Impact of Dietary Restriction on Brain Aging and Neurodegenerative Disorders: Emerging Findings from Experimental and Epidemiological Studies

Summary: Calorie restriction helps mice and rat models of Alzheimer's, Parkinson's and stroke. 2-doxyglucose does too.

Interestingness: 2

Paper by Mark P Mattson in the Journal of Anti-Aging Medicine, Volume 2, Issue 4, Winter 1999.

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Rats and mice models of Alzheimer's disease (AD) did better when they were on a calorie restriction diet (CR). The same for Parkinson's disease (PD). Also for Huntington disease (HD). Also for rats given a stroke. I don't like the models, except the one for stroke, so I don't care much about these results.

They think this effect comes from over-expression of heat shock proteins (HSP-70) when glucose goes low. When given 2-deoxygluose (2-DG), a modified glucose that competes with glucose for the energy chain enzymes but is not able to be broken down properly (http://readingrejuvenationresearch.blogspot.com/2010/07/2-deoxy-d-glucose-feeding-in-rats.html), rats and mice also did better in the AD, PD and stroke models, even though they lived under all-you-can eat buffet conditions.

Finally, some lame-sounding correlation studies between caloric intake surveys with PD, AD and stroke are listed.
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Abstract follows:

Although dietary restriction (DR) extends life span and reduces levels of cellular oxidative stress in several different organ systems of laboratory rodents and monkeys, its impact on the brain is unknown. As is the case with age-related disorders in other organ systems (e.g., cardiovascular disease, diabetes, and many cancers), neurodegenerative disorders such as Alzheimer disease (AD), Parkinson disease (PD), and stroke involve increased levels of cellular oxidative stress and metabolic compromise. Recent studies of experimental rat and mouse models of AD, PD, and stroke have shown that DR increases resistance of neurons to dysfunction and degeneration. DR can attenuate age-related and disease-specific deficits in cognitive and motor functions in rodents. The available data suggest at least two possible mechanisms whereby DR protects neurons. One involves decreased levels of mitochondrial oxyradical production, and the second involves induction of the expression of "stress proteins" and neurotrophic factors. The latter mechanism is supported by data showing that the neuroprotective effect of DR can be mimicked by administration of 2-deoxyglucose to animals fed ad libitum. Recent findings in epidemiological studies of human populations suggest that individuals with a low daily calorie intake have reduced risk for AD and PD. Collectively, the available data suggest that DR may prove beneficial in reducing both the incidence and severity of neurodegenerative disorders in humans.

Rest of volume 2, Issue 3

The rest of issue 3 of 1999 consists of:

A review of the 28th Annual Meeting of the American Aging Association by RM Anson and MA Lane.

Two book reviews:

• "Understanding the process of aging: The roles of mitochondria, free radicals, and antioxidants", edited by Enrique Cadenas and Lester Packer. Very positive.
• "Towards prolongation of the healthy life span: Practical approaches to intervention", edited by Denham Harman, Robin Holliday and Mohsen Meydani. This is the collection of papers and posters for the 1997 meeting of the International Association of Biomedical Gerontology. Also very positive

The gerontology literature review:

• "Human embryonic stem-cell research: science and ethics", by Shirley J Wright, in American Scientist. Ethics of stem cell research.
• "Embryonic stem cells for medicine", by Roger A Pedersen, in Scientific American. Ethics of embryonic stem cells and cloning.

The usual other sections: literature watch and calendar. Web watch disappeared.

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.

Centrophenoxine Slows Down, but Does Not Reverse, Lipofuscin Accumulation in Cultured Cells

Summary: Centrophenoxine is not very interesting with regards to lipofuscin

Interestingness: 1

Paper by Alexei Terman and Martin Welander in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

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Lipofuscin is made up of the residues from lysosome degradation. Wikipedia claims it is the product of oxidised unsaturated fatty acids. It doesn't degrade with time in the body by itself, it just accumulates. The age-spots in old people are made of this.

Centrophenoxine is a treatement for senile dementia, which wikipedia claims improves memory and general cognition.

They tried using centrophenoxine to stop formation of, and to get rid of lipofuscin in rat heart cells exposed to high levels of oxigen (to accelerate lipofuscin production is my guess, since they only left it for a few weeks). It reduced formation by about half at what seems to me to be very high concentrations (almost a millimole), but did didly for removing already established lipofuscin particles or modifying number of autophagic vacuoles induced by leupeptin. They attribute the reduction effect on its anti-oxidant properties
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Abstract follows:

Centrophenoxine, a drug used in the treatment of senile dementia, has been suggested to retard, or even reverse, lipofuscin accumulation within postmitotic cells. However, a true capacity of centrophenoxine to eliminate already formed lipofuscin inclusions has not been convincingly demonstrated. Moreover, no evidence has been obtained regarding the possible mechanisms through which intracellular content of lipofuscin would be diminished by centrophenoxine. Here we show that (a) centrophenoxine at concentrations of 0.25 or 0.5 mM diminishes lipofuscin accumulation within cultured neonatal rat cardiac myocytes (by 44% or 51%, respectively, during a period of 2 weeks) when it was constantly present in the culture medium; (b) the same treatment of rat cardiac myocytes and AG-1518 human f ibroblasts, however, does not eliminate already formed lipofuscin inclusions; (c) the formation of autophagic vacuoles, and ensuing degradation of their contents, are not influenced by centrophenoxine. Thus, our results do not support the idea that centrophenoxine can reverse age-related accumulation of lipofuscin. The observed decrease of lipofuscin formation is probably due to the previously shown antioxidant properties of centrophenoxine.

Possible Influence of Metabolic Activity on Aging

Summary: Details of ATP production control mechanism in mitochondria

Interestingness: 4

Paper by Bernhard Kadenbach, Elisabeth Bender, Annette Reith, Andreas Becker, Shahla Hammerschmidt, Icksoo Lee, Susanne Arnold and Maik Hüttemann in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

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This is more of a mitochondria biochem details piece, not directly related to aging. Most of it is too detailed for me to summarise or keep in memory or even follow.

Some interesting bits at the front that are not usually spelt out: out of the 13 proteins that mtDNA codes for, seven code for parts (out of 45) of NADH (nicotinamide adenine dinucleotide, protonated form) dehydrogenase (aka complex I), one for ubiquinol-cytochrome c oxidoreductase (aka complex III) (out of 11), three for cytochrome c oxidase (aka complex IV) (out of 13), and two for ATP synthase (out of some number I couldn't find). There's 5-10 mtDNA copies per mitochondrion, and 100-1000 mitochondria per cell.

It then describes two separate mechanisms of respiratory control. The first being due to the stimulation of ATP synthase by ADP triggering a lower proton motive force (deltaP) which trigger the proton pumps of the respiratory chain (NADH dehydrogenase, cytochrome c oxidoreductase and cytochrome c oxidase), kind of like an inverted system I think, with the final step pressuring the steps that come before it, but I imagine talking about the order here is completely wrong, they all happen at the same time. The second being due to the ATP/ADP ratio, with high ATP/ADP intramitochondrial ratio triggering a shut down of cytochrome c oxidase. This second method of control is bypassed by the presence of certain molecules, including 3,5-diiodo-L-thyronine, suggested as the mechanism of the short-term effects of thyoroid hormones, and palmitate (but not stearate, oleate or arachidonate).

The paper then does some studies showing that cAMP-dependent phosphorilation of complex IV enhances this ATP/ADP ratio control mechanism, and mitochondrial protein phosphatases reverse this enhancement. This second effect is shown mainly by adding a potassium fluoride which acts as a phosphatase inhibitor, and seeing the cAMP effect be stronger.

They also confirmed that it is mostly one mutant species of mtDNA that dominates a muscle fiber. They mapped a common deletion of mtDNA, probably that mtDNA4977 that was seen a couple of posts ago, and its occurrence varied between 0 and 0.06%, but corresponded with the bits of tissue that had malfunctioning complex IV.

They then speculate on how this phosphorilation/dephosphorilation mechanism is usually in balance, and is controlled by stressors and how when the ATP/ADP control mechanism is working, the proton gradient voltage is lower, and so less leakage of protons across the membrane occur, and less reactive oxide species are produced, and how this would be normally bypassed in a high caloric diet by the presence of palmitic acid, but the chain of reasoning is long and requires more concentration than I was willing to give it.

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Abstract follows:

The mitochondrial hypothesis on aging suggests stochastic stomatic mutations of mitochondrial DNA (mtDNA) as an important cause of respiratory-defective cells and the decline of energetic capabilities with increasing age. Reactive oxygen species (ROS), which are produced in the respiratory chain under stress conditions, are assumed to cause deletions and/or mutations of mtDNA. Using quantitative PCR, the stochastic distribution of the "common deletion" of mtDNA in human skeletal muscle tissue is shown. Recent data suggest that in vivo, under normal conditions, respiration is controlled by the intramitochondrial ATP/ADP ratio, via interaction of the nucleotides with subunit IV of cytochrome c oxidase, representing the rate-limiting step of the respiratory chain. Kinetic data are presented indicating that this "second mechanism of respiratory control" is turned on by cAMP-dependent phosphorylation of the enzyme and turned off by mitochondrial protein phosphatases. It is proposed that dephosphorylation of cytochrome c oxidase via "deleterious stress signals" results in increased mitochondrial membrane potentials and stimulated production of ROS in the mitochondrial respiratory chain. As a consequence, mutations of mtDNA would increase and aging would be accelerated. The inhibition of cytochrome c oxidase at high ATP/ADP ratios can also be abolished by low concentrations of free palmitate and high substrate pressure in the respiratory chain, supporting the notion that low caloric diet supports longevity.

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.

Segmental Nature of Age-Associated, Skeletal Muscle Mitochondrial Abnormalities Necessitates Three-Dimensional Analyses

Summary: Mitochondria with abnormal electron transport chain activity are grouped along the fibre in muscle tissue

Interestingness: 4

Paper by Nathan L Van Zeeland, Jonathan Wanagat, Marisol E Lopez and Judd M Aiken in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

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They looked at low cytochrome c oxidase (COX, complex IV) activity and high succinate dehydrogenase (SDH, complex II) activity in muscle tissue, which are supposedly common markers for age-related mitochondrial abnormalities. They are colocated with mitochondrial DNA (mtDNA) deletion (mtDNA4977). Also, muscle fibres with these abnormal mitochondrial activity are more commonly atrophied/have much lower cross-sections in rhesus monkeys. Part of the COX enzyme is encoded in the mtDNA, while all of the SDH enzyme is encoded in the nuclear DNA. (That explains why COX activity goes down, but why does the SDH activity go up?)

They measured COX and SDH activity in muscles of old (3-year old) rat and old (33 year old) rhesus monkey, making 200 slices across the muscle fibre so that they got a cross-section of the muscle at each slice. Each slice was about 10 microns thick, and they followed the muscle for about 1.6 millimetres in the monkey and 2 in the rat.

They found that the mutations were grouped along each muscle fibre. They found that in their sample, 3% of the rat's fibers had abnormal activity at some point along its length, and 0.31% of the monkey's (a 25-year old monkey though, not sure what happened to the other monkey), and contrasted these with the much lower values they would have gotten if they would have just sliced at one point (about six times lower). Through some dodgy extrapolation, they claim that 50% of the muscles fibers in the rat's case would be abnormal at some point if they had followed it through the whole length of the muscle, although they say that further studies by them point the number to be closer to 25%
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Abstract follows:

Age-associated electron transport system (ETS) abnormalities in skeletal muscle are distributed in a mosaic and segmental fashion; thus, histological techniques examining a single cross-section of tissue underestimate the number of fibers harboring such mitochondrial abnormalities. Analyses of consecutive cross-sections along the length of a muscle are necessary to determine the absolute number of ETS abnormal fibers within a given skeletal muscle. Two hundred serial cross-sections of old rat and rhesus monkey skeletal muscle were obtained by cryostat sectioning. Sections were stained and examined for cytochrome c oxidase and succinate dehydrogenase activity at regular intervals spanning a 1,600-micrometre region of muscle. All fibers staining negative for cytochrome c oxidase activity or hyperreactive for succinate dehydrogenase activity were then followed along their lengths to determine the extent of the ETS abnormal regions. ETS abnormalities in both animal models were found to be distributed in localized regions of individual muscle fibers (i.e., segmental). Examination of fibers along their length lead to a fourfold increase in detection of rat muscle fibers bearing mitochondrial abnormalities. In situ histological techniques that examine numerous sections at multiple positions along the length of skeletal muscles are particularly well suited for determining numbers and assessing the cellular impact of skeletal muscle fibers harboring age-related mitochondrial abnormalities.