Michael V. Miceli

Zinc protects against oxidative damage in cultured human retinal pigment epithelial cells.

This study was undertaken to determine whether bioavailable zinc can influence the effects of oxidative stress on cultured human retinal pigment epithelial (RPE) cells. RPE cells were maintained for 7 d in culture medium containing 14 microM total zinc, or in medium containing 0.55 microM total zinc. After 1 week, MTT assays were performed to determine the relative cytotoxicity of H2O2 or paraquat on RPE cells. Conjugated dienes and thiobarbituric acid reactive substances (TBARS) were measured in RPE cells treated with 0, 0.5 mM H2O2, 10 microM FeSO4 + 0.5 mM H2O2 or 10 microM FeSO4 + xanthine/xanthine oxidase for 24 h or paraquat for 7 d. Oxidized proteins were determined by the formation of carbonyl residues. The antioxidants metallothionein, catalase, superoxide dismutase, and glutathione peroxidase were also measured. The MTT assays showed that zinc protected cultured RPE from the toxicity of H2O2 and paraquat. RPE cells in 0.55 microM zinc medium contained higher levels of TBARS, conjugated dienes and protein carbonyls due to the oxidative stresses, compared to cells in 14 microM zinc. Catalase and MT content were reduced in cells cultured in 0.55 microM zinc medium and were reduced additionally when treated with above stresses. Superoxide dismutase activity increased in 0.55 microM zinc medium in response to these stresses. Our results show RPE cells cultured in zinc-reduced medium are more susceptible to oxidative insult.

Experimental Transplantation of Human Retinal Pigment Epithelial Cells on Collagen Substrates

We studied the use of human retinal pigment epithelial cells cultured on a collagen support as a potential transplantation therapy to replace diseased or damaged retinal pigment epithelium. Using a transvitreal approach, we transplanted human retinal pigment epithelial cells attached to either a sheet of noncross-linked or cross-linked type I collagen into the subretinal space of New Zealand white rabbits, whose eyes lack pigment. Animals were killed after six weeks, and the eyes were fixed for light microscopy. The results demonstrated that, in eyes receiving the noncross-linked collagen support, a layer of pigmented donor retinal pigment epithelium was visible within the subretinal space, with a normal-appearing retina and no evidence of proliferative vitreoretinopathy or graft rejection. We believe this method may be applicable to replace dysfunctional retinal pigment epithelial cells in humans.

Molecular and bioenergetic differences between cells with African versus European inherited mitochondrial DNA haplogroups: Implications for population susceptibility to diseases

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

Loss of mitochondrial membrane potential triggers the retrograde response extending yeast replicative lifespan.

In the budding yeast Saccharomyces cerevisiae, loss of mitochondrial DNA (rho0) can induce the retrograde response under appropriate conditions, resulting in increased replicative lifespan (RLS). Although the retrograde pathway has been extensively elaborated, the nature of the mitochondrial signal triggering this response has not been clear. Mitochondrial membrane potential (MMP) was severely reduced in rho0 compared to rho+ cells, and RLS was concomitantly extended. To examine the role of MMP in the retrograde response, MMP was increased in the rho0 strain by introducing a mutation in the ATP1 gene, and it was decreased in rho+ cells by deletion of COX4. The ATP1-111 mutation in rho0 cells partially restored the MMP and reduced mean RLS to that of rho+ cells. COX4 deletion decreased MMP in rho+ cells to a value intermediate between rho+ and rho0 cells and similarly increased RLS. The increase in expression of CIT2, the diagnostic gene for the retrograde response, seen in rho0 cells, was substantially suppressed in the presence of the ATP1-111 mutation. In contrast, CIT2 expression increased in rho+ cells on deletion of COX4. Activation of the retrograde response results in the translocation of the transcription factor Rtg3 from the cytoplasm to the nucleus. Rtg3–GFP translocation to the nucleus was directly observed in rho0 and rho+ cox4Δ cells, but it was blunted in rho0 cells with the ATP1-111 mutation. We conclude that a decrease in MMP is the signal that initiates the retrograde response and leads to increased RLS.

Mitochondrial DNA Variants Mediate Energy Production and Expression Levels for CFH, C3 and EFEMP1 Genes: Implications for Age-Related Macular Degeneration

Background Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD). Methodology/Principal Findings Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism. Conclusion/Significance Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.

HRAS1 and LASS1 with APOE are associated with human longevity and healthy aging.

The search for longevity‐determining genes in human has largely neglected the operation of genetic interactions. We have identified a novel combination of common variants of three genes that has a marked association with human lifespan and healthy aging. Subjects were recruited and stratified according to their genetically inferred ethnic affiliation to account for population structure. Haplotype analysis was performed in three candidate genes, and the haplotype combinations were tested for association with exceptional longevity. An HRAS1 haplotype enhanced the effect of an APOE haplotype on exceptional survival, and a LASS1 haplotype further augmented its magnitude. These results were replicated in a second population. A profile of healthy aging was developed using a deficit accumulation index, which showed that this combination of gene variants is associated with healthy aging. The variation in LASS1 is functional, causing enhanced expression of the gene, and it contributes to healthy aging and greater survival in the tenth decade of life. Thus, rare gene variants need not be invoked to explain complex traits such as aging; instead rare congruence of common gene variants readily fulfills this role. The interaction between the three genes described here suggests new models for cellular and molecular mechanisms underlying exceptional survival and healthy aging that involve lipotoxicity.

Zinc content of human retinal pigment epithelium decreases with age and macular degeneration, but superoxide dismutase activity increases

We previously reported an age-related decline of catalase activity and metallothionein (MT) in human retinal pigment epithelium (RPE) isolated from donor eyes. MT content and catalase activity also were shown to decrease in RPE cells cultured in zinc-deficient medium. We now sought to determine whether zinc content and superoxide dismutase activity change with age and signs of macular degenerative disease in isolated human RPE. Eyes from 57 donors were graded with respect to disease by the presence or absence of visible drusen and macular pigment changes. RPE was collected from macular and peripheral regions. Tissues from 39 donors were separated into soluble and pigment granule fractions and zinc content determined by flame atomic absorption spectroscopy. RPE from 18 separate donors was used for SOD activity analysis. Total RPE zinc was 9% lower in eyes from donors >70 years of age compared to that from donors age 70 and showed a greater decline in the soluble fraction of macular RPE in eyes with signs of macular disease. Total SOD activity increased significantly (P < .05) with donor age. We conclude that zinc content decreased dramatically in the soluble fraction of macular RPE with age and signs of macular degeneration. Total SOD activity correlated negatively with changes in zinc content. It remains to be determined whether the age-related decline in macular zinc is causal with respect to previously observed decreases in cellular MT and catalase.

Influence of zinc on selected cellular functions of cultured human retinal pigment epithelium

Zinc is a necessary micronutrient, usually abundant in human RPE. Our study was undertaken to determine the effects of short-term, zinc deficiency on human retinal pigment epithelium (RPE) using a culture model of fetal human RPE cells. Human fetal RPE cells were isolated and cultured in Coons modified Hams F-12 medium. For zinc depletion studies, cells were cultured for 1 week in Chelex-treated Dulbeccos modified Eagles medium containing low (0.25 microM) or physiologic (11 microM) total zinc concentrations as determined by flame atomic absorption spectroscopy. Protein synthesis was determined by incorporation of 35S-cysteine/methionine and labeled proteins analysed by polyacrylamide gel electrophoresis. Several cell parameters and enzymes were significantly reduced below control when cultured in low zinc: zinc content (40%), proliferation (63%), protein/well (50%), catalase activity (68%), alkaline phosphatase activity (61%), alpha-mannosidase activity (68%), and metallothionein (82%). No statistically significant decline was seen in acid phosphatase activity, superoxide dismutase activity, glutathione peroxidase activity and dexamethasone induction of metallothionein. Zinc repletion (100 microM, 1 h) increased catalase and alpha-mannosidase activities from 32% and 33% of control to 75% and 73%, respectively. Cycloheximide did not inhibit this short-term zinc-induced repletion of catalase or alpha-mannosidase. Protein synthesis in low zinc medium was depressed, but not significantly, as shown by incorporation of radiolabeled 35S-cysteine/methionine into newly synthesized proteins. The effects of zinc deficiency in cultured human RPE are selective. Adequate intracellular zinc was required for maximal activity of some enzymes. The dependence of catalase activity on zinc was not predicted and may help explain the observed decline in catalase activity seen with age in RPE. Our model of zinc deficiency should prove useful in elucidating the complex effects of zinc deficiency and repletion in human RPE.

Zinc uptake by primate retinal pigment epithelium and choroid

We studied zinc uptake by nonhuman primate retinal pigment epithelium (RPE) and choroid, using 65Zn as a probe. With intravenously administered 65ZnCl2, virtually all detectable tracer was lost from the plasma after 20 hours but the pigment epithelium-choroid showed prominent uptake and retention. Plasma concentrations of oral 65ZnO remained high 20 hours after feeding. Uptake and retention of orally administered 65Zn as 65ZnO from the bloodstream by the RPE/choroid was avid in both young and old animals. Excretion in urine and feces was minimal. All pigmented ocular tissues took up and retained 65Zn. A survey of total zinc content of human and nonhuman primate ocular tissues showed that the pigmented tissues had consistently higher concentrations of zinc. Our results demonstrate for the first time direct uptake and retention of zinc from the blood by primate RPE and other ocular tissues.

Human Retinal Transmitochondrial Cybrids with J or H mtDNA Haplogroups Respond Differently to Ultraviolet Radiation: Implications for Retinal Diseases

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 for growth rates and gene expression profiles. The UV-treated and untreated J cybrids had higher growth rates compared to H cybrids. Before treatment, J cybrids showed lower expression levels for CFH, CD55, IL-33, TGF-A, EFEMP-1, RARA, BCL2L13 and BBC3. At 120 hours after UV-treatment, the J cybrids had decreased CFH, RARA and BBC3 levels but increased CD55, IL-33 and EFEMP-1 compared to UV-treated H cybrids. Conclusion/Significance In cells with identical nuclei, the cellular response to sub-lethal UV-radiation is mediated in part by the mtDNA haplogroup. This supports the hypothesis that differences in growth rates and expression levels of complement, inflammation and apoptosis genes may result from population-specific, hereditary SNP variations in mtDNA. Therefore, when analyzing UV-induced damage in tissues, the mtDNA haplogroup background may be important to consider.