Cystinosin-LKG rescues cystine accumulation and decreases apoptosis rate in cystinotic proximal tubular epithelial cells

Abstract

Background: Nephropathic cystinosis is a lysosomal storage disease that is caused by mutations in the CTNS gene encoding a cystine/proton symporter cystinosin and an isoform cystinosin-LKG which is generated by an alternative splicing of exon 12. We have investigated the physiological role of the cystinosin-LKG that is widely expressed in epithelial tissues. Methods: We have analyzed the intracellular localization and the function of the cystinosin-LKG conjugated with DsRed (cystinosin-LKG-RFP) in Madin-Darby canine kidney cells (MDCK II) and in proximal tubular epithelial cells carrying a deletion of the CTNS gene (cystinotic PTEC), respectively. results: Cystinosin-LKG-RFP colocalized with markers of lysosomes, late endosomes and was also expressed on the apical surface of polarized MDCK II cells. Moreover, immuneelectron microscopy images of MDCK II cells overexpressing cystinosin-LKG-RFP showed stacked lamellar membranes inside perinuclear lysosomal structures. To study the role of LKG-isoform, we have investigated cystine accumulation and apoptosis that have been described in cystinotic cells. Cystinosin-LKG decreased cystine levels by approximately 10-fold similarly to cystinosin-RFP. The levels of TNFα-and actinomycin D-inducted apoptosis dropped in cystinotic cells expressing LKG-isoform. This effect was also similar to the main isoform. conclusion: Our results suggest that cystinosin-LKG and cystinosin move similar functional activities in cells.n ephropathic cystinosis (NC) is a rare lysosomal storage disease. It is caused by defective activity of cystinosin, a proton/cystine symporter that is ubiquitously expressed on lysosomal membranes, which leads to cystine accumulation into lysosomes and crystal formations in many tissues (1). One of the first symptoms of NC is renal Fanconi syndrome, secondary to a global dysfunction of proximal tubular cells. During their lifetimes, patients with NC develop several others symptoms in particular, if they are not well treated with cysteamine (2,3).Recent works have demonstrated an involvement of cystinosin in lysosomal kinetics and exocytosis (4), vesicle trafficking (4,5), autophagy, (6) and in sensing cell oxidative state (7). Studies using a cystinotic mouse model have shown decreased expression of the multi-ligand receptors megalin and cubilin in proximal tubules; this was associated with cell dedifferentiation and apoptosis (5,8). Also, enhanced cell death due to activation of proapoptotic signals probably occurs in other tissues (9-11).Cystinosin (accession number: NP_004928) is composed of seven transmembrane domains that bear two lysosomal targeting motifs (GYDQL and YFPQA) and seven N-glycosylation sites in the amino-terminal region (12). The aceview database reports various alternative splicing transcripts for the CTNS gene that encodes the cystinosin (http://www.ncbi.nlm.nih. gov/IEB/Research/Acembly). In 2008, we have identified a 400 amino acid cystinosin isoform that derives from alternative splicing of CTNS gene exon 12 (accession number: NP_001026...

Are the Clinical Presentations (Phenotypes) of Gitelman’s and Bartter’s Syndromes Gene Mutations Driven by Their Effects on Intracellular pH, Their “pH” Enotype?

“…Nephropathic cystinosis is a lysosomal disorder caused by functional defects of CTNS, a lysosomal 7-transmembrane protein H + -driven cystine transporter that mediates cystine efflux into the cytosol. Taranta et al characterized cystinosin protein isoforms resulting in alternative splicing of exon 12, which directs the protein to other cell compartments, including the plasma membrane, the Golgi apparatus, the endoplasmic reticulum (ER), and cytosolic vesicles resembling endosomes [12].…”

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

“…Cystinotic cells are known to be more sensitive to pro-apoptotic stimuli than CTNS +/+ cells [ 8 , 9 , 10 , 11 ]. We performed as secondary screening an imaging-based high content screening using the Prestwick chemical library to identify drugs that, in addition to reducing cystine content, also protect cystinotic cells from apoptosis.…”

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

“…50 In addition, lack of megalin expression may also indicate cell dedifferentiation, as shown in mPTCs by Raggi et al 21 Therefore, and irrespective of the underlying mechanism, restored expression of megalin after in vitro and in vivo treatments in our experimental settings indicates that luteolin may be efficient in treating the renal Fanconi syndrome of cystinosis and is not at odds with the report by Janssens et al 49 In conclusion, luteolin was selected from the positive hits of an HTS aimed at improving autophagy because of its additional effects on oxidation, apoptosis, and inflammation, all of which are altered in cystinosis. [8][9][10][11][14][15][16][17]32,45,[51][52][53] Moreover, luteolin belongs to the flavonoid family, similar to genistein, which we recently showed to ameliorate lysosomal cystine content and lysosomal compartment distribution in cystinosis, likely through stimulation of the TFEB pathway. 27 Flavonoids have been proposed for the treatment of other lysosomal storage diseases 54,55 and luteolin has been shown to have a very good safety profile in humans.…”

“…In particular, mechanisms leading to PTC dysfunction are probably not solely related to cystine accumulation because renal Fanconi syndrome is not improved by cysteamine. In this respect, recent studies have identified several defects, including enhanced apoptosis, [8][9][10][11] mitochondrial dysfunction, 12,13 oxidative stress, [14][15][16][17] aberrant autophagy, [18][19][20] endo-lysosomal dysfunction, 21,22 and decreased expression of megalin and cubilin. 21,23 Among these, altered autophagy likely plays a pivotal role.…”