Molecular phenotype of a human lymphoblastoid cell-line homoplasmic for the np 7445 deafness-associated mitochondrial mutation
Molecular phenotype of a human lymphoblastoid cell-line homoplasmic for the np 7445 deafness-associated mitochondrial mutationFiona M. Reid+, Anja Rovio1, Ian J. Holt2 and Howard T. Jacobs1,*
Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, 56 Dumbarton Rd, Glasgow G11 6NU, UK, 1Institute of Medical Technology, University of Tampere, PO Box 607, 33101 Tampere, Finland and 2Department of Biochemical Medicine, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, UK
Received October 18, 1996;Revised and Accepted December 3, 1996
We have studied mitochondrial gene expression and metabolic function in a human lymphoblastoid cell-line homoplasmic for the np 7445, deafness-associated mitochondrial DNA mutation. The mutation maps to the 3' termini of the oppositely oriented genes encoding cytochrome oxidase subunit I (COI) and tRNA-ser(UCN). In comparison with control lymphoblastoid cells, we detected a marked depletion (>60%) of tRNA-ser(UCN). There was, however, no significant impairment of respiratory function, no alteration to the structure or abundance of COI mRNA or its precursors, and no detectable abnormality of mitochondrial protein synthesis. We also found considerable tissue-variation in the abundance of tRNA-ser(UCN). We propose that the tissue-specific phenotype associated with this mutation results from an inherent deficiency in the processing of the mutant pre-tRNA, that becomes limiting for protein synthesis only in a restricted set of cells of the auditory system in which the tRNA is, for other reasons, already at a critically low level.
Human disorders associated with mutations of mitochondrial DNA (mtDNA) show a bafflingly diverse range of symptoms, affecting one or more of the many tissues heavily dependent on respiratory function (see ref. 1 for review). Generally, the precise nature of the mutation has some bearing on the pathological phenotype, although in no case is the relationship between mitochondrial genotype and phenotype fully understood. We recently described a mutation at np 7445 of the human mitochondrial genome (2 ), in members of a pedigree with maternally inherited, bilateral, sensorineural hearing loss. The mutation was absent from over 600 control subjects, and has subsequently been observed in a second pedigree, with a similar phenotype (3 ). The two pedigrees are unrelated, as they exhibit a series of distinct mtDNA sequence polymorphisms (3 ,4 ).
The np 7445 mutation alters a nucleotide pair that has functions on both strands (5 ): as the terminal nucleotide of the stop codon of cytochrome oxidase subunit I (COI), and as the site of 3' processing of the precursor of tRNA-ser(UCN). As regards COI, it is presumably silent, altering the AGA stop codon to AGG, but could conceivably affect COI mRNA at the level of processing or stability. A more likely effect of the mutation would be in the maturation or function of tRNA-ser(UCN). Although np 7445 was originally proposed (5 ) to be the 3' terminal nucleotide of tRNA-ser(UCN), structural studies of bovine mitochondrial tRNA-ser(UCN) (see ref. 6 ) and alignments with the sequences of other vertebrate mtDNAs indicate that the correct structure for this tRNA places np 7445 one nucleotide beyond its 3' end.
Mitochondrial tRNA sequences, by virtue of their structure, are believed to play an essential role in the processing of the primary, polycistronic transcripts of the genome. These tRNA structures appear to be recognized by 5' and 3' tRNA-processing endonucleases (7 ), the resulting cleavages creating most of the termini of mitochondrial mRNAs and rRNAs, in addition to those of tRNAs: the so called punctuation model (8 ). By virtue of its location, adjacent to the site of such a cleavage, the mutation at np 7445 may therefore affect the 3' maturation of tRNA-ser(UCN). If the mutation alters the specificity of 3' cleavage it might also influence the translational properties of the tRNA, for example, by affecting the efficiency or specificity of aminoacylation of the tRNA. The 12S rRNA mitochondrial mutation at np 1555, which results in hearing loss via interaction with aminoglycoside antibiotics (9 ), maps to a site in the ribosome known to be involved in translational fidelity (10 ).
The extreme tissue-specificity of the phenotype associated with the np 7445 mutation suggested that it might be difficult to establish any biochemical abnormality in cultured cells. However, a modest biochemical phenotype has been demonstrated in cells from patients with homoplasmic mutations at np 3460 (11 ) or np 11778 (12 ) that are also associated with a tissue-specific sensorineural phenotype, Leber's Hereditary Optic Neuropathy (LHON). In this paper we present the results of a series of experiments to characterize the state of mitochondrial function in a lymphoblastoid cell-line, created by Epstein-Barr Virus (EBV) transformation of lymphocytes from the index patient carrying the np 7445 mutation (2 ), and whose mtDNA had been completely sequenced (4 ).
In order to study the effects of the np 7445 mutation on mitochondrial function, a permanent cell-line was created by EBV-transformation of patient-derived lymphocytes. After verification of the mitochondrial genotype of the cell-line, it was subjected to a series of tests to establish whether mitochondrial metabolism or gene expression was altered in any systematic way in comparison with wild-type cells.
DNA was prepared from the patient-derived lymphoblastoid cell-line CT001, and from the control lymphoblastoid line 01MC. In order to check that the mutant mtDNA was established in the patient-derived cell-line, and that the control line selected for comparison was wild-type at np 7445, a 662 bp DNA fragment spanning the COI/tRNA-ser(UCN) gene junction was amplified from both DNA samples, and digested with XbaI (Fig. 1 ). The PCR product from 01MC cell DNA was completely cut to the predicted digestion products of 400 and 262 bp, whereas that from CT001 cell DNA remained uncut, confirming that the cell-line is homoplasmic, within the detection limits, for the mutation originally described in the patient.
Three tests were applied to determine whether the patient-derived cells were impaired in respiration compared with control cells. Firstly, lactate production was measured, by assaying the ratio of lactate to pyruvate in spent medium. The production of elevated levels of lactate is held to be diagnostic for respiratory impairment, both in cultured cells and in patients (19 ,20 ), as it indicates increased dependence on glycolysis for ATP generation. Secondly, respiratory chain activities (SCCR, i.e. complex II+III and COX, complex IV) were measured directly in cell lysates. Thirdly, the growth impairment resulting from the substitution of galactose for glucose in the culture medium, which places cells under respiratory stress (21 ,22 ), was determined by the use of the vital stain MTT (13 ,14 ).
The metabolic data are summarized in Table 1 ; the results of growth experiments are described below. Lactate to pyruvate ratios after 48 h of culture were derived from three independent experiments. The mean values obtained for the two cell-lines differed by less than the range of experimental variation. Similarly, the absolute values for SCCR and COX specific activities, as well as the ratio of the two, based on the means of duplicate experiments, differed little between control and patient-derived cells. Note that these values are lower than those reported in the literature for mitochondrial proteins (e.g. ref. 23 ), because they are based on whole-cell extracts. We conclude that the patient-derived cell-line CT001 shows no evidence of a respiratory defect, at least under the culture conditions used.
Growth on galactose is believed to place cells under respiratory stress, due to flux limitations in the galactose-utilization pathway that oblige the cell to use respiration in order to obtain sufficient ATP for survival. Table 2 shows mean doubling times on glucose-and galactose-containing media for each cell-line, based on the combined results of two experiments. All MTT assays were carried out in triplicate. Both cell-lines grew more slowly in galactose medium, but the patient-derived cells grew better than control cells in both media, and their relative growth impairment in galactose was only slightly greater than that of the control cells. This supports the view that the np 7445 mutation is not significantly limiting the ability of lymphoid cells to survive conditions of mild respiratory stress.
Mitochondrial protein synthesis was assessed in CT001 and 01MC cells by pulse labelling with 35S-labelled amino acids, in the presence of emetine, a specific inhibitor of cytosolic translation. The patterns of translation products from the two lines were indistinguishable, based on SDS-PAGE/fluorography (Fig. 2 ). The synthesis of the major bands, tentatively assigned by comparison with published data (24 ), was inhibited by 1 mM chloramphenicol (not shown), confirming their identity as mitochondrial translation products. We conclude that under the conditions used, the np 7445 mutation does not significantly interfere with mitochondrial protein synthesis in lymphoblastoid cells.
Table 2 Growth of patient-derived and control cells in galactose medium
Parameter
CT001 cells (patient)
01MC cells (control)
Mean doubling time in glucose medium (hours)a
32
56
Mean doubling time on galactose medium (hours)
55
80
Relative growth rate on galactose/glucoseb
0.58
0.70
aCalculated from MTT assay as slope of plot of log2(OD570) against time.bInverse ratio of mean doubling times.
The above data indicate that the np 7445 mutation, as expected for a mutation associated with a highly tissue-specific phenotype, is not limiting for mitochondrial translation or respiratory function in lymphoblastoid cells. Nevertheless, we reasoned that it might produce a global effect on the synthesis, structure, stability or function of one or both of the mitochondrial RNAs in or adjacent to which it maps [COI mRNA or tRNA-ser(UCN)], but which is not functionally limiting in lymphoblastoid cells. To do this, we isolated total RNA from CT001 and 01MC cells, and examined the structure and representation of these transcripts by Northern blot hybridization, using a variety of appropriate probes (Fig. 3 ). A double-stranded probe for sequences entirely within the COI coding region (derived from a PCR product obtained using primers FR29/FR30) detected a major transcript of 1.75 kb from both cell-lines (Fig. 3 a), that corresponds with the COI mRNA, as well as a less abundant transcript of 1.95 kb, probably representing the precursor or alternate form of COI mRNA that includes an upstream leader of four antisense tRNA sequences (25 ). Both of these were detected by a strand-specific probe for the COI coding strand. After normalization with respect to the G3PDH loading control (Fig. 4 ) the representation of both of these major transcripts is very similar in the two cell-lines. The pattern and representation of minor, higher molecular weight transcripts is also very similar (Fig. 3 b), after normalization for loading. A transcript of 4.2 kb (arrowed) did not appear reproducibly on all blots, and is probably an artefact (compare Fig. 3 b and c). Additional Northern blot analysis (Fig. 4 ) indicated that representation of COI mRNA is relatively low in both of these lymphoblastoid lines, compared with a lung carcinoma line, mammary gland, ovary or testis.
By contrast, when the same blot was re-probed specifically for tRNA-ser(UCN), using a strand-specific probe derived from the HJ07/HJ08 PCR product (Fig. 5 ), CT001 cells had a clear deficiency in the steady-state level of this tRNA, compared with 01MC cells (Fig. 3 c), after controlling for differential loading. In three separate experiments (Figs 3 c and 4 , plus other data not shown), the level of tRNA-ser(UCN) in CT001 cells was determined densitometrically to be always in the range of 35-40% of its level in 01MC cells, regardless of whether normalization was to the nuclear-coded mRNA G3PDH or to mitochondrially encoded 12S rRNA or COI mRNA. This suggests that the efficiency of processing, or the stability of this tRNA is inherently reduced in the patient-derived cells carrying the np 7445 mutation.
The above findings indicate a specific alteration in gene expression but not in mitochondrial metabolic function in lymphoblastoid cells, associated with the np 7445 mutation. These observations suggest a mechanistic model for how this mutation, and perhaps others, might cause a highly tissue-specific sensorineural disorder. In the following paragraphs we review the arguments that support such a model.
The failure to find any significant impairment of mitochondrial respiratory metabolism in patient-derived lymphoblastoid cells homoplasmic for the np 7445 mutation is entirely consistent with the fact that the mutation is associated with a highly tissue-specific pathology. There is, furthermore, no evidence for any impairment to blood cell function in individuals carrying the mutation. The mutation is homoplasmic in many individuals in both pedigrees studied (3 ,26 ), some of whom show no evidence of any disorder, indicating that it is not counter-selected at the level of the whole organism. Lymphoid cells carrying a 70% load of deleted mtDNA have been previously reported as unimpaired in oxidative phosphorylation (27 ). Nevertheless, a mild biochemical phenotype is evident in lymphoblastoid cells from individuals carrying the np 1555 12S rRNA mutation (22 ). Since hearing loss is absent or mild in many members of the np 7445 pedigree studied by us it is possible that nuclear background influences both clinical and biochemical phenotype. It may therefore prove instructive to compare CT001 cells with cells from a patient from the second pedigree to be identified, harbouring the np 7445 mutation, since most members of this latter family were more severely affected (3 ).
The degree of tissue-variation in tRNA-ser(UCN) representation, when normalized against either nuclear-coded or mitochondrially encoded reference RNAs, is unexpected. Some of it may be due to minor differences in methods of RNA preparation in our laboratory compared with the commercial source of some of the RNAs (Clontech), leading to different amounts of recovery of short RNAs. However, the same protocol (18 ) was followed in both cases, and such arguments should not apply to RNA samples prepared in parallel by us (i.e. from all of the cultured cells). The values for placenta are probably unreliable, due to weak signals, but even discounting this, a variation of some 5-fold is evident when tRNA-ser(UCN) levels in different cell-types are compared with a nuclear-encoded reference RNA (G3PDH mRNA). More dramatic variation is evident if the pattern of tissue-variation in tRNA-ser(UCN) levels is compared with that in COI mRNA levels: whereas the mitochondrial tRNA is at lower abundance in breast, ovary and testis than in lymphoid cells, the opposite is true for COI message, and both are high in lung carcinoma cells. We also observed some variation in tRNA-ser(UCN) levels in different control lymphoblastoid lines, as already indicated. Although these are transformed and uncloned cell-lines, hence developmental differences could account for some of this variation, it is also possible that inherent nuclear genotypic variation could be playing a role, by affecting the parameters of mitochondrial transcription, RNA processing and turnover. The question of variation in mitochondrial tRNA levels in different tissues and between individuals warrants more general investigation, as it could underlie a number of pathological phenomena associated with mitochondrial tRNA gene mutations. In particular it would be worth studying affected and unaffected members of both pedigrees carrying the np 7445 mutation, to determine whether differences in tRNA-ser(UCN) levels attributable to nuclear genetic background might correlate with expression of the disease phenotype in this case. This is highly plausible, given that in CT001 cells the depletion of tRNA-ser(UCN) is just significant compared with a panel of control cell lines: minor differences in mtRNA and protein synthesis could alter this level above or below a critical threshold. Alternatively, the expression of the disorder may depend on an as yet unidentified environmental factor.
A decreased level of tRNA-ser(UCN) in cells carrying a mutation that lies beyond the 3' terminus of the tRNA must logically be due primarily to an alteration in RNA synthesis, rather than turnover of the mature RNA (though increased turnover of the precursor could be the mechanism). The most likely explanation would be an impairment in 3' processing of pre-tRNA-ser(UCN). Two other mitochondrial tRNA mutations, at np 3243 and np 3302, both in the gene for tRNA-leu(UUR), appear to impair tRNA maturation, resulting in the accumulation of a precursor-like RNA (28 ,29 ). Careful inspection of the long exposure of the RNA blot shown in Figure 3 a and b does not reveal any transcripts that are elevated in CT001 cells, that could represent an unprocessed precursor of tRNA-ser(UCN). However, all higher molecular weight transcripts from this region that were reproducibly detected with double-stranded probes also hybridized with a probe specific for the COI coding strand, indicating that the steady-state level of the tRNA-ser(UCN) precursor is either below the limit of detection in both normal and patient-derived cells, or else that it is heterogeneous.
Our data indicate that a significantly decreased tRNA-ser(UCN) level is found in cells from a patient carrying the np 7445 mutation. This is consistent with the proposition that the site of the mutation, at the 3' boundary of the tRNA, could affect the efficiency, or conceivably the specificity of its processing. In lymphoid cells, however, tRNA-ser(UCN) appears to be a relatively abundant mitochondrial transcript, and even in patient-derived cells of this type it is still more abundant than in some other tissues from controls (e.g. mammary gland). This is fully consistent with the absence of any discernible effect on mitochondrial protein synthesis in patient-derived lymphoid cells. We propose that a defect in mitochondrial protein synthesis will only occur in a tissue-type where the level of mitochondrial tRNA-ser(UCN) is already so low, as a result of the tissue-specific expression of nuclear components of the mitochondrial gene expression machinery, that the further impairment to its synthesis resulting from the mutation depresses the level below a critical threshold. A prediction from this hypothesis is that the level of mitochondrial tRNA-ser(UCN), or some other component of the mitochondrial translation machinery that interacts with it, will be very low in cochlear hair cells (or in auditory neurons innervating them), a proposition that is, in principle, testable. Direct investigation of the affected tissue will be essential, in order to draw firm conclusions regarding the effects of this mutation. In addition, the discovery of a second tRNA-ser(UCN) mutation at np 7472, in a pedigree with a predominant phenotype of deafness (30 ), raises the important question of whether this also causes a depletion in tRNA-ser(UCN) levels.
A permanent lymphoblastoid cell-line was obtained by EBV transformation of lymphocytes from patient III-29 (index case) of the pedigree described previously (2 ). This line, designated CT001, was created by the European Collection of Animal Cell Cultures, Porton Down (ECACC), from a blood sample taken from the patient. A control lymphobastoid cell-line (01MC), derived similarly from a female without systemic disease, was provided by Dr M. Crouch (Department of Medical Genetics, Yorkhill Hospital, Glasgow). Six additional control lymphoblastoid cell-lines (AB48, AB132, AB233, AE7, AG110 and AG130) were provided by ECACC. Except where indicated in figure legends, lymphoblastoid cell-lines were maintained in RPMI 1640 medium (including 2 mM glutamine, 100 U/ml penicillin and 100 [mu]g/ml streptomycin) + 10% heat-inactivated fetal bovine serum. Cell growth rates were measured by the use of the vital dye MTT [3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide: Ref. (13 )]. A homogeneous solution which can be measured for optical density is produced by the addition of SDS (14 ).
Oligonucleotides, corresponding with segments of the human mtDNA sequence as described below, were purchased from Oswel (Edinburgh) or Cruachem (Glasgow), or were synthesized by phosphoramidite chemistry, using an Applied Biosystems PCRmate. To facilitate subsequent strand-specific labelling, primers FR29, FR31 and HJ08 were 5' biotinylated. The sequences of the oligonucleotides, and their co-ordinates on the human mitochondrial genome (5 ) were as follows:
DNA was prepared from growing cultures of lymphoblastoid cells, or from control blood samples, as described previously (2 ). Primers FR31 and FR32 were used to amplify a 662 bp segment of human mtDNA across the COI/tRNA-ser(UCN) junction (see Fig. 5 ), in quadruplicate reactions (2 ). PCR products were purified using the Promega `Magic' PCR purification system, and 1 [mu]g of each was digested with 10 U XbaI (BRL). Digestion products were analysed by electrophoresis through 1% agarose gels. PCR products for use as probes in Northern blots were amplified from control blood DNA samples, using the primers indicated below.
Lactate production by cell-lines was measured by assaying spent medium for lactate and pyruvate, and computing the ratio of the two metabolites, as adapted from Noll (15 ) and as described previously (16 ). Briefly, pyruvate production was determined by measuring the oxidation of NADH, in the presence of LDH, when combined with RPMI 1640 medium after 48 h incubation with cultured cells. Lactate was measured similarly except that the reduction of NAD was determined in the presence of hydrazine hydrate 0.1% (v/v).
Cytochrome c oxidase (COX) and succinate-cytochrome c reductase (SCCR) activities were measured in whole cell lysates as described previously (16 ), based on the method of Wharton and Tzagoloff (17 ). Enzyme activities were converted to specific activities, using protein concentrations derived from the Bradford assay (Bio-Rad).
RNA was extracted from cultures of lymphoblastoid cells by the guanidinium isothiocyanate/acid phenol method (18 ). RNA prepared similarly from A549 human lung carcinoma cells and from human placenta (villous trophoblast) was supplied, respectively, by Dr G.S.C.Dance, and by Ms S.Burridge and Dr R.G.Sutcliffe. RNA from human mammary gland, ovary and testis was purchased as ethanol precipitates from Clontech. Dried RNA pellets were resuspended in water, electrophoresed in formaldehyde/1% agarose gels and transferred to nylon filter membranes (Pall Biodyne `B') by capillary blotting in 20* SSC. RNA blots were hybridized overnight in a buffer containing 50% formamide, 5* SSC, 1% Denhardt's solution and 5% dextran sulfate, at 43oC [or 37oC, when probed for tRNA-ser(UCN)]. Double-stranded probes were synthesized by random hexanucleotide primer extension, using appropriate PCR products as template, in the presence of [[alpha]-32P]dATP (ICN; 3000 Ci/mmol), and heat denatured before use. An RT-PCR product, synthesized by Ms S.Burridge and Dr R.G.Sutcliffe from human testis RNA using customized primers (Clontech), was used to prepare a probe for human glyceraldehyde-3-phosphate dehydrogenase mRNA. Single-stranded probes for COI mRNA and tRNA-ser(UCN) were synthesized from strand-separated PCR products, created, respectively, using primers FR29/FR30 and HJ07/HJ08 on control blood DNA. The template strand was immobilized on Dynabeads (Dynal), and the complementary strand probe synthesized by Klenow DNA polymerase (Promega) in the presence of [[alpha]-32P]dATP (ICN; 3000 Ci/mmol), using FR30 or HJ07 as primer. The single-stranded probe was eluted from the magnetic beads by heat denaturation. Blots were finally washed at 55oC in 0.1* SSC, 0.1% SDS. Prior to re-hybridization, blots were stripped at 95-99oC in 0.1* SSC, 0.1% SDS. Signals were quantitated by laser-scanning densitometry, using autoradiographic exposures.
Cells (106)were incubated for 1 h at 37oC in 100 [mu]l methionine-free RPMI 1640 medium, containing emetine at 10-1000 [mu]M, with or without 1 mM chloramphenicol. An aliquot of 25 [mu]Ci 35S-labelled methionine/cysteine mixture (NEN `Expre35S35S' label; 1000 Ci/mmol) was added, and the cells incubated for a further 1 h. Cells were then washed in PBS and lysed in 20 [mu]l of SDS sample buffer. After denaturation at 100oC for 10 min, samples were analysed by SDS-PAGE in 12% polyacrylamide gels, stained with Coomassie blue, and visualized by fluorography using `Amplify' (Amersham).
We are grateful to Mr G. Vernham and colleagues for taking the blood sample which was used to create cell-line CT001, and to Dr M. Crouch for providing the control cell-line 01MC. We thank Marion Stone for technical assistance, and our colleagues in Glasgow, Tampere and within the EU Network on Mitochondrial Biogenesis in Development and Disease for useful discussions. This paper is dedicated to the memory of Anne Lusk, who also provided technical assistance for the work. The work was supported by grants from the Scottish Office Home and Health Department, the Juselius Foundation, the European Union and the Medical Research Fund of Tampere University Hospital. IJH is a Royal Society University Research Fellow.
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*To whom correspondence should be addressed+Present address: Leukaemia Research Laboratories, Glasgow Royal Infirmary, Glasgow G4 0SF, UK
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