Mechanisms of Mitochondrial DNA Deletion Formation.

Mitochondrial DNA (mtDNA) encodes a subset of genes which are essential for oxidative phosphorylation. Deletions in the mtDNA can ablate a number of these genes and result in mitochondrial dysfunction, which is associated with bona fide mitochondrial disorders. Although mtDNA deletions are thought to occur as a result of replication errors or following double-strand breaks, the exact mechanism(s) behind deletion formation have yet to be determined. In this review we discuss the current knowledge about the fate of mtDNA following double-strand breaks, including the molecular players which mediate the degradation of linear mtDNA fragments and possible mechanisms of recircularization. We propose that mtDNA deletions formed from replication errors versus following double-strand breaks can be mediated by separate pathways.We are grateful for support from the National Institutes of Health Grants 1R01AG036871, 5R01EY010804, and 1R01NS079965, the Muscular Dystrophy Association and the Champ Foundation (to C.T.M.); and the Medical Research Council Grant MC_UU_00015/4 (to M.M.)

Mitochondrial DNA Fate and Deletion Formation Following Double-Strand Breaks

Mitochondrial DNA (mtDNA) deletions have been identified in patients with bona-fide mitochondrial disorders as well as implicated in normal aging. The mechanism of how these deletions form is still debated – some possibilities include formation as a result of errors in replication, double-strand breaks (DSBs), or during mtDNA repair. Due to the multi-copy nature of mtDNA, usually following DSB, there is a rapid degradation of linear mtDNA fragments instead of repair. The goals of this study were to first understand which nucleases play a role in mtDNA degradation following DSBs and second to generate and characterize murine cellular models harboring large mtDNA deletions. For the first aim we used mitochondrially-targeted restriction endonucleases to create DSBs in the mtDNA in ex vivo and in vivo mouse models of exonuclease-deficient polymerase gamma (Polg), known as the mutator mouse. Although there was a rapid degradation of linear mtDNA fragments in the wild-type samples, this degradation was impaired in the mutator samples. This impaired degradation was only found in exonuclease-deficient POLG, but not polymerase-deficient POLG. One consequence of the persistence of these linear mtDNA fragments was the formation of mtDNA rearrangements or deletions, which amplified when the partially-deleted mtDNA contained the origins of replication. For the second aim, we fused synaptosomes from the cortex of a mouse expressing the mitochondrial-targeted restriction endonuclease PstI with mouse ρ0 cells to generate cell lines with high levels of mtDNA deletions. Clones harboring the “PstI Deletion” had decreased cell growth, decreased steady-state mitochondrial protein levels, altered supercomplex formation, decreased protein synthesis, and decreased respiration. Additionally, there was a heteroplasmy-dependence to the measured parameters. To use these cell lines for the development of a genetic therapy we generated a mitoTALEN which was able to shift mtDNA heteroplasmy in the PstI Deletion clones, and this shift was stable over time. Together, these studies give a new role to POLG in degrading linear mtDNA fragments following DSBs and provide us with a novel tool for studying mtDNA deletions.</p

Mitochondrial DNA damage and reactive oxygen species in neurodegenerative disease

Mitochondria are essential organelles within the cell where most ATP is produced through oxidative phosphorylation (OXPHOS). A subset of the genes needed for this process are encoded by the mitochondrial DNA (mtDNA). One consequence of OXPHOS is the production of mitochondrial reactive oxygen species (ROS), whose role in mediating cellular damage, particularly in damaging mtDNA during ageing, has been controversial. There are subsets of neurons that appear to be more sensitive to ROS-induced damage, and mitochondrial dysfunction has been associated with several neurodegenerative disorders. In this review, we will discuss the current knowledge in the field of mtDNA and neurodegeneration, the debate about ROS as a pathological or beneficial contributor to neuronal function, bona fide mtDNA diseases, and insights from mouse models of mtDNA defects affecting the central nervous system

Mitochondrial DNA heteroplasmy in disease and targeted nuclease-based therapeutic approaches

Mitochondrial DNA (mtDNA) encodes a subset of the genes which are responsible for oxidative phosphorylation. Pathogenic mutations in the human mtDNA are often heteroplasmic, where wild-type mtDNA species co-exist with the pathogenic mtDNA and a bioenergetic defect is only seen when the pathogenic mtDNA percentage surpasses a threshold for biochemical manifestations. mtDNA segregation during germline development can explain some of the extreme variation in heteroplasmy from one generation to the next. Patients with high heteroplasmy for deleterious mtDNA species will likely suffer from bona-fide mitochondrial diseases, which currently have no cure. Shifting mtDNA heteroplasmy toward the wild-type mtDNA species could provide a therapeutic option to patients. Mitochondrially targeted engineered nucleases, such as mitoTALENs and mitoZFNs, have been used in vitro in human cells harboring pathogenic patient-derived mtDNA mutations and more recently in vivo in a mouse model of a pathogenic mtDNA point mutation. These gene therapy tools for shifting mtDNA heteroplasmy can also be used in conjunction with other therapies aimed at eliminating and/or preventing the transfer of pathogenic mtDNA from mother to child

Long Term Flux Profile of Implanon Birth Control Implant

Hormonal birth control methods have become increasingly popular since 2000, as the technology becomes more convenient for users, moving from daily pills, to weekly patches, to yearly implants. Implanon is an example of a long-term birth control implant. The goal of this project is to create an accurate computational model of Implanon’s hormone diffusion over its standard prescription length: three years. Because Implanon is intended for long-term use, any potential improvements in the drug or release mechanism take several years to clinically test. It is time and cost inefficient to develop several new designs and test them all over this long time period. Instead, we can eliminate those designs that fail to reach specifications in our computer model and be more confident in the clinical success of those that function properly in our computer model, thus reducing the number of clinical trials needed and the time and money spent. We created a two-dimensional cross section model of the Implanon implant and surrounding tissue under mass transfer conditions model using COMSOL Multiphysics software. We confirmed our model’s accuracy with comparisons to published Implanon hormone release rates at six weeks, one year, two years, and three years. Our model’s hormone release rate was found to stay within a factor of 10 of the published data at all critical time periods. This data is significant in that it has the potential to expedite the hormone modification process. There were several assumptions made in the model’s schematic design as well as material properties and boundary parameters. It is recommended that more in vivo experimentation and data gathering on Implanon implant placement and hormone diffusivity be conducted to improve this model’s accuracy

Lack of Parkin Anticipates the Phenotype and Affects Mitochondrial Morphology and mtDNA Levels in a Mouse Model of Parkinson's Disease

is the most common gene mutated in monogenic recessive familial cases of Parkinson's disease (PD). Pathogenic mutations cause a loss of function of the encoded protein Parkin. ParkinKO mice, however, poorly represent human PD symptoms as they only exhibit mild motor phenotypes, minor dopamine metabolism abnormalities, and no signs of dopaminergic neurodegeneration. Parkin has been shown to participate in mitochondrial turnover, by targeting damaged mitochondria with low membrane potential to mitophagy. We studied the role of Parkin on mitochondrial quality control by knocking out Parkin in the PD-mito- I mouse (males), where the mitochondrial DNA (mtDNA) undergoes double-strand breaks only in dopaminergic neurons. The lack of Parkin promoted earlier onset of dopaminergic neurodegeneration and motor defects in the PD-mito- I mice, but it did not worsen the pathology. The lack of Parkin affected mitochondrial morphology in dopaminergic axons and was associated with an increase in mtDNA levels (mutant and wild type). Unexpectedly, it did not cause a parallel increase in mitochondrial mass or mitophagy. Our results suggest that Parkin affects mtDNA levels in a mitophagy-independent manner. Parkinson's disease is characterized by progressive motor symptoms due to the selective loss of dopaminergic neurons in the substantia nigra. Loss-of-function mutations of Parkin cause some monogenic forms of Parkinson's disease, possibly through its role in mitochondrial turnover and quality control. To study whether Parkin has a role in the context of mitochondrial damage, we knocked out Parkin in a mouse model in which the mitochondrial DNA is damaged in dopaminergic neurons. We found that the loss of Parkin did not exacerbate the parkinsonian pathology already present in the mice, but it was associated with an increase in mtDNA levels (mutant and wild-type) without altering mitochondrial mass. These results shed new light on the function of Parkin

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

Hypoxia Promotes Mitochondrial Complex I Abundance via HIF-l[alpha] in Complex III and Complex IV Deficient Cells

Murine fibroblasts deficient in mitochondria respiratory complexes III (CHI) and IV (CIV) produced by either the ablation of Uqcrfs1 (encoding for Rieske iron sulfur protein, RISP) or Cox10 (encoding for protoheme IX farnesyltransferase, COX10) genes, respectively, showed a pleiotropic effect in complex I (CI). Exposure to 1-5% oxygen increased the levels of CI in both RISP and COX10 KO fibroblasts. De novo assembly of the respiratory complexes occurred at a faster rate and to higher levels in 1% oxygen compared to normoxia in both RISP and COX10 KO fibroblasts. Hypoxia did not affect the levels of assembly of Oil in the COX10 KO fibroblasts nor abrogated the genetic defect impairing CIV assembly. Mitochondrial signaling involving reactive oxygen species (ROS) has been implicated as necessary for HIF-1[alpha] stabilization in hypoxia. We did not observe increased ROS production in hypoxia. Exposure to low oxygen levels stabilized HIF-1[alpha] and increased CI levels in RISP and COX10 KO fibroblasts. Knockdown of HIF-1[alpha] during hypoxic conditions abrogated the beneficial effect of hypoxia on the stability/assembly of CI. These findings demonstrate that oxygen and HIF-1[alpha] regulate the assembly of respiratory complexes.Academi