Brain, Vol. 123, No. 1, 93-104,
January 2000
© 2000 Oxford University Press
Apoptosis in mitochondrial encephalomyopathies with mitochondrial DNA mutations: a potential pathogenic mechanism
Institute of Neurology, Catholic University, Rome, Italy
Correspondence to:
Serenella Servidei, Istituto di Neurologia, Università Cattolica del S. Cuore, Largo A. Gemelli 8, 00168 Rome, Italy
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Abstract |
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Mitochondrial encephalomyopathies caused by mitochondrial DNA (mtDNA) defects are a genetically and phenotypically heterogeneous group of disorders. The site, percentage and distribution of mutations do not explain the overall clinical heterogeneity that is found. Apoptosis (programmed cell death) is an evolutionarily conserved mechanism that is essential for tissue development and homeostasis. Dysregulation of apoptosis has been implicated in the pathogenesis of various human diseases, such as cancer and autoimmune and neurodegenerative disorders. Recent in vitro evidence has indicated the central role of mitochondria in the apoptotic process. We investigated the occurrence of apoptosis in muscle biopsies of 36 patients carrying different mtDNA mutations and four patients with inclusion body myositis and mitochondrial abnormalities. Apoptotic features, mainly localized in cytochrome c oxidase-negative fibres, were observed in muscle fibres of patients carrying a high percentage of single mtDNA deletions (>40%) and of tRNA point mutations (>70%). By contrast, no apoptotic changes were observed in inclusion body myositis and in patients carrying mutations of mtDNA structural genes. Our study suggests that apoptosis is not simply a means whereby cells with dysfunctional mitochondria are eliminated, but that it seems to play a role in the pathogenesis of mitochondrial disorders associated with mtDNA defects affecting mitochondrial protein synthesis. The imbalance and relative abundances of nuclear-encoded and mtDNA-encoded subunits may favour cytochrome c inactivation and release. Cytochrome c, together with respiratory chain dysfunction, could activate apoptotic pathways that, in turn, inhibit the rate of mitochondrial translation and the importation of nuclear-encoded mitochondrial protein precursors. This vicious circle may amplify the biochemical defects and tissue damage and contribute to the modulation of clinical features.
apoptosis; mitochondrial encephalomyopathies; mtDNA mutations
COX = cytochrome c oxidase; LHON = Leber hereditary optic neuropathy; MELAS = mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes; MERRF = myoclonic epilepsy and ragged red fibres; mtDNA = mitochondrial DNA; MNGIE = myogastrointestinal encephalopathy; NARP = neuropathy ataxia retinitis pigmentosa; PBS = phosphate-buffered saline; PEO = progressive external ophthalmoplegia; RRF = ragged red fibres; TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Mitochondrial encephalomyopathies represent an expanding group of clinically heterogeneous disorders associated with mitochondrial DNA (mtDNA) mutations or nuclear gene defects (Wallace, 1992
; Morgan-Hughes, 1994; Di Mauro and Bonilla, 1997; Di Mauro and Schon, 1998). The complexity of mitochondrial metabolism, its central role in energy production, its dual genetic control (mitochondrial and nuclear DNA) and some unique features of mitochondrial genetics may explain the exceptional clinical heterogeneity of mitochondrial disorders. In sharp contrast to the rapid progress of knowledge in molecular genetics, our understanding of the pathogenic mechanisms that lead to the wide biochemical and clinical variability is still far from clear. Heteroplasmy, the threshold effect, and the site, percentage and inter- and intra-tissue distribution of the mtDNA mutations contribute to the phenotype but do not explain the overall clinical heterogeneity that is also present within the same genetic defect. Moreover, while a nuclear defect may lead to specific enzymatic impairment, the correlation between mtDNA mutations, respiratory chain dysfunction and clinical outcome is less obvious. The altered synthesis of mitochondrial proteins—the consequence of mtDNA defects—may have various causes, such as abnormal mRNA processing, the accumulation of anomalous RNA transcripts, the impairment of transcript binding to ribosomes, altered aminoacylation and incorrect amino acid-tRNA conjugation (Di Mauro and Bonilla, 1997; Schon, 1997; Di Mauro and Schon, 1998). Whatever the mechanism, the final common step in mitochondrial encephalomyopathies is a defect of energy production resulting from respiratory chain impairment. Cellular necrosis is seldom observed in these disorders. Other mechanisms, such as incorrect assembly of the defective enzyme complex or complexes with other mitochondrial proteins, the production of free radicals and ways of cell death other than necrosis such as apoptosis, may be postulated to explain the phenotypic variability and the severity of mitochondrial diseases.
Apoptosis (programmed cell death) is an evolutionarily conserved mechanism that is essential for tissue development and homeostasis. It requires the activation of specific genes that lead to a series of distinctive morphological and biochemical features. These changes include the activation of cellular proteases (caspases), mitochondrial depolarization, chromatin condensation, oligonucleosomal DNA degradation and cell volume loss or cell fragmentation without elicitation of an inflammatory response (White, 1996; Salvasen and Dixit, 1997; Hetts, 1998).
Apoptotic features in human pathology are documented in neoplasms, autoimmune diseases, stroke and some neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and familial amyotrophic lateral sclerosis (Hetts, 1998).
In human muscle pathology, apoptotic nuclei and apoptosis-related proteins have been demonstrated in atrophic fibres in neurogenic muscle atrophy (Fidziamska et al., 1990; Mirabella et al., 1996; Tews and Goebel, 1997).
Mitochondria have recently been found to have a leading role in the triggering and mediation of apoptosis. Experimental studies in vitro have shown that the disruption of the mitochondrial transmembrane potential (
) and the release of some mitochondrial proteins (cytochrome c and apoptosis inducing factor) into the cytoplasm are able to initiate and activate different apoptotic pathways (Liu et al., 1996; Kroemer et al., 1997; Kluck et al., 1997; Zhivotosky et al., 1998).
In order to discover whether apoptosis plays a part in mediating tissue damage in human mitochondrial diseases, we investigated the presence of DNA fragmentation and the expression of apoptosis-associated proteins (Fas, p75 and caspase-3) in muscle biopsies of patients with mtDNA point mutations and deletions.
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Patients
We studied muscle biopsies obtained, with informed consent, from 36 patients of different ages (2 months to 69 years) affected by various mitochondrial disorders with heterogeneous mtDNA defects, four patients with inclusion body myositis and mitochondrial abnormalities and 10 patients who had proved to be free of muscle disease. Diagnosis of all mitochondrial patients was based on clinical, biochemical and molecular genetic studies (their clinical, morphological, biochemical and genetic features are summarized in Table 1
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Five patients had MERRF (myoclonic epilepsy and ragged red fibres) with an mtDNA mutation at nucleotide 8344 in the tRNALys gene (patients 1–5); six patients had the A3243G transition in the tRNALeu (UUR) gene, three of them with typical mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) (patients 6, 7 and 11), one with progressive external ophthalmoplegia (PEO) (patient 8), one with cardiomyopathy (patient 9) and one with PEO and cardiomyopathy (patient 10); 16 patients had a single large mtDNA deletion associated with multisystem Kearns–Sayre syndrome (patients 12–14), Pearson's syndrome (patient 15), encephalopathy (patient 16) or PEO (patients 17–27); four patients (patients 28–31) had PEO with multiple mtDNA deletions, two with autosomal dominant transmission and two sporadic cases; one patient with myoneurogastrointestinal encephalopathy (MNGIE) syndrome carried a 1 bp deletion (patient 32); two patients (patients 33 and 34) had PEO associated with a T4285C (tRNAIle) and G5521A (tRNATrp) point mutation, respectively; patient 35 had neuropathy ataxia retinitis pigmentosa (NARP) syndrome associated with a T8993C point mutation in the ATPase 6 gene; patient 36 had Leber hereditary optic neuropathy (LHON) associated with a G11778A mutation in the ND4 gene.
Mitochondrial myopathy, ranging from absent (NARP and LHON) to marked (Table 1
), was defined morphologically by the presence of a variable number of succinate dehydrogenase strongly reactive ragged red fibres (RRF) and of cytochrome c oxidase (COX)-negative non-RRF.
The four inclusion body myositis patients with typical nuclear and cytoplasmic filamentous inclusions in vacuolated fibres (Griggs et al., 1995) were chosen because of the presence of numerous RRF and COX-negative fibres. In these patients, electron microscopy showed proliferation in the size and number of mitochondria with abnormal shape and paracrystalline inclusions. A very low number of mtDNA deletions was demonstrated in inclusion body myositis patients by the PCR (polymerase chain reaction), but not by Southern blotting.
TUNEL and immunohistochemistry
The terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) technique was used for the detection of nuclear DNA fragmentation in situ. Frozen muscle sections from patients and controls were incubated under the same coverslip with TUNEL reaction mixture, and incorporated fluorescein-dUTP was detected by using alkaline phosphatase-conjugated anti-fluorescein antibodies according to the manufacturer's instructions (In Situ Cell Death Detection Kit, Boehringer, Mannheim, Germany). Negative experimental controls were incubated with label solution without terminal transferase instead of TUNEL reaction mixture, while a positive control was set up by preincubating muscle sections with DNase I for 10 min before the TUNEL procedure. TUNEL-positive nuclei were counted in at least 100 muscle fibres per section and biopsies were classified in six groups depending upon the percentage of muscle fibres harbouring apoptotic nuclei (0–3%, 3–5%, 5–25%, 25–50%, 50–75%, 75–100%). Moreover, the number of TUNEL-positive nuclei was correlated with the age of the patient at the time of biopsy, the presence of myopathy, the severity of the phenotype, the biochemical defect and the type and percentage of mtDNA mutation (Table 1). In order to evaluate the relative number of TUNEL-positive nuclei within individual fibres and their correlation with COX-negative fibres, TUNEL was also performed on muscle sections after histochemistry for COX and was followed by nuclear staining with Hoechst 33258. Unfixed 10 µm muscle sections adjacent to those analysed by TUNEL were processed for immunocytochemistry as follows. Sections were dried at room temperature, fixed in cold acetone and pretreated with 0.3% H2O2 in PBS (phosphate-buffered saline) to quench endogenous peroxidase activity, rinsed in PBS and incubated with 10% normal serum (goat or rabbit depending on the secondary antibodies used) for 60 min to mask non-specific adsorption sites. Sections were then incubated for 1 h at room temperature with one of the following antibodies: mouse monoclonal antibody against human Fas/APO-1 (Calbiochem, Cambridge, Mass., USA, diluted 1 : 50); rabbit polyclonal antibody against human Fas (C-20, Santa Cruz, Calif., USA, diluted 1 : 200) and p75-NTR (Promega, Madison, Wis., USA, diluted 1 : 100); and goat polyclonal anti-human caspase-3 antibody (Santa Cruz, diluted 1 : 200). In control experiments the primary antibodies were omitted or replaced by preimmune sera. After several rinses in PBS, the sections were incubated with the appropriate biotinylated secondary antibodies (goat anti-mouse, goat anti-rabbit or rabbit anti-goat IgG), washed in PBS and then incubated with the avidin–biotin peroxidase complex according to the manufacturer's instructions (Vectastain ABC; Vector Laboratories, Burlingame, Calif., USA). Peroxidase staining was obtained by incubating the sections in 0.075% DAB (3,3-diaminobenzidine) and 0.002% H2O2 in 50 mM Tris buffer (pH 7.6) for 10 min.
Electron microscopy
For electron microscopy, muscle samples were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), postfixed in 1% osmium tetraoxide, dehydrated in a graded ethanol series and embedded in Epon 812. Ultrathin sections were cut and stained with uranyl acetate and lead citrate, and examined with a Philips 208S electron microscope.
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Results |
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TUNEL labelling showed that myonuclei containing double-stranded DNA fragments in situ, indicative of apoptosis, were present in variable amount in the muscle biopsies of patients with mtDNA point mutations and deletions (Table 1
and Fig. 1). Apoptotic nuclei were commonly observed in fibres with a non-pathological appearance; the few TUNEL-positive nuclei located in the rare muscle fibres exhibiting histological changes characteristic of necrosis were not included in the counts. No TUNEL-positive myonuclei were observed in negative experimental controls, and only rare, isolated positive nuclei (<2%) were present in normal muscle.
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The presence of TUNEL-positive myonuclei significantly correlated with increased p75, Fas and caspase-3 immunoreactivity in all biopsies studied (Table 1
). Immunoreactivities for p75 (Fig. 2A) and Fas (Fig. 3) were localized mainly in the plasmalemma, while intense caspase-3 immunostaining was observed in the cytoplasm (Fig. 2C–F). In addition to the surface membrane staining, diffuse or granular cytoplasmic immunopositivity for both p75 and Fas was observed within myofibres (Figs 2A and 3). In all biopsies, including normal muscles, p75 and Fas immunoreactivities were detected in intramuscular nerve twigs (Fig. 2B) and blood vessels, respectively. In control experiments, when primary antibodies were omitted or replaced by non-immune sera, the immunoreaction did not take place. In the 10 control biopsies we did not find any significant apoptotic features. In four inclusion body myositis patients with marked mitochondrial abnormalities, there were no TUNEL-positive myonuclei, while Fas was expressed on the surface membrane of a variable number (5–25%) of muscle fibres and inflammatory cells (not shown).
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Patients carrying a single mtDNA deletion with <40% of mutated mtDNA molecules had only sporadic muscle fibres with apoptotic traits, and all these patients showed a mild phenotype (patients 17–23). When the percentage of mutated mtDNA exceeded 40%, apoptotic features were present in a significant number of fibres. TUNEL-positive nuclei, Fas, p75 and caspase-3 were expressed in 25–50% of fibres which had 40–62% of mutated mtDNA molecules (patients 12–14,16, 24–26); in one patient with 65% mutated genomes, apoptotic nuclei were expressed in 50–75% of fibres (patient 27) (Figs 1B, 2A and D and 3C
); in another patient with 78% mutated genomes and a very severe phenotype, virtually all fibres appeared apoptotic (patient 15) (Figs 1A, 2C and 3A). One patient with MNGIE carrying a 1 bp deletion also showed 50–75% TUNEL-positive fibres and 25–50% of fibres positive for Fas, p75 and caspase-3 (patient 32). Four patients with PEO syndrome and multiple mtDNA deletions, with mild neurological impairment and myopathy, showed TUNEL-positive nuclei and Fas, p75 and caspase-3 expression in 3–5% of fibres (patients 28–31).
tRNA point mutations causing mainly MERRF, MELAS and PEO syndromes, with a moderate to severe neurological phenotype, were associated with a major degree of apoptosis in the biopsies when the percentage of mutated genomes was >73% (patients 1–7 and 9–11) (Figs 1C and 2E and F). The most severely affected patient with MERRF, who carried 73% of mutated mtDNAs (patient 1) (Fig. 3D) had >50% of muscle fibres displaying TUNEL, FAS, p75 and caspase-3 positivity. In one patient with mild PEO and 54% mutated mtDNA, there were no clear signs of apoptosis; apoptotic nuclei were rare, immunostaining for caspase-3 was negative and <25% of fibres expressed Fas and p75 immunoreactivities (patient 8).
T8993C and G11778A point mutations, involving the structural genes for ATPase 6 and ND4, respectively, causing NARP and LHON syndromes, were not associated with significant apoptotic signs, even if expressed in high amounts (97–100%); only rare apoptotic nuclei were present, and Fas and p75 positivity was seen in a minority of fibres (patient 34) (Fig. 1D).
Major apoptotic features correlated with severe dysfunction of the respiratory chain, mainly affecting complexes I and IV, but they were present even in patients with a mild biochemical impairment if the muscle fibres carried a high percentage of mutated mtDNA (Table 1).
By performing TUNEL, COX histochemistry and nuclear staining with Hoechst 33258 (Fig. 4) on the same sections, either single or multiple TUNEL-positive nuclei were observed within individual muscle fibres on a given section, but all nuclei visible with Hoechst 33258 were TUNEL-labelled only in rare COX-negative areas of longitudinal fibres (Fig. 4A and B). The majority of TUNEL-positive nuclei were present within COX-negative fibres, especially in patients with a high percentage of single mtDNA deletions or tRNA point mutations. However, TUNEL-positive nuclei were also seen in COX-positive fibres (Fig. 4C and D) and RRF did not express apoptotic signs at a significantly higher rate than non-RRF. Moreover, in spite of numerous COX-negative fibres, the rare apoptotic nuclei observed in patients with multiple mtDNA deletions were not preferentially located in the COX-negative fibres (Fig. 4E and F).
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Sex and age at the time of muscle biopsy did not appear to influence the apoptotic phenotype.
In TUNEL-positive muscle biopsies, electron microscopy showed, in addition to the abnormalities of number and structure of mitochondria, myofibres containing nuclei with highly condensed chromatin (Fig. 5). There were no signs of necrosis in the muscle fibres with abnormal nuclei. Interestingly, there was no constant correlation between mitochondrial abnormalities and myonuclei with morphological changes indicative of apoptosis. In fact, myonuclei with irregular shape and condensed chromatin were present either within muscle fibres with an otherwise normal morphology or in association with an increased number of dense mitochondria.
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Discussion |
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In muscle biopsies from a large group of patients with mitochondrial diseases and heterogeneous mtDNA defects we demonstrated the significant presence of apoptotic features associated with specific phenotypes and a high percentage of mutated mitochondrial genomes.
Mitochondria are the main cellular source of ATP, the primary generators of reactive oxygen species and an important storage site for calcium homeostasis. In recent years several lines of evidence have indicated a critical role for mitochondria and mitochondrial proteins in the control of the apoptotic process: (i) the disruption precedes by far the typical nuclear signs of apoptosis—the condensation and margination of chromatin and the cleavage and fragmentation of genomic DNA (Zamzami et al., 1956; Marchetti et al., 1996; Kroemer et al., 1997); (ii) mitochondria release proteins, such as cytochrome c and AIF, that activate proteases responsible for DNA fragmentation (Liu et al., 1996; Kluck et al., 1997; Higuchi et al., 1997; Yang et al., 1997); (iii) the uncoupling of oxidative phosphorylation and the respiratory chain inhibitors induce apoptosis in vitro (Wolvetang et al., 1994; Marton et al., 1997); (iv) excessive free radical production (reactive oxygen species can alter the external mitochondrial membrane and facilitate the release of cytochrome c and AIF) increases apoptosis (Richter et al., 1995; Slater et al., 1995; Yoneda et al., 1995); (v) the inner and external membranes of mitochondria host a set of proteins belonging to the Bcl-2 superfamily that are important regulators of the apoptotic machinery (Yang et al., 1997; Kluck et al., 1997; Narita et al., 1998).
In mitochondrial myopathies, apoptosis may represent a selective mechanism for the elimination of cells with dysfunctional mitochondria and excessive free radical production or, on the contrary, may produce or amplify cell damage.
The impact of nuclear apoptosis on the functionality and viability of single cells is more difficult to evaluate in multinucleated and partly regenerating tissues such as muscle than in mononucleated, highly regenerating tissues. In fact, DNA fragmentation within isolated nuclei in syncytial muscle cells does not imply apoptosis of the entire fibre cell, and the time course of the apoptotic changes may differ from the classical apoptosis observed in other cell types, such as lymphocytes, thymocytes, fibroblasts and tumour cells. However, it is conceivable that individual myofibres may be severely affected when apoptotic myonuclei exceed a certain critical number. Unlike muscular dystrophies and inflammatory myopathies, the occurrence of muscle necrosis and regeneration in mitochondrial myopathies is quite rare. This strengthens the significance of apoptosis as a pathogenic mechanism of tissue damage in mitochondrial disorders, and the focal distribution of TUNEL-positive nuclei may explain the long survival of the affected fibres.
A variable percentage of TUNEL-positive fibres has been demonstrated previously by us and others in muscle of patients with mitochondrial myopathies (Mirabella et al., 1998; Monici et al., 1998). However, TUNEL positivity per se does not always indicate apoptosis. The expression on muscle cells of other markers of the apoptotic machinery, such as p75, Fas and caspase-3, further supports the specificity of the findings obtained with TUNEL. The overexpression of Fas and caspase-3 observed in TUNEL-positive fibres of patients with specific mtDNA defects suggests that the apoptotic pathway starting with Fas and ending with downstream activation of caspases may be a real model of cell death in some human mitochondrial diseases.
In fact, in patients carrying single mtDNA deletions or point mutations in tRNA genes (tRNALys, tRNALeu(UUR), tRNAIle, tRNATrp) the degree of apoptosis in muscle matched the number of mutated genomes and the severity of both mitochondrial myopathy and the neurological phenotype. MERRF-8344 (tRNALys), MELAS-3243 (tRNALeu(UUR)) and disorders associated with single mtDNA deletions account for the great majority of mitochondrial encephalomyopathies. The absence or reduction of the translation of all mtDNA-encoded mRNAs is the consequence of mtDNA defects involving at least one tRNA (Schon, 1997). By contrast, we found only modest or no signs of apoptosis in mitochondrial diseases associated with point mutations in structural genes, such as NARP and LHON, in spite of the presence of an extremely high percentage of mutated genomes in muscle (97–100%). In these disorders, mtDNA mutations do not affect overall mitochondrial protein synthesis but selectively involve a single subunit of a specific enzyme complex—ATPase in NARP and complex I in LHON.
This evidence suggests that only mtDNA abnormalities that impair mitochondrial protein synthesis can induce apoptosis when the percentage of mutated mtDNAs exceeds a threshold: ~40% in the case of single deletions and 70% in the case of tRNA point mutations. Our in vivo data are in agreement with a previous in vitro demonstration of the increased susceptibility to apoptosis, mediated by overexpression of Fas, of cybrids with a high percentage of mtDNAs carrying a single deletion or a point mutation in the tRNAIle gene (Asoh et al., 1996).
The paucity of apoptotic features in myopathies associated with multiple mtDNA deletions is puzzling. Multiple deletions, in fact, also impair mitochondrial protein synthesis and are detected in high amounts in muscle. The formation of multiple deletions of mtDNA in mitochondrial disorders that are inherited in a Mendelian manner is still unclear, and is probably due to a defective nuclear gene that appears to increase the frequency of mtDNA rearrangements (Schon, 1997). It is intriguing to hypothesize that the faulty communication between nuclear and mitochondrial genomes in these disorders may also induce intracellular proteins to inhibit apoptosis that would otherwise be activated by the presence of multiple mtDNA deletions.
Low percentages of multiple deletions and COX-negative fibres in muscle are often observed as accompanying phenomena in inclusion body myositis (Griggs et al., 1995), and have also been considered to be an age-dependent manifestation in normal old individuals. We did not find any apoptotic features in four patients with inclusion body myositis who had a level of mitochondrial abnormalities similar to or higher than that in the experimental group. Furthermore, there was no correlation between apoptosis and age at biopsy in all series of patients and controls.
Apoptosis is an active process that requires energy for its accomplishment, but a reduction in the level of ATP makes cells more susceptible to programmed cell death (Richter et al., 1996; Leist et al., 1997). There is, in fact, evidence that the induction of selective respiratory chain deficiencies makes cells more vulnerable to Fas-mediated apoptosis (Asoh et al., 1996) and that respiratory chain inhibitors induce apoptosis in vitro (Wolvetang et al., 1994; Marton et al., 1997; Higuchi et al., 1998).
In mitochondrial disorders associated with deletions or tRNA mutations there is partial, multiple involvement of all complexes of respiratory chain bearing mitochondrial subunits, although this involvement is sometimes so slight as to be hardly detectable by biochemical assays in vitro (Table 1). Our data demonstrate that, with few exceptions, in all patients with respiratory chain dysfunction, mainly involving complexes I and IV, muscle fibres are more prone to undergo apoptosis. However, even in the absence of a clear biochemical deficiency, apoptotic features are present when the percentage of mutated mtDNA exceeds the threshold.
In positive patients, although a rough correlation between the severity of mitochondrial myopathy and apoptosis could be established, RRF did not show more apoptotic features than non-RRF. However, especially in patients with a high percentage of single mtDNA deletions or tRNA point mutations and severe biochemical impairment, apoptotic nuclei were mainly located in COX-negative fibres. Nevertheless, apoptotic nuclei were scarce in multiple mtDNA deletions in spite of the presence of numerous COX-negative fibres. These findings, along with the negative results obtained in inclusion body myositis, suggest that apoptosis is not simply a way to eliminate cells that accumulate abnormal mitochondria, but is specifically induced in muscle fibres of patients with certain mtDNA abnormalities.
It is possible, in fact, that in mtDNA mutations affecting protein synthesis the imbalance and relative abundance of nuclear-encoded versus mtDNA-encoded subunits favour cytochrome c inactivation and release. Cytochrome c is a soluble protein loosely attached to the inner mitochondrial membrane and is an essential component of the mitochondrial respiratory chain. It has been recognized to play an important role in apoptosis signalling (Krippner et al., 1996; Liu et al., 1996) and has been demonstrated to be able to induce apoptosis-activating caspase-3 directly (Yang et al., 1997; Zou et al., 1997; Scaffidi et al., 1998; Zhivotovsky et al., 1998), independently of mitochondrial transmembrane depolarization (Liu et al., 1996; Yang et al., 1997, Bossy-Wetzel et al., 1998).
Cytochrome c, reactive oxygen species and dysfunction of the respiratory chain may all contribute to apoptosis, which in turn further inhibits the maturation of mitochondrial protein precursors encoded in the nucleus and the rate of mitochondrial translation (Mignotte et al., 1990; Vayssière et al., 1994; Mitsui et al., 1996), thus amplifying the biochemical defect, the production of reactive oxygen species and mitochondrial damage. This vicious circle is maintained and amplified in postmitotic non-regenerating tissues. Thus, apoptosis, which may represent a cure for mitochondrial disorders (Wolvetang et al., 1994; Asoh et al., 1996) through the specific killing of cells that accumulate mutant mtDNA, appears instead to be deleterious in perennial tissues, such as muscle, brain and heart. In agreement with this view is the example of Pearson's syndrome. This disorder, associated with a single deletion of mtDNA, is characterized by refractory sideroblastic anaemia and the vacuolization of bone marrow precursors. Death occurs in early childhood and the few surviving patients later develop Kearns-Sayre syndrome. The clinical improvement of blood dyscrasia in these patients is probably due to the elimination of affected blood cell precursors that carry a high percentage of mtDNA deletions; however, some remain to accumulate in muscle, the heart and the brain, giving rise to the Kearns-Sayre syndrome (Di Mauro and Bonilla, 1997). We demonstrated that virtually all nuclei were apoptotic in muscle from a patient with Pearson's syndrome. This suggests that apoptosis could be the mechanism by which dysfunctional, highly proliferative blood cell precursors are eliminated, unlike muscle and postmitotic cells, which cannot be easily removed and replaced.
In mitochondrial encephalomyopathies associated with mtDNA mutations, selected areas of a given tissue or specific subsets of cells are more prone to be affected clinically, even in the presence of the same number of mutant genomes. Apoptosis may be triggered by a variety of cell death signals, and there are multiple paths of commitment to death and different susceptibilities of diverse cells to the various stimuli (Hetts, 1998; Peter and Krammer, 1998; Scaffidi et al., 1998). Thus, apoptosis may help to explain the uneven tissue involvement of mitochondrial disorders.
In conclusion, apoptosis seems to represent a potential pathogenic mechanism of muscle tissue damage and to play a role in modulating the clinical expression of some mitochondrial disorders. Further studies are needed to clarify the sequence of the different events and to establish whether a single or several apoptotic pathways contribute to the heterogeneity of phenotypes even among patients carrying the same mtDNA mutation.
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Acknowledgments |
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This work was supported by Telethon-Italy, grant 1121 and MURST ex 40%.
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Received June 25, 1999. Accepted July 26, 1999.
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