Relationship between DNA repair and aging


The aging of animal brain and tissue is a major cause of functional decline in old age and is a causative agent of many diseases like Alzheimer's and Pakinson's disease. The DNA repair mechanism maintains the genome stability. Many function like oxidation stress (old age) reduced mitochondrial functions, toxic byproduct s damage to DNA. The DNA repair mechanism decreases its capabilities with increasing age. The miRNA also play a vital role in regulating the life. The miRNA expression profile is established by RT-PCR and the expression is compared with young and old animals. The expression profile revealed that decrease expression of miRNA with age. The miRNA expressions changes have potential to be used as diagnostic tool of aged related diseases and will also help to establishing the anti aging strategies. The old individual is also more associated with stroke and slow recovery after stroke. In aged brain the development of infarct area is more as compare to young ones. Moreover the effect of caloric restriction on aging process was studied. The animal with control diet live longer than the animal with ad libitum feed. The gene expression is noted by using caloric restriction as parameter revealed that the caloric restriction prevents most of the alterations in gene expression, which change with age. Caloric restrictions delay the aging process by doing metabolic shift by increasing protein turnover and by altering the macromolecule damage.

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The main object of this project is to sort out the strategies and the mechanism that let the human aging to perform normal body functions more effectively. Main focus would be on the DNA damage repair, effects of caloric restriction, (CR) on aging process for example prokaryotes over the energy deprivation by the process of autophagy and also by the slowing down the metabolism and mechanical activities, but eukaryotes by regulation of networking and degrading the muscle protein to provide amino acid to body to use it for the process of glucogenesis. Other purposes are effects of aging on the expression of micro RNA of target genes, single strand repair ability of body with aging, functional recovery after stroke in aged subject also is the main focus of project. At molecular level, the change that occur with increasing age take place gradually , how these changes can be detected at genomic and proteomics level. The theory behind the aging process is that it is due to the oxidative deterioration of different critical molecular in the body and these critical molecules are including protein, lipids as well as nucleic acids. The damage to the DNA occurs when there is accumulation of reactive oxygen in the body or due to the insufficiency of antioxidant defensive. Mostly the oxidative damage to the DNA in elderly subject is due to mutation and there mutations are controlled by the process of base excision repair pathways. The base excision repair is promoted by DNA glycolysis that identifies and excises the concerned lesions.

DNA repair mechanism in old individuals

The experiments was taken on the Caenorhabditis elegans (C. elegans) that is a multi-cellular organism encodes two DNA- glycosylases, that are UNG-1 and other is NTH-1.(Morinage at al. , 2009) C. elegans NTH-1 has it ability to excise one oxidative purine. The Nucleotide excision repair is another mechanism to repair the purine that is oxidized. ( Reardon J et al 1997). Nucleotide excision repair pathway defective (mice) changes its gene expression in segmental progeroid phynotypes.( Kirkwood TB, 2005). For example the nucleotide excision repair defective Csbm/xpa mice has high antioxidant responses through suppression of insulin growth factor 1. Actually the base excision repair (BER) pathway is referred pathway for the repair of DNA which is damage due to reactive oxygen. A study reveals that in the BER and NER are the two ways to heal the mutations of S. cerevisiea.

Multification of DNA by BER occurs through five steps. (1) DNA glycosylase excise the damage base and apurinic apyrimidic (AP) site formation. (2) phosphor-diester bond cleavage at AP site with the help of AP lyase or AP -endo-nuclease (3) removal of base or other interfering chemical group following (4) filling of gap and (5) ligation. (Fortinip et al, 2003).

In the very first step of BER, identification and excision of altered base by DNA glycosylase took place. The human enzyme APE1 originates the excision of 8-oxoGua and eC. (Privezentzevev et al., 2001). The rate of excision process can be increased by 400 fold by increasing enzyme proficiency on the damage DNA other protein like XRcc1 (found on damaged DNA site and it stays there until the ligation, it control further stages of BER. An enzyme known as polyADP ribose polymerase it binds to the DNA free ends and saves their degradation. (Schreiber V et al., 2002).

Rehabilitation in old individual after stroke using rats as model.

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DNA damage due to aging effects differently in different tissues of body, most the outcomes of DNA damage are loss of cells and also loss of damaged DNA gene expression. The loss of expression has been reported both at mRNA and protein level. The damage of DNA due to oxidation is most important. The effects of aging on muscle tissue are loss of strength of muscles, physical power and stamina diminishes in function with increasing age in animal species. The tissue of muscles contains multinucleated myofibers. Loss of integrity of muscle tissues due to the DNA damage in animals has been reported many times. The damage in heart and skeletal muscles is due to oxidative DNA damage 8-OHdG level increase in these muscles. This accumulation of 8-OHdG has been reported in the kidney, liver and brain tissues of rat and mouse, but in human the accumulation of 8-OHdG has been reported in skeletal muscles only (Mecocci et al,. 1999). Talking generally the catalase enzyme is responsible for the removal of hydrogen peroxide and other reactive oxygen and in this way it minimizes the damage to tissue due to this reactive oxygen. So for minimizing the aging effects one have to up-regulate the expression of the enzyme catalase in skeletal muscles.

The aging of brain is due to cognitive decrease in elderly, when the aging begins in life is undefined, computing the loss of gene expression from a group of individuals from 26 to 106 years old. The genes are main playing agents in vesicular transport, synaptic plasticity and mitochondrial functions. Mostly the DNA damage is found on the promoter region of gene with limited expression in aged cortex and oxidative damage also affects these promoters.

For investigation of age-dependent expression of gene the experiments were conducted in which RNA was taken from human brain after postmortem and this RNA was analyzed by using Affymetrix gene chips. To assess the gene expression in aged individuals the transcriptome profile was taken and compared and Pearson correction coefficient was used to drive the measure of similarity between two ages. The group groups equal and more than 42 years were seemed to have most homologous results and pattern of gene expressions and other group of 273 years was also seen to have homologous pattern and the group also inversely proportional to each other. On the other hand the middle aged groups have grater heterogeneity like some individuals have similarity with younger group and some individual have similarity with older group.

Genes play vital role in plasticity and other synaptic functions. Many receptors of TP2B2 neurotransmitters which play their role in synaptic plasticity, showed reduced expression after age 40, these receptors are GluR1 AMPA, NMDA, R2A receptor and also subunit of GABA. The expression of other genes that regulates synaptic vesicles VAMP I, synapsin II, RAB3A and SNAPS. Expression of many other genes which regulate signal transduction system having their role in long term potentiating and also in memory storage were reduced, more important are calcium signaling system, (calmodulin I and CAM kinase II α).

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This is a summary of time duration of recovery in aged and young rats. Both groups had good functional capabilities on first post surgical days, young animals show fast recovery and recovery is extended in old animals.

The other system with reduced expression in aged subject are calbindin 1 and 2 (calcium bind protein), calcium activated transcriptional factors MEF2C and calcium pomp ATP2B2. Furthermore protein kinase C membrane and mitogen activated protein also reduced in expression. The genes that play role in protein transport and genes involved in stabilizing microtubules and axon transport were reduced in their expression. Increased expressions of several genes were also found in aged human frontal cortex. These genes include the genes that control stress response, protein folding genes (α crystalline), paraoxonase, glutathione peroxidase (anti-oxidant defense genes), genes that control BER, immune and inflammatory response and also TNF α.

It is hypothesized that specific genes are targeted by reactive oxygen (oxidative DNA damage) and within that specific genes the promoter region is especially more targeted because promoter region have high G+C ratio and which is very sensitive to oxidative DNA damage. So practically spot out oxidative damage to specific region on assay was devised (Pfeifer et al,. 1996). The isolation of genomic DNA was using carried out under conditions that save in vitro oxidation, the isolated DNA glycosylase (FPG). Single stranded breaks were achieved at apurinic site by using FPG, ensuring it to remain resistant against PCR amplification. So in this way after quantification PCR, the damage to specific region was pointed out by comparing the cleaved and uncleaved PCR product. It was proved that the damage to promoter region is more in aged subjects. The oxidative DNA damage was appeared in many cases above 40 and it was much pronounced in cases above 70.

miRNA expression variability with increasing age.

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The expression of miRNA is also varying with increasing life, so a survey was conducted to find out the pattern of miRNA expression in different age group. The miRNome RT-PCR was used for this purpose the age associated expression of miRNA was checked into two different groups, young and old groups. So after experiments it was found that there were 9 miRNAs which are down-regulated (reduced expression) with increasing age. The miRNA is a major regulator associated with age. The C. elegans change expression of miRNA with increasing age in mouse. In mouse expression of 31 miRNAs reduced and 17 miRNA increase with age. The miRNA which are up-regulated are seem to be associated with mitochondrial expression (Wang et al,. 2009). The miRNAs which were up-regulated seems to be associated with mRNA which regeneration and detoxification of liver so it is now suggested that miRNA, are very important life span regulators.

Genomic response of old rats after stroke.

The recovery after unilateral stroke is poor in aged subjects as compare to young's. The reduced transcriptional activity may be the one of many reasons of reduced recovery in old subjects, so gene expression was analyzed in the periinfarct and contra-lateral parts of young (3 months) and old (18 months) rats (Wagner et al,. 2008) the recovery is much better in young rats within two weeks after stroke. To know this mechanism of recovery DNA array technology was used. Gene expression differences and changes both in young and old rats was identify to check the change in gene expression after middle cerebral artery occlusion (MCAO) that give the clues about the transcriptional events that may be the reason of reduced recovery after stroke in old rats.

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The graph is showing the expression of genes with time. The genes up-regulated and down-regulated more than 1.5 fold in periinfarct.

The micro dissected tissues of brain were used to isolate total RNA, that RNA was purified to use in cDNA array and RT-PCR quantification. Total 442 genes were studied representing growth factors. The level of apoptosis gene Casp7 was high in aged rat's sensory-motor cortex in the control groups. This may result that in aged rats the apoptosis was increased. The mRNA levels for enzymes responsible for reactive oxygen catalase and superoxide dismutase were also decreased in aged rat's brain, which is a clue that aged brains have very less capabilities to remove free radicals. The level of protein 7 mRNA (fatty acid binding protein) whose transcription is up-regulated after axons injury was also high which is clue of damaged myelin sheets. Sixty one genes were analyzed; these genes were regulated differently in post-ischemic rat brain (figure 1). The 28 genes among them were up-regulated. Among these up-regulated genes 11 genes were in both age groups and in remaining genes 9 were up-regulated in young and 8 were up-regulated in old groups. A total of twenty genes were down-regulated. It can then be inferred from this data that after three days of stroke the age related transcriptional effects in periinfarcted region were (1) differential regulation of apoptotic genes (2) a fifty percent decrease in regulation of stem cell related genes. A third day of ischemia the hemisphere of young rats were more active then old rats at transcriptional level. It was also noted that apoptosis related genes were down regulated and also hypoxia related genes were also not regulated. At fourteen day after stroke the continuous down-regulation in gene which are related with stem cells was noted in the sensory motor cortex of aged rats

Up-regulation of gene expression in young rats.

During the very first week of temporary occlusion of cerebral artery several genes related to hypoxia are up-regulated in young rats. The glutathione peroxidase was high, which is the result from the gene related to anti oxidant defense system. During the energy restriction the various genes are up-regulated these genes include up-coupling protein 2 (Ucp2) protein cystain B was also up-regulated which is necessary for the neuro protection. During the second week after stroke the expression of two genes were found high these genes are included Gpx1 and Ucp2. Furthermore the antioxidant abilities were also increased by high level of glutathione peroxidase, which is high due to high gene expression; other genes that are necessary for the tissue recovery were also up-regulated. The growth factor β1 in young rats and Pleiotropic cytokine (Tgfb1) also up-regulated after the injury, at day third after injury the genes related to apoptosis was not up-regulated but after the fourteen day the genes related to apoptosis like ataxia were up-regulated. The up-regulation of fibroblast growth factor was also accomplished with up-regulation of nerve growth factor β and insulin like growth factor. The up-regulation of mRNA of procollagen type1, α was also noted. The procollagen type1, α (col1a1) is a compulsory gene required for vascular remodeling.

Down regulation of gene expression young rats.

After the second week of stroke many other genes also returned to control levels those genes are Igfr1, Fgf22, Gata2 and Fzd8 and many other genes also up-regulated. The genes that were up-regulated these genes include cystatin C which is needed for the glial development. In very first week several other genes were also down regulated which were involved in axonal growth, neurogenesis, dendritic injury, cell adhesion, axonal growth and growth neurofilament light chain which is involved in axonal growth was down regulated too.

The genes of morphogenetic protein receptor 2 responsible for dendritic genesis and dendritic injury also down regulated. The integrinβ5, intercellular adhesion molecule 5, α and δ catenins and vitronectin receptor are other down-regulated genes. Many genes were also down regulated in the second week of stroke; these genes are fibroblast growth factor receptor1 which is responsible for axonal growth and a very impotent gene Bhlhb2 which is responsible for regulation of neural differentiations during the development, neurite outgrowth and neural plasticity. The glucose phosphate isomerase is housekeeping genes having neurotrophic properties. The level of neuropeptide Y also decreased. The neuropeptide Y is responsible for energy balance and regulation in body.

Up-regulations of gene expression in aged rats.

Due to temporary cerebral artery occlusion the several genes were up-regulated in aged rats which were related to hypoxia in perilesional cortex. Due to occlusion the ribosomal protein 2 was up-regulated which acts as a substrate for protein arginine methyltransferase under normal condition and initializes the asymmetric dimethyl arginine formation. The ribosomal protein 2 formed by the astrocytes that accumulate in the perilesional area of old rats.

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The genes with average fold induction are more then 1.5 and lower or equal then 0.66 are chosen. Genes are listed alphabetically and up-down regulated order.

During old age the up-regulation of anti-oxidative defense setup was noticed but the level of this up regulation is low in aged rats as compare to young rats. The up-regulation of this anti oxidative defense is accomplished by the providing the energy. Which is the responsibility of Ucp2 gene which provide the mitochondrial glutathione. The up-regulation of this Ucp2 gene was also up-regulated after stroke in aged rats. The cystatin B (cstb) is non-caspase cystcin protease inhibitor and is responsible of protecting neurons from apoptopic death. The cstb was also up-regulated during the second week the Gpx1 and Ucp2 genes remain up-regulated and is also provided with CAT and SOD was not supplemented for antioxidative defense. But in young rats following the third day of stroke there was a strong up-regulation of DNA damaged associated with apoptosis and gene cycle arrest. In old rats the up-regulations of genes took place. Periodically these genes were DNA damaged inducible 45α, Hus homologous, transformed mouse 3t3 cell, telengiectasis mutated homologous, tumor necrosis factor and caspase 7. These genes are responsible for DNA damage inducible apoptosis. The up-regulation of these genes might be due to the decrease in expression of the gene that is related to anti apoptosis. These genes involved are Traf1, Trp53 and Traf4.

Furthermore other genes that are related to stroke like prostaglandin E synthatase 3, receptor type C, protein tyrosine phosphate, leukocyte common antigen and interleukin 6 was up-regulated only in old rats not in young rats. Due to inflammation the genes for transforming growth factor receptor type 1 was also up-regulated.

Down regulation after stroke

Several genes after stroke was also down regulated in old rats these genes were shh, which is a axonal chemo attractant chromogranim A encoding genes, prp4, tubule α 1, Bmpr2, protease 43 and microtubuline protein 2. The neural survival factor in hhb and the gene responsible for the neural migration was also down regulated. During the second week the other genes that down regulated were Fgfr1 required for axonal growth, cateninα2, cateninβ2 and Npy which is required for the energy balance regulation. Now coming to the effect of caloric restriction at cellular and molecular level the mainly the gene expression the skeletal muscles of mice was used (Cheol et al,. 1999). The oligonucleotide array was used to reveal the gene expression which indicate marked reduced expression in biosynthetic and metabolic genes, but caloric restriction prevent many alterations completely or incompletely, hence caloric restriction slowly down or cease the aging process by the mechanism of metabolic shift by increasing macromolecular damage.

Aging is continuous and irreversible physiological decline in the multi-cellular organisms, of which the molecular mechanism are almost unknown some postulates and hypothesis are genetic instability due to cumulative damage to DNA, reduced gene expression due to epi-genetic alterations, telomere shortening, oxidative damage to some very important macromolecules by the reactive oxygen. Many studies revealed that 25 to 50 percent caloric restriction without deficiency of essential nutrients result in less age associated change (pathological and physiological) which give clues about its mechanism that can increase DNA repair capability, lowering metabolic changes, and by reducing oxidative stress.

To check the effect of caloric restriction on the profile of gene expression of aging the 76% of calories fed to control group from the young age, and the animals remain on the same dietary regimen until the 30th month and animal were killed and analyzed for changes and the compression of these mice with control and caloric restriction revealed that the caloric restriction has completely cease the change in gene expression (age associated) and 34% changes were suppressed partially. For major ceases of genes that show consistent alterations in the gene expression 84% of these changes were reduced by caloric restrictions completely or partially. Caloric restriction acts in a mode that it reprograms the metabolic pathways by shifting the transcription by energy metabolism and increase the biosynthesis process and protein turnover. Caloric restriction increase the expression of 51 genes higher as compare to the animals of same age in control diet and 19% of genes are related to energy metabolism in this class. This shift of energy metabolism is an evident that the glucose-6-phosphate, IIP-2 and transketolase. The direction of fructose changes from glycolysis to glucose-6-phosphate which is a biosynthesis precursor. Surprisingly the same pathway and adaptations was observed in transcriptional reprogramming in Saccharomyces cerevisiae with the dietary switch to aerobic respiration by diminution of glucose.

Transketolase, modify and manage the non oxidative way of pentose phosphate pathway and supply NADPH and minimize the power of many antioxidant system. Caloric restriction also associated with fatty acid metabolism and it induce the transcription of fatty acid synthetase and also PPAR delta which is a powerful mediator of peroxisome. More surprisingly the caloric restriction may also establish the higher level of insulin sensitivity by the action of insulin sensitivity by the action of glucose dependent insulin sensitizer. Caloric restriction also increase the expression of glutamine synthetase, thymidylate turnover alongkinase and purine nucleoside phosphorylase. Caloric restriction can induce 16% of expression of transcripts and increase the protein turn over along with elongation factor. Proteosome activated PA28, 1 gamma, translocon associated protein, 26S ribosomal protein L23. Caloric restriction is associated with 1.6 fold greater down regulation of 57 genes among these 57 genes 12% of genes were stress and DNA repair system associated.