Dietary Calorie Restriction from Adulthood Through Old Age in Rats: Improved DNA Polymerase ß and DNA Gap Repair Activity in Cortical Neurons.

It is well established now that dietary calorie restriction (CR) leads to extension of life span in many species, although the exact mechanism of this effect is still eluding. In the present study, we examined the effect of 40 % CR imposed during a prolonged period of life span (from 6 to 30 months) of rats on the activity of DNA polymerase ß (pol ß) in view of its role in short gap base excision DNA repair and template driven primer extension. DNA pol ß activity is very low at this late age. However, cortical neuronal extracts prepared from CR rats of 30 months age showed significantly higher pol ß protein levels and activity when compared to control 30 month old rats. Yet, one-nucleotide gap repair in old control neurons and an improved efficiency in CR neurons could be visualized only after supplementation of the extracts with T4 DNA ligase indicating the lack of CR affect on ligase activity. No impressive primer extension activity is seen either in the CR or old control neurons. These results are taken to convey that extended CR through adult life leads to improved pol ß activity and therefore, pol ß dependent DNA gap repair activity.

Age-related decline in DNA polymerase ß activity in rat brain and tissues.

Fidelity of DNA polymerases is vital for maintaining genomic integrity. Deficient DNA repair leads to age related disorders or cancer. If the age at which the decline in activity of predominant DNA repair enzymes starts is identified, and the deficient proteins supplemented, then the manifestation of these diseases can be delayed promoting healthy aging. DNA polymerase ß (pol ß) is a predominant repair enzyme in brain. DNA pol ß activity declines with age in rat brain/neurons but the exact age during the life time of rat when this decline begins is not known, and comparison of this activity was not made between post mitotic and proliferating tissues therefore the pattern of pol ß with age was studied in rat brain and tissues. The decline in pol ß activity started between 30 and 45 days postnatal in all the tissues. Post mitotic tissues showed pronounced decline than the proliferating tissues.

Free radical induced oxidative damage to DNA: relation to brain aging and neurological disorders.

Free radicals are produced in cells by cellular metabolism and by exogenous agents. These species react with biomolecules in cells and one of the important targets is DNA. This kind of damage, often referred to as oxidative DNA damage, has consequences in various organs and particularly in brain, in view of its high metabolic activity and oxygen consumption. The consequences include mutagenesis of various kinds ranging from simple oxidation of bases to large deletions through single and double strand breaks. In brain, because of its post-mitotic nature, oxidative damage to DNA is seen more often at the level of bases. A major route for repairing oxidative damage to bases is base excision repair (BER). It is increasingly becoming apparent that defects in repairing oxidative DNA damage can lead to a number of neurological disorders like Alzheimer and Parkinson. Our recent studies have clearly demonstrated that BER is highly compromised in brain cells with increasing age and this could well be one of the major causative factors for normal aging and the associated deteriorating mental conditions, including certain neurological abnormalities.

On the inhibitory affect of some dementia drugs on DNA polymerase Beta activity.

Some drugs are routinely prescribed for dementia that sets in either due to normal ageing or due to neurodegenerative disorders. We have studied the effect of three of these drugs, Donepezil hydrochloride, Rivastigmine tartrate and Nootropyl, on the activity of DNA polymerases beta, a crucial enzyme in the base excision repair pathway, the most important mode of DNA repair in brain. All the three drugs inhibited DNA polymerase beta activity to varying degrees although the affects of Donepezil being the least and inconsistent. The drugs preferentially bind to and inhibit the activities of 8 kDa domain of DNA polymerase beta that is known to possess the dRP lyase activity. The function of 31 kDa domain dealing with template driven addition of nucleotides at 3' end of the primer is not adversely affected. The inhibitory action of most widely used dementia drugs on DNA repair potential signifies that pharma sector needs to consider this aspect especially while designing drugs targeted towards brain.

DNA double strand break repair in brain: reduced NHEJ activity in aging rat neurons.

Linearised pUC 19 DNA with cohesive, blunt and non-matching ends, generated by prior treatment with different restriction enzymes was presented as substrate to measure the NHEJ activity to repair DNA double strand breaks in extracts prepared from isolated neurons from neonatal, young adult and old rat cerebral cortex. Highest end joining activity was noticed with the substrates having cohesive 3' overhang (PstI) or 5' overhang (EcoRI) ends and this activity is significantly reduced with age. However, blunt and non-matching ends were very poorly repaired at all ages. Further, the end joining activity in neurons is not faithful and sequence changes occur during the repair process. Also, the end joining activity in old neuronal extracts, but not in young extracts, was found to decline very rapidly with time of cold storage. These findings, the first of their kind, thus demonstrate that neuronal cells have the capacity to repair DNA double strand breaks through error prone NHEJ mode and that the cohesive end joining activity decreases with age of the animal.

DNA-polymerase alpha, beta, delta and epsilon activities in isolated neuronal and astroglial cell fractions from developing and aging rat cerebral cortex.

The relative proportions of DNA-polymerases alpha, beta, delta and epsilon (pols alpha, beta, delta and epsilon ) activities in isolated neuronal and astroglial cell fractions from developing, adult and aging rat brain cerebral cortex, were examined. This was achieved through a protocol that takes advantage of the reported differential sensitivities of different DNA-polymerases towards certain inhibitors like butylphenyl and butylanilino nucleotide analogs, 2',3'-dideoxythymidine triphosphate (ddTTP), monoclonal antibody of human alpha polymerase and the use of two template primers as substrates. The results indicate that while DNA-polymerase beta (pol beta) is the predominant enzyme, significant levels of DNA-polymerases alpha and delta/epsilon (pols alpha and delta/epsilon ) are also present in both cell types at all the post-natal ages studied. A notable difference regarding the relative abundance of DNA-polymerases other than beta is the higher percentage of pol delta/epsilon in neurons and a more sustained pol alpha activity through the life span in astroglia. The presence of detectable proportion of DNA-polymerases other than beta (particularly the delta/epsilon type) may be taken to indicate their role in long patch base excision repair as well as in other modes of DNA repair.

Age-dependent decline of DNA base excision repair activity in rat cortical neurons.

Synthetic oligonucleotide duplexes containing a single uracil (U) or 8-oxoguanine (8-oxoG) were used as a model substrates to assess the base excision repair (BER) ability of neuronal extracts prepared from the cerebral cortex of young (7 days), adult (180 days) and old (720 days) rats. Our results demonstrate that BER activity in neurons markedly declines with age. The decline in BER could be attributed to decrease in the expression levels and activities of BER enzymes. Supplementing neuronal extracts with uracil DNA-glycosylase (UDG), 8-oxoguanine DNA glycosylase (OGG1), apurinic endonuclease 1, pol ß and T(4) DNA ligase independently could not restore the loss of BER activity in adult and old neuronal extracts. However, supplementation of pol ß in combination of T(4) DNA ligase to neuronal extract, improved the BER in adult and old neuronal extracts. Additional supplementation of the extracts with UDG or OGG1 apart from pol ß and T(4) DNA ligase, or with all pure enzymes restored very markedly the loss of BER in aging neurons. These results suggest the age-dependent decline in BER is due to an overall deficiency of the various factors needed for BER but pol ß and DNA ligase seem to be the most limiting factors.

Topoisomerase IIß regulates base excision repair capacity of neurons.

Topoisomerase IIß (TopoIIß), an enzyme involved in DNA rearrangements, is predominantly present in brain and its levels are shown to decrease with age. This study characterizes the function of TopoIIß in regulating BER (base excision repair) activity. TopoIIß deficient granule neurons (CGNT⁻) show greater sensitivity to N-ethyl N-nitroso urea (ENU)-mediated DNA damage. The cell-free extracts of TopoIIß knockdown cells (ECGNT⁻) show a significant decrease in G-U BER activity during ENU-treatment as well as during recovery, suggesting that TopoIIß promotes G-U BER activity. Since G-U BER activity is not affected in the presence of ICRF-193, catalytic inhibitor of TopoIIß, the activity of enzyme per se may not be participating in BER activity. Further characterization of the activities of BER enzymes present in ECGNT⁻ shows that uracil DNA-glycosylase (UDG) and ligase (LIG) activities decrease significantly in both ENU treatment and recovery. Supplementation of TopoIIß to ECGNT⁻ does not restore ligation activity and ICRF-193 does not influence the LIG activity. These results suggest a role, at least an indirect one, of TopoIIß in the repair of ENU-mediated strand breaks via BER pathway including the activities of UDG and LIG.

Studies on the molecular correlates of genomic stability in rat brain cells following Amalakirasayana therapy.

Adult Wistar NIN (WNIN) rats (6 months old) of both sexes were orally fed Amalakirasayana at a dose of 4.5 g per kg body weight, five days in a week. The Amalakirasayana was prepared by Arya Vaidya Sala, Kottakkal, Kerala, India, which is considered as gold standard. After 3, 9 and 15 months of such therapeutic regime, rats were sacrificed and various tissues including brain were removed. Isolated cell suspensions of neurons and astroglia were prepared from the cerebral cortex. DNA damage, as a prime indicator of the status of genomic stability was measured in terms of single (SSBs) and double strand breaks (DSBs) through (a). The "comet" assay and (b). The biochemical methods utilizing the unique properties of Escherichia coli DNA polymerase I (pol I) and calf thymus terminal transferase. The results convincingly indicate that while in control animals, there was a distinct increase in DNA damage with age in neurons and astrocytes, rasayana fed animals showed significantly less DNA damage in brain cells demonstrating beneficial effects of Rasayana therapy towards maintenance of genomic stability. DNA-damage may be the proximal cause of aging and strategies to reduce the rate of aging could be based on this fact.

Dietary calorie restriction, DNA-repair and brain aging.

It is now well established, in many species, that dietary calorie restriction confers beneficial effects like slowing down many age dependent processes and extending the lifespan. There are indications that this phenomenon may be applicable even in non-human primates and humans. However the precise mechanism through which these effects are achieved is not known. Since decreasing DNA repair has been correlated with increasing age, information available on the effect of dietary calorie restriction on DNA repair potential in different species, including humans, is reviewed with special emphasis on brain in view of its uniqueness and the age related appearance of several neurodegenerative disorders. There is considerable evidence to indicate that calorie restriction reduces the rate of, among other things, the age dependent decrease in DNA repair potential thus leading to a better maintenance of genomic integrity. In brain also dietary calorie restriction is found to improve the activities of some enzymes supposedly involved in DNA repair. It is suggested that one of the lifespan extending mechanisms of calorie restriction may be to channel the limited energy resource available to maintain a process like DNA repair rather than towards reproductive and anabolic activities.

The presence of 3'-5' exonuclease activity in rat brain neurons and its role in template-driven extension of 3'-mismatched primers by DNA-polymerase beta in aging neurons.

A three-step reaction strategy has been developed to examine the mechanism of extension of a mismatched primer in an oligoduplex substrate by rat neuronal extracts and DNA polymerase beta. The results revealed that in the case of duplexes with a mismatch at 3'-end of primer, significant extension by DNA polymerase beta has taken place only after the removal of the mismatched base, thus indicating the presence of a proof reading 3'-5' exonuclease activity in neuronal extracts of all ages. A closer examination of the neuronal exonuclease activity revealed that bases are excised from the 3' end in a sequential and nonspecific manner, although initial excision of a mismatched base was slightly faster. Further, the excision efficiency is seen to decrease with the age of the animal but apparently does not go below a critical level so as to become a rate-limiting factor for the DNA-repair activity.