Interplay between lysosomal, mitochondrial and death receptor pathways during manganese-induced apoptosis in glial cells.

Manganese (Mn) is an essential trace metal which plays a critical role in brain physiology by acting as a cofactor for several enzymes. However, upon overexposure, Mn preferentially accumulates within the basal ganglia leading to the development of a Parkinsonism known as Manganism. Data from our group have proved that Mn induces oxidative stress-mediated apoptosis in astrocytoma C6 cells. In the present study we described how cathepsins impact on different steps of each apoptotic cascade. Evidence obtained demonstrated that Mn generates lysosomal membrane permeabilization (LMP) and cathepsin release. Both cathepsins B (Ca-074 Me) and D (Pepstatin A) inhibitors as well as Bafilomycin A1 prevented caspases-3, -7, -8 and -9 activation, FasL upregulation, Bid cleavage, Δφm disruption and cytochrome c release. Results from in vivo studies showed that intrastriatal Mn injection increased cathepsin D levels from corpus striatum and substantia nigra pars compacta. Our results point to LMP and lysosomal cathepsins as key mediators in the apoptotic process triggered by Mn. These findings highlight the relevance of targeting the lysosomal pathway for Manganism therapy.

The autophagic- lysosomal pathway determines the fate of glial cells under manganese- induced oxidative stress conditions.

Manganese (Mn) overexposure is frequently associated with the development of a neurodegenerative disorder known as Manganism. The Mn-mediated generation of reactive oxygen species (ROS) promotes cellular damage, finally leading to apoptotic cell death in rat astrocytoma C6 cells. In this scenario, the autophagic pathway could play an important role in preventing cytotoxicity. In the present study, we found that Mn induced an increase in the amount and total volume of acidic vesicular organelles (AVOs), a process usually related to the activation of the autophagic pathway. Particularly, the generation of enlarged AVOs was a ROS- dependent event. In this report we demonstrated for the first time that Mn induces autophagy in glial cells. This conclusion emerged from the results obtained employing a battery of autophagy markers: a) the increase in LC3-II expression levels, b) the formation of autophagic vesicles labeled with monodansylcadaverine (MDC) or LC3 and, c) the increase in Beclin 1/ Bcl-2 and Beclin 1/ Bcl-X(L) ratio. Autophagy inhibition employing 3-MA and mAtg5(K130R) resulted in decreased cell viability indicating that this event plays a protective role in Mn- induced cell death. In addition, mitophagy was demonstrated by an increase in LC3 and TOM-20 colocalization. On the other hand, we proposed the occurrence of lysosomal membrane permeabilization (LMP) based in the fact that cathepsins B and D activities are essential for cell death. Both cathepsin B inhibitor (Ca-074 Me) or cathepsin D inhibitor (Pepstatin A) completely prevented Mn- induced cytotoxicity. In addition, low dose of Bafilomycin A1 showed a similar effect, a finding that adds evidence about the lysosomal role in Mn cytotoxicity. Finally, in vivo experiments demonstrated that Mn induces injury and alters LC3 expression levels in rat striatal astrocytes. In summary, our results demonstrated that autophagy is activated to counteract the harmful effect caused by Mn. These data is valuable to be considered in future research concerning Manganism therapies.

Neurohormone melatonin prevents cell damage: effect on gene expression for antioxidant enzymes.

It is well known that porphyrins cause a toxic light-mediated effect due to their capability to generate free radicals. Several reports have proved that melatonin is a potent free radical scavenger. The aim of this work has been to study the ability of melatonin to prevent the cell damage caused by porphyrins in the Harderian gland of female Syrian hamsters. Cell injury was evaluated estimating the percentage of damaged cells found in the gland and analyzing the degree of this damage at ultrastructural level. To explain the mechanism by which this hormone could prevent the cell damage caused by porphyrins, its capability to both decrease porphyrin synthesis and increase the mRNA levels for antioxidant enzymes was evaluated. Our results demonstrate that melatonin administration decreases the percentage of damaged cells, porphyrin synthesis, and aminolevulinate synthase (ALA-S) mRNA levels and increases the mRNA levels for manganese superoxide-dismutase and copper-zinc superoxide dismutase. When observed under an electron microscope, the lesions in the clear cells of the treated females were much less severe than in the corresponding cells of the control animals. Melatonin exerts a cytoprotective effect by inhibiting the ALA-S gene expression (and so porphyrin synthesis) and by raising the mRNA levels for several antioxidant enzymes.

Castration increases cell damage induced by porphyrins in the Harderian gland of male Syrian hamster. Necrosis and not apoptosis mediates the subsequent cell death.

It is known that the Harderian gland of male Syrian hamster synthesizes a much smaller amount of porphyrins than the gland of the female and that castration greatly increases this synthesis. We have studied in this experimental model the behavior of the different classes of secretory cells and their role in the synthesis of porphyrins, attempting to clarify the participation of these compounds in the cell damage leading to the formation of clear cells previously described in the gland of females. We have also investigated the mechanism underlying the death of these secretory cells after porphyrin accumulation (necrosis vs apoptosis). To achieve this, we have utilized the following techniques: (a) morphometrical; (b) ultrastructural; (c) biochemical (fluorescence spectrophotometry); and (d) molecular (DNA nick-end labeling in methacrylate sections and dot blot analysis). The glands from male hamsters (serving as control) present a very low rate of damaged cells that progressively rises after castration. This rise runs parallel to that of porphyrin synthesis, porphyrin deposits, and the decrease of Type II secretory cells. The damage and subsequent death of the secretory cells in the gland is produced by the deposit of porphyrins in the mitochondrial membrane. This porphyrin accumulation leads to a complete mitochondrial destruction that finally results in cell death and its secretion into the lumen. We finally conclude that this event is not a physiological cell death (apoptosis) but the consequence of the toxic accumulation of porphyrins (necrosis).

High delta-aminolevulinic acid uptake in rat cerebral cortex: effect on porphyrin biosynthesis.

The response of nerve cells to high exogenous aminolevulinic acid (ALA) concentrations was studied by examining the changes in its uptake and in porphyrin biosynthesis. ALA was shown to be taken up by cerebral cortex particles by a non-saturable process. As opposed to other previously described experimental systems, it was also observed that 84-87% of porphyrins formed was found within the cells. Exposure of cerebral cortex particles to high exogenous ALA (0.8-4.0 mM) showed that ALA can be accumulated in relatively high concentrations in brain cells (21.04 +/- 1.05 nmol/mg protein). Under these experimental conditions, porphyrin biosynthesis was found to be markedly inhibited (52%). 2.4 mM ALA caused an initial stimulation of glucose uptake after 1 hr incubation and a later fall to below control values, being consistent with the fact that acute porphyric crisis could be precipitated by the action of ALA on energy metabolism. ALA toxicity could be due both to its accumulation in the cells and to deficient heme concentrations, with an additional effect on glucose metabolism. These findings provide the basis for a useful brain tissue model to investigate the nature of the metabolic mechanisms occurring in acute intermittent porphyria (AIP) patients.

Porphyrin accumulation in the harderian glands of female Syrian hamster results in mitochondrial damage and cell death.

BACKGROUND: The Harderian glands of female Syrian hamsters contain very high concentrations of protoporphyrin (in the range of micrograms per mg of tissue) which accumulate in the tubulo-alveoli of the gland. We have studied the process of synthesis, accumulation, and secretion of this cyclic compound by the secretory cells of the hamster Harderian glands. METHODS: The animals used were female Syrian hamster of 15, 35, 75, 180, and 360 days of age. Items first examined were (1) percentage of the "clear cells," (2) area occupied by intraluminal porphyrins, and (3) histological characteristics of "clear cells" by light and transmission electron microscopy (TEM). In a second study the total content of porphyrins was determined. Finally, the levels of mRNA for the enzyme aminolevulinate synthase (ALV-S) were measured. RESULTS: In the glands of female hamsters, both the tissue concentration and the intraluminal area occupied by protoporphyrin correlate with the appearance of a special type of cell (clear cells) which show signs of cell degeneration. In addition, the expression of the gene for ALV-S, which is the limiting enzyme in porphyrin production, also parallels the relative number of clear cells. Analyzed under TEM, these clear cells display dilated mitochondria and short and swollen endoplasmic reticulum cisternae. In a late phase of necrosis, the nuclear envelope appears disorganized with scarce chromatin. The mitochondria undergo complete destruction, resulting in electron-dense bacillary formations which progressively coalesce in large and dense areas of protoporphyrin. The cell dies after this accumulation, being secreted by a "cytogen" mechanism. CONCLUSIONS: In view of our results, the Harderian gland of female Syrian hamster may provide a useful model for the study of the mechanism by which the anomalous accumulation of protoporphyrin induces cell damage in human protoporphyria.

Studies on uroporphyrinogen biosynthesis in pig liver.

Porphobilinogen-deaminase (PBG-D) and PBG-D-isomerase complex (PBG-D-I) from pig liver were isolated and partially purified. Uroporphyrinogen I and III formation was found to be linear with time and protein concentration. Optimal pH was about 7.4 and 7.6-7.8 for PBG-D and PBG-D-I complex, respectively. Some properties of the isolated enzymes were studied. Molecular mass determination gave a value of 40,000 Da for PBG-D and 50,000 Da for the complex. Both enzymes exhibited classical Michaelis-Menten kinetics. Km and Vmax parameters were estimated. The effect of several divalent cations, ammonia and thiol reagents was also investigated. The differential action of some of these chemicals on PBG-D and PBG-D-I system would suggest that PBG-D and isomerase may not be only physically adjacent but actually associated.

Involvement of free and enzyme-bound intermediates in the reaction mechanism catalyzed by the bovine liver immobilized porphobilinogen deaminase. Proof that they are substrates for cosynthetase in uroporphyrinogen III biosynthesis.

The detection and accumulation of tetrapyrrole intermediates synthesized by the action of bovine liver porphobilinogen deaminase immobilized to Sepharose 4B is reported. Employing Sepharose-deaminase preparations, two phases in uroporphyrinogen I synthesis as a function of time were observed, suggesting the accumulation of free and enzyme-bound intermediates, the concentration and distribution of which were time dependent. The deaminase-bound intermediate behaves as a substrate in uroporphyrinogen I synthesis whereas the free intermediates produce enzyme inhibition. The tetrapyrrole intermediate bound to the Sepharose-enzyme is removed from the protein by the binding of porphobilinogen. Free as well as enzyme-bound intermediates are shown to be substrates for cosynthetase with formation of 80% uroporphyrinogen III.

Immobilized uroporphyrinogen I synthetase from Rhodopseudomonas palustris.

Rhodopseudomonas palustris uroporphyrinogen I synthetase (URO-S) has been chemically attached to Sepharose 4B and some of its properties have been studied. When 7-8 mg protein/ml activated Sepharose was used, immobilized URO-S retained 45% of the activity of the original soluble preparation, with a coupling yield of 66% after a period of 15 h. Optimal incubation conditions for the activity of gel-enzyme were determined. Unlike the soluble enzyme, the Sepharose-bound URO-S showed a biphasic substrate saturation curve, indicating that a protein conformational change had occurred during the process of immobilization. Immobilized URO-S stored at 4 degrees C for 35 days retained 90% of activity and when repeatedly used, up to 5 times, retained 48% of the original activity. Attachment of URO-S to Sepharose led to an enhanced thermal stability.

Porphobilinogenase from Rhodopseudomonas palustris.

1. Porphobilinogenase (PBGase) from Rp. palustris has been isolated and some properties of a partially purified fraction were studied. 2. PBGase has an optimum pH of 7.4 when activity was expressed in terms of porphyrins formed and two pH maxima at 7.4 and 8.5 when activity was based on the amount of PBG consumed. 3. Cyclotetramerization rate and distribution of reaction products were not affected either by the presence or absence of oxygen. 4. Two PBGase active species of mol. wt 115,000 and 50,000 were found, by means of gel filtration through a calibrated Sephadex G-100 column. 5. Kinetic data show the existence of positive cooperative effects for porphyrin formation, while a hyperbolic behaviour for PBG consumption was observed.

Human red cell porphobilinogen deaminase. A simpler method of purification and some unusual properties.

A simpler method for purifying human red cell deaminase, using a mixture of n-butanol and chloroform, which denatures hemoglobin, followed by ammonium sulphate fractionation, heat treatment, Sephadex G-100 and DEAE-cellulose chromatography, yielding a 3400 fold purified enzyme is described. Some properties of purified deaminase were studied. The enzyme seems to have a strict requirement for oxygen, neither PBG consumption nor uroporphyrinogens formation were measured under anaerobiosis. Uroporphyrinogens formation was linear with both protein and time over a wide range of enzyme concentration and up to 2 h. The optimum pH was 7.4 and the mol. wt was 40,000 +/- 4000. The enzyme was heat-stable and increased its activity by heating. Ammonium and hydroxylamine ions inhibited the reaction. K+ and Na+ ions did not greatly affect activity, while most divalent cations tested significantly diminished uroporphyrinogen formation and to a lesser degree PBG consumption. Direct plots of velocity against PBG concentration were hyperbolic, however double-reciprocal plots were non-linear, Hill plots gave an n value of 2 and Eadie plots were bell-shaped, indicating the existence of weakly positive cooperative effect between 2 binding sites for PBG per molecule of deaminase.