Alteration of ADP-ribosylation in aging rat brain astrocytes

DNA damage and the enzyme poly(ADP-ribose)polymerase(PARP)associated with the pathogenesis of numerous age-related neurodegenerative disorders.Astrocytes play crucial roles in both support metabolic functions and cell viability of the brain.PARP regulates DNA damage and repair in the brain cells.In this study PARP activity and DNA strand break were investigated in the astrocytes isolated from young and aged rat brain.Three and 30-month-old rats were killed by decapitation and brains were removed onto an ice cooled glass plate.Astrocytes were isolated by sucrose density gradient centrifugation and glutamine synthetase(GS)served as a marker of the astrocytes lineage.The specific activity of PARP was assayed in permeabilized cells by measuring the incorporation of the ADP-ribose moiety of[3H]NAD into the nuclear acceptor proteins.The rate of DNA strand breaks was determined using a fluorescent dye and monitored spectrofluorimetry.An increase(about 75%)in the PARP activity was observed in the whole homogenates of aged rats,whereas this rise was more pronounced(about 360%)when the reaction was measured in the purified astrocyte preparations.The amount of DNA strand breaks was also higher in the astrocytes isolated from the aged brain as compared to that of young levels.The close relationship between the level of DNA strand breaks and PARP activity in the astrocytes suggest that these cells are susceptible to the metabolic alterations in aging.It is concluded that the astrocytes PARP might be considered as a therapeutic target for combating age related neurodegenerative disorders.

Age related interaction of dopamine and serotonin synthesis in striatal synaptosomes

Tyrosine hydroxylase and tryptophan hydroxylase are key rate limiting enzymes in the biosynthesis of dopamine and serotonin,respectively.Since both enzymes are active in striatum,and affected by age,this study was undertaken to investigate interaction between dopamine and serotonin synthesis in brain striatal synaptosomes of aging rat.Male Wistar rats(3 and 30 month old)were killed by decapitation and brain striatal synaptosomes were prepared by discontinuous Ficoll/sucrose gradient technique.Synaptosomes were incubated in the presence of added pargiline(monoamineoxidase inhibitor),dopamine or serotonin synthesized during 25 min was measured by HPLC,employing electrochemical detection.Dopamine synthesis in synaptosomes prepared from young animals was markedly inhibited by addition of 5μM serotonin concentrations(30%)and increasing serotonin concentrations up to 50μM caused only a smaller additional inhibition.Dopamine synthesis in synaptosomes obtained from old rats was signifi cantly lower than that of youg animals and addition of serotonin concentrations up to 50μM had little effect on these preparations.In case of serotonin synthesis,exogenously added 5μM dopamine inhibited serotonin synthesis in the synaptosomes of both ages by about 40%,whereas with higher concentration of dopamine(10-50μM)the rate of inhibition was highly pronounced in old rats as compared to that of young animals.It is concluded that dopamine and serotonin interaction may be signifi cant,and that these should be considered in long-term treatments of Parkinson’s disease with L-DOPA.