The mitochondrial T1095C mutation increases gentamicin-mediated apoptosis

Mitochondrion 12 (2012) 465–471

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The mitochondrial T1095C mutation increases gentamicin-mediated apoptosis Hakan Muyderman a,⁎, Neil R. Sims a, Masashi Tanaka b, Noriyuki Fuku b, Ravinarayan Raghupathi c, Dominic Thyagarajan d, e a

Discipline of Medical Biochemistry and Centre for Neuroscience, School of Medicine, Flinders University, Adelaide, South Australia, Australia Department of Genomics for Longevity and Health, Tokyo Metropolitan Institute of Gerontology, Japan Discipline of Human Physiology and Centre for Neuroscience, Flinders University, Adelaide, South Australia, Australia d Department of Neurology, Flinders Medical Centre, Adelaide, South Australia, Australia e Department of Neurology, Monash University, Clayton, Victoria, Australia b c

a r t i c l e

i n f o

Article history: Received 15 March 2012 Received in revised form 13 June 2012 Accepted 19 June 2012 Available online 24 June 2012 Keywords: Mitochondrial DNA T1095C 12SrRNA Aminoglycosides Apoptosis Mitochondrial parkinsonism

a b s t r a c t We have previously reported a heteroplasmic mtDNA mutation (T1095C) in the 12SrRNA gene of an Italian family with features of maternally-inherited parkinsonism, antibiotic-mediated deafness and peripheral neuropathy. In the present study, we demonstrate that a transmitochondrial cybrid line derived from the proband of this family shows selective depletion of mitochondrial glutathione and decreases in the activity of complex II/III. Moreover, when exposed to an aminoglycoside antibiotic these cells responded with a ten-fold increase in the number of apoptotic cells compared to controls. These results support a pathogenic role for the T1095C mutation and indicate that the mutation increases the risk for aminoglycoside-induced toxicity. © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction We previously reported a heteroplasmic mtDNA mutation T1095C in the 12SrRNA gene in a family of Southern Italian origin with variable features of maternally-inherited parkinsonism, deafness after treatment with aminoglycosides and peripheral neuropathy (Thyagarajan et al., 2000). The same mutation was later found in different proportions of heteroplasmy in affected members of an Italian pedigree with maternally-inherited non-syndromic deafness (Tessa et al., 2001). In two of the affected members from this pedigree, the deafness was thought to be aminoglycoside-induced. In neither of these reported pedigrees did the mutant load in DNA extracted from blood correlate with the degree of deafness. A T1095C mutation variant has also been discovered in several Chinese patients with auditory neuropathy, in some cases associated with aminoglycoside exposure, (Li et al., 2005; Wang et al., 2005; H. Zhao et al., 2004; L. Zhao et al., 2004) but an extended measurement of heteroplasmy or pedigree analysis was not performed in those cases. In three Chinese pedigrees with the T1095C mutation the probands had deafness following aminoglycoside administration but there was extremely low prevalence of deafness in matrilineal family members bearing the T1095C mutation. These findings led the authors ⁎ Corresponding author at: Discipline of Medical Biochemistry, School of Medicine, Flinders University, G.P.O. Box 2100, 5001, Adelaide, SA, Australia. E-mail address: hakan.muyderman@flinders.edu.au (H. Muyderman).

to conclude that the T1095C mutation on its own was insufficient to produce deafness but that an additional factor, perhaps the aminoglycoside, was required (Dai et al., 2006). These small studies suffer from ascertainment bias and large population studies are not available for the T1095C mutation. However recent population prevalence studies of the better studied mutation, A1555G, also support the conclusion that the penetrance of deafness‐associated mutations in 12SrRNA is low, but may increase with age (Bitner-Glindzicz et al., 2009; Vandebona et al., 2009). Interestingly, the A1555G and the T1095C mutation have been found co-segregating in a profoundly deaf child, raising the possibility that the T1095C mutation could modulate the penetrance of the A1555G mutation (Dai et al., 2008). Our previous report of the T1095C mutation supported a pathogenic effect. The mutation theoretically disrupts the secondary structure of a highly-conserved helix in stem-loop structure, close to the P site and we hypothesized that the mutation affects mitochondrial protein synthesis. A moderate decline in electron transport chain activity in lymphoblasts from the patient supported this hypothesis (Thyagarajan et al., 2000). Several lines of evidence indicated that this mutation is pathogenic: 1) heteroplasmy 2) segregation with at least deafness in maternal relatives 3) the presence of a respiratory chain defect 4) the absence of the mutation in a large number of controls 5) occurrence in individuals of different ethnic origins and 6) the disruption of the 12SrRNA secondary structure. In addition, the 12SrRNA gene appears to be a “hotspot” for mitochondrially mediated deafness. Two mutations (A→G at nucleotide

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(nt) 1555 (Prezant et al., 1993) and C→G at nt 1494 (H. Zhao et al., 2004; L. Zhao et al., 2004), which forms a non-canonical RNA base pair with nt 1555) in the penultimate helix of the 12SrRNA, a component of the A site, has been found in many ethnic groups and geographical regions in association with deafness ranging from a severe congenital form to late onset of progressive hearing loss. Of interest, the affected individuals are highly susceptible to the ototoxic effects of aminoglycosides (Fischel-Ghodsian, 1999). In bacterial hybrid ribosomes in which the central 34-nt part of the rRNA helix (Thyagarajan et al., 2000) is identical to that of the mutated human mitochondrial decoding site, both these mutations increase amino acid misincorporation in cell free translation assays (Hobbie et al., 2008a, 2008b), and cause the ribosomal decoding site to be excessively susceptible to translational infidelity induced by the aminoglycoside tobramycin. Length variations in the region of nt 961, (Bacino et al., 1995) and a T→G mutation at this nucleotide (Li et al., 2005) in the 12SrRNA gene have also been associated with aminoglycoside-induced deafness and non-syndromic hearing loss respectively. To further investigate the potential pathogenicity of this mutation, we fully sequenced the mitochondrial genome, evaluated key mitochondrial properties and, under conditions of aminoglycoside exposure, assessed cytotoxic death in a transmitochondrial cybrid line derived from the proband of our original family with Parkinsonism, deafness and neuropathy. Because of the suggested role for oxidative damage in gentamicin-induced deafness (Walker et al., 1999; Wullner et al., 1999) the content of glutathione was assessed in the cytosol and mitochondria of the cybrid. Depletion of this major cellular antioxidant can be induced by oxidative stress (Wullner et al., 1999) and can increase susceptibility of cells to oxidative damage (Muyderman et al., 2004, 2007). The study shows that our patient is most likely within a subgroup of the haplogroup HV, and that the presence of the T1095C mutation reduces mitochondrial anti-oxidant defenses and substantially increases apoptotic cell death in response to aminoglycoside treatment. 2. Methods 2.1. Materials All of the chemicals were of analytical grade and were obtained from Sigma (St. Louis, MO) unless stated otherwise. Tissue culture reagents were obtained from Invitrogen (San Diego, CA). Propidium iodide (PI) and the Annexin-V-Fluos kit were from Molecular Probes (Leiden, The Netherlands), and Hoechst 33258 was from Calbiochem (La Jolla, CA). The 143BTK-206 rho zero cell line was a gift from Dr Ian Trounce, University of Melbourne, Australia. 2.2. Cell types and culture conditions Immortalized lymphoblastoid cell lines derived from one affected member of the South Italian family and two genetically unrelated control individuals with comparable age were grown in RPMI 1640 medium with 10% fetal bovine serum, 0.03% glutamine, 0.2% sodium bicarbonate and 0.002% beta mercaptoethanol. Antibiotics were not added. The 143B TK-206 cell line was grown in DMEM (containing 4.5 mg of glucose and 100 μg pyruvate and 50 μg uridine per ml and bromodeoxyuridine), supplemented with 5% fetal bovine serum. The cybrid cell line constructed with enucleated lymphoblastoid cell lines was maintained in the same medium as the 143B TK-206 cell line except that dialyzed serum was used and pyruvate and uridine were not added (“selection medium”). No antibiotics were used. The lymphoblastoid cells derived from one affected subject and two control individuals were enucleated, and subsequently fused to a large excess of mtDNA-less 143B 206 rho zero cells, derived from the 143B.TK cell line. For enucleation, 1×107 lymphoblasts were resuspended in 20 ml of medium in 50 ml sterile round bottom polycarbonate centrifuge tubes, pre-equilibrated at 37 °C in a 5% CO2 incubator. Cytochalasin B was added at a final concentration of 20 μg/ml. The tubes were centrifuged at

44,000 ×g in a Sorvall JA20 rotor for 70 min at 30 °C and the upper two bands containing cytoplast and karyplasts were carefully removed, pelleted at 1000 ×g for 10 min, washed and resuspended in the lymphoblast growth medium and kept at room temperature. To this, 2×106 trypsinized rho zero cells were added and the mixture co-pelleted at 5000 ×g for 10 min. The supernatant was removed and the pellet overlaid with 0.5 ml of PEG 1500 (Roche, Indianapolis, USA) for 60 s. Excess PEG was removed and the pellet resuspended in 10 ml pre-warmed selection media. Cells were plated at a density of 2×105 cells per 10 cm dish. Between 15 and 25 days after fusion, 10 presumptive mitochondrial cybrids were isolated using cloning rings and subsequently analyzed for the presence and level of the T1095C mutation. 2.3. Separation of mitochondrial and cytosolic components Mitochondria were isolated as described previously (Muyderman et al., 2004). In short, cells were harvested by mild trypsinization (0.05% (w/v) trypsin and 0.02% EDTA; 3–5 min), washed twice and samples for determining total glutathione content were removed. The pellet was resuspended in “isolation medium” (10 mM Tris, 1 mM EDTA, 0.32 M sucrose, pH 7.4) containing 0.2 mg/ml digitonin and centrifuged (15,000 ×g) for 5 min. The supernatant was removed and used as the “cytoplasmic fraction”. To produce a crude “mitochondrial fraction,” the pellet was washed twice with isolation medium with centrifugation after each wash. By this method 96 ± 2% of total cellular citrate synthase activity (a mitochondrial marker) and 1.7 ± 1.2% of the total lactate dehydrogenase (LDH) activity (a cytoplasmic marker) were recovered in the mitochondrial fraction (n = 3). The cytoplasmic fraction contained 99± 1% of the LDH activity and no detectable citrate synthase activity (n = 3). 2.4. Sequencing of mtDNA DNA was extracted using an automated DNA extraction system MagExtractor MFX-2000 (Toyobo, Osaka, Japan) and the entire mitochondrial genome was amplified in a two-step PCR procedure and then sequenced as previously described (Tanaka et al., 1996). The sequence reactions were performed by the use of the second PCR template, and a BigDye Terminator Cycle Sequence Ready Reaction Kit version 1.0, 3.0, or 3.1 (Applied Biosystems, Foster City, CA). The following PCR conditions were used: an initial denaturation step at 96 °C for 5 min, followed by 25 cycles of denaturation at 96 °C for 10 s, annealing at 50 °C for 5 s, and extension at 62 °C for 4 min. After the sequence reaction, excess dye terminators were removed by gel filtration on a MultiScreen-PCR HV Plate (Millipore). The purified DNA samples were dried and suspended in the template suppression reagent or formamide from Applied Biosystems. The dissolved DNA samples were heated at 95 °C for 2 min for denaturation, and then immediately cooled on ice. Sequences were analyzed with an automated DNA sequencer Prism 310 or 377 from Applied Biosystems. Sequence analysis was performed by use of Sequencing Analysis Program version 4.1 software (Applied Biosystems). Complete sequences were aligned, assembled, and compared with the program Sequencher 4.1 (Gene Codes, Ann Arbor, MI). Each of the mtDNA sequences was compared with the original Cambridge sequence (Anderson et al., 1981) and the revised Cambridge reference sequence (rCRS) (Andrews et al., 1999). 2.5. Determination of glutathione content Samples were treated with 2 M perchloric acid containing 4 mM EDTA for 3 min, centrifuged, and neutralized with 2 M potassium bicarbonate and 0.3 M 3-(N-morpholino)-propanesulfonic acid. Total (reduced plus oxidized) glutathione was determined spectrophotometrically using the procedure of Akerboom and Sies (1981). The assay measured the rate of formation of 5-thio-2-nitrobenzoate from

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5,5′-dithio-bis(2-nitrobenzoate) in the presence of NADPH and glutathione reductase. 2.6. Measurements of citrate synthase, LDH and respiratory chain complexes Citrate synthase activity was measured at 410 nm using a Shimadzu UV-3000 spectrophotometer at 30 °C as described by Shepherd and Garland (1969). The reaction buffer contained 100 mM Tris–HCl, pH 8.1, 0.1 mM 5,5′-dithio-bis(2-nitrobenzoic acid), 0.3 mM acetyl-CoA, and 0.1% (v/v) Triton X-100. The assay was initiated by the addition of oxaloacetic acid (final concentration, 0.05 mM). Lactate dehydrogenase activity (LDH) was determined spectrophotometrically from the oxidation of NADH in the presence of pyruvate (Vassult, 1983). Release into the supernatant was determined as a percentage of the total lactate dehydrogenase activity, which was measured after treating cells for 10 min with 0.25% Triton X-100. The activity of nicotinamide adenine dinucleotide (NADH)ubiquinone oxidoreductase (complex I) was determined spectrophotometrically at 340 nm by following the rotenone-sensitive oxidation of NADH in the presence of ubiquinone-1 (Muyderman et al., 2004; Ragan et al., 1987). Succinate-cytochrome c oxidoreductase (complexes II–III) activity was determined by measuring the reduction of cytochrome c in the presence of sodium succinate, (King, 1967) and cytochrome c oxidase (complex IV) activity was assessed from the oxidation of reduced cytochrome c (Muyderman et al., 2004; Wharton and Tzagoloff, 1967). All measurements were made using a Shimadzu UV-3000 spectrophotometer (Shimadzu Scientific Instruments, Rydalmere, New South Wales, Australia). Enzyme activities were calculated as nanomoles per minute per mg protein. Results are presented as relative changes compared with untreated control cybrids not carrying the T1095 mutation. 2.7. Measurement of Caspase-3 activation Caspase-3 activity was determined using a colorimetric assay kit (Sigma) in accordance with the manufacturer's instructions. In short, cytosol extracts were prepared by repeated cycles of freezing and thawing of the cells in 100 μl of lysis buffer (50 mM HEPES, pH 7.4, 5 mM CHAPS, 5 mM DTT) per 10 7 cells. After centrifugation of the cell lysate at 16,000 ×g for 10 min at 4 °C, 50 μl of the supernatant was assayed using a Shimadzu UV-3000 spectrophotometer. Caspase 3‐ mediated hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide results in the release of the p-nitroaniline (pNA) moiety

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which was detected at 405 nm. Caspase 3 activity was calculated as μmol pNA released per minute per mg protein and is expressed as change in absorbance/min/mg protein. 2.8. Staining of cells with annexin V, PI, and Hoechst 33258 Apoptotic cell death was evaluated using an annexin-V-Fluo kit which assesses the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane lipid bilayer. PI (25 μM; 20 min) was used to visualize dead cells and Hoechst 33258 (1:1000) was used to visualize chromatin condensation and apoptotic bodies if present. Staining procedures were as previously described (Muyderman et al., 2004). Annexin V-positive/PI-negative cells were considered to be apoptotic, whereas cells that were only PI-positive were seen as necrotic. The small proportion of cells that were both annexin-V positive and PI-positive were considered apoptotic only if there was also evidence of apoptotic bodies or condensed chromatin. Images were taken from nine predefined areas per coverslip. Three of these were randomly chosen and analyzed. Total and apoptotic nuclei were counted, and the data are presented as percentage annexin V positive cells of the total number of cells. 2.9. Protein determination Cell suspensions and subcellular fractions were solubilized in 2 M NaOH, and the protein content was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. 2.10. Statistical analyses Results are presented as mean ± SD. Individual values were determined as the average of at least five separate measurements from three (or more) identically treated individual cultures. Statistical analyses were performed on raw data by one-way ANOVA followed by Student–Newman–Keuls test. 3. Results Complete sequencing of mtDNA extracted from the transmitochondrial cell line from our patient is compared with the revised Cambridge sequence (Andrews et al., 1999) in Table 1 and was identical with that of the donor. Other than the variant at nt 1095, our individual has none of the sequence motifs characterizing the haplogroup M11

Table 1 Variants from the revised Cambridge sequence (rCSR) found in the mtDNA sequence of a cybrid bearing the T1095C mutation. The replacement as nt 2706 and 7028 place this mtDNA within the haplogroups HV and H. Wt = wild type, AA = amino acid, TS = transition, Ins = insertion, Del = deletion, syn = synonymous, nsyn = non-synonymous, con = conserved, v = variable. Assignable haplogroup

Nuc no.

Wt

Mutant

Type

Gene

Too many rCRS rCRS rCRS rCRS (H2) Debatable rCRS (H2) HV vs. H Too many rCRS (H2)

195 263 303 311 750 1095 1438 2706 3106 4769 5452 7028 8410 8860 15326 15440 15519 16311 16348

T A – – A T A A C A C C C A A T T T C

C G C C G C G G – G T T T G G C C C T

TS TS Ins Ins TS TS TS TS Del TS TS TS TS TS TS TS TS TS TS

MajorNCR1 MajorNCR1 MajorNCR1 MajorNCR1 12S rRNA 12S rRNA 12S rRNA 16S rRNA 16S rRNA ND2 ND2 CO1 ATP8 ATP6 Cytb Cytb Cytb MajorNCR2 MajorNCR2

HV vs. H Not reported rCRS (H2) rCRS (H2) D4b1 V1 (coble) Not reported

AA no.

Wild AA

Mutant AA

Sens

Conservation

100 328 375 15 112 194 232 258

Met Thr Ala Pro Thr Thr Leu Leu

Met Met Ala Pro Ala Ala Leu Pro

syn nsyn syn syn nsyn nsyn syn nsys

var var con var var var var var

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(6531, 7642, 8108, 9950, 11969, 13074, 146, 215, 318, 326) (Tanaka et al., 2004). She does have the replacements at nts 2706 and 7028, thus placing her with the haplogroups HV and V according to the classical mtDNA phylogenetic map (http://www.mitomap.org/mitomap-phylogeny.pdf). Haplogroup HV is characterized by replacements at 14766, and preHV by replacements at 11719. Our individual has neither of these, so she belongs to a sub-haplogroup within the haplogroup HV. Our subject does not have the 12,705 replacement, so that she belongs to neither macrohaplogroup N nor M. We found 2 previously unreported synonymous or non-coding region variants, (C16348T and C8410T). There were also 2 rare nonsynonymous mutations. The first, previously unreported is C5452T in ND2 results in a substitution of Thr328 with Met. The mutation is in helix j of ND2 (Fig. 1A). This is not conserved among 61 mammalian species. The second, T15519C in Cytb results in a substitution of Leu258 with Pro. This mutation has been previously reported (Friedlaender et al., 2005; Howell et al., 2004; Sun et al., 2006) in the mtDNA phylogenetic tree and substitution with Pro is common among mammalian mtDNAs. Thus the likelihood that any of these mutations is pathogenic is very low. We also sequenced the non-coding region of mtDNA from nt15780-16569 in the 2 control cell lines to ensure that the sequences were closely related and that differences in respiratory chain activity could not be explained by large differences in haplogroup. In the two controls we found the same variants 16134C>T, (characterizing haplogroup U4a1), 16356T>C charactering (haplogroup U4) and 16519T>C, a very common sporadic variation. We conclude that all 3 lines were derived from individuals from closely related European haplogroups.

3.1. Assessment of mitochondrial function The specific activity of citrate synthase was assessed to test for possible differences between the cybrids in the number of mitochondria or in the synthesis or degradation of a key mitochondrial protein.

Table 2 The glutathione content of the T1095C cybrid and control cybrids expressed as nmol glutathione per mg total cell protein. Mitochondrial values are expressed relative to protein content in this fraction. There were no significant differences between the cells in total and cytosolic glutathione. However, mitochondrial glutathione was significantly decreased in the T1095C-carrying cybrids compared to control cells. Values are shown as mean ± SD. ⁎b0.01, ANOVA. n= 10.

Control 1 Control 2 T1095C

Total cellular content

Cytosolic content

Mitochondrial content

18.7 ± 1.8 18.6 ± 2.2 19.5 ± 2.1

16.8 ± 3.2 16.4 ± 2.8 18 ± 1.5

3.56 ± 0.2 3.69 ± 0.2 2.84 ± 0.3⁎

There was no significant difference in citrate synthase activity between the T1095C cybrids and either of the controls (23.1 ± 6 versus 24.8 ± 7 and 22.5 ± 8 nmol/min/mg protein; mean ± SD, n = 10). The results reported in subsequent studies, have been expressed relative to total cellular protein only. In each experiment, the values were also calculated based on citrate synthase activity. Not surprisingly, given the similar specific activities of citrate synthase in the three cybrids, these analyses did not lead to different conclusions for any of the investigations. Thus, the results expressed relative to citrate synthase activity have not been presented. Total cellular glutathione, cytosolic and mitochondrial glutathione content were measured to provide an assessment of the status of a key defense against oxidative stress (Table 2). Total cell or cytosolic glutathione were not significantly different between the cybrid lines. Mitochondrial glutathione made up 15% of the total pool in the control cybrids consistent with findings in other cells (Fernandez-Checa et al., 1998; Griffith and Meister, 1985; Sun et al., 2006). This glutathione pool was significantly reduced to 79% of the control cybrids in cells carrying the T1095C mutation (Table 2). Assessment of respiratory chain function in cells carrying the T1095C mutation showed a 30% reduction in complex II/III activity

Fig. 1. A. Secondary structure diagram of E. coli 16S RNA. The position of the mutations in helix 24 referred to in the text are highlighted in red and the position of the T1095C mutation in blue. B. The tertiary structure of T. thermophilus 16S RNA bound to tRNA Phe. Helix 24 is indicated in orange and the position of the mutation in blue. mRNA is shown in yellow.

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Fig. 2. Activities of (A) complex I and complexes II. III and (B) complex IV in the cybrid line carrying the T1095 mutation and in the two control cybrids. A statistically significant lower activity of complex II/II was seen in the T1095 cells compared to each of the controls. (Pb 0.001; one-way ANOVA with Student–Newman–Keuls test, n=5). Values are shown as mean±SD.

compared to controls (p b 0.01; Fig. 2). No significant differences were observed in complex I or complex IV activities.

3.2. Gentamicin induced cytotoxicity in T1095C cybrids Necrotic and apoptotic cell death were assessed by measuring caspase-3 activity, annexin V immunoreactivity, nuclear morphology, nuclear PI incorporation and release of LDH to the culture medium after exposure to gentamicin. No difference in necrotic cell death, as assessed with PI and LDH, was detected between the T1095C cells and cells not carrying the mutation when exposed to gentamicin concentrations of 1–10 mg/ml (n=5; not shown). For subsequent studies, a concentration of 5 mg/ml for 18 h was used as this concentration did not produce any detectable cytotoxicity in control cell lines. This dose has also been shown to be insufficient to ordinarily produce vestibular or cochlear damage in in vivo studies. At this concentration, caspase-3 activation was 216±16% of that of the controls (pb 0.01; n=5; Fig. 3) and the percentage of annexin V positive/PI negative cells was increased ten-fold in the T1095C cell line (pb 0.001; n=5; Fig. 4). Together these results clearly demonstrate a substantially increased susceptibility towards gentamicin in cells expressing the T1095C mutation that resulted in a large increase in apoptotic, but not necrotic, cell death.

Fig. 3. Exposure to 5 mg/ml gentamicin for 18 h produced a significantly lager increase in caspase-3 activity in the cybrid carrying the T1095C mutation than in the control cybrids as shown by the increase in AC-DeVD-pNA cleavage (p b 0.001, ANOVA with Student–Newman–Keuls test, n = 5). Under basal conditions the values were below 0.050 ΔABS 405 nm/μg protein for each of the cybrids and controls and did not differ significantly between them (n = 5).

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Fig. 4. There was a ten-fold increase in the number of annexin V+/PI− cells in response to 5 mg/ml gentamicin for 18 h as assessed 24 h after the initiation of the exposure in cells carrying the T1095C mutation compared to controls. The number of nuclei exhibiting features of apoptosis was increased eight-fold. In untreated cells, the proportion of annexin V+/PI−, or cells with nuclear morphology suggesting apoptosis, were below 4% in all cell populations (⁎⁎⁎pb 0.001, ANOVA with Student–Newman–Keuls test, n=5). Values are shown as mean±SD.

4. Discussion The present study describes that the heteroplasmic T1095C mutation in the 12SrRNA gene of the mitochondrial genome results in selective changes in mitochondrial anti-oxidant content and substantially increases caspase-3-mediated apoptosis in response to the aminoglycocide antibiotic gentamicin. Complete sequencing of the mitochondrial genome of the proband, and of the cybrids carrying the T1095A mutation, did not reveal any other mutation likely to explain this pathogenicity. Mutations in the mitochondrial 12SrRNA are generally associated with aminoglycoside-mediated neuro- and ototoxicity and the current findings strongly support the likelihood that this mutation is responsible for features of aminoglycoside-induced deafness. In our previous paper, we predicted that the T1095C would change the secondary structure of the highly evolutionarily conserved helix 25, thought to be close to the P site and involved in the initiation of mitochondrial protein synthesis. Experimental support for this was available from the mutational analysis of the homologous region 16SrRNA in Escherichia coli, showing reduced association between the 30S and 50S subunits, decreased IF3 binding, and reduced protein synthesis (Tapprich and Hill, 1986; Tapprich et al., 1989). Further insights from the crystal structure of the ribosome of Thermus thermophilus (Wimberly et al., 2000; Yusupov et al., 2001) (Fig. 1B) have supported the prediction that the T1095C mutation could interfere with mitochondrial protein synthesis. This is reinforced by recent analysis of the random mutants in the 16S ribosome of E. coli (Yassin et al., 2005), in which 3 dominantly inherited moderately deleterious mutations were found in helix 24 of E. coli, close to the position homologous to the T1095C mutation (Fig. 1A). In experiments replacing the bacterial 16S helix 44 (location of the A1555G and C1494T deafness mutations) with a mutated helix, the susceptibility of cells carrying the mitochondrial hybrid ribosomes to several antibiotics increased by 4- to 16-fold, and aminoglycoside-induced misreading was increased (Hobbie et al., 2008a). The central domain of the 30S RNA contains nine helical elements folded in the shape of a W (Wimberly et al., 2000). The long stack of helices 21–22–23 form the outer arms of the W. Helices 22, 23 and 24 form the bulk of the platform of the central domain. The tip of the platform consists of helix 23 B and 24a which contain the tightly packed functionally important hairpin loops (690 and 790 loops). There is a sharp bend between helix 23 and 23 B and between 24 and 24A. Our mutation is located in the middle of helix 24 (Figure B). Helix 24 is involved in several 30S–50S important intersubunit bridges (B2b, B2c and B7b) involving RNA–RNA or RNA–protein interactions (Yusupov et al., 2001). Helix 24 and the important 790 loop appear to be involved in class III conformational changes resulting in a tightening of the base of decoding site in the channel where the mRNA is bound and where

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the interaction between the A and P site codon–anticodon interactions take place possibly explaining the miscoding effects of aminoglycoside antibiotics (Yusupov et al., 2001). As complex III incorporates subunits encoded by the mtDNA, the decrease in the activity of complex II/III in the T1095C cybrids could be a direct result of altered protein synthesis in the mitochondria due to the presence of this mutation. Nevertheless, we cannot exclude the possibility that the observed rare non-synonymous T15519C change in cytochrome B could be responsible for this effect. However, mutations affecting the cytochrome B gene often lead to more pronounced reductions in oxidative phosphorylation resulting in a variety of clinical presentations, none of which were present in the studied family. Moreover, the likelihood that the T1095C mutation is responsible for the change in complex III activity is further supported by the moderate decline in ETC activity we could demonstrate in lymphoblasts from the patient in our previous study (Thyagarajan et al., 2000). Even small alterations in respiratory chain complexes have the potential to increase susceptibility to exogenous stressors. Mitochondria are a major source of superoxide and related reactive oxygen species both in normal cells and in diseased states and multiple components of mitochondria are capable of generating reactive oxygen species (Andreyev et al., 2005; Brand, 2010). Although the relative contribution of individual sites in intact cells is not well defined, superoxide production at complexes I and III are thought to play an important role (Andreyev et al., 2005; Brand, 2010). For example, the Qo semiquinone site of complex III normally generates superoxide that is released into both the matrix and cytosol (Brand, 2010; Muller et al., 2004). Thus, alterations in the properties of this respiratory chain complex arising from impaired synthesis of mitochondrial subunits could lead to increase superoxide production resulting in oxidative stress and contributing to the decreases in the mitochondrial glutathione pool seen in the T1095C cybrids. In support of this suggestion, similar moderate and selective decreases in complex III activity in mitochondria from failing hearts following myocardial infarction were found to be associated with a three-fold increase in the generation of reactive oxygen species derived from complex III (Heather et al., 2010). The underlying mechanisms for aminoglycoside-mediated cell death are not fully understood but an increasing amount of data suggests that generation of reactive oxygen species is directly involved in this type of cytotoxicity (Walker et al., 1999). Interestingly, it seems that a major site of this free radical generation is the mitochondria in which aminoglycosides have been shown to greatly increase hydrogen peroxide production (Walker and Shah, 1987). This is consistent with a primarily mitochondrial localization of a defect enhancing aminoglycoside toxicity and is supported by studies in which treatments aimed at maintaining mitochondrial antioxidant status, or more generally at countering oxidative stress, have been shown to protect against gentamicin-mediated nephrotoxicity (Chiu et al., 2008). More directly relevant to the present study, treatment with aspirin has been reported to lower the incidence of gentamicin-induced hearing loss, an effect attributed to the anti-oxidant properties of the aspirin (Chen et al., 2007; Sha et al., 2006). Thus, the selective losses of mitochondrial glutathione observed in our study provide a possible explanation for their increased susceptibility to gentamicin treatment. Glutathione is a central component in the antioxidant defenses of cells, acting both to directly detoxify reactive oxygen species and as a substrate for several peroxidises (Dringen, 2000). This antioxidant tri-peptide is synthesized in the cytosol and transported into the mitochondria. Depletion of this mitochondrial antioxidant pool is associated with dysfunction and loss of viability in cells challenged with a range of oxidative stresses (Muyderman et al., 2004, 2007; Wullner et al., 1999). Selective losses of the mitochondrial glutathione pool, similar to that observed in the mutant cybrids, have been reported under pathological conditions in the liver (Colell et al., 1998) and in the brain (Anderson and Sims, 2002) and proposed to contribute to tissue damage in these conditions. One major role of mitochondrial glutathione is in the

reactions catalyzed by glutathione peroxidase that are involved in removal of hydrogen peroxide. Thus, the partial loss of mitochondrial glutathione could contribute to an increased cytotoxicity when the T1095C cybrids are responding to increases in mitochondrial hydrogen peroxide production induced by exposure to gentamicin. It has been suggested that the T1095C variant may be a population polymorphism, common enough to be found in controls and not necessarily the cause of aminoglycoside-induced deafness (Yao et al., 2006). This is because the T1095C variant, together with 10 other variants, characterizes the haplogroup M11, a basal branch of the East Asian mtDNA phylogeny (Kong et al., 2003; Tanaka et al., 2004). However, a mitochondrial genetic study of deafness in the Chinese population failed to find the T1095C mutation in 364 Chinese controls (H. Zhao et al., 2004; L. Zhao et al., 2004). Moreover, sequencing analysis in the present study clearly excludes the possibility that the T1095C is specific for the M11 haplotype. The precise haplogroup of our proband is not certain, but we can place it with haplogroup HV and V. Thus the variant has arisen independently at least once, and, given the European ancestry of the patients described by Tessa et al. (2001), probably more than once. In both cases this mutation was associated with deafness and the variant was heteroplasmic. In this regard, T1095C may be a deafness mutation analogous to the A1555G mutation, which has arisen independently in different ancestries, but has low penetrance in Chinese populations. In addition to aminoglycoside-induced deafness we described an association with maternally-inherited levodopa-responsive parkinsonism in our original report on the T1095C mutation. However to know if the T1095C variant is indeed a risk factor for parkinsonism, an epidemiological study in the haplogroup M11, characterized by the T1095C variant, would be invaluable and detailed neurological examinations in cohorts of patients carrying the T1095C mutation would be required. This is outside the scope of the present study. Thus, we are still uncertain if parkinsonism is a direct manifestation of the T1095C mutation. In conclusion, we propose that, like the A1555G mutation which may become pathogenic after an individual is exposed to aminoglycoside antibiotics, the T1095C mutation is another gene variant conferring on the individual a susceptibility to aminoglycoside-induced deafness. Acknowledgments This research was supported by grants from the National Health and Medical Research Council (Australia), the Flinders Medical Centre Foundation and Flinders University. The experiments were conducted under Flinders Clinical Research Committee Approval 193/056. References Akerboom, T., Sies, H., 1981. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol. 373–382. Anderson, M.F., Sims, N.R., 2002. The effects of focal ischemia and reperfusion on the glutathione content of mitochondria from rat brain subregions. J. Neurochem. 81, 541–549. Anderson, S., Bankier, A.T., Barrell, B.G., de Bruijn, M.H., Coulson, A.R., Drouin, J., Eperon, I.C., Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J., Staden, R., Young, I.G., 1981. Sequence and organization of the human mitochondrial genome. Nature 290, 457–465. Andrews, R.M., Kubacka, I., Chinnery, P.F., Lightowlers, R.N., Turnbull, D.M., Howell, N., 1999. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat. Genet. 23, 147. Andreyev, A.Y., Kushnareva, Y.E., Starkov, A.A., 2005. Mitochondrial metabolism of reactive oxygen species. Biochemistry (Mosc) 70, 200–214. Bacino, C., Prezant, T.R., Bu, X., Fournier, P., Fischel-Ghodsian, N., 1995. Susceptibility mutations in the mitochondrial small ribosomal RNA gene in aminoglycoside induced deafness. Pharmacogenetics 5, 165–172. Bitner-Glindzicz, M., Pembrey, M., Duncan, A., Heron, J., Ring, S.M., Hall, A., Rahman, S., 2009. Prevalence of mitochondrial 1555A–>G mutation in European children. N. Engl. J. Med. 360, 640–642. Brand, M.D., 2010. The sites and topology of mitochondrial superoxide production. Exp. Gerontol. 45, 466–472. Chen, Y., Huang, W.G., Zha, D.J., Qiu, J.H., Wang, J.L., Sha, S.H., Schacht, J., 2007. Aspirin attenuates gentamicin ototoxicity: from the laboratory to the clinic. Hear. Res. 226, 178–182.

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