Emilio de Juan

Impairment of mitochondrial oxidative phosphorylation in the brain of aged mice

To elucidate the role of mitochondrial oxidative phosphorylation in neuronal aging, we have studied the activity of the respiratory complexes in the brain of young, adult and old mice. In synaptic mitochondria, we found a significant decrease in complexes IV (29%, P < 0.001) and V (21%, P < 0.01) in old as compared with adult mice. Nonsynaptic mitochondria also showed a senescent decrease in complexes I (15%, P < 0.01), II + III (34%, P < 0.01) and IV (17%, P < 0.01) activities. These findings suggest a dysfunction in mitochondrial oxidative phosphorylation in brain aging.

N-Acetylcysteine protects against age-related decline of oxidative phosphorylation in liver mitochondria

Since it has been proposed that oxygen radical inactivation of key enzymes plays a critical role in cell aging, we have investigated the effects of a thiolic free radical scavenger on the oxidative phosphorylation enzymes of liver mitochondria from female OF-1 mice. At 48 weeks of age a control group was fed standard food pellets and another group received pellets containing 0.3% (w/w) of N-acetylcysteine. A 24-week treatment resulted in a significant increase in the specific activities of complex I, IV and V in the hepatic mitochondria of the N-acetylcysteine-treated animals as compared to aged controls.

Age-related changes in glutathione and lipid peroxide content in mouse synaptic mitochondria: Relationship to cytochrome c oxidase decline

To elucidate the contribution of lipid peroxides and glutathione to brain aging, we have carried out a comparative study of their contents in synaptic mitochondria from young (10-week-old), adult (24-week-old), and senescent (72-week-old) mice. In synaptic mitochondria, there is a significant decline in lipid peroxide content (P < 0.01) and cytochrome c oxidase activity (P < 0.001) in senescent as compared to adult and young mice. By contrast, glutathione concentration showed an increase in senescent (P < 0.05) in comparison to the other age groups. Moreover, there was a significant inverse correlation between age and lipid peroxide content (r = -0.5, P < 0.001) or cytochrome c oxidase-specific activity (r = -0.58, P < 0.001). We suggest that this age-dependent decrease in lipid peroxide content in synaptic mitochondria may be the result of an age-related decline in the activity of the electron transport chain, with concomitant decrease in oxyradical production, in the senescent organelles.

Microgravity effects on Drosophila melanogaster behavior and aging. Implications of the IML-2 experiment

Earlier Space experiments had indicated that young male Drosophila flies exposed to microgravity showed an acceleration in aging. In a 14.5-day Space Shuttle Flight we sent 300 young male flies with the purpose of confirming these findings and to establish whether changes in the behavior of the flies were responsible for the effect in accordance with the proposal that alterations in mitochondrial metabolism may be involved in the aging response. By repeatedly video-recording, we have found a very marked increase in the locomotor activity of the fruitflies in Space. The males showed an accelerated aging response upon recovery, both in terms of physiological vitality assays (mating and negative geotaxis) and of life-span curves. The involvement of mitochondrial metabolism is also suggested by the finding of a greater decrease in mitochondrial 16S ribosomal RNA in the microgravity exposed flies than in ground controls. On the other hand, a parallel 1 x g centrifuge control did not show such differences in the life-span curves when compared to flies exposed to a similar centrifugation on the ground. Drosophila females also increased their locomotor activity but did not show differential changes in the life-span curves. These results are discussed in terms of the current mechanisms of aging in multicellular eukaryotic organisms.

Stimulation by the antioxidant thioproline of the lymphocyte functions of old mice

Diet supplementation with thioproline (thiazolidine-4-carboxylic acid), an intracellular sulfhydryl antioxidant and free radical scavenger, may slow the aging process of metazoans and prolongs their life span. In the present experiment Swiss mice fed thioproline (0.07%, w/w) from 13 to 22 months of age were used. Six- and 22-month-old mice fed standard diet were used as controls. Two important functions of lymphocytes, the proliferative response to the mitogen Concanavalin A (Con A) and the mobility, both spontaneous and directed to a chemoattractant gradient (chemotaxis), were analyzed in lymphocytes from axillary nodes, spleen and thymus. Mobility and chemotaxis were studied by Boydens technique, using filters of 3 microns pore diameter, 3 h of incubation and fmet-leu-phe at 10(-8) M as chemoattractant. The proliferative response was estimated as 3H-thymidine incorporation in lymphocytes incubated for 72 h in the presence of Con A (1 and 5 micrograms/ml). The results show a decrease in mobility, chemotaxis and lymphoproliferative response in old mice in comparison to adults. However, a significant increase in these functions was observed in old mice fed thioproline. The advantage of using this antioxidant for immunostimulation during aging, a stage of life characterized by a decreased immune response, is discussed.

Effects of the space environment on Drosophila melanogaster development. Implications of the IML-2 experiment.

One hundred and sixty Drosophila females laid several thousands of embryos during the 14.5 days of the IML-2 spaceflight. The progeny were either recovered frozen (embryos at final stages of development and larvae), or maintained alive developing further until adulthood. All embryos, larvae, pupae and imagoes recovered were normal in morphology and function. Results from earlier experiments were reproduced in IML-2 with a better experimental design. We confirm that in Space there is a stimulation of oogenesis and that development is slightly delayed when compared to that of synchronous parallel ground controls. Nevertheless, it is clear from the accompanying 1 x g flight control centrifuge and from the 1.4 x g ground centrifuge samples, that centrifugation itself can produce similar effects, emphasizing the importance of reevaluating the role of the 1 x g on-flight controls. The results emphasize the apparent paradox that simple cellular model systems in microgravity show alterations in many fundamental processes such as those involved in cell signalling, while development, relying heavily on these key cellular mechanisms can proceed quite normally in the absence of gravity. The effects on development are small and more the consequence of a reaction to the abnormal Space environment in general than a specific effect of microgravity.

Effect of magnetically simulated zero-gravity and enhanced gravity on the walk of the common fruitfly

Understanding the effects of gravity on biological organisms is vital to the success of future space missions. Previous studies in Earth orbit have shown that the common fruitfly (Drosophila melanogaster) walks more quickly and more frequently in microgravity, compared with its motion on Earth. However, flight preparation procedures and forces endured on launch made it difficult to implement on the Earths surface a control that exposed flies to the same sequence of major physical and environmental changes. To address the uncertainties concerning these behavioural anomalies, we have studied the walking paths of D. melanogaster in a pseudo-weightless environment (0g*) in our Earth-based laboratory. We used a strong magnetic field, produced by a superconducting solenoid, to induce a diamagnetic force on the flies that balanced the force of gravity. Simultaneously, two other groups of flies were exposed to a pseudo-hypergravity environment (2g*) and a normal gravity environment (1g*) within the spatially varying field. The flies had a larger mean speed in 0g* than in 1g*, and smaller in 2g*. The mean square distance travelled by the flies grew more rapidly with time in 0g* than in 1g*, and slower in 2g*. We observed no other clear effects of the magnetic field, up to 16.5 T, on the walks of the flies. We compare the effect of diamagnetically simulated weightlessness with that of weightlessness in an orbiting spacecraft, and identify the cause of the anomalous behaviour as the altered effective gravity.

Glucose deprivation increases aspartic acid release from synaptosomes of aged mice

To investigate the possible existence of age-related changes in excitatory amino acid release in brain, and the influence of glucose deprivation on this process, we have determined the release of endogenous aspartate and glutamate from synaptosomes freshly isolated from the cerebrum of young (12 months old) and aged (24 months old) mice. We found that there are no age-related changes in the synaptosomal release of aspartic and glutamic acids. However, in the absence of glucose in the medium of incubation aspartate and glutamate release was higher in old than in young animals (P < 0.05, and P = 0.09 respectively). Our results suggest that the ability of cerebral synaptosomes to release glutamate and aspartate remains functionally intact in old cerebrum, but there is an age-dependent dysfunction in this process linked to energy metabolism disturbances.

Arthropod model systems for studying complex biological processes in the space environment

Three arthropod systems are discussed in relation to their complementary and potential use in Space Biology. In a next biosatellite flight, Drosophila melanogaster pre-adapted during several months to different g levels will be flown in an automatic device that separates parental from first and second generations. In the same flight, flies will be exposed to microgravity conditions in an automatic unit in which fly motility can be recorded. In the International Microgravity Laboratory-2, several groups of Drosophila embryos will be grown in Space and the motility of a male fly population will be video-recorded. In the Biopan, an ESA exobiology facility that can be flown attached to the exterior of a Russian biosatellite, Artemia dormant gastrulae will be exposed to the space environment in the exterior of the satellite under a normal atmosphere or in the void. Gastrulae will be separated in hit and non-hit populations. The developmental and aging response of these animals will be studied upon recovery. With these experiments we will be able to establish whether exposure to the space environment influences arthropod development and aging, and elaborate on some of the cellular mechanisms involved which should be tested in future experiments.