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mtDNA replicative potential remains constant during ageing: polymerase [gamma] activity does not correlate with age related cytochrome oxidase activity decline in platelets
Introduction
Materials And Methods
Template preparation
Subjects for study
Platelet extraction
White blood cell (WBC) extraction
Intact nuclei extraction
Preparation of tissue homogenates
CS assay
Complex IV activity measurement
Polymerase assay
Considerations for assay conditions
Results
General specifications of the Pol[gamma] assay
Validation of mitochondrial Pol[gamma] assay specificity
Analysis of CS activity in platelets
Analysis of CytOx activity in platelets
Analysis of Pol[gamma] activity in platelets
Discussion
Acknowledgements
References
mtDNA replicative potential remains constant during ageing: polymerase [gamma] activity does not correlate with age related cytochrome oxidase activity decline in platelets
ABSTRACT
INTRODUCTION
The processive component of the mitochondrial DNA (mtDNA) replicative machinery has been known as a distinct biochemical entity since the 1960s (1). Recent studies of mtDNA polymerase [gamma] (Pol[gamma]) (2,3), suggest that early observations of a nuclear isoform most likely resulted from contamination of nuclear extracts due to perinuclear distribution of actively replicating mitochondria (4). Replication of mtDNA is now known to occur via a polymerase complex that is of a nuclear genetic origin with a 140 kDa catalytic subunit (Pol[gamma]) encoded by the nuclear Pol[gamma] gene located on chromosome 15q24 (2).
The first associations of mtDNA replication with clinical expression of oxidative phosphorylation (OxPhos) deficiency were made serendipitously during treatment of patients with AIDS: development of peripheral neuropathies in AIDS patients after 8-12 weeks treatment with 2[prime]3[prime]-dideoxy cytosine (ddC) caused decreased total cellular mtDNA levels (5). Further, myopathies involving mtDNA depletion developed in AIDS patients treated with 2[prime]-deoxy 3[prime]-azido thymidine (AZT) as a result of Pol[gamma] inhibition (6,7). This link was further established using tissue from patients with mitochondrial diseases: inhibition of mtDNA replication in fibroblasts cultured from Kearns-Sayre Syndrome (KSS) patients' tissue (8) by dideoxy nucleotide analogues caused a precipitation of mtDNA deletion from heteroplasmic mtDNA via wild type mtDNA depletion. Further reinforcing the significance of Pol[gamma] processive function to bioenergetic output, at least two independent studies have implicated quantitative defects of mtDNA replication in mtDNA depletion and consequent bioenergetic deficiency (9,10).
Age-related accumulation of mtDNA mutations is a potential factor contributing to the overall decline in OxPhos during the ageing process (11,12). Reactive oxygen species (ROS) are generated by OxPhos and result in the conversion of 2[prime]-deoxyguanosine (2[prime]-dG) to the 8[prime]-hydroxy 2[prime]-deoxyguanosine (8[prime]-OH 2[prime]-dG) analogue in mtDNA (13). In human mitochondria, this form of damage has been associated with point mutation (replicative mismatch) and mtDNA fragmentation (14). Age-related increases in the amount of 8[prime]-OH 2[prime]-dG have been observed in human skeletal muscle (15), heart (16) and brain (17). Inhibition of mtDNA replication by AZT at ~10% (1.0 mg/kg/day) of dosage administered to patients with AIDS showed significant (25%) conversion of 2[prime]-dG to 8[prime]-OH 2[prime]-dG over a 4 week period in mouse liver mtDNA (18). These findings collectively establish a potential link between age-related OxPhos decline and the mtDNA replicative process. Inefficient mitochondrial respiration can result in the exposure of mtDNA to increased oxidative stress and it has been postulated that this may be a factor contributing to cell damage in patients with mitochondrial myopathy (19,20). In addition to causing mtDNA damage (8[prime]-OH 2[prime]-dG) free radicals arising from oxidative stress also affect proteins within the mitochondrial matrix. The potential for increased mutation rates via oxidative stress adds perspective to elevated levels of mtDNA polymorphism detected in patients with mitochondrial disease (21,22). Whilst it is apparent that there is no overt effect on Pol[gamma] activity by most free radical adducts in DNA during replication, an inhibitory effect was observed at abasic sites (23) and it remains unclear as to whether total cellular Pol[gamma] activity is affected by ROS or other factors during ageing.
The recent purification of a mitochondrial oxidative damage-specific endonuclease (mtODE) in rat liver (24) almost certainly verifies the existence of a mtDNA repair system. These authors propose a hierarchical relationship between repair and replication, with repair of oxidative adducts preferentially taking place prior to the replication of the mutated DNA. This point is further strongly emphasised by a recent demonstration of Pol[gamma] as one major component of an mtDNA abasic site repair mechanism in Xenopus laevis (25). Given the demonstrations that AZT and ddC-inhibited mtDNA replication cause OxPhos defects and increase oxidative stress (elevated levels of 8[prime]-OH 2[prime]-dG), we wished to investigate the possibility that Pol[gamma] activity contributes to OxPhos profiles during ageing.
The Pol[gamma] assay system described in this communication is capable of utilising convenient amounts of any patient material. This means that large-scale purifications of Pol[gamma] are not required to study suspected defects of mtDNA replication in patients. The assay differentiates Pol[gamma] activity from nuclear polymerase activities, thereby extending its applicability from the platelets used in this study to other homogenates containing nucleated cells. In this communication we explored possible relationships between platelet derived Pol[gamma] activity and subject age, CytOx activity and mitochondrial mass [citrate synthase (CS) activity] to establish its possible involvement in age-related OxPhos decline.
MATERIALS AND METHODS
Template preparation
Poly(rA) (Sigma Chemical Co.) was suspended at 5 mg/ml in 10 mM Tris-HCl pH 7.4 made up using water treated with diethyl pyrocarbonate (DEPC-H2O) to eliminate possible RNAase contamination. Reverse-phase cartridge-purified Oligo (dT)20 (Gibco BRL) was suspended at 2.0 µg/µl in DEPC-H2O. Poly(rA):Oligo(dT)20 template was prepared by combining the two nucleic acids, (rA):(dT) molar ratio of 5:1 at a final concentration [relative to poly(rA)] of 1 µg/µl with 4 U/µl (final concentration) RNAsin RNAase inhibitor (Promega) in a 600 µl final volume using 10 mM Tris pH 7.0. The amount of template used in each assay was calculated to contain a ~3500-fold excess of RNase inhibitor required to inhibit the amount of RNase isolated from a mass of pancreas equivalent to the platelet mass used per assay (26).
Subjects for study
Whole blood (10 ml) was obtained with consent from individuals undergoing neurosurgical procedures or attending clinics for ongoing non-mitochondrial neurological conditions. Blood was collected in standard sodium-EDTA vacutainers (Becton-Dickson). Muscle and other tissues were obtained post mortem for the preparation of mitochondrial extracts and homogenates. In all instances samples were obtained in accordance with institutional ethics committee guidelines and approval.
Platelet extraction
Platelets were extracted from the 10 ml whole blood by differential centrifugation using a `gentle' protocol designed to minimise platelet activation as follows. The blood was centrifuged for 15 min at 110 g (room temperature). The platelet-rich serum was then removed (2-2.5 ml) under sterile conditions with attention being paid to not disturbing the red cell-serum interface. The platelet-rich serum was then centrifuged for 5 min at 2000 g at room temperature. The supernatant was removed using sterile technique and reintroduced into the test tube containing the non-serum blood fractions. This reconstituted `platelet minus' (P-) blood was then stored at 4°C until white cell extraction could proceed. The platelet pellet was resuspended in 1 ml of autoclaved Platelet Wash Buffer consisting of 4.3 mM K2HPO4, 4.3 mM Na2HPO4, 113 mM NaCl, 24.4 mM NaH2PO4 and 5.5 mM glucose at pH 6.5. The suspension was then transferred to a weighed centrifuge tube and spun for 5 min at 2000 g at room temperature to repellet the platelets. The supernatant was then aspirated, discarded, and platelet pellet mass determined. The platelet pellet was suspended at a concentration of 100 mg/ml in a 1× polymerase buffer consisting of 50 mM Tris pH 8.0 and 125 mM KCl, pH 7.5 and stored at -80°C awaiting assay. For polymerase assay, the 100 mg/ml platelet suspension was diluted to 50 mg/ml by the addition of an equal volume of 1× polymerase buffer with 6% (w/v) Triton X-100 to give a 3% final concentration of Triton X-100 in the platelet suspension prior to assay. This suspension was then centrifuged at 157 000 g, 4°C for 10 min (Beckman Tl-100 rotor) to remove particulate matter or intact platelets prior to storage at -80°C. Each polymerase assay replicate utilised 500 µg (10 µl) of these platelets. Prior to platelet lysis, the 100 mg/ml suspensions were assessed for contaminating nucleated cells. Such contamination usually consisted of <15 nucleated cells/10 µl of platelets (volume used per assay).
White blood cell (WBC) extraction
WBCs were prepared by differential phase centrifugation using Ficoll-Paque (Pharmacia Biotech) for B lymphocyte transformation with Epstein-Barr virus or for use as a source of cell nuclei or Pol[gamma] activity directly. To 10 ml of P- reconstituted blood (as described above), a 50% volume (5 ml) of sterile 1× phosphate buffered saline (PBS; ICN Flow) was added and 8 ml of this was carefully layered onto 2.5 ml of sterile Ficoll-Paque. The blood fractions were resolved by centrifugation at 800 g for 20 min at room temperature after which the WBC layer was removed, resuspended in 5 ml of1× PBS, and the suspension centrifuged for 5 min at 400 g at room temperature to pellet the WBC. The supernatant was discarded and cells resuspended either in 1× polymerase buffer at 50 mg/ml (as for platelets) for assay or nuclei extraction or in 2 ml of1× RPMI 1640 medium (Gibco) supplemented with 10% (v/v) fetal calf serum (FCS; Gibco), 100 U/ml penicillin-streptomycin (Trace), 2 mM l-glutamine, 1 mM Na-pyruvate and 50 µg/ml uridine (all from Sigma) for transformation. Cells in growth media were then transformed in duplicate sets with EBV. Cells prepared for polymerase assay were stored at -80°C until required while cells to be used for nuclei extraction were used immediately and frozen only at the completion of nuclei extraction.
Intact nuclei extraction
Cell nuclei were prepared either from WBCs directly or from transformed lymphoblast cultures by the same method. Cells were suspended at a concentration of 2 × 107 cells/ml in ice-cold buffer consisting of 10 mM Tris pH 7.5 and 0.5 mM MgCl2. This suspension was checked by light microscopy and staining with Trypan Blue for cell/nuclear integrity prior to sonication using a Branson 250 Sonifier at the lowest output setting for 1 s intervals. The extent of cell lysis was monitored between sonications to ensure that enough cell lysis had occurred, but not at the expense of intact nuclei. The optimal degree of sonification was determined separately in each case. The intact nuclei were pelletted by 1 min centrifugation at 700 g, 4°C. The nuclear pellet was washed once (200 µl/2 × 108 starting cells) in 10 mM HEPES pH 7.0, 1.5 mM MgCl2, 10 mM KCl pH 7.5 and 0.5 M DTT. Cell nuclei were suspended in 1× polymerase buffer at a final concentration of ~105 nuclei/ml (usually ~100 µl) and stored at -80°C until required.
Preparation of tissue homogenates
Tissues other than blood were obtained post mortem, frozen in liquid N2 and stored at -80°C until required. Small portions were shredded in a mortar pre-cooled on dry ice and ground to a fine powder with a pestle. The powder was transferred to a pre-weighed polypropylene tube and the tissue mass determined. A quantity of polymerase buffer or homogenisation buffer, pH 7.4 consisting of 225 mM mannitol, 75 mM sucrose and 100 mM EDTA (MSE) was added such that the final tissue concentration was 100 mg/ml. This suspension was thoroughly suspended and centrifuged at 157 000 g, 4°C for 15 min (Beckman Tl-100 rotor) to remove particulate matter before storage at -80°C awaiting assay.
CS assay
CS activity in the Triton X-100 solubilised platelet preparations was measured by 412 nm spectrophotometric detection of CoA 2-nitro 5-thiobenzoate (CoA-TNB2) formation during the reaction between Ellmans reagent (DTNB), Acetyl CoA and Oxaloacetic acid to form CoA-TNB2 ([epsis] =14.5/mM) and citrate essentially as described elsewhere (27). A total 250-500 µg of platelets were used per assay duplicate. CS activities were expressed in nmol CoA-TNB2 formed/min/mg platelets and used to correct for mitochondrial matrix equivalence (mt-eq) of Pol[gamma] activity.
Complex IV activity measurement
Prior to assay, reduced cytochrome c was prepared by suspension of 200 mg cytochrome c (Sigma) in 2 ml solution of 0.5 M sodium ascorbate. Reduced cytochrome c was resolved from the mixture by passage through a 20 ml Sephadex G-25 column equilibrated with 50 ml reaction buffer (pH 6.0) consisting of 0.1 M 2-N-morpholino ethanesulfonic acid (MES) and 10 µM EDTA. Cytochrome oxidase (CytOx) activity was measured by spectrophotometric determination of reduced cytochrome c oxidation at 550 nm as follows. To 1 ml of reaction buffer was added a sufficient volume of reduced cytochrome c to produce a stable A550 of ~0.5. The oxidation of cytochrome c was calculated ([epsis] =18.5/mM) and CytOx activity expressed in units of nmol cytochrome c oxidised/min/mg platelets.
Polymerase assay
With the exception of parameters varied for testing, the optimised protocol for the assay of mtDNA Pol[gamma] in crude platelet preparations is as follows. The 50 µl of 100 mg/ml platelet preparations (as described in these methods) were diluted to a final concentration of 50 mg/ml by the addition of an equal volume of polymerase buffer/6% Triton X-100. Individual reaction mixtures consisted of 50 mM Tris pH 8.0, 125 mM KCl (i.e. 1× polymerase buffer), 0.5 mM [3H]dTTP (specific activity 200 mCi/mmol), 10 µg/ml BSA, 150 mM NaCl, 2.5 mM dithiothreitol, 10 µg/ml Aphidicolin (Sigma), 0.5 mM MnCl2, 50 mM KH2PO4 pH 7.5, and 20 µg/ml [relative to poly(A)] poly(rA):oligo(dT)20 at a relative molar ratio of 5:1 [poly(rA):oligo(dT)20]. Quadruplicate assay mixtures were incubated at 37°C for 1 h and stopped by the addition of 100 µg of Escherichia coli tRNA (10 µl) and 4 vol (240 µl) of ice-cold 20% TCA/NaPP consisting of trichloroacetic acid (TCA; 20% m/v) with 40 mM sodium pyrophosphate. The polymerised product was allowed to precipitate for 5 min on ice and collected by centrifugation at 14 500 g for 30 min at 4°C. The supernatants were discarded and pellets thoroughly washed twice with 400 µl of 20% ice-cold TCA/NaPP. The washed pellets were suspended in 400 µl of deionised water and incorporated activity counted using Ecolite scintillant (ICN Flow). At least three concordant results (<10% deviation) from the quadruplicate assays were required for analysis. Assays not utilising platelet homogenates were as described in this method but with no addition of Triton X-100.
Considerations for assay conditions
The assay described in this work was specifically designed for crude homogenates which inevitably contain a mixture of enzymes other than the Pol[gamma]. As such, the assay must take into account any factors that may influence the function of the mitochondrial Pol[gamma] enzyme. A number of considerations towards this end were made.
Template integrity. Essentially, the template used for this assay is a DNA/RNA hybrid. The use of Mn2+ as a cofactor for the enzyme was preferred to Mg2+ because Mg2+ is the cofactor required for RNAase H activity which digests DNA/RNA hybrids (28) and because Mg2+ is also the cofactor for the 5[prime]-3[prime] exonuclease activity of mitochondrial Pol[gamma] (29,30). Also recently identified in mammalian mitochondria is endonuclease G (Endo G), capable of digesting both single- and double-stranded DNA but with a preference for RNA (31,32). Possible contamination by this activity was suppressed within this system by utilising non-permissive concentrations of KCl (125 mM) and NaCl (150 mM). The monovalent cation concentrations used in this assay were sufficient to suppress the activity of further endonucleases reported in mitochondrial systems (24,33) capable of digesting the template or product of this assay. Specificity of assay to Pol[gamma]. Aphidicolin derived from Cephalosporium aphidicola, inhibits mammalian DNA Pol[alpha], -[delta] and -[epsis] and other B family polymerases and was incorporated into the assay to eradicate any nuclear non-[gamma] DNA polymerase contaminating the mitochondrial Pol[gamma] activity. A nuclear Pol[gamma] activity described in early studies (34) showed subtle, if any, differences to the enzyme extracted from isolated mitochondria (35). The specific localisation of Pol[gamma] to mitochondria was demonstrated by anti-Pol[gamma] antibody staining in recent studies (3,4). Elegant immunohistochemical data showing that mtDNA replication by Pol[gamma] occurs mainly in mitochondria in close proximity to the nucleus (4) supports the likelihood that the `nuclear' or otherwise `cytoplasmic' Pol[gamma] observed in early studies resulted from artefactual co-purification of the mitochondrial enzyme in nuclear extracts. This contingency was, however, accommodated in this assay by use of enucleated cells (platelets) and inclusion of 50 mM KH2PO4 pH 7.5 (34,35). Further differentiation of the mitochondrial Pol[gamma] activity from nuclear DNA polymerases was achieved by preferential enhancement of the mitochondrial Pol[gamma] activity using 125 mM KCl in conjunction with the 50 mM KH2PO4 pH 7.5 (34,36), poly(rA):oligo(dT)20 (34,36) and the use of 150 mM NaCl (1). Contamination of platelet preparations by nuclei was assessed by light microscopy as outlined in the platelet preparation method. The specificity of the assay to mitochondrial Pol[gamma] was ascertained by assay of polymerase activity in intact nuclear preparations utilising between 100 and 1000 times the amount of nuclei determined to contaminate the volume of platelets used per assay (10 µl).RESULTS
General specifications of the Pol[gamma] assay
One major feature of the assay described in this study was the suspension of platelets in Triton X-100. This factor significantly increased the activity recovered from the 50 mg/ml platelet preparations. Sonication of platelets without Triton X-100 for up to 8 min (10 s/min × 48 min) failed to liberate any significant level of Pol[gamma] activity (data not shown; Fig.
Figure 1. Effect of Triton X-100. (A) The effect of Triton X-100 on platelet Pol[gamma] activity is shown expressed as pmol dTTP incorporated/min/mg platelets. The optimal concentration of Triton X-100 in the platelets was 3% (v/v). This represents a final Triton X-100 concentration of 0.6% during assay. Absence of Triton X-100 from the platelet suspensions resulted in minimal recovery of Pol[gamma] activity, even with up to 8 min sonication (5s/30s × 48 min; data not shown). (B) The effect of Triton X-100 was determined on CS activity. At the 3% concentration optima for Pol[gamma], no adverse effect was observed. The data shown are expressed as relative units. No significant drop in platelet Pol[gamma] activity was observed with whole blood storage over a 35 h period at room temperature (25°C; data not shown). Pol[gamma] assay of independent platelet preparations from a single individual varied by <10% (Table 1). No apparent drop in activity was observed after 6 months storage of platelets at -80°C, although inter-replicate variation did increase (Table 1). The Pol[gamma] activity in the platelet preparations was seen to vary markedly; however, with multiple thaw cycles, some samples losing almost their entire activity after just two cycles (data not shown). To minimise possible loss of Pol[gamma] activity with storage and freeze-thaw cycles, platelets subject to this analysis were assayed within 3 months of isolation, frozen, and thawed once only. Table 1. Miscellaneous properties of platelet Pol[gamma] activity
Validation of mitochondrial Pol[gamma] assay specificity
Platelets are the enucleate `progeny' of megakaryocytes and are formed by cytoplasmic budding of the parent cell. As such, they make an ideal model for the study of mitochondrial Pol[gamma] primarily because the need to account for nuclear polymerase activity is minimised. The [dTTP] Km for the Pol[gamma] present in the platelet homogenate was 150 µM (Fig.
Figure 2. (A) Platelet Pol[gamma] kinetics. Pol[gamma] activity was measured using separate aliquots of a single platelet extraction and varying amounts of substrate (dTTP). The [dTTP] Km for the enzyme in this suspension was 150 µM and the Vmax was 11.8 pmol/min/mg platelets. (B) Pol[gamma] assay in muscle homogenate. The Pol[gamma] activity was measured in an aliquot of a 100 mg/ml muscle homogenate as described in the Materials and Methods. The amount of homogenate used to obtain the activity shown corresponds to an equivalent of 4.5 mg starting material, with each point being the average of two readings each of 500 µg. The specificity of the assay to Pol[gamma] allows it to be used for the assay of homogenates containing nucleated cells and mitochondrial homogenates alike (as for WBC in Table 1). Pol[gamma] activity was measured in liver, kidney, heart, psoas and placental mitochondrial preparations (data not shown) in addition to the platelets and muscle homogenate detailed in this communication. No DNA polymerase activity was observed in intact nuclei extracts. Of particular importance regarding the specificity of this assay was the result that no incorporation of dTTP was evident using intact nuclei as a source of DNA polymerase activity (Table 1). This shows that the assay is indeed specific for the Pol[gamma] activity in cells and extends its application to other tissue homogenates such as muscle, which contain nucleated cells. This is illustrated in Figure 
Analysis of CS activity in platelets
CS is the Krebs cycle enzyme which catalyses the conversion of acetyl CoA and oxaloacetate to citrate as the first step of the TCA cycle in the mitochondrial matrix. It is used as a marker enzyme to establish sample to sample equivalence with respect to mt-eq. The population studied in this work was subdivided according to age into two groups <51 (young) and >50 (elderly) years of age and their respective CS activities measured (Fig.
Figure 3. (A) Comparison of CS activity in aged and young subjects. The studied population was subdivided according to age into two groups of <51 (young) and >50 (elderly) years of age. No age-wise difference in CS activity was observed (P < 0.82; t). (B) Age-related CytOx deficiency. Cytochrome activity was measured in the elderly and young subject groups as described in the Materials and Methods. Activity is expressed relative to CS (nmol/mg/min) activity to correct for mt-eq between individuals. A 24% deficiency (P < 0.01; t) was evident in the CytOx activity of the elderly group (1.76 ± 0.12 nmol/min/mt-eq) compared to the young subject group (2.30 ± 0.16 nmol/min/mt-eq). The same result was obtained using activities expressed in terms of platelet mass. CytOx activity showed a significant inverse correlation with age (P < 0.002; P). (C) Platelet Pol[gamma] activity and age. No difference in Pol[gamma] activity was evident between the young and elderly subjects of this study. Activity was measured as described in methods and expressed in terms of mt-eq (CS activity) as pmol dTTP incorporated/min/mt-eq.
Analysis of CytOx activity in platelets
CytOx represents Complex IV of the five respiratory complexes embedded in the mitochondrial matrix membrane. It is a critical point of the oxidative electron transport chain performing the functions of transmembrane proton translocation and reduction of O2 to H2O. Human CytOx consists of 13 subunits, only three of which are encoded by the mtDNA. Age related decline in CytOx function has been widely reported over the past decade (11). The elderly and young groups' platelets were assayed for CytOx function and a 24% comparative deficiency (P < 0.01; t) was observed in the elderly group (Fig.
Analysis of Pol[gamma] activity in platelets
The two age-wise groups studied did not differ in their respective Pol[gamma] activities to any degree of significance (Fig.
Figure 4. (A) No correlation between Pol[gamma] and CS activities. Pol[gamma] and CS activities did not correlate (P < 0.22; P), indicating that Pol[gamma] processive levels are largely independent of mitochondrial number. (B) No correlation between Pol[gamma] and CytOx activities. Pol[gamma] activity showed no correlation with CytOx activity in the subjects analysed (P < 0.53; P). Both activities were expressed in terms of mt-eq using CS activity. (C) No correlation between Pol[gamma] and age. No correlation was observed between Pol[gamma] activity and subject age, suggesting little likelihood of any involvement in the age-related decline in CytOx activity observed in this population. The results shown here expressed in terms of mt-eq did not differ when expressed in terms of activity per mg platelets.
DISCUSSION
In this study we have used an in vitro assay to investigate the possible contribution that Pol[gamma] activity may make towards age-related OxPhos decline. The assay adapted to platelets in this study, displayed good repeatability between independent platelet preparations from a single subject and provided a useful tool for the assessment of readily obtainable amounts of patient tissues. Possibly resulting from the presence of factors in the homogenate, the Km of the enzyme for dTTP in crude platelet homogenate was ~10 times (150 µM) the Kd determined (14 µM) for purified human Pol[gamma] (38), with a Vmax of 11.8 pmol/min/mg. The increased Km observed for the platelet homogenate compared to the human enzyme purified from transformed cells (38) and bovine heart (42) most likely reflects the heterogeneity of the platelet preparation or the possible existence of tissue-specific isoforms. The enzyme was inhibited by EtBr and no activity was detected in intact nuclear preparations containing between 100 and 1000 times the nuclei that were observed to contaminate the platelet preparations. Triton X-100 was observed to increase the recovered activity in platelet preparations (3% Triton X-100 v/v in platelets but 0.6% in assay) by at least 10-fold, although this also created a certain degree of difficulty regarding background levels. The activity was stable over 35 h storage of whole blood at room temperature, with no activity loss over 6 months storage at -80°C provided freeze-thaw cycles were prevented. Multiple freeze-thaw cycles resulted in considerable loss of activity in some samples.
The in vitro assay described here was applied to study the function of the mtDNA polymerase found in platelets from a group of elderly (n = 19) and young (n = 24) individuals. In addition to Pol[gamma] activity, CS and CytOx activities of the studied individuals were assayed to provide comparative markers of mt-eq and bioenergetic capacity, respectively. The significant 24% age-related decline in CytOx activity (P < 0.01; t) observed in the elderly subjects compared to the young subjects (Fig.
The absence of any correlation between CS and Pol[gamma] activities (Fig.
The lack of correlation between Pol[gamma] and CytOx activities described here is consistent with the apparent independence of mtDNA copy number and CytOx activity previously observed by others (46). Rapid increases in mammalian mitochondrial protein demands are primarily met by increased transcription rather than by increased mtDNA levels (47). Due to this, mitochondrial transcription has the potential to buffer mtDNA copy number fluctuations to preserve overall bioenergetic output. Such buffering potential is illustrated by 250% increased mitochondrial transcription coinciding with 50% depletion of mtDNA in human skeletal muscle from patients with type II diabetes (48). Further evidence of this is presented in a recent report of age-related increase in total mtDNA content of brain (49). In the latter work, the age-related increase in mtDNA content coincided with a decrease in mtRNA levels and was proposed to occur as an inefficient compensatory mechanism to maintain the normal levels of mtRNA transcripts, a finding which is also consistent with the small increase in Pol[gamma] activity in the elderly group described here (Fig.
The preservation of Pol[gamma] activity levels during ageing documented in this study indicates that mtDNA replicative potential remains constant throughout life. Coupled to this finding and reinforcing the unlikely correlation between Pol[gamma] activity and age-related bioenergetic decline, CytOx activity showed a significant age-related decline which was not reflected by Pol[gamma] activity in the subjects analysed. These results do not preclude the possibility of transient Pol[gamma] inhibition during crucial stages of cell function requiring major increases of bioenergetic output and/or Pol[gamma] activity. Such episodes should empirically compromise mtDNA integrity and OxPhos in a manner similar to that demonstrated in AZT studies and will not be detected under assay conditions as described here.
In addition to providing further evidence that constitutive expression of the Pol[gamma] gene is not regulated by mitochondrial biogenesis and that other factors largely influence the rate at which mtDNA is replicated (3,42), our results extend to exclude bioenergetic status as a Pol[gamma] gene regulatory factor. There are, however, two cell cycle/developmental stages during which elevated levels of Pol[gamma] activity are required. (i) Simultaneous Pol[gamma] and CytOx activity increase has been observed in rabbit spleen lymphocytes only immediately before and matching the expansion of ATP levels to supplement energy requirements for cell division in early S phase (50): AZT has been shown to arrest cell division at S phase in bone marrow and this cytotoxicity can be reversed in human bone marrow progenitor cell cultures by uridine supplementation (required by cells with OxPhos dysfunction for pyrimidine synthesis). (ii) Quantitative levels of Pol[gamma] transcripts coincide with cellular mtDNA level fluctuations in X.laevis during vitellogenesis (45). In terms of the latter scenario, mtDNA deletogenesis as a sporadic event prior to blastulation in human development (51) may result from transient processive Pol[gamma] deficiency which precipitates low level deleted species from mtDNA heteroplasmy in a manner similar to AZT in human KSS fibroblasts (8). These points of the cell cycle/development thus represent small windows of opportunity for transient deficiency of Pol[gamma] activity to exert an overall influence on bioenergetic output in daughter cells during cell division. Under this hypothesis, the stage at which such insults occur may determine whether bioenergetic defects manifest as clinical syndromes during life or as more passive sub-clinical age-related degenerative processes in isolated tissue/organ groups: it is empirically more likely that sporadic mtDNA mutation at the origin of development where mtDNA copy numbers are relatively low will lead to specific or multiple-tissue dysfunction than at later stages in development.
There is evidence that cells with OxPhos deficiency are subject to comparatively high levels of oxidative stress (19,20). Whilst 8[prime]-OH 2[prime]-dG adducts resulting from OxPhos-mediated free radicals does not affect Pol[gamma] processive function, ROS-generated abasic sites on template decrease Pol[gamma] activity (23), possibly by invoking the repair mechanism consisting of mtDNA ligase, mitochondrial AP endonuclease and Pol[gamma] activities recently described in X.laevis mitochondria (25). Furthermore, any direct inhibitory effect of ROS on the enzyme itself remains to be defined. The specific mechanism by which observed increases in 8[prime]-OH 2[prime]-dG adducts occur with Pol[gamma] inhibition remains unclear and beyond the scope of the present study. Both in instances of gross mtDNA rearrangements and point mutation, however, it appears from our results that the Pol[gamma] processive function is a passive bystander of the major mutagenic process in operation within the mitochondrial matrix. The AZT studies give rise to the possibility that deficiency of Pol[gamma] activity during replication may augment the mutational environment within the mitochondrial matrix and perhaps cause preferential amplification of deleted subspecies. The elevated mutation and/or polymorphic rates observed in tissues from ageing individuals (52) and patients with bioenergetic deficiency (21,22), respectively, are more likely, however, to arise from the inefficient repair of lesions than from the processive component of Pol[gamma]. Some understanding of this may be gained by further studies into the hierarchy apparent between mtDNA transcription, replication and repair (24) and study of mtDNA replication and/or repair fidelity in the mitochondrion.
The assay used in this study of Pol[gamma] involvement in age-related bioenergetic decline has demonstrated an applicability and relevance to issues involving mtDNA metabolism and associated defects. The applicability of this assay to homogenates containing nucleated cells facilitates access to this important metabolic information using realistic amounts of any tissue source. In particular, this assay is applicable to investigations into the involvement of Pol[gamma] activity in mtDNA depletion related OxPhos deficiency.
ACKNOWLEDGEMENTS
The authors wish to express a special appreciation of Prof. Lawrie Austin's involvement in their work: his valuable insight, technical expertise and informed discussion have been significant factors in the resolution of the many biochemical issues encountered in this study. Drs John Kanellis and Russell Auwardt are most gratefully thanked for their diligent assistance with sample collection. Thanks are also extended to the many volunteers who kindly donated their blood for this study. The work described in this communication was supported by project grant #034206 from the NH&MRC of Australia.
REFERENCES
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