Mitochondrial Cluster Research Articles

A novel role of the mitochondrial iron-sulfur cluster assembly protein ISCU-1/ISCU in longevity and stress response.

As an ancient cellular co-factor ubiquitously present in all domains of life, nearly all iron-sulfur ([Fe-S]) clusters are assembled in the mitochondrion. Although multiple mitochondrion-derived signalings are known to be key players in longevity regulation, whether the mitochondrial [Fe-S] cluster assembly machinery modulates lifespan is previously unknown. Here, we find that ISCU-1, the C. elegans ortholog of the evolutionarily conserved iron-sulfur cluster (ISC) assembly machinery central protein ISCU, regulates longevity and stress response. Specifically, ISCU-1 accelerates aging in the intestine. Moreover, we identify the Nrf2 transcription factor SKN-1 and a nuclear hormone receptor NHR-49 as the downstream factors of ISCU-1. Lastly, a mitochondrial outer membrane protein phosphatase PGAM-5 appears to link ISCU-1 to SKN-1 and NHR-49 in lifespan regulation. Together, we have identified a novel function of mitochondrial ISC assembly machinery in longevity modulation and stress response.

Mitochondrial Enzymes of the Urea Cycle Cluster at the Inner Mitochondrial Membrane.

Mitochondrial enzymes involved in energy transformation are organized into multiprotein complexes that channel the reaction intermediates for efficient ATP production. Three of the mammalian urea cycle enzymes: N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase 1 (CPS1), and ornithine transcarbamylase (OTC) reside in the mitochondria. Urea cycle is required to convert ammonia into urea and protect the brain from ammonia toxicity. Urea cycle intermediates are tightly channeled in and out of mitochondria, indicating that efficient activity of these enzymes relies upon their coordinated interaction with each other, perhaps in a cluster. This view is supported by mutations in surface residues of the urea cycle proteins that impair ureagenesis in the patients, but do not affect protein stability or catalytic activity. We find the NAGS, CPS1, and OTC proteins in liver mitochondria can associate with the inner mitochondrial membrane (IMM) and can be co-immunoprecipitated. Our in-silico analysis of vertebrate NAGS proteins, the least abundant of the urea cycle enzymes, identified a protein-protein interaction region present only in the mammalian NAGS protein—“variable segment,” which mediates the interaction of NAGS with CPS1. Use of super resolution microscopy showed that NAGS, CPS1 and OTC are organized into clusters in the hepatocyte mitochondria. These results indicate that mitochondrial urea cycle proteins cluster, instead of functioning either independently or in a rigid multienzyme complex.

Bola3 Regulates Beige Adipocyte Thermogenesis via Maintaining Mitochondrial Homeostasis and Lipolysis

Mitochondrial iron-sulfur (Fe-S) cluster is an important cofactor for the maturation of Fe-S proteins, which are ubiquitously involved in energy metabolism; however, factors facilitating this process in beige fat have not been established. Here, we identified BolA family member 3 (Bola3), as one of 17 mitochondrial Fe-S cluster assembly genes, was the most significant induced gene in the browning program of white adipose tissue. Using lentiviral-delivered shRNA in vitro, we determined that Bola3 deficiency inhibited thermogenesis activity without affecting lipogenesis in differentiated beige adipocytes. The inhibition effect of Bola3 knockdown might be through impairing mitochondrial homeostasis and lipolysis. This was evidenced by the decreased expression of mitochondria related genes and respiratory chain complexes, attenuated mitochondrial formation, reduced mitochondrial maximal respiration and inhibited isoproterenol-stimulated lipolysis. Furthermore, BOLA3 mRNA levels were higher in human deep neck brown fat than in the paired subcutaneous white fat, and were positively correlated with thermogenesis related genes (UCP1, CIDEA, PRDM16, PPARG, COX7A1, and LIPE) expression in human omental adipose depots. This study demonstrates that Bola3 is associated with adipose tissue oxidative capacity both in mice and human, and it plays an indispensable role in beige adipocyte thermogenesis via maintaining mitochondrial homeostasis and adrenergic signaling-induced lipolysis.

Tissue Imaging and Quantification Relying on Endogenous Contrast.

Cell-matrix interactions play an important role in regulating a variety of essential processes in multicellular organisms, and are closely associated with numerous diseases. Modified interactions have major effects upon key features of both cells and extracellular matrix (ECM), and a thorough understanding of changes in these features can lead to critically important insights of diseases as well as the identification of effective therapeutic targets. Here, we summarize recent advances in quantitative, optical imaging of cellular metabolism and ECM spatial organization using endogenous sources of contrast. Specifically, we focus on the two-photon excited fluorescence (TPEF) imaging of autofluorescent cellular coenzymes, NAD(P)H and FAD, for the extraction of metabolic information described by optical biomarkers including cellular redox state, NAD(P)H fluorescence lifetime, and mitochondrial clustering. We show representative applications in assessing adipose tissue function and detecting malignant lesions in human skin, and further demonstrate that a combination of these optical metrics can provide complementary insights into the underlying biological mechanisms. In addition, we review the development of quantitative analysis methods to extract spatial orientation and organization metrics of collagen fibers, a major ECM component, and demonstrate applications of these approaches in two and three dimensions in several diseases, including would healing, osteoarthritis and cancer, as well as assessments of matrix remodeling in hormone-regulated engineered breast tissues. Finally, we summarize this chapter and discuss important research directions that we expect will evolve in the near future.

Mammalian mitochondrial iron-sulfur cluster biogenesis and transfer and related human diseases.

As a cofactor, iron-sulfur (Fe-S) cluster binds to proteins or enzymes that play important roles in various important biological processes, including DNA synthesis and repair, mitochondrial function, gene transcription and translation. In mammals, the core components involved in Fe-S cluster biosynthesis are considered to include the scaffold protein ISCU, cysteine desulfurase NFS1 and its accessory proteins ISD11 and ACP, and frataxin (FXN). Proteins involved in Fe-S cluster transfer have been found to include HSC20/HSPA9, as chaperone system, and Fe-S cluster carriers. The biosynthesis and transfer of Fe-S clusters to Fe-S recipients require fine-tune regulation. Recently, significant progress has been made in the structure and mechanism of mitochondrial Fe-S biosynthesis and transfer. Based on, especially, the development of DNA sequencing technology, bioinformatics, and gene editing technology, diseases caused by mutations of Fe-S cluster-related genes have been revealed in recent years, promoting the rapid development in the field of Fe-S and human health. This review focuses on the function of genes involved in Fe-S cluster biosynthesis and transfer and on the diseases caused by the mutations of the related genes. Finally, some questions we are facing are raised, new hypotheses presented, and the perspectives discussed.

4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins

Numerous iron-sulfur (Fe-S) proteins with diverse functions are present in the matrix and respiratory chain complexes of mitochondria. Although [4Fe-4S] clusters are the most common type of Fe-S cluster in mitochondria, the molecular mechanism of [4Fe-4S] cluster assembly and insertion into target proteins by the mitochondrial iron-sulfur cluster (ISC) maturation system is not well-understood. Here we report a detailed characterization of two late-acting Fe-S cluster-carrier proteins from Arabidopsis thaliana, NFU4 and NFU5. Yeast two-hybrid and bimolecular fluorescence complementation studies demonstrated interaction of both the NFU4 and NFU5 proteins with the ISCA class of Fe-S carrier proteins. Recombinant NFU4 and NFU5 were purified as apo-proteins after expression in Escherichia coli. In vitro Fe-S cluster reconstitution led to the insertion of one [4Fe-4S]2+ cluster per homodimer as determined by UV-visible absorption/CD, resonance Raman and EPR spectroscopy, and analytical studies. Cluster transfer reactions, monitored by UV-visible absorption and CD spectroscopy, showed that a [4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer is effective in transferring [4Fe-4S]2+ clusters to both NFU4 and NFU5 with negligible back reaction. In addition, [4Fe-4S]2+ cluster-bound ISCA1a/2, NFU4, and NFU5 were all found to be effective [4Fe-4S]2+ cluster donors for maturation of the mitochondrial apo-aconitase 2 as assessed by enzyme activity measurements. The results demonstrate rapid, unidirectional, and quantitative [4Fe-4S]2+ cluster transfer from ISCA1a/2 to NFU4 or NFU5 that further delineates their respective positions in the plant ISC machinery and their contributions to the maturation of client [4Fe-4S] cluster-containing proteins.

Mitoptosis, Twenty Years After.

In 1999 V. P. Skulachev proposed the term "mitoptosis" to refer to the programmed elimination of mitochondria in living cells. According to the initial thought, mitoptosis serves to protect cells from malfunctioning of the damaged mitochondria. At the same time, a new mechanism of the complete mitochondria elimination was found under the conditions of massive mitochondrial damage associated with oxidative stress. In this experimental model, mitochondrial cluster formation in the perinuclear region leads to the formation of "mitoptotic body" surrounded by a single-layer membrane and subsequent release of mitochondria from the cell. Later, it was found that mitoptosis plays an important role in various normal and pathological processes that are not necessarily associated with the mitochondrial damage. It was found that mitoptosis takes place during cell differentiation, self-maintenance of hematopoietic stem cells, metabolic remodelling, and elimination of the paternal mitochondria in organisms with the maternal inheritance of the mitochondrial DNA. Moreover, the associated with mitoptosis release of mitochondrial components into the blood may be involved in the transmission of signals between cells, but also leads to the development of inflammatory and autoimmune diseases. Mitoptosis can be attributed to the asymmetric inheritance of mitochondria in the division of yeast and some animal cells, when the defective mitochondria are transferred to one of the newly formed cells. Finally, a specific form of mitoptosis appears to be selective elimination of mitochondria with deleterious mutations in whole follicular ovarian cells in mammals. During formation of the primary follicle, the mitochondrial DNA copy number is significantly reduced. After division, the cells that receive predominantly mitochondria with deleterious mutations in their mtDNA die, thereby reducing the likelihood of transmission of these mutations to offspring. Further study of the mechanisms of mitoptosis in normal and pathological conditions is important both for understanding the processes of development and aging, and for designing therapeutic approaches for inflammatory, neurodegenerative and other diseases.

Archaeobiogeography of extinct rice rats (Oryzomyini) in the Lesser Antilles during the Ceramic Age (500 BCE–1500 CE)

During the Ceramic Age (500 BCE–1500 CE), Lesser Antilles rice rats (Tribe Oryzomyini) made up a significant portion of the diet of Caribbean islanders. Archaeological excavations across the archipelago resulted to the discovery of large quantities of remains from to these now extinct taxa. It offers a unique opportunity to investigate the past biogeography of this taxon of high cultural and ecological importance. We have studied 1140 first lower molars originating from 40 archaeological sites across eleven islands of the Lesser Antilles archipelago using two-dimensional geometric morphometric approaches to establish spatiotemporal patterns relying on phenotypic variations. This study identified three morphological groups, present in all chrono-cultural periods, that were geographically restricted and consistent with published ancient mitochondrial DNA clusters. These three geographically-separate groups likely represent three distinct genera of rice rats. The first group includes specimens from the North of the archipelago (Saint-Martin, Saba, Saint-Eustatius, Saint-Kitts, and Nevis) and likely referable to as Pennatomys sp.; the second, occurring in the South (Martinique), is assigned to Megalomys desmarestii; and the third corresponds to specimens from the center of the Lesser Antilles (Antigua, Barbuda, Marie-Galante, and Guadeloupe) and likely corresponds to Antillomys sp. These oryzomyine morphotypes are present during all studied periods and support an older presence of these rodents in the region. Our results are congruent with ancient DNA studies that favor the hypothesis of a natural introduction of the group in the archipelago before settlement of human populations. Moreover, the observed phenotypic homogeneity and stability over the 2000 years of Pre-Columbian occupation suggests that rice rats were not part of long-distance inter-island exchanges by humans. Instead, rice rat human consumption was likely based on in-situ hunting of local populations.

MAVS is energized by Mff which senses mitochondrial metabolism via AMPK for acute antiviral immunity

Mitochondria are multifunctional organelles that produce energy and are critical for various signaling pathways. Mitochondrial antiviral signaling (MAVS) is a mitochondrial outer membrane protein essential for the anti-RNA viral immune response, which is regulated by mitochondrial dynamics and energetics; however, the molecular link between mitochondrial metabolism and immunity is unclear. Here we show in cultured mammalian cells that MAVS is activated by mitochondrial fission factor (Mff), which senses mitochondrial energy status. Mff mediates the formation of active MAVS clusters on mitochondria, independent of mitochondrial fission and dynamin-related protein 1. Under mitochondrial dysfunction, Mff is phosphorylated by the cellular energy sensor AMP-activated protein kinase (AMPK), leading to the disorganization of MAVS clusters and repression of the acute antiviral response. Mff also contributes to immune tolerance during chronic infection by disrupting the mitochondrial MAVS clusters. Taken together, Mff has a critical function in MAVS-mediated innate immunity, by sensing mitochondrial energy metabolism via AMPK signaling.

Coordinated Mis-Splicing of Multiple Mitochondrial Iron Metabolism Genes Causes Ring Sideroblast Formation in SF3B1-Mutant MDS

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous gain-of-function mutations in a core U2 spliceosome factor SF3B1 are present in ~25% of myelodysplastic syndrome (MDS) patients. Strikingly, SF3B1 mutations are present in ~80% of MDS with ring sideroblasts (MDS-RS) patients suggesting a causal connection between mutant SF3B1 and ring sideroblasts (RS), erythroid precursors with iron-laden mitochondria (Yoshida et al. Nature 2011, Papaemmanuil et al.NEJM 2011). However, the mechanism by which SF3B1 mutations cause RS formation remains poorly understood since existing models of SF3B1-mutant MDS do not recapitulate RS formation in vitro. We have established an induced pluripotent stem cell (iPSC) model of MDS-RS that recapitulates mutant SF3B1-mediated mis-splicing and in vitro ring sideroblast formation. We reprogrammed SF3B1-mutant and SF3B1-wild-type iPSCs from individual MDS-RS patients enabling internal normalization to the isogenic normal clone (Hsu et al. Blood 2019). We established expandable multipotential HPC lines by conditional expression of five transcription factors (5F-HPC), followed by an 18-day erythroid differentiation. We monitored RS formation during the erythroid differentiation of 5F-HPCs using Prussian blue histological staining. RS formation increased during terminal erythropoiesis and peaked at 25-40% on day 18 in SF3B1-mutant lines, whereas SF3B1-wild-type lines showed no detectable RS formation. The frequency of RS was similar in isogenic SF3B1-only and SF3B1/EZH2 co-mutant cells suggesting that the mutant SF3B1 is sufficient to drive RS formation. To identify mis-splicing events that contribute to RS formation, we performed RNA-sequencing and splicing analysis of SF3B1-mutant and SF3B1-wild-type iPSCs at three stages of erythroid differentiation: CD34+ progenitor, CD71+ early erythroblast, and CD71+Glycophorin A+ erythroblast. Global isoform usage was dramatically altered during erythropoiesis, but was more similar in stage-matched SF3B1-mutant and SF3B1-wild-type cells suggesting that mutant SF3B1 selectively mis-splices a subset of transcripts. We identified 2300 transcripts with >10% mis-splicing in SF3B1-mutant lines, and only 120 transcripts with more significant >40% mis-splicing. Of these, TMEM14C and PPOX, inner mitochondrial membrane components of the heme synthesis pathway were strongly mis-spliced throughout erythroid differentiation, consistent with previous studies (Conte et al.BJHaem 2015, Shiozawa et al. Nat Commun 2018). The transcript levels of PPOX but not TMEM14C were reduced in SF3B1-mutant lines as a result of mis-splicing. The expression of ABCB7, a mitochondrial iron sulfur cluster biogenesis component mutated in inherited X-linked sideroblastic anemia (Allikmets et al. Hum Mol Genet 1999), was also reduced in SF3B1-mutant cells as expected (Shiozawa et al. Nat Commun 2018, Dolatshad et al. Leukemia 2016). To investigate the role of these mis-splicing events in ring sideroblast formation, we performed lentiviral overexpression of TMEM14C, PPOX, and ABCB7, in SF3B1-mutant 5F-HPCs and quantified RS formation during late stages of erythroid differentiation. Overexpression of TMEM14C and ABCB7 in SF3B1-mutant cells partially but not completely rescued RS formation compared to luciferase control. These findings confirm the long-standing hypothesis that mis-splicing of mitochondrial iron metabolism genes causes RS formation. Furthermore, these findings suggest that RS formation in MDS is a multigenic event caused by coordinated but incomplete mis-splicing of several critical iron metabolism genes. Disclosures No relevant conflicts of interest to declare.

Regulation of Parkin-dependent mitophagy by Bcl-2-associated athanogene (BAG) family members.

Regulation of Parkin-dependent mitophagy by Bcl-2-associated athanogene (BAG) family members.

2-Ethyl-3-hydroxy-6-methylpyridine nitroxy succinate as a multifunctional hybrid structure

The effect of 2-ethyl-3-hydroxy-6-methylpyridine nitroxy succinate (1) on heart, brain, and liver tissues of experimental animals (C57/b1 mice) was studied by EPR. It was shown that compound 1, like nicorandil, increases the nitric oxide production in cells and, like mexidol, protects iron-sulfur clusters of the mitochondrial respiratory chain in heart, brain, and liver tissues of animals against oxidative damage. Experimental evidence was obtained that under oxidative conditions, mitochondrial iron-sulfur clusters are maintained in the reduced functional state due to the presence of succinate (as nitroxy succinic acid), as a metabolite of the tricarboxylic acid cycle, in compound 1.

CONGENITAL MYOPATHIES 2: P.98 A dog model for centronuclear myopathy carrying the most common DNM2 mutation

Centronuclear myopathies (CNM) are clinically and genetically heterogeneous disorders characterized by muscle atrophy and abnormal nuclear centralization in muscle fibers. Autosomal dominant CNM primarily results from mutations in DNM2, encoding the GTPase dynamin 2, a key regulator of membrane remodelling and endocytosis. The clinical presentation of the patients correlates with the position of the mutation, and ranges from severe neonatal hypotonia and respiratory distress to milder adult-onset forms. The most common mutation c.1393C>T (R465W) is found in 25% of all cases, and affected individuals typically show moderate generalized muscle weakness, ptosis, and ophthalmoplegia. Functional investigations suggest that the DNM2 mutations induce a gain-of-function, and the genetic or pharmacological downregulation of dynamin 2 efficiently antagonizes the disease development in the mouse model. Here we characterize a spontaneous CNM dog model, and we provide phenotypical, genetic, histological, and ultrastructural data through a longitudinal study over a period of 18 months. We identified the common DNM2 mutation R465W in a Border collie, and crossing with a healthy Beagle dam resulted in four affected offspring carrying the mutation, and a single healthy dog without the mutation. The affected dogs presented with mildly progressive muscle weakness from the age of 4 months, and MRI revealed muscle atrophy in particular of the paravertebral and masticatory muscles. Noteworthy, histological anomalies preceded the clinical signs, and the CNM-typical hallmarks myofiber atrophy, endomysial fibrosis, central nuclei, necklace fibers, and mitochondrial clustering were seen in biopsies at 2 months of age. Electron microscopy performed at 6 months of age detected mitochondrial anomalies, and functional investigations confirmed mitochondrial dysfunction. Overall, DNM2 dogs manifested a slowly progressive congenital myopathy and recapitulated the histological picture of the human disorder. The present study also provides insight into the sequence of events leading to muscle dysfunction. There is currently no treatment for DNM2-related CNM, and the canine model represents a valuable tool to validate pre-clinical treatment options on a large mammal based on proof-of-concepts recently obtained in the mouse.

Testing species hypotheses for Fridericia magna, an enchytraeid worm (Annelida: Clitellata) with great mitochondrial variation

BackgroundDeep mitochondrial divergences were observed in Scandinavian populations of the terrestrial to semi-aquatic annelid Fridericia magna (Clitellata: Enchytraeidae). This raised the need for testing whether the taxon is a single species or a complex of cryptic species.ResultsA total of 62 specimens from 38 localities were included in the study, 44 of which were used for species delimitation. First, the 44 specimens were divided into clusters using ABGD (Automatic Barcode Gap Discovery) on two datasets, consisting of sequences of the mitochondrial markers COI and 16S. For each dataset, the worms were divided into six not completely congruent clusters. When they were combined, a maximum of seven clusters, or species hypotheses, were obtained, and the seven clusters were used as input in downstream analyses. We tested these hypotheses by constructing haplowebs for two nuclear markers, H3 and ITS, and in both haplowebs the specimens appeared as a single species. Multi-locus species delimitation analyses performed with the Bayesian BPP program also mainly supported a single species. Furthermore, no apparent morphological differences were found between the clusters. Two of the clusters were partially separated from each other and the other clusters, but not strongly enough to consider them as separate species. All 62 specimens were used to visualise the Scandinavian distribution, of the species, and to compare with published COI data from other Fridericia species.ConclusionWe show that the morphospecies Fridericia magna is a single species, harbouring several distinct mitochondrial clusters. There is partial genetic separation between some of them, which may be interpreted as incipient speciation. The study shows the importance of rigorous species delimitation using several independent markers when deep mitochondrial divergences might give the false impression of cryptic speciation.

Cardiolipin-deficient cells have decreased levels of the iron–sulfur biogenesis protein frataxin

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays an important role in mitochondrial bioenergetics. Previous studies in the yeast model have indicated that CL is required for optimal iron homeostasis, which is disrupted by a mechanism not yet determined in the yeast CL mutant, crd1Δ. This finding has implications for the severe genetic disorder, Barth syndrome (BTHS), in which CL metabolism is perturbed because of mutations in the CL-remodeling enzyme, tafazzin. Here, we investigate the effects of tafazzin deficiency on iron homeostasis in the mouse myoblast model of BTHS tafazzin knockout (TAZ-KO) cells. Similarly to CL-deficient yeast cells, TAZ-KO cells exhibited elevated sensitivity to iron, as well as to H2O2, which was alleviated by the iron chelator deferoxamine. TAZ-KO cells exhibited increased expression of the iron exporter ferroportin and decreased expression of the iron importer transferrin receptor, likely reflecting a regulatory response to elevated mitochondrial iron. Reduced activities of mitochondrial iron-sulfur cluster enzymes suggested that the mechanism underlying perturbation of iron homeostasis was defective iron-sulfur biogenesis. We observed decreased levels of Yfh1/frataxin, an essential component of the iron-sulfur biogenesis machinery, in mitochondria from TAZ-KO mouse cells and in CL-deleted yeast crd1Δ cells, indicating that the role of CL in iron-sulfur biogenesis is highly conserved. Yeast crd1Δ cells exhibited decreased processing of the Yfh1 precursor upon import, which likely contributes to the iron homeostasis defects. Implications for understanding the pathogenesis of BTHS are discussed.

Abstract 554: Reverse-mode Mitochondrial Na + / Ca2+ Mitochondrial Exchange, Not the Mcu, is the Primary Mode of Ca2+ Import Into the Mitochondria During Ischemia/ Reperfusion in Neonatal Cardiac Myocytes

Ca 2+ entry via the Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload under physiological conditions but is thought to contribute to cell death during ischemia-reperfusion (I/R) injury. We have previously shown that mitochondrial membrane potential (ΔΨm) instability contributes to early-reperfusion arrhythmias and contractile dysfunction; however, the role of mitochondrial Ca 2+ (mCa 2+ ) uptake in triggering ΔΨm oscillation is unclear. Here, by acutely knocking out MCU, we examine whether MCU-mediated mCa 2+ uptake is required to trigger ΔΨm loss or oscillation during early reperfusion in neonatal mouse ventricular myocyte (NMVM) monolayers. We monitored mCa 2+ (with MitoCam) and ΔΨm (with TMRM) in WT(MCU fl/fl ) and MCU-KO (MCU fl/fl +AdCre) NMVMs during in vitro I/R (previously described by Solhjoo et al JMCC, 2015) by confocal microscopy. Image sequences were analyzed for changes in mCa 2+ and ΔΨm by segmenting individual cells in ImageJ. To quantify ΔΨm oscillations in mitochondrial clusters during reperfusion, a new wavelet-transform-based analysis was developed using MATLAB’s wavelet toolbox. Surprisingly, our findings demonstrate that MCU knockout does not significantly alter mCa 2+ import during I/R, nor does it affect ΔΨm recovery during Reperfusion. In fact, MCU-KO moderately shortened the latency to Ischemic ΔΨm depolarization. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCLX) with CGP-37157 suppressed the mCa 2+ increase during Ischemia. Moreover, blocking mNCLX also did not affect ΔΨm recovery during Reperfusion or the frequency of ΔΨ m oscillations, confirming that mitochondrial ΔΨm instability on reperfusion is not triggered by mCa 2+ . Interestingly, inhibition of mitochondrial electron transport stabilized ΔΨm oscillations during reperfusion. The findings are consistent with mCa 2+ overload being mediated by reverse-mode mNCLX activity and support ROS-induced ROS release as the primary trigger of ΔΨm instability during reperfusion injury.

Perinuclear mitochondrial clustering, increased ROS levels, and HIF1 are required for the activation of HSF1 by heat stress.

The heat shock response (HSR) is a conserved cellular defensive response against stresses such as temperature, oxidative stress and heavy metals. A significant group of players in the HSR is the set of molecular chaperones known as heat shock proteins (HSPs), which assist in the refolding of unfolded proteins and prevent the accumulation of damaged proteins. HSP genes are activated by the HSF1 transcription factor, a master regulator of the HSR pathway. A variety of stressors activate HSF1, but the key molecular players and the processes that directly contribute to HSF1 activation remain unclear. In this study, we show that heat shock induces perinuclear clustering of mitochondria in mammalian cells, and this clustering is essential for activation of the HSR. We also show that this perinuclear clustering of mitochondria results in increased levels of reactive oxygen species in the nucleus, leading to the activation of hypoxia-inducible factor-1α (HIF-1α). To conclude, we provide evidence to suggest that HIF-1α is one of the crucial regulators of HSF1 and that HIF-1α is essential for activation of the HSR during heat shock.

Endolysosomal Targeting of Mitochondria Is Integral to BAX-Mediated Mitochondrial Permeabilization during Apoptosis Signaling.

Mitochondrial outer membrane permeabilization (MOMP) is a core event in apoptosis signaling. However, the underlying mechanism of BAX and BAK pore formation remains incompletely understood. We demonstrate that mitochondria are globally and dynamically targeted by endolysosomes (ELs) during MOMP. In response to pro-apoptotic BH3-only protein signaling and pharmacological MOMP induction, ELs increasingly form transient contacts with mitochondria. Subsequently, ELs rapidly accumulate within the entire mitochondrial compartment. This switch-like accumulation period temporally coincides with mitochondrial BAX clustering and cytochrome c release. Remarkably, interactions of ELs with mitochondria control BAX recruitment and pore formation. Knockdown of Rab5A, Rab5C, or USP15 interferes with EL targeting of mitochondria and functionally uncouples BAX clustering from cytochrome c release, while knockdown of the Rab5 exchange factor Rabex-5 impairs both BAX clustering and cytochrome c release. Together, these data reveal that EL-mitochondrial inter-organelle communication is an integral regulatory component of functional MOMP execution during cellular apoptosis signaling.

Increasing ovarian NAD+ levels improve mitochondrial functions and reverse ovarian aging

Loss of follicles together with decreased oocyte quality and quantity contribute to age-associated ovarian senescence and infertility. Although underlying mechanisms for ovarian senescence are still unknown, mitochondrial dysfunctions have been reported. Here, we showed age-dependent decreases in ovarian Nicotinamide Adenine Dinucleotide (NAD+) levels in mice whereas supplementing aging mice with nicotinamide riboside (NR), an NAD+ precursor, increased ovarian NAD+ content. We found that increases in ovarian NAD+ levels in aging mice led to increased number of ovarian follicles and ovulatory potential as well as increased live birth rate. NR supplementation also reduced levels of reactive oxygen species and decreased spindle anomalies in aging oocytes, together with increased mitochondrial membrane potential (ΔΨm) and decreased mitochondrial clustering. In addition, NR supplementation improved ovarian mitochondrial energy metabolism. Our data suggested that supplementation with NAD+ precursors in vivo and in vitro could be potential therapeutic approaches for treating age-related ovarian infertility.

Involvement of Mrs3/4 in Mitochondrial Iron Transport and Metabolism in Cryptococcus neoformans.

Mitochondria play a vital role in iron uptake and metabolism in pathogenic fungi, and also influence virulence and drug tolerance. However, the regulation of iron transport within the mitochondria of Cryptococcus neoformans, a causative agent of fungal meningoencephalitis in immunocompromised individuals, remains largely uncharacterized. In this study, we identified and functionally characterized Mrs3/4, a homolog of the Saccharomyces cerevisiae mitochondrial iron transporter, in C. neoformans var. grubii. A strain expressing an Mrs3/4-GFP fusion protein was generated, and the mitochondrial localization of the fusion protein was confirmed. Moreover, a mutant lacking the MRS3/4 gene was constructed; this mutant displayed significantly reduced mitochondrial iron and cellular heme accumulation. In addition, impaired mitochondrial iron-sulfur cluster metabolism and altered expression of genes required for iron uptake at the plasma membrane were observed in the mrs3/4 mutant, suggesting that Mrs3/4 is involved in iron import and metabolism in the mitochondria of C. neoformans. Using a murine model of cryptococcosis, we demonstrated that an mrs3/4 mutant is defective in survival and virulence. Taken together, our study suggests that Mrs3/4 is responsible for iron import in mitochondria and reveals a link between mitochondrial iron metabolism and the virulence of C. neoformans.