- Email: [email protected]
Extracellular vesicles secreted by brain tumor stem cells are rich in mitochondrial function-associated proteins
Abstracts
Abstract 87 Protein replacement therapy for mitochondrial disorders Presenter: Haya Lorberboum-Galski M. Rapoporta, D. Marcusa, A. Saadaa, T. Erlicha, R. Hadada, H. Greif b, M. Lichtensteina, H. Lorberboum-Galskia a Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel–Canada (IMRIC), Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel b BioBlast-Pharma Ltd., Israel Modern medicine offers no cure for genetic mitochondrial disorders and the usual treatment is mostly palliative. We developed a novel concept for the treatment of mitochondrial disorders using Cell/Organelle-Directed Protein Replacement Therapy; the delivery of a wild type mitochondrial protein/enzyme directly to its subcellular location and into its natural complexes, in the form of a fusion protein. Our approach is to fuse a wild type mitochondrial protein, including the Mitochondrial targeting Sequence (MTS), with the delivery peptide TAT [HIV-transactivator of transcription (TAT) peptide], which will lead the protein/enzyme into the cells and their mitochondria, where it will substitute for the mutated endogenous protein. We tested this novel approach using a number of mitochondrial proteins, implicated in mitochondrial human diseases: Lipoamide Dehydrogenase (LAD), C6ORF66 and Frataxin were evaluated in vitro, in patients' cells and in vivo, in mouse models. TAT-MTSMitochondrial fusion proteins are rapidly and efficiently internalizing into cells and their mitochondria, both in patients' cells and in mice tissues, including the brain. Treatment with the new TAT-MTSMitochondrial fusion proteins, improves mitochondrial functions and life span in animal models. Interestingly, when we replaced the MTS sequence of the exogenous protein with a heterologous MTS sequence, both mitochondrial penetration and biological activity significantly increased. This novel approach may open new inroads in the management of many incurable mitochondrial diseases.
doi:10.1016/j.mito.2015.07.096
Abstract 88 Assessing mitochondrial function in OC1 and PK1 cells treated with gentamicin and MitoQ Presenter: Carolyn O. Dirain Carolyn O. Dirain, Patrick J. Antonelli, Kevin P. Raisch Department of Otolaryngology, College of Medicine, University of Florida, Gainesville, FL 32610, USA Body of Abstract: Objective: To assess mitochondrial bioenergetics in cells concurrently treated with gentamicin and the mitochondria-targeted antioxidant mitoquinone (MitoQ), which may prevent gentamicin ototoxicity. Materials and methods: LLC-PK1 and HEI-OC1 cells, renal and auditory cell lines, respectively, were used in this study. Both cell lines are commonly used in studies evaluating nephrotoxic and ototoxic drugs, such as gentamicin. Mitochondrial bioenergetics was assessed by measuring oxygen consumption rates using the Seahorse XF-24™ flux analyzer in untreated LLC-PK1 and HEI-OC1 cells and cells exposed to low (100 μM) or high (2000 μM) dose gentamicin for 24 h, with and without 0.5 μM MitoQ. Results: In HEI-OC1 cells, basal respiration rate was not different in untreated cells and in cells treated with low dose gentamicin alone, MitoQ alone, or cells concurrently treated with low dose gentamicin and MitoQ (p = 0.85). However, HEI-OC1 cells that were concurrently treated with low dose gentamicin and MitoQ have
S35
lower maximal respiratory capacity (p = 0.0002) and ATP-linked respiration (p = 0.008). Additionally, these cells have increased proton leak (p b 0.0001), indicating a decrease in mitochondrial efficiency. On the other hand, mitochondrial function was not different in untreated LLC-PK1 cells and cells treated with gentamicin alone, MitoQ alone or co-incubated with low dose gentamicin and MitoQ (p N 0.05). Treatment of cells with 2 mM gentamicin or 2 mM gentamicin + MitoQ were too toxic for both cell lines as later determined by lactate dehydrogenase assay. Thus, these were excluded from further analyses. Conclusions: Although antioxidants are considered generally safe, the combination of gentamicin and the antioxidant MitoQ holds the potential to disrupt mitochondrial function. This suggests that patients who are receiving both agents simultaneously must take precautions to monitor toxicity. Key words: Mitochondrial function, MitoQ, Antioxidant, Gentamicin, Aminoglycoside, Ototoxicity
doi:10.1016/j.mito.2015.07.097
Abstract 90 Extracellular vesicles secreted by brain tumor stem cells are rich in mitochondrial function-associated proteins Presenter: Jeroen de Vrij Jeroen de Vrija, Theo M. Luidera, Marike L.D. Broekmanb, René de Cooa a Department of Neurology, Erasmus Medical Center, Rotterdam, The Netherlands b Department of Neurosurgery, University Medical Center Utrecht, The Netherlands Body of Abstract: Many cell types secrete extracellular vesicles (EVs) (“exosomes”, “shedding vesicles”), which contain cell-derived components (incl. RNAs and proteins), and play important roles in both normal physiology and pathophysiology. In cancers, EVs appear to be an important mechanism for the tumor to modify the environment. Functional effects include vascularization, immunesystem suppression, and pre-metastatic/migratory niche formation. We and others have shown that these effects are prominent in glioblastoma multiforme (GBM), the most aggressive type of brain tumors. Interestingly, comparison between GBM stem cells (GSCs) and non-stem cells (established U87-MG cell line) revealed more aggressive EVs for the stem cells, i.e. higher amounts of EV secretion and stronger effects on recipient cells. Proteome profiling demonstrated a large degree of similarity between the GSC EVs and the U87-MG EVs: the EVs were rich in membrane- and endosomeassociated proteins, and scarce in nuclear- and cytoskeletonassociated proteins. Remarkably, the GSC-derived EVs appeared to be rich in specific types of mitochondrial function-associated proteins, which were undetectable or at low levels detected in the cells. This was not observed for the U87-MG EVs. Six proteins were significantly enriched in the GSC EVs (% EV versus % cell, Fisher exact test), including glutamate dehydrogenase 1, Catalase, and Gap junction alpha-1 protein. These findings may point toward (i) a biological role of EV secretion by tumor stem cells by transferring mitochondrial proteins to recipient cells, and (ii) possibility to specifically detect EVs of tumor stem cells, which may be of great value for EV-based diagnostics that are currently under development.
doi:10.1016/j.mito.2015.07.098
Abstract 87 Protein replacement therapy for mitochondrial disorders Presenter: Haya Lorberboum-Galski M. Rapoporta, D. Marcusa, A. Saadaa, T. Erlicha, R. Hadada, H. Greif b, M. Lichtensteina, H. Lorberboum-Galskia a Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel–Canada (IMRIC), Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel b BioBlast-Pharma Ltd., Israel Modern medicine offers no cure for genetic mitochondrial disorders and the usual treatment is mostly palliative. We developed a novel concept for the treatment of mitochondrial disorders using Cell/Organelle-Directed Protein Replacement Therapy; the delivery of a wild type mitochondrial protein/enzyme directly to its subcellular location and into its natural complexes, in the form of a fusion protein. Our approach is to fuse a wild type mitochondrial protein, including the Mitochondrial targeting Sequence (MTS), with the delivery peptide TAT [HIV-transactivator of transcription (TAT) peptide], which will lead the protein/enzyme into the cells and their mitochondria, where it will substitute for the mutated endogenous protein. We tested this novel approach using a number of mitochondrial proteins, implicated in mitochondrial human diseases: Lipoamide Dehydrogenase (LAD), C6ORF66 and Frataxin were evaluated in vitro, in patients' cells and in vivo, in mouse models. TAT-MTSMitochondrial fusion proteins are rapidly and efficiently internalizing into cells and their mitochondria, both in patients' cells and in mice tissues, including the brain. Treatment with the new TAT-MTSMitochondrial fusion proteins, improves mitochondrial functions and life span in animal models. Interestingly, when we replaced the MTS sequence of the exogenous protein with a heterologous MTS sequence, both mitochondrial penetration and biological activity significantly increased. This novel approach may open new inroads in the management of many incurable mitochondrial diseases.
doi:10.1016/j.mito.2015.07.096
Abstract 88 Assessing mitochondrial function in OC1 and PK1 cells treated with gentamicin and MitoQ Presenter: Carolyn O. Dirain Carolyn O. Dirain, Patrick J. Antonelli, Kevin P. Raisch Department of Otolaryngology, College of Medicine, University of Florida, Gainesville, FL 32610, USA Body of Abstract: Objective: To assess mitochondrial bioenergetics in cells concurrently treated with gentamicin and the mitochondria-targeted antioxidant mitoquinone (MitoQ), which may prevent gentamicin ototoxicity. Materials and methods: LLC-PK1 and HEI-OC1 cells, renal and auditory cell lines, respectively, were used in this study. Both cell lines are commonly used in studies evaluating nephrotoxic and ototoxic drugs, such as gentamicin. Mitochondrial bioenergetics was assessed by measuring oxygen consumption rates using the Seahorse XF-24™ flux analyzer in untreated LLC-PK1 and HEI-OC1 cells and cells exposed to low (100 μM) or high (2000 μM) dose gentamicin for 24 h, with and without 0.5 μM MitoQ. Results: In HEI-OC1 cells, basal respiration rate was not different in untreated cells and in cells treated with low dose gentamicin alone, MitoQ alone, or cells concurrently treated with low dose gentamicin and MitoQ (p = 0.85). However, HEI-OC1 cells that were concurrently treated with low dose gentamicin and MitoQ have
S35
lower maximal respiratory capacity (p = 0.0002) and ATP-linked respiration (p = 0.008). Additionally, these cells have increased proton leak (p b 0.0001), indicating a decrease in mitochondrial efficiency. On the other hand, mitochondrial function was not different in untreated LLC-PK1 cells and cells treated with gentamicin alone, MitoQ alone or co-incubated with low dose gentamicin and MitoQ (p N 0.05). Treatment of cells with 2 mM gentamicin or 2 mM gentamicin + MitoQ were too toxic for both cell lines as later determined by lactate dehydrogenase assay. Thus, these were excluded from further analyses. Conclusions: Although antioxidants are considered generally safe, the combination of gentamicin and the antioxidant MitoQ holds the potential to disrupt mitochondrial function. This suggests that patients who are receiving both agents simultaneously must take precautions to monitor toxicity. Key words: Mitochondrial function, MitoQ, Antioxidant, Gentamicin, Aminoglycoside, Ototoxicity
doi:10.1016/j.mito.2015.07.097
Abstract 90 Extracellular vesicles secreted by brain tumor stem cells are rich in mitochondrial function-associated proteins Presenter: Jeroen de Vrij Jeroen de Vrija, Theo M. Luidera, Marike L.D. Broekmanb, René de Cooa a Department of Neurology, Erasmus Medical Center, Rotterdam, The Netherlands b Department of Neurosurgery, University Medical Center Utrecht, The Netherlands Body of Abstract: Many cell types secrete extracellular vesicles (EVs) (“exosomes”, “shedding vesicles”), which contain cell-derived components (incl. RNAs and proteins), and play important roles in both normal physiology and pathophysiology. In cancers, EVs appear to be an important mechanism for the tumor to modify the environment. Functional effects include vascularization, immunesystem suppression, and pre-metastatic/migratory niche formation. We and others have shown that these effects are prominent in glioblastoma multiforme (GBM), the most aggressive type of brain tumors. Interestingly, comparison between GBM stem cells (GSCs) and non-stem cells (established U87-MG cell line) revealed more aggressive EVs for the stem cells, i.e. higher amounts of EV secretion and stronger effects on recipient cells. Proteome profiling demonstrated a large degree of similarity between the GSC EVs and the U87-MG EVs: the EVs were rich in membrane- and endosomeassociated proteins, and scarce in nuclear- and cytoskeletonassociated proteins. Remarkably, the GSC-derived EVs appeared to be rich in specific types of mitochondrial function-associated proteins, which were undetectable or at low levels detected in the cells. This was not observed for the U87-MG EVs. Six proteins were significantly enriched in the GSC EVs (% EV versus % cell, Fisher exact test), including glutamate dehydrogenase 1, Catalase, and Gap junction alpha-1 protein. These findings may point toward (i) a biological role of EV secretion by tumor stem cells by transferring mitochondrial proteins to recipient cells, and (ii) possibility to specifically detect EVs of tumor stem cells, which may be of great value for EV-based diagnostics that are currently under development.
doi:10.1016/j.mito.2015.07.098