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.eB to deliver the missing gene to the CNS of these mice. In both cases, we observed survival extension from 5-6 to more than 15 months, with no detectable disease phenotypes. Likewise, molecular and cellular phenotypes were mostly recovered in the treated mice. Surprisingly, these remarkable phenotypic improvements were achieved with only ~30% of neurons expressing the transgene from the AAV-PHP.eB vector in the conditions used. These findings suggest that neurons lacking OXPHOS are protected by the surrounding neuronal environment and that partial compensation for neuronal OXPHOS loss can have disproportionately positive effects.

PI3K signaling promotes formation of lipid-laden foamy macrophages at the spinal cord injury site.

After spinal cord injury (SCI), infiltrating macrophages undergo excessive phagocytosis of myelin and cellular debris, forming lipid-laden foamy macrophages. To understand their role in the cellular pathology of SCI, investigation of the foamy macrophage phenotype in vitro revealed a pro-inflammatory profile, increased reactive oxygen species (ROS) production, and mitochondrial dysfunction. Bioinformatic analysis identified PI3K as a regulator of inflammation in foamy macrophages, and inhibition of this pathway decreased their lipid content, inflammatory cytokines, and ROS production. Macrophage-specific inhibition of PI3K using liposomes significantly decreased foamy macrophages at the injury site after a mid-thoracic contusive SCI in mice. RNA sequencing and in vitro analysis of foamy macrophages revealed increased autophagy and decreased phagocytosis after PI3K inhibition as potential mechanisms for reduced lipid accumulation. Together, our data suggest that the formation of pro-inflammatory foamy macrophages after SCI is due to the activation of PI3K signaling, which increases phagocytosis and decreases autophagy.

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. Here we describe the development of a mitochondrial-targeted ARCUS (mitoARCUS) nuclease designed to target one of the most common pathogenic mtDNA mutations, m.3243A>G. mitoARCUS robustly eliminated mutant mtDNA without cutting wild-type mtDNA, allowing for shifts in heteroplasmy and concomitant improvements in mitochondrial protein steady-state levels and respiration. In vivo efficacy was demonstrated using a m.3243A>G xenograft mouse model with mitoARCUS delivered systemically by adeno-associated virus. Together, these data support the development of mitoARCUS as an in vivo gene-editing therapeutic for m.3243A>G-associated 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. Here we report the adaptation of a gene editing platform based on the I-CreI meganuclease known as ARCUS®. These mitochondrial-targeted meganucleases (mitoARCUS) have a relatively small size, are monomeric, and can recognize sequences differing by as little as one base pair. We show the development of a mitoARCUS specific for the mouse m.5024C>T mutation in the mt-tRNAAla gene and its delivery to mice intravenously using AAV9 as a vector. Liver and skeletal muscle show robust elimination of mutant mtDNA with concomitant restoration of mt-tRNAAla levels. We conclude that mitoARCUS is a potential powerful tool for the elimination of mutant mtDNA.

Biallelic loss-of-function variations in PRDX3 cause cerebellar ataxia.

Peroxiredoxin 3 (PRDX3) belongs to a superfamily of peroxidases that function as protective antioxidant enzymes. Among the six isoforms (PRDX1-PRDX6), PRDX3 is the only protein exclusively localized to the mitochondria, which are the main source of reactive oxygen species. Excessive levels of reactive oxygen species are harmful to cells, inducing mitochondrial dysfunction, DNA damage, lipid and protein oxidation and ultimately apoptosis. Neuronal cell damage induced by oxidative stress has been associated with numerous neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Leveraging the large aggregation of genomic ataxia datasets from the PREPARE (Preparing for Therapies in Autosomal Recessive Ataxias) network, we identified recessive mutations in PRDX3 as the genetic cause of cerebellar ataxia in five unrelated families, providing further evidence for oxidative stress in the pathogenesis of neurodegeneration. The clinical presentation of individuals with PRDX3 mutations consists of mild-to-moderate progressive cerebellar ataxia with concomitant hyper- and hypokinetic movement disorders, severe early-onset cerebellar atrophy, and in part olivary and brainstem degeneration. Patient fibroblasts showed a lack of PRDX3 protein, resulting in decreased glutathione peroxidase activity and decreased mitochondrial maximal respiratory capacity. Moreover, PRDX3 knockdown in cerebellar medulloblastoma cells resulted in significantly decreased cell viability, increased H2O2 levels and increased susceptibility to apoptosis triggered by reactive oxygen species. Pan-neuronal and pan-glial in vivo models of Drosophila revealed aberrant locomotor phenotypes and reduced survival times upon exposure to oxidative stress. Our findings reveal a central role for mitochondria and the implication of oxidative stress in PRDX3 disease pathogenesis and cerebellar vulnerability and suggest targets for future therapeutic approaches.

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. This transmitochondrial cytoplasmic hybrid (cybrid) technology has helped the field understand the mechanism of several pathogenic mutations. In this chapter, we describe procedures to obtain transmitochondrial cybrids.

Impaired mitophagy links mitochondrial disease to epithelial stress in methylmalonyl-CoA mutase deficiency.

Deregulation of mitochondrial network in terminally differentiated cells contributes to a broad spectrum of disorders. Methylmalonic acidemia (MMA) is one of the most common inherited metabolic disorders, due to deficiency of the mitochondrial methylmalonyl-coenzyme A mutase (MMUT). How MMUT deficiency triggers cell damage remains unknown, preventing the development of disease-modifying therapies. Here we combine genetic and pharmacological approaches to demonstrate that MMUT deficiency induces metabolic and mitochondrial alterations that are exacerbated by anomalies in PINK1/Parkin-mediated mitophagy, causing the accumulation of dysfunctional mitochondria that trigger epithelial stress and ultimately cell damage. Using drug-disease network perturbation modelling, we predict targetable pathways, whose modulation repairs mitochondrial dysfunctions in patient-derived cells and alleviate phenotype changes in mmut-deficient zebrafish. These results suggest a link between primary MMUT deficiency, diseased mitochondria, mitophagy dysfunction and epithelial stress, and provide potential therapeutic perspectives for MMA.

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. rAAV9-Ndufs3 delivered systemically into 15- to 18-day-old mice effectively restored NDUFS3 levels in skeletal muscle, precluding the development of the myopathy. To test the ability of rAAV9-mediated gene replacement to revert muscle function after disease onset, we also treated post-symptomatic, 2-month-old mice. The injected mice showed a remarkable improvement of the mitochondrial myopathy and biochemical parameters, which remained for the duration of the study. Our results showed that muscle pathology could be reversed after restoring complex I, which was absent for more than 2 months. These findings have far-reaching implications for the ability of muscle to tolerate a mitochondrial defect and for the treatment of mitochondrial myopathies.

Mitochondrial methionyl N-formylation affects steady-state levels of oxidative phosphorylation complexes and their organization into supercomplexes.

N-Formylation of the Met-tRNAMet by the nuclearly encoded mitochondrial methionyl-tRNA formyltransferase (MTFMT) has been found to be a key determinant of protein synthesis initiation in mitochondria. In humans, mutations in the MTFMT gene result in Leigh syndrome, a progressive and severe neurometabolic disorder. However, the absolute requirement of formylation of Met-tRNAMet for protein synthesis in mammalian mitochondria is still debated. Here, we generated a Mtfmt-KO mouse fibroblast cell line and demonstrated that N-formylation of the first methionine via fMet-tRNAMet by MTFMT is not an absolute requirement for initiation of protein synthesis. However, it differentially affected the efficiency of synthesis of mtDNA-coded polypeptides. Lack of methionine N-formylation did not compromise the stability of these individual subunits but had a marked effect on the assembly and stability of the OXPHOS complexes I and IV and on their supercomplexes. In summary, N-formylation is not essential for mitochondrial protein synthesis but is critical for efficient synthesis of several mitochondrially encoded peptides and for OXPHOS complex stability and assembly into supercomplexes.

SCO2 mutations cause early-onset axonal Charcot-Marie-Tooth disease associated with cellular copper deficiency.

Recessive mutations in the mitochondrial copper-binding protein SCO2, cytochrome c oxidase (COX) assembly protein, have been reported in several cases with fatal infantile cardioencephalomyopathy with COX deficiency. Significantly expanding the known phenotypic spectrum, we identified compound heterozygous variants in SCO2 in two unrelated patients with axonal polyneuropathy, also known as Charcot-Marie-Tooth disease type 4. Different from previously described cases, our patients developed predominantly motor neuropathy, they survived infancy, and they have not yet developed the cardiomyopathy that causes death in early infancy in reported patients. Both of our patients harbour missense mutations near the conserved copper-binding motif (CXXXC), including the common pathogenic variant E140K and a novel change D135G. In addition, each patient carries a second mutation located at the same loop region, resulting in compound heterozygote changes E140K/P169T and D135G/R171Q. Patient fibroblasts showed reduced levels of SCO2, decreased copper levels and COX deficiency. Given that another Charcot-Marie-Tooth disease gene, ATP7A, is a known copper transporter, our findings further underline the relevance of copper metabolism in Charcot-Marie-Tooth disease.

Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized.

In recent years mitochondrial DNA (mtDNA) has transitioned to greater prominence across diverse areas of biology and medicine. The recognition of mitochondria as a major biochemical hub, contributions of mitochondrial dysfunction to various diseases, and several high-profile attempts to prevent hereditary mtDNA disease through mitochondrial replacement therapy have roused interest in the organellar genome. Subsequently, attempts to manipulate mtDNA have been galvanized, although with few robust advances and much controversy. Re-engineered protein-only nucleases such as mtZFN and mitoTALEN function effectively in mammalian mitochondria, although efficient delivery of nucleic acids into the organelle remains elusive. Such an achievement, in concert with a mitochondria-adapted CRISPR/Cas9 platform, could prompt a revolution in mitochondrial genome engineering and biological understanding. However, the existence of an endogenous mechanism for nucleic acid import into mammalian mitochondria, a prerequisite for mitochondrial CRISPR/Cas9 gene editing, remains controversial.

NO control of mitochondrial function in normal and transformed cells.

Nitric oxide (NO) is a signaling molecule with multiple facets and involved in numerous pathological process, including cancer. Among the different pathways where NO has a functionally relevant participation, is the control of mitochondrial respiration and biogenesis. NO is able to inhibit the electron transport chain, mainly at Complex IV, regulating oxygen consumption and ATP generation, but at the same time, can also induce increase in reactive oxygen and nitrogen species. The presence of reactive species can induce oxidative damage or participate in redox signaling. In this review, we discuss how NO affects mitochondrial respiration and mitochondrial biogenesis, and how it influences the development of mitochondrial deficiency and cancer. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

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. One of the earliest phenotypes was accelerated thymus shrinkage by apoptosis and differentiation into adipose tissue, mimicking age-related thymic involution. This phenotype was accompanied by increased ROS and activation of cell cycle arrest proteins. Treatment with antioxidants improved the phenotype but the knocking out of p21 or p53 did not. Our results demonstrate that transient mtDNA DSBs can accelerate aging of certain tissues by increasing ROS. Surprisingly, this mtDNA DSB-associated senescence phenotype does not require p21/p53, even if this pathway is activated in the process.

Sustained AMPK activation improves muscle function in a mitochondrial myopathy mouse model by promoting muscle fiber regeneration.

Acute pharmacological activation of adenosine monophosphate (AMP)-kinase using 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) has been shown to improve muscle mitochondrial function by increasing mitochondrial biogenesis. We asked whether prolonged AICAR treatment is beneficial in a mouse model of slowly progressing mitochondrial myopathy (Cox10-Mef2c-Cre), and whether the compensatory mechanism is indeed an increase in mitochondrial biogenesis. We treated the animals for 3 months and found that sustained AMP-dependent kinase activation improved cytochrome c oxidase activity, rescued the motor phenotype and delayed the onset of the myopathy. This improvement was observed whether treatment started before or after the onset of the disease. We found that AICAR increased skeletal muscle regeneration thereby decreasing the levels of deleted Cox10-floxed alleles. We conclude that although increase in mitochondrial biogenesis and other pathways may contribute, the main mechanism by which AICAR improves the myopathy phenotype is by promoting muscle regeneration.

Enhanced Transcriptional Activity and Mitochondrial Localization of STAT3 Co-induce Axon Regrowth in the Adult Central Nervous System.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor central to axon regrowth with an enigmatic ability to act in different subcellular regions independently of its transcriptional roles. However, its roles in mature CNS neurons remain unclear. Here, we show that along with nuclear translocation, STAT3 translocates to mitochondria in mature CNS neurons upon cytokine stimulation. Loss- and gain-of-function studies using knockout mice and viral expression of various STAT3 mutants demonstrate that STAT3's transcriptional function is indispensable for CNS axon regrowth, whereas mitochondrial STAT3 enhances bioenergetics and further potentiates regrowth. STAT3's localization, functions, and growth-promoting effects are regulated by mitogen-activated protein kinase kinase (MEK), an effect further enhanced by Pten deletion, leading to extensive axon regrowth in the mouse optic pathway and spinal cord. These results highlight CNS neuronal dependence on STAT3 transcriptional activity, with mitochondrial STAT3 providing ancillary roles, and illustrate a critical contribution for MEK in enhancing diverse STAT3 functions and axon regrowth.

Cre recombinase activity is inhibited in vivo but not ex vivo by a mutation in the asymmetric spacer region of the distal loxP site.

The cre/loxP recombination system is a valuable tool used to generate tissue specific genomic rearrangements in mouse models. The deletion of a region of interest flanked by two loxP sites is accomplished by the recombinase (cre) enzyme, which binds to the inverted repeat segments of two loxP sites and recognition of a conserved TA sequence in the asymmetric central spacer region "ATAACTTCGTATA -NNNTANNN-TATACGAAGTTAT. In vivo, we found that a single T to C mutation at position 4 of the central spacer region in the distal (3') loxP site, completely inhibited the recombination reaction in two conditional mouse models. These mice were generated using a mitochondrial methionyl-tRNA formyltransferase (Mtfmt) gene targeted construct and cre transgene under the control of tissue-specific promoters: calcium/calmodulin-dependent kinase II alpha (Camk2a-cre) and myosin light polypeptide 1 (Myl1-cre). Surprisingly, transient transfection of a plasmid expressing cre in dermal fibroblasts derived from the same mutant floxed Mtfmt((loxP/loxP)) mice line, successfully deleted the region of interest. This study demonstrates the sequence specificity required in vivo, the possibility of bypassing this limitation by expressing high levels of cre recombinase ex vivo and raises concerns related to the quality control of large scale production of gene targeted constructs and mice. genesis 53:695-700, 2015. © 2015 Wiley Periodicals, Inc.

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.

Mitochondrial alterations during carcinogenesis: a review of metabolic transformation and targets for anticancer treatments.

Mitochondria play important roles in multiple cellular processes including energy metabolism, cell death, and aging. Regulated energy production and utilization are critical in maintaining energy homeostasis in normal cells and functional organs. However, mitochondria go through a series of morphological and functional alterations during carcinogenesis. The metabolic profile in transformed cells is altered to accommodate their fast proliferation, confer resistance to cell death, or facilitate metastasis. These transformations also provide targets for anticancer treatment at different levels. In this review, we discuss the major modifications in cell metabolism during carcinogenesis, including energy metabolism, apoptotic and autophagic cell death, adaptation of tumor microenvironment, and metastasis. We also summarize some of the main metabolic targets for treatments.

RETRACTED: Bezafibrate improves mitochondrial function in the CNS of a mouse model of mitochondrial encephalopathy.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This retraction was suggested by the University of Cologne Investigation committee and seconded by the authors who the journal was able to contact (Wenz, T., Dillon, L., Diaz, F., Hida, A., and Moraes, C.T.). Following an investigation of the last author, Dr. Tina Wenz, by the University of Cologne, Germany, the university determined that data presented in this article have been inappropriately manipulated https://www.portal.uni-koeln.de/9015.html?&tx_news_pi1%5Bnews%5D=4335&tx_news_pi1%5Bcontroller%5D=News&tx_news_pi1%5Baction%5D=detail&cHash=1deb8399d7f796d65ca9f6ae4764a1ce. Specifically, western blot images in Figure 5F (tubulin in cortex), 2F (COXI in hippocampus) and 3B (Sod2 in hippocampus) were re-used from an earlier article published elsewhere [Increased muscle PGC-1alpha expression protects from sarcopenia and metabolic disease during aging" Wenz T, Rossi SG, Rotundo RL, Spiegelman BM, and Moraes CT. Proc Natl Acad Sci U S A. 2009;106:20405-10, doi: 10.1073/pnas.0911570106] representing different experimental findings. Therefore, whether or not the main conclusions are still valid, the authors request retraction of this publication because the scientific integrity of the study was compromised. The authors sincerely apologize to the scientific community.