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Current Progress with Mammalian Models of Mitochondrial DNA Disease

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Current Progress with Mammalian Models of Mitochondrial DNA Disease Received: 24 July 2020 Revised: 19 October 2020 Accepted: 21 October 2020 DOI: 10.1002/jimd.12324 REVIEW ARTICLE Current progress with mammalian models of mitochondrial DNA disease James Bruce Stewart1,2,3 1Max Planck Institute for Biology of Ageing, Cologne, Germany Abstract 2Wellcome Centre for Mitochondrial Mitochondrial disorders make up a large class of heritable diseases that cause Research, Faculty of Medical Sciences, a broad array of different human pathologies. They can affect many different Newcastle University, Newcastle upon organ systems, or display very specific tissue presentation, and can lead to ill- Tyne, UK ness either in childhood or later in life. While the over 1200 genes encoded in 3Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle the nuclear DNA play an important role in human mitochondrial disease, it upon Tyne, UK has been known for over 30 years that mutations of the mitochondria's own Correspondence small, multicopy DNA chromosome (mtDNA) can lead to heritable human James Bruce Stewart, Wellcome Centre diseases. Unfortunately, animal mtDNA has resisted transgenic and directed for Mitochondrial Research, Faculty of genome editing technologies until quite recently. As such, animal models to Medical Sciences, Newcastle University, Framlington Place, Newcastle upon Tyne aid in our understanding of these diseases, and to explore preclinical therapeu- NE2 4HH, UK. tic research have been quite rare. This review will discuss the unusual proper- Email: [email protected] ties of animal mitochondria that have hindered the generation of animal Funding information models. It will also discuss the existing mammalian models of human mtDNA Marie-Sklodowska-Curie ITN European disease, describe the methods employed in their generation, and will discuss Training Network, Grant/Award Number: recent advances in the targeting of DNA-manipulating enzymes to the mito- EU 721757; Max-Planck-Gesellschaft; United Mitochondrial Disease chondria and how these may be employed to generate new models. Foundation, Grant/Award Number: 13-053R KEYWORDS animal models, heteroplasmy, homoplasmy, mitochondrial disease, mitochondrial DNA Communicating Editor: Avihu Boneh 1 | INTRODUCTION products function within the mitochondria but are encoded by the nuclear DNA. Current estimates are that 1100 to Mitochondrial diseases constitute a range of heritable dis- 1500 nuclear encoded genes3 are translated by cytosolic orders that, through various mechanisms, end up leading ribosomes, and the protein products are then targeted to to mitochondrial dysfunction. Despite all of these dis- the various subcompartments of the mitochondria. eases leading to a similar cellular disorder, a staggering As a consequence of their proteobacterial origin, array of variable disease syndromes, tissue specificities, mitochondria maintain their own small genome (the age of onset, and prognostic outcomes for the patient are mtDNA), which in animals, exists in multiple copies, known.1,2 Further complicating the diagnosis and under- which vary by different cell types.4 Animal mtDNA typi- standing of these disorders, the causative mutations can cally encodes subunits of mitochondrial respiratory be due to mutations of both the maternally inherited complexes I, III, and IV (EC 7.1.1.2, 7.1.1.8, 7.1.1.9), plus mitochondrial DNA (mtDNA) itself, or by genes whose the mitochondrial ATP synthase (complex V, EC 7.1.2.2). This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Author. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM. J Inherit Metab Dis. 2020;1–18. wileyonlinelibrary.com/journal/jimd 1 2 STEWART To express these genes, the mtDNA also encodes two of 5 the rRNA subunits of their own ribosomes and a mini- Synopsis mized array of 22 tRNAs to carry out the translation6 within the mitochondrial matrix. The mitochondrial To date, animal mitochondrial DNA has remained resistant to genome engineering genome shows evidence of the loss of these mtDNA technologies for the generation of animal encoded RNAs that are present in more ancestral-like 7 models of these rare diseases; this review mtDNA genomes. For instance, animal mitochondrial will discuss the currently available mam- ribosomes have evolved to utilize a tRNA in place of the malian models for mtDNA disease, how 5S RNA,8 they have also evolved a protein-only, multi- they were generated, and future prospects subunit RNaseP enzyme9 (EC 3.1.26.5), and they gener- for enhancing these resources. ally maintain only the minimal set of tRNAs to decode the modified mitochondrial genetic code.4 For over 30 years, we have known that mutations of research field has been quite resourceful, and a number of the small, 16.5 kb mitochondrial genome are also impor- nondirect methods have been used to generate relevant tant causes of diseases.10,11 Mutations by base changes, animal models for research. This review will summarize small indels or by large deletions or partial duplication of these advances, and highlight the small but growing num- the mtDNA sequence are also known to lead to mito- ber of animal models with pathological mtDNA mutations. chondrial disorders.2 The high copy number and lack of a coordinated mtDNA replication cycle leads to another unusual feature of mtDNA, where two or more variants 1.1 | ANIMAL MODELS IN of the mtDNA can coexist within the same organism, cell, MITOCHONDRIAL DISEASE RESEARCH or even mitochondrion in a condition known as heter- oplasmy.12 In most of the mtDNA derived disorders, a The selection of the animal model always remains a topic high relative proportion of the mutation is necessary to of much discussion. Each model organism must be lead to the disease state (referred to as the mitochondrial weighed by the research question at hand. There has threshold effect13). In some cases, the disease alleles can been a long history of biomedical research using mouse be the only allele in the cell, tissue, or organ in a state models, and for the nuclear-encoded mitochondrial referred to as homoplasmy, such as seen in some mito- genes, these have been the models of choice in many pre- chondrial disease alleles leading to Leber Optic Atrophy vious studies (for reviews of models with nuclear genes (LHON, OMIM #535000). involved in mitochondrial disease, see References 16-20), While each specific disease is quite rare in the popula- a trend that will continue with the ease of CRISPR-based tion, when summed together, frequency estimates for nuclear genome editing techniques.21 As a mammalian childhood mitochondrial diseases can be as high as model, mice are short lived, breed prolifically, have a 1:6700,2 and up to 1:4300 in adults.14 As with all rare dis- long history of transgenic manipulation of the nuclear eases, the infrequency of patients leads to many difficul- DNA and, from a purely phylogenetic perspective, share ties in uncovering the molecular basis of disease and with rats the closest genetic relationship of a model hampers recruitment in clinical trials.15 The rarity of organism to humans and other primates (Figure 1). Yet these disorders unfortunately also provides economic dis- this genetic closeness does not always result in the best incentives for corporate investment in preclinical and model of a human disease. clinical trials. In addition, the striking tissue specificity in For instance, a very clinically relevant mouse model mitochondrial diseases leads to many instances where of Leigh's syndrome generated by disrupting the Complex cell lines, through genetic manipulation or when derived I NADH dehydrogenase-ubiquinone-FeS 4 (NDUFS4) from patient fibroblasts, can be aphenotypic, or not gene leads to a mouse model displaying ataxia, reveal relevant biochemistry that underlies the pathology encephalomyopathy, and other features that model the of specific organ systems. Thus, the field has had a long human disorder.23 This model is now heavily used in pre- interest in animal models to move forward our under- clinical research.24-27 In contrast, mutations in the Surfeit standing of these disorders, and for preclinical research Locus Protein 1 (SURF1) gene, a cytochrome c oxidase into interventions and cures. assembly factor, in patients also leads to a Leigh syn- Despite a long history of nuclear transgenesis, and drome presentation with associated complex IV defi- recent advances in genome editing, animal mtDNA ciency.28,29 Yet a SURF1 knockout mouse line utilizing remains resistant to transgenic manipulation, and as such, the loxP-cre recombination system showed little patho- there are only a handful of models with relevant disease logical phenotype and in one study, with knockout mice mutations available. Fortunately, the mitochondrial that actually out-lived their wild-type littermates.30 This STEWART 3 FIGURE 1 Representative cladogram of the genetic divergence in animals models mentioned in this review, which are used in mitochondrial disease research. At the nodes are estimated genetic divergence times (mya = million years ago), with confidence intervals in brackets extracted from http://www.timetree.org/ (June 28, 2020).22 Branch lengths are only representative, and not to scale. Images from http://phylopic.org was despite a 30% to 50% reduction in complex IV (reviewed in36), and recent work with targeted restriction enzyme activity
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