Mechanism of cystine reaccumulation by cystinotic fibroblastsin vitro

It is well established that when cystine-depleted cystinotic cells are cultured in cystine-containing medium, they reaccumulate cystine within their lysosomes more rapidly than when cultured in cystine-free medium. This has been a puzzling result, since the lysosome membrane of cystinotic cells is impermeable to cystine. To probe the mechanism of cystine reaccumulation, we have measured reaccumulation in the presence of colchicine, an inhibitor of pinocytosis, or of glutamate, a competitive inhibitor of cystine transport into human fibroblasts. Colchicine had no effect, thus eliminating pinocytosis as a putative mechanism for cystine translocation from the culture medium to the lysosomes. Glutamate, however, strongly inhibited cystine reaccumulation. It is concluded that the true mechanism is as follows. 1. Exogenous cystine crosses the plasma membrane on the cystine-glutamate porter. 2. Cystine is reduced in the cytoplasm by GSH. 3. The cysteine that is generated enters the lysosome, where it becomes cystine by participating in the reduction of cystine residues during intralysosomal proteolysis, or by autoxidation.     

Lysosomal cystine transport. Effect of intralysosomal pH and membrane potential

The regulation of lysosomal cystine transport was studied using cystine dimethyl ester-loaded lysosomes isolated from human diploid fibroblasts. Net efflux from normal fibroblast lysosomes was compared to that from lysosomes of cystinotic fibroblasts, which contain an inherited mutation decreasing lysosomal cystine transport. This exodus of cystine from normal fibroblast lysosomes was greater than from cystinotic fibroblast lysosomes. When lysosomes were incubated with both 5 mM MgCl2 and 2 mM ATP (Mg/ATP), the amount of lysosomal cystine lost from normal lysosomes doubled, but the amount of cystine lost from cystinotic lysosomes remained small. This effect of Mg/ATP on cystine loss from lysosomes isolated from normal fibroblasts was abolished when either carbonyl cyanide m-chlorophenylhydrazone or N-ethylmaleimide was present, suggesting that the effect of Mg/ATP was mediated by the action of a lysosomal proton-translocating ATPase. Addition of KCl, RbCl, or NaCl to normal lysosomes caused smaller increases in cystine exodus. A variety of experimental conditions altered lysosomal pH, membrane potential, and the cystine lost from normal fibroblast lysosomes. These same experimental conditions produced similar alterations in the lysosomal pH and membrane potential of cystinotic fibroblast lysosomes without a comparable alteration in cystine loss. These results have led us to propose a model in which the transport of cystine out of the normal lysosome is regulated by both the lysosomal membrane potential gradient and the transmembrane pH gradient.     

The effects of decreased growth temperature on the cystine content of cystinotic fibroblasts

Cystinotic fibroblasts transferred from 37 degrees C to 28 degrees C accumulated additional cystine over the period from 4 to 7 days of incubation at 28 degrees C, after which the additional cystine was lost; warming (to 37 degrees C) of cells with elevated cystine stores led to rapid cystine loss. These results, taken together with previously published data showing cystine release from cystinotic fibroblasts incubated at above-normal temperature, are interpreted as indicating the presence in the cystinotic fibroblast lysosome membrane of a cystine-porter whose efficacy is increased by an increase in membrane fluidity. This porter may be the residual activity of the cystine porter that is known to be deficient in cystinosis, or it may be a second as yet unrecognized porter. It is further proposed that this porter is responsible for the presumed efflux of cystine from cystinotic lysosomes.     

Uptake of cystine by cystine-depleted fibroblasts from patients with cystinosis

[³⁵S]L-cystine uptake was measured in cultured skin fibroblasts from patients with nephropathic cystinosis, pretreated with cysteamine to deplete their cystine pools. The uptake was greater in the cystinotic cells than in normal cells. The data suggest that the enhanced [³⁵S]-cystine uptake observed in cystinotic cells is not a consequence of disulfide exchange with stored cystine and may be related to the underlying abnormality in this enigmatic disorder.     

Exchange of cystine and glutamate across plasma membrane of human fibroblasts

It is found that both the inward and outward transport of cystine and glutamate through the plasma membrane of cultured human fibroblasts is mediated mostly by a single transport system. Cystine and glutamate at one side of the membrane stimulate the passage of these amino acids present at the other side of the membrane. When the concentration of intracellular glutamate is reduced to near zero, cystine hardly enters the cell, and likewise the release of glutamate from the cell ceases when cystine is absent in the medium. Homocysteate and alpha-aminoadipate share this transport system and, when added, similarly participate in the transport process. Since the intracellular pool of cystine is negligibly small whereas that of glutamate is very large, the physiologic flows via this system are the entry of cystine and the exodus of glutamate coupled together. Measurements of the rate of uptake of cystine into the cells and the rate of release of glutamate from the cells indicate that the entry of cystine and the exodus of glutamate occur at a ratio close to 1:1. Since cystine is known to behave as an anionic form in this transport, it is concluded that the transport system for cystine and glutamate in plasma membrane of human fibroblasts is a kind of an anion-exchanging agency.     

Impaired clearance of free cystine from lysosome-enriched granular fractions of I-cell-disease fibroblasts.

Cultured fibroblasts from patients with I-cell disease (mucolipidosis II) accumulate excessive amounts of free cystine, similarly to cells from patients with nephropathic cystinosis, a disorder of lysosomal cystine transport. To clarify whether the intralysosomal accumulation of cystine in I-cell-disease fibroblasts was due to a defective disposal mechanism, we measured the rates of clearance of free [35S]cystine from intact normal, cystinotic and I-cell-disease fibroblasts. Loss of radioactivity from the two mutant cell types occurred slowly (t 1/2 = 500 min) compared with the rapid loss from normal cells (t 1/2 = 40 min). Lysosome-rich granular fractions isolated from three different cystine-loaded normal, cystinotic and I-cell-disease fibroblast strains were similarly examined for non-radioactive cystine egress. Normal granular fractions lost cystine rapidly (mean t 1/2 = 43 min), whereas cystinotic granular fractions did not lose any cystine (mean t 1/2 = infinity). I-cell-disease granular fractions displayed prolonged half-times for cystine disposal (mean = 108 min), suggesting that I-cell-disease fibroblasts, like cystinotic cells, possess a defective carrier mechanism for cystine transport.     

Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions.

Normal leucocyte lysosome-rich granular fractions exhibited counter-transport of cystine, confirming that cystine transport across the lysosomal membrane is carrier-mediated. The trans-activation of cystine transport was temperature-dependent but relatively independent of the external Na+ or K+ concentration in phosphate buffer. Counter-transport, measured as uptake of exogenous [3H]cystine, increased with increasing intralysosomal cystine content up to approx. 3 nmol of half-cystine/unit of hexosaminidase activity. The amount of [3H]cystine entering lysosomes loaded with unlabelled cystine decreased when unlabelled cystine was added to the extralysosomal medium. Lysosomal cystine counter-transport was stereospecific for the L-isomer. Cystathionine, cystamine and cysteamine-cysteine mixed disulphide gave evidence of sharing the lysosomal cystine-transport system, although at lower activity than cystine. Other tested amino acids, including arginine, glutamate and homocystine, were inactive in this system. Nine leucocyte lysosome-rich preparations from eight different cystinotic patients displayed virtually no counter-transport of cystine, conclusively establishing that a carrier-mediated system for cystine transport is dysfunctional in cystinotic lysosomes.     

Comparative study of cystine clearance in cystinotic and I-cell fibroblasts upon exposure to cystine dimethyl ester

I-cell fibroblasts can accumulate cystine at levels comparable to those seen in homozygous cystinotic fibroblasts. Cystine accumulation in cystinosis is accounted for cystine clearance defect in situ. To unravel the question whether the same clearance defect or two different mechanisms cause cystine accumulation in I-cell disease, we used the cystine loading technique upon exposure of skin fibroblasts to radioactive cystine dimethyl ester. Normal, cystinotic and I-cell fibroblasts were exposed to radioactive cystine dimethyl ester, and the clearance of the generated radioactive cystine was measured. Cystinotic cells showed a marked defect in cystine clearance in situ, as compared to normal fibroblasts. In I-cell fibroblasts, we observed slow hydrolysis of cystine dimethyl ester to cystine, indicating low esterase activity, but no defect in clearance of the generated cystine. Cysteine production from the exogenous cystine dimethyl ester, presumably by cytoplasmic hydrolysis of the generated cystine, is normal in I-cell fibroblasts. Thus, our results indicate that, unlike cystinosis, there is no cystine clearance defect in situ for cystine in I-cell disease, and probably unrelated mechanisms cause cystine storage in cystinosis and I-cell disease.     

The effect of chloroquine on the metabolism of [35S]cystine in normal and cystinotic human skin fibroblasts.

The present study concerns the effect of the lysosomotropic drug chloroquine on the uptake and metabolism of [35S]cystine in vitro by normal human fibroblasts and those from patients suffering from the lysosomal storage disease cystinosis. When the cells were cultured with [35S]cystine for periods in excess of 4 h, it was found that chloroquine considerably increased (up to 30-fold) the labelling of the intracellular cystine pool in cystinotic cells, with no increase or a much smaller increase in normal cells. For this effect chloroquine had an optimum concentration of 20 microM, with a small effect still being noticeable at 1 microM. A quinoline analogue, 4-(dimethylaminoethylamino)-7-iodoquinoline, had a similar effect to chloroquine. However, NH4Cl at concentrations of between 100 microM and 50 mM showed either no effect (at the lower concentrations) or a depression of intracellular cystine labelling (at the higher concentrations). The differences between the effects of the quinolines on cystinotic acid normal cells were not due to differences in total cell uptake of drug.     

Stem cell microvesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro

Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100-400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNS(Red) transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.     

Detection and characterization of carrier-mediated cationic amino acid transport in lysosomes of normal and cystinotic human fibroblasts. Role in therapeutic cystine removal?

The discovery of a trans-stimulation property associated with lysine exodus from lysosomes of human fibroblasts has enabled us to characterize a system mediating the transport of cationic amino acids across the lysosomal membrane of human fibroblasts. The cationic amino acids arginine, lysine, ornithine, diaminobutyrate, histidine, 2-aminoethylcysteine, and the mixed disulfide of cysteine and cysteamine all caused trans-stimulation of the exodus of radiolabeled lysine from the lysosomal fraction of human fibroblasts at pH 6.5. In contrast, neutral and acidic amino acids did not affect the rate of lysine exodus. trans-Stimulation of lysine exodus was observed over the pH range from 5.5 to 7.6, was specific for the L-isomer of the cationic amino acid, and was intolerant to methylation of the alpha-amino group of the amino acid. The lysosomotropic amine, chloroquine, greatly retarded lysine exodus, whereas the presence of sodium ion was without effect. The specificity and lack of Na+ dependence of this lysosomal transport system is similar to that of System y+ present on the plasma membrane of human fibroblasts. In addition, we find cystine exodus from the lysosomal fraction of cystinotic human fibroblasts to be greatly retarded as compared to that of normal human fibroblasts with half-times of exodus similar to those reported for the lysosomes of cystinotic and normal human leukocytes (Gahl, W. A., Tietze, F., Bashan, N., Steinherz, R., and Schulman, J. D. (1982) J. Biol. Chem. 257, 9570-9575). In contrast, normal and cystinotic human fibroblasts did not show any differences with regard to lysine efflux or its trans-stimulation by cationic amino acids. An important mechanism by which cysteamine treatment of cystinosis allows cystine escape from lysosomes may be the ability of the mixed disulfide of cysteine and cysteamine formed by sulfhydryl-disulfide exchange to migrate by this newly discovered system mediating cationic amino acid transport.     

Proton-translocating ATPase and lysosomal cystine transport

A proton-translocating ATPase was identified in highly purified lysosomes from Epstein-Barr virus-transformed human lymphoblasts. Activity of this ATPase caused acidification of highly purified, fluorescein isothiocyanate dextran-loaded lysosomes and correlated with the ATP-dependent efflux of lysosomal cystine. The lysosomal ATPase was distinct from mitochondrial F1-ATPase in its responses to a variety of inhibitors. Although ATP-dependent lysosomal cystine efflux is not demonstrable in cultured lymphoblasts from individuals with nephropathic cystinosis, ATPase activity and acidification in lysosomes from these cells is comparable to that in noncystinotic lysosomes. ATPase activity in lymphoblasts from normal individuals was 543 +/- 79 nmol/mg/min while in lymphoblasts from cystinotic individuals this activity was 541 +/- 25 nmol/mg/min. ATP-dependent acidification of lysosomes from normals was -0.5 +/- 0.1 pH units compared to -0.5 +/- 0.1 pH units in cystinotic lysosomes. Activity of the lysosomal proton-translocating ATPase is a necessary, but not sufficient, condition for lysosomal cystine efflux.     

Important differences in cationic amino acid transport by lysosomal system c and system y+ of the human fibroblast

Superficial similarities led us to extend our designation for the transport of the plasma membrane for cationic amino acids, y+, to the lysosomal system also serving for such amino acids. Further study on the purified lysosomes of human skin fibroblasts leads us now to redesignate the lysosomal system as c (for cationic), rather than y+, to emphasize important contrasts. Lysosomal uptake of arginine at pH 7.0 was linear during the first 2 min, but attained a steady state in 6 min. This arginine uptake was Na+-independent and was tripled in rate when the lysosomes had first been loaded with the cationic amino acid analog, S-2-aminoethyl-L-cysteine. Uptake was slowed to one-third when 2 mM MgATP was added to the incubation mixture. The following differences in cationic amino acid influx between lysosomal System c and the plasma membrane System y+ became apparent: 1) arginine influx is increased 10-fold by raising the external pH from 5.0 to 7.0. This effect favors net entry of cationic amino acids under the H+ gradient prevailing in vivo. In contrast, arginine uptake across the plasma membrane is insensitive to pH changes in this range. 2) The Km of arginine uptake by lysosomal System c, 0.32 mM, is eight times that for System y+ arginine uptake by the fibroblast. 3) Certain neutral amino acids in the presence of Na+ are accepted as surrogate substrates by System y+, but not by lysosomal system c. 4) Cationic amino acids in which the alpha-amino group is monomethylated or the distal amino group is quaternary, also D-arginine, are recognized by lysosomal System c, whereas System y+ has little affinity for these analogs. This broader substrate specificity of lysosomal system c led us to discover that thiocholine serves to deplete accumulated cystine from cystinotic fibroblasts as effectively as does the therapeutic agent, cysteamine. The quaternary nitrogen of thiocholine renders the mixed disulfide formed when it reacts with cystine unsatisfactory as a substrate for System y+.     

Role of proton dissociation in the transport of cystine and glutamate in human diploid fibroblasts in culture

The effect of extracellular pH on the transport interaction of cystine and glutamate in cultured human diploid cells was examined over the pH range of 5.8-8.0. The initial rates of uptake of cystine increased with an increase in pH and glutamate potently inhibited the cystine uptake independently of pH. The uptake of glutamate was almost invariable within the pH range, but it was inhibited by cystine in a pH-dependent manner; the inhibition increased with an increase in pH. Regardless of pH, the uptake of cystine and glutamate was strongly inhibited by alpha-aminoadipate, alpha-aminopimelate, and homocysteate. From the pK values of cystine and other amino acids, it is suggested that cystine is transported in the same ionic form as is glutamate.     

Cystine and dibasic amino acid uptake by opossum kidney cells

The characteristics of the uptake of L-cystine by the continuous opossum kidney cell line, OK, were examined. Uptake of cystine is rapid and, in contrast to other continuous cultured cell lines, these cells retain the cystine/dibasic amino acid transport system which is found in vivo and in freshly isolated kidney tissue. Confluent monolayers of cells also fail to show the presence of the cystine/glutamate transport system present in LLC-PK1 cells, fibroblasts, and cultured hepatocytes. Uptake of cystine occurs via a high-affinity saturable process which is independent of medium sodium concentration. The predominant site of cystine transport is across the apical cell membrane. The intracellular concentration of GSH far exceeds that of cystine with a ratio greater than 100:1 for GSH:cysteine. Incubation of cells for 5 minutes with a physiological level of labelled cystine resulted in the labelling of 66% and 5% of the total intracellular cysteine and glutathione, respectively. The ability of these cells to reflect the shared cystine/dibasic amino acid transport system makes them a suitable model for investigation of the cystine carrier which is altered in human cystinuria.     

Abnormal mitochondrial autophagy in nephropathic cystinosis

Cystinosis, which is characterized by lysosomal accumulation of cystine in many tissues, was the first known storage disorder caused by defective metabolite export from the lysosome. The molecular and cellular mechanisms underlying nephropathic cystinosis, the most severe form, which exhibits generalized proximal tubular dysfunction and progressive renal failure, remain largely unknown. We used renal proximal tubular epithelial (RPTE) cells and fibroblasts from patients with three clinical variants of cystinosis: nephropathic, intermediate and ocular to explore the specific injury mechanism in nephropathic cystinosis. We demonstrate enhanced autophagy of mitochondria, increase in apoptosis and mitochondrial dysfunction in the nephropathic cystinosis phenotype. Furthermore, specific inhibition of autophagy results in significant attenuation of cell death in nephropathic cystinosis. This study provides ultrastructural and functional evidence of abnormal mitochondrial autophagy in nephropathic cystinosis, which may contribute to renal Fanconi syndrome and progressive renal injury.Key words: cystinosis, autophagy, mitochondria, kidney, lysosome, apoptosis, cell death, mitophagyCystinosis is an autosomal recessive metabolic disorder caused by mutations in the CTNS gene, which encodes a 7-transmembrane domain protein, cystinosin, a lysosomal cystine transporter. Cystinosis belongs to the family of lysosomal storage disorders (LSDs) characterized by the tissue accumulation of cystine crystals leading to multiple organ dysfunction. The three types of cystinosis, i.e., nephropathic (classic renal and systemic disease), intermediate (a late-onset variant of nephropathic cystinosis) and non-nephropathic (clinically affecting only the cornea) are allelic disorders caused by CTNS mutations. Children affected with nephropathic cystinosis present with the Fanconi syndrome and usually develop progressive renal failure within the first decade of life. The mechanism linking lysosomal cystine storage to pathological manifestations, in particular to the prominent proximal tubular defect and renal injury, remains unclear. Renal injury in nephropathic cystinosis may not simply be caused just by cystine accumulation, as disruption of the ctns gene in mice induces cystine storage in many tissues but does not result in signs of tubulopathy or renal failure; renal injury is not seen in other human forms of cystinosis and progressive renal injury occurs despite cystine depletion therapy.The purpose of our study was to investigate the specific mechanism leading to tubulopathy and end stage renal injury in nephropathic cystinosis. We used primary fibroblast and renal proximal tubular epithelial (RPTE) cells derived from patients with three clinical phenotypes of cystinosis. Our data show an abnormal increase in macroautophagy (hereafter referred to as autophagy), specific to the nephropathic variant of cystinosis. We also demonstrate that specific inhibition of autophagy rescues cell death in nephropathic cystinotic RPTE cells. Our results indicate that mitochondrial autophagy may be a critical mechanism contributing to renal Fanconi syndrome and progressive renal injury in nephropathic cystinosis.Abnormal autophagy was also recently observed in other types of lysosomal storage diseases (LSD). However, our study provides the first evidence supporting the extensive involvement of autophagy in nephropathic cystinosis pathogenesis. Abundant vacuolization and abnormal mitochondria are detected by electron microscopy (EM) in nephropathic cystinotic cells. Additionally, elevated levels of LC3-II and Beclin 1 are also observed in nephropathic cystinotic RPTE cells, indicating a role of Beclin 1-mediated autophagy in cystinosis. These results altogether establish an abnormal increase in autophagy in nephropathic cystinotic cells.Renal biopsies from patients with nephropathic cystinosis can reveal abnormally large mitochondria, but the relevance of this finding and other ultrastructural abnormalities is unclear. Our study further demonstrates a significant decrease in mitochondrial ATP generation with an increase in reactive oxygen species (ROS) in cystinotic cells. To further dissect the association of abnormal mitochondria with increased autophagy in cystinosis, we carefully examined the electron micrographs at higher magnifications. We discovered various stages of degradation of mitochondria by autophagy (hereafter referred to as mitophagy). To further validate mitophagy in cystinosis, we used an immunofluorescence (IF) approach to capture colocalization images of LC3, LAMP-2 (lysosomal marker) and ATP5H (mitochondrial marker). Intriguingly, an increase in LAMP-2 perinuclear staining is detected by IF assay in cystinotic cells. This observation may also denote enhanced active autophagy as LAMP-2 is involved in lysosomal biogenesis and/or the fusion between autophagosomes and lysosomes. Alternatively, LAMP-2 accumulation could be a manifestation of retarded autophagic flux in cystinotic cells. A decreased ability of lysosomes to fuse with autophagosomes has been reported in various LSDs. However, the colocalization of LC3 and LAMP-2 in nephropathic cystinotic RPTE cells argues against this possibility. Nevertheless, the possibility of autophagic flux blockade after autophagosome-lysosome fusion leading to detrimental effects is yet to be investigated. Interestingly, previously published EM reports of the renal biopsies of patients with nephropathic cystinosis show only the nucleus and a thin rim of cytoplasm as remnants in a proximal tubular cell, while mitochondria and lysosomes completely disappear.Conventionally, autophagy has been suggested as a cytoprotective mechanism to ensure cell survival during starvation. In contrast, several forms of cell death have been associated with the appearance of autophagic vesicles. To gain insight into the role of autophagy as regards to cell death or cell survival in nephropathic cystinosis, we used 3-methyladenine (3MA), a specific inhibitor of autophagy and assayed cell viability and apoptosis in cystinotic cells. Increased apoptosis has been previously reported in cultured cystinotic fibroblasts and RPTE cells. Treatment with 3MA in cystinotic cells significantly rescues cell death, thus suggesting a synergistic role of apoptosis and autophagy in cystinosis.In conclusion, as illustrated in Figure 1, we speculate that there is a multifaceted impact of autophagy in nephropathic cystinosis as follows: (1) the mechanism linking autophagy to lysosomal cystine or apoptosis in cystinotic cells could potentially be related to lysosomal membrane permeabilization (LMP), proposed as an early step in apoptosis in cystinosis. We hypothesize that abnormal induction of autophagy besides providing more cargo to be digested in the lysosomes, leads to increased fusion of autophagosomes with cystine-laden lysosomes, rendering them more sensitive to membrane destabilization, and thus making them readily enter the apoptotic pathway; (2) the second most important question is the link between abnormal mitochondria and mitophagy in cystinosis. A decreased level of cytosolic glutathione in cystinotic cells is one of the known factors responsible for generating damaged mitochondria. Our data also indicate an impairment of complex I activity, an increase in ROS and a decrease in mitochondrial ATP generation in cystinotic cells. We hypothesize that the abnormal induction of autophagy leads to depletion of mitochondria, forcing cells to enter the ‘starvation mode,’ thereby leading to an uncontrolled autophagy and cell death; (3) the third key question yet to be answered is the link between autophagy and renal injury in nephropathic cystinosis. Skeletal muscles and neuronal tissues are the primary organs where autophagy is physiologically enhanced. Recently, it has been shown that mouse kidneys exert a high level of autophagy under basal conditions, influencing the susceptibility to glomerular disease and renal failure. Thus, we postulate an organ- and tissue-specific effect of abnormally induced autophagy in nephropathic cystinosis, causing severe injury to kidneys leading to loss of renal function, ultimately culminating in end-stage renal disease.Open in a separate windowFigure 1A schematic view of the interplay between autophagy, abnormal mitochondria and cell death in cystinosis. Abnormal induction of autophagy, typically mitophagy, forces cells into a starvation mode leading to cell death; and renders cystine-laden lysosomes sensitive to lysosomal membrane permeabilization (LMP) making it readily enter the apoptosis pathway. A potential block in autophagic flux, after autophagosome-lysosome fusion, remains to be elucidated. Preferential severe kidney damage in nephropathic cystinosis may be due to the tissue- and organ-specific injury effect of autophagy.The recent progress in autophagy research has increased the need for additional studies so that we can fully understand the underlying pathological mechanisms and the significance of the lysosomal cell death axis in lysosomal storage disorders.     

Upregulation of the Rab27a-Dependent Trafficking and Secretory Mechanisms Improves Lysosomal Transport,Alleviates Endoplasmic Reticulum Stress,and Reduces Lysosome Overload in Cystinosis

Cystinosis is a lysosomal storage disorder caused by the accumulation of the amino acid cystine due to genetic defects in the CTNS gene, which encodes cystinosin, the lysosomal cystine transporter. Although many cellular dysfunctions have been described in cystinosis, the mechanisms leading to these defects are not well understood. Here, we show that increased lysosomal overload induced by accumulated cystine leads to cellular abnormalities, including vesicular transport defects and increased endoplasmic reticulum (ER) stress, and that correction of lysosomal transport improves cellular function in cystinosis. We found that Rab27a was expressed in proximal tubular cells (PTCs) and partially colocalized with the lysosomal marker LAMP-1. The expression of Rab27a but not other small GTPases, including Rab3 and Rab7, was downregulated in kidneys from Ctns^−/− mice and in human PTCs from cystinotic patients. Using total internal reflection fluorescence microscopy, we found that lysosomal transport is impaired in Ctns^−/− cells. Ctns^−/− cells showed significant ER expansion and a marked increase in the unfolded protein response-induced chaperones Grp78 and Grp94. Upregulation of the Rab27a-dependent vesicular trafficking mechanisms rescued the defective lysosomal transport phenotype and reduced ER stress in cystinotic cells. Importantly, reconstitution of lysosomal transport mediated by Rab27a led to decreased lysosomal overload, manifested as reduced cystine cellular content. Our data suggest that upregulation of the Rab27a-dependent lysosomal trafficking and secretory pathways contributes to the correction of some of the cellular defects induced by lysosomal overload in cystinosis, including ER stress.     

Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells

Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.     

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.     

Cystinosin, the protein defective in cystinosis, is a H(+)-driven lysosomal cystine transporter.

Cystinosis is an inherited lysosomal storage disease characterized by defective transport of cystine out of lysosomes. However, the causative gene, CTNS, encodes a seven transmembrane domain lysosomal protein, cystinosin, unrelated to known transporters. To investigate the molecular function of cystinosin, the protein was redirected from lysosomes to the plasma membrane by deletion of its C-terminal GYDQL sorting motif (cystinosin-DeltaGYDQL), thereby exposing the intralysosomal side of cystinosin to the extracellular medium. COS cells expressing cystinosin-DeltaGYDQL selectively take up L-cystine from the extracellular medium at acidic pH. Disruption of the transmembrane pH gradient or incubation of the cells at neutral pH strongly inhibits the uptake. Cystinosin-DeltaGYDQL is directly involved in the observed cystine transport, since this activity is highly reduced when the GYDQL motif is restored and is abolished upon introduction of a point mutation inducing early-onset cystinosis. We conclude that cystinosin represents a novel H(+)-driven transporter that is responsible for cystine export from lysosomes, and propose that cystinosin homologues, such as mammalian SL15/Lec35 and Saccharomyces cerevisiae ERS1, may perform similar transport processes at other cellular membranes.