Mouse mtDNA Research Articles

Endonuclease G is dispensable for sperm mitochondrial DNA elimination during spermatogenesis in mice.

Maternal inheritance of mitochondrial DNA (mtDNA) is a widespread phenomenon in eukaryotes. Our earlier research indicated that sperm mtDNA is removed prior to fertilization in mice, and Endonuclease G (ENDOG) orchestrates the degradation of sperm mitochondria in Caenorhabditis elegans. However, the mechanisms underlying sperm mtDNA disposal in mammals remain poorly understood. To investigate the potential role of ENDOG in sperm mtDNA elimination, we created Endog knockout (Endog-/-) mice. Our findings revealed that Endog-/- mice maintained normal spermatogenesis and fertility. Most strikingly, we detected no substantial discrepancy in sperm mtDNA copy number between Endog-/- and control mice. Furthermore, we noted that sperm mtDNA copy numbers were unchanged in both less motile and motile sperm isolated by Percoll gradient centrifugation from Endog-/- and control mice. Taken together, our results indicate that ENDOG is not essential for spermatogenesis or the elimination of sperm mtDNA in mice.

Single-Mitochondrion Sequencing Uncovers Distinct Mutational Patterns and Heteroplasmy Landscape in Mouse Astrocytes and Neurons.

Mitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting 'normal' mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes In this study we amplified mt-genomes from 1,645 single mitochondria (mts) isolated from mouse single astrocytes and neurons to 1. determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome, 2. assess differences in mtDNA SNVs between neurons and astrocytes, and 3. Study cosegregation of variants in the mouse mtDNA. 1. The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. 2. Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G>A and 9419:C>T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. 3. Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461. This study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants.

Mitochondrial DNA drives neuroinflammation through the cGAS-IFN signaling pathway in the spinal cord of neuropathic pain mice.

Neuroinflammation is pivotal in the development of neuropathic pain (NeP). While mitochondrial deoxyribonucleic acid (mtDNA) and cyclic GMP-AMP synthase (cGAS) are recognized for inducing inflammation in various neurological disorders, their involvement in NeP remains ambiguous. In this study, we examined: (1) the changes in mtDNA and cGAS in mice with NeP induced by chronic constriction injury (CCI) of the sciatic nerve, whether mtDNA triggers inflammation via the cGAS signaling; (2) the effects of RU.521, a cGAS antagonist, on CCI-induced nociception (allodynia and hyperalgesia) and relative inflammatory protein expression; (3) the activation of microglia and the cGAS-IFN pathway mediated by mtDNA in BV2 cell; (4) the effect of RU.521 on mtDNA-induced inflammatory response in BV2 cells. Results revealed reduced mtDNA levels in the sciatic nerve but increased levels in the spinal cord of CCI mice, along with elevated cGAS expression and inflammatory factors. RU.521 alleviated nociceptive behaviors in CCI mice, possibly by normalizing cGAS levels and suppressing inflammation. Neuron-derived mtDNA provoked cellular activation and upregulated cGAS signaling in BV2 cells. Additionally, RU.521 and DNase I effectively inhibited cGAS-induced inflammation. These findings underscore the critical role of mtDNA accumulation and mtDNA-mediated cGAS signaling in NeP development after peripheral nerve injury.

Quantitative analysis of mitochondrial DNA in porcine-mouse cloned embryos.

The aim of the research is to identify that porcine oocytes can function as recipients for interspecies cloning and have the ability to develop to blastocysts. Furthermore each mitochondrial DNA (mtDNA) in interspecises cloned embryos was analyzed. For the study, mouse-porcine and porcine-porcine cloned embryos were produced with mouse fetal fibroblasts (MFF) and porcine fetal fibroblasts (PFF), respectively, introduced as donor cells into enucleated porcine oocytes. The developmental rate and cell numbers of blastocysts between intraspecies porcine-porcine and interspecies mouse-porcine cloned embryos were compared and real-time polymerase chain reaction (PCR) was performed for the estimate of mouse and porcine mtDNA copy number in mouse-porcine cloned embryos at different stages.There was no significant difference in the developmental rate or total blastocyst number between mouse-porcine cloned embryos and porcine-porcine cloned embryos (11.1 ± 0.9%, 25 ± 3.5 vs. 10.1 ± 1.2%, 24 ± 6.3). In mouse-porcine reconstructed embryos, the copy numbers of mouse somatic cell-derived mtDNA decreased between the 1-cell and blastocyst stages, whereas the copy number of porcine oocyte-derived mtDNA significantly increased during this period, as assessed by real-time PCR analysis. In our real-time PCR analysis, we improved the standard curve construction-based method to analyze the level of mtDNA between mouse donor cells and porcine oocytes using the copy number of mouse beta-actin DNA as a standard. Our findings suggest that mouse-porcine cloned embryos have the ability to develop to blastocysts in vitro and exhibit mitochondrial heteroplasmy from the 1-cell to blastocyst stages and the mouse-derived mitochondria can be gradually replaced with those of the porcine oocyte in the early developmental stages of mouse-porcine cloned embryos.

Circadian disruption and psychostimulants dysregulates plasma acute-phase proteins and circulating cell-free mitochondrial DNA

BackgroundPrevious studies have indicated a close link between the inflammatory response, exacerbated by circadian disruption and psychostimulants such as cocaine and methamphetamine (METH). Indicators of this inflammation include cortisol and acute-phase proteins (APPs) like C-reactive protein (CRP), complement C3 (C3), and serum amyloid A (SAA). The connection between these inflammation markers and circulating mitochondrial DNA (mtDNA) has been gaining attention. However, the specific influence of cocaine and METH on APP, cortisol, and mtDNA levels in mice with disturbed circadian rhythm has yet to be explored, which is the main aim of this research. MethodsIn our study, we employed 10–12-week-old male C57BL/6J mice, which underwent an imposed 6-h phase advance every six days for a total of eight cycles. This process led to the formation of mice with disrupted circadian rhythm and sleep disorders (CRSD). We administered 11 dosages of cocaine and METH 15 mg/kg and 20 mg/kg, respectively to these CRSD mice over the course of 22 days. Quantitative assessments of CRP, C3, SAA, cortisol, and cell-free circulating mtDNA were conducted using enzyme-linked immunosorbent assay (ELISA), Western Blot, and quantitative real-time polymerase chain reaction (qRT-PCR) techniques. ResultsThe experiment revealed that disruption in circadian rhythm alone or cocaine or METH on their own increased CRP, C3, SAA, and cortisol levels in comparison with the control group. CRSD mice, exposed to cocaine and METH, showed a significant rise in CRP, C3, and SAA, while those without exposure remained stable. We also found a reduction in circulating cell-free mtDNA in all CRSD mice, regardless of cocaine and METH exposure. ConclusionsThe findings of our study affirm that the levels of CRP, C3, SAA, and cortisol, which reflect inflammation, are enhanced by circadian disruption, cocaine, and METH, and these levels show a strong correlation with the content of circulating cell-free mtDNA. Furthermore, it also shows the potential link between the disruption of the circadian clock and the inflammatory response triggered by cocaine and METH.

DdCBE mediates efficient and inheritable modifications in mouse mitochondrial genome

Critical mutations of mitochondrial DNA (mtDNA) generally lead to maternally inheritable diseases that affect multiple organs and systems; however, it was difficult to alter mtDNA in mammalian cells to intervene in or cure mitochondrial disorders. Recently, the discovery of DddA-derived cytosine base editor (DdCBE) enabled the precise manipulation of mtDNA. To test its feasibility for in vivo use, we selected several sites in mouse mtDNA as DdCBE targets to resemble the human pathogenic mtDNA G-to-A mutations. The efficiency of DdCBE-mediated mtDNA editing was first screened in mouse Neuro-2A cells and DdCBE pairs with the best performance were chosen for in vivo targeting. Microinjection of the mRNAs of DdCBE halves in the mouse zygotes or 2-cell embryo successfully generated edited founder mice with a base conversion rate ranging from 2.48% to 28.51%. When backcrossed with wild-type male mice, female founders were able to transmit the mutations to their offspring with different mutation loads. Off-target analyses demonstrated a high fidelity for DdCBE-mediated base editing in mouse mtDNA both in vitro and in vivo. Our study demonstrated that the DdCBE is feasible for generation of mtDNA mutation models to facilitate disease study and for potential treatment of mitochondrial disorders.

Intra-individual state-dependent comparison of plasma mitochondrial DNA copy number and IL-6 levels in patients with bipolar disorder

BackgroundPatients with bipolar disorder (BD) have increased plasma IL-6 levels, which are higher in depressed BD (dBD) than remitted BD (rBD). However, the mechanism that differentiates the cytokine levels between dBD and rBD is not understood. First, we determined whether brain-derived mtDNA can be detected in plasma using neuron-specific mutant Polg1 transgenic (Tg) mice. Second, we investigated whether the plasma circulating cell-free mitochondrial DNA (ccf-mtDNA) differentiate the cytokine levels between dBD and rBD. MethodsMouse plasma ccf-mtDNA levels were measured using real-time PCR targeting two regions of the mtDNA (CO1 and d-loop) in Tg mice and non-Tg littermates. Human plasma ccf-mtDNA levels were measured using real-time PCR targeting two regions of the mtDNA (ND1 and ND4) and IL-6 levels were evaluated in 10 patients in different states (depressed and remitted) of BD in a longitudinal manner and 10 healthy controls. ResultsThe mouse plasma CO1/D-loop ratio was significantly lower in Tg than non-Tg mice (P = 0.0029). Human plasma ccf-mtDNA copy number, ND4/ND1 ratio, and IL-6 levels were not significantly different between dBD and rBD. Human plasma ccf-mtDNA levels showed a nominal significant correlation with delusional symptoms (P = 0.033, ρ = 0.68). LimitationsA larger sample size is required to generalize the results and to determine whether plasma ccf-mtDNA is associated with systemic inflammation. ConclusionsTg mice revealed that brain-derived mtDNA could be present in peripheral blood. The present findings did not coincide with our hypothesis that plasma ccf-mtDNA differentiates the cytokine levels between dBD and rBD.

A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA.

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational ‘hot-spots’ or ‘cold-spots’. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.

Ancient mitochondrial DNA connects house mice in the British Isles to trade across Europe over three millennia

BackgroundThe earliest records in Britain for the western European house mouse (Mus musculus domesticus) date from the Late Bronze Age. The arrival of this commensal species in Britain is thought to be related to human transport and trade with continental Europe. In order to study this arrival, we collected a total of 16 ancient mouse mandibulae from four early British archaeological sites, ranging from the Late Bronze Age to the Roman period.ResultsFrom these, we obtained ancient mitochondrial DNA (mtDNA) house mouse sequences from eight house mice from two of the sites dating from the Late Bronze to Middle Iron Age. We also obtained five ancient mtDNA wood mouse (Apodemus spp.) sequences from all four sites. The ancient house mouse sequences found in this study were from haplogroups E (N = 6) and D (N = 2). Modern British house mouse mtDNA sequences are primarily characterised by haplogroups E and F and, much less commonly, haplogroup D.ConclusionsThe presence of haplogroups D and E in our samples and the dating of the archaeological sites provide evidence of an early house mouse colonisation that may relate to Late Bronze Age/Iron Age trade and/or human expansion. Our results confirm the hypothesis, based on zooarchaeological evidence and modern mtDNA predictions, that house mice, with haplogroups D and E, were established in Britain by the Iron Age and, in the case of haplogroup E, possibly as early as the Late Bronze Age.

MtDNA-STING Pathway Promotes Necroptosis-Dependent Enterocyte Injury in Intestinal Ischemia Reperfusion

Background: Intestinal ischemia reperfusion (I/R) injury is the important pathogenesis for acute intestinal barrier disruption. The STING signaling is associated with gut homeostasis and barrier integrity. However, the biological function and regulation of STING signaling in intestinal I/R injury are not yet fully understood. As the ligand of STING signaling, the mitochondrial DNA (mtDNA) has been found to be associated with necroptosis. It still remains unknown whether mtDNA-STING signaling triggers intestinal necroptosis in intestinal I/R injury. Methods: The levels of circulating receptor interacting protein kinase 3 (RIPK3) were first collected to explore the role of necroptosis in critically ill patients with intestinal injury. Furthermore, the relationship between mtDNA-STING signaling and necroptosis was explored through establishing a murine model of intestinal I/R injury in wild-type mice and STING knockout mice. Primary intestinal epithelial cells (IECs) were isolated to investigate downstream signaling of STING leading to intestinal necroptosis. Findings: In critically ill patients with intestinal injury, circulating RIPK3 was significantly increased and had a positive correlation with markers of enterocyte injury. Moreover, the levels of circulating mtDNA were also associated with the levels of circulating RIPK3. After the intraperitoneal injection of mtDNA in mice, necroptosis signaling was remarkably activated and the inhibition of necroptosis alleviated mtDNA-induced intestinal injury. Furthermore, STING knockout mice showed an alleviated necroptosis. In intestinal I/R injury, mtDNA released from IECs was released and necroptosis was also triggered, companied with a significant decrease of RIPK3 in the intestine. STING knockout mice markedly attenuated intestinal necroptosis and intestinal I/R injury. Finally, we found that mtDNA-mediated STING signaling triggered necroptosis through synergistic IFN and TNF-α signaling in primary IECs. Interpretation: Our results indicated that mtDNA-STING signaling can contribute to intestinal I/R injury by promoting IEC neroptosis. STING-mediated both IFN and TNF-α signaling can trigger intestinal nercroptosis. Funding Statement: This work was supported by the National Natural Science Foundation of China (81801971, 81772052), Distinguished Scholars Foundation of Jiangsu Province (JCRCB2016006). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The collection of human blood samples was approved by the Institutional Review Board Ethics Committee at Jinling Hospital. Before collecting blood samples, all patients and healthy adult volunteers provided written informed consent. The care and use of the animals were approved by Jinling Hospital Animal Care Committee.

Abstract 397: Myeloma derived mitochondrial DNA activates TLR9 driven pro-tumoral expansion

Abstract Multiple myeloma (MM) is reported to cause 85,000 global deaths per year which is estimated to double by 2040. MM is an incurable malignancy of antibody (Ig) secreting differentiated B cells (plasma cells) characterized by the accumulation and localisation of tumor cells in the bone marrow microenvironment (BMM). Mitochondrial genome (mtDNA) contains CpG DNA repeats and CpG DNA has been shown to activate and induce proliferation of memory B cells to secrete Ig. Here we determine if malignant plasma cells maintain their proliferative advantage by releasing mtDNA into the tumour microenvironment which acts as feedback mechanism to induce further proliferation and Ig production by the malignant plasma cell. Human MM cell lines U266 and MM1S were engrafted into NSG immunocompromised mice. Blood sample from control and engrafted mice were collected at various times and plasma was isolated and spun at 15,000g to remove cell debris and large extracellular vesicles. Human and mouse mtDNA was determine by real-time PCR. Results show that human mtDNA can be detected in the plasma from mice engrafted with U266 and MM1S at 2 weeks and 3 weeks post injection of cells. Next, CpG oligonucleotides were used to determine if MM cells can be activated by mtDNA CpG repeats. Primary MM, U226 and MM1S cells were all treated with CpG and CpG control for 4 and 24 hours. Real-time PCR was then used to detect the pro-survival signature showing that IL-1beta, IL-6 and IL-8 were all upregulated by CpG. We then analysed primary MM and MM cell lines for TLR9 expression by flow cytometry and showed that all samples tested had high levels of cytosolic TLR9 expression. Using TLR9 antagonist (ODN TTAGGG (A151)) we show that primary MM cells and MM cell lines have reduced IL-6 and IL-8 expression and decreased cell proliferation. Finally, U266 and MM1S transduced with TLR9 knockdown lentivirus had reduced engraftment, as measured by bioluminescence, in NSG mice. Here we show that MM release mtDNA in vivo and this can induce a tumour directed proliferative response through the engagement of TLR9. This process is necessary for optimum tumor growth and forms part of the malignant phenotype of MM. Citation Format: Aisha Jibril, Jayna J. Mistry, Prakrit Kumar, Jamie A. Moore, Charlotte Hellmich, Cesar Gomez, Kristian Bowles, Stuart A. Rushworth. Myeloma derived mitochondrial DNA activates TLR9 driven pro-tumoral expansion [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 397.

MtDNA sequence features associated with 'selfish genomes' predict tissue-specific segregation and reversion.

Mitochondrial DNA (mtDNA) encodes cellular machinery vital for cell and organism survival. Mutations, genetic manipulation, and gene therapies may produce cells where different types of mtDNA coexist in admixed populations. In these admixtures, one mtDNA type is often observed to proliferate over another, with different types dominating in different tissues. This ‘segregation bias’ is a long-standing biological mystery that may pose challenges to modern mtDNA disease therapies, leading to substantial recent attention in biological and medical circles. Here, we show how an mtDNA sequence’s balance between replication and transcription, corresponding to molecular ‘selfishness’, in conjunction with cellular selection, can potentially modulate segregation bias. We combine a new replication-transcription-selection (RTS) model with a meta-analysis of existing data to show that this simple theory predicts complex tissue-specific patterns of segregation in mouse experiments, and reversion in human stem cells. We propose the stability of G-quadruplexes in the mtDNA control region, influencing the balance between transcription and replication primer formation, as a potential molecular mechanism governing this balance. Linking mtDNA sequence features, through this molecular mechanism, to cellular population dynamics, we use sequence data to obtain and verify the sequence-specific predictions from this hypothesis on segregation behaviour in mouse and human mtDNA.

Transfer of Xenomitochondria Containing the Entire Mouse Mitochondrial Genome into a Genetically Modified Yeast Expressing Mitochondrial Transcription Factor A.

Recently, it was reported that entire mammalian mtDNA genomes could be transplanted into the mitochondrial networks of yeast, where they were accurately and stably maintained without rearrangement as intact genomes. Here, it was found that engineered mtDNA genomes could be readily transferred to and steadily maintained in the mitochondria of genetically modified yeast expressing the mouse mitochondrial transcription factor A (Tfam), one of the mitochondrial nucleoid proteins. The transferred mtDNA genomes were stably retained in the Tfam-expressing yeast cells for many generations. These results indicated that the engineered mouse mtDNA genomes introduced in yeast mitochondria could be relocated into the mitochondria of other cells and that the transferred genomes could be maintained within a mitochondrial environment that is highly amenable to mimicry of the biological conditions in mammalian mitochondria.

Sequence conservation of mitochondrial (mt)DNA during expansion of clonal mammary epithelial populations suggests a common mtDNA template in CzechII mice.

One major foundation of cancer etiology is the process of clonal expansion. The mechanisms underlying the complex process of a single cell leading to a clonal dominant tumor, are poorly understood. Our study aims to analyze mitochondrial DNA (mtDNA) for somatic single nucleotide polymorphisms (SNPs) variants, to determine if they are conserved throughout clonal expansion in mammary tissues and tumors. To test this hypothesis, we took advantage of a mouse mammary tumor virus (MMTV)-infected mouse model (CzechII). CzechII mouse mtDNA was extracted, from snap-frozen normal, hyperplastic, and tumor mammary epithelial outgrowth fragments. Next generation deep sequencing was used to determine if mtDNA “de novo” SNP variants are conserved during serial transplantation of both normal and neoplastic mammary clones. Our results support the conclusion that mtDNA “de novo” SNP variants are selected for and maintained during serial passaging of clonal phenotypically heterogeneous normal cellular populations; neoplastic cellular populations; metastatic clonal cellular populations and in individual tumor transplants, grown from the original metastatic tumor. In one case, a mammary tumor arising from a single cell, within a clonal hyperplastic outgrowth, contained only mtDNA copies, harboring a deleterious “de novo” SNP variant, suggesting that only one mtDNA template may act as a template for all mtDNA copies regardless of cell phenotype. This process has been attributed to “heteroplasmic-shifting”. A process that is thought to result from selective pressure and may be responsible for pathogenic mutated mtDNA copies becoming homogeneous in clonal dominant oncogenic tissues.

Superoxide Drives PI3 Kinase Mediated Mitochondria Transfer from the Bone Marrow Microenvironment to Hematopoietic Stem Cells in Response to Salmonella Typhimurium

Hematopoietic stem cells (HSC) undergo rapid expansion in response to stress stimuli. Specifically, there is rapid expansion of leukocytes in response to pathogenic bacteria which underpins the mammalian response to infection. Presently, the mechanisms by which HSC metabolism is regulated in response to the challenges of pathogenic stress are not fully understood. Here we investigate the bioenergetic processes which facilitate the HSCs expansion in response to infection. We created 2 animal models to study mitochondrial transfer from bone marrow cells to HSCs. The first is a xenograft humanized mouse model in which human CD34+ cord blood (CB) were transplanted into NSG (huNSG) to study mouse mtDNA transfer into cell sorted humanhematopoietic stem cells (huHSC) (mCD45-, hCD34+, hCD38-, hCD45RA-, hCD90+, hCD49f+). The second took advantage of strain specific SNP in the COX3 and ND3 region between NSG (A/C) and C57BL/6 (G/T) mice. Here we transplanted C57BL/6 derived lineage negative hematopoietic progenitor cells into NSG recipient animals and then the chimeric NSG mice were treated with lipopolysaccharide (LPS). We then isolated the HSCs by cell sorting (LN-, CD117+, Sca1+, CD48-, CD150+ and CD34+) and performed mitochondria analysis on the hematopoietic progenitors. In both transplant models we detected recipient mtDNA in donor HSC post infection (11.2% +/-4.1% of total mtDNA from recipient after LPS treatment).HSC expansion and differentiation (as measured by colony forming cell assay and Ki-67 staining) was shown for Salmonella Typhimurium (S.typhimurium)and LPS treated animals. Seahorse mitochondrial stress analysis confirmed increased oxygen consumption levels in hematopoietic stem and progenitor cells (HSPC) from LPS (2 hours) and S.typhimurium(72 hours) treated C57BL/6. We confirmed that this was not due to mitochondrial biogenesis by showing that TFAM was not upregulated in HSPC in response to LPS at 2 hours. In previous work we have reported that transfer of functional mitochondria from the BM to AML is driven by AML derived NOX2 dependent ROS (Marlein, Blood 2017). To determine if ROS drives mitochondrial transfer from bone marrow cells to HSCs, we treated huNSG with with L-buthionine-sulfoximine (BSO), a GSH biosynthesis inhibitor, for 2 hours. Mouse mtDNA (12.3% +/-4.8) was detected in huHSC from BSO treated huNSG mice but not in the control huHSC. Moreover, the ROS scavenger N-acetyl-cysteine (NAC) was shown to reduce mtDNA transfer in LPS treated mice. We next investigated which cell of the BM donates their mitochondria during stress hematopoiesis. To do this we cultured bone marrow stromal cells (BMSC), osteoblast and macrophages with HSPC in the presence of H2O2. Transfer of mtDNA was observed from BMSC to HSPC, but we did not see mitochondria transfer from either macrophages or osteoblasts to HSPC. In addition, in vivo mitochondrial levels were reduced in the BMSCs (CD105+, CD140a+ CD31-, Ter119-, CD45-) of LPS treated C57BL/6 compared to control animals and this reduction was inhibited by the addition of NAC. Finally, ROS has been shown to drive PtdIns(3,4,5)P3 (PIP3) oxidation of PTEN and subsequent activation of PI3K (Covey, Oncogene 2007). We foundthat AKT phosphorylation was elevated in both the BMSC and the HSC of LPS treated animals when compared to the untreated controls, andin vivoadministration ofidelalisib (a PI3 Kinase delta inhibiting drug), decreased mitochondrial transfer from the BMSC to HSC. Our data indicates that infection induced ROS drives PI3 kinase mediated mitochondrial transfer from the BM microenvironment to HSCs. This process is an early physiologic event in the mammalian response to acute bacterial infection and results in bioenergetic changes which underpin emergency granulopoiesis. Disclosures Bowles: Janssen: Research Funding; Abbvie: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.

Dexamethasone does not prevent malignant cell reintroduction in leukemia patients undergoing ovarian transplant: risk assessment of leukemic cell transmission by a xenograft model.

Does dexamethasone (DXM) incubation avoid the reintroduction of leukemic malignant cells after ovarian tissue retransplantation in vivo? DXM incubation prior to retransplantation of ovarian tissue does not prevent reintroduction of leukemic cells. Retransplantation of cryopreserved ovarian cortex from patients diagnosed with acute lymphoblastic leukemia (ALL) involves a risk of reintroducing malignant cells. DXM treatment is effective at inducing leukemic cell death in vitro. This was an experimental study where ovarian cortex fragments from patients with ALL were randomly allocated to incubation with or without DXM (n = 11/group) and grafted to 22 immunodeficient mice for 6months. In a parallel experiment, 22 immunodeficient mice were injected i.p. with varying amounts of RCH-ACV ALL cells (human leukemia cell line) and maintained for 4months. Cryopreserved ovarian fragments from patients with ALL were exposed in vitro to 0.4μM DXM or basal media (control) prior to xenograft into ovariectomized severe combined immunodeficiency (SCID) mice (experiment 1). After 6months of monitoring, leukemia cell contamination was assessed in ovarian grafts and mouse organs by histology, PCR (presence of mouse mtDNA and absence of p53 were together considered a negative result for the presence of human cells) and detection of immunoglobulin monoclonality and specific ALL markers if present in the patient.In experiment 2, a series of 22 immunodeficient female mice was injected with specific doses of the leukemia cell line RCH-ACV (103 - 5 × 106, n = 4/group) to assess the engraftment competence of the SCID model. ALL metastatic cells were detected, by PCR, in five DXM-treated and one control human ovarian tissue graft as well as in a control mouse liver, although malignant cell infiltration was not detected by histology in any sample after 6months. In total, minimal residual disease was present in three DXM-treated and three control mice.RCH-ACV cells were detected in liver and spleen samples after the injection of as little as 103 cells, although only animals receiving 5 × 106 cells developed clinical signs of disease and metastases. This is an experimental study where the malignant potential of leukemic cells contained in human ovarian tissues has been assessed in immunodeficient mice. These results indicate that DXM incubation prior to retransplantation of ovarian tissue does not prevent reintroduction of leukemic cells. Therefore, caution should be taken in retransplanting ovarian tissue from patients with leukemia until safer systems are developed, as leukemic cells present in ovarian grafts were able to survive, proliferate and migrate after cryopreservation and xenograft. Funded by the Regional Valencian Ministry of Education (PROMETEO/2018/137) and by the Spanish Ministry of Economy and Competitiveness (PI16/FIS PI16/01664 and PTQ-16-08222 for S.H. participation). There are no competing interests.

Intramitochondrial transfer and engineering of mammalian mitochondrial genomes in yeast

Mitochondrial genomes (mtDNA) depend on the nuclear genome with which they have evolved to provide essential replication functions and have been known to replicate as xenotransplants only in the cells of closely related species. We now report that complete mouse mitochondrial genomes can be stably transplanted into the mitochondrial network in yeast devoid of their own mtDNA. Our analyses of these xenomitochondrial yeast cells show that they are accurately replicating intact mouse mtDNA genomes without rearrangement and that these mtDNA genomes have the same overall topology as the mtDNA present in the mouse mitochondrial network (i.e., circular monomers). Moreover, non-mtDNA replication and selection sequences required for maintaining the mitochondrial genomes in bacterial hosts are dispensable in these yeast mitochondria and could be efficiently and seamlessly removed by targeted homologous recombination within the mitochondria. These findings demonstrate that the yeast mtDNA replication system is capable of accurately replicating intact mammalian mtDNA genomes without sequence loss or rearrangement and that yeast mitochondria are a highly versatile host system for engineering complete mammalian mitochondrial genomes.

Stressed Hematopoiesis Induces Mitochondrial Trafficking to Hematopoietic Stem Cells

IntroductionThe bone marrow microenvironment regulates the production of both hematopoietic and non-hematopoietic cells for the maintenance of blood production under normal and stress conditions. Intercellular mitochondrial transfer has recently been reported in an acute myeloid leukemia as well as models of lung inflammation. In the context of acute bacterial infection the hematopoietic system needs to drive the granulocytic response necessary for host survival. Therefore, we hypothesis that under stressed hematopoiesis, mitochondria move from the non-hematopoietic cells of the bone marrow microenvironment to the hematopoietic stem/progenitor cells (HSPC) to rapidly support and sustain the host response to bacterial infection.MethodsC57/BL6 mice were injected with lipopolysaccharide (LPS) for 16 hours or infected with salmonella for 72 hours. The mice were sacrificed after 16 hours and the BM harvested and analyzed using flow cytometry for HSPC mitochondrial content using MitoTracker Green FM. Human cord blood CD34+ cells were engrafted into 3 to 4 week old NSG mice (hu-NSG). After 3 months engraftment of human cord blood CD34+ cells was verified by flow cytometry. Hu-NSG mice were then injected with LPS. HSPC populations were isolated by cell sorting for human CD34, CD38, CD45RA, CD90 and CD49f. Quantification of mitochondrial DNA (mtDNA) transfer was undertaken using Taqman qPCR with species or strain specific probes. Human CD34+ cells obtained from patient cord blood, with informed consent and under approval from the United Kingdom (UK) National Health Service Health Research Authority. Animal experiments were conducted with approval from the UK Home Office and University of East Anglia Animal Welfare and Ethical Review BoardResultsFirst, we quantified mitochondria content in the HSPC populations before and after LPS treatment or salmonella infection and quantified multipotent progenitor cells (MPP), total hematopoietic stem cell (HSC), long-term (LT-HSC) and short-term (ST-HSC) hematopoietic stem cells. MPP, HSC and ST HSC, but not the LT-HSC population, have increased mitochondria content in response to LPS treatment or salmonella infection. This corresponded with an increase frequency of MPP, HSC, and ST HSC. Next, we quantified levels of mitochondrial transfer within the bone marrow by analyzing the percentage of mouse mitochondria in the human HSC and MPP population in response to LPS treatment. Results show that mouse mtDNA was detected in the human HSC and human MPP populations of hu-NSG animals treated with LPS. No mouse mtDNA was detected in human HSC and human MPP populations in untreated hu-NSG animals.ConclusionHere we show that acute bacterial infection and LPS drive mitochondrial transfer from the bone marrow microenvironment to HSPC populations. We do not observe this occurring in comparator baseline unstressed hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Reversing wrinkled skin and hair loss in mice by restoring mitochondrial function

Mitochondrial DNA (mtDNA) depletion is involved in mtDNA depletion syndromes, mitochondrial diseases, aging and aging-associated chronic diseases, and other human pathologies. To evaluate the consequences of depletion of mtDNA in the whole animal, we created an inducible mtDNA-depleter mouse expressing, in the polymerase domain of POLG1, a dominant-negative mutation to induce depletion of mtDNA in various tissues. These mice showed reduced mtDNA content, reduced mitochondrial gene expression, and instability of supercomplexes involved in oxidative phosphorylation (OXPHOS) resulting in reduced OXPHOS enzymatic activities. We demonstrate that ubiquitous depletion of mtDNA in mice leads to predominant and profound effects on the skin resulting in wrinkles and visual hair loss with an increased number of dysfunctional hair follicles and inflammatory responses. Development of skin wrinkle was associated with the significant epidermal hyperplasia, hyperkeratosis, increased expression of matrix metalloproteinases, and decreased expression of matrix metalloproteinase inhibitor TIMP1. We also discovered markedly increased skin inflammation that appears to be a contributing factor in skin pathology. Histopathologic analyses revealed dysfunctional hair follicles. mtDNA-depleter mice also show changes in expression of aging-associated markers including IGF1R, KLOTHO, VEGF, and MRPS5. mtDNA-repleter mice showed that, by turning off the mutant POLG1 transgene expression, mitochondrial function, as well as the skin and hair pathology, is reversed to wild-type level. To our knowledge that restoration of mitochondrial functions can reverse the skin and hair pathology is unprecedented.

Accurate estimation of 5-methylcytosine in mammalian mitochondrial DNA

Whilst 5-methylcytosine (5mC) is a major epigenetic mark in the nuclear DNA in mammals, whether or not mitochondrial DNA (mtDNA) receives 5mC modification remains controversial. Herein, we exhaustively analysed mouse mtDNA using three methods that are based upon different principles for detecting 5mC. Next-generation bisulfite sequencing did not give any significant signatures of methylation in mtDNAs of liver, brain and embryonic stem cells (ESCs). Also, treatment with methylated cytosine-sensitive endonuclease McrBC resulted in no substantial decrease of mtDNA band intensities in Southern hybridisation. Furthermore, mass spectrometric nucleoside analyses of highly purified liver mtDNA preparations did not detect 5-methyldeoxycytidine at the levels found in the nuclear DNA but at a range of only 0.3–0.5% of deoxycytidine. Taken together, we propose that 5mC is not present at any specific region(s) of mtDNA and that levels of the methylated cytosine are fairly low, provided the modification occurs. It is thus unlikely that 5mC plays a universal role in mtDNA gene expression or mitochondrial metabolism.