Potential compounds for the treatment of mitochondrial disease.

INTRODUCTION: Mitochondrial diseases are a group of heterogeneous disorders for which no curative therapy is currently available. Several drugs are currently being pursued as candidates to correct the underlying biochemistry that causes mitochondrial dysfunction. SOURCES OF DATA: A systematic review of pharmacological therapeutics tested using in vitro, in vivo models and clinical trials. Results presented from database searches undertaken to ascertain compounds currently being pioneered to treat mitochondrial disease. AREAS OF AGREEMENT: Previous clinical research has been hindered by poorly designed trials that have shown some evidence in enhancing mitochondrial function but without significant results. AREAS OF CONTROVERSY: Several compounds under investigation display poor pharmacokinetic profiles or numerous off target effects. GROWING POINTS: Drug development teams should continue to screen existing and novel compound libraries for therapeutics that can enhance mitochondrial function. Therapies for mitochondrial disorders could hold potential cures for a myriad of other ailments associated with mitochondrial dysfunction such as neurodegenerative diseases.

Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids.

Mitochondrila DNA (mtDNA) is the only extrachromosomal DNA in humans. It is a small (16.5 kb) genome which encodes 13 essential peptides of the respiratory chain, two rRNAs and 22 tRNAs. Defects of this genome are now recognized as important causes of disease and may take the form of point mutations or rearrangements. There is no effective treatment for patients with mtDNA mutations. In the majority of patients with mtDNA defects, both mutant and wild-type molecules are present in the same cell-a phenomenon known as intracellular heteroplasmy. In addition, in the presence of heteroplasmy there is a threshold whereby a certain level of mutant mtDNA is necessary before the disease becomes biochemically and clinically apparent. Based on the presence of heteroplasmy and the recessive nature of these mutations, we believe it will be possible to treat patients by selectively inhibiting the replication of the mutant mtDNA, thereby allowing propagation of only the wild-type molecule. To confirm the validity of such an approach we synthesised peptide nucleic acids (PNAs) complementary to human mtDNA templates containing a deletion breakpoint or single base mutation, both mutations well documented to cause disease. Using an in vitro replication run-off assay under physiological conditions, the antigenomic PNAs specifically inhibited replication of mutant but not wild-type mtDNA templates. Furthermore, we have shown uptake of these PNAs into cultured human myoblasts. We believe that we have therefore established the potential value of antigenomic PNA therapy for patients with heteroplasmic mtDNA disorders.

Progress and prospects: gene therapy for mitochondrial DNA disease.

Defects of the mitochondrial genome cause a wide variety of clinical disorders. Except for rare cases where surgery or transplant is indicated, there is no effective treatment for patients. Genetic-based therapies are consequently being considered. On account of the difficulties associated with mitochondrial (mt) transfection, alternative approaches whereby mitochondrial genes can be engineered and introduced into the nucleus (allotopic expression) are being attempted with some success, at least in cultured cells. Defects in the activities of multi-subunit complexes of the oxidative phosphorylation apparatus have been circumvented by the targeted expression of simple single subunit enzymes from other species (xenotopic expression). Although far from the clinic, these approaches show promise. Similarly, nuclear transfection with genes encoding restriction endonucleases or sequence-specific zinc finger-binding proteins destined for mitochondria has also proved successful in targeting mtDNA-borne pathogenic mutations. This is particularly important, as mutated mtDNA is often found in cells that also contain normal copies of the genome, a situation termed heteroplasmy. Shifting the levels of heteroplasmy towards the normal mtDNA has become the goal of a variety of invasive and non-invasive methods, which are also highlighted in this review.

Mitochondrial DNA--all things bad?

Mutations in mitochondrial DNA (mtDNA) are undoubtedly associated with a diverse spectrum of human disorders. More controversially, it has been claimed that they accumulate during ageing, and that they are responsible for an age-related decline in bioenergetic function and tissue viability. Here, we review the evidence for this assertion, concluding that claims for the age-accumulation of mtDNA mutations are based largely on non-quantitative methods, and that no clear, functional deficit of mitochondrial respiration has been shown to result from such lesions in aged individuals. The mitochondrial theory of ageing, however attractive in principle, is supported by very little hard evidence.

Mammalian mitochondrial genetics: heredity, heteroplasmy and disease.

Mammalian mitochondrial DNA (mtDNA) is present at high copy number (10(3)-10(4) copies) in virtually all cells of the body. The mitochondrial genome shows strict maternal inheritance and the vast majority of copies are identical at birth (homoplasmy). Occasionally, a subpopulation of mtDNA molecules carry a pathogenic mutation. When this heteroplasmic mtDNA is present during embryogenesis, it can lead to a variety of clinical symptoms predominantly affecting muscle and nerve, but also affecting other tissues. While the importance of mitochodrial heteroplasmy in human disease is unquestioned, we remain largely ignorant of many fundamental aspects of mitochondrial genetics. How do mutations arise and can they be repaired, what influences the segregation and fixation of heteroplasmic mtDNA, do levels of heteroplasmy fluctuate during life, is it possible to modulate these levels by external intervention and, finally, can we predict the segregation and transmission of a mutant genome? The aim of this article is to summarize and discuss recent observations that have addressed several of these fundamental issues and to reiterate how much we still have to learn about mitochondrial genetics.

The inheritance of mitochondrial DNA heteroplasmy: random drift, selection or both?

The mammalian mitochondrial genome (mtDNA) is a small double-stranded DNA molecule that is exclusively transmitted down the maternal line. Pathogenic mtDNA mutations are usually heteroplasmic, with a mixture of mutant and wild-type mtDNA within the same organism. A woman harbouring one of these mutations transmits a variable amount of mutant mtDNA to each offspring. This can result in a healthy child or an infant with a devastating and fatal neurological disorder. Understanding the biological basis of this uncertainty is one of the principal challenges facing scientists and clinicians in the field of mitochondrial genetics.

SCID mice containing muscle with human mitochondrial DNA mutations. An animal model for mitochondrial DNA defects.

Defects of the mitochondrial genome are important causes of disease. Despite major advances in our investigation of patients, there is no effective therapy. Progress in this area is limited by the absence of any animal models in which we can evaluate treatment. To develop such a model we have injected human myoblasts into the tibialis anterior of SCID mice after inducing necrosis. After injection of normal human myoblasts, regenerating fibers expressed human beta-spectrin, confirming they were derived from fusion of human myoblasts. The stability of the muscle fibers was inferred by demonstrating the formation of motor end plates on the regenerating fibers. In addition, we show the presence of human cytochrome c oxidase subunit II, which is encoded by the mitochondrial genome, in the regenerated fibers. After injection of human myoblasts containing either the A8344G or the T8993C heteroplasmic mitochondrial DNA mutations, human beta-spectrin positive fibers were found to contain the mutation at a similar level to the injected myoblasts. These studies highlight the potential value of this model for the study of mitochondrial DNA defects.

Linked oligodeoxynucleotides show binding cooperativity and can selectively impair replication of deleted mitochondrial DNA templates.

Mutations in mitochondrial DNA (mtDNA) cause a spectrum of human pathologies, which predominantly affect skeletal muscle and the central nervous system. In patients, mutated and wild-type mtDNAs often co-exist in the same cell (mtDNA heteroplasmy). In the absence of pharmacological therapy, a genetic strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by selective hybridisation of a nucleic acid derivative to the single-stranded replication intermediate, allowing propagation of the wild-type genome and correction of the associated respiratory chain defect. Previous studies have shown the efficacy of this anti-genomic approach in vitro, targeting pathogenic mtDNA templates with only a single point mutation. Pathogenic molecules harbouring deletions, however, present a more difficult problem. Deletions often occur at the site of two short repeat sequences (4-13 residues), only one of which is retained in the deleted molecule. With the more common larger repeats it is therefore difficult to design an anti-genomic molecule that will bind selectively across the breakpoint of the deleted mtDNA. To address this problem, we have used linker-substituted oligodeoxynucleotides to bridge the repeated residues. We show that molecules can be designed to bind more tightly to the deleted as compared to the wild-type mtDNA template, consistent with the nucleotide sequence on either side of the linker co-operating to increase binding affinity. Furthermore, these bridging molecules are capable of sequence-dependent partial inhibition of replication in vitro.

Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease.

The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium-PNA (ph-PNA) conjugates of 3.4-4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph-PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph-PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph-PNA uptake. The ph-PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease 'myoclonic epilepsy and ragged red fibres' (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph-PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph-PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA replication. In summary, we have developed a new strategy for targeting PNA oligomers to mitochondria and used it to determine the effects of PNA on mutated mtDNA replication in cells. This work presents new approaches for the manipulation of mtDNA replication and expression, and will assist in the development of therapies for mtDNA diseases.

Sodium channel mRNAs at the neuromuscular junction: distinct patterns of accumulation and effects of muscle activity.

Voltage-gated sodium channels (VGSCs) are highly concentrated at the neuromuscular junction (NMJ) in mammalian skeletal muscle. Here we test the hypothesis that local upregulation of mRNA contributes to this accumulation. We designed radiolabeled antisense RNA probes, specific for the "adult" Na(V)1.4 and "fetal" Na(V)1.5 isoforms of VGSC in mammalian skeletal muscle, and used them in in situ hybridization studies of rat soleus muscles. Na(V)1.4 mRNA is present throughout normal adult muscles but is highly concentrated at the NMJ, in which the amount per myonucleus is more than eightfold greater than away from the NMJ. Na(V)1.5 mRNA is undetectable in innervated muscles but is dramatically upregulated by denervation. In muscles denervated for 1 week, both Na(V)1.4 and Na(V)1.5 mRNAs are present throughout the muscle, and both are concentrated at the NMJ. No Na(V)1.5 mRNA was detectable in denervated muscles stimulated electrically for 1 week in vivo. Neither denervation nor stimulation had any significant effect on the level or distribution of Na(V)1.4 mRNA. We conclude that factors, probably derived from the nerve, lead to the increased concentration of VGSC mRNAs at the NMJ. In addition, the expression of Na(V)1.5 mRNA is downregulated by muscle activity, both at the NMJ and away from it.

Developmental regulation of tissue-specific isoforms of subunit VIa of beef cytochrome c oxidase.

The switching of the subunit VIa isoforms of cytochrome c oxidase has been followed in heart tissue during bovine development both by transcript levels and in terms of the incorporation of L- (liver) and H- (heart) polypeptides into mitochondria. In early fetuses, e.g., 60-days development, there are high levels of VIaL transcript and high levels of the VIaL polypeptide incorporated into mitochondria. In late fetuses (after 200 days), the levels of VIaL transcript are still high, with less but still significant amounts of VIaL polypeptide present in comparison to adult heart in which the amount of this isoform is negligible. As the proportion of VIaL transcript is reduced, the proportion of VIaH transcript increases along with the amount of the VIaH isoform in mitochondria. These data indicate isoform switching during late fetal development. The presence of COLBP (cytochrome oxidase liver isoform binding protein) (Preiss, T. and Lightowlers, R.N. (1993) J. Biol. Chem. 268, 10659-10667) was examined at different developmental stages. COLBP binding activity was observed in hearts of late fetuses but not found in adult heart tissue, providing a correlation between the presence of this factor and the presence of the VIaL polypeptide in mitochondria.

The tissue-specific RNA-binding protein COLBP is differentially regulated during myogenesis.

Cytochrome c oxidase L-form transcript-binding protein (COLBP) activity parallels the tissue-specific mRNA expression of bovine cytochrome c oxidase liver isopeptides. A similar RNA-binding activity is found in human myoblast and liver Hep G2 cell homogenates. Human COLBP activity, however, is not present in myotubes or adult skeletal muscle. It is proposed that COLBP down-regulation during muscle cell differentiation may underlie oxidase isoform switching during myogenesis.

Isolation of a cDNA specifying subunit VIIb of human cytochrome c oxidase.

Human cytochrome c oxidase (COX) is a complex of 13 subunits: three are encoded by mitochondrial DNA and ten by nuclear DNA. We have now isolated a full-length cDNA specifying subunit VIIb, the last remaining uncharacterized nuclear-encoded subunit cDNA of human COX. The cDNA encodes a deduced 80-aa polypeptide, including a 24-amino acid (aa) N-terminal leader sequence and a 56-aa mature polypeptide with 82% identity to mature bovine COX VIIb. Southern blot hybridization of human muscle genomic DNA showed multiple hybridizing bands, implying the presence of a large coxVIIb gene family, including a potential processed pseudogene.

The proton pore in the Escherichia coli F0F1-ATPase: substitution of glutamate by glutamine at position 219 of the alpha-subunit prevents F0-mediated proton permeability.

Three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli were produced by site-directed mutagenesis. These mutations directed the substitution of Glu-219 by Gln, or of Lys-203 by Ile, or of Glu-196 by Ala. Strains carrying either the Lys-203 or Glu-196 substitutions showed growth characteristics indistinguishable from the coupled control strain. Properties of membrane preparations from these strains were also similar to those from the coupled control strain. The substitution of Glu-219 by Gln resulted in a strain which was unable to utilise succinate as sole carbon source and had a growth-yield characteristic of an uncoupled strain. Membrane preparations of the Glu-219 mutant were proton impermeable and the F1-ATPase activity was inhibited by about 50% when membrane-bound. The results are discussed with reference to a previously proposed intramembranous proton pore involving subunits a and c.

Mutational analysis of the function of the a-subunit of the F0F1-APPase of Escherichia coli.

In a model proposed for the structure of the a-subunit of the Escherichia coli F0F1-ATPase (Howitt, S.M., Gibson, F. and Cox, G.B. (1988) Biochim. Biophys. Acta 936, 74-80), a cluster of charged residues, including one arginine and four aspartic acid residues, lie on the periplasmic side of the membrane. On the cytoplasmic side, three pairs of lysine residues and an arginine residue are present. Site-directed mutagenesis was used to investigate the roles of these residues. It was found that none was directly involved in the proton pore. However, the substitutions of Asp-124 or Asp-44 by asparagine or Arg-140 by glutamine had similar effects in that the membranes from such mutants from which the F1-ATPase was removed were proton-impermeable. A combination of the Asp-44 mutation with either the Asp-124 or Arg-140 mutations in the same strain resulted in complete loss of oxidative phosphorylation. It was tentatively concluded that Asp-124 and Arg-140 form a salt bridge, as did Asp-44 with an unknown residue, and these salt bridges were concerned with the maintenance of correct a-subunit structure. Further support for this conclusion was obtained when second site revertants of a Glu-219 to histidine mutant were found to have either histidine or leucine replacing Arg-140. Thus, the lack of the Asp-124/Arg-140 salt bridge might enable repositioning of the helices of the a-subunit such that His-219 becomes a functional component of the proton pore.

The proton pore in the Escherichia coli F0F1-ATPase: a requirement for arginine at position 210 of the a-subunit.

Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F0F1-ATPase complex and these plasmids were used to transform strain AN727 (uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F1-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62-69).