title: Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells.
abstract: Inheritance of one of three primary mutations at positions 11778, 3460 or 14484 of the mitochondrial genome in subunits of Complex I causes Leber's Hereditary Optic Neuropathy (LHON), a specific degeneration of the optic nerve, resulting in bilateral blindness. It has been unclear why inheritance of a systemic mitochondrial mutation would result in a specific neurodegeneration. To address the neuron-specific degenerative phenotype of the LHON genotype, we have created cybrids using a neuronal precursor cell line, Ntera 2/D1 (NT2), containing mitochondria from patient lymphoblasts bearing the most common LHON mutation (11778) and the most severe LHON mutation (3460). The undifferentiated LHON-NT2 mutant cells were not significantly different from the parental cell control in terms of mtDNA/nDNA ratio, mitochondrial membrane potential, reactive oxygen species (ROS) production or the ability to reduce Alamar Blue. Differentiation of NT2s resulted in a neuronal morphology and neuron-specific pattern of gene expression, and a 3-fold reduction in mtDNA/nDNA ratio in both mutant and control cells; however, the differentiation protocol yielded significantly less LHON cells than controls, by 30%, indicating either a decreased proliferative potential or increased cell death of the LHON-NT2 cells. Differentiation of the cells to the neuronal form also resulted in significant increases in ROS production in the LHON-NT2 neurons versus controls, which is abolished by rotenone, a specific inhibitor of Complex I. We infer that the LHON genotype requires a differentiated neuronal environment in order to induce increased mitochondrial ROS, which may be the cause of the reduced NT2 yield; and suggest that the LHON degenerative phenotype may be the result of an increase in mitochondrial superoxide which is caused by the LHON mutations, possibly mediated through neuron-specific alterations in Complex I structure.
authors.affiliation: Department of Molecular Biosciences, One Shields Avenue, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
authors.forename: Alice
authors.initials: A
authors.lastname: Wong
authors.forename: Lucia
authors.initials: L
authors.lastname: Cavelier
authors.forename: Heather E
authors.initials: HE
authors.lastname: Collins-Schramm
authors.forename: Michael F
authors.initials: MF
authors.lastname: Seldin
authors.forename: Michael
authors.initials: M
authors.lastname: McGrogan
authors.forename: Marja-Liisa
authors.initials: ML
authors.lastname: Savontaus
authors.forename: Gino A
authors.initials: GA
authors.lastname: Cortopassi
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000473:pathology; D001766:Blindness / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D002460:Cell Line; D004272:DNA, Mitochondrial / Q000235:genetics; D006801:Humans; D008564:Membrane Potentials; D009154:Mutation; D009474:Neurons / Q000187:drug effects / Q000473:pathology*; D029242:Optic Atrophy, Hereditary, Leber / Q000235:genetics* / Q000473:pathology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
chemical_list: D004272:DNA, Mitochondrial; D017382:Reactive Oxygen Species; D014475:Uncoupling Agents; D012402:Rotenone