PEGylated derivatives of cystamine as enhanced treatments for nephropathic cystinosis

The genetic disease, nephropathic cystinosis is characterized by lysosomal accumulation of the amino acid cystine. Crystallization of cystine in affected organs, if untreated, results in mortality of the affected individuals by their middle to late teens. The only approved treatment for cystinosis is administration of cysteamine. However, cysteamine is associated with an offending odor and taste and this, coupled to a rapid first pass metabolism and a 6 h dosing regimen, suggest a clear need to improve the therapy. A number of PEGylated derivatives of cystamine, the disulfide counterpart of cysteamine, have been synthesised and evaluated in cultured cystinotic fibroblasts for toxicity and efficacy. All of the tested compounds were non-cytotoxic and displayed a remarkable depletion of intralysosomal cystine.     

Intralysosomal cystine accumulation in mice lacking cystinosin,the protein defective in cystinosis

Cystinosis is an autosomal recessive disorder characterized by an accumulation of intralysosomal cystine. The causative gene, CTNS, encodes cystinosin, a seven-transmembrane-domain protein, which we recently showed to be a lysosomal cystine transporter. The most severe and frequent form of cystinosis, the infantile form, appears around 6 to 12 months, with a proximal tubulopathy (de Toni-Debré-Fanconi syndrome) and ocular damage. End-stage renal failure is reached by 10 years of age. Accumulation of cystine in all tissues eventually leads to multisystemic disease. Treatment with cysteamine, which reduces the concentration of intracellular cystine, delays disease progression but has undesirable side effects. We report the first Ctns knockout mouse model generated using a promoter trap approach. We replaced the last four Ctns exons by an internal ribosome entry site-betagal-neo cassette and showed that the truncated protein was mislocalized and nonfunctional. Ctns(-/-) mice accumulated cystine in all organs tested, and cystine crystals, pathognomonic of cystinosis, were observed. Ctns(-/-) mice developed ocular changes similar to those observed in affected individuals, bone defects and behavioral anomalies. Interestingly, Ctns(-/-) mice did not develop signs of a proximal tubulopathy, or renal failure. A preliminary therapeutic trial using an oral administration of cysteamine was carried out and demonstrated the efficiency of this treatment for cystine clearance in Ctns(-/-) mice. This animal model will prove an invaluable and unique tool for testing emerging therapeutics for cystinosis.     

A potential new prodrug for the treatment of cystinosis: design, synthesis and in-vitro evaluation

Nephropathic cystinosis is a rare autosomal recessive disease characterised by raised lysosomal levels of cystine in the cells of most organs. The disorder is treated by regular administration of the aminothiol, cysteamine, an odiferous and unpleasant tasting compound that along with its metabolites is excreted in breath and sweat, leading to poor patient compliance. In an attempt to improve patient compliance a series of novel prodrugs has been designed and evaluated as a potential new treatment for nephropathic cystinosis. The first of the prodrugs tested, 3a, was found to decrease the levels of intracellular cystine in cystinotic fibroblasts. This is the first report of a potential new therapeutic treatment for nephropathic cystinosis since the advent of cysteamine bitartrate.     

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.     

Synthesis and in vitro evaluation of novel pro-drugs for the treatment of nephropathic cystinosis

As part of our continuing work to obtain new pro-drugs for the treatment of nephropathic cystinosis, a number of glutaric and succinic acid derivatives of cystamine have been designed, synthesised and biologically evaluated in vitro. These compounds have been designed as odourless and tasteless pro-drugs which will release multiple molecules of cysteamine upon administration. All of the synthesised compounds evaluated in this study were non-cytotoxic and displayed a greater ability than cysteamine to deplete the levels of cystine in cultured fibroblasts.     

Cysteamine prevents and reverses the inhibition of pyruvate kinase activity caused by cystine in rat heart

Cystinosis is a disorder associated with excessive lysosomal cystine accumulation secondary to defective cystine efflux. Patients affected by this disease develop a variable degree of symptoms depending on the involved tissues. Accumulation of cystine in myocardium may lead to heart failure. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that thiolic enzymes like pyruvate kinase (PK) may be altered by disulfides like cystine, the main objective of the present study was to investigate the effect of cystine on PK activity in the heart of developing rats. We performed kinetic studies and investigated the effects of reduced glutathione (GSH), a biologically occurring thiol groups protector, and cysteamine, the drug used for cystinosis treatment, on the enzyme activity. We observed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. We also observed that GSH and cysteamine fully prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits PK activity by oxidation of the sulfhydryl groups of the enzyme. Although there is no definite proof of cystine within cytoplasm, there is indirect proof t it is able to escape lysosomes and come in contact with PK. Considering that cysteamine is used in patients with cystinosis because it causes parenchymal organ cystine depletion, the present data provide a possible new effect for this drug.     

Cysteamine prevents and reverses the inhibition of creatine kinase activity caused by cystine in rat brain cortex

Cystinosis is a disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Cystine accumulation provokes a variable degree of symptoms depending on the involved tissues. Adult patients may present brain cortical atrophy. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that brain damage may be developed by energy deficiency, creatine kinase is a thiolic enzyme crucial for energy homeostasis, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on creatine kinase activity in total homogenate, cytosolic and mitochondrial fractions of the brain cortex from 21-day-old Wistar rats. We performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol group protector, and cysteamine, the drug used for cystinosis treatment, to better understand the effect of cystine on creatine kinase activity. Results showed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. GSH partially prevented and reversed CK inhibition caused by cystine and cysteamine fully prevented and reversed this inhibition, suggesting that cystine inhibits creatine kinase activity by interaction with the sulfhydryl groups of the enzyme. Considering that creatine kinase is a crucial enzyme for brain cortex energy homeostasis, these results provide a possible mechanism for cystine toxicity and also a new possible beneficial effect for the use of cysteamine in cystinotic patients.     

Antioxidant Effect of Cysteamine in Brain Cortex of Young Rats

Cysteamine is a cystine-depleting drug used in the treatment of cystinosis, a metabolic disorder caused by deficiency of the lysosomal cystine carrier. As a result, cystine accumulates within lysosomes in many tissues and organs, including the nervous system. Studies with cystine dimethyl ester loaded cells suggest that cystine might induce apoptosis through oxidative stress. Our objective was to investigate the effects of co-administration of cysteamine with the oxidant cystine dimethyl ester on several parameters of oxidative stress in the brain cortex of rats. Animals were injected with 1.6 μmol/g cystine dimethyl ester and/or 0.26 μmol/g body weight cysteamine. Cystine dimethyl ester induced lipoperoxidation, protein carbonylation, and stimulated superoxide dismutase, glutathione peroxidase and catalase activities, probably through the formation of free radicals. Cysteamine prevented those effects, possibly increasing cellular thiol pool and acting as a scavenger of free radicals. These results suggest that the antioxidant effect of cysteamine may be important in the treatment of cystinosis.     

Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development

The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus-oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 0.3mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.     

Endo-Lysosomal Dysfunction in Human Proximal Tubular Epithelial Cells Deficient for Lysosomal Cystine Transporter Cystinosin

Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC) deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles.     

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.     

FISH diagnosis of the common 57-kb deletion in <Emphasis Type="Italic">CTNS</Emphasis> causing cystinosis

Cystinosis is an autosomal recessive lysosomal storage disease caused by mutations in CTNS. The most prevalent CTNS mutation, a 57-kb deletion, occurs in ~60% of patients in the United States and northern Europe and removes exons 1–9, most of exon 10, the CTNS promoter region, and all of an adjacent gene of unknown function called CARKL. CTNS codes for the lysosomal cystine transporter, whose absence leads to intracellular cystine accumulation, widespread cellular destruction, renal Fanconi syndrome in infancy, renal glomerular failure in later childhood, and other systemic complications. Because treatment with oral cysteamine can prevent or delay these complications significantly, early and accurate diagnosis is critical. This study describes the generation of fluorescence in situ hybridization (FISH) probes for the 57-kb deletion in CTNS, enabling cytogenetics laboratories to test for this common mutation. The probes would also be able to detect a less frequent 11.7-kb deletion. A blinded study was performed using multiplex PCR analysis as the gold standard to determine the presence or absence of the 57-kb deletion. The FISH probes, evaluated on 12 lymphoblastoid cell lines from singly deleted, doubly deleted, and nondeleted patients, made the correct diagnosis in every case. This appears to be the first FISH-based diagnostic method described for any lysosomal storage disorder. It can assist in the antenatal and perinatal diagnosis of cystinosis and promote earlier salutary therapy with cysteamine.     

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.     

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.     

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.     

Germline DNA copy number variation in individuals with Argyrophilic grain disease reveals CTNS as a plausible candidate gene

Argyrophilic grain disease (AGD) is a progressive neurodegenerative disease of the human brain that has never been associated to a particular gene locus. In the present study, we report the results of a CNV investigation in 29 individuals whose anatomopathologic investigation of the brain showed AGD. Rare CNVs were identified in six patients (21%), in particular a 40 kb deletion at 17p13.2 encompassing the CTNS gene. Homozygote mutations in CTNS are known to cause cystinosis, a disorder characterized by the intralysosomal accumulation of cystine in all tissues. We present the first CNV results in individuals presenting AGD and a possible candidate gene implicated in the disorder.     

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.     

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.     

Identification and Characterisation of the Murine Homologue of the Gene Responsible for Cystinosis, Ctns

Background  

Cystinosis is an autosomal recessive disorder characterised by an intralysosomal accumulation of cystine, and affected individuals progress to end-stage renal failure before the age of ten. The causative gene, CTNS, was cloned in 1998 and the encoded protein, cystinosin, was predicted to be a lysosomal membrane protein.     

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.