Mitophagy promotes the stemness of bone marrow-derived mesenchymal stem cells

Previous studies demonstrated that mitochondrial fission arguments the stemness of bone marrow-derived mesenchymal stem cells (BMSCs). Because mitophagy is critical in removing damaged or surplus mitochondrial fragments and maintaining mitochondrial integrity, the present study was undertaken to test the hypothesis that mitophagy is involved in mitochondrial fission-enhanced stemness of BMSCs. Primary cultures of rat BMSCs were treated with tyrphostin A9 (TA9, a potent inducer of mitochondrial fission) to increase mitochondrial fission, which was accompanied by enhanced mitophagy as defined by increased co-staining of MitoTracker Green for mitochondria and LysoTracker Deep Red for lysosomes, as well as the increased co-localization of autophagy markers (LC3B, P62) and mitochondrial marker (Tom20). A mitochondrial uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was used to promote mitophagy, which was confirmed by an increased co-localization of mitochondrial and lysosome biomarkers. The argumentation of mitophagy was associated with enhanced stemness of BMSCs as defined by increased expression of stemness markers Oct4 and Sox2, and enhanced induction of BMSCs to adipocytes or osteocytes. Conversely, transfection of BMSCs with siRNA targeting mitophagy-essential genes Pink1/ Prkn led to diminished stemness of the stem cells, as defined by depressed stemness markers. Importantly, concomitant promotion of mitochondrial fission and inhibition of mitophagy suppressed the stemness of BMSCs. These results thus demonstrate that mitophagy is critically involved in mitochondrial fission promotion of the stemness of BMSCs.

Parámetros metabólicos, antioxidantes y competencia para el desarrollo embrionario de ovocitos bovinos madurados in vitro con L-Carnitina

Se evaluó el efecto antioxidante y metabólico de la L-Carnitina (L-C) durante la maduración in vitro de ovocitos bovinos sobre parámetros asociados a la calidad: cantidad relativa de lípidos, generación de especies reactivas de oxígeno (EROs), niveles de glutatión reducido (GSH), actividad mitocondrial y la competencia para el desarrollo embrionario posterior a la fertilización. Los complejos cúmulo ovocito fueron madurados por 24 h con y sin L-C (3.8 mM), e incubados con el fluoróforo específico para cada parámetro, Rojo Nilo (lípidos), Diclorofluoresceina diacetato (EROs), Monoclorobimane (GSH), Mitotracker Green (mitocondria). La intensidad de la fluorescencia fue analizada con el software ImageJ y normalizada al grupo control de ovocitos madurados sin LCarnitina. Para determinar la competencia del ovocito para el desarrollo embrionario, los ovocitos madurados fueron fertilizados y cultivados por 8 días. En los ovocitos madurados con L-C se encontró una disminución del 8.1% de la cantidad relativa de lípidos y en la generación de EROs del 41.6%; mientras que la actividad mitocondrial aumentó en 160% con respecto al control, y mejoró la cinética y el porcentaje de blastocistos. Sin embargo, los niveles de GSH no se afectaron con la L-C. Los resultados soportan el efecto benéfico de la L-C durante la maduración in vitro del ovocito bovino para mejorar parámetros antioxidantes y metabólicos que se reflejan en la producción in vitro de embriones bovinos.

3071 Cell Survival in Corneal Endothelial Dystrophies

OBJECTIVES/SPECIFIC AIMS: Purpose - The goal of this study is to understand how loss of the membrane protein SLC4A11 alters endothelial cell metabolism thereby producing Corneal Endothelial Dystrophy. Studies from our lab indicated that glutamine-dependent mitochondrial dysfunction is one of the outcomes of SLC4a11 loss. In the current study, we ask if autophagy and mitophagy pathways and the signaling pathways that regulate these processes are altered in SLC4a11 KO cells. METHODS/STUDY POPULATION: Methods – Immortalized mouse WT and SLC4a11 KO cell lines were incubated in DMEM with and without 0.5mM glutamine for 6 hours. In order to assess mitophagy, cells were stained using Lysotracker Red and Mitotracker Green. Colocalization co-efficients of red and green channels were obtained for at least 35 cells using Zeiss-Zen Pro software. Student’s t-test was used to determine statistical significance. For Western Blots, antibodies against LC3b, AMPK, pAMPK, and b-actin were used to examine autophagy flux and potential signaling pathways that regulate autophagy. RESULTS/ANTICIPATED RESULTS: Results – In the presence of glutamine, the colocalization co-efficient of Lysotracker Red and Mitotracker Green channels was significantly increased in KO cells (0.74 ±0.18) relative to WT (0.58±0.20) with a p-value ≤0.0024. In the absence of glutamine, the colocalization co-efficient was reversed, for KO cells 0.54 ±0.14 and for WT cells 0.77±0.0.16 with a p-value ≤0.0001, suggesting increased mitophagy by glutamine in KO cells. Western Blots indicated that glutamine increased autophagy flux, as indicated by increased levels of LC3b following bafilomycin A treatment in KO cells. Concomitantly, there was an increase in pAMPK/AMPK levels suggesting a potential mechanism for increased mitophagy. DISCUSSION/SIGNIFICANCE OF IMPACT: Conclusion and Future studies –Our data indicates enhanced mitophagy as well as autophagy in SLC4a11 KO cells. Future studies will determine whether these processes regulate cell survival in mouse models of corneal endothelial dystrophies.

All-Trans Retinoic Acid (ATRA) up-Regulates Cell Surface CD38 Expression Which Promotes Pro-Tumoral Mitochondrial Trafficking from Stromal Cells to Multiple Myeloma

Background Multiple myeloma (MM) is malignancy highly reliant on its microenvironment. In this study, we investigated whether mitochondrial transfer occurred between bone marrow stromal cells (BMSC) and malignant plasma cells. We then used our observations as a platform to investigate the mechanisms controlling pro-tumoral mitochondrial transfer with a view to identifying druggable targets. Methods Primary MM cells were obtained from patients' bone marrow after informed consent and under approval from the United Kingdom Health Research Authority. Animal experiments were conducted under approvals from the UK Home Office and the University of East Anglia Animal Welfare and Ethics Review Board. Primary BMSC were also obtained from patient bone marrow, using adherence and characterised using flow cytometry. Mitochondrial transfer was assessed using two methods; a MitoTracker Green based staining of the BMSC (in-vitro), rLV.EF1.AcGFP-Mem9 labelling of the MM plasma membrane with MitoTracker CMXRos staining of the BMSC (in-vitro) and an in vivo MM NSG xenograft model. CD38 expression on MM cells was tested after ATRA treatment, using RT-qPCR and flow cytometry. Mitochondrial transfer levels were assessed when CD38 was over expressed using ATRA or inhibited using lentivirus targeted shRNA. Results We report that mitochondria are transferred from BMSC to MM cells. First, we cultured MM cells on MitoTracker Green labelled BMSC and found increased MitoTracker Green fluorescence in the MM cells. We then transduced MM with rLV.EF1.AcGFP-Mem9 lentivirus and stained BMSC with MitoTracker CMXRos and used wide field microscopy to show MM derived tunnelling nanotubles (TNT) formed between MM cells and BMSC, with red mitochondria located within the GFP-tagged TNT. Next, we engrafted the MM cell lines MM1S and U266 into NSG mouse, after isolation we detected the presence of mouse mitochondrial DNA in the purified MM population. Together, these data show that mitochondria are transferred from BMSC to MM cells. We next analysed OXPHOS levels in MM cells grown on BMSC, using the seahorse extracellular flux assay. We found that the MM cells had increased levels of OXPHOS after culture with BMSC, which was also the case for MM cell lines analysed after isolation from NSG mice, showing the micro-environment of MM can alter the metabolism of the malignant cell. To examine whether the mitochondrial transfer process was controlled by CD38, we knocked down CD38 in MM cells using lentiviral targeted shRNA. We found reduced levels of mitochondrial transfer in CD38KD MM cells, with a consequent reduction of OXPHOS in the malignant cells. Finally, as ATRA has previously been shown to increase CD38 expression in AML, we next quantified CD38 mRNA and surface glycoprotein level on malignant plasma cells with and without ATRA treatment. We found ATRA increased CD38 expression at the mRNA and protein levels and this resulted in an increase in mitochondrial transfer from BMSC to MM cells. Conclusion Here we show that CD38 mediated mitochondrial transfer in the MM micro-environment forms part of the malignant phenotype of multiple myeloma. This finding develops our understanding of the mechanisms which underpin the efficacy of CD38 directed therapy in MM. Disclosures No relevant conflicts of interest to declare.

Botanical Alkyl Hydroquinone Derivative HQ17(3) Induces Calcium-Associated Mitochondrial Damage and Mitophagy to Exert Ctotoxicity to Philadelphila Chromosome(+) ALL Cells

Introduction: Acute lymphoblastic leukemias (ALLs) harboring t(9;22)(Ph+-ALL) are very high risk (VHR) ALL displaying poor clinical outcome irrespective of intensive chemotherapies plus tyrosine kinase inhibitor (TKI) treatment. HQ17(3)[10'(Z),13'(E),15'(E)-heptadecatrienyl hydroquinone] isolated from sap of the lacquer tree showed rapid (within 24hrs) and potent cytotoxic effect at micromolar concentration on several ALL cell lines, including Imatinib-refractory Ph+-ALL SUP-B15 cells, but spared normal PB leukocytes, and showed nontoxic in experimental rats after 28-day injection. Therefore HQ17(3) presents as a potential anti-leukemic agents and provide a platform for exploring anti-leukemic adjuvants. Our previous study showed HQ17(3)-induced rapid cell demise, characterized by oxidative stress, mitochondrial membrane potential disturbance, loss of membrane integrity, and nuclear DNA fragmentation. HQ17(3)-induced cell death is a caspase-independent program, and is different from the RIP1-mediated controlled necroptosis since both pan-caspase inhibitor and RIP-1 inhihitor failed to protect SUP-B15 cells from death. The ER stress markers (chaperon Grp78 and phosphorylated-eIF2α) were up-regulated as early as 5hrs after HQ17(3) treatment. Here we aim to illustrate the characters of the HQ17(3)-induced non-classical death on Ph+-SUP-B15 cells, focus on ER stress-associated mitochondrial Ca2+ homeostasis. Methods: Cell death and changes of mitochondria in response to HQ17(3) w/wo inhibitors were analyzed. Cells were stained by Annexin V/PI and analyzed by flow cytometry for cell death. Mitochondria mass, mitochondrial Ca2+ accumulation was detected by fluorescent Mitotracker Green and Rhod-2 probes, respectively. Mitochondrial superoxide was measured by Mitosox stain. Western blot analysis was used to analyze MFN1/2, OPA1 (mitochondrial markers). Nuclear accumulation of apoptosis inducing factor (AIF), co-localization of mitochondrial COX-IV and LC3-II (mitophagy) were revealed by immunofluorescence stain and confocal microscopy. Results: We showed mitochondrial Ca2+ accumulation at the early time when ER stress occurred (Fig 1), accompanied by mitochondrial superoxide elevation, followed by loss of mitochondrial membrane potential (MMP) and nuclear translocation of apoptosis-inducing factor (AIF). HQ17(3) treatment lead to decreased mitochondrial proteins MFN1/2 and OPA1, while Mitotracker Green stain showed significant loss of mitochondrial mass preceded cell death, indicating damaged mitochondria underwent fission followed by mitophagy. Immunofluorescence stain showed evidence of mitophagy (COX IV and LC3B co-localization). Calpain-1 inhibitor PD150606 blocked AIF nuclear translocation but only slightly reduced the HQ17(3)-induced cell death (Fig 2). Further, Ca2+ chelator Bapta-AM prevented mitochondrial superoxide production, MMP loss, mitophagy (Fig 3), and rescued cell death (Fig 1) more effectively. Conclusion: In Ph+-ALL SUP-B15 cells, HQ17(3) induce ER stress by yet-defined mechanism, this mobilizes Ca2+ to mitochondria and acts in multi-facet: a) results in AIF cleavage and translocation to mediate nuclear chromatin fragmentation, b) Ca2+-overload leads to oxidative stress and perturbs mitochondria integrity, c) damaged mitochondria trigger extensive mitophagy and cell death ensues. Therefore, agents that help elicit similar intricate effector network associated with ER/mitochondria stress will have potential to be adjuvants in aiding control of the Ph+ VHR-ALL cells refractory to conventional chemotherapies and TKI regime. Disclosures No relevant conflicts of interest to declare.

Salvianolate Protects Hepatocytes from Oxidative Stress by Attenuating Mitochondrial Injury

Salvianolate is widely used to treat angiocardiopathy in clinic in China, but its application in liver diseases remains unclear. Our study aims to investigate the effect of Salvianolate on rat hepatic injury by protecting hepatocyte mitochondria. To evaluate the effects of Salvianolate on injured hepatocytes, alpha mouse liver 12 (AML-12) cells were induced with hydrogen peroxide (H2O2) and treated with Salvianolate. Cell viability and MitoTracker Green for mitochondria and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanide iodine (JC-1) levels and cytochrome C (Cyto-C) expressions were detectedin vitro. To identify the effect of Salvianolate on protecting against mitochondria injury, male Wistar rats were injected with carbon tetrachloride (CCl4) and treated with Salvianolate (40 mg·kg−1). Serum liver function, parameters for peroxidative damage, hematoxylin and eosin (H&E) staining, and transmission electron microscope (TEM) of hepatocyte mitochondria were assayed. Our results showed that Salvianolate effectively protected hepatocytes, increased mitochondria vitality, and decreased Cyto-C expressionsin vitro. Besides, Salvianolate alleviated the liver function, attenuated the indicators of peroxidation, and relieved the mitochondria injuryin vivo. In conclusion,Salvianolateis effective in protecting hepatocytes from injuryin vitroandin vivo, and the mechanism might be related to its protective effect on hepatocyte mitochondria against oxidative stress.

Differential Segregation Patterns of Sperm Mitochondria in Embryos of the Blue Mussel (Mytilus edulis)

In Mytilus, females carry predominantly maternal mitochondrial DNA (mtDNA) but males carry maternal mtDNA in their somatic tissues and paternal mtDNA in their gonads. This phenomenon, known as doubly uniparental inheritance (DUI) of mtDNA, presents a major departure from the uniparental transmission of organelle genomes. Eggs of Mytilus edulis from females that produce exclusively daughters and from females that produce mostly sons were fertilized with sperm stained with MitoTracker Green FM, allowing observation of sperm mitochondria in the embryo by epifluorescent and confocal microscopy. In embryos from females that produce only daughters, sperm mitochondria are randomly dispersed among blastomeres. In embryos from females that produce mostly sons, sperm mitochondria tend to aggregate and end up in one blastomere in the two- and four-cell stages. We postulate that the aggregate eventually ends up in the first germ cells, thus accounting for the presence of paternal mtDNA in the male gonad. This is the first evidence for different behaviors of sperm mitochondria in developing embryos that may explain the tight linkage between gender and inheritance of paternal mitochondrial DNA in species with DUI.