Lysosomal Cystine Transporter Research Articles

Investigation on Cross-correction of Cystinosis through Genetically Engineered Cells Secreting Cystinosin

Background: Cystinosis is a rare inherited lysosomal storage disease (LSD), caused by a mutation in the Cystinosin Lysosomal Cystine Transporter (CTNS). Novel therapies and strategies are needed to improve patients' clinical conditions and quality of life. Objectives and Methods: This study assessed whether CTNS can be secreted, and investigated a method to enhance its secretion, by adding a secretion signal to the N-terminus. Human Embry-onic Kidney (HEK) 293 cells were transfected with the resulting construct. The amount of protein secreted was then measured. Uptake by monolayer cultures of cystinotic cells and enzyme activity were also assessed. Results: The recombinant protein could effectively be secreted, and the secretion signal slightly further increased its secretion. The secreted recombinant protein was taken up by cystinotic cells, and, after internalization, still retained its biological activity. Conclusion: Optimization of the proposed method to increase the secretion of CTNS would pro-vide new insights into the production of recombinant proteins for medical and industrial use. Further identification and screening of alternative signalling peptides and cell types can maximise the secretion and production of recombinant CNTS, to be used as a therapeutic agent in human healthcare.

Omic Studies on In Vitro Cystinosis Model: siRNA-Mediated CTNS Gene Silencing in HK-2 Cells

Cystinosis is an autosomal recessive disease caused by mutations in the CTNS gene encoding a protein called cystinosine, which is a lysosomal cystine transporter. Disease-causing mutations lead to accumulation of cystine crystals in the lysosomes, thereby causing dysfunction of vital organs. Determination of the increased leukocyte cystine level is one of the most used methods for diagnosis. However, this method is expensive, difficult to perform, and may yield different results in different laboratories. In this study, a disease model was created with CTNS gene-silenced HK2 cells, which can mimic cystinosis in cell culture, and multiomics methods (ie, proteomics, metabolomics, and fluxomics) were implemented at this cell culture to investigate new biomarkers for the diagnosis. CTNS-silenced cell line exhibited distinct metabolic profiles compared with the control cell line. Pathway analysis highlighted significant alterations in various metabolic pathways, including alanine, aspartate, and glutamate metabolism; glutathione metabolism; aminoacyl-tRNA biosynthesis; arginine and proline metabolism; beta-alanine metabolism; ascorbate and aldarate metabolism; and histidine metabolism upon CTNS silencing. Fluxomics analysis revealed increased cycle rates of Krebs cycle intermediates such as fumarate, malate, and citrate, accompanied by enhanced activation of inorganic phosphate and ATP production. Furthermore, proteomic analysis unveiled differential expression levels of key proteins involved in crucial cellular processes. Notably, peptidyl-prolyl cis–trans isomerase A, translation elongation factor 1-beta (EF-1beta), and 60S acidic ribosomal protein decreased in CTNS-silenced cells. Additionally, levels of P0 and tubulin α-1A chain were reduced, whereas levels of 40S ribosomal protein S8 and Midasin increased. Overall, our study, through the utilization of an in vitro cystinosis model and comprehensive multiomics approach, led to the way toward the identification of potential new biomarkers while offering valuable insights into the pathogenesis of cystinosis.

Starry-like Cornea: A Case of Ocular Cystinosis

Ocular cystinosis is a rare autosomal recessive disorder caused by mutations in the CTNS gene, which encodes a lysosomal cystine transporter protein. This results in the accumulation of cystine crystals in various ocular structures, leading to a range of ocular manifestations. The incidence of cystinosis is estimated to be 1 in 100,000 to 200,000 live births, with a higher prevalence in certain populations such as those of European descent. We report the case of a 5-year-old child with ocular cystinosis. The ophthalmological examination revealed a photophobic child with a visual acuity of 3/10 in both eyes (Pigassou scale), and diffuse stromal crystal deposits over the entire cornea in both eyes. The rest of the examination was unremarkable. The patient was referred to pediatrics for work-up of storage disease and was diagnosed with ocular and nephrological cystinosis. The patient was able to start general treatment with Mercaptamine with improvement in renal function, but was unable to obtain local treatment due to lack of funds. The patient is still being followed in our clinic with stable corneal involvement. Ocular cystinosis is a very rare genetic disorder. There are three main types of cystinosis: nephropathic cystinosis and non-nephropathic cystinosis. Nephropathic cystinosis divides further on infantile and intermediate. The most common ocular manifestation of cystinosis is corneal cystine crystal deposit, which typically presents in the first year of life and can lead to photophobia, tearing, and decreased visual acuity. The corneal crystals can also cause recurrent erosions, which can be very painful. The severity of corneal involvement can range from mild punctate deposition to severe confluent crystal accumulation that can lead to corneal scarring and vision loss. Cysteamine drops, which are a form of cysteamine hydrochloride, can help dissolve the cystine crystals and improve corneal clarity, prevent further vision loss, and reduce the frequency of recurrent erosions. Early diagnosis and treatment are crucial in preventing further ocular damage in individuals with cystinosis. Regular ophthalmologic examinations should be conducted to monitor for ocular manifestations and initiate treatment as early as possible. A multidisciplinary approach is necessary, involving ophthalmologists, nephrologists, and other specialists, to manage the systemic manifestations of cystinosis.

Clinical and genetic characteristics of Tunisian children with infantile nephropathic cystinosis.

Nephropathic cystinosis is an autosomal recessive disease caused by a mutation in the CTNS gene which encodes cystinosin, a lysosomal cystine transporter. The spectrum of mutations in the CTNS gene is not well defined in the North African population. Here, we investigated twelve patients with nephropathic cystinosis belonging to eight Tunisian families in order to analyze the clinical and genetic characteristics of Tunisian children with infantile nephropathic cystinosis. Clinical data were collected retrospectively. Molecular analysis of the CTNS gene was performed by Sanger sequencing. We describe a new splicing mutation c.971-1G > C in the homozygous state in 6/12 patients which seems to be a founder mutation. The reported deletion of 23nt c.771_793 Del (p.Gly258Serfs*30) was detected in a homozygous state in one patient and in a heterozygous compound state with the c.971-1G > C mutation in 3/12 patients. Two of 12 patients have a deletion of exons 4 and 5 of the CTNS gene. None of our patients had the most common 57-kb deletion. The mutational spectrum in the Tunisian population is different from previously described populations. Thus, a molecular diagnostic strategy must be implemented in Tunisia, by targeting as a priority the common mutations described in this country. Such a strategy will allow a cost-effective diagnosis confirmation as well as early administration of treatment with oral cysteamine. A higher resolution version of the Graphical abstract is available as Supplementary information.

Deficiency of the sedoheptulose kinase (Shpk) does not alter the ability of hematopoietic stem cells to rescue cystinosis in the mouse model

Cystinosis is an autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin, and leading to multi-organ degeneration including kidney failure. A clinical trial for cystinosis is ongoing to test the safety and efficacy of transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) ex vivo gene-modified to introduce functional CTNS cDNA. Preclinical studies in Ctns−/− mice previously showed that a single HSPC transplantation led to significant tissue cystine decrease and long-term tissue preservation. The main mechanism of action involves the differentiation of the transplanted HSPCs into macrophages within tissues and transfer of cystinosin-bearing lysosomes to the diseased cells via tunneling nanotubes. However, a major concern was that the most common cystinosis-causing mutation in humans is a 57-kb deletion that eliminates not only CTNS but also the adjacent sedopheptulose kinase SHPK/CARKL gene encoding a metabolic enzyme that influences macrophage polarization. Here, we investigated if absence of Shpk could negatively impact the efficiency of transplanted HSPCs to differentiate into macrophages within tissues and then to prevent cystinosis rescue. We generated Shpk knockout mouse models and detected a phenotype consisting of perturbations in the pentose phosphate pathway (PPP), the metabolic shunt regulated by SHPK. Shpk−/− mice also recapitulated the urinary excretion of sedoheptulose and erythritol found in cystinosis patients homozygous for the 57-kb deletion. Transplantation of Shpk−/−-HSPCs into Ctns−/− mice resulted in significant reduction in tissue cystine load and restoration of Ctns expression, as well as improved kidney architecture comparable to WT-HSPC recipients. Altogether, these data demonstrate that absence of SHPK does not alter the ability of HSPCs to rescue cystinosis, and then patients homozygous for the 57-kb deletion should benefit from ex vivo gene therapy and can be enrolled in the ongoing clinical trial. However, because of the limits inherent to animal models, outcomes of this patient population will be carefully compared to the other enrolled subjects.

DYNC1LI2 regulates localization of the chaperone-mediated autophagy receptor LAMP2A and improves cellular homeostasis in cystinosis

ABSTRACT The dynein motor protein complex is required for retrograde transport but the functions of the intermediate-light chains that form the cargo-binding complex are not elucidated and the importance of individual subunits in maintaining cellular homeostasis is unknown. Here, using mRNA arrays and protein analysis, we show that the dynein subunit, DYNC1LI2 (dynein, cytoplasmic 1 light intermediate chain 2) is downregulated in cystinosis, a lysosomal storage disorder caused by genetic defects in CTNS (cystinosin, lysosomal cystine transporter). Reconstitution of DYNC1LI2 expression in ctns -/- cells reestablished endolysosomal dynamics. Defective vesicular trafficking in cystinotic cells was rescued by DYNC1LI2 expression which correlated with decreased endoplasmic reticulum stress manifested as decreased expression levels of the chaperone HSPA5/GRP78, and the transcription factors ATF4 and DDIT3/CHOP. Mitochondrial fragmentation, membrane potential and endolysosomal-mitochondrial association in cystinotic cells were rescued by DYNC1LI2. Survival of cystinotic cells to oxidative stress was increased by DYNC1LI2 reconstitution but not by its paralog DYNC1LI1, which also failed to decrease ER stress and mitochondrial fragmentation. DYNC1LI2 expression rescued the localization of the chaperone-mediated autophagy (CMA) receptor LAMP2A, CMA activity, cellular homeostasis and LRP2/megalin expression in cystinotic proximal tubule cells, the primary cell type affected in cystinosis. DYNC1LI2 failed to rescue phenotypes in cystinotic cells when LAMP2A was downregulated or when co-expressed with dominant negative (DN) RAB7 or DN-RAB11, which impaired LAMP2A trafficking. DYNC1LI2 emerges as a regulator of cellular homeostasis and potential target to repair underlying trafficking and CMA in cystinosis, a mechanism that is not restored by lysosomal cystine depletion therapies. Abbreviations: ACTB: actin, beta; ATF4: activating transcription factor 4; CMA: chaperone-mediated autophagy; DYNC1LI1: dynein cytoplasmic 1 light intermediate chain 1; DYNC1LI2: dynein cytoplasmic 1 light intermediate chain 2; ER: endoplasmic reticulum; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; LIC: light-intermediate chains; LRP2/Megalin: LDL receptor related protein 2; PTCs: proximal tubule cells; RAB: RAB, member RAS oncogene family; RAB11FIP3: RAB11 family interacting protein 3; RILP: Rab interacting lysosomal protein

Renal and Extra Renal Manifestations in Adult Zebrafish Model of Cystinosis.

Cystinosis is a rare, incurable, autosomal recessive disease caused by mutations in the CTNS gene. This gene encodes the lysosomal cystine transporter cystinosin, leading to lysosomal cystine accumulation in all cells of the body, with kidneys being the first affected organs. The current treatment with cysteamine decreases cystine accumulation, but does not reverse the proximal tubular dysfunction, glomerular injury or loss of renal function. In our previous study, we have developed a zebrafish model of cystinosis through a nonsense mutation in the CTNS gene and have shown that zebrafish larvae recapitulate the kidney phenotype described in humans. In the current study, we characterized the adult cystinosis zebrafish model and evaluated the long-term effects of the disease on kidney and extra renal organs through biochemical, histological, fertility and locomotor activity studies. We found that the adult cystinosis zebrafish presents cystine accumulation in various organs, altered kidney morphology, impaired skin pigmentation, decreased fertility, altered locomotor activity and ocular anomalies. Overall, our data indicate that the adult cystinosis zebrafish model reproduces several human phenotypes of cystinosis and may be useful for studying pathophysiology and long-term effects of novel therapies.

An international cohort study spanning five decades assessed outcomes of nephropathic cystinosis

Nephropathic cystinosis is a rare disease secondary to recessive mutations of the CTNS gene encoding the lysosomal cystine transporter cystinosin, causing accumulation of cystine in multiple organs. Over the years, the disease has evolved from being a fatal condition during early childhood into a treatable condition, with patients surviving into adulthood. Data on cystinosis are limited by the rarity of the disease. Here, we have investigated factors associated with kidney and growth outcome in a very large cohort of 453 patients born between 1964 and 2016 and followed in Belgium, Germany, Austria, France, Italy, Spain, The Netherlands, Turkey and United Kingdom. From the 1970s to the 1990s, the median increase in kidney survival was 9.1 years. During these years, cysteamine, a cystine-depleting agent, was introduced for the treatment of cystinosis. Significant risk factors associated with early progression to end-stage kidney disease assessed by Cox proportional multivariable analysis included delayed initiation of cysteamine therapy and higher mean leucocyte cystine levels. No significant effect on kidney function was observed for gender, pathogenic variant of the CTNS gene, and the prescription of indomethacin or renin angiotensin system blockers. Significantly improved linear growth was associated with early use of cysteamine and lower leukocyte cystine levels. Thus, our study provides strong evidence in favor of early diagnosis and optimization of cystine depletion therapy in nephropathic cystinosis.

Cell-Based Phenotypic Drug Screening Identifies Luteolin as Candidate Therapeutic for Nephropathic Cystinosis.

Mutations in the gene that encodes the lysosomal cystine transporter cystinosin cause the lysosomal storage disease cystinosis. Defective cystine transport leads to intralysosomal accumulation and crystallization of cystine. The most severe phenotype, nephropathic cystinosis, manifests during the first months of life, as renal Fanconi syndrome. The cystine-depleting agent cysteamine significantly delays symptoms, but it cannot prevent progression to ESKD and does not treat Fanconi syndrome. This suggests the involvement of pathways in nephropathic cystinosis that are unrelated to lysosomal cystine accumulation. Recent data indicate that one such potential pathway, lysosome-mediated degradation of autophagy cargoes, is compromised in cystinosis. To identify drugs that reduce levels of the autophagy-related protein p62/SQSTM1 in cystinotic proximal tubular epithelial cells, we performed a high-throughput screening on the basis of an in-cell ELISA assay. We then tested a promising candidate in cells derived from patients with, and mouse models of, cystinosis, and in preclinical studies in cystinotic zebrafish. Of 46 compounds identified as reducing p62/SQSTM1 levels in cystinotic cells, we selected luteolin on the basis of its efficacy, safety profile, and similarity to genistein, which we previously showed to ameliorate other lysosomal abnormalities of cystinotic cells. Our data show that luteolin improves the autophagy-lysosome degradative pathway, is a powerful antioxidant, and has antiapoptotic properties. Moreover, luteolin stimulates endocytosis and improves the expression of the endocytic receptor megalin. Our data show that luteolin improves defective pathways of cystinosis and has a good safety profile, and thus has potential as a treatment for nephropathic cystinosis and other renal lysosomal storage diseases.

Microvesicle delivery of a lysosomal transport protein to ex vivo rabbit cornea

Therapeutic use of transmembrane proteins is limited because of irreversible denaturation when away from their native lipid membrane. Mutations in lysosomal membrane transport proteins cause many lethal disorders including cystinosis which results from mutations in CTNS, which codes for the lysosomal cystine transport protein, cystinosin. Cystinosin-deficient fibroblasts, including keratocytes (corneal fibroblasts) accumulate lysosomal cystine. Cystinosis patients develop highly painful corneal cystine crystals, resulting in severe visually debilitating photophobia. The only available therapy is daily treatment with cysteamine eye drops. We have previously shown that microvesicles containing functional cystinosin are spontaneously produced by infecting Spodoptera frugiperda cells (Sf9) with baculovirus containing human wt CTNS. Infecting Sf9 cells for 3 days at a MOI of 1 yields 1011microvesicles /ml with a modal diameter of 90 nm. Addition of these vesicles to cultures of cystinotic fibroblasts produces cystine depletion over the course of 96 h, which persists for 2 weeks. In this paper we show that addition of such microvesicles containing cystinosinGFP to ex vivo rabbit ocular globes yields punctate perinuclear green fluorescence in the corneal keratocytes. These results support potential therapeutic use of these cystinosin containing microvesicles in treating cystinotic corneal keratopathy with the advantage of administering twice/month instead of daily topical administration.

Regulation of the chaperone‐mediated autophagy receptor LAMP2A by DYNC1LI2 in cystinosis

The dynein motor protein complex is required for retrograde transport but the specific functions of the intermediate and light chains that form the cargo‐binding complex are not elucidated and the importance of individual subunits in the maintenance of cellular homeostasis is unknown. 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. Increased levels of intra‐lysosomal cystine lead to cell malfunction, which is especially manifested in the kidney epithelial proximal tubule cells (PTCs). Many cellular defects in cystinosis are independent of lysosomal overload, suggesting that cystinosin regulates cellular processes independently of its role as a cystine transporter. We showed that cystinotic cells (Ctns−/−) are characterized by vesicular transport defects, increased endoplasmic reticulum (ER) stress and susceptibility to oxidative stress. Here, using a mRNA array and immunoblot assays, we show that the dynein subunit, intermediate chain 2 (DYNC1LI2) is downregulated in cystinotic cells. Reconstitution of the expression of DYNC1LI2 in Ctns−/− fibroblasts re‐established endolysosomal dynamics quantified by Total Internal Reflection Fluorescence microscopy (TIRFM). Rescue of vesicular trafficking by DYNC1LI2 correlated with decreased ER stress manifested as decreased levels of the chaperone Grp78 in cystinotic cells. Mitochondrial fragmentation in cystinotic fibroblasts was also reduced by DYNC1LI2 expression. Survival of cystinotic cells to oxidative stress insult was increased by DYNC1LI2 but not by its paralog DYNC1LI1, which also failed to reestablish endolysosomal trafficking, or to decrease ER stress levels and mitochondrial fragmentation. Cystinotic (CTNS‐KO) PTCs, generated using CRISPR‐Cas9, showed defective endolysosomal trafficking and decreased expression of the Rab‐GTPase Rab11 carrier of the chaperone‐mediated autophagy receptor LAMP2A and the endocytic receptor megalin, both mislocalized in cystinosis. Restoring DYNC1LI2 expression rescued the localization of LAMP2A and restored the expression of megalin at the plasma membrane of CTNS‐KO cells. Expression of GFP‐DYNC1LI2 failed to rescue phenotypes in cystinotic cells (Ctns−/−) when co‐expressed with dominant negative (DN) RAB7/RAB11 but not DN‐Rab5 and in correlation with this finding, TR‐FRET assays show that DYNC1LI2 associates with RILP, a RAB7 effector and Rab11‐FIP4, a Rab11 effector. The rescue of the increased ER stress phenotype by DYNC1LI2 in cystinotic cells was blocked by downregulation of LAMP2A expression. Our results suggest that DYNC1LI2 reconstitution improves cellular homeostasis in a LAMP2A‐dependent manner highlighting DYNC1LI2 as a possible target molecule to improve cellular function in cystinosis.Support or Funding InformationNIHCRF

Chaperone-Mediated Autophagy Upregulation Rescues Megalin Expression and Localization in Cystinotic Proximal Tubule Cells.

Cystinosis is a lysosomal storage disorder caused by defects in CTNS, the gene that encodes the lysosomal cystine transporter cystinosin. Patients with nephropathic cystinosis are characterized by endocrine defects, defective proximal tubule cell (PTC) function, the development of Fanconi syndrome and, eventually, end-stage renal disease. Kidney disease is developed despite the use of cysteamine, a drug that decreases lysosomal cystine overload but fails to correct overload-independent defects. Chaperone-mediated autophagy (CMA), a selective form of autophagy, is defective in cystinotic mouse fibroblasts, and treatment with cysteamine is unable to correct CMA defects in vivo, but whether the vesicular trafficking mechanisms that lead to defective CMA in cystinosis are manifested in human PTCs is not currently known and whether PTC-specific mechanisms are corrected upon CMA upregulation remains to be elucidated. Here, using CRISPR-Cas9 technology, we develop a new human PTC line with defective cystinosin expression (CTNS-KO PTCs). We show that the expression and localization of the CMA receptor, LAMP2A, is defective in CTNS-KO PTCs. The expression of the lipidated form of LC3B, a marker for another form of autophagy (macroautophagy), is decreased in CTNS-KO PTCs indicating decreased autophagosome numbers under basal conditions. However, the autophagic flux is functional, as measured by induction by starvation or by blockage using the v-ATPase inhibitor bafilomycin A, and by degradation of the macroautophagy substrate SQSTM1 under starvation and proteasome-inhibited conditions. Previous studies showed that LAMP2A accumulates in Rab11-positive vesicles in cystinotic cells. Here, we show defective Rab11 expression, localization and trafficking in CTNS-KO PTCs as determined by confocal microscopy, immunoblotting and TIRFM. We also show that both Rab11 expression and trafficking in cystinotic PTCs are rescued by the upregulation of CMA using small-molecule CMA activators. Cystinotic PTCs are characterized by PTC de-differentiation accompanied by loss of the endocytic receptor megalin, and megalin recycling is regulated by Rab11. Here we show that megalin plasma membrane localization is defective in CTNS-KO PTCs and its expression is rescued by treatment with CMA activators. Altogether, our data support that CMA upregulation has the potential to improve PTC function in cystinosis.

Short-cut diagnostic tool in cystinosis: Bone marrow aspiration.

Cystinosis is a rare metabolic genetic disorder caused by a mutation in cystinosin lysosomal cystine transporter (CTNS). The diagnosis of nephropathic cystinosis (NC) is made by observing corneal cystine crystals and/or measuring the cystine content of leukocytes. CTNS mutation analysis confirms the diagnosis of cystinosis, but leukocyte cystine measurement and CTNS analysis have not been widely available, and cystine crystals in the cornea may not be apparent in the first months of life. Cystine crystal deposition can be seen in the bone marrow earlier than corneal deposition, in patients with NC. Ten patients with cystinosis diagnosis were enrolled in the study. Medical records were reviewed retrospectively to collect demographic and clinical data such as age at diagnosis, disease presentation, parental consanguinity, family history, corneal cystine deposition, leukocyte cystine level, bone marrow cystine deposition, presence of renal failure, follow-up time and prognosis. Cystine crystals were seen in all of the patients' fresh bone marrow aspiration samples. Eight patients had corneal cystine deposition. Leukocyte cystine measurement could have been performed in four patients who had come from another center. Complications such as pulmonary hypertension and idiopathic intracranial hypertension (IIH) were observed in two patients. Bone marrow aspiration might be an easy and short-cut diagnostic tool for NC especially when it is not possible to measure fibroblast cystine content. Additionally some rare complications such as pulmonary hypertension and IIH can be encountered during the course of NC.

Impact of Cystinosin Glycosylation on Protein Stability by Differential Dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations have been reported, leading to varying disease severity, often in correlation with residual cystinosin activity as a transporter and with maintenance of its protein-protein interactions. In this study, we focus on the ΔITILELP mutation, the only mutation reported that sometimes leads to severe forms, inconsistent with its residual transported activity. ΔITILELP is a deletion that eliminates a consensus site on N66, one of the protein's seven glycosylation sites. Our hypothesis was that the ΔITILELP mutant is less stable and undergoes faster degradation. Our dynamic stable isotope labeling by amino acids in cell culture (SILAC) study clearly showed that wild-type cystinosin is very stable, whereas ΔITILELP is degraded three times more rapidly. Additional lysosome inhibition experiments confirmed ΔITILELP instability and showed that the degradation was mainly lysosomal. We observed that in the lysosome, ΔITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway, similar to the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies showed that ΔITILELP is partially retained at the endoplasmic reticulum (ER). We proposed that the ΔITILELP mutation causes protein misfolding, ER retention and inability to be processed in the Golgi apparatus, and we demonstrated that ΔITILELP carries high-mannose glycans on all six of its remaining glycosylation sites. We found that the high turnover of ΔITILELP, because of its immature glycosylation state in combination with low transport activity, might be responsible for the phenotype observed in some patients.

The renal Fanconi syndrome in cystinosis: pathogenic insights and therapeutic perspectives.

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. It is caused by a defect in the lysosomal cystine transporter, cystinosin, which results in an accumulation of cystine in all organs. Despite the ubiquitous expression of cystinosin, a renal Fanconi syndrome is often the first manifestation of cystinosis, usually presenting within the first year of life and characterized by the early and severe dysfunction of proximal tubule cells, highlighting the unique vulnerability of this cell type. The current therapy for cystinosis, cysteamine, facilitates lysosomal cystine clearance and greatly delays progression to kidney failure but is unable to correct the Fanconi syndrome. This Review summarizes decades of studies that have fostered a better understanding of the pathogenesis of the renal Fanconi syndrome associated with cystinosis. These studies have unraveled some of the early molecular changes that occur before the onset of tubular atrophy and identified a role for cystinosin beyond cystine transport, in endolysosomal trafficking and proteolysis, lysosomal clearance, autophagy and the regulation of energy balance. These studies have also led to the identification of new potential therapeutic targets and here, we outline the potential role of stem cell therapy for cystinosis and provide insights into the mechanism of haematopoietic stem cell-mediated kidney protection.

TRPV1 dysfunction in cystinosis patients harboring the homozygous 57 kb deletion.

Cystinosis is a rare autosomal recessive disorder characterized by lysosomal cystine accumulation due to loss of function of the lysosomal cystine transporter (CTNS). The most common mutation in cystinosis patients of Northern Europe consists of a 57-kb deletion. This deletion not only inactivates the CTNS gene but also extends into the non-coding region upstream of the start codon of the TRPV1 gene, encoding the capsaicin- and heat-sensitive ion channel TRPV1. To evaluate the consequences of the 57-kb deletion on functional TRPV1 expression, we compared thermal, mechanical and chemical sensitivity of cystinosis patients with matched healthy controls. Whereas patients heterozygous for the 57-kb deletion showed normal sensory responses, homozygous subjects exhibited a 60% reduction in vasodilation and pain evoked by capsaicin, as well as an increase in heat detection threshold. Responses to cold, mechanical stimuli or cinnamaldehyde, an agonist of the related nociceptor channel TRPA1, were unaltered. We conclude that cystinosis patients homozygous for the 57-kb deletion exhibit a strong reduction of TRPV1 function, leading to sensory deficiencies akin to the phenotype of TRPV1-deficient mice. These deficits may account for the reported sensory alterations and thermoregulatory deficits in these patients, and provide a paradigm for life-long TRPV1 deficiency in humans.

Ca2+ signalling in human proximal tubular epithelial cells deficient for cystinosin

Nephropathic cystinosis is an autosomal recessive lysosomal storage disorder caused by loss-of-function mutations in the CTNS gene coding for the lysosomal cystine transporter, cystinosin. Recent studies have demonstrated that, apart from cystine accumulation in the lysosomes, cystinosin-deficient cells, especially renal proximal tubular epithelial cells are characterized by abnormal vesicle trafficking and endocytosis, possible lysosomal dysfunction and perturbed intracellular signalling cascades. It is therefore possible that Ca2+ signalling is disturbed in cystinosis, as it has been demonstrated for other disorders associated with lysosomal dysfunction, such as Gaucher, Niemann-Pick type C and Alzheimer’s diseases. In this study we investigated ATP-induced, IP3-induced and lysosomal Ca2+ release in human proximal tubular epithelial cells derived from control and cystinotic patients. No major dysregulation of intracellular Ca2+ dynamics was found, although ATP-induced Ca2+ release appeared slightly sensitized in cystinotic cells compared to control cells. Hence, these subtle changes in Ca2+ signals elicited by agonists may contribute to the pathogenesis of the disease.

Cystinosis in Eastern Turkey.

This study was conducted to investigate CTNS (cystinosin, lysosomal cystine transporter) gene mutations and the clinical spectrum of nephropathic cystinosis among patients diagnosed with the disease in a single center in Turkey. Patients' clinical and laboratory data were extracted from an electronic health registry. Molecular CTNS gene analysis was performed using either next-generation sequencing or Sanger sequencing. Eleven patients (age range: 1.5-12 years) from nine families were identified. The presenting complaint was growth retardation in seven patients; polydipsia and polyuria in three patients; and vomiting in two patients. At presentation, electrolyte loss was noted in all patients, of which eight patients presented with metabolic acidosis, and three patients presented with metabolic alkalosis. All patients also presented with proteinuria and glucosuria, and four patients developed varying degrees of renal insufficiency, for which peritoneal dialysis was initiated in one patient. Cystine crystals were detected via ocular examination in one patient at presentation. No cystine crystals were detected among patients who underwent bone marrow aspiration. In the CTNS gene, a p.T7FX7 (c.18-21del4bp) mutation was detected in three patients, whereas a p.E227E (c.681 G>A) (homozygous) mutation was detected in eight patients. We detected two distinct mutations, p.T7FX7 (c.18-21del4bp) and p.E227E (c.681 G>A) (homozygous), in the CTNS gene in 11 patients with cystinosis from the East Anatolian region of Turkey. Patients with a homozygous c.681 G>A (p.E227E) mutation are more likely to develop chronic renal failure and should be monitored closely, whereas patients with a p.T7FX7 (c.18-21del4bp) mutation have a milder phenotype. Additionally, metabolic alkalosis does not exclude cystinosis, although cystinosis is a cause of proximal renal tubular acidosis.

32. Towards a Phase I Clinical Trial for Cystinosis

Cystinosis is an autosomal recessive lysosomal storage disorder characterized by the accumulation of cystine in the lysosomes leading to cystine crystals formation. The gene involved, CTNS, encodes a lysosomal cystine transporter, cystinosin. Cystinosis leads to a renal Fanconi's syndrome before the age of one characterized by polyuria and nutrients loss, and to multi-organ degeneration especially the eyes and kidneys. The cysteamine treatment allows the exit of cystine out of the lysosomes but only delays the evolution of the disease. We showed that transplantation of wild-type Hematopoietic Stem and Progenitor Cells (HSPCs) in a lethally irradiated Ctns−/− mouse leads to cystine content decrease in every tissues tested and kidney, eye and thyroid function and structure improvement in treated mice compared to mock-treated mice. An autologous HSPCs gene therapy approach has then been developed with Ctns−/− HSPCs gene-modified ex vivo to express a functional CTNS cDNA using the lentiviral vector pCCL-CTNS. Tissue cystine decrease and kidney function rescue were observed with this strategy. The toxicology and pharmacology studies required by the FDA are in progress with a targeted Vector Copy Number (VCN) included between 1 and 3. The in vitro studies, Colony Forming Unit assay and In Vitro Immortalization assay, have been completed using peripheral blood CD34+ cells from five healthy donors and four cystinosis patients. The in vivo studies are in progress: Ctns−/− HSPCs are isolated and transduced with pCCL-CTNS and transplanted into primary Ctns−/− mice and 6-months later their bone marrow cells are transplanted into secondary Ctns−/− recipients. Primary and secondary mice are carefully monitored and comprehensive histological, biochemical, molecular and clinical analyses are performed at 6 months post-transplant. So far, seven primary and one secondary Ctns−/− mice transplanted with pCCL-CTNS-transduced Ctns−/− HSPCs have reached the 6 month post-transplantation time point. The primary mice had a mean VCN of 1.713 and the secondary 2.04. Clinical evaluations, histopathology and Vector Integration Site (VIS) analyses revealed no adverse event so far suggesting a good safety profile of our product. Moreover, cystine content was significantly decreased in all tissues tested. Analysis of the remaining primary and secondary recipient mice is in progress and these data will be included in an Investigational New Drug (IND) for a phase 1 clinical trial for autologous transplantation of pCCL-CTNS-modified CD34+ HSPCs in patients with nephropathic cystinosis, For the design and conduct of the future clinical trial, the Cystinosis Stem Cell and Gene Therapy Consortium was recently created and is composed of experts in cystinosis, bone marrow transplant and gene therapy. The clinical grade pCCL-CTNS virus preparation is about to be produced at the Gene Therapy Resources Program (GTRP), Clinical Grade Lentivirus Vector Core directed by Dr. Kenneth Cornetta who prepared the Good Manufactory Practice-comparable (GMPc) virus used for the pharmacology/toxicology studies. We are currently preparing the documents necessary for the IND such as the clinical protocol, the toxicology/pharmacology report, the Chemistry, Manufacturing and Controls (CMC) report, etc. This clinical trial will represent the first stem cell and gene therapy treatment strategy for cystinosis.

Renal Fanconi Syndrome Is Caused by a Mistargeting-Based Mitochondriopathy.

We recently reported an autosomal dominant form of renal Fanconi syndrome caused by a missense mutation in the third codon of the peroxisomal protein EHHADH. The mutation mistargets EHHADH to mitochondria, thereby impairing mitochondrial energy production and, consequently, reabsorption of electrolytes and low-molecular-weight nutrients in the proximal tubule. Here, we further elucidate the molecular mechanism underlying this pathology. We find that mutated EHHADH is incorporated into mitochondrial trifunctional protein (MTP), thereby disturbing β-oxidation of long-chain fatty acids. The resulting MTP deficiency leads to a characteristic accumulation of hydroxyacyl- and acylcarnitines. Mutated EHHADH also limits respiratory complex I and corresponding supercomplex formation, leading to decreases in oxidative phosphorylation capacity,mitochondrial membrane potential maintenance, and ATP generation. Activity of the Na(+)/K(+)-ATPase is thereby diminished, ultimately decreasing the transport activity of the proximal tubule cells.