Pyruvate protection against beta-amyloid-induced neuronal death: role of mitochondrial redox state

The mechanism by which beta-amyloid protein (A beta) causes degeneration in cultured neurons is not completely understood, but several lines of evidence suggest that A beta-mediated neuronal death is associated with an enhanced production of reactive oxygen species (ROS) and oxidative damage. In the present study, we address whether supplementation of glucose-containing culture media with energy substrates, pyruvate plus malate (P/M), protects rat primary neurons from A beta-induced degeneration and death. We found that P/M addition attenuated cell death evoked by beta-amyloid peptides (A beta(25-35) and A beta(1-40)) after 24 hr treatment and that this effect was blocked by alpha-ciano-3-hydroxycinnamate (CIN), suggesting that it requires mitochondrial pyruvate uptake. P/M supply to control and A beta-treated neuronal cultures increases cellular reducing power, as indicated by the ability to reduce the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The early increases in ROS levels, measured by dichlorofluorescein (DCF) fluorescence, and caspase-3 activity that follow exposure to A beta were notably reduced in the presence of P/M. These results place activation of caspase-3 most likely downstream of oxidative damage to the mitochondria and indicate that mitochondrial NAD(P) redox status plays a central role in the neuroprotective effect of pyruvate. Inhibition of respiratory chain complexes and mitochondrial uncoupling did not block the early increase in ROS levels, suggesting that A beta could initiate oxidative stress by activating a source of ROS that is not accesible to the antioxidant defenses fueled by mitochondrial substrates.     

Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation

The glutamate–glutamine cycle faces a drain of glutamate by oxidation, which is balanced by the anaplerotic synthesis of glutamate and glutamine in astrocytes. De novo synthesis of glutamate by astrocytes requires an amino group whose origin is unknown. The deficiency in Aralar/AGC1, the main mitochondrial carrier for aspartate–glutamate expressed in brain, results in a drastic fall in brain glutamine production but a modest decrease in brain glutamate levels, which is not due to decreases in neuronal or synaptosomal glutamate content. In vivo ¹³C nuclear magnetic resonance labeling with ¹³C₂acetate or (1-¹³C) glucose showed that the drop in brain glutamine is due to a failure in glial glutamate synthesis. Aralar deficiency induces a decrease in aspartate content, an increase in lactate production, and lactate-to-pyruvate ratio in cultured neurons but not in cultured astrocytes, indicating that Aralar is only functional in neurons. We find that aspartate, but not other amino acids, increases glutamate synthesis in both control and aralar-deficient astrocytes, mainly by serving as amino donor. These findings suggest the existence of a neuron-to-astrocyte aspartate transcellular pathway required for astrocyte glutamate synthesis and subsequent glutamine formation. This pathway may provide a mechanism to transfer neuronal-born redox equivalents to mitochondria in astrocytes.     

Effects of chronic nimodipine on working memory of old rats in relation to defects in synaptosomal calcium homeostasis

The present study was designed to investigate whether chronic (from 12 to 23 months of age) dietary treatment with the L-type Ca²⁺ channel blocker nimodipine (30 mg/kg body weight) enhances the cognitive behavior of aged animals and whether such a treatment would have long-term effects on the mechanisms of Ca²⁺ regulation in synaptic terminals from the aged rat brain. Cognitive behavior was evaluated in an 8-arm radial maze in 6 test series comprising a total of 105 test sessions, with intervals of no training between series. Nimodipine-treated rats performed better than vehicle-treated, aged-matched controls in all the test series, making more correct choices every time a new series was initiated. However, differences between nimodipine- and vehicle-treated rats were most remarkable in the last three test series, when the rats were 19 to 22 months. In these series 74% of the nimodipine-treated rats were able to perform the task in 4 to 9 test sessions whereas only 12%, 14% or none of the control rats learned the task. To study Ca²⁺ regulation in synaptosomes derived from cerebral cortex and hippocampus, we analyzed

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accumulation as well as the levels of the Ca²⁺-binding proteins calbindin-D28K and calreticulin by Western blotting. Nimodipine administration had no effect on hippocampal synaptosomes but increased the levels of calbindin-D28K and calreticulin in cerebral cortex preparations. These results indicate that chronic nimodipine treatment from 12 to 23 months of age prevents age-induced learning deficits without showing any signs of toxicity, and that these effects are associated with a small increase in the levels of synaptosomal Ca²⁺-binding proteins from cerebral cortex. The up-regulation of these proteins might provide a link between the long-term effects of nimodipine on gene expression and learning ability in old rats.     

Prevalencia de la fibrilación auricular desconocida y la no tratada con anticoagulantes. Estudio AFABE

Introduction and objectives

Atrial fibrillation constitutes a serious public health problem because it can lead to complications. Thus, the management of this arrhythmia must include not only its treatment, but antithrombotic therapy as well. The main goal is to determine the proportion of cases of undiagnosed atrial fibrillation and the proportion of patients not being treated with oral anticoagulants.

Methods

A multicenter, population-based, retrospective, cross-sectional, observational study. In all, 1043 participants over 60 years of age were randomly selected to undergo an electrocardiogram in a prearranged appointment. Demographic data, CHA₂DS₂-VASc and HAS-BLED scores, international normalized ratio results, and reasons for not receiving oral anticoagulant therapy were recorded.

Results

The overall prevalence of atrial fibrillation was 10.9% (95% confidence interval, 9.1%-12.8%), 20.1% of which had not been diagnosed previously. In the group with known atrial fibrillation, 23.5% of those with CHA₂DS₂-VASc≥2 were not receiving oral anticoagulant therapy, and 47.9% had a HAS-BLED score≥3. The odds ratio for not being treated with oral anticoagulation was 2.04 (95% confidence interval, 1.11-3.77) for women, 1.10 (95% confidence interval, 1.05-1.15) for more advanced age at diagnosis, and 8.61 (95% confidence interval 2.38-31.0) for a CHA₂DS₂-VASc score<2. Cognitive impairment (15.2%) was the main reason for not receiving oral anticoagulant therapy.

Conclusions

The prevalence of previously undiagnosed atrial fibrillation in individuals over 60 years of age is 20.1%, and 23.5% of those who have been diagnosed receive no treatment with oral anticoagulants.Full English text available from:www.revespcardiol.org/en     

Cytosolic reducing power preserves glutamate in retina

Glutamate in neurons is an important excitatory neurotransmitter, but it also is a key metabolite. We investigated how glutamate in a neural tissue is protected from catabolism. Flux analysis using ¹³C-labeled fuels revealed that retinas use activities of the malate aspartate shuttle to protect >98% of their glutamate from oxidation in mitochondria. Isolation of glutamate from the oxidative pathway relies on cytosolic NADH/NAD⁺, which is influenced by extracellular glucose, lactate, and pyruvate.Glutamate is especially important as a metabolite because it is required for the synthesis of glutathione, other amino acids, and proteins. Glutamate also is a key intermediate in glutamine-dependent anaplerosis, now known to be a principal source of citric acid cycle intermediates in cancer cells (1).When it is released as a neurotransmitter at brain synapses, glutamate that escapes from the synapse is taken up by astrocytes. There it is converted to glutamine and is delivered back to neurons in a process called the “glutamate/glutamine cycle” (2). Uptake of glutamate and conversion to glutamine within astrocytes stimulates glycolysis and synthesis of lactate. Astrocytes export the lactate to neurons as fuel in a process called the “astrocyte neuron lactate shuttle” (ANLS) (3).Synaptic terminals of rod and cone photoreceptors have characteristics that appear incompatible with the ANLS. The photoreceptor terminal is enriched with transporters for reuptake of glutamate (4), and it encapsulates the synapse. It is unlikely that much glutamate can escape the synapse before being sequestered back into the photoreceptor. We initiated a study to evaluate the role of ANLS in retina. However, the unusual metabolic features of retina revealed a surprising feature of neuronal metabolism, that >98% of glutamate is protected from catabolism. We investigated this protection and show here that the protection is provided by activities associated with the metabolic pathway known as the “malate aspartate shuttle” (MAS) (shown schematically in Fig. 1).Open in a separate windowFig. 1.How the malate aspartate shuttle isolates glutamate. The glutamate/α-ketoglutarate cycle in retina isolates the carbon atoms of glutamate from the oxidative pathway inside mitochondria. In this report we demonstrate the influence that cytosolic reducing power and Aralar/AGC1 activity have on this pathway. ASP, aspartate; OAA, oxaloacetate; MAL, malate; GLU, glutamate; AKG, α-ketoglutarate; OGC, oxoglutarate carrier, IPM, interphotoreceptor matrix.MAS activity regenerates cytosolic NAD⁺ that is needed to support glycolysis. To do so, it uses two important transporters to trap the reducing power from cytosolic NADH and shuttle it into the mitochondrial matrix. One transporter is the neuronal aspartate/glutamate carrier (AGC1 or Aralar) (Fig. 1, orange circle); the other transporter is the oxoglutarate carrier (OGC) (Fig. 1, light blue circle). AGC1 transports glutamate from the cytoplasm into the mitochondrial matrix in exchange for aspartate from the matrix (Fig. 1). OGC transports α-ketoglutarate from the matrix into the cytoplasm in exchange for malate from the cytoplasm (Fig. 1) (5). An important consequence of MAS activity is that it diverts metabolic flux in mitochondria away from succinyl CoA, succinate, and fumarate (Fig. 1). Most importantly, glutamate that completes a MAS cycle functions as a catalyst for the importation of reducing power into the mitochondria. The carbon atoms of glutamate are isolated from the oxidative pathway in the mitochondrial matrix. To determine the extent of that isolation in a neuronal tissue, we used ¹³C-labeled fuels to identify metabolic networks in mouse retinas and quantify their metabolic flux.     

Role of aralar, the mitochondrial transporter of aspartate-glutamate, in brain N-acetylaspartate formation and Ca(2+) signaling in neuronal mitochondria

Aralar, the Ca(2+)-dependent mitochondrial aspartate-glutamate carrier expressed in brain and skeletal muscle, is a member of the malate-aspartate NADH shuttle. Disrupting the gene for aralar, SLC25a12, in mice has enabled the discovery of two new roles of this carrier. On the one hand, it is required for synthesis of brain aspartate and N-acetylaspartate, a neuron-born metabolite that supplies acetate for myelin lipid synthesis; and on the other, it is essential for the transmission of small Ca(2+) signals to mitochondria via an increase in mitochondrial NADH.     

Comparison between developmental and senescent changes in enzyme activities linked to energy metabolism in rat heart

The developmental and senescent patterns of a number of heart enzyme activities linked to energy metabolism have been studied in rats aged between 4 days and 21 months. A morphometric study of mitochondrial volume fractions and numbers has been also carried out. Developmental changes result in a rise of most mitochondrial enzymes (NADP⁺-isocitrate dehydrogenase, malic enzyme, succinate dehydrogenase, citrate synthase) and mitochondrial volume fractions. Exceptions are NAD⁺-isocitarte dehydrogenase, which declines from 4 days onwards, and NAD⁺-malate dehydrogenase, which declines and then rises over the same period. Senescent changes follow two different trends. While pyruvate kinase and those mitochondrial enzymes lying between citrate formation and isocitrate oxidation (citrate synthase, NADP⁺- and NAD⁺-isocitrate dehydrogenases) decline to some degree, mitochondrial succinate dehydrogenase and NAD⁺-malate dehydrogenase activities increase over the same period. This could point towards a partial impairment of Krebs cycle function, and a reduced energy-producing capacity in the aged rat heart.     

Pyruvate kinase and aspartate-glutamate carrier distributions reveal key metabolic links between neurons and glia in retina

Symbiotic relationships between neurons and glia must adapt to structures, functions, and metabolic roles of the tissues they are in. We show here that Müller glia in retinas have specific enzyme deficiencies that can enhance their ability to synthesize Gln. The metabolic cost of these deficiencies is that they impair the Müller cell’s ability to metabolize Glc. We show here that the cells can compensate for this deficiency by using metabolites produced by neurons. Müller glia are deficient for pyruvate kinase (PK) and for aspartate/glutamate carrier 1 (AGC1), a key component of the malate-aspartate shuttle. In contrast, photoreceptor neurons express AGC1 and the M2 isoform of pyruvate kinase, which is commonly associated with aerobic glycolysis in tumors, proliferating cells, and some other cell types. Our findings reveal a previously unidentified type of metabolic relationship between neurons and glia. Müller glia compensate for their unique metabolic adaptations by using lactate and aspartate from neurons as surrogates for their missing PK and AGC1.Aerobic glycolysis is a metabolic adaptation that proliferating cells use to meet anabolic demands (1, 2). In tumors, it is called the “Warburg effect.” Tumors convert ∼90% of Glc they consume to lactate (Lac). The brain converts only 2–25% of the Glc it uses to Lac (3).In retinas of vertebrate animals, energy is produced in a way that resembles tumor metabolism more than brain metabolism. Aerobic glycolysis accounts for 80–96% of Glc used by retinas (4–7). Retinas are made up of neurons and glia (8). The outermost layer is occupied by photoreceptors (PRs). The inner layers are a diverse collection of signal processing neurons. Müller glia spans the thickness of the retina. The site of aerobic glycolysis in retina has not been established.Exchange of fuels is an important part of the relationship between neurons and glia (9–12). Transfer of metabolites between intracellular compartments also is important. Glycolysis is supported by reoxidation of cytosolic NADH, which can be catalyzed by lactate dehydrogenase (LDH) or by the malate-aspartate shuttle (MAS). PRs and other neurons in retinas express aspartate/glutamate carrier 1 (AGC1; also known as “Aralar”) (13), a mitochondrial aspartate/glutamate carrier that has a key role in the MAS. However, Müller cells (MCs) are AGC1-deficient (13). The significance of the distribution of AGC1 has been enigmatic.Aerobic metabolism in tumors is linked to expression of the M2 isoform of pyruvate kinase, PKM2 (14, 15). Pyruvate kinase (PK) catalyzes the final step in glycolysis, synthesis of Pyr (16). Liver (PKL) and erythrocyte (PKR) isoforms are splice variants of the PKLR gene, and PKM1 and PKM2 are splice variants of the PKM gene. A unique feature of PKM2 is that it is responsive to allosteric and posttranslational regulators (16). PKM2 expression in cancer cells correlates with reduced yield of ATP from Glc and accumulation of glycolytic intermediates. PKM2 also favors synthesis of Ser and pentose phosphate pathway intermediates that support anabolic activity (17).Association of PKM2 with aerobic glycolysis in tumors motivated us to explore the relationship between PKM2 and aerobic glycolysis in retina. We found that PKM2 is abundant and only in the outer retina, implicating PRs as a primary site of aerobic glycolysis. Unexpectedly, we also found MCs are deficient for all isoforms of PK.The deficiency of MCs for AGC1 and PK led us to investigate metabolic relationships between PRs and MCs. We found cultured MCs have an impaired ability to metabolize Glc, but they metabolize Asp effectively. Our findings reveal that metabolic benefits of down-regulating PK and AGC1 in MCs have led to specific metabolic adaptations in the retina. Rather than use Glc to fuel their mitochondria, MCs use Lac and Asp from neurons as surrogates for their missing PK and AGC1 activities.     

Differential effects of age on the pathways of calcium influx into nerve terminals

Calcium accumulation by synaptosomes decreases during ageing and this is partly due to an impaired calcium uptake by mitochondria (Brain Research, 378 (1986) 36-48). In the present work we have sought to define that effect of age on the pathways of K+-stimulated calcium influx. The plasma membrane potential of synaptosomes incubated at different K+ concentrations in choline-based or sodium-based media monitored with TPP+ did not change significantly with age. 45Ca uptake was reduced by around 20% in 24-vs 3-month-old rats at high K+ concentrations in both choline- and sodium-based media. However, the internal free calcium concentration in K+-depolarized synaptosomes estimated by the quin-2 method was found to be higher in 24- than in 3-month-old rats. When the apparent calcium permeabilities (P'Ca) in choline-based media were calculated from the corresponding calcium uptake values, membrane potentials and internal calcium concentration, it was found that the P'Ca values from old rats were only slightly lower than those of adults over the whole range of membrane potentials. The contribution of the Na/Ca exchanger to 45Ca uptake was estimated at different voltages by subtracting the normalized calcium uptake values obtained in choline media from those in Na media. The 'estimated' Na/Ca exchange was found to decrease markedly with age. Our results suggest that under our experimental conditions the apparent calcium permeability of synaptosomes is only modestly decreased during ageing. However, the operation of 45Ca/Na exchange is markedly reduced maybe as a result of alterations of the exchanger itself or due to changes in the concentration of internal Na or other ions.     

Mitochondrial transporters as novel targets for intracellular calcium signaling

Ca(2+) signaling in mitochondria is important to tune mitochondrial function to a variety of extracellular stimuli. The main mechanism is Ca(2+) entry in mitochondria via the Ca(2+) uniporter followed by Ca(2+) activation of three dehydrogenases in the mitochondrial matrix. This results in increases in mitochondrial NADH/NAD ratios and ATP levels and increased substrate uptake by mitochondria. We review evidence gathered more than 20 years ago and recent work indicating that substrate uptake, mitochondrial NADH/NAD ratios, and ATP levels may be also activated in response to cytosolic Ca(2+) signals via a mechanism that does not require the entry of Ca(2+) in mitochondria, a mechanism depending on the activity of Ca(2+)-dependent mitochondrial carriers (CaMC). CaMCs fall into two groups, the aspartate-glutamate carriers (AGC) and the ATP-Mg/P(i) carriers, also named SCaMC (for short CaMC). The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle, and citrin, the liver AGC, is also a member of the urea cycle. Both types of CaMCs are activated by Ca(2+) in the intermembrane space and function together with the Ca(2+) uniporter in decoding the Ca(2+) signal into a mitochondrial response.