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Transmitochondrial embryonic stem cells containing pathogenic mtDNA mutations are compromised in neuronal differentiation.

ABSTRACT: Defects of the mitochondrial genome (mtDNA) cause a series of rare, mainly neurological disorders. In addition, they have been implicated in more common forms of movement disorders, dementia and the ageing process. In order to try to model neuronal dysfunction associated with mitochondrial disease, we have attempted to establish a series of transmitochondrial mouse embryonic stem cells harbouring pathogenic mtDNA mutations.Transmitochondrial embryonic stem cell cybrids were generated by fusion of cytoplasts carrying a variety of mtDNA mutations, into embryonic stem cells that had been pretreated with rhodamine 6G, to prevent transmission of endogenous mtDNA. Cybrids were differentiated into neurons and assessed for efficiency of differentiation and electrophysiological function.Neuronal differentiation could occur, as indicated by expression of neuronal markers. Differentiation was impaired in embryonic stem cells carrying mtDNA mutations that caused severe biochemical deficiency. Electrophysiological tests showed evidence of synaptic activity in differentiated neurons carrying non-pathogenic mtDNA mutations or in those that caused a mild defect of respiratory activity. Again, however, neurons carrying mtDNA mutations that resulted in severe biochemical deficiency had marked reduction in post-synaptic events.Differentiated neurons carrying severely pathogenic mtDNA defects can provide a useful model for understanding how such mutations can cause neuronal dysfunction.

SUBMITTER: Kirby DM

PROVIDER: S-EPMC2730481 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Transmitochondrial embryonic stem cells containing pathogenic mtDNA mutations are compromised in neuronal differentiation.

Kirby D M DM Rennie K J KJ Smulders-Srinivasan T K TK Acin-Perez R R Whittington M M Enriquez J-A JA Trevelyan A J AJ Turnbull D M DM Lightowlers R N RN

Cell proliferation 20090623 4

<h4>Objectives</h4>Defects of the mitochondrial genome (mtDNA) cause a series of rare, mainly neurological disorders. In addition, they have been implicated in more common forms of movement disorders, dementia and the ageing process. In order to try to model neuronal dysfunction associated with mitochondrial disease, we have attempted to establish a series of transmitochondrial mouse embryonic stem cells harbouring pathogenic mtDNA mutations.<h4>Materials and methods</h4>Transmitochondrial embry ...[more]

PMID: 19552636

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Project description:Mitochondrial dysfunction is associated with several retinal degenerative diseases including Age-related Macular Degeneration (AMD). Human mitochondrial DNA (mtDNA) haplogroups are inherited from a common ancestral clan and are defined by specific sets of genetic differences. The purpose of this study was to determine and compare the effects of mtDNA haplogroups H and J on transcriptome regulation and cellular resilience to oxidative stress in human RPE cytoplasmic hybrid (cybrid) cell lines in vitro. ARPE-19 cybrid cell lines containing mtDNA haplogroups H and J were created by fusing platelets obtained from normal individuals containing H and J haplogroups with mitochondria-deficient (Rho0) ARPE-19 cell lines. These cybrids were exposed to oxidative stress using 300 μM hydrogen peroxide (H2O2), following which mitochondrial structural dynamics was studied at varying time points using the mitochondrial markers - TOMM20 (Translocase of Outer Mitochondrial Membrane 20) and Mitotracker. To evaluate mitochondrial function, levels of ROS, ΔΨm and [Ca2+]m were measured using flow cytometry, and ATP levels were measured using luminescence. The H and J cybrid cell transcriptomes were compared using RNAseq to determine how changes in mtDNA regulate gene expression. Inflammatory and angiogenic markers were measured using Luminex assay to understand how these mtDNAs influenced cellular response to oxidative stress. Actin filaments' morphology was examined using confocal microscopy. Following exposure to H2O2 stress, the J cybrids showed increased mitochondrial swelling and perinuclear localization, disturbed fission and fusion, increased calcium uptake (p < 0.05), and higher secreted levels of TNF-α and VEGF (p < 0.001), compared to the H cybrids. Calcium uptake by J cybrids was reduced using an IP3R inhibitor. Thirteen genes involved in mitochondrial complex I and V function, fusion/fission events, cellular energy homeostasis, antioxidant defenses, and inflammatory responses, were significantly downregulated with log2 fold changes ranging between -1.5 and -5.1. Actin levels were also significantly reduced in stressed J cybrids (p ≤ 0.001) and disruption in actin filaments was observed. Thirty-eight genes involved in mitochondrial and cellular support functions, were upregulated with log2 fold changes of +1.5 to +5.9 in J cybrids compared to H cybrids. Our results demonstrate significant structural and functional differences between mtDNA haplogroups H vs. J -containing cybrid cells. Our study suggests that the J mtDNA haplogroup can alter the transcriptome to increase cellular susceptibility to stress and retinal degenerations.

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Transmitochondrial embryonic stem cells containing pathogenic mtDNA mutations are compromised in neuronal differentiation.

Publications

Transmitochondrial embryonic stem cells containing pathogenic mtDNA mutations are compromised in neuronal differentiation.

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