Elena Bogónez

Age-associated decrease of SIRT1 expression in rat hippocampus. Prevention by late onset caloric restriction

We have studied the effect of aging and late onset caloric restriction (CR) on the expression of SIRT1 in hippocampus and cerebral cortex of the rat. Quantitative analysis showed that there is a significant reduction of SIRT1 protein levels in hippocampus with aging. Late onset, moderate CR prevented the deleterious effect of aging on SIRT1 content. Examination of SIRT1 immunoreactivity in coronal sections from hippocampus supported these results, and confirmed that old animals are able to respond to the beneficial effects of CR by regulating SIRT1 protein expression. Differences in the amounts of SIRT1 transcripts among animal groups were not found, which suggest that post-transcriptional mechanisms could be involved in the effects of aging and CR on SIRT1 expression.

Cytosolic and mitochondrial calcium in synaptosomes during aging

Synaptosomal [Ca2+]i levels increase during aging, particularly in the old rat hippocampus, both under basal conditions and after high K depolarization. This is probably the result of age-dependent modifications in calcium buffering and extrusion systems rather than due to increased calcium influx, since calcium uptake through synaptosomal voltage gated calcium channels decreases in old animals. The calcium binding capacity of the cytosolic compartment (i.e, that excluded from mitochondria and endoplasmic reticulum) of synaptosomes was markedly reduced in old rats. Calcium compartmentation in synaptosomal mitochondria, is also reduced during aging, and this is associated with a decrease in activity of the mitochondrial calcium uniporter. Taken together, these modifications point towards a clear deterioration of the cell calcium homeostatic mechanisms towards increased [Ca2+]i in old age, specially under conditions of high calcium loads, a situation that may exacerbate neuronal vulnerability to excitotoxicity.

Reduction of K+‐Stimulated 45Ca2+ Influx in Synaptosomes with Age Involves Inactivating and Noninactivating Calcium Channels and Is Correlated with Temporal Modifications in Protein Dephosphorylation

Abstract: The voltage‐dependent calcium uptake in rat brain synaptosomes was measured under conditions in which [Ca2+]o/[Na+]i exchange was minimized to characterize the voltage‐sensitive calcium channels from rats of different ages. In solutions of CaCl2 concentrations of <500 μM, the initial (5‐s) calcium uptake declined by ∼ 20–50% in 12‐ and 24‐month‐old rats relative to 3‐month‐old adults. Depolarization of synaptosomes from 3‐month‐old rats in a calcium‐free medium or in the presence of 0.5 mM CaCl2 led to an exponential decline of the calcium uptake rate after 20 s (voltage‐ or voltage‐and‐calcium‐dependent inactivation) to ∼ 66 and 34% of the initial value with a t1/2 of 1.6 or 0.7 s, respectively. The presence of 1 μM nifedipine resulted in a 15–25% reduction of 45Ca2+ uptake rates, which appeared to affect noninactivating calcium channels, but addition of the calcium channel agonist Bay K 8644 was without effect. In 24‐month‐old rats, inactivation of 45Ca2+ uptake in calcium‐free media was nondetectable, and in the presence of 0.5 mM CaCl2, the rate and extent of inactivation were also much lower than in 3‐month‐old animals (the t1/2 was 0.9 s, and the calcium uptake rate at 20 s was 55% of its initial value). Moreover, the presence of 1 μM nifedipine was without effect on initial calcium uptake or inactivation in synaptosomes from 24‐month‐old rats. These results indicate that the decrease in calcium channel‐mediated 45Ca2+ uptake involves an inhibition or block of both dihydropyridine‐resistant and ‐sensitive calcium channels. It is interesting that the large decrease in calcium uptake via calcium channels that occurs in 24‐month‐old rats is correlated with a slower dephosphorylation of the synaptosomal phosphoprotein P96, a process that is intimately related to the opening and ensuing calcium flow through voltage‐gated calcium channels.

Age-related changes in calcium homeostatic mechanisms in synaptosomes in relation with working memory deficiency.

Aging is associated with alterations in different systems that govern neuronal calcium homeostasis. This study was designed to determine whether any of these alterations may contribute to the decline in spatial working memory that is observed in old rats. Several parameters [initial (5 s) and steady state (15 min) 45Ca2+ uptake, FCCP-releaseable 45Ca2+, [Ca2+]i levels, depolarization-induced phosphoprotein (P97, PP65, P42) dephosphorylation and acetylcholine levels and release) involved in calcium homeostasis/signaling were determined in whole brain synaptosomes derived from adult (9-month-old) and old (24-month-old) rats that were evaluated for spatial memory performance in the eight-arm radial maze. The neurochemical analysis indicated that both the 9- and 24-month-old rats were impaired with respect to 3-month-old animals. When learners (animals reaching criterion; RC) were compared to memory impaired rats (MI), it was found that the FCCP-releaseable 45Ca2+ of synaptosomes, that reflects mitochondrial calcium, was lower in the MI than the RC rats and was correlated with the behavioral performance of the rats in their first testing sessions. The results suggest that the loss of calcium uptake capacity in synaptic mitochondria during aging may be associated with impaired working memory in old animals.

Development of Insulin Resistance During Aging: Involvement of Central Processes and Role of Adipokines

Aging in mammals associates with the development of peripheral insulin resistance. Additionally, adiposity usually increases with aging and this could play a relevant role in the gradual impairment of insulin action. In fact, fat accretion leads to changes in the expression and circulating concentrations of factors originated in adipose tissue like leptin, resistin and inflammatory cytokines which have been shown to modulate insulin signaling in insulin target tissues acting both, directly or through the central nervous system. Even insulin action on peripheral target tissues has been recently demonstrated to be partially mediated by its central action, suggesting that a decrease in central insulin action could be involved in the development of peripheral insulin resistance. In the present review we analyze the available research data on aging-associated insulin resistance making emphasis in the following aspects: 1) The time-course of development of overall insulin resistance and the evolution of changes in circulating adipokines; 2) The effect of caloric restriction and the decrease of adiposity in insulin action; 3) The influence of changes in the central action of factors like leptin or insulin in the development and maintenance of insulin resistance during aging.

Regulation by Ammonium of Glutamate Dehydrogenase (NADP+) from Saccharomyces cerevisiae

The activity of glutamate dehydrogenase (NADP+) (EC 1.4.1.4; NADP-GDH) of Saccharomyces cerevisiae is decreased under conditions in which intracellular ammonia concentrations increases. A high internal ammonia concentration can be obtained (a) by increasing the ammonium sulphate concentration in the culture medium, and (b) by growing the yeast either in acetate + ammonia media, where the pH of the medium rises during growth, or in heavily buffered glucose + ammonia media at pH 7.5. Under these conditions cellular oxoglutarate concentrations do not vary and changes in NADP-GDH activity appear to provide a constant rate of oxoglutarate utilization. The following results suggest that the decrease in NADP-GDH activity in ammonia-accumulating yeast cells is brought about by repression of synthesis: (i) after a shift to high ammonium sulphate concentrations, the number of units of activity per cell decreased as the inverse of cell doubling; and (ii) the rate of degradation of labelled NADP-GDH was essentially the same in ammonia-accumulating yeast cells and in controls, whereas the synthesis constant was much lower in the ammonia-accumulating cells than in the controls.

Ammonia accumulation in acetate-growing yeast

During growth on acetate, the pH of yeast cultures rises from 5.8 to around 7-8 in the stationary phase. This was found to result from acetic acid uptake and accompanying H+ loss. In addition, acetate-growing yeast were found to accumulate ammonia. The influence of pH on ammonia transport and accumulation was studied with the analogue [14C]methylamine with the following results. (a) Methylamine uptake kinetics from 0.1-50 mM were consistent with a single-component uptake system (NH+4 permease) at pH values more acidic than 6.5, and with a two-component system (NH+4 permease and NH3 diffusion) above pH 7.5. (b) Equilibrium accumulation of methylamine was found to increase with increasing pH. (c) Methylamine efflux from methylamine-loaded cells increased as the external pH decreased. It was concluded from measurements of the internal pH under various culture conditions that the accumulation of ammonia in acetate-growing alkaline cultures resulted from the sum of two processes: (1) an energy-driven NH+4 transport; and (2) NH3 diffusion dependent on the delta pH.

The activity of synaptosomal calcium channels is inversely correlated with working memory performance in memory impaired, aged rats

Aged, memory-impaired rats do not learn an 8-arm radial maze task but differ in their performance along testing. The aim of this study was to determine whether any of the systems that govern calcium homeostasis in synaptosomes may be related to that difference in performance. A negative correlation between initial (5 s) K(+)-stimulated 45Ca2+ uptake and the behavioral scores from the last testing sessions was obtained K(+)-stimulated 45Ca2+ uptake showed also a negative correlation with an improvement score that evaluates the progress made by the rat along testing. The results support the notion that calcium inflow through synaptosomal voltage gated calcium channels in old rats is inversely correlated with their behavior. This may explain the beneficial effects of organic calcium channel blockers on behavioral performance in aged animals.

Pyruvate dehydrogenase dephosphorylation in rat brain synaptosomes and mitochondria : evidence for a calcium-mediated effect in response to depolarization, and variations due to ageing

The phosphorylation state of P42, the phosphorylated, catalytically inactive, alpha-subunit of pyruvate dehydrogenase (PDH), decreased markedly (42.4%) in response to K(+)-depolarization of synaptosomes. The dephosphorylation was rapid (5-15 s), calcium-dependent and could also be observed in isolated mitochondria exposed to a rise in extramitochondrial calcium, suggesting that P42 dephosphorylation may act as a calcium sensor in the mitochondrial matrix. The depolarization-dependent dephosphorylation rate of P42 was decreased in synaptosomes derived from 24-month-old animals with respect to 3-month-old adults. The relevance of these results in terms of PDH activation during ageing is discussed.

Conditions Restricting Depolarization-Dependent Calcium Influx in Synaptosomes Reveal a Graded Response of P96 Dephosphorylation and a Transient Dephosphorylation of P65

Abstract: Temporal changes in the phosphorylation level of synaptosomal phosphoproteins following depolarization of synaptosomes were investigated under conditions restricting calcium influx. High‐K+ depolarization in media of low [Na+]o (32 mM during preincubation and depolarization) at pH 6.5 resulted in a pronounced fall in the cytosolic free calcium concentration transient, and in a reduction in the initial K+‐stimulated 45Ca2+ uptake and endogenous acetylcholine release relative to the values obtained with control synaptosomes (preincubated and depolarized in Na+‐based media). This reduction was paralleled by a decrease in the rate of dephosphorylation of the synaptosomal protein P96. A slower dephosphorylation of P96 also was observed on exposure to 20 μM veratridine at 0.5 mM external calcium. Our results indicate that, similar to synapsin I phosphorylation, P96 dephosphorylation shows a graded response to the amount of calcium entering the presynaptic terminal. Depolarization of synaptosomes under conditions restricting the influx of calcium revealed a transient dephosphorylation (reversed within 10 s) of the phosphoprotein P65. The possible significance of this finding to the process of neurotransmitter release is discussed.