Sphingomyelin-induced inhibition of the plasma membrane calcium ATPase causes neurodegeneration in type A Niemann-Pick disease.

Niemann-Pick disease type A (NPA) is a rare lysosomal storage disorder characterized by severe neurological alterations that leads to death in childhood. Loss-of-function mutations in the acid sphingomyelinase (ASM) gene cause NPA, and result in the accumulation of sphingomyelin (SM) in lysosomes and plasma membrane of neurons. Using ASM knockout (ASMko) mice as a NPA disease model, we investigated how high SM levels contribute to neural pathology in NPA. We found high levels of oxidative stress both in neurons from these mice and a NPA patient. Impaired activity of the plasma membrane calcium ATPase (PMCA) increases intracellular calcium. SM induces PMCA decreased activity, which causes oxidative stress. Incubating ASMko-cultured neurons in the histone deacetylase inhibitor, SAHA, restores PMCA activity and calcium homeostasis and, consequently, reduces the increased levels of oxidative stress. No recovery occurs when PMCA activity is pharmacologically impaired or genetically inhibited in vitro. Oral administration of SAHA prevents oxidative stress and neurodegeneration, and improves behavioral performance in ASMko mice. These results demonstrate a critical role for plasma membrane SM in neuronal calcium regulation. Thus, we identify changes in PMCA-triggered calcium homeostasis as an upstream mediator for NPA pathology. These findings can stimulate new approaches for pharmacological remediation in a disease with no current clinical treatments.

The regulation of neuronal mitochondrial metabolism by calcium.

Calcium signalling is fundamental to the function of the nervous system, in association with changes in ionic gradients across the membrane. Although restoring ionic gradients is energetically costly, a rise in intracellular Ca(2+) acts through multiple pathways to increase ATP synthesis, matching energy supply to demand. Increasing cytosolic Ca(2+) stimulates metabolite transfer across the inner mitochondrial membrane through activation of Ca(2+) -regulated mitochondrial carriers, whereas an increase in matrix Ca(2+) stimulates the citric acid cycle and ATP synthase. The aspartate-glutamate exchanger Aralar/AGC1 (Slc25a12), a component of the malate-aspartate shuttle (MAS), is stimulated by modest increases in cytosolic Ca(2+) and upregulates respiration in cortical neurons by enhancing pyruvate supply into mitochondria. Failure to increase respiration in response to small (carbachol) and moderate (K(+) -depolarization) workloads and blunted stimulation of respiration in response to high workloads (veratridine) in Aralar/AGC1 knockout neurons reflect impaired MAS activity and limited mitochondrial pyruvate supply. In response to large workloads (veratridine), acute stimulation of respiration occurs in the absence of MAS through Ca(2+) influx through the mitochondrial calcium uniporter (MCU) and a rise in matrix [Ca(2+) ]. Although the physiological importance of the MCU complex in work-induced stimulation of respiration of CNS neurons is not yet clarified, abnormal mitochondrial Ca(2+) signalling causes pathology. Indeed, loss of function mutations in MICU1, a regulator of MCU complex, are associated with neuromuscular disease. In patient-derived MICU1 deficient fibroblasts, resting matrix Ca(2+) is increased and mitochondria fragmented. Thus, the fine tuning of Ca(2+) signals plays a key role in shaping mitochondrial bioenergetics.

Regulation of cytosolic free calcium concentration by intrasynaptic mitochondria.

By the use of digitonin permeabilized presynaptic nerve terminals (synaptosomes), we have found that intrasynaptic mitochondria, when studied "in situ," i.e., surrounded by their cytosolic environment, are able to buffer calcium in a range of calcium concentrations close to those usually present in the cytosol of resting synaptosomes. Adenine nucleotides and polyamines, which are usually lost during isolation of mitochondria, greatly improve the calcium-sequestering activity of mitochondria in permeabilized synaptosomes. The hypothesis that the mitochondria contributes to calcium homeostasis at low resting cytosolic free calcium concentration ([Ca2+]i) in synaptosomes has been tested; it has been found that in fact this is the case. Intrasynaptic mitochondria actively accumulates calcium at [Ca2+]i around 10(-7) M, and this activity is necessary for the regulation of [Ca2+]i. When compared with other membrane-limited calcium pools, it was found that depending on external concentration the calcium pool mobilized from mitochondria is similar or even greater than the IP3- or caffeine-sensitive calcium pools. In summary, the results presented argue in favor of a more prominent role of mitochondria in regulating [Ca2+]i in presynaptic nerve terminals, a role that should be reconsidered for other cellular types in light of the present evidence.

The influence of age on the calcium-efflux pathway and matrix calcium buffering power in brain mitochondria.

The variations with age of the ruthenium red-insensitive calcium efflux rate have been studied in rat brain mitochondria. Both H+- and Na+-dependent effluxes are decreased with age when expressed as a function of calcium taken up in mitochondria incubated in the presence of 0.8 mM inorganic phosphate (Pi) and 0.2 mM ADP. However, the age-dependent differences in calcium efflux rates disappear when mitochondria are incubated in the absence of ADP and Pi. It is suggested that the decrease in efflux rate observed with age corresponds to an increased calcium buffering power of the mitochondrial matrix due to an increase in mitochondrial Pi. The causes of the increased Pi accumulation in old-rat-brain mitochondria are yet unknown but possibly not due to differences in the Pi efflux. The results suggest that the age-dependent lowering of the free calcium concentration in the brain mitochondrial matrix together with the reduced activity of the calcium uniporter (Vitórica, J. and Satrústegui, J. (1986) Brain Research 378, 36-48) could lead to an impaired activation of mitochondrial dehydrogenases after a rise in cytosolic calcium.

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.

Measurement of 'in situ' mitochondrial membrane potential in Ehrlich ascites tumor cells during aerobic glycolysis.

(1) A method is presented for continuous and simultaneous monitoring of the 'in situ' mitochondrial membrane potential (delta psi m) and respiration rate of Ehrlich ascites tumor cells. The method involves permeabilization of the plasma membrane, achieved by treatment with low digitonin concentration, and the use of a TPP+ selective electrode attached to an oxygraph vessel. Binding of the probe inside the cells was analyzed assuming a proportional relationship between the amount of bound TPP+ and the free concentration of the lipophilic cation. (2) Evidence is reported that the addition of glucose to digitonin-permeabilized Ehrlich ascites tumor cells causes a decrease of mitochondrial membrane potential that coincided with a transient enhancement of the respiration rate and remained unchanged during the subsequent Crabtree effect. We have characterized the effect of glucose on delta psi m by determining its dependent on the glycolytic pathway and its sensitivity towards oligomycin. The mutual relationships between glucose and ADP effects on the mitochondrial membrane potential were also studied. A plausible mechanism underlying the depolarization of mitochondrial membrane induced by glucose is presented.

Affinity chromatography purification of mitochondrial inner membrane proteins with calcium transport activity.

Immobilized calcium affinity chromatography was used to obtain a preparation enriched in calcium transporters from Triton X-100 extracts of rat liver mitochondria inner membranes (PPCT). The PPCT were reconstituted into preformed asolectin liposomes which contained 120 mM KCl as internal high K+ medium. 45Ca2+ uptake into proteoliposomes was studied under conditions favoring electrophoretic uptake, and H+i/45Ca2+o or Na+i/45Ca2+o exchange, to test for the presence of the three calcium transport modes present in mitochondria. 45Ca2+ uptake in liposomes was studied in parallel. Na+i/45Ca2+o exchange activity was not detectable. H+i/45Ca2+o exchange activity measured in the presence of a pH gradient (acid inside) obtained after suspension in low K medium in the presence of nigericin, was 100-200 nmoles 45Ca2+ per mg protein in 30 s. 45Ca2+ uptake in voltage-dependent assays (a K+ diffusion membrane potential induced by valinomycin in the presence of methylamine) was not electrophoretic since it was stimulated by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and probably due to secondary Ca2+/H+ countertransport. H+i/45Ca2+o uptake showed a saturable component at around 80 microM Ca and was coupled to an increase in internal pH in pyranine-loaded PPCT proteoliposomes. 45Ca2+ uptake in PPCT proteoliposomes could also be driven by a pH gradient obtained by raising external pH in high K+ medium. The results are consistent with the presence of a functional nH+/Ca2+ antiporter. Polyclonal antibodies raised against the PPCT were able to immunoprecipitate the H+/45Ca2+ uptake activity and recognized two major bands in the PPCT with molecular masses of about 66 kDa and 55 kDa. This is the first report of a partial purified protein(s) which may represent the H+/Ca2+ exchanger of the inner mitochondrial membrane, and represents an important step towards its identification.

Modulation of presynaptic calcium homeostasis by nitric oxide.

In the present work, we have adapted established microfluorimetric techniques based on the calcium indicator Fura-2, for the study of synaptosomal calcium homeostasis regulation in an immobilized synaptosomal preparation from the rat hippocampus. With this tool, we have addressed the actions of two proposed interneuronal messengers, nitric oxide (NO) and arachidonic acid (AA). NO donors (sodium nitroprusside, SNP and hydroxylamine, HX) and AA induced an increase in depolarization-induced calcium transients (both in magnitude and duration). However, resting calcium levels were not modified by NO, whereas AA application resulted in an steady increase in Ca. The effects of SNP were blocked when EGTA was present between depolarizations, suggesting that a minimum level of internal calcium load is required for NO effects. The effects of NO on Cai transients are persistent up to 90 min after drug application, and could be involved in some of the forms of synaptic plasticity where NO plays a role.

Effect of Quin-2 on 45Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i.

The Na+/Ca2+ exchanger of squid axons, barnacle muscle and sarcolemma requires micromolar intracellular calcium for activation in the Na+i/Ca2+o exchange mode ('reverse' Na+/Ca2+ exchange). The requirement for [Ca2+]i has been demonstrated with the use of intracellular calcium buffers, such as Quin-2, to inhibit Na+i/Ca2+o exchange. However, the inhibition of Na+i/Ca2+o exchange in mammalian nerve terminals loaded with Quin-2 has not been observed [7], suggesting a lower sensitivity to low [Ca2+]i for this system. In contrast, the results reported herein indicate that 45Ca2+ uptake in synaptosomes through Na+i/Ca2+o exchange is inhibited by Quin-2 much in the same way as it is in the squid, provided that synaptosomes are preincubated in low Ca2+ medium to avoid saturation of Quin-2. Under these conditions, 45Ca2+ efflux via Ca2+i/Ca2+o exchange is also inhibited. Our results indicate that the Na+i/Ca2+o and Ca2+i/Ca2+o modes of the Na+/Ca2+ exchanger from rat brain synaptosomes require intracellular calcium for activation. However, because no clear relationship between the observed [Ca2+]i values and the inhibition of Na+i/Ca2+o exchange has been found, it is suggested that localised submembrane calcium concentrations not detected by the [Ca2+]i probe might regulate the exchanger.

Prolactin increases cytosolic free calcium concentration in hepatocytes of lactating rats.

PRL at a physiological concentration (10(-8) M) produced a very rapid and transient increase in 45Ca efflux in freshly isolated hepatocytes, which reached the highest value within 5 min and returned to baseline level after 20 min. PRL-induced 45Ca2+ efflux resulted in a loss of 15% of total cell calcium, which was similar to that found in vasopressin-treated cells. However, in contrast with the PRL effect, 45Ca2+ efflux induced by vasopressin was sustained. We demonstrate by using two different approaches, glycogen phosphorylase-a activation and direct cytosolic calcium concentration [( Ca2+]i) measurements, that PRL elicits a [Ca2+]i increase. The treatment of hepatic cells with PRL caused a 4-fold stimulation in glycogen phosphorylase-alpha activity after 2 min of PRL addition. Direct [Ca2+]i determination in fluo-3-loaded hepatocytes showed a 11% increase after 5 min of PRL addition. Similar data were observed in hepatocytes stimulated either with vasopressin (10(-7) M) or calcium ionophore A23187 (200 nM). The increase in [Ca2+]i promoted by PRL was independent of extracellular calcium or voltage-operated calcium channels. The data demonstrate that calcium is involved in the intracellular signaling of PRL in liver cells and that PRL initiates its action by a Ca2+ mobilization from the intracellular stores.

In vivo insulin-dependent glucose uptake of specific tissues is decreased during aging of mature Wistar rats.

Aging has been associated with peripheral insulin resistance in both humans and rats. However, the specific tissues that become insensitive to insulin before glucose homeostasis is altered remain to be elucidated. In the present work we studied the glucose metabolic index of a number of tissues known to be insulin sensitive in 3- and 24-month-old Wistar rats by measuring 2-deoxy-D-[1-3H]glucose uptake both under euglycemic-hyperinsulinemic conditions and in the basal state. Analysis of the glucose infusion rate to maintain normoglycemia during the clamp confirmed that the old rats show overall insulin resistance at both saturating and subsaturating insulin concentrations. The maximal response of glucose uptake to insulin as well as insulin sensitivity in red and white quadriceps were unaltered in old rats. In contrast, glucose uptake by soleus and diaphragm was poorly stimulated in old animals, and a marked decrease in insulin sensitivity was observed in both tissues. In heart, only the sensitivity to the hormone, not the maximal response, was impaired in old rats. In white adipose tissue, no significant stimulation was detected. We conclude that during aging in Wistar rats and before fasting plasma insulin and glucose levels become altered, specific tissues develop insulin resistance, whereas other remain insulin sensitive. We postulate that fat tissue plays a qualitative important role in eliciting the insulin resistance in old animals. Due to the metabolic characteristics of the aged Wistar rat, the changes reported might reflect what occurs in nonobese elderly humans, nongenetically committed to develop type 2 diabetes.

Altered cell calcium regulation in synaptosomes and brain cells of the 30-month-old rat: prominent effects in hippocampus.

A deficient regulation of neuronal cytosolic calcium levels has been suggested to play a role in the pathogenesis of neurodegeneration. However, evidence for an alteration in cytosolic calcium regulation in old age is at present controversial. The present work was aimed at studying whether changes in synaptosomal calcium homeostasis in 30-month-old rats are uniform throughout the brain or affect specific brain regions. A second question addressed in this work is whether the effect of ageing on calcium homeostasis is restricted to the nerve terminal or a more general process affecting also cell bodies. To study these questions cytosolic calcium regulation was studied in parallel in synaptosomes and a preparation of acutely dissociated brain cells obtained from different regions of 3- and 30-month-old rats. 45Ca2+ accumulation and distribution in mitochondria (assessed as FCCP-releasable 45Ca2+) was also studied. Mean [Ca2+]i obtained at rest and after high K+ depolarization were unchanged in cerebral cortex synaptosomes but increased in hippocampal synaptosomes at 30 months. Resting [Ca2+]i also increased with age in hippocampal, but not cerebral cortex cells, whereas the increase in [Ca2+]i obtained by depolarization was larger in both brain regions. Calcium compartmentation in mitochondria from hippocampal neurons incubated under high K+ conditions was also decreased with ageing. An altered calcium regulation in cell bodies and synaptic terminals in the hippocampus may be involved in the development of functional impairments in the hippocampal formation.

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.

Increased basal gluconeogenesis in the aged rat.

Post-absorptive gluconeogenesis from lactate measured in vivo increases 3-fold in 24-month-old rats compared to 3-month-old animals. Fractional lactate turnover rates showed no significant differences between the two groups of animals. Lower plasma glucose concentrations and insulin-glucagon ratios may explain the increase in gluconeogenesis observed in aged rats.

Vanadate fully stimulates insulin receptor substrate-1 associated phosphatidyl inositol 3-kinase activity in adipocytes from young and old rats.

Vanadate stimulates adipocyte 2-deoxyglucose transport and GLUT-4 translocation to the membrane through an insulin receptor-independent but wortmannin-inhibitable pathway. Vanadate stimulates PI 3-kinase in anti-IRS-1 immunoprecipitates and the binding between IRS-1 and the p85alpha subunit of PI 3-kinase. In insulin-resistant adipocytes from old rats vanadate fully stimulates IRS-1-associated PI 3-kinase, but partially activates glucose uptake. We conclude that: (a) vanadate stimulates 2-deoxyglucose uptake using a pathway that converges with that of insulin at the level of PI 3-kinase; and (b) adipocytes from old rats are defective in the insulin pathway at steps located both upstream and downstream of PI 3-kinase.

Lithium protects cultured neurons against beta-amyloid-induced neurodegeneration.

The deposition of beta-amyloid peptide (A beta), the hyperphosphorylation of tau protein and the death of neurons in certain brain regions are characteristic features of Alzheimer's disease. It has been proposed that the accumulation of aggregates of A beta is the trigger of neurodegeneration in this disease. In support of this view, several studies have demonstrated that the treatment of cultured neurons with A beta leads to the hyperphosphorylation of tau protein and neuronal cell death. Here we report that lithium prevents the enhanced phosphorylation of tau protein at the sites recognized by antibodies Tau-1 and PHF-1 which occurs when cultured rat cortical neurons are incubated with A beta. Interestingly, lithium also significantly protects cultured neurons from A beta-induced cell death. These results raise the possibility of using chronic lithium treatment for the therapy of Alzheimer's disease.

Age-dependent modifications of rat heart succinate dehydrogenase.

The activity of rat heart succinate dehydrogenase (SDH) increases 1.7-fold in old animals (Vitorica, J., Cano, J., Satrústegui, J. and Machado, A., Mech. Ageing Dev., 16 (1981) 105-116). This increase is due to an increase in enzyme protein itself because: (a) the activation state of the enzyme does not vary with age; and (b) the increase of activity is paralleled by an increase in immunoprecipitable SDH in old rat heart mitochondria. SDH from old rat heart mitochondria differs in a number of ways from that of young animals: (a) The km value for succinate increases with age. (b) The thermostability decreases, and the activation energy in the 20-40 degrees C interval is higher in old animals. (c) The breaking points of the Arrhenius plots of SDH are shifted to higher values. (d) Reactivity towards N-ethylmaleimide in succinate protected mitochondria decreases with age.

Variations due to hyperoxia and ageing in the activities of glutathione S-transferase and NADPH-cytochrome c reductase.

We have studied the influence of hyperoxia and ageing on the activities of NADPH-cytochrome c reductase and glutathione S-transferase in different rat organs. Lung glutathione S-transferase activity increases markedly in 5-day-old pups exposed to hyperoxia, as observed for the O2- scavenging enzyme, superoxide dismutase. The levels of NADPH-cytochrome c reductase increase as well but after a 3-day lag period. In the liver, there is a pronounced decrease of both activities in 24-month-old rats, but at 12 months the activity of glutathione S-transferase increases whereas that of NADPH cytochrome c reductase activity decreases with respect to 3 months. The pattern of variations with age of NADPH cytochrome c reductase is similar in liver and brain. However the behaviour of brain glutathione S-transferase parallels that of the liver enzyme only up to 12 months. Thereafter the brain activity is maintained at a high level. These observations open the possibility that the high glutathione S-transferase levels in the old rat brain might be involved in protection towards oxidative alterations during ageing.

Impairment of glutamate uptake and absence of alterations in the energy-transducing ability of old rat brain mitochondria.

The proton electrochemical gradient has been measured in old brain mitochondria isolated from 2- or 24-month-old rats with the use of different respiratory substrates. With succinate as substrate, neither the respiratory rate, membrane potential or delta pH varied with age, indicating that the dielectric strength of the mitochondrial membrane was unaltered in old animals. The ohmic behavior of the membrane was tested in experiments in which the respiratory rate was partially inhibited by malonate, and was found to be unchanged with age. When glutamate plus malate were used as substrates, the respiratory rate was substantially reduced, and a drastic decrease in glutamate uptake was observed in old rat brain mitochondria.

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+-isocitrate 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.