The Experts below are selected from a list of 276 Experts worldwide ranked by ideXlab platform
James P. Bennett - One of the best experts on this subject based on the ideXlab platform.
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Cybrid models of Parkinson's disease show variable mitochondrial biogenesis and genotype-respiration relationships
Experimental neurology, 2009Co-Authors: Paula M. Keeney, Lisa D. Dunham, Caitlin K. Quigley, Stephanie L. Morton, Kristen E. Bergquist, James P. BennettAbstract:Sporadic Parkinson's disease (sPD) is a nervous system-wide disease that presents with a bradykinetic movement disorder and frequently progresses to include depression and cognitive impairment. Cybrid models of sPD are based on expression of sPD platelet mitochondrial DNA (mtDNA) in neural cells and demonstrate some similarities to sPD brains. In sPD and CTL Cybrids we characterized aspects of mitochondrial biogenesis, mtDNA genomics, composition of the respirasome and the relationships among isolated mitochondrial and intact cell respiration. Cybrid mtDNA levels varied and correlated with expression of PGC-1 alpha, a transcriptional co-activator regulator of mitochondrial biogenesis. Levels of mtDNA heteroplasmic mutations were asymmetrically distributed across the mitochondrial genome; numbers of heteroplasmies were more evenly distributed. Neither levels nor numbers of heteroplasmies distinguished sPD from CTL. sPD cybrid mitochondrial ETC subunit protein levels were not altered. Isolated mitochondrial complex I respiration rates showed limited correlation with whole cell complex I respiration rates in both sPD and CTL Cybrids. Intact cell respiration during the normoxic-anoxic transition yielded K(m) values for oxygen that directly related to respiration rates in CTL but not in sPD cell lines. Both sPD and CTL cybrid cells are substantially heterogeneous in mitochondrial genomic and physiologic properties. Our results suggest that mtDNA depletion may occur in sPD neurons and could reflect impairment of mitochondrial biogenesis. Cybrids remain a valuable model for some aspects of sPD but their heterogeneity mitigates against a simple designation of sPD phenotype in this cell model.
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The cybrid model of sporadic Parkinson's disease
Experimental neurology, 2009Co-Authors: Patricia A. Trimmer, James P. BennettAbstract:Abstract Parkinson's disease (PD) is the eponym attached to the most prevalent neurodegenerative movement disorder of adults, derived from observations of an early nineteenth century physician and paleontologist, James Parkinson, and is now recognized to encompass much more than a movement disorder clinically or dopamine neuron death pathologically. Most PD (∼ 90%) is sporadic (sPD), is associated with mitochondrial deficiencies and has been studied in cell and animal models arising from the use of mitochondrial toxins that unfortunately have not predicted clinical efficacy to slow disease progression in humans. We have extensively studied the cytoplasmic hybrid (“cybrid”) model of sPD in which donor mtDNAs are introduced into and expressed in neural tumor cells with identical nuclear genetic and environmental backgrounds. sPD Cybrids demonstrate many abnormalities in which increased oxidative stress drives downstream antioxidant response and cell death activating signaling pathways. sPD Cybrids regulate mitochondrial ETC genes and gene ontology families like sPD brain. sPD Cybrids spontaneously form Lewy bodies and Lewy neurites, linking mtDNA expression to neuropathology, and demonstrate impaired organelle transport in processes and reduced mitochondrial respiration. Our recent studies show that near-infrared laser light therapy normalizes mitochondrial movement and can stimulate respiration in sPD cybrid neurons, and mitochondrial gene therapy can restore respiration and stimulate mitochondrial ETC gene and protein expression. sPD Cybrids have provided multiple lines of circumstantial evidence linking mtDNA to sPD pathogenesis and can serve as platforms for therapy development. sPD cybrid models can be improved by the use of non-tumor human stem cell-derived neural precursor cells and by an introduction of postmortem brain mtDNA to test its causality directly.
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altered intracellular signaling and reduced viability of alzheimer s disease neuronal Cybrids is reproduced by β amyloid peptide acting through receptor for advanced glycation end products rage
Molecular and Cellular Neuroscience, 2005Co-Authors: Isaac G Onyango, Jeremy B Tuttle, James P. BennettAbstract:Abstract Activation of apoptosis by increased production of amyloid beta peptides (Aβ) has been implicated in neuronal cell death of Alzheimer's disease (AD). We used mitochondrial transgenic cybrid models of sporadic AD (SAD), which overproduce Aβ compared to control (CTL) Cybrids, to investigate the effects of endogenously generated Aβ on intracellular signaling pathways and viability. Reducing SAD Aβ production with γ-secretase inhibition altered the total phosphorylation profile of SAD cybrid to one similar to CTL Cybrids and enhanced viability to ∼CTL cybrid levels. Treating CTL Cybrids with exogenous Aβ or conditioned media (CM) from SAD Cybrids activated the signaling pathways active in SAD Cybrids under basal condition and decreased viability. Antibodies against receptor for advanced glycation end products (RAGE) blocked Aβ-induced activation of the p38, JNK pathways, and NF-κB in CTL Cybrids and offered protection against the neurotoxic effects of Aβ. Expression of SAD mitochondrial genes in Cybrids activates stress-related signaling pathways and reduces viability. This SAD phenotype is produced by endogenously generated Aβ and can be replicated by exogenous Aβ acting through RAGE.
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cyclical mitochondrial δψm fluctuations linked to electron transport f0f1 atp synthase and mitochondrial na ca 2 exchange are reduced in alzheimer s disease Cybrids
Mitochondrion, 2005Co-Authors: Christine Thiffault, James P. BennettAbstract:Abstract Reduced complex IV, increased oxidative stress and beta amyloid peptide secretion in Alzheimer's disease (AD) can be replicated in cybrid models. We characterized cyclical mitochondrial ΔΨ M fluctuations (‘flickering’) in neuroblastoma cells and AD/CTL Cybrids. Flickering was blocked by ATP-synthase inhibition, was not observed in rho 0 cells and was not blocked by antioxidant treatment. Flickering was not affected by the Ca +2 uniporter antagonist Ru360 but was eliminated by BAPTA or CGP37137 blockade of the mitochondrial Na + /Ca +2 exchanger. AD cybrid mitochondria showed reduced flickering. Flickering seems to represent coupling of ΔΨ M to F 0 F 1 ATP-synthase; reduction of flickering in AD Cybrids suggests dysfunction of this coupling.
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activation of p38 and n acetylcysteine sensitive c jun nh2 terminal kinase signaling cascades is required for induction of apoptosis in parkinson s disease Cybrids
Molecular and Cellular Neuroscience, 2005Co-Authors: Isaac G Onyango, Jeremy B Tuttle, James P. BennettAbstract:Cytoplasmic hybrid cells (Cybrids) are created by selective amplification of mitochondrial genes against constant nuclear genetic and environmental backgrounds. Cybrids from patients with sporadic Parkinson's disease (PD) recapitulate disease features such as decreased complex I activity, increased oxidative stress, elevated activation of NF-kappaB, and production of Lewy body inclusions. We examined the activation of signaling pathways and NF-kappaB in PD Cybrids after exposure to MAPK inhibitors and/or the antioxidant N-acetylcysteine (NAC). Under basal replicating conditions, PD Cybrids have decreased viability that is associated with increased DNA condensation and poly-ADP ribose polymerase (PARP) cleavage as well as elevated p38 and JNK activity. Pharmacological inhibition of oxidative stress diminished the elevated p38, JNK activity and PARP cleavage, and enhanced PD cybrid viability. PD mitochondrial genes expressed in Cybrids stimulate pro-apoptotic cell signaling and biochemistry through oxidative stress. These results support development of antioxidative therapeutics for PD.
Yuri Yu. Gleba - One of the best experts on this subject based on the ideXlab platform.
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Self-fertile Cybrids Nicotiana tabacum (+ Hyoscyamus aureus) with a nucleo-plastome incompatibility.
Theoretical and Applied Genetics, 2002Co-Authors: Mikhajlo K Zubko, Elena I Zubko, Yuri Yu. GlebaAbstract:Cytoplasmic hybrids (Cybrids) in a novel inter-generic combination, Nicotiana tabacum (+Hyoscyamus aureus), were generated by fusion of protoplasts from a plastome tobacco albino mutant (line R100a1) and γ-irradiated green protoplasts of H. aureus. Cybrids possessed a plastome of H. aureus and a rearranged mitochondrial DNA. The Cybrids displayed a syndrome of nucleo-plastome incompatibility expressed as a partial chlorophyll-deficiency of cotyledonary and true leaves at the early stage of vegetative development of plants grown from seeds in soil. During later development, the plants restored a normal green coloration. This character is phenotypically indistinguishable from the same syndrome in previously generated Cybrids N. tabacum (+H. nigrum). In contrast to the Cybrids N. tabacum (+H. nigrum), Cybrids N. tabacum (+H. aureus) were self-fertile, and did not manifest other features that were interpreted as nucleo-mitochondrial incompatibilities in N. tabacum (+H. nigrum) plants. Therefore, the Cybrids N. tabacum (+H. aureus) present a self-propagating system of Nicotiana (+Hyoscyamus) nucleo-plastome incompatibility in its pure form.
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self fertile Cybrids nicotiana tabacum hyoscyamus aureus with a nucleo plastome incompatibility
Theoretical and Applied Genetics, 2002Co-Authors: Mikhajlo K Zubko, Elena I Zubko, Yuri Yu. GlebaAbstract:Cytoplasmic hybrids (Cybrids) in a novel inter-generic combination, Nicotiana tabacum (+Hyoscyamus aureus), were generated by fusion of protoplasts from a plastome tobacco albino mutant (line R100a1) and γ-irradiated green protoplasts of H. aureus. Cybrids possessed a plastome of H. aureus and a rearranged mitochondrial DNA. The Cybrids displayed a syndrome of nucleo-plastome incompatibility expressed as a partial chlorophyll-deficiency of cotyledonary and true leaves at the early stage of vegetative development of plants grown from seeds in soil. During later development, the plants restored a normal green coloration. This character is phenotypically indistinguishable from the same syndrome in previously generated Cybrids N. tabacum (+H. nigrum). In contrast to the Cybrids N. tabacum (+H. nigrum), Cybrids N. tabacum (+H. aureus) were self-fertile, and did not manifest other features that were interpreted as nucleo-mitochondrial incompatibilities in N. tabacum (+H. nigrum) plants. Therefore, the Cybrids N. tabacum (+H. aureus) present a self-propagating system of Nicotiana (+Hyoscyamus) nucleo-plastome incompatibility in its pure form.
Russell H. Swerdlow - One of the best experts on this subject based on the ideXlab platform.
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Cytoplasmic hybrid (cybrid) cell lines as a practical model for mitochondriopathies
Redox biology, 2014Co-Authors: Heather M. Wilkins, Steven M. Carl, Russell H. SwerdlowAbstract:Cytoplasmic hybrid (cybrid) cell lines can incorporate human subject mitochondria and perpetuate its mitochondrial DNA (mtDNA)-encoded components. Since the nuclear background of different cybrid lines can be kept constant, this technique allows investigators to study the influence of mtDNA on cell function. Prior use of Cybrids has elucidated the contribution of mtDNA to a variety of biochemical parameters, including electron transport chain activities, bioenergetic fluxes, and free radical production. While the interpretation of data generated from cybrid cell lines has technical limitations, Cybrids have contributed valuable insight into the relationship between mtDNA and phenotype alterations. This review discusses the creation of the cybrid technique and subsequent data obtained from cybrid applications.
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oxidative stress involvement in α synuclein oligomerization in parkinson s disease Cybrids
Antioxidants & Redox Signaling, 2009Co-Authors: Raquel A Esteves, Russell H. Swerdlow, Daniela M Arduino, Catarina R Oliveira, Sandra M. CardosoAbstract:Mitochondrial dysfunction, oxidative stress, and alpha-synuclein oligomerization occur in Parkinson disease (PD). We used an in vitro PD cybrid approach that models these three phenomena specifically to evaluate the impact of mitochondria-derived oxidative stress on alpha-synuclein oligomerization. Compared with control cybrid cell lines, reactive oxygen species (ROS) production and protein oxidative stress markers were elevated in PD Cybrids. The antioxidants CoQ(10) and GSH attenuated changes in PD cybrid peroxide, protein carbonyl, and protein sulfhydryl levels. Elevated PD cybrid alpha-synuclein oligomer levels were also attenuated by CoQ(10) and GSH. In PD Cybrids, alpha-synuclein oligomerization was activated via a complex I-mediated increase in the free tubulin/polymerized tubulin ratio. CoQ(10) but not GSH increased complex I activity, restored ATP to control levels, and normalized the PD cybrid free tubulin/polymerized tubulin ratio. Overall, we conclude that two different antioxidants can decrease alpha-synuclein oligomerization whether by improving mitochondrial function or by preventing protein carbonylation or both. We conclude that mitochondrial dysfunction can induce alpha-synuclein oligomerization via ATP depletion-driven microtubule depolymerization and via ROS increase-driven protein oxidation.
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mitochondrial function in parkinson s disease Cybrids containing an nt2 neuron like nuclear background
Mitochondrion, 2008Co-Authors: Raquel A Esteves, Russell H. Swerdlow, Catarina R Oliveira, Filipa A Domingues, Luisa I Ferreira, Cristina Januario, Sandra M. CardosoAbstract:Mitochondria likely play a role in Parkinson’s disease (PD) neurodegeneration. We modelled PD by creating cytoplasmic hybrid (cybrid) cell lines in which endogenous mitochondrial DNA (mtDNA) from PD or control subject platelets was expressed within human teratocarcinoma (NT2) cells previously depleted of endogenous mtDNA. Complex I activity was reduced in both PD cybrid lines and in the platelet mitochondria used to generate them. Under basal conditions PD Cybrids had less ATP, more LDH release, depolarized mitochondria, less mitochondrial cytochrome c, and higher caspase 3 activity. Equivalent MPP + exposures are more likely to trigger
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Mitochondria in Cybrids containing mtDNA from persons with mitochondriopathies
Journal of neuroscience research, 2007Co-Authors: Russell H. SwerdlowAbstract:The cytoplasmic hybrid (cybrid) technique allows investigators to express selected mitochondrial DNA (mtDNA) sequences against fixed nuclear DNA (nDNA) backgrounds. Cybrids have been used to study the effects of known mtDNA mutations on mitochondrial biochemistry, mtDNA-nDNA inter-species compatibility, and mtDNA integrity in persons without mtDNA mutations defined previously. This review discusses events leading up to creation of the cybrid technique, as well as data obtained via application of the cybrid strategies listed above. Although interpreting cybrid data requires awareness of technique limitations, valuable insights into mtDNA genotype-functional phenotype relationships are suggested.
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Mitochondrial abnormalities in cybrid cell models of sporadic Alzheimer's disease worsen with passage in culture.
Neurobiology of disease, 2004Co-Authors: Patricia A. Trimmer, Russell H. Swerdlow, W. Davis Parker, Paula M. Keeney, M.kate Borland, Frederic A. Simon, Jatanna Almeida, Janice P. Parks, James P. BennettAbstract:We created and studied new cybrid cell lines from sporadic Alzheimer's disease (SAD) or control (CTL) subjects to assess mitochondrial abnormalities just after metabolic selection ("early passage") and again six passages later ("late passage"). Cytochrome oxidase (CO) activities in early passage SAD Cybrids created independently from the same platelet samples were highly correlated. Early passage SAD and CTL Cybrids showed equivalent mitochondrial morphologies. Late passage SAD Cybrids showed increased mitochondrial number, reduced mitochondrial size, and an approximately eightfold increase in morphologically abnormal mitochondria. Deficiency of SAD cybrid mitochondrial membrane potentials (DeltaPsi(M)) increased with passage. Mitochondrial bromodeoxyuridine (BrdU) uptake to estimate mitochondrial DNA (mtDNA) synthesis did not change with passage in CTL but increased in SAD Cybrids. With time in culture, SAD mtDNA appears to replicate faster in Cybrids, yielding cells with relative worsening of bioenergetic function. Metabolically deleterious SAD mitochondrial genes, like those in yeast, may have a replicative advantage over nondeleterious mitochondrial genes that assume dominance in CTL Cybrids.
Marilyn Chwa - One of the best experts on this subject based on the ideXlab platform.
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age related macular degeneration amd transmitochondrial Cybrids protected from cellular damage and death by human retinal progenitor cells hrpcs
Stem Cells International, 2021Co-Authors: Daniel Azzam, Marilyn Chwa, Kevin Schneider, Cristina M. Kenney, Janghyeon Cho, Chinhui Hsiang, Henry Klassen, Jing YangAbstract:Purpose. One of the leading causes of irreversible blindness worldwide, age-related macular degeneration (AMD) is a progressive disorder leading to retinal degeneration. While several treatment options exist for the exudative form of AMD, there are currently no FDA-approved treatments for the more common nonexudative (atrophic) form. Mounting evidence suggests that mitochondrial damage and retinal pigment epithelium (RPE) cell death are linked to the pathogenesis of AMD. Human retinal progenitor cells (hRPCs) have been studied as a potential restorative therapy for degenerative conditions of the retina; however, the effects of hRPC treatment on retinal cell survival in AMD have not been elucidated. Methods. In this study, we used a cell coculture system consisting of hRPCs and AMD or age-matched normal cybrid cells to characterize the effects of hRPCs in protecting AMD Cybrids from cellular and mitochondrial damage and death. Results. AMD Cybrids cocultured with hRPCs showed (1) increased cell viability; (2) decreased gene expression related to apoptosis, autophagy, endoplasmic reticulum (ER) stress, and antioxidant pathways; and (3) downregulation of mitochondrial replication genes compared to AMD Cybrids without hRPC treatment. Furthermore, hRPCs cocultured with AMD Cybrids showed upregulation of (1) neuronal and glial markers, as well as (2) putative neuroprotective factors, responses not found when hRPCs were cocultured with age-matched normal Cybrids. Conclusion. The current study provides the first evidence that therapeutic benefits may be obtainable using a progenitor cell-based approach for atrophic AMD. Our results suggest that bidirectional interactions exist between hRPCs and AMD Cybrids such that hRPCs release trophic factors that protect the Cybrids against the cellular and mitochondrial changes involved in AMD pathogenesis while, conversely, AMD Cybrids upregulate the release of these neuroprotective factors by hRPCs while promoting hRPC differentiation. These in vitro data provide evidence that hRPCs may have therapeutic potential in atrophic AMD.
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Differential effects of risuteganib and bevacizumab on AMD cybrid cells.
Experimental eye research, 2020Co-Authors: Kevin Schneider, Marilyn Chwa, Shari R. Atilano, Zixuan Shao, John Park, Hampar L. Karageozian, Vicken H. Karageozian, M. Cristina KenneyAbstract:Abstract Purpose Intravitreal injections of anti-vascular endothelial growth factor (VEGF) treatments are currently used to treat wet age-related macular degeneration (AMD), diabetic retinopathy, and macular edema. Chronic, repetitive treatments with anti-VEGF may have unintended consequences beyond the inhibition of angiogenesis. Most recently, clinical trials have been conducted with risuteganib (RSG, Luminate®), which is anti-angiogenic and has neuroprotective and anti-inflammatory properties. Mitochondrial damage and dysfunction play a major role in development of AMD. Transmitochondrial Cybrids are cell lines established by fusing human retinal pigment epithelial (RPE) cells that are Rho0 (lacking mtDNA) with platelets isolated from AMD subjects or age-matched normal subjects. Cybrid cell lines have identical nuclei but mitochondria from different subjects, enabling investigation of the functional consequences of damaged AMD mitochondria. The present study compares the responses of AMD Cybrids treated with bevacizumab (Bmab, Avastin®) versus risuteganib (RSG, Luminate®). Methods Cybrids were created by fusing mtDNA depleted ARPE-19 cells with platelets from AMD or age-matched normal patients. AMD (n = 5) and normal (n = 3) Cybrids were treated for 48 h with or without 1x clinical dose of 1.25 mg/50 μl (25,000 μg/ml) of Bmab or 1.0 mg/50 μl (20,000 μg/ml) of RSG. Cultures were analyzed for levels of cleaved caspase 3/7 and NucLight Rapid Red staining (IncuCyte® Live Cell Imager), mitochondrial membrane potential (ΔΨm, JC1 assay) or reactive oxygen species (ROS, H2DCFDA assay). Expression levels of genes related to the following pathways were analyzed with qRT-PCR: Apoptosis (BAX, BCL2L13, CASP-3, -7, -9); angiogenesis (VEGFA, HIF1α, PDGF); integrins (ITGB-1, -3, -5, ITGA-3, -5, -V); mitochondrial biogenesis (PGC1α, POLG); oxidative stress (SOD2, GPX3, NOX4); inflammation (IL-6, -18, -1β, IFN-β1); and signaling (P3KCA, PI3KR1). Statistical analyses were performed using GraphPad Prism software. Results The untreated AMD Cybrids had significantly higher levels of cleaved caspase 3/7 compared to the untreated normal Cybrids. The Bmab-treated AMD Cybrids showed elevated levels of cleaved caspase 3/7 compared to untreated AMD or RSG-treated AMD Cybrids. The Bmab-treated Cybrids had lower ΔΨm compared to untreated AMD or RSG-treated AMD Cybrids. The ROS levels were not changed with Bmab or RSG treatment. Results showed that Bmab-treated Cybrids had higher expression levels of inflammatory (IL-6, IL1-β), oxidative stress (NOX4) and angiogenesis (VEGFA) genes compared to untreated AMD, while RSG-treated Cybrids had lower expression levels of apoptosis (BAX), angiogenesis (VEGFA) and integrin (ITGB1) genes. Conclusions These data suggest that the mechanism(s) of action of RSG, an integrin regulator, and Bmab, a recombinant monoclonal antibody, affect the AMD RPE cybrid cells differently, with the former having more anti-apoptosis properties, which may be desirable in treating degenerative ocular diseases.
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Differential effects of cisplatin on cybrid cells with varying mitochondrial DNA haplogroups.
PeerJ, 2020Co-Authors: Sina Abedi, Marilyn Chwa, Shari R. Atilano, Gregory Yung, Kunal Thaker, Steven D. Chang, Kevin Schneider, Nitin Udar, Daniela A. Bota, M. Cristina KenneyAbstract:Author(s): Abedi, Sina; Yung, Gregory; Atilano, Shari R; Thaker, Kunal; Chang, Steven; Chwa, Marilyn; Schneider, Kevin; Udar, Nitin; Bota, Daniela; Kenney, M Cristina | Abstract: BackgroundDrug therapy yields different results depending on its recipient population. Cisplatin, a commonly used chemotherapeutic agent, causes different levels of resistance and side effects for different patients, but the mechanism(s) are presently unknown. It has been assumed that this variation is a consequence of differences in nuclear (n) DNA, epigenetics, or some external factor(s). There is accumulating evidence that an individual's mitochondrial (mt) DNA may play a role in their response to medications. Variations within mtDNA can be observed, and an individual's mtDNA can be categorized into haplogroups that are defined by accumulations of single nucleotide polymorphisms (SNPs) representing different ethnic populations.MethodsThe present study was conducted on transmitochondrial cytoplasmic hybrids (Cybrids) that possess different maternal-origin haplogroup mtDNA from African (L), Hispanic [A+B], or Asian (D) backgrounds. Cybrids were created by fusing Rho0 ARPE-19 cells (lacking mtDNA) with platelets, which contain numerous mitochondria but no nuclei. These cybrid cells were cultured to passage five, treated with cisplatin, incubated for 48 h, then analyzed for cell metabolic activity (tetrazolium dye (MTT) assay), mitochondrial membrane potential (JC-1 assay), cytotoxicity (lactate dehydrogenase (LDH) assay), and gene expression levels for ALK, BRCA1, EGFR, and ERBB2/HER2.ResultsResults indicated that untreated Cybrids with varying mtDNA haplogroups had similar relative metabolic activity before cisplatin treatment. When treated with cisplatin, (1) the decline in metabolic activity was greatest in L (27.4%, p l 0.012) l D (24.86%, p = 0.0001) and [A+B] Cybrids (24.67%, p = 0.0285) compared to untreated Cybrids; (2) mitochondrial membrane potential remained unchanged in all Cybrids (3) LDH production varied between Cybrids (L g[A+B], p = 0.0270). (4) The expression levels decreased for ALK in L (p l 0.0001) and [A+B] (p = 0.0001) Cybrids but not in D Cybrids (p = 0.285); and decreased for EGFR in [A+B] Cybrids (p = 0.0246) compared to untreated Cybrids.ConclusionOur findings suggest that an individual's mtDNA background may be associated with variations in their response to cisplatin treatment, thereby affecting the efficiency and the severity of side effects from the treatment.
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Effects of bevacizumab, ranibizumab, and aflibercept on phagocytic properties in human RPE Cybrids with AMD versus normal mitochondria.
Experimental eye research, 2018Co-Authors: Sina Abedi, Marilyn Chwa, Kevin Schneider, M. Cristina KenneyAbstract:Abstract Purpose A critical biological function of retina pigment epithelium (RPE) cells is phagocytosis of photoreceptor outer segment (POS) disc membranes. Mitochondrial damage and dysfunction are associated with RPE cells of age-related macular degeneration (AMD) retinas. In this study, we use a transmitochondrial cybrid model to compare the phagocytic properties of RPE cells that contain AMD mitochondria versus age-matched normal mitochondria and their response to treatment with anti-vascular endothelial growth factor (VEGF) drugs: bevacizumab, ranibizumab, and aflibercept. Methods Cybrids, which are cell lines with identical nuclei but mitochondria (mt) from different subjects, are created by fusing mtDNA depleted ARPE-19 cells with platelets from AMD or age-matched normal patients. AMD (n = 5) and normal (n = 5) Cybrids were treated with 1 μm fluorescent latex beads (1.52 × 107 beads/mL) and either 2.09 μM of bevacizumab, 2.59 μM of ranibizumab, or 5.16 μM of aflibercept. These doses of anti-VEGF drugs are equivalent to intravitreal injections given to AMD patients with choroidal neovascularization. Flow cytometry was performed using the ImageStreamX Mark II to assess phagocytic bead-uptake. The average fold values for bead-uptake and SEM were calculated using GraphPad Prism software. Results Normal Cybrids showed decreased bead-uptake with a fold value of 0.65 ± 0.103 (p = 0.01) after treatment with bevacizumab, 0.80 ± 0.034 (p = 0.0003) with ranibizumab, and 0.81 ± 0.053 (p = 0.007) with aflibercept compared to the untreated normal Cybrids (baseline fold of 1). The bevacizumab-treated, ranibizumab-treated, and aflibercept-treated AMD Cybrids had decreased bead-uptake with a fold value of 0.71 ± 0.061 (p = 0.001), 0.70 ± 0.101 (p = 0.02), and 0.74 ± 0.125 (p = 0.07), respectively, compared to the untreated AMD Cybrids (baseline fold of 1). Conclusions Our initial findings showed that when treated with bevacizumab and ranibizumab, both AMD Cybrids and age-matched normal Cybrids had a significant decrease in bead-uptake. A similar decrease in bead-uptake was found in normal Cybrids treated with aflibercept and while the AMD values trended lower, they were not significant. This data suggests that anti-VEGF drugs can cause loss of phagocytic function.
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Human Retinal Transmitochondrial Cybrids with J or H mtDNA Haplogroups Respond Differently to Ultraviolet Radiation: Implications for Retinal Diseases
2016Co-Authors: Deepika Malik, Tiffany Hsu, Payam Falatoonzadeh, Mohamed Tarek, Marilyn Chwa, Shari R. Atilano, Claudio Ramirez, Anthony B. Nesburn, David S. Boyer, Michal S. JazwinskiAbstract:Background: It has been recognized that cells do not respond equally to ultraviolet (UV) radiation but it is not clear whether this is due to genetic, biochemical or structural differences of the cells. We have a novel cybrid (cytoplasmic hybrids) model that allows us to analyze the contribution of mitochondrial DNA (mtDNA) to cellular response after exposure to sub-lethal dose of UV. mtDNA can be classified into haplogroups as defined by accumulations of specific single nucleotide polymorphisms (SNPs). Recent studies have shown that J haplogroup is high risk for age-related macular degeneration while the H haplogroup is protective. This study investigates gene expression responses in J Cybrids versus H Cybrids after exposure to sub-lethal doses of UV-radiation. Methodology/Principal Findings: Cybrids were created by fusing platelets isolated from subjects with either H (n = 3) or J (n = 3) haplogroups with mitochondria-free (Rho0) ARPE-19 cells. The H and J Cybrids were cultured for 24 hours, treated with 10 mJ of UV-radiation and cultured for an additional 120 hours. Untreated and treated Cybrids were analyzed fo
Mikhajlo K Zubko - One of the best experts on this subject based on the ideXlab platform.
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Self-fertile Cybrids Nicotiana tabacum (+ Hyoscyamus aureus) with a nucleo-plastome incompatibility.
Theoretical and Applied Genetics, 2002Co-Authors: Mikhajlo K Zubko, Elena I Zubko, Yuri Yu. GlebaAbstract:Cytoplasmic hybrids (Cybrids) in a novel inter-generic combination, Nicotiana tabacum (+Hyoscyamus aureus), were generated by fusion of protoplasts from a plastome tobacco albino mutant (line R100a1) and γ-irradiated green protoplasts of H. aureus. Cybrids possessed a plastome of H. aureus and a rearranged mitochondrial DNA. The Cybrids displayed a syndrome of nucleo-plastome incompatibility expressed as a partial chlorophyll-deficiency of cotyledonary and true leaves at the early stage of vegetative development of plants grown from seeds in soil. During later development, the plants restored a normal green coloration. This character is phenotypically indistinguishable from the same syndrome in previously generated Cybrids N. tabacum (+H. nigrum). In contrast to the Cybrids N. tabacum (+H. nigrum), Cybrids N. tabacum (+H. aureus) were self-fertile, and did not manifest other features that were interpreted as nucleo-mitochondrial incompatibilities in N. tabacum (+H. nigrum) plants. Therefore, the Cybrids N. tabacum (+H. aureus) present a self-propagating system of Nicotiana (+Hyoscyamus) nucleo-plastome incompatibility in its pure form.
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self fertile Cybrids nicotiana tabacum hyoscyamus aureus with a nucleo plastome incompatibility
Theoretical and Applied Genetics, 2002Co-Authors: Mikhajlo K Zubko, Elena I Zubko, Yuri Yu. GlebaAbstract:Cytoplasmic hybrids (Cybrids) in a novel inter-generic combination, Nicotiana tabacum (+Hyoscyamus aureus), were generated by fusion of protoplasts from a plastome tobacco albino mutant (line R100a1) and γ-irradiated green protoplasts of H. aureus. Cybrids possessed a plastome of H. aureus and a rearranged mitochondrial DNA. The Cybrids displayed a syndrome of nucleo-plastome incompatibility expressed as a partial chlorophyll-deficiency of cotyledonary and true leaves at the early stage of vegetative development of plants grown from seeds in soil. During later development, the plants restored a normal green coloration. This character is phenotypically indistinguishable from the same syndrome in previously generated Cybrids N. tabacum (+H. nigrum). In contrast to the Cybrids N. tabacum (+H. nigrum), Cybrids N. tabacum (+H. aureus) were self-fertile, and did not manifest other features that were interpreted as nucleo-mitochondrial incompatibilities in N. tabacum (+H. nigrum) plants. Therefore, the Cybrids N. tabacum (+H. aureus) present a self-propagating system of Nicotiana (+Hyoscyamus) nucleo-plastome incompatibility in its pure form.
