Mitochondrial degeneration after organic phosphate poisoning in prosimian primates

Summary The degenerative reaction of mitochondria to tricresylphosphate (TCP) poisoning in spinal ganglion cells of Slow Loris (Nycticebus coucang coucang) were studied with the electron microscope. In neurones of animals treated with TCP, mitochondria display various stages of alterations which confirm mitochondrial involvement in TCP poisoning. The role of degenerated mitochondria in the formation of neuronal lipofuscin is discussed. It is suggested that the lipofuscin granule is a metabolic product inherently related to mitochondrial degeneration, irrespective of the primary cause: ageing or intoxication.Fellow of the Deutscher Akademischer Austauschdienst on study leave from the Medical Faculty, University of SingaporeA grant from the Deutsche Forschungsgemeinschaft (Gl. 28/20) is gratefully acknowledgedThe skilfull technical assistance of Mr. Tajuddin b.M. Ali, Mr. P. Gopal, Mr. R. Dungan and Mrs. C. Weinrichter is gratefully acknowledged     

A novel hypothesis of lipofuscinogenesis and cellular aging based on interactions between oxidative stress and autophagocytosis.

Based on a series of experiments, using cultured postmitotic neonatal rat cardiac myocytes as a model system, we present a novel hypothesis of lipofuscin formation. This hypothesis proposes that lipofuscin is formed within secondary lysosomes due to an interplay of two processes, the production of partially reduced oxygen species by mitochondria and the autophagocytotic degradation within secondary lysosomes. Specifically, it is proposed that H2O2 generated by mitochondria and other organelles permeates into the lumen of secondary lysosomes, which contain iron derived from cellular structures undergoing intralysosomal degradation. The interaction between reactive ferrous iron and H2O2 results, via Fenton-type mechanisms, in the generation of hydroxyl free radicals (OH), inducing lipid peroxidation and eventually leading to intermolecular cross-linking and lipofuscin formation. Additionally, mitochondria undergoing intralysosomal decomposition might continue for a certain period to produce superoxide anion radicals (O2-) and thus also H2O2. This model of lipofuscinogenesis could satisfactorily explain the variations observed in the rates of lipofuscinogenesis among different postmitotic cell types in various species. Such variations might arise from a variety of factors including differences in the efficiency of the 'anti-oxidative shield', rate of H2O2 generation, amount of chain-breaking antioxidants, mode of intralysosomal iron chelation, rate of autophagocytosis as well as degree of efficiency of the intralysosomal hydrolytic enzymes.     

Quantification of Ceroid and Lipofuscin in Skeletal Muscle

Ceroid and lipofuscin are autofluorescent granules thought to be generated as a consequence of chronic oxidative stress. Because ceroid and lipofuscin are persistent in tissue, their measurement can provide a lifetime history of exposure to chronic oxidative stress. Although ceroid and lipofuscin can be measured by quantification of autofluorescent granules, current methods rely on subjective assessment. Furthermore, there has not been any evaluation of variables affecting quantitative measurements. The article describes a simple statistical approach that can be readily applied to quantitate ceroid and lipofuscin. Furthermore, it is shown that several factors, including magnification tissue thickness and tissue level, can affect precision and sensitivity. After optimizing for these factors, the authors show that ceroid and lipofuscin can be measured reproducibly in the skeletal muscle of dystrophic mice (ceroid) and aged mice (lipofuscin).     

Human and rat brain lipofuscin proteome

The accumulation of an autofluorescent pigment called lipofuscin in neurons is an invariable hallmark of brain aging. So far, this material has been considered to be waste material without particular relevance for cellular pathology. However, two lines of evidence argue that lipofuscin may play a yet unidentified role for pathological cellular functions: (i) Genetic forms of premature accumulation of similar autofluorescent material in neuronal ceroid lipofuscinosis indicate a direct disease-associated link to lipofuscin; (ii) Retinal pigment epithelium cell lipofuscin is mechanistically linked to age-associated macular degeneration. Here, we purified autofluorescent material from the temporal and hippocampal cortices of three different human individuals by a two-step ultracentrifugation on sucrose gradients. For human brain lipofuscin, we could identify a common set of 49 (among > 200 total) proteins that are mainly derived from mitochondria, cytoskeleton, and cell membrane. This brain lipofuscin proteome was validated in an interspecies comparison with whole brain rat lipofuscin (total > 300 proteins), purified by the same procedure, yielding an overlap of 32 proteins (64%) between lipofuscins of both species. Our study is the first to characterize human and rat brain lipofuscin and identifies high homology, pointing to common cellular pathomechanisms of age-associated lipofuscin accumulation despite the huge (40-fold) difference in the lifespan of these species. Our identification of these distinct proteins will now allow research in disturbed molecular pathways during age-associated dysfunctional lysosomal degradation.     

Estimation of fluorescence lifetime of lipofuscin fluorophores contained in lipofuscin granules of retinal pigment epithelium of human cadaver eyes without signs of pathology

The fluorescence lifetimes of lipofuscin fluorophores contained in chloroform extracts from retinal pigment epithelium (RPE) of human cadaver eyes without signs of pathology were evaluated by single photon counting. The comparison of fluorescence lifetimes of N-retinylidene-N-retinylethanolamine (A2E) and its photooxidation and photodegradation products has been carried out. It was shown that the contribution of A2E to the total fluorescence of chloroform extract from lipofuscin granules is not major. The results are important for the improvement of noninvasive diagnostic method of degenerative diseases of the retina and RPE—fundus autofluorescence (FAF).     

Inhibition of autophagy with 3-methyladenine results in impaired turnover of lysosomes and accumulation of lipofuscin-like material

Autophagy (which includes macro-, micro-, and chaperone-mediated autophagy) is an important biological mechanism for degradation of damaged/obsolete macromolecules and organelles. Ageing non-dividing cells, however, progressively accumulate oxidised proteins, defective organelles and intralysosomal lipofuscin inclusions, suggesting inherent insufficiency of autophagy. To learn more about the role of macroautophagy in the turnover of organelles and lipofuscin formation, we inhibited autophagic sequestration with 3-methyladenine (3 MA) in growth-arrested human fibroblasts, a classical model of cellular ageing. Such treatment resulted in a dramatic accumulation of altered lysosomes, displaying lipofuscin-like autofluorescence, as well as in a moderate increase of mitochondria with lowered membrane potential. The size of the late endosomal compartment appeared not to be significantly altered following 3 MA exposure. The accumulation of lipofuscin-like material was enhanced when 3 MA administration was combined with hyperoxia. The findings suggest that macroautophagy is essential for normal turnover of lysosomes. This notion is supported by reports in the literature of lysosomal membrane proteins inside lysosomes and/or late endosomes, as well as lysosomes with active hydrolases within autophagosomes following vinblastine-induced block of fusion between lysosomes and autophagosomes. The data also suggest that specific components of lysosomes, such as membranes and proteins, may be direct sources of lipofuscin.     

Lipofuscin and N-retinylidene-N-retinylethanolamine (A2E) accumulate in retinal pigment epithelium in absence of light exposure: their origin is 11-cis-retinal

The age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been associated with the development of retinal diseases, particularly age-related macular degeneration and Stargardt disease. A major component of lipofuscin is the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E). The current model for the formation of A2E requires photoactivation of rhodopsin and subsequent release of all-trans-retinal. To understand the role of light exposure in the accumulation of lipofuscin and A2E, we analyzed RPEs and isolated rod photoreceptors from mice of different ages and strains, reared either in darkness or cyclic light. Lipofuscin levels were determined by fluorescence imaging, whereas A2E levels were quantified by HPLC and UV-visible absorption spectroscopy. The identity of A2E was confirmed by tandem mass spectrometry. Lipofuscin and A2E levels in the RPE increased with age and more so in the Stargardt model Abca4(-/-) than in the wild type strains 129/sv and C57Bl/6. For each strain, the levels of lipofuscin precursor fluorophores in dark-adapted rods and the levels and rates of increase of RPE lipofuscin and A2E were not different between dark-reared and cyclic light-reared animals. Both 11-cis- and all-trans-retinal generated lipofuscin-like fluorophores when added to metabolically compromised rod outer segments; however, it was only 11-cis-retinal that generated such fluorophores when added to metabolically intact rods. The results suggest that lipofuscin originates from the free 11-cis-retinal that is continuously supplied to the rod for rhodopsin regeneration and outer segment renewal. The physiological role of Abca4 may include the translocation of 11-cis-retinal complexes across the disk membrane.     

Formation of enlarged mitochondria in a liver cell line in response to a synthetic glucocorticoid

For a number of years it has been recognized that glucocorticoids cause alterations in liver cell morphology (6, 9). Several investigators have shown that in liver in vivo mitochondria can be enlarged to many times their normal volume by treatment with cortisone (13, 15). There is a concomitant decrease in mitochondrial number, and the results of Kimberg and Loeb suggest that this is due to mitochondrial fusion (7). However, the exact mechanism whereby mitochondrial volume is altered and whether in fact cortisone is the direct causal agent are not known due to the complexity of studying these questions in a whole animal system. We have found that dexamethasone sodium phosphate (dex), a synthetic glucocorticoid, causes the formation of enlarged mitochondria in a liver cell line RLC-GAI, which grows in defined medium. In this paper we present our observations on the amount of enlargement that occurs after 5 days of treatment. The formation of enlarged mitochondria is reversible upon removal of the hormone from the medium, and we have attempted to determine whether "mitochondrial" or "nonmitochondrial" inhibitors are more effective in blocking the return of mitochondria to their normal size when the hormone is removed.     

Effect of energy metabolism inhibitors on the morphometric parameters and 3-dimensional structure of L-cell mitochondria

It has been shown by stereological analysis that the earlier discovered changes in the structure of mitochondria in cyanide treated L-cells (decrease in numerical density of mitochondria, increase in volume density of mitochondria, and surface density of mitochondrial membranes) are prevented by oligomycin, and they do not occur in the presence of oligomycin and protonophorous uncoupler carbonylcyanide m-chlorphenyl hydrazone applied separately. Proceeding from three-dimension reconstructed mitochondrial models it has been shown that cyanide treatment of L-cells for 23 hours causes a transformation of mitochondria as discrete column-like structures into a network of mitochondrial reticulum oriented from the nucleus to the periphery of the cell. After the treatment of L-cells with cyanide together with oligomycin, or with oligomycin and protonophore applied separately, the mitochondria retain the structure of discrete column-like for mations characteristic of the control cells. It is assumed that the functioning ATP-system is a physiological prerequisite of the formation of mitochondrial reticulum under conditions of the inhibited respiratory energy metabolism in the cell.     

Increased autophagic degradation of mitochondria in Alzheimer disease

Extensive literature exists supporting a role for mitochondrial dysfunction and oxidative damage in the pathogenesis of Alzheimer disease. Mitochondria are a major source of intracellular reactive oxygen species and are themselves particularly vulnerable to oxidative stress. It has been recently shown that the immunoreactivity of lipoic acid and cytochrome oxidase-1, two mitochondrial markers, is increased in the cytoplasm of pyramidal neurons in Alzheimer disease cases compared with controls. Furthermore, lipoic acid was found to be strongly associated with granular structures and, by ultrastructure analysis, shown to be localized in mitochondria, cytosol and, importantly, in organelles identified as autophagic vacuoles. Lipoic acid was also found associated with the electron dense core of lipofuscin in the brains of Alzheimer disease cases but not in controls, whereas cytochrome oxidase-1 immunoreactivity was limited to mitochondria and cytosol in both Alzheimer and control cases. These data suggest that mitochondria are key targets of increased autophagic degradation in Alzheimer disease. The study of autophagy in Alzheimer disease could clarify the mechanisms underlying this neurodegenerative disorder and, eventually, help in the development of new therapeutic strategies.     

Spatial structures in mitochondrial suspension induced by cation efflux

The formation of spatial structures in a thin unstirred layer of a mitochondrial suspension has been studied. It is shown that the structure formation depends on the state of the ion-transporting systems of mitochondria and that pattern development coincides with the activation of cation efflux from preloaded mitochondria. Spatial structure formation is an energy-dependent process and is suppressed by respiratory chain inhibitors. Patterning is also inhibited by EGTA, EDTA and ruthenium red, reflecting the requirement for divalent cation translocation in mitochondria for the studied phenomenon.     

Electron microscopic study of the epiphysis in the hen in different age periods

Electron microscopic investigation of the epiphysis in 1 and 2 years old hens revealed that pineocytes are rich in mitochondria, ribosomes, granular endoplasmic network, Golgi apparatus, synapses. In contrast to 1-year hens, large amounts of lipofuscin granules and lysosomes were found in older animals. The data obtained reveal intensive hormone formation and oxidation-reduction processes in the epiphysis of birds.     

Localisation of islet amyloid peptide in lipofuscin bodies and secretory granules of human B-cells and in islets of type-2 diabetic subjects

Summary Islet amyloid peptide (or diabetes-associated peptide), the major component of pancreatic islet amyloid found in type-2 diabetes, has been identified by electronmicroscopic immunocytochemistry in pancreatic B-cells from five non-diabetic human subjects, and in islets from five type-2 diabetic patients. The greatest density of immunoreactivity for islet amyloid peptide was found in electrondense regions of some lysosomal or lipofuscin bodies. The peptide was also localised by quantification of immunogold in the secretory granules of B-cells, and was present in cytoplasmic lamellar bodies. Acid phosphatase activity was also demonstrated in these organelles. Immunoreactivity for insulin was found in some lysosomes. These results suggest that islet amyloid peptide is a constituent of normal pancreatic B-cells, and accumulates in lipofuscin bodies where it is presumably partially degraded. In islets from type-2 diabetic subjects, amyloid fibrils and lipofuscin bodies in B-cells showed immunoreactivity for the amyloid peptide. Abnormal processing of the peptide within B-cells could lead to the formation of islet amyloid in type-2 diabetes.     

Inhibition by hydroxymalonate of malate dependent biosynthesis of progesterone in the mitochondrial fraction of human term placenta

It has been shown that the conversion of cholesterol to progesterone by human term placental mitochondria incubated in the presence of malate or fumarate was inhibited by hydroxymalonate—an inhibitor of malic enzyme. No inhibition was observed when mitochondria were incubated in the presence of citrate or isocitrate. The degree of inhibition by hydroxymalonate of partly purified NAD(P)-linked malic enzyme activity was identical to that of both malate dependent pyruvate and progesterone formation by intact mitochondria. These data strongly support a previous suggestion that malic enzyme plays an important role in the malate dependent progesterone biosynthesis by human placental mitochondria.     

Lipofuscin-formation in retinal pigment epithelial cells is reduced by antioxidants.

The accumulation of lipofuscin by retinal pigment epithelium may be an important feature in the pathogenesis of age-related macular degeneration, suggesting the possibility that this common cause of blindness might be prevented or delayed by antioxidants. In support of this idea, we now report significantly reduced formation of lipofuscin when the antioxidant substances lutein, zeaxanthin, lycopene (carotenoids), or alpha-tocopherol were added to rabbit and bovine (calf) retinal pigment epithelial (RPE) cells exposed to normobaric hyperoxia (40%) and photoreceptor outer segments. Rabbit and calf RPE cells were grown for 2 weeks with addition of one of the test substances every 48 h. The cellular uptake of carotenoids and alpha-tocopherol was assayed by HPLC after 2 weeks. The lipofuscin-content was measured by static fluorometry (rabbit cells) or by image analysis (calf cells). Both rabbit and calf RPE showed similar results with significantly lower amounts of lipofuscin in antioxidant-treated cells. The effect of carotenoids is especially interesting, since the result is not dependent on their protective effect against photo-oxidative reactions. The chain-breaking abilities of these antioxidants in peroxidative reactions of lipid membranes and quenching of free radicals seem to be of importance for inhibition of lipofuscin formation.     

The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis.

Cellular manifestations of aging are most pronounced in postmitotic cells, such as neurons and cardiac myocytes. Alterations of these cells, which are responsible for essential functions of brain and heart, are particularly important contributors to the overall aging process. Mitochondria and lysosomes of postmitotic cells suffer the most remarkable age-related alterations of all cellular organelles. Many mitochondria undergo enlargement and structural disorganization, while lysosomes, which are normally responsible for mitochondrial turnover, gradually accumulate an undegradable, polymeric, autofluorescent material called lipofuscin, or age pigment. We believe that these changes occur not only due to continuous oxidative stress (causing oxidation of mitochondrial constituents and autophagocytosed material), but also because of the inherent inability of cells to completely remove oxidatively damaged structures (biological 'garbage'). A possible factor limiting the effectiveness of mitochondial turnover is the enlargement of mitochondria which may reflect their impaired fission. Non-autophagocytosed mitochondria undergo further oxidative damage, resulting in decreasing energy production and increasing generation of reactive oxygen species. Damaged, enlarged and functionally disabled mitochondria gradually displace normal ones, which cannot replicate indefinitely because of limited cell volume. Although lipofuscin-loaded lysosomes continue to receive newly synthesized lysosomal enzymes, the pigment is undegradable. Therefore, advanced lipofuscin accumulation may greatly diminish lysosomal degradative capacity by preventing lysosomal enzymes from targeting to functional autophagosomes, further limiting mitochondrial recycling. This interrelated mitochondrial and lysosomal damage irreversibly leads to functional decay and death of postmitotic cells.     

Effect of alpha-tocopherol and some metal chelators on lipofuscin accumulation in cultured neonatal rat cardiac myocytes

The objective of this study was to test further the hypothesis that oxidative stress is a major causal factor in lipofuscin formation. We have previously shown that cultured cardiac myocytes constitute a suitable model system for the study of factors influencing lipofuscinogenesis. The specific aim of the present study was to elucidate the effects of the chain-breaking free radical scavenger alpha-tocopherol, and the chelators desferrioxamine, EDTA and DTPA on the accumulation of lipofuscin. The effects were examined at different degrees of oxidative stress, obtained by varying the ambient oxygen concentration from 5 to 40%. Lipofuscin was quantified by microspectrofluorometry. Lipofuscin-specific, yellow autofluorescence increased with time in culture, and with enhanced oxidative stress. Increasing concentration of alpha-tocopherol, up to 40 microM, had an inhibitory effect on lipofuscin accumulation that was most pronounced at high oxidative stress. Desferrioxamine and DTPA, both caused a pronounced reduction in lipofuscin formation, while EDTA had no significant effect. The findings are interpreted to support the concept that oxidative stress is a causal factor in lipofuscinogenesis, and that lipofuscin is a product of autophagocytosed, membrane-rich material subjected to free radical-induced, metal-catalyzed peroxidation, fragmentation, and polymerization within the lysosomal vacuome.     

Germinal proto-mitochondria from rat liver

A number of properties of the smallest (less than 0.2 μm) germinal proto-mitochondria (PRMC) from rat liver have been studied. These PRMC were obtained by filtering the light fraction of hepatic mitochondria (MC) through calibrated millipore membranes. Germinal PRMC contain in general the same proteins as MC. However, they have the reduced content of flavoproteins and zero cytochrome oxidase. Germinal PRMC, in contrast to MC, almost does not contain the “aging pigment” - lipofuscin. They have DNA; the DNA/protein ratio in them is much higher than in MC, i.e. they are poor in protein. The obtained results support the earlier assumption that MC in specialized animal cells can arise from germinal PRMC - particles smaller than 0.2 μm containing DNA. It is assumed that the DNA molecules enter to cytoplasm during degradation of old MC serves as a seed for the formation of PRMC (with the connection of nuclear DNA).     

Carnosine and protein carbonyl groups: a possible relationship

Carnosine has been shown to react with low-molecular-weight aldehydes and ketones and has been proposed as a naturally occurring anti-glycating agent. It is suggested here that carnosine can also react with ("carnosinylate") proteins bearing carbonyl groups, and evidence supporting this idea is presented. Accumulation of protein carbonyl groups is associated with cellular ageing resulting from the effects of reactive oxygen species, reducing sugars, and other reactive aldehydes and ketones. Carnosine has been shown to delay senescence and promote formation of a more juvenile phenotype in cultured human fibroblasts. It is speculated that carnosine may intracellularly suppress the deleterious effects of protein carbonyls by reacting with them to form protein-carbonyl-carnosine adducts, i.e., "carnosinylated" proteins. Various fates of the carnosinylated proteins are discussed including formation of inert lipofuscin and proteolysis via proteosome and RAGE activities. It is proposed that the anti-ageing and rejuvenating effects of carnosine are more readily explainable by its ability to react with protein carbonyls than its well-documented antioxidant activity.     

Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations

Recent evidence suggests that the ability of ceramides to induce apoptosis is due to a direct action on mitochondria. Mitochondria are known to contain enzymes responsible for ceramide synthesis and hydrolysis and mitochondrial ceramide levels have been shown to be elevated prior to the mitochondrial phase of apoptosis. Ceramides have been reported to induce the release of intermembrane space proteins from mitochondria, which has been linked to their ability to form large channels in membranes. The aim of this study was to determine if the membrane concentration of ceramide required for the formation of protein permeable channels is within the range that is present in mitochondria during the induction phase of apoptosis. Only a very small percentage of the ceramide actually inserts into the mitochondrial membranes. The permeability of the mitochondrial outer membrane correlates directly with the level of ceramide in the membrane. Importantly, the concentration of ceramide at which significant channel formation occurs is consistent with the level of mitochondrial ceramide that occurs during the induction phase of apoptosis (4 pmol ceramide/nanomole phospholipid). Similar results were obtained with short- and long-chain ceramide. Ceramide channel formation is specific to mitochondrial membranes in that no channel formation occurs in the plasma membranes of erythrocytes even at concentrations 20 times higher than those required for channel formation in mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway by which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis.