Jake Jacobson

Mechanisms of Life Span Extension by Rapamycin in the Fruit Fly Drosophila melanogaster

Summary The target of rapamycin (TOR) pathway is a major nutrient-sensing pathway that, when genetically downregulated, increases life span in evolutionarily diverse organisms including mammals. The central component of this pathway, TOR kinase, is the target of the inhibitory drug rapamycin, a highly specific and well-described drug approved for human use. We show here that feeding rapamycin to adult Drosophila produces the life span extension seen in some TOR mutants. Increase in life span by rapamycin was associated with increased resistance to both starvation and paraquat. Analysis of the underlying mechanisms revealed that rapamycin increased longevity specifically through the TORC1 branch of the TOR pathway, through alterations to both autophagy and translation. Rapamycin could increase life span of weak insulin/Igf signaling (IIS) pathway mutants and of flies with life span maximized by dietary restriction, indicating additional mechanisms.

Expression and Modulation of an NADPH Oxidase in Mammalian Astrocytes

Amyloid β peptides generate oxidative stress in hippocampal astrocytes through a mechanism sensitive to inhibitors of the NADPH oxidase [diphenylene iodonium (DPI) and apocynin]. Seeking evidence for the expression and function of the enzyme in primary hippocampal astrocytes, we confirmed the expression of the subunits of the phagocyte NADPH oxidase by Western blot analysis and by immunofluorescence and coexpression with the astrocyte-specific marker glial fibrillary acidic protein both in cultures and in vivo. Functional assays using lucigenin luminescence, dihydroethidine, or dicarboxyfluorescein fluorescence to measure the production of reactive oxygen species (ROS) demonstrated DPI and apocynin-sensitive ROS generation in response to the phorbol ester PMA and to raised [Ca2+]c after application of ionomycin or P2u receptor activation. Stimulation by PMA but not Ca2+ was inhibited by the protein kinase C (PKC) inhibitors staurosporine and hispidin. Responses were absent in transgenic mice lacking gp91phox. Expression of gp91phox and p67phox was increased in reactive astrocytes, which showed increased rates of both resting and stimulated ROS generation. NADPH oxidase activity was modulated by intracellular pH, suppressed by intracellular alkalinization, and enhanced by acidification. The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone suppressed basal ROS generation but markedly increased PMA-stimulated ROS generation. This was independent of mitochondrial ROS production, because it was unaffected by mitochondrial depolarization with rotenone and oligomycin. Thus, the NADPH oxidase is expressed in astrocytes and is functional, activated by PKC and intracellular calcium, modulated by pHi, and upregulated by astrocyte activation. The astrocytic NADPH oxidase is likely to play important roles in CNS physiology and pathology.

Imaging mitochondrial function in intact cells.

Publisher Summary This chapter describes the imaging mitochondrial function in intact cells. Mitochondria play a central role in cell life and cell death. Mitochondria not only generate ATP, the major currency used by cells in energy-requiring processes, but also house a range of synthetic enzymes, including enzymes involved in heme and steroid biosynthesis. Mitochondria are intimately involved in shaping the spatiotemporal characteristics of intracellular [Ca 2+ ] c signaling, and they house several proteins that play a key role in the regulation of programmed cell death, or apoptosis. The discovery that proteins critically involved in the initiation and regulation of apoptotic cell death are housed within mitochondria and may be released, either accidentally as a consequence of cell injury or in a regulated fashion at the onset of programmed cell death, has set the mitochondrion center stage as a key player in major aspects of modern cell biology and medicine.

Interplay between mitochondria and cellular calcium signalling

Mitochondria are increasingly ascribed central roles in vital cell signalling cascades. These organelles are now recognised as initiators and transducers of a range of cell signals, including those central to activation and amplification of apoptotic cell death. Moreover, as the main source of cellular ATP, mitochondria must be responsive to fluctuating energy demands of the cell. As local and global fluctuations in calcium concentration are ubiquitous in eukaryotic cells and are the common factor in a dizzying array of intra- and inter-cellular signalling cascades, the relationships between mitochondrial function and calcium transients is currently a subject of intense scrutiny. It is clear that mitochondria not only act as local calcium buffers, thus shaping spatiotemporal aspects of cytosolic calcium signals, but that they also respond to calcium uptake by upregulating the tricarboxylic acid cycle, thus reacting metabolically to local signalling. In this chapter we review current knowledge of mechanisms of mitochondrial calcium uptake and release and discuss the consequences of mitochondrial calcium handling for cell function, particularly in conjunction with mitochondrial oxidative stress.

Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential: implications for assays of cardiolipin and mitochondrial mass.

Cardiolipin, a polyunsaturated acidic phospholipid, is found exclusively in bacterial and mitochondrial membranes where it is intimately associated with the enzyme complexes of the respiratory chain. Cardiolipin structure and concentration are central to the function of these enzyme complexes and damage to the phospholipid may have consequences for mitochondrial function. The fluorescent dye, 10 nonyl acridine orange (NAO), has been shown to bind cardiolipin in vitro and is frequently used as a stain in living cells to assay cardiolipin content. Additionally, NAO staining has been used to measure the mitochondrial content of cells as dye binding to mitochondria is reportedly independent of the membrane potential. We used confocal microscopy to examine the properties of NAO in cortical astrocytes, neonatal cardiomyocytes and in isolated brain mitochondria. We show that NAO, a lipophilic cation, stained mitochondria selectively. However, the accumulation of the dye was clearly dependent upon the mitochondrial membrane potential and depolarisation of mitochondria induced a redistribution of dye. Moreover, depolarisation of mitochondria prior to NAO staining also resulted in a reduced NAO signal. These observations demonstrate that loading and retention of NAO is dependant upon membrane potential, and that the dye cannot be used as an assay of either cardiolipin or mitochondrial mass in living cells.

Dynamics of the action of dFOXO on adult mortality in Drosophila

The insulin/insulin growth factor (IGF)‐like signaling (IIS) pathway has a conserved role in regulating lifespan in Caenorhabditis elegans, Drosophila and mice. Extension of lifespan by reduced IIS has been shown in C. elegans to require the key IIS target, forkhead box class O (FOXO) transcription factor, DAF‐16. dFOXO, the Drosophila DAF‐16 orthologue, is also an IIS target, and its overexpression in adult fat body increases lifespan. In C. elegans, IIS acts exclusively during adulthood to determine adult survival. We show here, using an inducible overexpression system, that in Drosophila continuous dFOXO overexpression in adult fat body reduces mortality rate throughout adulthood. We switched the IIS status of the flies at different adult ages and examined the effects of these switches on dFOXO expression and mortality rates. dFOXO protein levels were switched up or down by the inducible expression system at all ages examined. If IIS status is reversed early in adulthood, similar to the effects of another intervention that reduces adult mortality in Drosophila, dietary restriction (DR), there is a complete switch of subsequent mortality rate to that of flies chronically exposed to the new IIS regime. At this age, IIS thus acts acutely to determine risk of death. Mortality rates continued to respond to a switch in IIS status up to 4 weeks of adult age, but not thereafter. However, unlike DR, as IIS status was altered at progressively later ages, mortality rates showed incomplete switching and responded with progressively smaller changes. These findings indicate that alteration of expression levels of dFOXO may have declining effects on IIS status with age, that there could be some process that prevents or lessens the physiological response to a switch in IIS status or that, unlike DR, this pathway regulates aging‐related damage. The decreased mortality and increased lifespan of dFOXO overexpressing flies was uncoupled from any effect on female fecundity and from expression levels of Drosophila insulin‐like peptides in the brain.

Induction of mitochondrial oxidative stress in astrocytes by nitric oxide precedes disruption of energy metabolism.

Inhibition of the mitochondrial electron transport chain (ETC) ultimately limits ATP production and depletes cellular ATP. However, the individual complexes of the ETC in brain mitochondria need to be inhibited by ∼ 50% before causing significant depression of ATP synthesis. Moreover, the ETC is the key site for the production of intracellular reactive oxygen species (ROS) and inhibition of one or more of the complexes of the ETC may increase the rate of mitochondrial ROS generation. We asked whether partial inhibition of the ETC, to a degree insufficient to perturb oxidative phosphorylation, might nonetheless induce ROS production. Chronic increase in mitochondrial ROS might then cause oxidative damage to the ETC sufficient to produce prolonged changes in ETC function and so compound the defect. We show that the exposure of astrocytes in culture to low concentrations of nitric oxide (NO) induces an increased rate of O2·− generation that outlasts the presence of NO. No effect was seen on oxygen consumption, lactate or ATP content over the 4–6 h that the cells were exposed to NO. These data suggest that partial ETC inhibition by NO may initially cause oxidative stress rather than ATP depletion, and this may subsequently induce irreversible changes in ETC function providing the basis for a cycle of damage.

Biomarkers of aging in Drosophila

Low environmental temperature and dietary restriction (DR) extend lifespan in diverse organisms. In the fruit fly Drosophila, switching flies between temperatures alters the rate at which mortality subsequently increases with age but does not reverse mortality rate. In contrast, DR acts acutely to lower mortality risk; flies switched between control feeding and DR show a rapid reversal of mortality rate. Dietary restriction thus does not slow accumulation of aging‐related damage. Molecular species that track the effects of temperatures on mortality but are unaltered with switches in diet are therefore potential biomarkers of aging‐related damage. However, molecular species that switch upon instigation or withdrawal of DR are thus potential biomarkers of mechanisms underlying risk of mortality, but not of aging‐related damage. Using this approach, we assessed several commonly used biomarkers of aging‐related damage. Accumulation of fluorescent advanced glycation end products (AGEs) correlated strongly with mortality rate of flies at different temperatures but was independent of diet. Hence, fluorescent AGEs are biomarkers of aging‐related damage in flies. In contrast, five oxidized and glycated protein adducts accumulated with age, but were reversible with both temperature and diet, and are therefore not markers either of acute risk of dying or of aging‐related damage. Our approach provides a powerful method for identification of biomarkers of aging.

No extension of lifespan by ablation of germ line in Drosophila

Increased reproduction is frequently associated with a reduction in longevity in a variety of organisms. Traditional explanations of this ‘cost of reproduction’ suggest that trade-offs between reproduction and longevity should be obligate. However, it is possible to uncouple the two traits in model organisms. Recently, it has been suggested that reproduction and longevity are linked by molecular signals produced by specific reproductive tissues. For example, in Caenorhabditis elegans, lifespan is extended in worms that lack a proliferating germ line, but which possess somatic gonad tissue, suggesting that these tissues are the sources of signals that mediate lifespan. In this study, we tested for evidence of such gonadal signals in Drosophila melanogaster. We ablated the germ line using two maternal effect mutations: germ cell-less and tudor. Both mutations result in flies that lack a proliferating germ line but that possess a somatic gonad. In contrast to the findings from C. elegans, we found that germ line ablated females had reduced longevity relative to controls and that the removal of the germ line led to an over-proliferation of the somatic stem cells in the germarium. Our results contrast with the widely held view that it is downstream reproductive processes such as the production and/or laying of eggs that are costly to females. In males, germ line ablation caused either no difference, or a slight extension, in longevity relative to controls. Our results indicate that early acting, upstream reproductive enabling processes are likely to be important in determining reproductive costs. In addition, we suggest that the specific roles and putative patterns of molecular signalling in the germ line and somatic tissues are not conserved between flies and worms.

Evidence for Intracellular Aggregation of Hypericin and the Impact on its Photocytotoxicity in PAM 212 Murine Keratinocytes

Abstract We have assessed photoinduced toxicity of hypericin in PAM 212 murine keratinocytes and the relationship between concentration, incubation time and light fluence to evaluate the effect of intracellular aggregation at high concentrations. Confocal microscopy was used to establish the subcellular localization of hypericin at 5 and 50 μM and incubation times of 1 and 3 h. From fluorescence uptake time course studies, intracellular hypericin was demonstrated to exist predominantly in the monomeric form for up to 26 h incubation at 5 μM. However, there was a pronounced aggregation effect at 50 μM, with intracellular hypericin fluorescence levels initially showing an increase followed by a decrease with incubation time. This effect was subsequently shown to exert an effect on the phototoxicity of hypericin. On irradiation, the photocytotoxicity for 1 and 7 h incubation with 50 μM hypericin was comparable, whereas using 5 μM the photocytotoxicity showed good correlation with the intracellular fluorescence measurements at 1 and 7 h incubation.