Evolution of Aging: Do Mitochondria hold the key

[Pages:15]Aging: Do Mitochondria Hold the Key? Michelle Marraffini University of Florida

Department of Biology Mentor: John Aris

Keywords: Mitochondria fission/fusion; Mdivi-1; Pdr3; networked mitochondria

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Abstract Both fission and fusion events are important for mtDNA integrity and respiratory

function of mitochondria. Previous experiments have showed increased and prolonged networked (fused) mitochondrial morphology under conditions of calorie restriction, a known extender of yeast chronological lifespan. The goal of these experiments was to induce this network phenotype. Mdivi-1 has been discovered to inhibit mitochondrial fission in yeast, producing network morphology. We hypothesized that Mdivi-1 would inhibit mitochondrial fission producing an observable networked morphology. Several conditions were tested to maximize Mdivi-1 action, including media containing elevated leucine (100mg/L) and 2%, 1.2%, 1%, 0.8%, and 0.6% glucose or galactose levels. Cells in 1% glucose samples performed similar to the previously experiments with a mean of 43.5% of cells treated with Mdivi-1 showing network morphology. The galactose samples did not show a considerable effect of Mdivi-1. From this experiment it was shown that in the low glucose samples no network morphology was observed, which is different from previous reports. This may be due to the deletion of the PDR3 gene, which is used to test Mdivi-1 because it impairs drug resistance. More studies are needed to understand the conditions that are optimal for the effects of Mdivi-1 on mitochondrial morphology and chronological lifespan.

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Introduction Evolution of Aging

Why do organisms show visible signs of aging correlated to a decrease in likelihood of survival and further reproduction, when increasing these traits at any age should increase an individual's fitness? An individual's main goal is to survive long enough to pass on genes to the next generation. Large amounts of information about the complete puzzle of animal aging have come to light but we still do not understand it fully. For the complete picture we would need to know the molecular mechanism of age-related changes across broad cell types, factors that determine species specific longevity, and the evolutionary basis for aging in cells and organisms (Miquel, 1998). General Senescence Theories of Aging

Many theories of aging are based on the "wear-and-tear" hypothesis, that organisms and cells, similar to man-made machines, simply get old and gears become worn leading to the breakdown of the "machine" (organism) (Weissman, 1889). More recent theories of aging focus on the role of oxygen free radicals as a molecular basis for cellular "wear and tear". Harman hypothesized that senescence results from the sum of damage due to deleterious free radicals that occur continuously and throughout cells and tissues (1956). Alternatively, Greshman believed it was a result of a small, continuous slipping in the defenses against oxygen toxicity (1962). Both theories point to oxygen radicals as a senescence causing factor but fail to explain why there are no repair mechanisms to counteract the harmful effects of these radicals even though all cells expend a considerable amount of energy protecting themselves against oxidative damage (Miquel, 1998).

Evolution favors maximum mitochondrial function at the expense of the maintenance of the organelle by switching-off some of the repair systems in somatic cells to save energy for

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reproduction (the disposable soma hypothesis) (Kirkwood and Holliday, 1979). Kirkwood also suggests the error theory of aging, termed the "error catastrophe" hypothesis, which states that aging is the result of progressive breakdown in the accuracy of the protein-synthesizing machinery (1977; Orgel, 1963). Experiments show that the cell allocates most of its energy to division and then saves energy after differentiation by switching off the mechanisms that ensure high accuracy of translation (Kirkwood 1977). By turning off these mechanisms the cell risks accumulating deleterious errors that could lead to its death. Role of Mitochondria

It is believed that a fundamental mechanism for animal aging is the oxygen stress suffered by the mitochondrial genome. The oxygen stress-mitochondria mutation theory hypothesizes that cell senescence is due to genetic damage by oxygen radicals derived from mitochondrial membrane (Miquel, 1998). Experiments on fixed post-mitotic cells (permanently differentiated) have shown large mitochondria found commonly in dead cells, consistent with the view that aging results in an impairment of mitochondrial biogenesis (Miquel, 1998). Damage occurs to mitochondrial DNA (mtDNA) as a result of its highly mutagenic environment (in the matrix near the inner membrane where oxyradicals are generated). Unlike in the nuclear genome mtDNA is not protected by histones and multiple repair mechanisms (Miquel, 1998). It is believed that this damage to mitochondrial DNA occurs almost exclusively in differentiated somatic cells because they cannot regenerate their mitochondria as effectively as actively dividing cells. Also, differentiated cells may be exposed to higher oxygen-stress due to the high respiration levels needed to carry out their specialized functions (Miquel, 1998). Fission and Fusion Balance

Fission occurs early in apoptosis very close in time to cytochrome c release, yet apoptosis is only one condition that induces mitochondrial fission and is associated with mitochondrial

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fragmentation (Suen, Norris, and Youle, 2008). Several components of mitochondrial fission machinery, Drp1, Fis1, and Endophilin B1 are linked to programmed cell death progression (Tanaka and Youle, 2008). Down regulation of Drp1, in humans, (Dnm1 in yeast) delays mitochondrial fragmentation, cytochrome c release, caspase activation, and cell death (Suen, Norris, and Youle, 2008). Scheckhuber et al. (2006) found that in the senescent phase the mitochondrial were spherical, indicating that fragmentation increases during aging. This associated with age-related increase in Dnm1 expression and networked morphology correlated with an extension of lifespan in yeast (Scheckhuber et al., 2006). Bax concentrates on the mitochondrial surface as foci once the cell has committed to die causing cytochrome c release (Suen, Norris, and Youle Fig. 5, 2008). In vitro, mitochondrial division inhibitor (Mdivi-1) selectively inhibits the mitochondrial division dynamin by inhibiting Dnm1p assembly and GTPase activity (Cassidy-Stone et al., 2008; Suen, Norris, and Youle, 2008). It also blocks Bax/Bak-dependent cytochrome c release from the mitochondria which delays apoptosis (Cassidy-Stone et. al., 2008; Tanaka and Youle, 2008).

Fusion deficient yeast shows mitochondrial fragmentation, which leads to the loss of mtDNA by unknown mechanisms (Okamoto and Shaw, 2005). These cells cannot survive in media that requires respiration for energy production (Okamoto and Shaw, 2005). While cells defective in mitochondrial fission form interconnected, net-like mitochondria that are respiratory competent (Okamoto and Shaw, 2005). Cells lacking both pathways lose mtDNA at an elevated frequency when grown in media that does not require respiration. Thus, both fission and fusion events are important for mtDNA integrity and the respiratory function of mitochondria (Okamoto and Shaw, 2005). Previous experiments in our laboratory (Seo et al., Unpublished) have observed increased and prolonged network mitochondrial morphology under calorie restriction, a

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factor known to extend yeast chronological longevity. Calorie restriction can be accomplished either by limiting the carbon source concentration in the media or performing a water wash. Mdivi-1 and Similar Compounds

Mitochondrial division promotes cell death by an unknown mechanism and Mdivi-1 can inhibit mitochondrial division (Cassidy-Stone et. al., 2008; Tanaka and Youle, 2008). It is unknown if Mdivi-1 extends chronological longevity in yeast by inhibiting mitochondrial fission. To study Mdivi-1 in yeast, we looked at its effects on mitochondrial morphology and chronological longevity (longevity of a cell once it has stopped replicating, measured by optical density). We hypothesized that Mdivi-1 would inhibit mitochondrial fission producing an observable networked morphology under ideal conditions. If this is the case there would be an increase in the amount of network mitochondrial phenotype when compared to samples without the drug Mdivi-1, and this phenotype would correlate with increased longevity. To test this hypothesis, experiments were designed to determine the performance of Mdivi-1 under conditions that could be suitable for a future chronological lifespan study. Methods Yeast

Yeast strain YJPA76 (pdr1, pdr3) was transformed with plasmid pVT100U-mtGFP (which expresses mitochondrially targeted green florescent protein) to make the mitochondria visible with an epiflorescent microscope. Transformants were grown and streaked to single colonies under selection and after a third day of growth 30 ?C inoculated into 5 mL of SD media. After 24 hours growth at 30 ?C, cultures were diluted with 5 mL of the final media (at 30 ?C) and separated into two tubes of 5 mL each. The goal was for samples to reach mid log (OD600 ~ 0.25) growth while two samples were allowed to reach full log (OD600 ~1.0). Droplets of 200

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mM Mdivi-1 or DMSO (solvent) were added as a control. The final dilution in culture medium was 1/1000, which yielded a final concentration of Mdivi-1 of 200 ?M. Media

To study mitochondrial morphology, synthetic dextrose (SD) minimal media were prepared in 100 ml aliquots of synthetic base ("S90") (Sherman, 2002). To separate aliquots 2%, 1.2%, 1%, 0.8% or 0.6% (wt/vol) glucose were added. Amino acids histidine (20mg/L), lysine (30mg/L), and leucine (30mg/L or elevated to 100mg/L) and the antibiotic G418 were also added to the media (Sherman, 2002). To each aliquot, 100 ?l of 1.5M ammonium hydroxide solution was added to adjust the pH of the solution. Mitochondrial Morphology

To study the mitochondrial morphology, different SD solutions were made (Table 1 and 2). Transformed yeast were allowed to grow in each media for two hours. Mitochondrial morphology of approximately 50 cells per treatment were observed using a Zeiss Axiophot fluorescent microscope equipped with a 100x objective once a day for seven days. Mitochondria morphology was classified as network, linear, or punctuated based on previously established criteria (Fig. 1). Also, noted the number of cells not expressing GFP and those classified as dead through Erythrocin B staining (a drug that accumulates in the yeast cells after their death). The percentages of cells expressing network phenotype were calculated based on the total amount of living cells in the sample. Statistical Tests

The results of these experiments were analyzed using a single factor ANalysis Of VAriance between groups (ANOVA). This provides a statistical test to determine whether or not the means of the experimental treatments are significantly different. The null hypothesis for this test was that there are no differences between the means of any experimental treatments. If the

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probability value (p) is less then 0.05 the null hypothesis will be accepted. This test was performed on data sets from the two experiments. Each experiment aimed to find the optimal conditions for Mdiv-1 effectiveness. One tested the leucine levels and optical density at point of drug addition, while the other looked at the carbon source given to the yeast. Results Glucose and Optical Density Study

Samples grown in SD media with various experimental treatments showed means ranging from approximately 18% to approximately 44% percent networked mitochondria (Table 1. Fig. 2). Notably the treatment grown in SD containing 2% glucose, standard amounts of amino acids (histidine 20mg/L, lysine 30mg/L, and leucine 30mg/L), and 200 ?M Mdivi-1 showed the highest mean of percent network mitochondrial morphology with 43.59% (Table 1.). Treatments containing elevated levels of leucine (100mg/L) did not show an increase in their percentage of network mitochondria. The ANOVA performed revealed a p value of 0.7415 indicating that none of the treatments produced a differential network phenotype. Staining with erythrosine B revealed that the percentage of dead cells observed ranged from 5.4% to 86.3% on day five of the experiment. An Anova was not performed for this portion of the experiment due to lack of data. It is useful to make comparisons between the treatments with and without drug (ie treatment 1 and 2; 3 and 4; etc. Table 2). The 2% glucose treatment combined with the drug showed more cell death than the treatment at the same conditions without drug (samples 1 and 2, Table 2). Glucose and Galactose Study

Treatment one (grown in 1% glucose) had the highest mean of network morphology at 43.5% and more network than the DMSO control, which had 38.29% (Table 3). The treatments containing 1% galactose showed 24% network morphology with the addition of Mdivi-1 and

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