Formamidopyrimidine—DNA Glycosylase Targeted to Specific Organelles in C2C12 Cells

Summary: Targeting mitochondria or nucleus with an oxidised DNA base remover

Interestingness: 3

Paper by Karah A Street, Kerrie L. Hall, Patrick Murphy and Christi A Walter in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

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The follow up paper to this one could be very interesting. This one seems to show that they could target either the mitochondria, or the nucleus with a protein, formamidopyrimidine-DNA glycosylase (Fpg), that gets rid of 2-deoxy-8-hydroxyguanine (8-OHdG), a screwed up version of the guanine base and the most common oxidised base. 8-OHdG causes the guanines (G) to be replaced by thymine (T) (by the normal repair mechanism I think). Fpg gets rid of 8-OHdG by taking out the base and leaving the ribose chain. This is supposedly a part of one of the normal DNA fixing mechanisms, called the base excision repair (BER), where one protein gets rid of a mutated base, and another goes and inserts the right base in.

So, yes, they created two DNA vectors, inserted them into some mouse muscle cells, and mostly saw what they were looking for, with the nuclear DNA being expressed mostly in the nucleus, and the mitochondrial in the cytoplasm. The levels of the molecule seemed pretty low though, and didn't correlate with the number of copies they inserted.

No assessment of the amount of 8-OHdG damage in the DNAs after transfection was done. I assume that's part of the plan for future work.
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Abstract follows:

Mitochondrial respiration provides a major source of energy for eukaryotic cells. However, the energy-producing processes also generate reactive oxygen species, which in turn damage mitochondrial DNA found in the mitochondrial matrix. Due to its locale, mitochondrial DNA is more susceptible to oxidative damage than nuclear DNA. While mitochondria do have some DNA repair capabilities, particularly base excision repair, oxidative damage persists in mitochondrial DNA. Correlations have been demonstrated between increasing age and increased levels of oxidative damage and mitochondrial DNA mutations. The current experiments were designed to begin to more directly delineate the role oxidative damage in mitochondrial DNA plays in aging. The mouse myoblast cell line, C2C12, was transfected with vectors, which express formamidopyrimidine-DNA glycosylase-myc fusion protein (Fpg-myc) and which contain either a mitochondrial or nuclear localization signal. Positive transfectants display expression of fpg at the mRNA level and exhibit an increase in Fpg activity in a whole-cell protein extract using a Fpg activity assay. Immunofluorescence analyses confirm that the transfected vectors have Fpg-myc appropriately targeted to mitochondria or nuclei. These cell lines with specifically targeted Fpg-myc expression provide the tools to test the effects of increasing the levels of a DNA glycosylase in mitochondria and nuclei on oxidative damage in DNA.

Modeling the Role of Mitochondrial Mutations in Cellular Aging

Summary: Model of what happens if mitochondria with damaged DNA both reproduces and degrades slower than intact mitochondria, and how it fits observed data

Interestingness: 7

Paper by Axel Kowald and Thomas BL Kirkwood in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

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They start by claiming there is a problem with the then-present theory of how damaged mitochondria are preferentially disseminated/take over cells by noting that it, the theory, is inconsistent with experimental results that show that damaged mitochondria is more prevalent in senescent cells than in dividing cells, and that the cells, or at least the muscle fibres, are taken over by one mutant type of mitochondria (by one I mean one type per case, not one common type for all cases) like we just saw in the last post http://readingrejuvenationresearch.blogspot.com/2010/12/segmental-nature-of-age-associated.html. Those are the main problems they want to see if they can patch with their model.

Their model starts at de Grey's model http://readingrejuvenationresearch.blogspot.com/2010/01/proposed-refinement-of-mitochondrial.html that basically hypothesises that mutant mitochondria produce less holes in their membranes and so are degraded less often. They justify the apparent contradiction in mutant mitochondria producing less holes with the "well known" fact that they produce more radicals by saying that most radicals are O2.- radicals but only the perhydroxy radical (HO2.-) can rip protons from lipids. Mutant mitochondria have a lower proton gradient so they produce lower amounts of HO2.- even if they produce more O2.-. Would seem good to get actual measurements, but they say that these aren't available and that they would be hard to get.

They, instead, produce a model with two assumptions: the first is that damaged mitochondria are destroyed slower than ones with intact mtDNA, and secondly, one introduced by them, that damaged mitochondria grow slower, which they justify by the energy shortage produced by the lower proton gradient. They split mitochondria into six groups, for little membrane damage, medium membrane damage and high membrane damage, each with intact mtDNA or mutant mtDNA. Radicals can increase the level of membrane damage or switch the mitochondria from an intact to a damaged mtDNA state. They give different turnover rates for mitochondria in each of the membrane damage classes, independent of their mtDNA state. The corresponding half lives for each damage class are 10, 2 and 1 week for low, medium and high damage. They used a factor of 2 as the increase in rate of free radicals that a mutant mitochondria produces compared to intact mitochondria, that mutants produced membrane damage at a rate 10 times lower than intact, and that intact grew 5 times quicker. I guess these numbers were half-guesses, and probably important in the results they got.

The model replicates the features from experiments they were looking to replicate, with one mutant taking over cells, and senescent cells having larger proportion of mutants than dividing cells, due to cell replication being a purifier of mitochondria. This purification happens because of the growth advantage of the intact mitochondria. This effect dominates when large amounts of mitochondria are to be produced, as in dividing cells, but the rate of destruction dominates when few mitochondria are being synthesised. They have some graphs showing what happens when the parameters are very different: if the mitochondria destruction rate are a bit lower, the population eventually collapses, if they are much higher, they collapse very quickly, along with other graphs showing the effects of different rates of cell reproduction and how that affects mitochondria population and stability (quick enough cell reproduction can fix higher rates of mutation).

From the model they also predict differences in importance between telomere shortening and mitochondrial damage in vivo vs in vitro. They claim that because in vitro conditions cells are replicated quickly, their collection of mitochondria will be pure through the process talked about above, so they will reach their Hayflick limit with nary an issue in their mitochondria, while in vivo, where cells replicate more slowly, mitochondrial damage will accumulate earlier and keeping telomeres long will not have an effect on cell lifespan.

(Interesting little factoid in the paper that I didn't fit in anywhere else: oxygen radicals are estimated to amount to 1-4% of consumed oxygen which sounded like a lot)

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Abstract follows:

The mitochondrial theory of aging suggests that an accumulation of defective mitochondria leads to loss of cell viability. The challenge is to explain how mitochondrial defects accumulate within cells, and why this process is more evident in postmitotic than in dividing cells. We describe a new mathematical model incorporating two critical features: (a) defective mitochondria are turned over more slowly than intact ones, and (b) defective mitochondria suffer a growth disadvantage. We also model the effect of cell division on the accumulation of defective mitochondria. The results support the mitochondrial theory and explain many of the observed data. The relationship of the mitochondrial theory to the suggested role of telomere loss in cell replicative senescence is discussed. We suggest that because of differences in the kinetics of their impact on cells, these two mechanisms have different relative importance for in vivo and in vitro cell aging.

RNA Oxidation in Alzheimer and Parkinson Diseases

Summary: RNA is oxidised in some of Alzheimer's, Parkinson's and Down syndrome patients' neurons

Interestingness: 2

Paper by Akihiko Nunomura, George Perry, Jing Zhang, Thomas J Montine, Atsushi Takeda, Shigeru Chiba and Mark A Smith in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

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They measured 8-hydroxydeoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG) as markers for DNA and RNA oxidation respectively in an unknown number of brains of postmortem Alzheimer's (AD), Parkinson's (PD) and Down syndrome (DS) patients. They found more 8-OHG in some parts of the brains of some types of disease, and less in others, but the parts of the brain still don't mean much to me. In any case, here they are:

• More oxidation in the cytoplasm than in the nucleolus and nuclear envelope in the neurons of AD and DS, clean in controls
• No difference in cerebellum between AD, DS and controls
• RNA oxidation was the main thing being detected in AD and DS
• Less oxidation with increased amyloid beta (AB) and neurofibrillary tangles (NFT)
• Increased oxidation in substantia negra in PD, dementia with Lewy bodies (DLB), and multiple system atrophy-Parkinsonian type (MSA-P). More in PD than other two
• Both RNA and DNA oxidation in PD, DLB and MSA-P
• No increase in RNA oxidation in PD in cerebellum or cerebral cortex, but increase in cerebral cortex for DLB

They think the source of oxidation is damaged mitochondria spewing hydrogen peroxide, and it transforming to hydroxyl radicals through the Fenton reaction in the cytoplasm. They don't know what effect oxidation has on RNA's functionality or if it is important. Probably some translation issues with wrong base pairing.
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Abstract follows:

In Alzheimer and Parkinson diseases, oxidative alterations, affecting lipids, proteins, and DNA, have been described. Using an in situ approach to identify 8-hydroxyguanosine, an oxidized nucleoside, we recently identified RNA as a major target of oxidation in Alzheimer and Parkinson diseases as well as Down syndrome, where premature Alzheimer-like neuropathology is invariably found. RNA oxidation is localized to the neuronal populations potentially affected in these diseases. Together with the known mitochondrial dysfunction in Alzheimer and Parkinson diseases, the cytoplasmic predominance of neuronal 8-hydroxyguanosine supports mitochondria as the most likely source of reactive oxygen responsible for RNA oxidation. The consequence of oxidatively damaged RNA is not fully understood; however, the potential of oxidized RNA to cause errors in translation indicates a metabolic abnormality in neurodegenerative diseases.

Free Radical Theory of Aging: Increasing the Average Life Expectancy at Birth and the Maximum Life Span

Summary: The founder of the free radical theory of aging again summarising the results that back the theory, and theorising on what could help slow down this process.

Interestingness: 2

Paper by Denham Harman, MD, PhD, in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

(((This is a rewrite of the paper two issues ago, summarised here: http://readingrejuvenationresearch.blogspot.com/2010/09/aging-minimizing-free-radical-damage.html, with better editing and slightly abridged (no cool graphs). It was more interesting the first time around, but this version is more polished.

This one puts more emphasis on substances that could slow down the aging process. There are a couple mentioned in this one that weren't mentioned in the first one, but nothing particularly intesting.
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Abstract follows:

Continued improvements in general living conditions—e.g., better nutrition, medical care, and housing—during the past two millennia have increased average life expectancies at birth from about 30 years in ancient Rome to almost 80 years in the developed countries with no change in the maximum life span. Current average life expectancies at birth will be increased little by further improvements. The rate of accumulation of damage inflicted on us by our inherent aging process limits average life expectancy at birth under optimal living conditions to around 85 years and the maximum life span to about 122 years. The inherent aging process is caused by chemical reactions that arise in the course of normal metabolism. Attempts to significantly increase average life expectancies at birth and the maximum life span in the future, unlike in the past, will require an understanding of aging. The free radical theory of aging postulates that this process is caused by free radical reactions, largely initiated by superoxide radicals arising from the mitochondria at an increasing rate with age. Some measures based on the free radical theory of aging may further increase the life span without interfering with the activities of normal life include: (a) caloric restriction, (b) compounds that decrease O2 access to "electron-rich areas" of the mitochondria, and (c) substances that help to minimize mitochondrial damage. The foregoing are discussed briefly along with the amelioration of damaging reactions in early life that predispose to life-shortening diseases. The feasibility of the measures suggested above needs to be evaluated. This task should be both interesting and rewarding.

Aging: Minimizing Free Radical Damage

Summary: The founder of the free radical theory of aging summarising the results that back the theory, some nice graphs, and other interesting bits of speculation.

Interestingness: 6

Paper by Denham Harman, MD, PhD, in the Journal of Anti-Aging Medicine, Volume 2, Issue 1, Spring 1999.

(((This is a summary of the current state of the free radical theory of aging (FRTA) by the guy that is introduced as the father of the FRTA. I didn't like the way it was written. I'm going to skip big chunks of it)))

(((The paper starts with a series of mortality curves over age across time for women in Sweden from the 1750s to 1992. These are cool, even if I've seen them before. They show the mortality following Gompertz function with mortality going up exponentially after around age 50 with a doubling time of about 7 years. The slope of this exponential is the same in all curves. While mortality is much lower across all ages as we get closer to the present, the line goes exponential at a younger and younger age, so that the difference in mortality at ages 70 onwards is not that big across history. So, for example, the curve for the 1900s and 1920s seem to hit the exponential proper only at age 60, while the curve for 1992 seems to be on the exponential from age 40. The left hand side of the curves, that is, the bits before we hit the exponential growth, have declined massively across history. The text mentions that in that 1992 curve, only 1.1% of all females in Sweden die before age 28 (the date at which he puts the exponential rise starting) )))


(((It continues with a couple of life expectancy graphs from the 1950s to the present for male and females in Sweden, Switzerland, the USA and Japan. The first three going up by 1-2 years per decade and Japan by 3 years per decade, from a lower base. I don't really understand what these graphs or the previous mortality curves have to do with the main theme of the article, but I like them anyway)))

The free radical theory of aging (FRTA) says that all aging and death in all living things is based on the initiation of free radical reactions, the rate of which is determined by genetics and environment. This theory was later extended (((modified?))) to say that in mitochondria-containing living things, it is the rate of initiation of free radical reactions (FRR) in the mitochondria that determines their lifespan. FRRs can be classified into initiation, a propagation chain, and termination. An antioxidant usually refers to a compound that breaks the propagation chain, or, in general, any substance that delays or inhibits oxidation in low concentrations.

The major sources of radical reactions are:

• Respiratory chain
• Phagocytosis
• Prostaglandin synthesis
• Cytochrome P-450 system
• Nonenzymatic reactions of O2
• Ionising radiation

Defenses against damage caused by FRR are:

• Antioxidants. eg: tocopherols, carotenes
• Heme-containing peroxidases. eg: catalase
• Glutathione peroxidase
• Superoxide dismutases (SOD)
• DNA repair mechanisms

By the FRTA, slowing down FRRs would increase longevity. Studies backing this up include:

• Overexpression of superoxide dismutase and catalase in fruit flies extended life span by a third.
• Longer-lived strains of fruit flies, flatworms and bread mold have higher antioxidant enzyme activity than short-lived strains
• Addition of 2-mercaptoethylamine (2-MEA), an antioxidant, to food increased average lifespan of LAF1 mice by 29.2% (((no idea what the characteristics of LAF1 mice are)))
• 2-MEA addition to food of mice mothers before mating increased lifespan of their offsprings by 15% and 8% to male and female offsprings respectively

Decreasing initiation rates of endogenous FRRs would also lead to increased longevity. The rate can be reduced by caloric reduction, compounds that compete with O2 for access to electron-rich areas of the mitochondria, compounds that bind to the respiratory chain and stop the reaction with O2, and genetic regulation of mitochondrial superoxide creation. Cutting caloric intake of rats by 40% increased average lifespan by 40% and maximal life span by 49% (((Those numbers are higher than I'm usually accustomed to))). The study also suggests a lower rate of aging for rats under caloric restriction (((ie a lower gradient on the semilog plot of age vs mortality))) (((I think the suggested link is lower amount of products to oxidise => lower total load of FRR in mitochondria))).

Only study showing antioxidant to extend maximal lifespan of mice is 2-MEA, added at 0.25% w/w to the diet of BC3F mice extended mean and maximal lifespan by 13% and 12% respectively. The study hasn't been replicated. The reason that most antioxidants fail to extend lifepan is that they have toxic effects on mitochondria at lower concentrations than those needed to slow down FRRs significantly.

The paper continues by listing the possible effects of the FRTA on specific diseases. They are:

• Cancer, listing epidemiological studies suggesting vitamin C and fruits and vegetables having lower incidence
• Atherosclerosis, caused by lesions that would result in higher localised concentrations of oxidation products, and oxidation of polyunsaturatid lipids, and mentioning a study of vitamin E supplementation showing a decrease of 40% in coronary artery disease (((never heard of that one. will have to look it up)))
• Hypertension, mentioning a study of SOD targeted to endothelium cells lowering blood pressure in spontaneously hypertensive rats, but not in normal rats
• Alzheimer's disease, listing mutations in mtDNA, mutations in amyloid precursor protein (APP), and increases in levels of APP and SOD in Down's syndrome (((I don't get how the last two are meant to relate to FRTA)))
• Immune deficiency, saying some antioxidants increase immune responses
• Autoimmunity, with ethoxyquin fed to a mice used for studying autoimmune disease (NZB) increasing lifespan by 32%

The gender mortality gap is also supposedly explained by the FRTA via two different effects: one is the lower stores of iron in women prior to menopause leading to less FRRs catalysed by iron, and the second (((something I don't even understand enough to describe))).

The paper finishes by claiming that a large part of the increase in lifespan in the USA since the 1960s could be attributable to the widespread use of multi-vitamins by the population (((yeah riiiiiight))).


Abstract follows:

Aging is the accumulation of changes that increase the risk of death. The major contributors after age 28 years are the endogenous chemical reactions that, collectively, produce aging changes that exponentially increase the chances for disease and death with age. These reactions constitute the "inborn aging process." This process is the major risk factor for disease and death of the 98% to 99% of cohorts still alive at age 28 in developed countries, where living conditions are now near optimum.

The Free Radical Theory of Aging (FRTA) and, simultaneously, the discovery of the ubiquitous, important involvement of endogenous free radical reactions in the metabolism of biologic systems, arose in 1954 from a consideration of aging phenomena from the premise that a single common process, modifiable by genetic and environmental factors, was responsible for the aging and death of all living things. The FRTA postulates that the single common process is the initiation of free radical reactions. These reactions, however initiated, could be responsible for the progressive deterioration of biologic systems with time because of their inherent ability to produce random change. The theory was extended in 1972 with the suggestion that the life span was largely determined by the rate of free radical damage to the mitochondria.

The FRTA suggests the possibility that measures to decrease the rate of initiation and/or the chain length of free radical reactions may, at least in some cases, decrease the rate of reactions that produce aging changes without significantly depressing those involved in maintenance and function. Many studies support this possibility.

Applications of the FRTA have been fruitful. For example, it is a useful guide to efforts to increase the life span, and it provides plausible explanations for the aging phenomenon (e.g., the association of disease with age as well as insight into pathogenesis; the gender gap; the association between events in early life and late onset disease; and the shortening of telomeres with cell division). Further, it is reasonable to expect on the basis of animal and epidemiologic studies that the increasing population-wide use of antioxidant supplements and ingestion of foods high in antioxidant capacity over the past 40 years have helped to increase the functional life span of the population by contributing significantly to the decline in "free radical" diseases, to increases in the fraction of elderly, and to the decline in chronic disability in this group.