Mitochondrial DNA Stimulates TLR9-Dependent Neutrophil Extracellular Trap Formation in Primary Graft Dysfunction.

The immune system is designed to robustly respond to pathogenic stimuli but to be tolerant to endogenous ligands to not trigger autoimmunity. Here, we studied an endogenous damage-associated molecular pattern, mitochondrial DNA (mtDNA), during primary graft dysfunction (PGD) after lung transplantation. We hypothesized that cell-free mtDNA released during lung ischemia-reperfusion triggers neutrophil extracellular trap (NET) formation via TLR9 signaling. We found that mtDNA increases in the BAL fluid of experimental PGD (prolonged cold ischemia followed by orthotopic lung transplantation) and not in control transplants with minimal warm ischemia. The adoptive transfer of mtDNA into the minimal warm ischemia graft immediately before lung anastomosis induces NET formation and lung injury. TLR9 deficiency in neutrophils prevents mtDNA-induced NETs, and TLR9 deficiency in either the lung donor or recipient decreases NET formation and lung injury in the PGD model. Compared with human lung transplant recipients without PGD, severe PGD was associated with high levels of BAL mtDNA and NETs, with evidence of relative deficiency in DNaseI. We conclude that mtDNA released during lung ischemia-reperfusion triggers TLR9-dependent NET formation and drives lung injury. In PGD, DNaseI therapy has a potential dual benefit of neutralizing a major NET trigger (mtDNA) in addition to dismantling pathogenic NETs.

Rodents of Senegal and their role as intermediate hosts of Hydatigera spp. (Cestoda: Taeniidae).

Hydatigera (Cestoda: Taeniidae) is a recently resurrected genus including species seldom investigated in sub-Saharan Africa. We surveyed wild small mammal populations in the areas of Richard Toll and Lake Guiers, Senegal, with the objective to evaluate their potential role as intermediate hosts of larval taeniid stages (i.e. metacestodes). Based on genetic sequences of a segment of the mitochondrial DNA gene cytochrome c oxidase subunit 1 (COI), we identified Hydatigera parva metacestodes in 19 out of 172 (11.0%) Hubert's multimammate mice (Mastomys huberti) and one out of six (16.7%) gerbils (Taterillus sp.) and Hydatigera taeniaeformis sensu stricto metacestodes in one out of 215 (0.5%) Nile rats (Arvicanthis niloticus). This study reports epidemiological and molecular information on H. parva and H. taeniaeformis in West African rodents, further supporting the phylogeographic hypothesis on the African origin of H. parva. Our findings may indicate significant trophic interactions contributing to the local transmission of Hydatigera spp. and other parasites with similar life-cycle mechanisms. We therefore propose that further field investigations of rodent population dynamics and rodent-borne infectious organisms are necessary to improve our understanding of host-parasite associations driving the transmission risks of rodent parasites in West Africa.

Mitochondrial transgene expression via an artificial mitochondrial DNA vector in cells from a patient with a mitochondrial disease.

To achieve mitochondrial gene therapy, developing a mitochondrial transgene expression system that produces therapeutic proteins in mitochondria of disease cells is essential. We previously reported on the design of pCMV-mtLuc (CGG) containing a CMV promotor and a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system, and showed that the mitochondrial transfection of pCMV-mtLuc (CGG) resulted in the efficient production of the Nluc luciferase protein in human HeLa cells. This mitochondrial transfection was achieved using a MITO-Porter, a liposome-based carrier for delivering a cargo to mitochondria via membrane fusion. We report herein that mitochondrial transfection using the MITO-Porter results in mitochondrial transgene expression in G625A fibroblasts obtained from a patient with a mitochondrial disease. We investigated the effect of promoters and the basic structure of pCMV-mtLuc (CGG) on gene expression efficiency, and were able to construct a high performance mitochondrial DNA vector, pCMV-mtLuc (CGG) [hND4] that contains a human mitochondrial endogenous gene. We also constructed an RP/KALA-MITO-Porter composed of the KALA peptide (cell-penetrating peptide) with a mitochondrial RNA aptamer to enhance cellular uptake and mitochondrial targeting. Finally, the mitochondrial transfection of pCMV-mtLuc (CGG) [hND4] in G625A fibroblasts using the RP/KALA-MITO-Porter resulted in strong mitochondrial transgene expression.

Exogenous administration of mitochondrial DNA promotes ischemia reperfusion injury via TLR9-p38 MAPK pathway.

Previous studies have shown a role of mitochondrial DNA (mtDNA) in innate immunity. However, the specific role of mtDNA in acute myocardial infarction remains elusive. This study was designed to examine the damaging effect of mtDNA on cardiomyocytes. H9c2s cells were incubated with purified mtDNA or nuclear DNA with or without pretreatment by chloroquine, an inhibitor of Toll-like receptor 9(TLR9). The cell viability was tested by MTT. To demonstrate the toxicity of mtDNA, mtDNA fragments were injected into rats 10 min before ischemia for 30 min and reperfusion for 24 h. Infarct size was measured by TTC staining. Apoptosis of myocardium was detected by TUNEL staining and caspase-3 activity. The levels of TLR9, p-p38 MAPK, and p38 MAPK were detected by western blotting. The results showed that exogenous mtDNA reduced the viability of H9c2s cells and induced TLR9 expression, caspase 3 activation and p38 mitogen-activated protein kinase (MAPK) phosphorylation. However, these effects were inhibited by chloroquine. In contrast, nuclear DNA did not have these effects. Intravenous injection of mtDNA into rats aggravated ischemia-reperfusion injury and increased infarction area through TLR9-p38 MAPK activation. We concluded that mtDNA released into the circulation by AMI may has detrimental effect on myocardium through aggravating ischemia-reperfusion injury via TLR9-p38 MAPK pathway.

Intra-Peritoneal Administration of Mitochondrial DNA Provokes Acute Lung Injury and Systemic Inflammation via Toll-Like Receptor 9.

The pathogenesis of sepsis is complex. Mitochondrial dysfunction, which is responsible for energy metabolism, intrinsic apoptotic pathway, oxidative stress, and systemic inflammatory responses, is closely related with severe sepsis induced death. Mitochondria DNA (mtDNA) contain un-methylated cytosine phosphate guanine (CpG) motifs, which exhibit immune stimulatory capacities. The aim of this study was to investigate the role and mechanism of mtDNA release on lipopolysaccharide (LPS) induced acute lung injury (ALI) and systemic inflammation. Following LPS injection, plasma mtDNA copies peak at 8 h. Compared with wild-type (WT) mice, mtDNA in toll like receptor 4 knockout (TLR4 KO) mice were significantly decreased. MtDNA intra-peritoneal administration causes apparent ALI as demonstrated by increased lung injury score, bronchoalveolar lavage fluid (BALF) total protein and wet/dry (W/D) ratio; mtDNA injection also directly provokes systemic inflammation, as demonstrated by increased IL-1ß, IL-6, high-mobility group protein B1 (HMGB1) level; while nuclear DNA (nDNA) could not induce apparent ALI and systemic inflammation. However, compared with WT mice, TLR4 KO could not protect from mtDNA induced ALI and systemic inflammation. Specific TLR9 inhibitor, ODN 2088 pretreatment can significantly attenuate mtDNA induced ALI and systemic inflammation, as demonstrated by improved lung injury score, decreased lung wet/dry ratio, BALF total protein concentration, and decreased systemic level of IL-1ß, IL-6 and HMGB1. MtDNA administration activates the expression of p-P38 mitogen-activated protein kinases (MAPK) in lung tissue and specific TLR9 inhibitor pretreatment can attenuate this activation. Thus, LPS-induced mtDNA release occurs in a TLR4-dependent manner, and mtDNA causes acute lung injury and systemic inflammation in a TLR9-dependent and TLR4-independent manner.

Extracellular Mitochondria and Mitochondrial Components Act as Damage-Associated Molecular Pattern Molecules in the Mouse Brain.

Mitochondria and mitochondrial debris are found in the brain's extracellular space, and extracellular mitochondrial components can act as damage associated molecular pattern (DAMP) molecules. To characterize the effects of potential mitochondrial DAMP molecules on neuroinflammation, we injected either isolated mitochondria or mitochondrial DNA (mtDNA) into hippocampi of C57BL/6 mice and seven days later measured markers of inflammation. Brains injected with whole mitochondria showed increased Tnfα and decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation. Some of these effects were also observed in brains injected with mtDNA (decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation), and mtDNA injection also caused several unique changes including increased CSF1R protein and AKT phosphorylation. To further establish the potential relevance of this response to Alzheimer's disease (AD), a brain disorder characterized by neurodegeneration, mitochondrial dysfunction, and neuroinflammation we also measured App mRNA, APP protein, and Aß1-42 levels. We found mitochondria (but not mtDNA) injections increased these parameters. Our data show that in the mouse brain extracellular mitochondria and its components can induce neuroinflammation, extracellular mtDNA or mtDNA-associated proteins can contribute to this effect, and mitochondria derived-DAMP molecules can influence AD-associated biomarkers.

Intratracheal administration of mitochondrial DNA directly provokes lung inflammation through the TLR9-p38 MAPK pathway.

An increasing number of studies have focused on the phenomenon that mitochondrial DNA (mtDNA) activates innate immunity responses. However, the specific role of mtDNA in inflammatory lung disease remains elusive. This study was designed to examine the proinflammatory effects of mtDNA in lungs and to investigate the putative mechanisms. C57BL/6 mice were challenged intratracheally with mtDNA with or without pretreatment with chloroquine. Changes in pulmonary histopathology, cytokine concentrations, and phosphorylation levels of p38 MAPK were assayed at four time points. In in vitro experiments, THP-1 macrophages were pretreated or not pretreated with chloroquine, TLR9 siRNA, p38 MAPK siRNA, or SB203580 and then incubated with mtDNA. The levels of cytokines and p-p38 MAPK were detected by ELISA and Western blot, respectively. The intratracheal administration of mtDNA induced infiltration of inflammatory cells, production of proinflammatory cytokines (including IL-1ß, IL-6, and TNF-α), and activation of p38 MAPK. The chloroquine pretreatment resulted in an abatement of mtDNA-induced local lung inflammation. In vitro experiments showed that the exposure of THP-1 macrophages to mtDNA also led to a significant upregulation of IL-1ß, IL-6, and TNF-α and the activation of p38 MAPK. And these responses were inhibited either by chloroquine and TLR9 siRNA or by SB203580 and p38 MAPK siRNA pretreatment. The intratracheal administration of mtDNA induced a local inflammatory response in the mouse lung that depended on the interactions of mtDNA with TLR9 and may be correlated with infiltrating macrophages that could be activated by mtDNA exposure via the TLR9-p38 MAPK signal transduction pathway.

[MITO-Porter; a cutting-edge technology for mitochondrial gene therapy].

Gene therapy is an attractive strategy, for not only targeting nuclear genome, but the mitochondrial genome as well. Human mitochondrial DNA (mtDNA) encodes 13 subunits of the electron transport chain, 22 tRNAs, and 2 rRNAs and their mutations cause a wide range of mitochondrial diseases. Each cell contains hundreds to thousands of mtDNAs, and in the case of a diseased cell, the mitochondrion possesses both mutant mtDNA and wild-type mtDNA. It is generally accepted that the disease phenotype appears when the proportion of the pathogenic mutant mtDNA exceeds a certain threshold. Therefore, the suppression of mutant mtDNA or supplementing wild-type mtDNA will control the onset of mitochondrial disease. To achieve the transfection of an exogenous therapeutic gene to the mitochondrial matrix where mtDNA is transcribed and translated, it is necessary to transfer cargos through mitochondrial outer and inner membranes. Several methods have been examined for mitochondrial transfection, but a universal, wide-ranging transfection technique has yet not been established. We recently developed a mitochondrial targeting delivery system, namely the MITO-Porter. The MITO-Porter is liposomal nanocarrier with a mitochondrial fusogenic lipid composition. We reported that the MITO-Porter could deliver chemical compounds and proteins to the mitochondrial matrix via membrane fusion. In this review, we report (1) on the pharmacological enhancement of lecithinized superoxide dismutase (PC-SOD) using MITO-Porter, (2) the transcription activation of exogenous DNA by mitochondrial transcription factor A (TFAM), and (3) perspectives on a mitochondrial targeting device.

Progress and prospects: gene therapy for mitochondrial DNA disease.

Defects of the mitochondrial genome cause a wide variety of clinical disorders. Except for rare cases where surgery or transplant is indicated, there is no effective treatment for patients. Genetic-based therapies are consequently being considered. On account of the difficulties associated with mitochondrial (mt) transfection, alternative approaches whereby mitochondrial genes can be engineered and introduced into the nucleus (allotopic expression) are being attempted with some success, at least in cultured cells. Defects in the activities of multi-subunit complexes of the oxidative phosphorylation apparatus have been circumvented by the targeted expression of simple single subunit enzymes from other species (xenotopic expression). Although far from the clinic, these approaches show promise. Similarly, nuclear transfection with genes encoding restriction endonucleases or sequence-specific zinc finger-binding proteins destined for mitochondria has also proved successful in targeting mtDNA-borne pathogenic mutations. This is particularly important, as mutated mtDNA is often found in cells that also contain normal copies of the genome, a situation termed heteroplasmy. Shifting the levels of heteroplasmy towards the normal mtDNA has become the goal of a variety of invasive and non-invasive methods, which are also highlighted in this review.

Development of mitochondrial gene replacement therapy.

Many "classic" mitochondrial diseases have been described that arise from single homoplasmic mutations in mitochondrial DNA (mtDNA). These diseases typically affect nonmitotic tissues (brain, retina, muscle), present with variable phenotypes, can appear sporadically, and are untreatable. Evolving evidence implicates mtDNA abnormalities in diseases such as Alzheimer's, Parkinson's, and type II diabetes, but specific causal mutations for these conditions remain to be defined. Understanding the mtDNA genotype-phenotype relationships and developing specific treatment for mtDNA-based diseases is hampered by inability to manipulate the mitochondrial genome. We present a novel protein transduction technology ("protofection") that allows insertion and expression of the human mitochondrial genome into mitochondria of living cells. With protofection, the mitochondrial genotype can be altered, or exogenous genes can be introduced to be expressed and either retained in mitochondria or be directed to other organelles. Protofection also delivers mtDNA in vivo, opening the way to rational development of mitochondrial gene replacement therapy of mtDNA-based diseases.

Synthesis of trifunctional PNA-benzophenone derivatives for mitochondrial targeting, selective DNA binding, and photo-cross-linking.

Mutations in mitochondrial DNA (mtDNA) cause a variety of human pathologies. In many patients, mutated and wild-type mtDNAs coexist in the same cell, a situation termed mtDNA heteroplasmy. In the absence of standard therapies for these disorders, a genetic strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by the selective hybridization of a nucleic acid derivative, allowing propagation of the wild-type genome and correction of the associated defects. To allow for selective binding under physiological conditions, peptide nucleic acids (PNA) are being used. Two other problems, however, have to be resolved: mitochondrial import and attachment of the PNA to the target DNA to inhibit replication. Mitochondrial localization can be achieved by the addition of a caged lipophilic cation and addition of a photo-cross-linking reagent should facilitate covalent attachment. We therefore report the synthesis of benzophenone-PNA derivatives carrying a triphenylphosphonium moiety and demonstrate irreversible binding selectivity between two DNA molecules that differ by a single nucleotide.

Cationic bolasomes with delocalized charge centers as mitochondria-specific DNA delivery systems.

Since their first discovery during the end of the 1980s, the number of diseases found to be associated with a defect in the mitochondrial genome has grown significantly. However, despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is no satisfactory treatment available for the vast majority of patients. This is largely due to the fact that most of these patients have respiratory chain defects, i.e. defects that involve the final common pathway of oxidative metabolism, making it impossible to bypass the defect by giving alternative metabolic carriers of energy. These objective limitations of conventional biochemical treatment for patients with defects of mtDNA warrant the exploration of gene therapy approaches. However, mitochondrial gene therapy currently appears to be only theoretical and speculative. Any possibility for gene replacement is dependent on the use of a yet unavailable mitochondrial transfection vector. In this review we describe the current state of the development of mitochondrial DNA delivery systems. We also summarize our own efforts in exploring the properties of dequalinium, a cationic bolaamphiphile with delocalized charge centers, for the design of a vector suited for the transport of DNA to mitochondria in living cells.

Selective DNA release from DQAsome/DNA complexes at mitochondria-like membranes.

The number of diseases found to be associated with defects of the mitochondrial genome has grown significantly over the past decade (Wallace 1999). Despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is no satisfactory treatment available for the vast majority of patients and the exploration of gene therapeutic approaches is highly warranted. However, mitochondrial gene therapy still appears only theoretical and speculative. Any possibility for gene replacement depends on the use of a yet unavailable mitochondria-specific transfection vector. Mitochondria-specific vectors must posses two properties: they have to transport DNA to the side of mitochondria; they must not release DNA during endocytosis. Amphiphile compounds with delocalized cationic charge centers such as rhodamine 123 and the bolaamphiphile dequalinium have long been known to accumulate in mitochondria. Sufficient lipophilicity combined with delocalization of the positive charge to reduce the free energy change when moving from an aqueous to a hydrophobic environment are believed to be prerequisite for mitochondrial accumulation in response to the mitochondrial membrane potential. We have recently succeeded in preparing cationic vesicles made of dequalinium that we termed DQAsomes (Weissig et al. 1998a). We have shown that DQAsomes bind and protect DNA against DNase activity (Lasch et al. 1999). Based on the intrinsic property of dequalinium to preferentially accumulate in mitochondria in response to the electrochemical gradient at the mitochondrial membrane, we believe that DQAsomes can serve as a vector to deliver DNA to mitochondria in living cells. As a first step in the development of mitochondria-specific DNA delivery systems, we report here that DQAsome/DNA complexes selectively release DNA at cardiolipin-rich liposomes mimicking both the inner and the outer mitochondrial membrane. We demonstrate that DNA remains tightly associated with DQAsomes in the presence of an excess of anionic lipids other than cardiolipin.