection
For mutation detection, the following primers were used to amplify the region containing the 11778 mutation: forward 5'-CCCATCGCTGGGTCAATAGT-3' and reverse 5'-ATTTGATCAGGAGAACGTGG-3'. PCR conditions were as follows: 30 s denaturation at 95°C, 30 s annealing at 58°C, and 1 min NSextension at 72°C. The G
A mutation can be screened via two restriction enzyme digestions, SfaNI cleaves wild-type but not mutant (1), and MaeIII makes an additional cut in the mutant (31). The 3460 amplification used the following primers and PCR conditions: forward 5'-GCAGAGCCCGGTAATCGCATA-3' and reverse 5'-AAGGTCGGGGCGGTGATGTAG-3', same PCR conditions as above, except annealing was performed at 55°C. Hin1I cleaves the wild-type (32).
Microsatellite analysis
All samples were tested with 70 markers from the Marshfield screening set 8A (http://research.marshfieldclinic.org/genetics/). PCR consisted of 0.5 µl PCR buffer, 0.7 µl 2.5 mM dNTP mix (Pharmacia and Upjohn), 0.05 µl cDNA polymerase (Clonetech Advantage), 0.1 µl of 10 µM fluorescently labelled primer mix, 2.65 µl double distilled H20, and 1 µl DNA at 0.5 ng/µl. PCR conditions were as described (http://research.marshfieldclinic.org/genetics/). Approximately 0.5 µl PCR product (amount varied depending on marker optimization) was loaded onto a 6% denaturing polyacrylamide gel with Genescan-350 Tamra standard (Perkin-Elmer) and run at 25 W on an ABI 373 DNA sequencer. Gels were then analyzed with Genescan software and imported into Genotyper software (Perkin-Elmer) for electrophoretogram analysis. Genotyper’s automated analysis was used to initially genotype samples, and all genotypes were then verified by inspection.
Microscopy
Undifferentiated cells were grown on uncoated glass coverslips and differentiated cells were plated onto glass coverslips coated with astrocytes. The cells were imaged using an Olympus AX70 epifluorescent Provis microscope equipped with a SONY 3CCD video camera. Magnification and scale bars are indicated in the figure legends.
Mitochondrial and nuclear DNA quantitation
Quantitation of mtDNA and nDNA from control and mutant NT2 cells and neurons was performed using the LightCycler instrument (Roche). Cystic fibrosis primers were used to amplify a 460 bp single copy nuclear gene using the following primers: forward 5'-AGCAGAGTACCTGAAACAGGAA-3' and reverse 5'-AGCTTACCCATAGAGGAAACATAA-3'. Primers to the ND5 gene (13175–13501) were used to amplify the mitochondrial genome (forward 5'-AGGCGCTATCACCACTCTGTTCG-3' and reverse 5'-AACCTGTGAGGAAAGGTATTCCTG-3'). PCR was performed under the following conditions, after an initial 30 s denaturation at 95°C, 40 cycles of the following was used: 0 s for 95°C, 5 s for 60°C and 13 s for 72°C. Ten nanograms and 0.1 ng of DNA was added to amplify nDNA and mtDNA genes, respectively. To establish a standard curve for both nuclear and mitochondrial DNA, K562 DNA (Life Technologies) was used at optimal concentrations (0–100 ng for nuclear gene and 0–1 ng for mitochondrial gene). A standard curve was performed during every PCR run. Quantitation was determined by the cycle number at which fluorescent values reached threshold. One nanogram of DNA was assumed to have 150 nuclear copies and 300 000 mitochondrial copies.
RT–PCR
Total RNA was prepared from cells using the SV total RNA isolation system (Promega). To obtain cDNA, reverse transcriptase reactions were performed using 1 µg of RNA. The following primers were used to amplify tau, neurofilament medium chain (NFM), vimentin and GFAP: tau, forward 5'-ACAAGCTGACCTTCCGCGAG-3' and reverse 5'-ACAAACCCTGCTTGGCCAGG-3'; medium neurofilament (NFM), forward 5'-TCTGTAACCGTCACTCAAAAGG-3' and reverse 5'-CCGTTCTGTTTTGAAGCTGCC-3'; vimentin, forward 5'-GTCAGCAATATGAAAGTGTGGC-3' and reverse 5'-GGTAGTTAGCAGCTTCAACGG-3'; and GFAP, forward 5'- GAGGGACAATCTGGCACAGG-3' and reverse 5'-CAGCTGCTCCTGGAGTTCCC-3'. PCR conditions were as follows, denaturation at 95C for 30 s, annealing at 54°C (NFM, vimentin and GFAP) or at 58°C (tau) for 30 s, and extension at 72°C for 60 s.
Alamar blue assay
Alamar Blue reduction was measured in undifferentiated and differentiated cells following the manufacturer’s protocol (AccuMed International Companies, Westlake, OH). Briefly, 10 000 cells/well were plated in 96-well plates in 200 µl volume and 20 µl of the Alamar blue reagent was added to each well. Six hours later, fluorescence measurements were taken at 560 nm excitation and 590 nm emission.
Measurement of mitochondrial membrane permeability
Mitochondrial membrane permeability was measured in digitonin permeabilized cells using the assay described by Antonická et al. (32). Briefly, cells were harvested and washed three times in PBS, resuspended in KCl medium (80 mM KCl, 10mM Tris–HCl, 3 mM MgCl2, 1 mM EDTA, 5 mM KH2PO4 pH 7.4, 10 mM succinate and 1 µM rotenone, and incubated with digitonin (0.2 mg/ml) for 5 min on ice. The cells were washed once in KCl medium and incubated with 50 nM TMRM for 15 min at room temperature, after which, the cells were washed once and resuspended in KCl medium. To depolarize the membrane, 1 µM DNP was added to cells before analysis. Analysis was performed on the FACSort flow cytometer (Becton Dickinson, San Jose, CA), with a 488 nm argon laser, and data was acquired.
ROS measurements
ROS measurements were performed following the procedure of Degli Esposti and McLennan (24). Briefly, cells were harvested and resuspended in PBS (0.5 x 106 cells/ml) supplemented with 20 mM glucose and 2 µM DCFDA or DHE, and incubated at 37°C. Cells treated with mitochondrial inhibitors received 10 µM antimycin A or 10 µM rotenone. Fluorescence readings were taken at 2 h (485–530 nm for DCFDA and 530–620 nm for DHE). A standard curve of dichlorofluorescein was used for quantification purposes.
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ACKNOWLEDGEMENTS |
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We thank Drs Rebecca Hartley and Lupe Gonzalez for helpful discussions. This work was supported by USPHS grants EY12245,AG11967, AG16719, and core support by P30ES05707 to G.A.C.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 530 754 9665; Fax: +1 530 754 9342; Email: gacortopassi@ucdavis.edu
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