Ian A. Trounce

ASSESSMENT OF MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION IN PATIENT MUSCLE BIOPSIES, LYMPHOBLASTS, AND TRANSMITOCHONDRIAL CELL LINES

Publisher Summary This chapter discusses the methods of assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Investigation of oxidative phosphorylation (OX-PHOS) in mitochondrial diseases has traditionally focused on the muscle biopsy. Skeletal muscle biopsy remains a valuable resource for biochemical studies as it is an easily accessed tissue and milligram quantities of mitochondria can be isolated for a wide range of OX-PHOS investigations. However, in mitochondrial diseases, the postmitotic muscle fibers commonly show secondary OX-PHOS defects that may hinder investigations of primary defects. Transformed cell lines expressing OX-PHOS defects provide a powerful model system for further genetic and biochemical characterization of nuclear and mitochondrial DNA (mtDNA) mutants and the design and testing of therapeutic approaches. The approach to OX-PHOS investigation combines the identification of patients with OX-PHOS defects using more sensitive muscle biopsy studies followed by further biochemical characterization of primary defects in Epstein–Barr virus-transformed lymphoblasts. The chapter presents novel methods for the production of transmitochondrial cybrids using lymphoblastoid and osteosarcoma ρO cells as recipients. Suspension enucleation of cells combined with electrofusion allows the use of any cell type, including lymphoblasts, as mitochondrial donors in fusions with either lymphoblastoid or osteosarcoma ρo cells.

DECLINE IN SKELETAL MUSCLE MITOCHONDRIAL RESPIRATORY CHAIN FUNCTION: POSSIBLE FACTOR IN AGEING

State III (activated) mitochondrial respiration rates with pyruvate/malate, glutamate/malate, and succinate as substrates were assayed in isolated intact skeletal muscle mitochondria in 29 subjects aged 16-92 years. There was a significant negative correlation between respiration rate and age with all substrates tested. A similar trend was seen for respiratory enzyme activities assayed in muscle homogenate. These findings suggest a substantial fall in mitochondrial oxidative capacity in ageing muscle, which may contribute to reduced exercise capacity in elderly people. Mitochondrial respiratory failure may contribute to the ageing process in other organs.

A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoform of the adenine nucleotide translocator

In an attempt to create an animal model of tissue-specif ic mitochondrial disease, we generated ‘knockout’ mice deficient in the heart/muscle isoform of the adenine nucleotide translocator (Ant1). Histological and ultrastructural examination of skeletal muscle from Ant1 null mutants revealed ragged-red muscle fibers and a dramatic proliferation of mitochondria, while examination of the heart revealed cardiac hypertrophy with mitochondrial proliferation. Mitochondria isolated from mutant skeletal muscle exhibited a severe defect in coupled respiration. Ant1 mutant adults also had a resting serum lactate level fourfold higher than that of controls, indicative of metabolic acidosis. Significantly, mutant adults manifested severe exercise intolerance. Therefore, Ant1 mutant mice have the biochemical, histological, metabolic and physiological characteristics of mitochondrial myopathy and cardiomyopathy.

Copper-Dependent Inhibition of Human Cytochrome c Oxidase by a Dimeric Conformer of Amyloid-β1-42

In studies of Alzheimers disease pathogenesis there is an increasing focus on mechanisms of intracellular amyloid-β (Aβ) generation and toxicity. Here we investigated the inhibitory potential of the 42 amino acid Aβ peptide (Aβ1-42) on activity of electron transport chain enzyme complexes in human mitochondria. We found that synthetic Aβ1-42 specifically inhibited the terminal complex cytochrome c oxidase (COX) in a dose-dependent manner that was dependent on the presence of Cu2+ and specific “aging” of the Aβ1-42 solution. Maximal COX inhibition occurred when using Aβ1-42 solutions aged for 3-6 h at 30°C. The level of Aβ1-42-mediated COX inhibition increased with aging time up to ∼6 h and then declined progressively with continued aging to 48 h. Photo-induced cross-linking of unmodified proteins followed by SDS-PAGE analysis revealed dimeric Aβ as the only Aβ species to provide significant temporal correlation with the observed COX inhibition. Analysis of brain and liver from an Alzheimers model mouse (Tg2576) revealed abundant Aβ immunoreactivity within the brain mitochondria fraction. Our data indicate that endogenous Aβ is associated with brain mitochondria and that Aβ1-42, possibly in its dimeric conformation, is a potent inhibitor of COX, but only when in the presence of Cu2+. We conclude that Cu2+-dependent Aβ-mediated inhibition of COX may be an important contributor to the neurodegeneration process in Alzheimers disease.

Mitochondrial DNA mutations in human degenerative diseases and aging.

A wide variety of mitochondrial DNA (mtDNA) mutations have recently been identified in degenerative diseases of the brain, heart, skeletal muscle, kidney and endocrine system. Generally, individuals inheriting these mitochondrial diseases are relatively normal in early life, develop symptoms during childhood, mid-life, or old age depending on the severity of the maternally-inherited mtDNA mutation; and then undergo a progressive decline. These novel features of mtDNA disease are proposed to be the product of the high dependence of the target organs on mitochondrial bioenergetics, and the cumulative oxidative phosphorylation (OXPHOS) defect caused by the inherited mtDNA mutation together with the age-related accumulation mtDNA mutations in post-mitotic tissues.

Recharacterization of the RGC-5 Retinal Ganglion Cell Line

PURPOSE The transformed RGC-5 retinal ganglion cell line is used widely in glaucoma research. Increased resistance to glutamate was noted in published literature and led to the recharacterization of the RGC-5 cell line. METHODS Characterization of the RGC-5 cell line was performed by sequencing of a region of the nuclear Thy1 gene and mitochondrial DNA sequencing of a region of the d-loop and tRNA(Phe) gene. Marker expression was examined in undifferentiated cells, and cells differentiated with 50 microg/mL succinyl concanavalin A (S Con A) for 3 days. Glutamate sensitivity was examined in undifferentiated and S Con A differentiated cells by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 24-hours of glutamate treatment. RESULTS RGC-5 cells were found to be of mouse (Mus musculus), not rat (Rattus norvegicus), origin by mitochondrial and nuclear DNA analyses. RGC-5 DNA sequenced in a second laboratory was subsequently found to be of M. musculus origin. Cells stained positively for the neuronal markers beta-tubulin and PGP9.5 and for the microtubule-associated protein tau, but not for known markers of ganglion cells such as neurofilaments or Thy1.2, suggesting that they likely represented a lineage of mouse neuronal precursor cells. Differentiation with S Con A did not increase RGC-5 sensitivity to glutamate excitotoxicity or increase the expression of retinal or ganglion cell marker proteins. CONCLUSIONS Investigators using cells designated as RGC-5 should confirm the species to be of rat origin and retinal-specific marker expression before considering their use as retinal ganglion-like cells.

Mitochondrial Dysfunction and Glaucoma

Glaucoma is increasingly recognized as a neurodegenerative disorder, characterized by the accelerated loss of retinal ganglion cells (RGCs) and their axons. Open angle glaucoma prevalence and incidence increase exponentially with increasing age, yet the pathophysiology underlying increasing age as a risk factor for glaucoma is not well understood. Accumulating evidence points to age-related mitochondrial dysfunction playing a key role in the etiology of other neurodegenerative disorders including amyotrophic lateral sclerosis, Alzheimer and Parkinson disease. The 2 major functions of mitochondria are the generation of ATP through oxidative phosphorylation and the regulation of cell death by apoptosis. This review details evidence to support our hypothesis that age-associated mitochondrial dysfunction renders RGCs susceptible to glaucomatous injury by reducing the energy available for repair processes and predisposing RGCs to apoptosis. Eliciting the role of mitochondria in glaucoma pathogenesis may uncover novel therapeutic targets for protecting the optic nerve and preventing vision loss in glaucoma.

Use of Transmitochondrial Cybrids To Assign a Complex I Defect to the Mitochondrial DNA-Encoded NADH Dehydrogenase Subunit 6 Gene Mutation at Nucleotide Pair 14459 That Causes Leber Hereditary Optic Neuropathy and Dystonia

A heteroplasmic G-to-A transition at nucleotide pair (np) 14459 within the mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) gene has been identified as the cause of Leber hereditary optic neuropathy (LHON) and/or pediatric-onset dystonia in three unrelated families. This ND6 np 14459 mutation changes a moderately conserved alanine to a valine at amino acid position 72 of the ND6 protein. Enzymologic analysis of mitochondrial NADH dehydrogenase (complex I) with submitochondrial particles isolated from Epstein-Barr virus-transformed lymphoblasts revealed a 60% reduction (P < 0.005) of complex I-specific activity in patient cell lines compared with controls, with no differences in enzymatic activity for complexes II plus III, III and IV. This biochemical defect was assigned to the ND6 np 14459 mutation by using transmitochondrial cybrids in which patient Epstein-Barr virus-transformed lymphoblast cell lines were enucleated and the cytoplasts were fused to a mtDNA-deficient (p 0) lymphoblastoid recipient cell line. Cybrids harboring the np 14459 mutation exhibited a 39% reduction (p < 0.02) in complex I-specific activity relative to wild-type cybrid lines but normal activity for the other complexes. Kinetic analysis of the np 14459 mutant complex I revealed that the Vmax of the enzyme was reduced while the Km remained the same as that of wild type. Furthermore, specific activity was inhibited by increasing concentrations of the reduced coenzyme Q analog decylubiquinol. These observations suggest that the np 14459 mutation may alter the coenzyme Q-binding site of complex I.

Oxidative stress and mitochondrial dysfunction in glaucoma.

Mitochondrial dysfunction increases reactive oxygen species (ROS) production and when this overwhelms the cellular antioxidant defences, oxidative stress ensues. Oxidative stress is recognized as a common pathologic pathway in many neurodegenerative diseases. Recent reports have also demonstrated oxidative stress in ocular tissues derived from experimental glaucoma models and clinical samples. There is also accumulating evidence pointing to mitochondrial dysfunction being present in some glaucoma patients. Thus oxidative stress from mitochondrial dysfunction may also play a causal role in glaucoma. The mechanisms by which oxidative stress may induce retinal ganglion cell loss in glaucoma are not fully understood but could include direct neurotoxic effects from ROS or indirect damage from oxidative stress-induced dysfunction of glial cells. This review will consider the evidence for the presence of oxidative stress in glaucoma; the mechanisms by which oxidative stress may contribute to disease pathogenesis; and also consider therapeutic approaches that target oxidative stress as a means of protecting against optic nerve degeneration.

The Role of Glia, Mitochondria, and the Immune System in Glaucoma

Author(s): Tezel, Gulgun; Fourth ARVO/Pfizer Ophthalmics Research Institute Conference Working Group