NEK10 kinase ablation affects mitochondrial morphology, function and protein phosphorylation status.

Background: NEK10, a serine/threonine/tyrosine kinase belonging to the NEK (NIMA-related kinases) family, has been associated with diverse cellular processes. However, no specific target pathways have been identified. Our previous work knocking down NEK10 in HeLa cells suggested a functional association with mitochondria, as we observed altered mitochondrial morphology, mitochondrial oxygen consumption, mtDNA integrity, and reactive oxygen species levels.

Restoration of defective oxidative phosphorylation to a subset of neurons prevents mitochondrial encephalopathy.

Oxidative Phosphorylation (OXPHOS) defects can cause severe encephalopathies and no effective treatment exists for these disorders. To assess the ability of gene replacement to prevent disease progression, we subjected two different CNS-deficient mouse models (Ndufs3/complex I or Cox10/complex IV conditional knockouts) to gene therapy. We used retro-orbitally injected AAV-PHP.

High fat diet ameliorates mitochondrial cardiomyopathy in CHCHD10 mutant mice.

Mutations in CHCHD10, a mitochondrial protein with undefined functions, are associated with autosomal dominant mitochondrial diseases. Chchd10 knock-in mice harboring a heterozygous S55L mutation (equivalent to human pathogenic S59L) develop a fatal mitochondrial cardiomyopathy caused by CHCHD10 aggregation and proteotoxic mitochondrial integrated stress response (mtISR). In mutant hearts, mtISR is accompanied by a metabolic rewiring characterized by increased reliance on glycolysis rather than fatty acid oxidation.

mitoTALEN reduces the mutant mtDNA load in neurons.

Mutations within mtDNA frequently give rise to severe encephalopathies. Given that a majority of these mtDNA defects exist in a heteroplasmic state, we harnessed the precision of mitochondrial-targeted TALEN (mitoTALEN) to selectively eliminate mutant mtDNA within the CNS of a murine model harboring a heteroplasmic mutation in the mitochondrial tRNA alanine gene (m.5024C>T).

Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease.

Nuclease-mediated editing of heteroplasmic mitochondrial DNA (mtDNA) seeks to preferentially cleave and eliminate mutant mtDNA, leaving wild-type genomes to repopulate the cell and shift mtDNA heteroplasmy. Various technologies are available, but many suffer from limitations based on size and/or specificity. The use of ARCUS nucleases, derived from naturally occurring I-CreI, avoids these pitfalls due to their small size, single-component protein structure and high specificity resulting from a robust protein-engineering process.

Precise and simultaneous quantification of mitochondrial DNA heteroplasmy and copy number by digital PCR.

Mitochondrial DNA (mtDNA) is present in multiple copies and phenotypic consequences of mtDNA mutations depend on the mutant load surpassing a specific threshold. Additionally, changes in mtDNA copy number can impact mitochondrial ATP production, resulting in disease. Therefore, the precise determination of mtDNA heteroplasmy and copy number is crucial to the study of mitochondrial diseases.

Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo.

Diseases caused by heteroplasmic mitochondrial DNA mutations have no effective treatment or cure. In recent years, DNA editing enzymes were tested as tools to eliminate mutant mtDNA in heteroplasmic cells and tissues. Mitochondrial-targeted restriction endonucleases, ZFNs, and TALENs have been successful in shifting mtDNA heteroplasmy, but they all have drawbacks as gene therapy reagents, including: large size, heterodimeric nature, inability to distinguish single base changes, or low flexibility and effectiveness.

Manipulation of mitochondrial genes and mtDNA heteroplasmy.

Most patients with mitochondrial DNA (mtDNA) mutations have a mixture of mutant and wild-type mtDNA in their cells. This phenomenon, known as mtDNA heteroplasmy, provides an opportunity to develop therapies by selectively eliminating the mutant fraction. In the last decade, several enzyme-based gene editing platforms were developed to cleave specific DNA sequences.

Cybrid technology.

The study of the mitochondrial DNA (mtDNA) has been hampered by the lack of methods to genetically manipulate the mitochondrial genome in living animal cells. This limitation has been partially alleviated by the ability to transfer mitochondria (and their mtDNAs) from one cell into another, as long as they are from the same species. This is done by isolating mtDNA-containing cytoplasts and fusing these to cells lacking mtDNA.

Myopathy reversion in mice after restauration of mitochondrial complex I.

Myopathies are common manifestations of mitochondrial diseases. To investigate whether gene replacement can be used as an effective strategy to treat or cure mitochondrial myopathies, we have generated a complex I conditional knockout mouse model lacking NDUFS3 subunit in skeletal muscle. NDUFS3 protein levels were undetectable in muscle of 15-day-old smKO mice, and myopathy symptoms could be detected by 2 months of age, worsening over time.

Author Correction: MitoTALEN reduces mutant mtDNA load and restores tRNA levels in a mouse model of heteroplasmic mtDNA mutation.

In the version of this article originally published, there was an error in Fig. 1a. The m.

MitoTALEN reduces mutant mtDNA load and restores tRNA levels in a mouse model of heteroplasmic mtDNA mutation.

Mutations in the mitochondrial DNA (mtDNA) are responsible for several metabolic disorders, commonly involving muscle and the central nervous system. Because of the critical role of mtDNA in oxidative phosphorylation, the majority of pathogenic mtDNA mutations are heteroplasmic, co-existing with wild-type molecules. Using a mouse model with a heteroplasmic mtDNA mutation, we tested whether mitochondrial-targeted TALENs (mitoTALENs) could reduce the mutant mtDNA load in muscle and heart.

mitoTev-TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels.

Pathogenic mitochondrial DNA (mtDNA) mutations often co-exist with wild-type molecules (mtDNA heteroplasmy). Phenotypes manifest when the percentage of mutant mtDNA is high (70-90%). Previously, our laboratory showed that mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) can eliminate mutant mtDNA from heteroplasmic cells.

The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions.

Double-strand breaks in the mitochondrial DNA (mtDNA) result in the formation of linear fragments that are rapidly degraded. However, the identity of the nuclease(s) performing this function is not known. We found that the exonuclease function of the mtDNA polymerase gamma (POLG) is required for this rapid degradation of mtDNA fragments.

Transient mitochondrial DNA double strand breaks in mice cause accelerated aging phenotypes in a ROS-dependent but p53/p21-independent manner.

We observed that the transient induction of mtDNA double strand breaks (DSBs) in cultured cells led to activation of cell cycle arrest proteins (p21/p53 pathway) and decreased cell growth, mediated through reactive oxygen species (ROS). To investigate this process in vivo we developed a mouse model where we could transiently induce mtDNA DSBs ubiquitously. This transient mtDNA damage in mice caused an accelerated aging phenotype, preferentially affecting proliferating tissues.

MitoTALEN: A General Approach to Reduce Mutant mtDNA Loads and Restore Oxidative Phosphorylation Function in Mitochondrial Diseases.

We have designed mitochondrially targeted transcription activator-like effector nucleases or mitoTALENs to cleave specific sequences in the mitochondrial DNA (mtDNA) with the goal of eliminating mtDNA carrying pathogenic point mutations. To test the generality of the approach, we designed mitoTALENs to target two relatively common pathogenic mtDNA point mutations associated with mitochondrial diseases: the m.8344A>G tRNA(Lys) gene mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) and the m.

Selective elimination of mitochondrial mutations in the germline by genome editing.

Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA.

The use of mitochondria-targeted endonucleases to manipulate mtDNA.

For more than a decade, mitochondria-targeted nucleases have been used to promote double-strand breaks in the mitochondrial genome. This was done in mitochondrial DNA (mtDNA) homoplasmic systems, where all mtDNA molecules can be affected, to create models of mitochondrial deficiencies. Alternatively, they were also used in a heteroplasmic model, where only a subset of the mtDNA molecules were substrates for cleavage.

Manipulating mitochondrial genomes in the clinic: playing by different rules.

Recently, several publications have surfaced describing methods to manipulate mitochondrial genomes in tissues and embryos. With them, a somewhat sensationalistic uproar about the generation of children with 'three parents' has dominated the discussion in the lay media. It is important that society understands the singularities of mitochondrial genetics to judge these procedures in a rational light, so that this ongoing discussion does not preclude the helping of patients and families harboring mutated mitochondrial genomes.

Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs.

Mitochondrial diseases are commonly caused by mutated mitochondrial DNA (mtDNA), which in most cases coexists with wild-type mtDNA, resulting in mtDNA heteroplasmy. We have engineered transcription activator-like effector nucleases (TALENs) to localize to mitochondria and cleave different classes of pathogenic mtDNA mutations. Mitochondria-targeted TALEN (mitoTALEN) expression led to permanent reductions in deletion or point-mutant mtDNA in patient-derived cells, raising the possibility that these mitochondrial nucleases can be therapeutic for some mitochondrial diseases.

Emerging therapeutic approaches to mitochondrial diseases.

Mitochondrial diseases are very heterogeneous and can affect different tissues and organs. Moreover, they can be caused by genetic defects in either nuclear or mitochondrial DNA as well as by environmental factors. All of these factors have made the development of therapies difficult.

Intra- and inter-molecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks.

To investigate mtDNA recombination induced by multiple double strand breaks (DSBs) we used a mitochondria-targeted form of the ScaI restriction endonuclease to introduce DSBs in heteroplasmic mice and cells in which we were able to utilize haplotype differences to trace the origin of recombined molecules. ScaI cleaves multiple sites in each haplotype of the heteroplasmic mice (five in NZB and three in BALB mtDNA) and prolonged expression causes severe mtDNA depletion. After a short pulse of restriction enzyme expression followed by a long period of recovery, mitochondrial genomes with large deletions were detected by PCR.

Mitochondrial involvement in amyotrophic lateral sclerosis: trigger or target?

Despite numerous reports demonstrating mitochondrial abnormalities associated with amyotrophic lateral sclerosis (ALS), the role of mitochondrial dysfunction in the disease onset and progression remains unknown. The intrinsic mitochondrial apoptotic program is activated in the central nervous system of mouse models of ALS harboring mutant superoxide dismutase 1 protein. This is associated with the release of cytochrome-c from the mitochondrial intermembrane space and mitochondrial swelling.