Germline-soma Supply Mitochondria for mtDNA Inheritance in Mouse Oogenesis

Maternal transmission paradigm of mtDNA remains controversial in mammalian oogenesis. Germline-soma-to-oocyte communication by numerous transzonal nanotubes (TZTs) reminds whether intercellular mitochondrial transfer is associated with maternal inheritance. Here, we found that mouse oocytes egocentrically receive mitochondria via TZTs, which projected from germline-soma, to achieve 105 copies, instead of de novo synthesis of mtDNA subpopulation in growing oocytes. De novo assembled TZTs amongst germline-soma and oocytes accumulated mtDNA amounts of the oocytes in vitro. However, mitochondrial supplement from germline-soma gradually diminished along with oocyte growth and was terminated by meiosis resumption, in line with a decrease in the proportion of germline-soma with thriving mtDNA replication and FSH capture capability. Thus, germline-soma-to-oocyte mitochondrial transfer is responsible for mammalian mtDNA inheritance as well as oogenesis and aging.

Investigations of Intercellular Mitochondrial Transfer in Neural Cells by Applied Single Molecule Genotyping

<p>Over the past decade and a half, evidence for transfer of whole mitochondria between mammalian cells has emerged in the literature. The notion that mitochondria are restricted to the cell of origin has been overturned by this curious phenomenon, yet the physiological relevance of these transfer events remains unclear. This thesis investigates intercellular mitochondrial transfer in co-cultures of neural cells in vitro, to understand whether neural cells placed under stress demonstrate an enhanced rate of intercellular mitochondrial transfer. This would implicate the phenomenon as a cellular response to stress. Reliable techniques for quantitative study of intercellular mitochondrial transfer are limited so far in this field. To address this, a novel quantitative approach was developed to detect intercellular mitochondrial transfer, based on single molecule genotyping by target-primed rolling circle amplification. This enabled imaging of individual mitochondrial DNA molecules in situ, to detect those molecules which had moved between cells. Through this strategy, intercellular mitochondrial transfer was detected in new in vitro co-culture models. Primary murine pericytes derived from brain microvessels, were found to readily transfer mitochondria to a murine astrocyte cell line in vitro. Cisplatin, a DNA damaging agent; and chloramphenicol, a mitochondrial ribosome inhibitor, used to induce acute cellular injuries in the murine astrocyte cell line. These injuries were characterised and found to induce apoptosis, cause changes in growth characteristics, mitochondrial gene expression, and alter the metabolic phenotype of the cells. A derivative of the astrocyte cell line which completely lacks mitochondrial respiration, was found to model a chronic metabolic injury. As pericytes are prevalent throughout the brain, the pericyte/astrocyte co-culture model was selected to evaluate how the rate of intercellular mitochondrial transfer was altered, when the astrocytes were injured prior to co-culture. Through in situ single molecule genotyping and high throughput confocal microscopy, quantitative data was produced on how the rate of intercellular mitochondrial transfer was altered by injury in these models. The rate of intercellular mitochondrial transfer remained unaltered by chloramphenicol, however both cisplatin and the chronic metabolic injury model demonstrated reduced numbers of pericyte mitochondrial DNAs transferred into the injured astrocytes. These studies demonstrate successful application of a novel approach to study intercellular mitochondrial transfer and enable quantitative studies of this phenomenon.</p>

Investigations of Intercellular Mitochondrial Transfer in Neural Cells by Applied Single Molecule Genotyping

<p>Over the past decade and a half, evidence for transfer of whole mitochondria between mammalian cells has emerged in the literature. The notion that mitochondria are restricted to the cell of origin has been overturned by this curious phenomenon, yet the physiological relevance of these transfer events remains unclear. This thesis investigates intercellular mitochondrial transfer in co-cultures of neural cells in vitro, to understand whether neural cells placed under stress demonstrate an enhanced rate of intercellular mitochondrial transfer. This would implicate the phenomenon as a cellular response to stress. Reliable techniques for quantitative study of intercellular mitochondrial transfer are limited so far in this field. To address this, a novel quantitative approach was developed to detect intercellular mitochondrial transfer, based on single molecule genotyping by target-primed rolling circle amplification. This enabled imaging of individual mitochondrial DNA molecules in situ, to detect those molecules which had moved between cells. Through this strategy, intercellular mitochondrial transfer was detected in new in vitro co-culture models. Primary murine pericytes derived from brain microvessels, were found to readily transfer mitochondria to a murine astrocyte cell line in vitro. Cisplatin, a DNA damaging agent; and chloramphenicol, a mitochondrial ribosome inhibitor, used to induce acute cellular injuries in the murine astrocyte cell line. These injuries were characterised and found to induce apoptosis, cause changes in growth characteristics, mitochondrial gene expression, and alter the metabolic phenotype of the cells. A derivative of the astrocyte cell line which completely lacks mitochondrial respiration, was found to model a chronic metabolic injury. As pericytes are prevalent throughout the brain, the pericyte/astrocyte co-culture model was selected to evaluate how the rate of intercellular mitochondrial transfer was altered, when the astrocytes were injured prior to co-culture. Through in situ single molecule genotyping and high throughput confocal microscopy, quantitative data was produced on how the rate of intercellular mitochondrial transfer was altered by injury in these models. The rate of intercellular mitochondrial transfer remained unaltered by chloramphenicol, however both cisplatin and the chronic metabolic injury model demonstrated reduced numbers of pericyte mitochondrial DNAs transferred into the injured astrocytes. These studies demonstrate successful application of a novel approach to study intercellular mitochondrial transfer and enable quantitative studies of this phenomenon.</p>

Investigation of Mitochondrial Transfer between Human Erythroblasts

<p>The increasingly studied phenomenon of mitochondria transferring between cells contrasts the popular belief that mitochondria reside permanently within their cells of origin. Research has identified this process occurring in many tissues such as brain, lung and more recently within the bone marrow. This project aimed to investigate if mitochondria could be transferred between human erythroblasts, a context not previously studied. Tissue microenvironments can be modelled using co-culture systems. Fluorescence activated cell sorting and a highly sensitive Allele-Specific-Blocker qPCR assay were used to leverage mitochondrial DNA polymorphisms between co-cultured populations. Firstly, HL-60ρ₀ bone marrow cells, without mitochondrial DNA, deprived of essential nutrients pyruvate and uridine were co-cultured in vitro with HEL cells, a human erythroleukemia. Secondly, HEL cells treated with deferoxamine or cisplatin, were cocultured with parental HL-60 cells in vitro. Lastly, ex vivo co-cultures between erythroblasts differentiated from mononuclear cells in peripheral blood were conducted, where one population was treated with deferoxamine. Co-culture was able to improve recovery when HL-60ρ₀ cells were deprived of pyruvate and uridine. Improved recovery was similarly detected for HEL cells treated with deferoxamine after co-culture with HL-60 cells. Transfer of mitochondrial DNA did not occur at a detectable level in any co-culture condition tested. The high sensitivity of the allele-specific-blocker qPCR assay required completely pure populations to analyse, however this was not achieved using FACS techniques. In conclusion, results have not demonstrated but cannot exclude the possibility that erythroid cells transfer mitochondria to each other.</p>

Mitochondrial Transfer in Saccharomyces cerevisiae

<p>This thesis investigated mitochondrial transfer in Saccharomyces cerevisiae, between respiratory compromised B18p⁰ recipient and respiratory competent donor cells. The respiratory compromised strain had three red fluorescent proteins tagged to the membrane, nucleus and cytoplasm (triple RFP-B18p⁰) and is referred to as the B18p⁰ strain. B18p⁰ cells did not contain mitochondrial DNA, causing it to be respiratory compromised and required a fermentable carbon source, such as glucose/dextrose, for proliferation. The respiratory competent strain used had a green fluorescent protein tagged to the Tom70 mitochondrial protein (Tom70-GFP) and is referred to as the Tom70 strain. The Tom70 cells contained the nuclear encoded URA3 cassette, allowing for negative selectivity of this strain using 5-FOA. S. cerevisiae strains were co-cultured together in media containing only non-fermentable carbon sources (YPGE), plated on YPGE plates containing 5-FOA and colonies grown were distinguished post-co-culture based on their distinct phenotypic and genotypic characteristics. Fluorescent analysis of co-culture colonies revealed the presence of 5-FOA resistant Tom70 cells and some red B18p⁰ cells that had acquired the ability to grow on non-fermentable carbon sources. Genotypic analysis revealed that the majority of these red colonies had acquired mtDNA as well as the nuclear encoded, Tom70 specific URA3 cassette. Several permutations of co-cultures were performed, using different ratios of recipient and donor cells and single-gene deletion donor cells. Purified mitochondria from Tom70 cells were tried to be transferred into B18p⁰ cells using centrifugation forces to induce a higher occurrence frequency of mitochondrial transfer. Metabolic support experiments were conducted to investigate if the Tom70 strain could provide metabolic support to the B18p⁰ strain without mitochondrial transfer. Results indicate that no permutation induced potential mitochondrial transfer at a higher rate than others. However, results indicate that mitochondrial transfer did occur at low frequencies, potentially through the fusion of respiratory competent and respiratory compromised cells. Forced transfer did not increase the occurrence frequency of B18p⁰ cells to take up mitochondria and Tom70 cells did not provide metabolic support to B18p⁰ cells.</p>

MitoVisualize: A resource for analysis of variants in human mitochondrial RNAs and DNA

We present MitoVisualize, a new tool for analysis of the human mitochondrial DNA (mtDNA). MitoVisualize enables visualization of: (1) the position and effect of variants in mitochondrial transfer RNA (tRNA) and ribosomal RNA (rRNA) secondary structures alongside curated variant annotations, (2) data across RNA structures, such as to show all positions with disease-associated variants or with post-transcriptional modifications, and (3) the position of a base, gene or region in the circular mtDNA map, such as to show the location of a large deletion. All visualizations can be easily downloaded as figures for reuse. MitoVisualize can be useful for anyone interested in exploring mtDNA variation, though is designed to facilitate mtDNA variant interpretation in particular. MitoVisualize can be accessed via https://www.mitovisualize.org/. The source code is available at https://github.com/leklab/mito_visualize/.

Mitochondrial Transfer in Saccharomyces cerevisiae

<p>This thesis investigated mitochondrial transfer in Saccharomyces cerevisiae, between respiratory compromised B18p⁰ recipient and respiratory competent donor cells. The respiratory compromised strain had three red fluorescent proteins tagged to the membrane, nucleus and cytoplasm (triple RFP-B18p⁰) and is referred to as the B18p⁰ strain. B18p⁰ cells did not contain mitochondrial DNA, causing it to be respiratory compromised and required a fermentable carbon source, such as glucose/dextrose, for proliferation. The respiratory competent strain used had a green fluorescent protein tagged to the Tom70 mitochondrial protein (Tom70-GFP) and is referred to as the Tom70 strain. The Tom70 cells contained the nuclear encoded URA3 cassette, allowing for negative selectivity of this strain using 5-FOA. S. cerevisiae strains were co-cultured together in media containing only non-fermentable carbon sources (YPGE), plated on YPGE plates containing 5-FOA and colonies grown were distinguished post-co-culture based on their distinct phenotypic and genotypic characteristics. Fluorescent analysis of co-culture colonies revealed the presence of 5-FOA resistant Tom70 cells and some red B18p⁰ cells that had acquired the ability to grow on non-fermentable carbon sources. Genotypic analysis revealed that the majority of these red colonies had acquired mtDNA as well as the nuclear encoded, Tom70 specific URA3 cassette. Several permutations of co-cultures were performed, using different ratios of recipient and donor cells and single-gene deletion donor cells. Purified mitochondria from Tom70 cells were tried to be transferred into B18p⁰ cells using centrifugation forces to induce a higher occurrence frequency of mitochondrial transfer. Metabolic support experiments were conducted to investigate if the Tom70 strain could provide metabolic support to the B18p⁰ strain without mitochondrial transfer. Results indicate that no permutation induced potential mitochondrial transfer at a higher rate than others. However, results indicate that mitochondrial transfer did occur at low frequencies, potentially through the fusion of respiratory competent and respiratory compromised cells. Forced transfer did not increase the occurrence frequency of B18p⁰ cells to take up mitochondria and Tom70 cells did not provide metabolic support to B18p⁰ cells.</p>

Investigation of Mitochondrial Transfer between Human Erythroblasts

<p>The increasingly studied phenomenon of mitochondria transferring between cells contrasts the popular belief that mitochondria reside permanently within their cells of origin. Research has identified this process occurring in many tissues such as brain, lung and more recently within the bone marrow. This project aimed to investigate if mitochondria could be transferred between human erythroblasts, a context not previously studied. Tissue microenvironments can be modelled using co-culture systems. Fluorescence activated cell sorting and a highly sensitive Allele-Specific-Blocker qPCR assay were used to leverage mitochondrial DNA polymorphisms between co-cultured populations. Firstly, HL-60ρ₀ bone marrow cells, without mitochondrial DNA, deprived of essential nutrients pyruvate and uridine were co-cultured in vitro with HEL cells, a human erythroleukemia. Secondly, HEL cells treated with deferoxamine or cisplatin, were cocultured with parental HL-60 cells in vitro. Lastly, ex vivo co-cultures between erythroblasts differentiated from mononuclear cells in peripheral blood were conducted, where one population was treated with deferoxamine. Co-culture was able to improve recovery when HL-60ρ₀ cells were deprived of pyruvate and uridine. Improved recovery was similarly detected for HEL cells treated with deferoxamine after co-culture with HL-60 cells. Transfer of mitochondrial DNA did not occur at a detectable level in any co-culture condition tested. The high sensitivity of the allele-specific-blocker qPCR assay required completely pure populations to analyse, however this was not achieved using FACS techniques. In conclusion, results have not demonstrated but cannot exclude the possibility that erythroid cells transfer mitochondria to each other.</p>

Bone marrow derived-mesenchymal stem cell improves diabetes-associated fatty liver via mitochondria transformation in mice

Background Non-alcoholic fatty liver disease (NAFLD) has become a global epidemic disease. Its incidence is associated with type 2 diabetes mellitus (T2DM). Presently, there is no approved pharmacological agents specially developed for NAFLD. One promising disease-modifying strategy is the transplantation of stem cells to promote metabolic regulation and repair of injury. Method In this study, a T2DM model was established through 28-week high-fat diet (HFD) feeding resulting in T2DM-associated NAFLD, followed by the injection of bone marrow mesenchymal stem cells (BMSCs). The morphology, function, and transfer of hepatocyte mitochondria were evaluated in both vivo and in vitro. Results BMSC implantation resulted in the considerable recovery of increasing weight, HFD-induced steatosis, liver function, and disordered glucose and lipid metabolism. The treatment with BMSC transplantation was accompanied by reduced fat accumulation. Moreover, mitochondrial transfer was observed in both vivo and vitro studies. And the mitochondria-recipient steatotic cells exhibited significantly enhanced OXPHOS activity, ATP production, and mitochondrial membrane potential, and reduced reactive oxygen species levels, which were not achieved by the blocking of mitochondrial transfer. Conclusion Mitochondrial transfer from BMSCs is a feasible process to combat NAFLD via rescuing dysfunction mitochondria, and has a promising therapeutic effect on metabolism-related diseases.