Abstract Mitochondrial dysfunction is a hallmark of cellular aging, but whether age-associated cellular decline can be functionally reversed remains unclear. Here, we applied mitochondrial genome replacement to replicative senescent fibroblasts and aged T cells derived from mice and humans. In senescent fibroblasts, replacement with mitochondria from young cells extended proliferative lifespan, whereas replacement with aged mitochondria accelerated proliferative decline, indicating bidirectional modulation of aging-associated phenotypes. In aged mouse T cells, mitochondrial genome replacement restored proliferative capacity and significantly enhanced antitumor activity following adoptive transfer into tumor-bearing mice. Similarly, mitochondrial genome replacement in aged human T cells enhanced cytokine production and shifted the transcriptomic programs toward a more youthful state. Collectively, these findings identify mitochondrial genetic integrity as a functional regulator of aging-associated cellular states and support the emerging view that mitochondria actively influence cellular aging trajectories.

The detection of antidrug antibodies (ADAs) is important for monitoring patients with Fabry disease who are undergoing enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A (GLA) drugs, as ADAs can cause allergic reactions and reduce therapeutic efficacy. Various immunological methods, particularly enzymelinked immunosorbent assay (ELISA), are widely used to measure serum ADA titers in patients with Fabry disease. However, estimating and comparing results of ELISA is challenging because of the absence of a reference antibody. In this study, we developed a genetically engineered chimeric immunoglobulin G monoclonal antibody with mouse-derived variable regions that can react with recombinant GLA drugs and human constant regions that are necessary for recognition by the enzyme-conjugated antihuman signal antibody. Using this chimeric antibody as a reference, a new ELISA-based test was designed to measure anti-GLA antibody concentrations in serum samples. After validating the method, serum anti-GLA antibody titers were measured in 31 patients with Fabry disease, and three representative patients were monitored for 36 months after ERT initiation, achieving successful results, particularly in patients with high ADA titers.

Sephin1 was discovered as a protein phosphatase inhibitor, and its efficacy against neurodegenerative diseases has been confirmed. There are conflicting reports on whether inhibition of eIF2α dephosphorylation by PP1 holoenzyme with the protein phosphatase 1 regulatory subunit 15 A is the mechanism of action of Sephin1. In the present study, we found that Sephin1 significantly suppressed renal tubular cell death in an animal model of ER stress administered with tunicamycin. CHOP, which plays a central role in the ER stress-induced cell death pathway, requires nuclear translocation to act as a transcription factor to increase the expression of cell death-related genes. Sephin1 markedly suppressed this nuclear translocation of CHOP. To elucidate the molecular mechanism underlying the cell death suppressive effect of Sephin1, we used human renal tubular epithelial cells under ER stress with tunicamycin. Sephin1 reduced intracellular CHOP levels by promoting CHOP phosphorylation at Ser30, which led to protein degradation in UPS. Phosphorylated CHOP is generated by Thr172-phosphorylated activated AMPK, and Sephin1 increased phosphorylated AMPK. Phosphorylated AMPK is inactivated by PP2A through dephosphorylation of its Thr172, and Sephin1 inhibits the formation of the PP2A holoenzyme with the PP2A subunit B isoform delta. These results indicate that inhibition of PP2A holoenzyme formation is the molecular target of Sephin1 in this experimental system.

Peptide drug discovery has great potential, but the cell membrane is a major obstacle when the target is an intracellular protein-protein interaction (PPI). It is difficult to target PPIs with small molecules; indeed, there are no intervention tools that can target any intracellular PPI. In this study, we developed a platform that enables the introduction of peptides into cells via mRNA-based gene delivery. Peptide-length nucleic acids do not enable stable ribosome binding and exhibit little to no translation into protein. In this study, a construct was created in which the sequence encoding dihydrofolate reductase (DHFR) was placed in front of the sequence encoding the target peptide, together with a translation skipping sequence, as a sequence that meets the requirements of promoting ribosome binding and rapid decay of the translated protein. This enabled efficient translation from the mRNA encoding the target protein while preventing unnecessary protein residues. Using this construct, we showed that it can inhibit Drp1/Fis1 binding, one of the intracellular PPIs, which governs mitochondrial fission, an important aspect of mitochondrial dynamics. In addition, it was shown to inhibit pathological hyperfission, normalize mitochondrial dynamics and metabolism, and inhibit apoptosis of the mitochondrial pathway.

Polymicrobial sepsis still has a high mortality rate despite the development of antimicrobial agents, elaborate strategies to protect major organs, and the investment of numerous medical resources. Mitochondrial dysfunction, which acts as the center of energy metabolism, is clearly the basis of pathogenesis. Drugs that act on PGC1α, the master regulator of mitochondrial biosynthesis, have shown useful effects in the treatment of sepsis; therefore, we investigated the efficacy of ZLN005, a PGC1α agonist, and found significant improvement in overall survival in an animal model. The mode of action of this effect was examined, and it was shown that the respiratory capacity of mitochondria was enhanced immediately after administration and that the function of TFEB, a transcriptional regulator that promotes lysosome biosynthesis and mutually enhances PGC1α, was enhanced, as was the physical contact between mitochondria and lysosomes. ZLN005 strongly supported immune defense in early sepsis by increasing lysosome volume and acidity and enhancing cargo degradation, resulting in a significant reduction in bacterial load. ZLN005 rapidly acted on two organelles, mitochondria and lysosomes, against sepsis and interactively linked the two to improve the pathogenesis. This is the first demonstration that acidification of lysosomes by a small molecule is a mechanism of action in the therapeutic strategy for sepsis, which will have a significant impact on future drug discovery.

Mitochondrial dysfunction is observed in various conditions, from metabolic syndromes to mitochondrial diseases. Moreover, mitochondrial DNA (mtDNA) transfer is an emerging mechanism that enables the restoration of mitochondrial function in damaged cells. Hence, developing a technology that facilitates the transfer of mtDNA can be a promising strategy for the treatment of these conditions. Here, we utilized an ex vivo culture of mouse hematopoietic stem cells (HSCs) and succeeded in expanding the HSCs efficiently. Upon transplantation, sufficient donor HSC engraftment was attained in-host. To assess the mitochondrial transfer via donor HSCs, we used mitochondrial-nuclear exchange (MNX) mice with nuclei from C57BL/6J and mitochondria from the C3H/HeN strain. Cells from MNX mice have C57BL/6J immunophenotype and C3H/HeN mtDNA, which is known to confer a higher stress resistance to mitochondria. Ex vivo expanded MNX HSCs were transplanted into irradiated C57BL/6J mice and the analyses were performed at six weeks post transplantation. We observed high engraftment of the donor cells in the bone marrow. We also found that HSCs from the MNX mice could transfer mtDNA to the host cells. This work highlights the utility of ex vivo expanded HSC to achieve the mitochondrial transfer from donor to host in the transplant setting.

Bacterial infections still impose a significant burden on humanity, even though antimicrobial agents have long since been developed. In addition to individual severe infections, the f fatality rate of sepsis remains high, and the threat of antimicrobial-resistant bacteria grows with time, putting us at inferiority. Although tremendous resources have been devoted to the development of antimicrobial agents, we have yet to recover from the lost ground we have been driven into. Looking back at the evolution of treatment for cancer, which, like infectious diseases, has the similarity that host immunity eliminates the lesion, the development of drugs to eliminate the tumor itself has shifted from a single-minded focus on drug development to the establishment of a treatment strategy in which the de-suppression of host immunity is another pillar of treatment. In infectious diseases, on the other hand, the development of therapies that strengthen and support the immune system has only just begun. Among innate immunity, the first line of defense that bacteria encounter after invading the host, the molecular mechanisms of the phagolysosome pathway, which begins with phagocytosis to fusion with lysosome, have been elucidated in detail. Bacteria have a large number of strategies to escape and survive the pathway. Although the full picture is still unfathomable, the molecular mechanisms have been elucidated for some of them, providing sufficient clues for intervention. In this article, we review the host defense mechanisms and bacterial evasion mechanisms and discuss the possibility of host-directed therapy for bacterial infection by intervening in the phagolysosome pathway.

Sepsis, a systemic inflammatory response to pathogenic factors, is a difficult to treat life-threatening condition associated with cytokine and eicosanoid storms and multi-organ damage. Omega-3 polyunsaturated fatty acids, such as eicosapentaenoic (EPA) and docosahexaenoic acid, are the precursors of potent anti-inflammatory lipid mediators, including 17,18-epoxyeicosatetraenoic acid (17,18-EEQ), the main metabolite of EPA generated by cytochrome P450 epoxygenases. Searching for novel therapeutic or preventative agents in sepsis, we tested a metabolically robust synthetic analog of 17,18-EEQ (EEQ-A) for its ability to reduce mortality, organ damage, and pro-inflammatory cytokine transcript level in a mouse model of lipopolysaccharide (LPS)-induced endotoxemia, which is closely related to sepsis. Overall survival significantly improved following preventative EEQ-A administration along with decreased transcript level of pro-inflammatory cytokines. On the other hand, the therapeutic protocol was effective in improving survival at 48 hours but insignificant at 72 hours. Histopathological analyses showed significant reductions in hemorrhagic and necrotic damage and infiltration in the liver. In vitro studies with THP-1 and U937 cells showed EEQ-A mediated repression of LPS-induced M1 polarization and enhancement of IL-4-induced M2 polarization of macrophages. Moreover, EEQ-A attenuated the LPS-induced decline of mitochondrial function in THP-1 cells, as indicated by increased basal respiration and ATP production as well as reduction of the metabolic shift to glycolysis. Taken together, these data demonstrate that EEQ-A has potent anti-inflammatory and immunomodulatory properties that may support therapeutic strategies for ameliorating the endotoxemia.

In cells, mRNA synthesis and decay are influenced by each other, and their balance is altered by either external or internal cues, resulting in changes in cell dynamics. We previously reported that it is important that an array of mRNAs that shape a phenotype are degraded before cellular transitions, such as cellular reprogramming and differentiation. In adipogenesis, the interaction between DDX6 and 4E-T had a definitive impact on the pathway in the processing body (PB). We screened a library of α-helix analogs with an alkaloid-like backbone to identify compounds that inhibit the binding between DDX6 and 4E-T proteins, which occurs between the α-helix of structured and internally disordered proteins. IAMC-00192 was identified as a lead compound. This compound directly inhibited the interaction between DDX6 and 4E-T. IAMC-00192 inhibited the temporal increase in PB formation that occurs during adipogenesis and epithelial-mesenchymal transition (EMT) and significantly suppressed these cellular transitions. In the EMT model, the half-life of preexisting mRNAs in PBs was extended twofold by the compound. The novel inhibitor of RNA decay not only represents a potentially useful tool to analyze in detail the pathological conditions affected by RNA decay and how it regulates the pathological state. The identification of this inhibitor may lead to the discovery of a first-in-class RNA decay inhibitor drug.

In vivo tissue-engineered vascular grafts constructed in the subcutaneous spaces of graft recipients have functioned well clinically. Because the formation of vascular graft tissues depends on several recipient conditions, chemical pretreatments, such as dehydration by ethanol (ET) or crosslinking by glutaraldehyde (GA), have been attempted to improve the initial mechanical durability of the tissues. Here, we compared the effects of short-duration (10 min) chemical treatments on the mechanical properties of tissues. Tubular tissues (internal diameter, 5 mm) constructed in the subcutaneous tissues of beagle dogs (4 weeks, n = 3), were classified into three groups: raw tissue without any treatment (RAW), tissue dehydrated with 70% ET (ET), and tissue crosslinked with 0.6% GA (GA). Five mechanical parameters were measured: burst pressure, suture retention strength, ultimate tensile strength (UTS), ultimate strain (%), and Young’s modulus. The tissues were also autologously re-embedded into the subcutaneous spaces of the same dogs for 4 weeks (n = 2) for the evaluation of histological responses. The burst pressure of the RAW group (1275.9 ± 254.0 mm Hg) was significantly lower than those of ET (2115.1 ± 262.2 mm Hg, p = 0.0298) and GA (2570.5 ± 282.6 mm Hg, p = 0.0017) groups. Suture retention strength, UTS or the ultimate strain did not differ significantly among the groups. Young’s modulus of the ET group was the highest (RAW: 5.41 ± 1.16 MPa, ET: 12.28 ± 2.55 MPa, GA: 7.65 ± 1.18 MPa, p = 0.0185). No significant inflammatory tissue response or evidence of residual chemical toxicity was observed in samples implanted subcutaneously for four weeks. Therefore, short-duration ET and GA treatment might improve surgical handling and the mechanical properties of in vivo tissue-engineered vascular tissues to produce ideal grafts in terms of mechanical properties without interfering with histological responses.

PURPOSE:Autologous pericardium is an ideal material for cardiovascular reconstruction including pulmonary artery plasty. Despite the fact that dehydration by ethanol has been used to improve its surgical handling, the effects of the ethanol on mechanical properties of the pericardium have not been previously investigated. The effects of short-duration ethanol dehydration on the mechanical properties of porcine pericardium were evaluated.METHODS:Porcine pericardia (n = 3) were separated into three groups: the raw group with no treatments (RAW), the group immersed in 70% ethanol for 10 min (ET group), and the group immersed in 0.6% glutaraldehyde for 10 min (GA). We measured five parameters of mechanical properties as specified in ISO 7198.RESULTS:ET treatment improved surgical handling as well as GA treatment. There were no significant differences in burst pressure (P = 0.639), suture retention strength (P = 0.529), ultimate tensile strength (UTS; P = 0.486), or Young's modulus (P = 0.408). Only the ultimate strain of the GA group was significantly higher among the three groups (RAW: 33.34% ± 2.02%, ET: 37.48% ± 1.84%, GA: 44.74% ± 2.87%; P = 0.046).CONCLUSIONS:Short-duration ethanol dehydration did not compromise its mechanical properties while maintaining its surgical handling improvements.

Cell transplantation is expected to be another strategy to treat lysosomal diseases, having several advantages compared to enzyme replacement therapy, such as continuous enzyme secretion and one-time treatment to cure diseases. However, cell transplantation for lysosomal diseases holds issues to be resolved for the clinical field. In this study, we developed a new ex vivo gene therapy platform using a transplant pack, which consists of a porous membrane made of ethylene-vinyl alcohol in the pack-type and spheroids with scaffolds. These membranes have countless pores of less than 0.1 µm2 capable of secreting proteins, including alpha-galactosidase enzyme, and segregating the contents from the host immune system. When the packs were subcutaneously transplanted into the backs of green fluorescent protein (GFP) mice, no GFP-positive cells migrated to the transplanted pack in either autogenic or allogenic mice. The transplanted cells in the pack survived for 28 days after transplantation. When cells overexpressing alpha-galactosidase were used as donor cells for the packs and implanted into Fabry disease model mice, the accumulation of the alpha-galactosidase enzyme was also observed in the livers. In this study, we reported a new ex vivo therapeutic strategy combining macroencapsulation and cellular spheroids with scaffolds. This pack, macroencapsulated spheroids with scaffolds, can also be applied to other types of lysosomal diseases by modifying genes of interest.

Mitochondrial diseases currently have no cure regardless of whether the cause is a nuclear or mitochondrial genome mutation. Mitochondrial dysfunction notably affects a wide range of disorders in aged individuals, including neurodegenerative diseases, cancers, and even senescence. Here, we present a procedure to generate mitochondrial DNA-replaced somatic cells with a combination of a temporal reduction in endogenous mitochondrial DNA and coincubation with exogeneous isolated mitochondria. Heteroplasmy in mitochondrial disease patient-derived fibroblasts in which the mutant genotype was dominant over the wild-type genotype was reversed. Mitochondrial disease patient-derived fibroblasts regained respiratory function and showed lifespan extension. Mitochondrial membranous components were utilized as a vehicle to deliver the genetic materials into endogenous mitochondria-like horizontal genetic transfer in prokaryotes. Mitochondrial DNA-replaced cells could be a resource for transplantation to treat maternal inherited mitochondrial diseases.

Abstract Background We have previously applied in vivo tissue‐engineered vascular grafts constructed in patients’ subcutaneous spaces. However, since the formation of these vascular grafts depends on host health, their application is challenging in patients with suppressed regenerative ability. Therefore, the allogeneic implantation of grafts from healthy donors needs to be evaluated. This study aimed to fabricate allogeneic cardiovascular grafts in animals. Materials and methods Silicone rod molds were implanted into subcutaneous pouches in dogs; the implants, along with surrounding connective tissues, were harvested after four weeks. Tubular connective tissues were decellularized and stored before they were cut open, trimmed to elliptical sheets, and implanted into the common carotid arteries of another dog as vascular patches (n = 6); these were resected and histologically evaluated at 1, 2, and 4 weeks after implantation. Results No aneurysmal changes were observed by echocardiography. Histologically, we observed neointima formation on the luminal graft surface and graft wall cell infiltration. At 2 and 4 weeks after implantation, α‐SMA‐positive cells were observed in the neointima and graft wall. At 4 weeks after implantation, the endothelial lining was observed at the grafts’ luminal surfaces. Conclusion Our data suggest that decellularized connective tissue membranes can be prepared and stored for later use as allogeneic cardiovascular grafts.

The RNA decay pathway plays key regulatory roles in cell identities and differentiation processes. Although adipogenesis is transcriptionally and epigenetically regulated and has been thoroughly investigated, how RNA metabolism that contributes to the stability of phenotype-shaping transcriptomes participates in differentiation remains elusive. In this study, we investigated Ddx6, an essential component of processing bodies (PBs) that executes RNA decay and translational repression in the cytoplasm and participates in the cellular transition of reprogramming. Upon adipogenic induction, Ddx6 dynamically accumulated to form PBs with a binding partner, 4E-T, at the early phase prior to emergence of intracellular lipid droplets. In contrast, preadipocytes with Ddx6 knockout (KO) or 4E-T knockdown (KD) failed to generate PBs, resulting in significant suppression of adipogenesis. Transcription factors related to preadipocytes and negative regulators of adipogenesis that were not expressed under adipogenic stimulation were maintained in Ddx6-KO and 4E-T-KD preadipocytes under adipogenic induction. Elimination of Dlk1, a major negative regulator of adipogenesis, in 3T3L1 Ddx6-KO cells did not restore adipogenic differentiation capacity to any extent. Similar to murine cells, human primary mesenchymal stem cells, which can differentiate into adipocytes upon stimulation with adipogenic cocktails, required DDX6 to maturate into adipocytes. Therefore, RNA decay of the entire parental transcriptome, rather than removal of a strong negative regulator, could be indispensable for adipogenesis.

Acute myocardial infarction is a leading cause of death among single organ diseases. Despite successful reperfusion therapy, ischaemia reperfusion injury (IRI) can induce oxidative stress (OS), cardiomyocyte apoptosis, autophagy and release of inflammatory cytokines, resulting in increased infarct size. In IRI, mitochondrial dysfunction is a key factor, which involves the production of reactive oxygen species, activation of inflammatory signalling cascades or innate immune responses, and apoptosis. Therefore, intercellular mitochondrial transfer could be considered as a promising treatment strategy for ischaemic heart disease. However, low transfer efficiency is a challenge in clinical settings. We previously reported uptake of isolated exogenous mitochondria into cultured cells through co-incubation, mediated by macropinocytosis. Here, we report the use of transactivator of transcription dextran complexes (TAT-dextran) to enhance cellular uptake of exogenous mitochondria and improve the protective effect of mitochondrial replenishment in neonatal rat cardiomyocytes (NRCMs) against OS. TAT-dextran-modified mitochondria (TAT-Mito) showed a significantly higher level of cellular uptake. Mitochondrial transfer into NRCMs resulted in anti-apoptotic capability and prevented the suppression of oxidative phosphorylation in mitochondria after OS. Furthermore, TAT-Mito significantly reduced the apoptotic rates of cardiomyocytes after OS, compared to simple mitochondrial transfer. These results indicate the potential of mitochondrial replenishment therapy in OS-induced myocardial IRI.

Fabry disease is caused by a decrease in or loss of the activity of alpha-galactosidase, which causes its substrates globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) to accumulate in cells throughout the body. This accumulation results in progressive kidney injury due to glomerulosclerosis and in heart failure due to hypertrophy. Enzyme replacement therapy (ERT) has been used as the standard therapy for Fabry disease, but it causes a significant financial burden, and regular administration is inconvenient for patients. Because of the short half-life of alpha-galactosidase in vivo, therapeutic methods that can supplement or replace ERT are expected to involve continuous release of alpha-galactosidase, even at low doses. Cell transplantation therapy is one of these methods; however, its use has been hindered by the short-term survival of transplanted cells. CellSaic technology, which utilizes cell spheroids that form after cells are seeded simultaneously with a recombinant collagen peptide scaffold called a μ-piece, has been used to improve cell survival upon implantation. In this study, syngeneic murine embryonic fibroblasts were used to generate CellSaic that were transplanted into Fabry mice. These spheroids survived for 28 days in the renal subcapsular space with forming blood vessels. These results indicate CellSaic technology could be a platform to promote cellular graft survival and may facilitate the development of cell transplantation methods for lysosomal diseases.

[This corrects the article DOI: 10.1371/journal.pone.0177988.].

Mitochondrial heteroplasmy, which fundamentally means intracellular heterogeneity of mitochondrial DNA (mtDNA), has been measured in a group of cells, regardless of intercellular heterogeneity. Ordinal methods for mitochondrial heteroplasmy cannot discriminate between an intercellular homogenic population composed of cells with similar intracellular heterogeneity for mtDNA and an intercellular heterogenic population composed of cells with different rates of mutated mtDNA. A high-throughput method to determine mitochondrial heteroplasmy in a single cell was developed by using droplet digital PCR with TaqMan polymerase in this study. This technique revealed that there are three different cell populations of cultured fibroblasts derived from patients with mitochondrial disease carrying a mutation in the mtDNA; cells with homoplasmy of either mutated or healthy mtDNA; and cells mixed with mutated and healthy mtDNA. The presence of intercellular heterogeneity, even in uniformed cultured fibroblasts, suggests that heterogeneity should exist among different kinds of cells. The diagnosis of intercellular heterogeneity with respect to mitochondrial heteroplasmy by this methodology could provide novel insight into developing a treatment strategy for mitochondrial diseases.

Mitochondrial transfer has been recognized to play a role in a variety of processes, ranging from fertilization to cancer and neurodegenerative diseases as well as mammalian horizontal gene transfer. It is achieved through either exogeneous or intercellular mitochondrial transfer. From the viewpoint of evolution, exogeneous mitochondrial transfer is quite akin to the initial process of symbiosis between α-protobacterium and archaea, although the progeny have developed more sophisticated machinery to engulf environmental materials, including nutrients, bacteria, and viruses. A molecular-based knowledge of endocytosis, including macropinocytosis and endosomal escape involving bacteria and viruses, could provide mechanistic insights into exogeneous mitochondrial transfer. We focus on exogeneous mitochondrial transfer in this review to facilitate the clinical development of the use of isolated mitochondria to treat various pathological conditions. Several kinds of novel procedures to enhance exogeneous mitochondrial transfer have been developed and are summarized in this review.