Changes in the organization of the intermediate filament system of human fibroblasts in lysosomal storage diseases and their modeling]

The organization of the system of the vimentin intermediate filaments (IFs) in human fibroblasts in lysosomal storage diseases (Fabry's disease, mannosidosis) and their modelling has been studied in vitro. It was shown that during accumulation of nonhydrolyzable compounds, hypertrophy of the lysosomal compartment is accompanied by formation of ring-shaped bundles IFs, surrounding apparently these increased organelles. The changed organization of IFs is characteristic of polarised pathological cells in monolayer, and after repassage it is retained only at the spreading state; on transition from the discoid to extended cellular form there occurred the centrifugal shift of ring-shaped structures of IFs to active cell border and gradual restoration of radial fibrillar state of IFs. It is suggested that on intralysosomal storage of unsplit compounds reorganization of the vimentin-type IFs in ring-shaped structure is necessary for optimal distribution and stabilization into the cytoplasm of large amounts of increased lysosomes with exo- and endogenous contents. In condition of free spreading (i. e. with diminished cell density) the restoration of normal fibrillar IF organization may be due to the loss of considerable number of hypertrophied lysosomes; the involvement of lysosomal membrane in formation of active cellular border is not to be ruled out.     

The intracellular significance of pH, its dynamics and possible role in regulating cell functions]

The intracellular and intralysosomal pH were studied on living monolayer cell cultures SPEV (pig embryo kidney), CHC (Chinese hamster's fibroblasts), NGUK-1 (neurinoma of Gasser's gland of rat), and also on primary cell cultures of rat's hepatocytes and chick embryo fibroblasts. In cytoplasm and lysosomes of these cells were found out pH changes during cells cycle and cell cultivation. There was also showed a heterogeneity of different parts of cells and lysosomes, and circahoral pH oscillations were registered. So there was found a spatial and temporal pH mosaity of living cells. The probable role of intracellular pH changes via regulator of cell vital activity is discussed.     

Accumulation of weak bases in relation to intralysosomal pH in cultured human skin fibroblasts

The volume of the lysosomal compartment in cultured human skin fibroblasts was estimated from the distribution between the cells and the medium of tracer amounts of labelled methylamine and chloroquine, which accumulate in the lysosomes, 2,2-dimethyloxazolidine-2,4-dione, which accumulates in the soluble cytoplasmic compartment relative to the lysosomes, and sucrose, which is excluded by the cells. In a foetal fibroblast line, the fractional volume of the lysosomal compartment was $\text{0.044 ± 0.007}$ ($\text{n}\text{= 8}$). In fibroblasts from a patient with the I-cell disease, the fractional volume was 0.15.The fractional volume of the lysosomal compartment was used to calculate the intralysosomal pH from the accumulation of the weak bases in the cells. The mean value obtained was $\text{5.29 ± 0.04}$ ($\text{n}\text{= 8}$).In fibroblasts incubated with various concentrations of chloroquine, the fractional volume of the lysosomal compartment and the accumulation of chloroquine in the cells were used to calculate the concentration of chloroquine in the lysosomes. The intralysosomal concentration increased from 3 to 114 mM as the extracellular concentration increased from 1 to 100 μM. Concomitantly, the intralysosomal pH increased from 5.3 in the absence of chloroquine to 5.9 in the presence of 100 μM chloroquine. A similar increase in intralysosomal pH could be calculated in fibroblasts incubated with different concentrations of ammonia.     

A pH increase in human fibroblast lysosomes during accumulation of non-hydrolysable compounds

The changes in intralysosomal pH were measured in the stationary phase of normal human embryonic fibroblast growth under sucrose loading over a period of 6 to 120 hours and in cells with a typical lysosomal storage pathology, Fabry's disease, using a vital indicator dye, neutral red. It was shown that long-term hypertrophy of the lysosomal compartment during intracellular accumulation of non-hydrolysable compounds is concomitant with a pH increase, on the average, by 0.4 units. The highest values of pH (7.0-7.2) were seen in large-sized heterogeneic lysosomes of pathological cells. It is suggested that an increase in intralysosomal pH during accumulation of non-hydrolysable compounds leads to deterioration of conditions that are favourable for the acidic hydrolase function.     

Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH. We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions. The pH of the medium has no effect on the intralysosomal pH. Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH. However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form. Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH. This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A. The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.     

Effect of amines on the pH in the lysosomes of 3T3 cells

The measurements of intralysosomal pH under the action of the number of amines earlier reported to block the process of the initiation of cell proliferation (Nikolsky et al., 1984) were made on Swiss 3T3 cells. The intralysosomal pH (pH1) value was estimated by parameters of fluorescence of fluorescein-labeled dextran in single intact cells. The pHl value was equal to 4.7 +/- 0.2 for both actively growing and quiescent cells. The pH gradient between lysosomes and the cytoplasm was completely destroyed by monensin and partially by carbonylcyamide-m-chlorophenylhydrasone. Methylamine and chloroquine rapidly enhanced the pHl, value to 6.4-7.0. Dansylcadaverine, 5-methoxytryptamine and dimethylurea did not affect pHl value. Intracellular accumulation of dansylcadaverine was shown to be due to the existence of acidic compartments into the cell and highly decreased in the presence of monensin. A conclusion is made that the inhibition of mitogenic signal by amines cannot be unequivocally accounted for by increasing the pH in organelles involved in the intracellular processing of growth factors.     

Autophagosome maturation is impaired in Fabry disease

Fabry disease is a lysosomal storage disorder (LSD) caused by a deficiency in α-galactosidase A. The disease is characterized by severe major organ involvement, but the pathologic mechanisms responsible have not been elucidated. Disruptions of autophagic processes have been reported for other LSDs, but have not yet been investigated in Fabry disease. Renal biopsies were obtained from 5 adult male Fabry disease patients before and after 3 years of enzyme replacement therapy (ERT) with agalsidase alfa. Vacuole accumulation was seen in renal biopsies from all patients compared with control biopsies. Decreases in the number of vacuoles were seen after 3 years of ERT primarily in renal endothelial cells and mesangial cells. Measurement of the levels of LC3, a specific autophagy marker, in cultured cells from Fabry patients revealed increased basal levels compared to cells from non-Fabry subjects and a larger increase in response to starvation than seen in non-Fabry cells. Starvation in the presence of protease inhibitors did not result in a significant increase in LC3 in Fabry cells, whereas a further increase in LC3 was observed in non-Fabry cells, an observation that is consistent with impaired autophagic flux in Fabry disease. Overexpression of LC3 mRNA in Fabry fibroblasts compared to control cells is consistent with an upregulation of autophagy. Furthermore, LC3 and p62/SQSTM1 (that binds to LC3) staining in renal tissues and in cultured fibroblasts from Fabry patients supports impairment of autophagic flux. These findings suggest that Fabry disease is linked to a deregulation of autophagy.     

Further studies on the effect of chloroquine on the uptake, metabolism and intracellular translocation of [35S]cystine in cystinotic fibroblasts

The present study uses the lysosomotropic drug chloroquine to investigate the mechanisms by which exogenous [35S]cystine is able to label the intracellular (intralysosomal) cystine pool(s) in cystinotic fibroblasts. When cystinotic fibroblasts were labelled for short periods of time (8 h or less), chloroquine (20 microM) inhibited the labelling of the intracellular cystine pool(s). However, when the cells were labelled for longer periods of time (24 h or more) chloroquine stimulated the labelling of the intracellular cystine pool(s). The short-term effect was selectively abolished when the cells were washed free of chloroquine, while the long-term effect was selectively abolished when the medium was depleted of cystine. Two routes of translocation of exogenous cystine to the lysosomes could be defined. One route was fast, had a low capacity, was inhibited by chloroquine and increased with increasing medium pH, while the other route was slow, had a high capacity, was stimulated by chloroquine and was more active at low pH. The former pathway probably consisted of plasma membrane transport of cystine into the cytosol followed by direct or indirect transport into the lysosomes. The latter route possibly consisted of pinocytosis with fusion of the cystine-containing pinosomes with lysosomes.     

Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease

The synthesis and processing of the human lysosomal enzyme alpha-galactosidase A was examined in normal and Fabry fibroblasts. In normal cells, alpha-galactosidase A was synthesized as an Mr = 50,500 precursor, which contained phosphate groups in oligosaccharide chains cleavable by endoglucosaminidase H. The precursor was processed via ill-defined intermediates to a mature Mr 46,000 form. Processing was complete within 3-7 days after synthesis. In the presence of NH4Cl and in I-cell fibroblasts, the majority of newly synthesized alpha-galactosidase A was secreted as an Mr = 52,000 form. For comparison, the processing and stability of alpha-galactosidase A were examined in fibroblasts from five unrelated patients with Fabry disease, which is caused by deficient alpha-galactosidase A activity. In one cell line, synthesis of immunologically cross-reacting polypeptides was not detectable. In another, the synthesis, processing, and stability of alpha-galactosidase A was indistinguishable from that in normal fibroblasts. In a third Fabry cell line, the mutation retarded the maturation of alpha-galactosidase A. Finally, in two cell lines, alpha-galactosidase A polypeptides were synthesized that were rapidly degraded following delivery to lysosomes. These results clearly indicate that Fabry disease comprises a heterogeneous group of mutations affecting synthesis, processing, and stability of alpha-galactosidase A.     

Pinocytosis and degradation of exogenous proteins by cystinotic fibroblasts

Lysosomes of cystinotic human fibroblasts contain over 100-times the normal concentration of cystine. The high cystine concentration (probably in the millimolar range) might be expected to inhibit intralysosomal protein breakdown. A comparison of pinocytosis and degradation of five ¹²⁵I-labelled proteins (bovine serum albumin, formaldehyde-denatured bovine serum albumin, bovine pancreatic ribonuclease A and porcine lactate dehydrogenase isoenzymes H4 and M4) by human fibroblasts has been made, using one cystinotic and two normal cell-lines. The proteins each entered fibroblasts by adsorptive pinocytosis and were then degraded within the lysosomes by enzymes susceptible to leupeptin, the thiol-proteinase inhibitor. Each protein was captured by the fibroblasts at a characteristic rate, which was not different in cystinotic cells. Normal and cystinotic fibroblasts did not differ in their proteolytic capacity, as measured in extracts of disrupted cells. In intact fibroblasts, four of the five proteins were rapidly and fully digested following pinocytosis, in both cystinotic and normal cells. However, with formaldehyde-denatured albumin, the most resistant to degradation of the proteins tested, or with some other proteins in the presence of leupeptin, when the proteolytic capacity of lysosomes is diminished, intralysosomal degradation of pinocytosed protein was incomplete. Moreover, under these conditions, cystinotic cells demonstrated a lower rate of protein digestion than normal cells. It is concluded that pinocytic capture, rather than intralysosomal proteolysis, is commonly the rate-limiting step in the overall process of uptake and degradation of proteins by fibroblasts, and that intralysosomal cystine inhibits digestion of pinocytosed protein only in circumstances when degradation becomes the rate-limiting step.     

High intralysosomal levels of lipoprotein cholesterol influence the endocytic activity and acid hydrolase content of CH-23 fibroblasts

Lipoprotein extracts isolated from hyperlipemic rabbit serum were used to study the effects of abnormal lipid levels on the functions of the lysosomal system in Chinese hamster CH-23 fibroblasts. The ability of cells enriched with lipid to endocystose [³H]inulin was largely unimpaired and utilising density gradient fractionation procedures the fusion between incoming inulin and low density lysosomes ladened with esterified cholesterol could be demonstrated. Studies in which cells were exposed to short ‘burst’ of [³H]inulin indicated, however, that the rate of fusion between inulin and secondary lysosomes was reduced. The incorporation of lipid material also produced a rapid though transient increase in several acid hydrolase activities. The stimulus for increased enzyme activity does not appear to be the deposition of ingested lipid within the lysosomes but rather at some stage prior to this, possibly the formation of endocytic vesicles. The current findings suggest that the intralysosomal incorporation of lipid material which occurs in several pathological conditions has marked effects on lysosomal function.     

Biochemical mechanisms of aminoglycoside cell toxicity. I. The uptake of gentamicin by cultured skin fibroblasts and the alteration of lysosomal enzyme activities

In order to study the toxicity of aminoglycoside, human skin fibroblasts were used as a model for basic studies, since they are known to have a specific aminoglycoside-binding site and to translocate the drug into the cells. Following the exposure of fibroblasts to gentamicin for 3 days, the cells formed many osmiophilic lamellar materials (myeloid bodies) in the lysosomes, while the other cellular structures appeared to remain normal. Although gentamicin was intensively accumulated within the lysosomes, intralysosomal pH, determined by the fluorescence intensity ratio method using fluorescein-isothiocyanate-labeled dextran, did not alter. Among the lysosomal enzymes, the activities of six different glycosidases were unchanged. On the other hand, sphingomyelinase and acid lipase activities were greatly decreased, while phospholipase A activity was increased. These results indicate that the lipid metabolism of fibroblasts is altered by gentamicin treatment, and that perturbation of intralysosomal pH can not be the cause of the changes observed in cell lysosomal enzyme activities.     

Apparently normal extracellular acidic alpha-mannosidase in fibroblast cultures from patients with mannosidosis.

Fibroblasts from patients with mannosidosis, cultured in medium supplemented with fetal calf serum from which acidic alpha-mannosidase (alpha-D-mannoside mannohydrolase, E.C.3.2.1.24) has been removed, secreted a normal amount of apparently unaffected acidic alpha-mannosidase into fetal calf serum-free medium. Both the intracellular and extracellular acidic alpha-mannosidase activities were completely precipitated by antiserum to placenta alpha-mannosidase B. In contrast to the heat-lability of the intracellular acidic alpha-mannosidase and its low affinity for artificial mannoside substrate, the extracellular enzyme exhibited both normal thermostability and normal kinetics. Mixing experiments with the intercellular enzymes suggested that the decreased activity in the patients' fibroblasts is not the effect of an inhibitor or absence of an activator. However, incubation of the mannosidosis extracellular enzyme with either normal or patient cell lysate resulted in a partial loss of activity, whereas an additive value was observed with the normal extracellular enzyme. In contrast to normal culture medium, the medium from mannosidosis cell culture was unable to enhance the rate of reduction of intracellular radioactivity in mucolipidosis type II fibroblasts precultured in the presence of radiolabeled mannose. These findings suggest that the defect in mannosidosis is expressed only after the enzyme has been delivered to lysosomes and presumably undergone some form of processing there.     

Production in yeast of alpha-galactosidase A,a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease

A mammalian-like sugar moiety was created in glycoprotein by Saccharomyces cerevisiae in combination with bacterial alpha-mannosidase to produce a more economic enzyme replacement therapy for patients with Fabry disease. We introduced the human alpha-galactosidase A (alpha-GalA) gene into an S. cerevisiae mutant that was deficient in the outer chains of N-linked mannan. The recombinant alpha-GalA contained both neutral (Man(8)GlcNAc(2)) and acidic ([Man-P](1-2)Man(8)GlcNAc(2)) sugar chains. Because an efficient incorporation of alpha-GalA into lysosomes of human cells requires mannose-6-phosphate (Man-6-P) residues that should be recognized by the specific receptor, we trimmed down the sugar chains of the alpha-GalA by a newly isolated bacterial alpha-mannosidase. Treatment of the alpha-GalA with the alpha-mannosidase resulted in the exposure of a Man-6-P residue on a nonreduced end of oligosaccharide chains after the removal of phosphodiester-linked nonreduced-end mannose. The treated alpha-GalA was efficiently incorporated into fibroblasts derived from patients with Fabry disease. The uptake was three to four times higher than that of the nontreated alpha-GalA and was inhibited by the addition of 5 mM Man-6-P. Incorporated alpha-GalA was targeted to the lysosome, and hydrolyzed ceramide trihexoside accumulated in the Fabry fibroblasts after 5 days. This method provides an effective and economic therapy for many lysosomal disorders, including Fabry disease.     

Cell contact induces the synthesis of a lysosomal enzyme precursor in lymphocytes and its direct transfer to fibroblasts

The activity of a lysosomal enzyme, alpha-D-mannosidase (EC 3.2.1.24), increased markedly in normal lymphocytes when they were cultured together with fibroblasts from a patient with an inherited deficiency of this enzyme. Cell-to-cell contact was obligatory for this increase in activity, which also required new protein synthesis. The enzyme induced in the co-cultured lymphocytes was a high molecular weight form of alpha-D-mannosidase that was not detected in lymphocytes cultured alone, which had only the low molecular weight mature enzyme. It was this precursor form alone that was directly transferred to the mannosidosis fibroblasts, where it was present initially in organelles of low density. When the culture period was extended the lymphocyte precursor enzyme was transported to the heavy lysosomes in the recipient cells, and correctly processed to the functionally effective mature enzyme.     

Synthesis of lysosomal alpha-mannosidase in normal and mannosidosis fibroblasts

The biosynthesis and secretion of lysosomal alpha-mannosidase was studied in metabolically labelled fibroblasts from controls and two patients with mannosidosis. Normal fibroblasts secrete alpha-mannosidase as a 110kDa polypeptide. Intracellularly alpha-mannosidase is represented by several polypeptides with apparent Mrs ranging from 40 to 67kDa. In two mannosidosis cell lines none of intra- and extracellular polypeptides of alpha-mannosidase were detectable. The mannosidosis fibroblasts secreted acid alpha-mannosidase activity at one third of the normal rate. In contrast to normal cells the secretion was not enhanced by NH4C1 and the secreted activity was not immunoprecipitable, indicating that the acid alpha-mannosidase activity secreted by mannosidosis fibroblasts is not related to the lysosomal alpha-mannosidase.     

Intralysosomal iron: a major determinant of oxidant-induced cell death

As a result of continuous digestion of iron-containing metalloproteins, the lysosomes within normal cells contain a pool of labile, redox-active, low-molecular-weight iron, which may make these organelles particularly susceptible to oxidative damage. Oxidant-mediated destabilization of lysosomal membranes with release of hydrolytic enzymes into the cell cytoplasm can lead to a cascade of events eventuating in cell death (either apoptotic or necrotic depending on the magnitude of the insult). To assess the importance of the intralysosomal pool of redox-active iron, we have temporarily blocked lysosomal digestion by exposing cells to the lysosomotropic alkalinizing agent, ammonium chloride (NH(4)Cl). The consequent increase in lysosomal pH (from ca. 4.5 to > 6) inhibits intralysosomal proteolysis and, hence, the continuous flow of reactive iron into this pool. Preincubation of J774 cells with 10 mM NH(4)Cl for 4 h dramatically decreased apoptotic death caused by subsequent exposure to H(2)O(2), and the protection was as great as that afforded by the powerful iron chelator, desferrioxamine (which probably localizes predominantly in the lysosomal compartment). Sulfide-silver cytochemical detection of iron revealed a pronounced decrease in lysosomal content of redox-active iron after NH(4)Cl exposure, probably due to diminished intralysosomal digestion of iron-containing material coupled with continuing iron export from this organelle. Electron paramagnetic resonance experiments revealed that hydroxyl radical formation, readily detectable in control cells following H(2)O(2) addition, was absent in cells preexposed to 10 mM NH(4)Cl. Thus, the major pool of redox-active, low-molecular-weight iron may be located within the lysosomes. In a number of clinical situations, pharmacologic strategies that minimize the amount or reactivity of intralysosomal iron should be effective in preventing oxidant-induced cell death.     

Degradation of mucopolysaccharide in intact isolated lysosomes

The function of isolated lysosomes was studied by measuring mucopolysaccharide degradation. Cultured human diploid skin fibroblasts were grown in medium containing H235SO4 to label endogenous mucopolysaccharide. Lysosome containing preparations at various stages of purity were isolated from disrupted cells. These preparations degraded mucopolysaccharide as indicated by the release of radioactive sulfate. Degradation was temperature-dependent, required intact lysosomes, and was optimal when incubation was carried out at neutral pH in a buffer of low ionic strength. Lysosomes from Hurler fibroblasts were unable to carry out the degradative process. ATP at 0.5 mM was found to stimulate both the rate and the extent of mucopolysaccharide degradation; GTP, UTP, and CTP had similar effects, whereas the noncleavable ATP analog adenosine 5'-(beta gamma-imido)triphosphate gave no stimulation. The ATP stimulation was inhibited by nigericin. ATP also stimulated chloroquine accumulation in lysosomes, the magnitude of which was used to measure the change in intralysosomal pH. The presence of ATP was associated with acidification of lysosome pH by 0.23 units. Acetyl coenzyme A was also found to stimulate lysosome function. This reagent, however, had no effect on chloroquine accumulation and thus appears to stimulate mucopolysaccharide degradation by a mechanism different than that caused by ATP.     

Gangliosides are Transported from the Plasma Membrane to Intralysosomal Membranes as Revealed by Immuno-Electron Microscopy

A biotin-labeled derivative of the ganglioside GM1 (biotin-GM1) was used to study its transport along the endocytic pathway of cultured fibroblasts by immuno-electron microscopy. Using electron dense endocytic tracers we could demonstrate that late endosomes and lysosomes of these cells are long living organelles with a high content of internal membranes. Our studies show that during endocytosis the biotin-GM1 was transported to these intraendosomal and intralysosomal membranes. These observations support the hypothesis that glycosphingolipids (GSL) are preferentially degraded in intralysosomal vesicles.     

Disulphide reduction in lysosomes. The role of cysteine.

Published evidence indicates that cystine-containing proteins have their disulphide bonds reduced during proteolysis in lysosomes. However, the intralysosomal accumulation of cystine in the cells of patients with cystinosis has been seen as evidence that protein cystine residues are not reduced. The data are reconcilable and fully in harmony if it is postulated that cysteine from the cytoplasm is the physiological reducing agent.