Mitochondrial Transport Research Articles

Plasmodium falciparum OPA3-like protein (PfOPA3) is essential for maintenance of mitochondrial homeostasis and parasite proliferation.

Metabolic pathways and proteins responsible for maintaining mitochondrial dynamics and homeostasis in the Plasmodium parasite, the causative agent of malaria, remain to be elucidated. Here, we identified and functionally characterized a novel OPA3-like domain-containing protein in P. falciparum (PfOPA3). We show that PfOPA3 is expressed in the intraerythrocytic stages of the parasite and localizes to the mitochondria. Inducible knock-down of PfOPA3 using GlmS ribozyme hindered the normal intraerythrocytic cycle of the parasites; specifically, PfOPA3-iKD disrupted parasite development as well as parasite division and segregation at schizont stages, which resulted in a drastic reduction in the number of merozoites progenies. Parasites lacking PfOPA3 show severe defects in the development of functional mitochondria; the mitochondria showed reduced activity of mtETC but not ATP synthesis, as evidenced by reduced activity of complex III of the mtETC, and increased sensitivity for drugs targeting DHODH as well as complex III, but not to the drugs targeting complex V. Further, PfOPA3 downregulation leads to reduction in the level of mitochondrial proton transport uncoupling protein (PfUCP) to compensate reduced activity of complex III and maintain proton efflux across the inner membrane. The reduced activity of DHODH, which is responsible for pyrimidine biosynthesis required for nuclear DNA synthesis, resulted in a significant reduction in parasite nuclear division and generation of progeny. In conclusion, we show that PfOPA3 is essential for the functioning of mtETC and homeostasis required for the development of functional mitochondria as well as for parasite segregation, and thus PfOPA3 is crucial for parasite survival during blood stages.

Hem25p is required for mitochondrial IPP transport in fungi.

Coenzyme Q (CoQ, ubiquinone) is an essential cellular cofactor composed of a redox-active quinone head group and a long hydrophobic polyisoprene tail. How mitochondria access cytosolic isoprenoids for CoQ biosynthesis is a longstanding mystery. Here, via a combination of genetic screening, metabolic tracing and targeted uptake assays, we reveal that Hem25p-a mitochondrial glycine transporter required for haem biosynthesis-doubles as an isopentenyl pyrophosphate (IPP) transporter in Saccharomyces cerevisiae. Mitochondria lacking Hem25p failed to efficiently incorporate IPP into early CoQ precursors, leading to loss of CoQ and turnover of CoQ biosynthetic proteins. Expression of Hem25p in Escherichia coli enabled robust IPP uptake and incorporation into the CoQ biosynthetic pathway. HEM25 orthologues from diverse fungi, but not from metazoans, were able to rescue hem25∆ CoQ deficiency. Collectively, our work reveals that Hem25p drives the bulk of mitochondrial isoprenoid transport for CoQ biosynthesis in fungi.

SFXN3 is Associated with Poor Clinical Outcomes and Sensitivity to the Hypomethylating Therapy in Non-M3 Acute Myeloid Leukemia Patients.

DNA hypermethylation plays a critical role in the occurrence and progression of acute myeloid leukemia (AML). The mitochondrial serine transporter, SFXN3, is vital for onecarbon metabolism and DNA methylation. However, the impact of SFXN3 on the occurrence and progression of AML has not been reported yet. In this study, we hypothesized that SFXN3 indicates a poor prognosis and suggested tailored treatment for AML patients. We used GEPIA and TCGA repository data to analyze the expression of SFXN3 and its correlation with survival in AML patients. RT-qPCR was used to detect the SFXN3 level in our enrolled AML patients and volunteers. Additionally, Whole Genome Bisulfite Sequencing (WGBS) was used to detect the genomic methylation level in individuals. Through the TCGA and GEPIA databases, we found that SFXN3 was enriched in AML patients, predicting shorter survival. Furthermore, we confirmed that SFXN3 was primarily overexpressed in AML patients, especially non-M3 patients, and that high SFXN3 in non-M3 AML patients was found to be associated with poor outcomes and frequent blast cells. Interestingly, non-M3 AML patients with high SFXN3 levels who received hypomethylating therapy showed a higher CR ratio. Finally, we found that SFXN3 could promote DNA methylation at transcription start sites (TSS) in non-M3 AML patients. These sites were found to be clustered in multiple vital cell functions and frequently accompanied by mutations in DNMT3A and NPM1. In conclusion, SXFN3 plays an important role in the progression and hypermethylation in non-M3 AML patients and could be a potential biomarker for indicating a high CR rate for hypomethylating therapy.

The effect of insulin resistance on extracellular BCAA accumulation and SLC25A44 expression in a myotube model of skeletal muscle insulin resistance.

Insulin resistance is often accompanied by elevated circulating branched-chain amino acids (BCAA). We investigated the effects of insulin resistance on the mitochondrial BCAA transporter, SLC25A44, using a myotube model of insulin resistance. Insulin sensitivity and SLC25A44 expression were assessed via Western blot. Liquid chromatography-mass spectrometry was used to evaluate extracellular BCAA media content. Insulin resistance reduced pAkt activation following insulin stimulation but did not alter SLC25A44 expression. Under select conditions, insulin resistance led to the accumulation of extracellular BCAA.

Phylogenetic Analysis Guides Transporter Protein Deorphanization: A Case Study of the SLC25 Family of Mitochondrial Metabolite Transporters.

Homology search and phylogenetic analysis have commonly been used to annotate gene function, although they are prone to error. We hypothesize that the power of homology search in functional annotation depends on the coupling of sequence variation to functional diversification, and we herein focus on the SoLute Carrier (SLC25) family of mitochondrial metabolite transporters to survey this coupling in a family-wide manner. The SLC25 family is the largest family of mitochondrial metabolite transporters in eukaryotes that translocate ligands of different chemical properties, ranging from nucleotides, amino acids, carboxylic acids and cofactors, presenting adequate experimentally validated functional diversification in ligand transport. Here, we combine phylogenetic analysis to profile SLC25 transporters across common eukaryotic model organisms, from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, to Homo sapiens, and assess their sequence adaptations to the transported ligands within individual subfamilies. Using several recently studied and poorly characterized SLC25 transporters, we discuss the potentials and limitations of phylogenetic analysis in guiding functional characterization.

Mitochondrial transport of catalytic RNAs and targeting of the organellar transcriptome in human cells.

Mutations in the small genome present in mitochondria often result in severe pathologies. Different genetic strategies have been explored, aiming to rescue such mutations. A number of these strategies were based on the capacity of human mitochondria to import RNAs from the cytosol and designed to repress the replication of the mutated genomes or to provide the organelles with wild-type versions of mutant transcripts. However, the mutant RNAs present in mitochondria turned out to be an obstacle to therapy and little attention has been devoted so far to their elimination. Here, we present the development of a strategy to knockdown mitochondrial RNAs in human cells using the transfer RNA-like structure of Brome mosaic virus or Tobacco mosaic virus as a shuttle to drive trans-cleaving ribozymes into the organelles in human cell lines. We obtained a specific knockdown of the targeted mitochondrial ATP6 mRNA, followed by a deep drop in ATP6 protein and a functional impairment of the oxidative phosphorylation chain. Our strategy provides a powerful approach to eliminate mutant organellar transcripts and to analyse the control and communication of the human organellar genetic system.

Acanthamoeba castellanii Uncoupling Protein: A Complete Sequence, Activity, and Role in Response to Oxidative Stress.

Uncoupling proteins (UCPs) are mitochondrial inner membrane transporters that mediate free-fatty-acid-induced, purine-nucleotide-inhibited proton leak into the mitochondrial matrix, thereby uncoupling respiratory substrate oxidation from ATP synthesis. The aim of this study was to provide functional evidence that the putative Acucp gene of the free-living protozoan amoeba, A. castellanii, encodes the mitochondrial protein with uncoupling activity characteristic of UCPs and to investigate its role during oxidative stress. We report the sequencing and cloning of a complete Acucp coding sequence, its phylogenetic analysis, and the heterologous expression of AcUCP in the S. cerevisiae strain InvSc1. Measurements of mitochondrial respiratory activity and membrane potential indicate that the heterologous expression of AcUCP causes AcUCP-mediated uncoupling activity. In addition, in a model of oxidative stress with increased reactive oxygen species levels (superoxide dismutase 1 knockout yeasts), AcUCP expression strongly promotes cell survival and growth. The level of superoxide anion radicals is greatly reduced in the ΔSOD1 strain expressing AcUCP. These results suggest that AcUCP targeted to yeast mitochondria causes uncoupling and may act as an antioxidant system. Phylogenetic analysis shows that the A. castellanii UCP diverges very early from other UCPs, but clearly locates within the UCP subfamily rather than among other mitochondrial anion carrier proteins.

Post-translational modifications and protein quality control of mitochondrial channels and transporters.

Mitochondria play a critical role in energy metabolism and signal transduction, which is tightly regulated by proteins, metabolites, and ion fluxes. Metabolites and ion homeostasis are mainly mediated by channels and transporters present on mitochondrial membranes. Mitochondria comprise two distinct compartments, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM), which have differing permeabilities to ions and metabolites. The OMM is semipermeable due to the presence of non-selective molecular pores, while the IMM is highly selective and impermeable due to the presence of specialized channels and transporters which regulate ion and metabolite fluxes. These channels and transporters are modulated by various post-translational modifications (PTMs), including phosphorylation, oxidative modifications, ions, and metabolites binding, glycosylation, acetylation, and others. Additionally, the mitochondrial protein quality control (MPQC) system plays a crucial role in ensuring efficient molecular flux through the mitochondrial membranes by selectively removing mistargeted or defective proteins. Inefficient functioning of the transporters and channels in mitochondria can disrupt cellular homeostasis, leading to the onset of various pathological conditions. In this review, we provide a comprehensive overview of the current understanding of mitochondrial channels and transporters in terms of their functions, PTMs, and quality control mechanisms.

Triple Therapy with Metformin, Ketogenic Diet, and Metronomic Cyclophosphamide Reduced Tumor Growth in MYCN-Amplified Neuroblastoma Xenografts.

Neuroblastoma (NB) is a childhood cancer in which amplification of the MYCN gene is the most acknowledged marker of poor prognosis. MYCN-amplified NB cells rely on both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) for energy production. Previously, we demonstrated that a ketogenic diet (KD) combined with metronomic cyclophosphamide (CP) delayed tumor growth in MYCN-amplified NB xenografts. The anti-diabetic drug metformin (MET) also targets complex I of the OXPHOS system. Therefore, MET-induced disruptions of mitochondrial respiration may enhance the anti-tumor effect of CP when combined with a KD. In this study, we found that MET decreased cell proliferation and mitochondrial respiration in MYCN-amplified NB cell lines, while the combination of KD, MET, and low-dose CP (triple therapy) also reduced tumor growth and improved survival in vivo in MYCN-amplified NB xenografts. Gene ontology enrichment analysis revealed that this triple therapy had the greatest effect on the transcription of genes involved in fatty acid ß-oxidation, which was supported by the increased protein expression of CPT1A, a key mitochondrial fatty acid transporter. We suspect that alterations to ß-oxidation alongside the inhibition of complex I may hamper mitochondrial energy production, thus explaining these augmented anti-tumor effects, suggesting that the combination of MET and KD is an effective adjuvant therapy to CP in MYCN-amplified NB xenografts.

MiR-122-5p is involved in posttranscriptional regulation of the mitochondrial thiamin pyrophosphate transporter (SLC25A19) in pancreatic acinar cells.

Thiamin (vitamin B1) plays a vital role in cellular energy metabolism/ATP production. Pancreatic acinar cells (PACs) obtain thiamin from circulation and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. TPP is then taken up by the mitochondria via a carrier-mediated process that involves the mitochondrial TPP transporter (MTPPT; encoded by the gene SLC25A19). We have previously characterized different aspects of the mitochondrial carrier-mediated TPP uptake process, but nothing is known about its possible regulation at the posttranscriptional level. We address this issue in the current investigations focusing on the role of miRNAs in this regulation. First, we subjected the human (and rat) 3'-untranslated region (3'-UTR) of the SLC25A19 to three in-silico programs, and all have identified putative binding sites for miR-122-5p. Transfecting pmirGLO-hSLC25A19 3'-UTR into rat PAC AR42J resulted in a significant reduction in luciferase activity compared with cells transfected with pmirGLO-empty vector. Mutating as well as truncating the putative miR-122-5p binding sites in the hSLC25A19 3'-UTR led to abrogation of inhibition in luciferase activity in PAC AR42J. Furthermore, transfecting/transducing PAC AR42J and human primary PACs with mimic of miR-122-5p led to a significant inhibition in the level of expression of the MTPPT mRNA and protein as well as in mitochondrial carrier-mediated TPP uptake. Conversely, transfecting PAC AR42J with an inhibitor of miR-122-5p increased MTPPT expression and function. These findings show, for the first time, that expression and function of the MTPPT in PACs are subject to posttranscriptional regulation by miR-122-5p.NEW & NOTEWORTHY This study shows that the expression and function of mitochondrial TPP transporter (MTPPT) are subject to posttranscriptional regulation by miRNA-122-5p in pancreatic acinar cells.

FASN deficiency induces a cytosol-to-mitochondria citrate flux to mitigate detachment-induced oxidative stress

Fatty acid synthase (FASN) maintains de novo lipogenesis (DNL) to support rapid growth in most proliferating cancer cells. Lipogenic acetyl-coenzyme A (CoA) is primarily produced from carbohydrates but can arise from glutamine-dependent reductive carboxylation. Here, we show that reductive carboxylation also occurs in the absence of DNL. In FASN-deficient cells, reductive carboxylation is mainly catalyzed by isocitrate dehydrogenase-1 (IDH1), but IDH1-generated cytosolic citrate is not utilized for supplying DNL. Metabolic flux analysis (MFA) shows that FASN deficiency induces a net cytosol-to-mitochondria citrate flux through mitochondrial citrate transport protein (CTP). Previously, a similar pathway has been shown to mitigate detachment-induced oxidative stress in anchorage-independent tumor spheroids. We further report that tumor spheroids show reduced FASN activity and that FASN-deficient cells acquire resistance to oxidative stress in a CTP- and IDH1-dependent manner. Collectively, these data indicate that by inducing a cytosol-to-mitochondria citrate flux, anchorage-independent malignant cells can gain redox capacity by trading off FASN-supported rapid growth.

Mitochondrial transplantation rescues neuronal cells from ferroptosis

Ferroptosis is a type of oxidative cell death that can occur in neurodegenerative diseases and involves damage to mitochondria. Previous studies demonstrated that preventing mitochondrial dysfunction can rescue cells from ferroptotic cell death. However, the complexity of mitochondrial dysfunction and the timing of therapeutic interventions make it difficult to develop an effective treatment strategy against ferroptosis in neurodegeneration conditions. In this study, we explored the use of mitochondrial transplantation as a novel therapeutic approach for preventing ferroptotic neuronal cell death. Our data showed that isolated exogenous mitochondria were incorporated into both healthy and ferroptotic immortalized hippocampal HT-22 cells and primary cortical neurons (PCN). The mitochondrial incorporation was accompanied by increased metabolic activity and cell survival through attenuating lipid peroxidation and mitochondrial superoxide production. Further, the function of mitochondrial complexes I, III and V activities contributed to the neuroprotective activity of exogenous mitochondria. Similarly, we have also showed the internalization of exogenous mitochondria in mouse PCN; these internalized mitochondria were found to effectively preserve the neuronal networks when challenged with ferroptotic stimuli. The administration of exogenous mitochondria into the axonal compartment of a two-compartment microfluidic device induced mitochondrial transportation to the cell body, which prevented fragmentation of the neuronal network in ferroptotic PCN. These findings suggest that mitochondria transplantation may be a promising therapeutic approach for protecting neuronal cells from ferroptotic cell death.

The complexities of investigating mitochondria dynamics in multiple sclerosis and mouse models of MS.

Multiple sclerosis (MS) is a demyelinating, degenerating disorder of the central nervous system (CNS) that is accompanied by mitochondria energy production failure. A loss of myelin paired with a deficit in energy production can contribute to further neurodegeneration and disability in patients in MS. Mitochondria are essential organelles that produce adenosine triphosphate (ATP) via oxidative phosphorylation in all cells in the CNS, including neurons, oligodendrocytes, astrocytes, and immune cells. In the context of demyelinating diseases, mitochondria have been shown to alter their morphology and undergo an initial increase in metabolic demand. This is followed by mitochondrial respiratory chain deficiency and abnormalities in mitochondrial transport that contribute to progressive neurodegeneration and irreversible disability. The current methodologies to study mitochondria are limiting and are capable of providing only a partial snapshot of the true mitochondria activity at a particular timepoint during disease. Mitochondrial functional studies are mostly performed in cell culture or whole brain tissue, which prevents understanding of mitochondrial pathology in distinct cell types in vivo. A true understanding of cell-specific mitochondrial pathophysiology of MS in mouse models is required. Cell-specific mitochondria morphology, mitochondria motility, and ATP production studies in animal models of MS will help us understand the role of mitochondria in the normal and diseased CNS. In this review, we present currently used methods to investigate mitochondria function in MS mouse models and discuss the current advantages and caveats with using each technique. In addition, we present recently developed mitochondria transgenic mouse lines expressing Cre under the control of CNS specific promoters to relate mitochondria to disease in vivo.

Transcriptome Highlights Cannabinol Modulation of Mitophagy in a Parkinson's Disease In Vitro Model.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra and the accumulation of α-synuclein aggregates, known as Lewy bodies. It is known that mitochondria dysfunctions, including impaired localization, transport and mitophagy, represent features of PD. Cannabinoids are arising as new therapeutic strategies against neurodegenerative diseases. In this study, we aimed to evaluate the potential protective effects of cannabinol (CBN) pre-treatment in an in vitro PD model, namely retinoic acid-differentiated SH-SY5Y neuroblastoma cells treated with 1-methyl-4-phenylpyridinium (MPP+). With this aim, we performed a transcriptomic analysis through next-generation sequencing. We found that CBN counteracted the loss of cell viability caused by MPP+ treatment. Then, we focused on biological processes relative to mitochondria functions and found that CBN pre-treatment was able to attenuate the MPP+-induced changes in the expression of genes involved in mitochondria transport, localization and protein targeting. Notably, MPP+ treatment increased the expression of the genes involved in PINK1/Parkin mitophagy, while CBN pre-treatment reduced their expression. The results suggested that CBN can exert a protection against MPP+ induced mitochondria impairment.

Loss of ANT1 Increases Fibrosis and Epithelial Cell Senescence in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by progressive lung scarring and remodeling. Although treatments exist that slow disease progression, IPF is irreversible, and there is no cure. Cellular senescence, a major hallmark of aging, has been implicated in IPF pathogenesis, and mitochondrial dysfunction is increasingly recognized as a driver of senescence. Adenine nucleotide translocases (ANTs) are abundant mitochondrial ATP-ADP transporters critical for regulating cell fate and maintaining mitochondrial function. We sought to determine how alterations in ANTs influence cellular senescence in pulmonary fibrosis. We found that SLC25A4 (solute carrier family 25 member 4) (ANT1) and SLC25A5 (ANT2) expression is reduced in the lungs of patients with IPF, particularly within alveolar type II (AT2) cells, by single-cell RNA sequencing and tissue staining. Loss of ANT1 by siRNA in lung epithelial cells resulted in increased senescence markers such as β-galactosidase and p21, with a reduction in the ratio of nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide. Bleomycin-treated ANT1 knockdown cells also had increased senescence markers compared with bleomycin-treated control cells. Loss of ANT1 in AT2 cells resulted in a reduction in alveolar organoid growth, with an increase in p21 by staining. Global loss of ANT1 resulted in worse lung fibrosis and increased senescence in the bleomycin- and asbestos-induced mouse models of pulmonary fibrosis. In summary, loss of ANT1 contributes to IPF pathogenesis through mitochondrial dysfunction, increased senescence, and decreased regenerative capacity of AT2 cells, resulting in enhanced lung fibrosis. Modulation of ANTs presents a new therapeutic avenue that may alter cellular senescence pathways and limit pulmonary fibrosis.

The mitochondrially targeted peptide elamipretide (SS-31) improves ADP sensitivity in aged mitochondria by increasing uptake through the adenine nucleotide translocator (ANT)

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function.Graphical abstractELAM binds directly to ANT and ATP synthase and ELAM treatment improves ADP sensitivity, increases ATP production, and improves physiological function in old muscles. ADP (adenosine diphosphate), ATP (adenosine triphosphate), VDAC (voltage-dependent anion channel), ANT (adenine nucleotide translocator), H+ (proton), ROS (reactive oxygen species), NADH (nicotinamide adenine dinucleotide), FADH2 (flavin adenine dinucleotide), O2 (oxygen), ELAM (elamipretide), –SH (free thiol), –SSG (glutathionylated protein)

Syntaphilin Inactivation Can Enhance Axonal Mitochondrial Transport to Improve Spinal Cord Injury.

Mitochondria are important organelle of eukaryotic cells. They consists of a large number of different proteins that provide most of the ATP and supply power for the growth, function, and regeneration of neurons. Therefore, smitochondrial transport ensures that adequate ATP is supplied for metabolic activities. Spinal cord injury (SCI), a detrimental condition, has high morbidity and mortality rates. Currently, the available treatments only provide symptomatic relief for long-term disabilities. Studies have implicated mitochondrial transport as a critical factor in axonal regeneration. Hence, enhancing mitochondrial transports could be beneficial for ameliorating SCI. Syntaphilin (Snph) is a mitochondrial docking protein that acts as a "static anchor," and its inhibition enhances mitochondrial transports. Therefore, Snph as a key mediator of mitochondrial transports, may contribute to improving axonal regeneration following SCI. Herein, we examine Snph's biological effects and its relation to mitochondrial pathway. Then, we elaborate on mitochondrial transports after SCI, the possible role of Snph in SCI, and some possible therapeutic approaches by Snph.

Armcx1 attenuates secondary brain injury in an experimental traumatic brain injury model in male mice by alleviating mitochondrial dysfunction and neuronal cell death

Armcx1 is highly expressed in the brain and is located in the mitochondrial outer membrane of neurons, where it mediates mitochondrial transport. Mitochondrial transport promotes the removal of damaged mitochondria and the replenishment of healthy mitochondria, which is essential for neuronal survival after traumatic brain injury (TBI). This study investigated the role of Armcx1 and its potential regulator(s) in secondary brain injury (SBI) after TBI. An in vivo TBI model was established in male C57BL/6 mice via controlled cortical impact (CCI). Adeno-associated viruses (AAVs) with Armcx1 overexpression and knockdown were constructed and administered to mice via stereotactic cortical injection. Exogenous miR-223-3p mimic or inhibitor was transfected into cultured cortical neurons, which were then scratched to simulate TBI in vitro. It was found that Armcx1 expression decreased significantly, while miR-223-3p levels increased markedly in peri-lesion tissues after TBI. The overexpression of Armcx1 significantly reduced TBI-induced neurological dysfunction, neuronal cell death, mitochondrial dysfunction, and axonal injury, while the knockdown of Armcx1 had the opposite effect. Armcx1 was potentially a direct target of miR-223-3p. The miR-223-3p mimic obviously reduced the Armcx1 protein level, while the miR-223-3p inhibitor had the opposite effect. Finally, the miR-223-3p inhibitor dramatically improved mitochondrial membrane potential (MMP) and increased the total length of the neurites without affecting branching numbers. In summary, our results suggest that the decreased expression of Armcx1 protein in neurons after experimental TBI aggravates secondary brain injury, which may be regulated by miR-223-3p. Therefore, this study provides a potential therapeutic approach for treating TBI.

Inhibition of multiple uptake transporters in cardiomyocytes/mitochondria alleviates doxorubicin-induced cardiotoxicity

Doxorubicin (DOX) has been widely used to treat various tumors; however, DOX-induced cardiotoxicity limits its utilization. Since high accumulation of DOX in cardiomyocytes/mitochondria is the key reason, we aimed to clarify the mechanisms of DOX uptake and explore whether selectively inhibiting DOX uptake transporters would attenuate DOX accumulation and cardiotoxicity. Our results demonstrated that OCTN1/OCTN2/PMAT (organic cation/carnitine transporter 1/2 or plasma membrane monoamine transporter), especially OCTN2, played crucial roles in DOX uptake in cardiomyocytes, while OCTN2 and OCTN1 contributed to DOX transmembrane transport in mitochondria. Metformin (1–100 μM) concentration-dependently reduced DOX (5 μM for accumulation, 500 nM for cytotoxicity) concentration and toxicity in cardiomyocytes/mitochondria via inhibition of OCTN1-, OCTN2- and PMAT-mediated DOX uptake but did not affect its efflux. Furthermore, metformin (iv: 250 and 500 mg/kg or ig: 50, 100 and 200 mg/kg) could dose-dependently reduce DOX (8 mg/kg) accumulation in mouse myocardium and attenuated its cardiotoxicity. In addition, metformin (1–100 μM) did not impair DOX efficacy in breast cancer or leukemia cells. In conclusion, our study clarified the role of multiple transporters, especially OCTN2, in DOX uptake in cardiomyocytes/mitochondria; metformin alleviated DOX-induced cardiotoxicity without compromising its antitumor efficacy by selective inhibition of multiple transporters mediated DOX accumulation in myocardium/mitochondria.

Mrs4 loss of function in fungi during adaptation to the cystic fibrosis lung.

The genetic disease cystic fibrosis (CF) frequently leads to chronic lung infections by bacteria and fungi. We identified three individuals with CF with persistent lung infections dominated by Clavispora (Candida) lusitaniae. Whole-genome sequencing analysis of multiple isolates from each infection found evidence for selection for mutants in the gene MRS4 in all three distinct lung-associated populations. In each population, we found one or two unfixed, non-synonymous mutations in MRS4 relative to the reference allele found in multiple environmental and clinical isolates including the type strain. Genetic and phenotypic analyses found that all evolved alleles led to loss of function (LOF) of Mrs4, a mitochondrial iron transporter. RNA-seq analyses found that Mrs4 variants with decreased activity led to increased expression of genes involved in iron acquisition mechanisms in both low iron and replete iron conditions. Furthermore, surface iron reductase activity and intracellular iron were much higher in strains with Mrs4 LOF variants. Parallel studies found that a subpopulation of a CF-associated Exophiala dermatitidis infection also had a non-synonymous LOF mutation in MRS4. Together, these data suggest that MRS4 mutations may be beneficial during chronic CF lung infections in diverse fungi, perhaps, for the purposes of adaptation to an iron-restricted environment with chronic infections. IMPORTANCE The identification of MRS4 mutations in Clavispora (Candida) lusitaniae and Exophiala dermatitidis in individuals with cystic fibrosis (CF) highlights a possible adaptive mechanism for fungi during chronic CF lung infections. The findings of this study suggest that loss of function of the mitochondrial iron transporter Mrs4 can lead to increased activity of iron acquisition mechanisms, which may be advantageous for fungi in iron-restricted environments during chronic infections. This study provides valuable information for researchers working toward a better understanding of the pathogenesis of chronic lung infections and more effective therapies to treat them.