Residual Cystine Transport Activity for Specific Infantile and Juvenile CTNS Mutations in a PTEC-Based Addback Model.

Cystinosis is a rare, autosomal recessive, lysosomal storage disease caused by mutations in the gene CTNS, leading to cystine accumulation in the lysosomes. While cysteamine lowers the cystine levels, it does not cure the disease, suggesting that CTNS exerts additional functions besides cystine transport. This study investigated the impact of infantile and juvenile CTNS mutations with discrepant genotype/phenotype correlations on CTNS expression, and subcellular localisation and function in clinically relevant cystinosis cell models to better understand the link between genotype and CTNS function. Using CTNS-depleted proximal tubule epithelial cells and patient-derived fibroblasts, we expressed a selection of CTNSmutants under various promoters. EF1a-driven expression led to substantial overexpression, resulting in CTNS protein levels that localised to the lysosomal compartment. All CTNSmutants tested also reversed cystine accumulation, indicating that CTNSmutants still exert transport activity, possibly due to the overexpression conditions. Surprisingly, even CTNSmutants expression driven by the less potent CTNS and EFS promoters reversed the cystine accumulation, contrary to the CTNSG339R missense mutant. Taken together, our findings shed new light on CTNS mutations, highlighting the need for robust assessment methodologies in clinically relevant cellular models and thus paving the way for better stratification of cystinosis patients, and advocating for the development of more personalized therapy.

Nlrp2 deletion ameliorates kidney damage in a mouse model of cystinosis.

Cystinosis is a rare autosomal recessive disorder caused by mutations in the CTNS gene that encodes cystinosin, a ubiquitous lysosomal cystine/H+ antiporter. The hallmark of the disease is progressive accumulation of cystine and cystine crystals in virtually all tissues. At the kidney level, human cystinosis is characterized by the development of renal Fanconi syndrome and progressive glomerular and interstitial damage leading to end-stage kidney disease in the second or third decade of life. The exact molecular mechanisms involved in the pathogenesis of renal disease in cystinosis are incompletely elucidated. We have previously shown upregulation of NLRP2 in human cystinotic proximal tubular epithelial cells and its role in promoting inflammatory and profibrotic responses. Herein, we have investigated the role of NLRP2 in vivo using a mouse model of cystinosis in which we have confirmed upregulation of Nlrp2 in the renal parenchyma. Our studies show that double knock out Ctns-/- Nlrp2-/- animals exhibit delayed development of Fanconi syndrome and kidney tissue damage. Specifically, we observed at 4-6 months of age that animals had less glucosuria and calciuria and markedly preserved renal tissue, as assessed by significantly lower levels of inflammatory cell infiltration, tubular atrophy, and interstitial fibrosis. Also, the mRNA expression of some inflammatory mediators (Cxcl1 and Saa1) and the rate of apoptosis were significantly decreased in 4-6-month old kidneys harvested from Ctns-/- Nlrp2-/- mice compared to those obtained from Ctns-/-mice. At 12-14 months of age, renal histological was markedly altered in both genetic models, although double KO animals had lower degree of polyuria and low molecular weight proteinuria and decreased mRNA expression levels of Il6 and Mcp1. Altogether, these data indicate that Nlrp2 is a potential pharmacological target for delaying progression of kidney disease in cystinosis.

A Comparative Pharmacokinetic Study for Cysteamine-Containing Eye Drops as an Orphan Topical Therapy in Cystinosis.

Cystinosis is a low-prevalence lysosomal storage disease. The pathomechanism involves abnormal functioning of the cystinosine lysosomal cystine transporter (CTNS), causing intraliposomal accumulation of the amino acid cysteine disulfide, which crystallizes and deposits in several parts of the body. The most common ophthalmic complication of cystinosis is the deposition of "gold dust" cystine crystals on the cornea, which already occurs in infancy and leads to severe photosensitivity and dry eyes as it gradually progresses with age. In the specific treatment of cystinosis, preparations containing cysteamine (CYA) are used. The availability of commercialized eyedrops for the targeted treatment is scarce, and only Cystadrops® are commercially available with strong limitations. Thus, magistral CYA-containing compounded eyedrops (CYA-CED) could have a key role in patient care; however, a rationally designed comprehensive study on the commercialized and magistral products is still missing. This work aims to build up a comprehensive study about commercialized and magistral CYA eye drops, involving pharmacokinetic and physicochemical characterization (applying mucoadhesivity, rheology test, investigation of drug release, and parallel artificial membrane permeability assays), as well as ex vivo tests, well supported by statistical analysis.

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.

The CTNS-MTORC1 axis couples lysosomal cystine to epithelial cell fate decisions and is a targetable pathway in cystinosis.

Differentiation and fate decisions are critical for the epithelial cells lining the proximal tubule (PT) of the kidney, but the signals involved remain unknown. Defective cystine mobilization from lysosomes through CTNS (cystinosin, lysosomal cystine transporter), which is mutated in cystinosis, triggers the dedifferentiation and dysfunction of the PT cells, causing kidney disease and severe metabolic complications. Using preclinical models and physiologically relevant cellular systems, along with functional assays and a generative artificial intelligence (AI)-powered engine, we found that cystine storage imparted by CTNS deficiency stimulates Ragulator-RRAG GTPase-dependent recruitment of MTORC1 and its constitutive activation. In turn, this diverts the catabolic trajectories and differentiating states of PT cells toward growth and proliferation, disrupting homeostasis and their specialized functions. Therapeutic MTORC1 inhibition by using low doses of rapamycin corrects lysosome function and differentiation downstream of cystine storage and ameliorates PT dysfunction in preclinical models of cystinosis. These discoveries suggest that cystine may act as a lysosomal fasting signal that tailors MTORC1 signaling to direct fate decisions in the kidney PT epithelium, highlighting novel therapeutic paradigms for cystinosis and other lysosome-related disorders.

Impact of compliance to oral cysteamine treatment on the costs of Kidney failure in patients with nephropathic cystinosis in the United Kingdom.

BACKGROUND: Nephropathic Cystinosis (NC), a rare disease characterised by intra-lysosomal accumulation of cystine, results in progressive kidney failure (KF). Compliance to lifelong oral cysteamine, the only therapy, is often compromised. The relationship between compliance and costs of NC has not been previously formally assessed. The present study evaluates the impact of compliance on lifetime (direct) costs of treating KF in NC patients in the United Kingdom. METHODS: A three-state (KF-free, post-KF, death) partitioned survival model was developed for hypothetical 'Good Compliance' (GC) and 'Poor Compliance' (PC) cohorts. Survival in the KF-free state was determined by a published regression function of composite compliance score (CCS). The CCS is a summation of annual compliance scores (ACS) over treatment duration prior to KF. ACSs are indexed on annual (average) leukocyte cystine levels (LCL). The Poor Compliance cohort was defined to reflect NC patients in a previous study with a mean LCL of 2.35 nmols nmol half-cystine/mg protein over the study period - and an estimated mean ACS of 1.64 over a 13.4 year treatment duration. The Good Compliance cohort was assumed to have an ACS of 2.25 for 21 years. Major KF costs were evaluated - i.e., dialysis, kidney transplants, and subsequent monitoring. RESULTS: The mean CCS was 47 for the GC and 22 for the PC cohort respectively, corresponding to estimated lifetime KF costs of £92,370 and £117,830 respectively - i.e., a cost saving of £25,460/patient, or £1,005/patient for every 1-unit improvement in CCS. CONCLUSION: This analysis indicates that lifetime costs of KF in NC can be reduced through improved treatment compliance with oral cysteamine.

Dietary supplementation of cystinotic mice by lysine inhibits the megalin pathway and decreases kidney cystine content.

Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold vs control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by L- and D-stereoisomers. In cystinotic mice, 2-month diets with 5x-L-lysine and 5x-L-arginine were overall well tolerated, while 5x-D-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-D-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by D-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo.

ER-associated degradation in cystinosis pathogenesis and the prospects of precision medicine.

Cystinosis is a lysosomal storage disease that is characterized by the accumulation of dipeptide cystine within the lumen. It is caused by mutations in the cystine exporter, cystinosin. Most of the clinically reported mutations are due to the loss of transporter function. In this study, we identified a rapidly degrading disease variant, referred to as cystinosin(7Δ). We demonstrated that this mutant is retained in the ER and degraded via the ER-associated degradation (ERAD) pathway. Using genetic and chemical inhibition methods, we elucidated the roles of HRD1, p97, EDEMs, and the proteasome complex in cystinosin(7Δ) degradation pathway. Having understood the degradation mechanisms, we tested some chemical chaperones previously used for treating CFTR F508Δ and demonstrated that they could facilitate the folding and trafficking of cystinosin(7Δ). Strikingly, chemical chaperone treatment can reduce the lumenal cystine level by approximately 70%. We believe that our study conclusively establishes the connection between ERAD and cystinosis pathogenesis and demonstrates the possibility of using chemical chaperones to treat cystinosin(7Δ).

The Pitfall of White Blood Cell Cystine Measurement to Diagnose Juvenile Cystinosis.

Cystinosis is an autosomal recessive lysosomal storage disease, caused by mutations in the CTNS gene, resulting in multi-organ cystine accumulation. Three forms of cystinosis are distinguished: infantile and juvenile nephropathic cystinosis affecting kidneys and other organs such as the eyes, endocrine system, muscles, and brain, and adult ocular cystinosis affecting only the eyes. Currently, elevated white blood cell (WBC) cystine content is the gold standard for the diagnosis of cystinosis. We present a patient with proteinuria at adolescent age and corneal cystine crystals, but only slightly elevated WBC cystine levels (1.31 ½ cystine/mg protein), precluding the diagnosis of nephropathic cystinosis. We demonstrate increased levels of cystine in skin fibroblasts and urine-derived kidney cells (proximal tubular epithelial cells and podocytes), that were higher than the values observed in the WBC and healthy control. CTNS gene analysis shows the presence of a homozygous missense mutation (c.590 A > G; p.Asn177Ser), previously described in the Arab population. Our observation underlines that low WBC cystine levels can be observed in patients with juvenile cystinosis, which may delay the diagnosis and timely administration of cysteamine. In such patients, the diagnosis can be confirmed by cystine measurement in slow-dividing cells and by molecular analysis of the CTNS gene.

The TGM2 inhibitor cysteamine hydrochloride does not impact corneal epithelial and stromal wound healing in vitro and in vivo.

Corneal wound healing is integral for resolution of corneal disease or for post-operative healing. However, corneal scarring that may occur secondary to this process can significantly impair vision. Tissue transglutaminase 2 (TGM2) inhibition has shown promising antifibrotic effects and thus holds promise to prevent or treat corneal scarring. The commercially available ocular solution for treatment of ocular manifestations of Cystinosis, Cystaran®, contains the TGM2 inhibitor cysteamine hydrochloride (CH). The purpose of this study is to assess the safety of CH on corneal epithelial and stromal wounds, its effects on corneal wound healing, and its efficacy against corneal scarring following wounding. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC) were first used to quantify and localize TGM2 expression in the cornea. Subsequently, (i) the in vitro effects of CH at 0.163, 1.63, and 16.3 mM on corneal epithelial cell migration was assessed with an epithelial cell migration assay, and (ii) the in vivo effects of application of 1.63 mM CH on epithelial and stromal wounds was assessed in a rabbit model with ophthalmic examinations, inflammation scoring, color and fluorescein imaging, optical coherence tomography (OCT), and confocal biomicroscopy. Post-mortem assessment of corneal tissue post-stromal wounding included biomechanical characterization (atomic force microscopy (AFM)), histology (H&E staining), and determining incidence of myofibroblasts (immunostaining against α-SMA) in wounded corneal tissue. TGM2 expression was highest in corneal epithelial cells. Application of the TGM2 inhibitor CH did not affect in vitro epithelial cell migration at the two lower concentrations tested. At 16.3 mM, decreased cell migration was observed. In vivo application of CH at 57 mM was well tolerated and did not adversely affect wound healing. No difference in corneal scarring was found between CH treated and vehicle control eyes. This study shows that the TGM2 inhibitor CH, at the FDA-approved dose, is well tolerated in a rabbit model of corneal wound healing and does not adversely affect epithelial or stromal wound healing. This supports the safe use of this medication in Cystinosis patients with open corneal wounds. CH did not have an effect on corneal scarring in this study, suggesting that Cystaran® administration to patients with corneal wounds is unlikely to decrease corneal fibrosis.

Cystinosin-deficient rats recapitulate the phenotype of nephropathic cystinosis.

The lysosomal storage disease cystinosis is caused by mutations in CTNS, encoding the cystine transporter cystinosin, and in its severest form leads to proximal tubule dysfunction followed by kidney failure. Patients receive the drug-based therapy cysteamine from diagnosis. However, despite long-term treatment, cysteamine only slows the progression of end-stage renal disease. Preclinical testing in cystinotic rodents is required to evaluate new therapies; however, the current models are suboptimal. To solve this problem, we generated a new cystinotic rat model using CRISPR/Cas9-mediated gene editing to disrupt exon 3 of Ctns and measured various parameters over a 12-mo time course. Ctns-/- rats display hallmarks of cystinosis by 3-6 mo of age, as demonstrated by a failure to thrive, excessive thirst and urination, cystine accumulation in tissues, corneal cystine crystals, loss of LDL receptor-related protein 2 in proximal tubules, and immune cell infiltration. High levels of glucose, calcium, albumin, and protein were excreted at 6 mo of age, consistent with the onset of Fanconi syndrome, with a progressive diminution of urine urea and creatinine from 9 mo of age, indicative of chronic kidney disease. Kidney histology and immunohistochemistry showed proximal tubule atrophy and glomerular damage as well as classic "swan neck" lesions. Overall, Ctns-/- rats show a disease progression that more faithfully recapitulates nephropathic cystinosis than existing rodent models. The Ctns-/- rat provides an excellent new rodent model of nephropathic cystinosis that is ideally suited for conducting preclinical drug testing and is a powerful tool to advance cystinosis research.NEW & NOTEWORTHY Animal models of disease are essential to perform preclinical testing of new therapies before they can progress to clinical trials. The cystinosis field has been hampered by a lack of suitable animal models that fully recapitulate the disease. Here, we generated a rat model of cystinosis that closely models the human condition in a timeframe that makes them an excellent model for preclinical drug testing as well as being a powerful tool to advance research.

Evaluation of NACA and diNACA in human cystinosis fibroblast cell cultures as potential treatments for cystinosis.

BACKGROUND: Cystinosis is a rare autosomal recessive lysosomal storage disease, associated with high morbidity and mortality. Mutations in the CTNS gene disable a membrane protein responsible for the transport of cystine out of the lysosome. Loss of transporter function leads to intralysosomal cystine accumulation and long-term damage to various tissues and organs, including the kidneys, eyes, liver, muscles, pancreas, and brain. The only cystine-depletion therapy for treatment of cystinosis is cysteamine which requires frequent administration of high doses and often causes gastrointestinal pain as well as pungent sulfurous odor in patients. The current in vitro study evaluated antioxidants, N-acetylcysteine amide (NACA; NPI-001) and (2R,2R')-3,3'-disulfanediyl bis(2-acetamidopropanamide) (diNACA; NPI-002), as potential treatments for cystinosis. METHODS: Cytotoxicity of cysteamine, NACA and diNACA was evaluated in cultured human cystinotic fibroblasts (HCFs). HCFs were cultured in 96 well plates incubated for 0-72 h in the presence of 25, 50 or 75 µM each of either cysteamine, NACA or diNACA along with an untreated control. Media was removed and cell viability assessed. Next, cystine-depleting activities of cysteamine, NACA and diNACA were screened in HCFs cell culture utilizing an inexpensive, proven colorimetric assay. HCFs were seeded and allowed to reach approximately 80% confluence before the addition of the test articles: 50 µM of either cysteamine, NACA or diNACA in media along with an untreated control. HCFs were incubated, harvested, and cystine was reduced to cysteine, the concentration of which was then determined per quantity of protein compared to a cysteine standard. Statistically significant cystine depletion was determined by paired t-test versus untreated control (p < 0.05). RESULTS: Neither cysteamine, NACA nor diNACA at 25, 50 or 75 µM caused cytotoxicity in HCFs. Treatment with all tested concentrations (25, 50 or 75 µM) of either NACA or diNACA at 48 or 72 h resulted in statistically significant increases in cell viability, relative to untreated control, whereas the higher concentrations (50 or 75 µM) of cysteamine achieved statistical significance at both timepoints but not the lowest concentration (25 µM). All test articles depleted cystine from HCFs compared to control. NACA depletion of cystine was statistically superior to cysteamine at 6, 24 and 48 h and numerically greater at 72 h. DiNACA depletion of cystine was statistically superior to cysteamine at 6 and 48 h, slightly numerically greater at 24 h and slightly less at 72 h. CONCLUSIONS: NACA and diNACA were non cytotoxic to HCFs and significantly increased cell viability. Cystine reduction was determined as percent of control after incubation with 50 µM of NACA, diNACA or cysteamine in HCFs cell culture for 6, 24, 48 and 72 h. Of the three test articles, NACA exhibited most rapid and greatest potency in cystine reduction. Rank order potency for cystine reduction over time was observed, NACA > diNACA ≥ cysteamine. Therefore, further study of NACA and diNACA as potential treatments for cystinosis is warranted.

Cystinosis and two rare mutations in CTNS gene: two case reports.

BACKGROUND: Cystinosis is an autosomal recessive disorder characterized by an accumulation of the amino acid cystine in lysosomes throughout the body. Cystinosis is an inherited disease resulting from the failure of lysosomal cystine transport. The responsible gene, Cystinosin, Lysosomal Cystine Transporter (CTNS), encodes the lysosomal cystine carrier cystinosin. CASE PRESENTATION: In this case report, we reviewed the genetic basis of cystinosis and investigated two Iranian cases affected by cystinosis, one of which revealed a rare mutation in the CTNS gene. Two patients, 9-year-old (patient A) and 11-year-old (patient B) symptomatic Iranian females with renal insufficiency, were diagnosed with cystinosis on the basis of their clinical features and laboratory tests. After genetic counseling, blood samples were obtained from the patients and their parents. Genomic Deoxyribonucleic Acid (DNA) was extracted from whole blood, and mutation analysis was performed using polymerase chain reaction and sequencing methods for all exons of the CTNS gene. At least 148 different pathogenic and deleterious mutations in the CTNS gene have been reported to date. Owing to our patient's prominent clinical features of cystinosis, we carried out a targeted search for mutations in the CTNS gene. CONCLUSIONS: This led us to confirm the existence of a homozygous DNA variation c.257_258deletionCT (p.Ser86PhefsTer38) in exon 6 of the gene in patient A and another homozygous DNA variation, c.323delA (p.Q108RfsTer10), in the same exon in patient B. As expected, the mentioned mutation existed in both her parents in a heterozygous state. Variations c.257_258delCT and c.323delA reported in three Iranian patients in the CTNS gene are frameshifts, and truncating mutations that affect product function result in relatively mild symptoms of cystinosis. The present finding confirms previous research and proves the importance of the association of this gene rare mutations with cystinosis. Since reported mutations are rare, their previous reports in Iranian patients indicate the high frequency of these mutations in our region.

Molecular characterization of CTNS mutations in Tunisian patients with ocular cystinosis.

BACKGROUND: Ocular cystinosis is a rare autosomal recessive disorder characterized by intralysosomal cystine accumulation in renal, ophthalmic (cornea, conjunctiva), and other organ abnormalities. Patients with ocular cystinosis are mostly asymptomatic and typically experience mild photophobia due to cystine crystals in the cornea observed accidently during a routine ocular examination. The ocular cystinosis is associated with different mutations in CTNS gene. Cysteamine therapy mostly corrects the organ abnormalities. METHODS: This study was performed in collaboration with the department of ophthalmology of Farhat Hached Hospital. The Optical Coherence Tomography (OCT) of the cornea and retinal photography were used to search cystine crystals within the corneas and conjunctiva in eight Tunisian patients. Screening for the common 57-kb deletion was performed by standard multiplex PCR, followed by direct sequencing of the entire CTNS gene. RESULTS: The studied patients were found to have cystine crystal limited anterior corneal stroma and the conjunctiva associated with retinal crystals accumulation. CTNS gene sequencing disclosed 7 mutations: three missense mutations (G308R, p.Q88K, and p.S139Y); one duplication (C.829dup), one framshift mutation (p.G258f), one splice site mutation (c.681 + 7delC) and a large deletion (20,327-bp deletion). Crystallographic structure analysis suggests that the novel mutation p.S139Y is buried in a first transmembrane helix closed to the lipid bilayer polar region, introducing a difference in hydrophobicity which could affect the hydrophobic interactions with the membrane lipids. The second novel mutation p.Q88K which is located in the lysosomal lumen close to the lipid membrane polar head region, introduced a basic amino acid in a region which tolerate only uncharged residue. The third missense mutation introduces a positive change in nonpolar tail region of the phospholipid bilayer membrane affecting the folding and stability of the protein in the lipid bilayer. CONCLUSIONS: Our data demonstrate that impaired transport of cystine out of lysosomes is the most common, which is obviously associated with the mutations of transmembrane domains of cystinosine resulting from a total loss of its activity.

Urine-Derived Kidney Progenitor Cells in Cystinosis.

Nephropathic cystinosis is an inherited lysosomal storage disorder caused by pathogenic variants in the cystinosin (CTNS) gene and is characterized by the excessive shedding of proximal tubular epithelial cells (PTECs) and podocytes into urine, development of the renal Fanconi syndrome and end-stage kidney disease (ESKD). We hypothesized that in compensation for epithelial cell losses, cystinosis kidneys undertake a regenerative effort, and searched for the presence of kidney progenitor cells (KPCs) in the urine of cystinosis patients. Urine was cultured in a specific progenitor medium to isolate undifferentiated cells. Of these, clones were characterized by qPCR, subjected to a differentiation protocol to PTECs and podocytes and assessed by qPCR, Western blot, immunostainings and functional assays. Cystinosis patients voided high numbers of undifferentiated cells in urine, of which various clonal cell lines showed a high capacity for self-renewal and expressed kidney progenitor markers, which therefore were assigned as cystinosis urine-derived KPCs (Cys-uKPCs). Cys-uKPC clones showed the capacity to differentiate between functional PTECs and/or podocytes. Gene addition with wild-type CTNS using lentiviral vector technology resulted in significant reductions in cystine levels. We conclude that KPCs present in the urine of cystinosis patients can be isolated, differentiated and complemented with CTNS in vitro, serving as a novel tool for disease modeling.

A Personal History of Cystinosis by Dr. Jerry Schneider.

Cystinosis is a rare lysosomal storage disease that is tightly linked with the name of the American physician and scientist Dr. Jerry Schneider. Dr. Schneider (1937-2021) received his medical degree from Northwestern University, followed by a pediatrics residency at Johns Hopkins University and a fellowship in inherited disorders of metabolism. He started to work on cystinosis in J. Seegmiller's laboratory at the National Institutes of Health (NIH) and subsequently moved to the UC San Diego School of Medicine, where he devoted his entire career to people suffering from this devastating lysosomal storage disorder. In 1967, Dr. Schneider's seminal Science paper 'Increased cystine in leukocytes from individuals homozygous and heterozygous for cystinosis' opened a new era of research towards understanding the pathogenesis and finding treatments for cystinosis patients. His tremendous contribution transformed cystinosis from a fatal disorder of childhood to a treatable chronic disease, with a new generation of cystinosis patients being now in their 40th and 50th years. Dr. Schneider wrote a fascinating 'Personal History of Cystinosis' highlighting the major milestones of cystinosis research. Unfortunately, he passed away before this manuscript could be published. Fifty-five years after his first paper on cystinosis, the 'Personal History of Cystinosis' by Dr. Schneider is a tribute to his pioneering discoveries in the field and an inspiration for young doctors and scientists who have taken over the torch of cystinosis research towards finding a cure for cystinosis.

Defective Cystinosin, Aberrant Autophagy-Endolysosome Pathways, and Storage Disease: Towards Assembling the Puzzle.

Epithelial cells that form the kidney proximal tubule (PT) rely on an intertwined ecosystem of vesicular membrane trafficking pathways to ensure the reabsorption of essential nutrients-a key requisite for homeostasis. The endolysosome stands at the crossroads of this sophisticated network, internalizing molecules through endocytosis, sorting receptors and nutrient transporters, maintaining cellular quality control via autophagy, and toggling the balance between PT differentiation and cell proliferation. Dysregulation of such endolysosome-guided trafficking pathways might thus lead to a generalized dysfunction of PT cells, often causing chronic kidney disease and life-threatening complications. In this review, we highlight the biological functions of endolysosome-residing proteins from the perspectives of understanding-and potentially reversing-the pathophysiology of rare inherited diseases affecting the kidney PT. Using cystinosis as a paradigm of endolysosome disease causing PT dysfunction, we discuss how the endolysosome governs the homeostasis of specialized epithelial cells. This review also provides a critical analysis of the molecular mechanisms through which defects in autophagy pathways can contribute to PT dysfunction, and proposes potential interventions for affected tissues. These insights might ultimately accelerate the discovery and development of new therapeutics, not only for cystinosis, but also for other currently intractable endolysosome-related diseases, eventually transforming our ability to regulate homeostasis and health.

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

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.

Drug Repurposing in Rare Diseases: An Integrative Study of Drug Screening and Transcriptomic Analysis in Nephropathic Cystinosis.

Diagnosis and cure for rare diseases represent a great challenge for the scientific community who often comes up against the complexity and heterogeneity of clinical picture associated to a high cost and time-consuming drug development processes. Here we show a drug repurposing strategy applied to nephropathic cystinosis, a rare inherited disorder belonging to the lysosomal storage diseases. This approach consists in combining mechanism-based and cell-based screenings, coupled with an affordable computational analysis, which could result very useful to predict therapeutic responses at both molecular and system levels. Then, we identified potential drugs and metabolic pathways relevant for the pathophysiology of nephropathic cystinosis by comparing gene-expression signature of drugs that share common mechanisms of action or that involve similar pathways with the disease gene-expression signature achieved with RNA-seq.

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.