Nuclear DNA Background Research Articles

Mitochondrial DNA drives AD Phenotypes in Cell Models

AbstractBackgroundMitochondrial DNA (mtDNA) inheritance influences Alzheimer’s Disease (AD) risk. Adult children of mother’s afflicted with AD show decreased cognition and increased AD biomarkers (both peripheral and brain) at earlier ages than adult children with father’s afflicted with AD. These human derived data paired with prior in vitro work support a maternal inheritance association of AD risk. However, the exact mechanism linking mtDNA inheritance patterns and AD is not understood.MethodsCytoplasmic hybrid (cybrid) cells were derived by fusing platelet mitochondria with SH‐SY5Y cells devoid of mtDNA. Cybrid cells have the same nuclear DNA background with varying mtDNA sequences derived from human subjects. We examined mitochondrial function parameters in cybrids derived from 8 cognitively healthy (CH), 7 AD, and 5 mild cognitive impairment (MCI) subjects. Mitochondrial electron transport chain (ETC) function was measured using Seahorse XF technology. Mitochondrial membrane potential, mitochondrial superoxide, and mitochondrial calcium were measured using fluorescent assays (TMRE, MitoSox, and Rhod2AM). Mitophagy and mitochondrial biogenesis/mass were measured using adenoviral constructs (EGFP‐Cox8 and MitoTimer).ResultsCybrids derived from AD subjects had elevated mitochondrial superoxide production when compared to CH cybrid cells. MCI cybrids did not show changes to mitochondrial superoxide production. Mitochondrial membrane potential and mitochondrial calcium were not changed in MCI or AD cybrids compared to CH. AD cybrids, but not MCI cybrids, showed reduced flux through all ETC components, including complex I, II, III, and IV. Finally, AD cybrids showed increased mitophagy levels but no change in mitochondrial biogenesis. MCI cybrids did not reflect these changes. Data do indicate AD cybrids might have increased mitochondrial mass, but further assays are needed to verify these findings.ConclusionsTransfer of mtDNA from AD patients to cells harboring consistent nuclear DNA recapitulates mitochondrial phenotypes observed in AD. We are currently measuring AD biomarkers in these models and working to examine their correlation with clinical data from this cohort of research subjects.

Abstract 3103: TorqTM system improves liquid biopsy sample shipping stability

Abstract Quantifying circulating tumor DNA (ctDNA) markers for liquid biopsy testing often requires shipping blood samples to centralized laboratories. Mechanical damage to blood cells can occur during transport, especially if samples endure extensive vibration and temperature excursions. Such mechanical damage may result in Red Blood Cell (RBC) disruption known as in vitro hemolysis, as well as White Blood Cell (WBC) lysis leading to the release of nuclear DNA and several types of nucleases into the plasma. Nuclear DNA and nuclease contamination are the primary interfering factors impeding ctDNA recovery. Several commercial products are developed to address this issue by chemically stabilizing the cells in the tube during transport (e.g., the Streck cell-free DNA BCT™). Nevertheless, such products do not entirely resolve the problem, and cell lysis during shipping remains a complication. The Torq™ zero delay centrifuge system offers a novel solution by immediately centrifuging and separating plasma from the cell component at the point of collection, after the blood draw and prior to shipping, preventing ctDNA dilution from whole blood interferents. Here we compare hemolysis levels in blood samples collected and prepared by the Torq system and Streck BCTs following simulated shipping conditions including time, vibration, and temperature excursions. Blood samples from healthy individuals were collected into Torq ZDiscs™ and Streck BCTs. The plasma was immediately separated within the ZDiscs using the Torq ZDrive™ and transferred to microtubes. The specimens in Streck BCTs remained as whole blood, as per the manufacturer's instructions. Both sample types went through shipping simulation for 24 and 72 hours following ISTA 3A standards while experiencing temperature conditions of 4°C, 24°C, and 40°C. The blood within Streck BCTs was separated into plasma and cell components after reaching the designated time point, and all samples were inspected for hemolysis by quantifying hemoglobin content. Samples collected and processed with the Torq zero delay centrifuge system showed minimal hemolysis throughout the simulation and at all temperature conditions, whereas samples in the Streck BCTs exhibited significantly higher hemolysis, especially after 72 hours (p-value: 0.0001). Streck samples stored at 4°C and 40°C were notably susceptible to high hemolysis contamination. Compared to the Torq system, the Streck BCTs yielded significantly increased sample hemolysis following simulated shipping times and temperature excursions (both low and high), thereby increasing the likelihood of genomic contamination. The ZDiscs provide enhanced shipping resiliency and decreased nuclear DNA background, thus increasing the chance for rare mutation detection in liquid biopsy applications. Citation Format: Ali Rahimian, Kyungjin Hong, Clara Neal, Gabriella Iacovetti, Greg Sommer, Ulrich Schaff. TorqTM system improves liquid biopsy sample shipping stability [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3103.

Lithium influences whole-organism metabolic rate in Drosophila subobscura.

Lithium is widely used to treat bipolar disorder. However, the efficacy and vulnerability as to its side effects are known to differ. Although the specific biochemical mechanism of action is still elusive, lithium may influence mitochondrial function, and consequently, metabolism. Lithium exposure in this study was conducted on a unique set of mito-nuclear introgression lines of Drosophila subobscura to disentangle the independent effects of mitochondrial DNA (mtDNA) against a common nuclear DNA background. The study addressed three issues: (a) whether lithium has a dose-dependent effect on whole-organism metabolic rate, (b) whether mtDNA haplotypes show divergent metabolic efficiency measured by metabolic rate to lithium exposure and (c) whether lithium influences the whole-organism metabolic rate across sexes. The results confirm that lithium influenced the whole-organism metabolic rate, showing a subtle balance between efficacy and adverse effects within a narrow dose range. In addition, lithium exposure was found to influence metabolism differently based on mtDNA haplotypes and sex. This preliminary research may have a range of biological implications for the role of mitochondrial variability in psychiatric disease and treatment by contributing to the understanding and predicting of the lithium treatment response and risk for toxic side effects.

M.3243A > G-Induced Mitochondrial Dysfunction Impairs Human Neuronal Development and Reduces Neuronal Network Activity and Synchronicity

Epilepsy, intellectual and cortical sensory deficits, and psychiatric manifestations are the most frequent manifestations of mitochondrial diseases. How mitochondrial dysfunction affects neural structure and function remains elusive, mostly because of a lack of proper invitro neuronal model systems with mitochondrial dysfunction. Leveraging induced pluripotent stem cell technology, we differentiated excitatory cortical neurons (iNeurons) with normal (low heteroplasmy) and impaired (high heteroplasmy) mitochondrial function on an isogenic nuclear DNA background from patients with the common pathogenic m.3243A > G variant of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). iNeurons with high heteroplasmy exhibited mitochondrial dysfunction, delayed neural maturation, reduced dendritic complexity, and fewer excitatory synapses. Micro-electrode array recordings of neuronal networks displayed reduced network activity and decreased synchronous network bursting. Impaired neuronal energy metabolism and compromised structural and functional integrity of neurons and neural networks could be the primary drivers of increased susceptibility to neuropsychiatric manifestations of mitochondrial disease.

Abstract 5128: Changes in mitochondrial background affect nuclear DNA methylation

Abstract There is emerging evidence demonstrating that mitochondrial DNA (mtDNA) contributes to complex genetic diseases such as cancer. To test directly the role of mtDNA in tumorigenicity and metastasis, a new genetically engineered mouse model was created that contains nuclear DNA from one mouse strain and mtDNA contribution from different mouse strain(s). Taking advantage of maternal inheritance, Mitochondrial Nuclear Exchange (MNX) mice can be bred to mice sharing the same nuclear DNA background. As a result, MNX serves as an important tool to determine if and how mitochondrial genetic background contributes to cancer, negating the concern for mixing maternal and paternal nuclear DNA polymorphisms. MNX mice are healthy, breed normally, show no overt differences in cell cycle or apoptosis and are genetically stable for multiple (>10) generations. Nonetheless, they are distinct strains based upon behaviors and gene expression patterns. The mechanisms controlling gene expression in different MNX strains are not yet known; however, retrograde and anterograde cross-talk exists between the nuclear and mitochondrial genomes. Our hypothesis is that important epigenetic changes occur in the nuclear genome depending upon mitochondrial genetics. To test this hypothesis, we performed Meth-Seq using the Agilent Mouse SureSelect platform and Affymetrix gene expression arrays on four mouse brains (8 wk) that were pooled from different litters and cages of wild-type and MNX to minimize age, developmental, inter-individual and generational heterogeneity. Both differential DNA methylation and gene expression patterns were observed but analyses are ongoing to better define the observed differences. Together these observations provide novel insights into how mtDNA could be altering epigenetic regulation and thereby contribute to cancer pathogenesis. Support: Susan G. Komen for the Cure (SAC11037), Natl Fndn Cancer Res., Kansas Bioscience Authority, Steiner Family Fund. Citation Format: Carolyn J. Vivian, Amanda E. Brinker, Gerald C. Gooden, Christophe Legendre, Bodour Salhia, Danny R. Welch. Changes in mitochondrial background affect nuclear DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5128. doi:10.1158/1538-7445.AM2015-5128

A specific nuclear DNA background is required for high frequency lymphoma development in transmitochondrial mice with G13997A mtDNA.

We previously found that mouse mitochondrial DNA (mtDNA) with a G13997A mutation (G13997A mtDNA) controls not only the transformation of cultured lung carcinoma cells from poorly metastatic into highly metastatic cells, but also the transformation of lymphocytes into lymphomas in living C57BL/6 (B6) mice. Because the nuclear genetic background of the B6 strain makes the strain prone to develop lymphomas, here we examined whether G13997A mtDNA independently induces lymphoma development even in mice with the nuclear genetic background of the A/J strain, which is not prone to develop lymphomas. Our results showed that the B6 nuclear genetic background is required for frequent lymphoma development in mice with G13997A mtDNA. Moreover, G13997A mtDNA in mice did not enhance the malignant transformation of lung adenomas into adenocarcinomas or that of hepatocellular carcinomas from poorly metastatic into highly metastatic carcinomas. Therefore, G13997A mtDNA enhances the frequency of lymphoma development under the abnormalities in the B6 nuclear genome, and does not independently control tumor development and tumor progression.

Maternally transmitted mitochondrial DNA mutations can reduce lifespan.

We recently showed that germline transmission of mitochondrial DNA mutations via the oocyte cause aggravation of aging phenotypes in prematurely aging mtDNA mutator (PolgAmut/mut) mice. We discovered that 32% of these mice also exhibit stochastic disturbances of brain development, when maternal mtDNA mutations were combined with homozygosity for the PolgA mutation, leading to de novo somatic mtDNA mutations. Surprisingly, we also found that maternally transmitted mtDNA mutations can cause mild premature aging phenotypes also in mice with a wild-type nuclear DNA background. We now report that in addition to the early onset of aging phenotypes, these mice, burdened only by low levels of mtDNA mutations transmitted via the germline, also exhibit reduced longevity. Our data thus demonstrate that low levels of maternally inherited mtDNA mutations when present during development can affect both overall health and lifespan negatively.

Rise and Fall of a Hybrid Zone: Implications for the Roles of Aggression, Mate Choice, and Secondary Succession

Hybridization can be an important evolutionary force by generating new species and influencing evolution of parental species in multiple ways, including introgression and the consequences of hybrid vigor. Determining the ecological processes underlying evolution in hybrid zones is difficult however because it requires examining changes in both genotypic frequencies over time and corresponding ecological information, data that are rarely collected together. Here, we describe genetic and ecological aspects of a hybrid zone between the Eastern Fence Lizard, Sceloporus undulatus, and the Florida Scrub Lizard, Sceloporus woodi, occurring over at least 23 generations. The hybrid zone, discovered greater than 35 years ago using morphological characters, originally consisted of nearly even proportions of parental species and hybrids. Now, using genetic markers (species-diagnostic mtDNA sites and 6 nDNA microsatellite loci across a total of n = 117 individuals), we confirm not only that hybridization occurred but also that subsequent backcrossing has resulted in highly introgressed hybrids, with many hybrids containing mitochondrial DNA from one species on a nuclear DNA background of the other. Ecological aspects explaining these shifts in genetic composition include female mate choice, changes in habitat associated with secondary succession, and, most strongly, a hierarchy of male territorial advantage-ecological mechanisms likely to be involved in the emergence and disappearance of many animal hybrid zones. Our results suggest that genetic assimilation is not a significant threat to either species and that rather transient hybrid zones such as this may serve to increase genetic diversity and are candidates for causing genetic discordance in phylogeographic analyses.

Specific mtDNA Mutations in Mouse Carcinoma Cells Suppress Their Tumor Formation via Activation of the Host Innate Immune System

In mammalian species, mitochondrial DNA (mtDNA) with pathogenic mutations that induce mitochondrial respiration defects has been proposed to be involved in tumor phenotypes via induction of enhanced glycolysis under normoxic conditions (the Warburg effects). However, because both nuclear DNA and mtDNA control mitochondrial respiratory function, it is difficult to exclude the possible contribution of nuclear DNA mutations to mitochondrial respiration defects and the resultant expression of tumor phenotypes. Therefore, it is important to generate transmitochondrial cybrids sharing the same nuclear DNA background but carrying mtDNA with and without the mutations by using intercellular mtDNA transfer technology. Our previous studies isolated transmitochondrial cybrids and showed that specific mtDNA mutations enhanced tumor progression as a consequence of overproduction of reactive oxygen species (ROS). This study assessed whether mtDNA mutations inducing ROS overproduction always enhance tumor progression. We introduced mtDNA from senescence-accelerated mice P1 (SAMP1) into C57BL/6J (B6) mice-derived Lewis lung carcinoma P29 cells, and isolated new transmitochondrial cybrids (P29mtSAMP1 cybrids) that overproduced ROS. The inoculation of the cybrids into B6 mice unexpectedly showed that mtDNA from SAMP1 mice conversely induced tumor suppression. Moreover, the tumor suppression of P29mtSAMP1 cybrids in B6 mice occurred as a consequence of innate immune responses of the host B6 mice. Enzyme pretreatment experiments of P29mtSAMP1 cybrids revealed that some peptides encoded by mtDNA and expressed on the cell surface of P29mtSAMP1 cybrids induce increased IL-6 production from innate immune cells (dendritic cells) of B6 mice, and mediate augmented inflammatory responses around the tumor-inoculated environment. These observations indicate presence of a novel role of mtDNA in tumor phenotype, and provide new insights into the fields of mitochondrial tumor biology and tumor immunology.

Heteroplasmy of Mouse mtDNA Is Genetically Unstable and Results in Altered Behavior and Cognition

Maternal inheritance of mtDNA is the rule in most animals, but the reasons for this pattern remain unclear. To investigate the consequence of overriding uniparental inheritance, we generated mice containing an admixture (heteroplasmy) of NZB and 129S6 mtDNAs in the presence of a congenic C57BL/6J nuclear background. Analysis of the segregation of the two mtDNAs across subsequent maternal generations revealed that proportion of NZB mtDNA was preferentially reduced. Ultimately, this segregation process produced NZB-129 heteroplasmic mice and their NZB or 129 mtDNA homoplasmic counterparts. Phenotypic comparison of these three mtDNA lines demonstrated that the NZB-129 heteroplasmic mice, but neither homoplasmic counterpart, had reduced activity, food intake, respiratory exchange ratio; accentuated stress response; and cognitive impairment. Therefore, admixture of two normal but different mouse mtDNAs can be genetically unstable and can produce adverse physiological effects, factors that may explain the advantage of uniparental inheritance of mtDNA.

Mitochondrial DNA effects on fitness in Drosophila subobscura

We tested different fitness components on a series of conspecific mtDNA haplotypes, detected by RFLPs in Drosophila subobscura. Additionally, haplotype VIII, endemic to the Canary Islands, was tested upon its own native nuclear DNA background and upon that of the rest of mtDNAs tested herein. We found that both nuclear and mitochondrial DNA can have a significant effect upon their hosts' fitness, and that negative selection is one of the mechanisms that can intervene in this species' mtDNA haplotype pattern. We discuss the importance of this mechanism in relation to genetic drift, in the form of periodic population bottlenecks, and how the latter can enhance the former. We also detected a significant positive effect of haplotype VIII upon fitness that could explain in part the dominance of this endemic haplotype on some of the Canary Islands, and a mitochondrial heterosis involving this haplotype when on a foreign nuclear DNA background.

The innate immune system in host mice targets cells with allogenic mitochondrial DNA

Mitochondrial DNA (mtDNA) has been proposed to be involved in respiratory function, and mtDNA mutations have been associated with aging, tumors, and various disorders, but the effects of mtDNA imported into transplants from different individuals or aged subjects have been unclear. We examined this issue by generating trans-mitochondrial tumor cells and embryonic stem cells that shared the syngenic C57BL/6 (B6) strain-derived nuclear DNA background but possessed mtDNA derived from allogenic mouse strains. We demonstrate that transplants with mtDNA from the NZB/B1NJ strain were rejected from the host B6 mice, not by the acquired immune system but by the innate immune system. This rejection was caused partly by NK cells and involved a MyD88-dependent pathway. These results introduce novel roles of mtDNA and innate immunity in tumor immunology and transplantation medicine.