Biomedical subjects

Gino A Cortopassi

Publications and source records attributed to Gino A Cortopassi.

8 recordsLinked to original sources

PubMed2005

Frataxin deficiency alters heme pathway transcripts and decreases mitochondrial heme metabolites in mammalian cells.

Deficiency of the frataxin mRNA alters the transcriptome, triggering neuro- and cardiodegeneration in Friedreich's ataxia. We microarrayed murine frataxin-deficient heart tissue, liver tissue and cardiocytes and observed a transcript down-regulation to up-regulation ratio of nearly 2:1 with a mitochondrial localization of transcriptional changes. Combining all mouse and human microarray data for frataxin-deficient cells and tissues, the most consistently decreased transcripts were mitochondrial coproporphyrinogen oxidase (CPOX) of the heme pathway and mature T-cell proliferation 1, a homolog of yeast COX23, which is thought to function as a mitochondrial metallochaperone. Quantitative RT-PCR studies confirmed the significant down-regulation of Isu1, CPOX and ferrochelatase at 10 weeks in mouse hearts. We observed that mutant cells were resistant to aminolevulinate-dependent toxicity, as expected if the heme pathway was inhibited. Consistent with this, we observed increased cellular protoporphyrin IX levels, reduced mitochondrial heme a and heme c levels and reduced activity of cytochrome oxidase, suggesting a defect between protoporphyrin IX and heme a. Fe-chelatase activities were similar in mutants and controls, whereas Zn-chelatase activities were slightly elevated in mutants, supporting the idea of an altered metal-specificity of ferrochelatase. These results suggest that frataxin deficiency causes defects late in the heme pathway. As ataxic symptoms occur in other diseases of heme deficiency, the heme defect we observe in frataxin-deficient cells could be primary to the pathophysiological process.

Amino Acid Sequence↗

PubMed2005

Isolation of transcriptomal changes attributable to LHON mutations and the cybridization process.

Leber's hereditary optic neuropathy (LHON) is thought to be the most common disease resulting from mitochondrial DNA (mtDNA) point mutations, and transmitochondrial cytoplasmic hybrid (cybrid) cell lines are the most frequently used model for understanding the pathogenesis of mitochondrial disorders. We have used oligonucleotide microarrays and a novel study design based on shared transcripts to allocate transcriptomal changes into rho-zero-dependent, cybridization-dependent and LHON-dependent categories in these cells. The analysis indicates that the rho-zero process has the largest transcriptomal impact, followed by the cybridization process, and finally the LHON mutations. The transcriptomal impacts of the rho-zero and cybridization processes preferentially and significantly affect the mitochondrial compartment, causing upregulation of many transcripts involved in oxidative phosphorylation, presumably in response to the mtDNA depletion that occurs at the rho-zero step. Nine LHON-specific transcriptional alterations were shared among osteosarcoma cybrids and lymphoblasts bearing LHON mutations. Notably, the aldose reductase transcript was overexpressed in LHON cybrids and lymphoblasts. Aldose reductase is also overexpressed in diabetic retinopathy, leading to optic nerve and retinal complications. The LHON-specific increase in transcript level was confirmed by quantitative reverse transcription-polymerase chain reaction (RT-PCR), and a western blot confirmed a higher level of aldose reductase in mutant mitochondria. One product of aldose reductase is sorbitol, which has been linked to osmotic stress, oxidative stress and optic neuropathy, and sorbitol levels were increased in LHON cybrids. If these results are confirmed in patient tissues, aldose reductase inhibitors could have some therapeutic value for LHON.

Aldehyde Reductase↗

PubMed2002

High-throughput measurement of mitochondrial membrane potential in a neural cell line using a fluorescence plate reader.

Mutations in mitochondrial genes cause mitochondrial genetic disease, which is often associated with deficiency of the mitochondrial membrane potential (MMP). We present a high-throughput method for measuring MMP in intact neural cells using TMRM, a well-known potentiometric dye, in a 48-well plate format. Addition of known MMP depolarizing agents, FCCP or DNP, resulted in a time- and concentration-dependent decrease in fluorescence, which was saturable, whereas the addition of drugs that affect non-mitochondrial properties did not. A cell line deficient in mtDNA had decreased fluorescence, which was not further depleted by a depolarizing agent. The high-throughput results are similar to those produced by more time-consuming and low-throughput flow cytometry or microscopy methods. This plate-based system could facilitate the identification of cell-permeant small molecules (i.e., drugs) that modify MMP, which could be used to enhance mitochondrial function, and also for screening small populations of neural cells for mutations in nuclear or mtDNA genes that decrease MMP.

Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone↗

PubMed2002

A neutral theory predicts multigenic aging and increased concentrations of deleterious mutations on the mitochondrial and Y chromosomes.

Population genetic forces have molded the constitution of the human genome over evolutionary time, and some of the most important parameters are the initial frequency of the allele, p, the effective population size, Ne, and the selection coefficient, s. There is considerable agreement among evolutionary gerontologists that the amplitude of -s is small for alleles that are Deleterious In Late Life (DILL), and thus DILL traits are effectively neutral and should be fixed in the human population in relationship to Ne and p. Even higher rates of fixation of deleterious mutations are predicted to occur in the two nonrecombinant genomes in humans, i.e., the Y chromosome and the mitochondrial genome, as a consequence of their lower Ne than autosomes, and the predicted higher rate of fixation of deleterious alleles on the Y may explain the reduced average life span of males vs. females. The high probability of fixation of neutral and mildly deleterious mutations in the mitochondrial genome explains in part its fast rate of evolution, the high observed frequency of mitochondrial disease in relationship to this genome's small size, and may be the underlying reason for the transfer of mitochondrial genes over evolutionary time to the nucleus. The predicted higher concentration of deleterious mutations on the mitochondrial genome could have some leverage to cause more dysfunction than that predicted by mitochondrial gene number alone, because of the essential role of mitochondrial gene function in multisubunit complexes, the coupling of mitochondrial functions, the observation that some mtDNA sequences facilitate somatic mutation, and the likelihood of deleterious mutations either increasing the production of or the sensitivity to mitochondrial ROS.

Aging↗

PubMed2002

Fixation of deleterious alleles, evolution and human aging.

Human aging, like everything else in biology, only makes sense in the context of evolution. There is substantial agreement among evolutionary geneticists regarding the evolutionary underpinnings of one of the main questions of human aging i.e. 'Why do we age?', which involves the higher rate of fixation of alleles that cause deleterious post-reproductive phenotypes vs. alleles that cause deleterious pre-reproductive phenotypes. Even higher rates of fixation of deleterious mutations are predicted to occur on population genetic grounds in two non-recombinant genomes in humans i.e. the Y chromosome, and the mitochondrial genome. The added burden of deleterious mutations on the Y may explain the reduced average lifespan of males vs. females. A high predicted fixation rate of the mitochondrial genome may explain its fast rate of evolution, and the high frequency of mitochondrial disease in relationship to this genomes' small size, and may underlie the transfer of mitochondrial genes over evolutionary time to the nucleus. Thus a molecular evolutionary model of aging which focuses on increased fixation rates of (1) nuclear alleles which only have deleterious effects post-reproduction, (2) deleterious genes on the Y chromosome, and (3) deleterious genes on the mitochondrial genome, makes multiple testable predictions about the causes and mechanisms of aging.

Aging↗

PubMed2002

Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells.

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.

Animals↗

PubMed2001

Cells bearing mutations causing Leber's hereditary optic neuropathy are sensitized to Fas-Induced apoptosis.

Three prevalent mitochondrial DNA pathogenic mutations at positions 11778, 3460, and 14484, which affect different subunits of Complex I, cause retinal ganglion cell death and optic nerve atrophy in Leber's hereditary optic neuropathy (LHON). The cell death is painless and without inflammation, consistent with an apoptotic mechanism. We have investigated the possibility that the LHON mutation confers a pro-apoptotic stimulus and have tested the sensitivity of osteosarcoma-derived cybrid cells carrying the most common and severe mutations (11778 and 3460) to cell death induced by Fas. We observed that LHON cybrids were sensitized to Fas-dependent death. Control cells that bear the same mitochondrial genetic background (the J haplogroup) without the pathogenic 11778 mutation are no more sensitive than other controls, indicating that increased Fas-dependent death in LHON cybrids was induced by the LHON pathogenic mutations. The type of death was apoptotic by several criteria, including induction by Fas, inhibition by the caspase inhibitor zVAD-fmk (zVal-Ala-Asp-fluoro-methyl ketone), activation of DEVDase activity (Asp-Glu-Val-Asp protease), specific cleavage of caspase-3, DNA fragmentation, and increased Annexin-V labeling. These data indicate that the most common and severe LHON pathogenic mutations 11778 and 3460 predispose cells to apoptosis, which may be relevant for the pathophysiology of cell death in LHON, and potential therapy.

Amino Acid Chloromethyl Ketones↗

PubMedDate not supplied

Frataxin, iron-sulfur clusters, heme, ROS, and aging.

A deficiency in mitochondrial frataxin causes an increased generation of mitochondrial reactive oxygen species (ROS), which may contribute to the cell degenerative features of Friedreich's ataxia. In this work the authors demonstrate mitochondrial iron-sulfur cluster (ISC) defects and mitochondrial heme defects, and suggest how both may contribute to increased mitochondrial ROS in lymphoblasts from human patients. Mutant cells are deficient in the ISC-requiring mitochondrial enzymes aconitase and succinate dehydrogenase, but not in the non-ISC mitochondrial enzyme citrate synthase; also, the mitochondrial iron-sulfur scaffold protein IscU2 co-immunoprecipitates with frataxin in vivo. Presumably as a consequence of the iron-sulfur cluster defect, cytochrome c heme is deficient in mutants, as well as heme-dependent Complex IV. Mitochondrial superoxide is elevated in mutants, which may be a consequence of cytochrome c deficiency. Hydrogen peroxide, glutathione peroxidase activity, and oxidized glutathione (GSSG) are each elevated in mutants, consistent with activation of the glutathione peroxidase pathway. Mutant status blunted the effects of Complex III and IV inhibitors, but not a Complex I inhibitor, on superoxide production. This suggests that heme defects late in the electron transport chain of mutants are responsible for increased mutant superoxide. The impact of ISC and heme defects on ROS production with age are discussed.

Aging↗