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Definitions

Nephropathic cystinosis: 10 th anniversary of the CTNS gene Elena Levtchenko, MD, PhD University Hospital Leuven, Belgium. Definitions. Cystinosis an autosomal recessive disease caused by lysosomal accumulation of cystine due to defective exodus of cystine out of the lysosomes Cystinosis

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  1. Nephropathic cystinosis: 10th anniversary of the CTNS geneElena Levtchenko, MD, PhDUniversity Hospital Leuven, Belgium

  2. Definitions • Cystinosis • an autosomal recessive disease caused by lysosomal accumulation of cystine due to defective exodus of cystine out of the lysosomes • Cystinosis • incidence ~1:200,000 • clustering in some populations • Cystinosis • most common cause of inherited generalized proximal tubular dysfunction (renal Fanconi syndrome)

  3. Clinical forms • Infantile form: • Fanconi syndrome ~ 3-6 months • end stage renal disease (ESRD) ~ 10 years • “Late-onset” (juvenile) form: • later onset (often during puberty) • mild tubulopathy, more pronounced proteinuria, (even in nephrotic range) • later progression to ESRD • Ocular form • Overlap between ocular and juvenile forms (Servais et al. 2008) 3

  4. Extra-renal involvement Eye • photophobia • kerathopathy • retinal blindness Endocrine organs • hypothyroidism • diabetes mellitus • male hypogonadism Neuromuscular disease • myopathy Neurological complaints • epilepsy • mental deterioration • cerebellar and pyramidal signs • stroke-like episodes Liver disease, exocrine pancreas deficiency Gahl et al. 2002 4

  5. CAT- transporter Treatment with cysteamine cystinosin cytoplasm cytoplasm cysteine –cysteamine cystine cysteamine cysteine NH2 COOH CH CH2 S S CH2 CH NH2 COOH lysosome NH2 COOH CH CH2 S S CH2 CH2 NH2 SH CH2 CH NH2 COOH HS CH2 CH2 NH2 lysosome + + cystine cysteine transporter 5

  6. Efficiency of cysteamine in cystinosis • Cysteamine postpones the deterioration of the renal function (Markello et al. 1993) • Cysteamine protects extra-renal organs and should be administered also after renal transplantation (Gahl et al. 2002) • Topical cysteamine drops (0.5%) dissolve corneal cystine crystals (Tsilou et al. 2002). New viscous cysteamine eye preparations (Bozdag et al. 2008) • BUT: • Cysteamine does not reverse renal Fanconi syndrome • Many patients still develop ESRD and extra-renal complications (at later age) • Major compliance problems due to: • - dose regimen every 6 hours (daily dose 1.3-1.95 g/m2) • - gastro-intestinal complaints (Dohil et al. 2006) • - bad breath and sweat odor due to formation of dimethylsulfide • (Besouw et al. 2007)

  7. cystinosis heterozygotes controls Cystine dysposal from granular fraction of normal, heterozygous and cystinotic cells loaded with [35S] cystine dimethyl ester

  8. Cystinosin: predicted structure GYDQL YFPQA CTNS gene structure (17p13, 23 kb) TAG ATG 1 2 3c 44 5 6 7 8 9 10 11 12 Chergui et al. 2001 Most common mutation in European population: 57 kb deletion: CTNS gene, CARKL gene (Town et al. 1998, Wamelink et al. 2008) 9

  9. Functional assay of cystinosin + H+ [35S] - C-S-S-C N C C cytoplasm lysosome N Kalatzis et al. 2001, 2004

  10. Expression of Cystinosin-LKG is not restricted to lysosomes Taranta et al. 2008

  11. Pathogenesis of cystinosis • How cystinosin defects lead to cell dysfunction in cystinosis is still unknown • Existing hypotheses from in vitro studies: • Cystinosin dysfunction leads to defective gluthation synthesis in cystinosis  enhanced oxidative stress leading to cell damage (Levtchenko et al. 2005, Mannucci et al. 2006, Laube et al. 2006) • Increased sensitivity of cystinotic cells to apoptotic stimuli due to cysteinylation of protein kinase C (Park et al. 2002, 2006)  enhanced cystinotic cell death • ATP deficiency  cell dysfunction and Fanconi syndrome (Coor et al. 1991, Ben-Nun et al. 1993, Foreman et al. 1995, Wilmer et al. 2006) • No prove in humans or in ctns -/- mice model

  12. nucleus cystine O2-. interstitium lumen cytosole lysosome influence on gene expression cysteine  cystine cystinosin oxidative redox state cystine reductase GCS   cystine - LKG - LKG cystine -glut-cys  GSSG/GSH GS H2O2  GSSG  GSH Cysteinylation of PKC- oxoglutarate carrier dicarboxilate carrier glucose GSH reductase mitochonrion glycolytic pathway GSH  GSSG GSH H2O2 GSH peroxidase citric acid cycle O2-. Stimulation of apoptosis SOD ATP ADP + P pyruvate NADH FADH2 e- I II III IV V pyruvate carrier H+ H+ O2-. e- H+ SOD Disturbed ATP metabolism  lipid peroxides H2O2 O2-. 14

  13. Possible functions of cystinosin-LKG • Cystinosin-LKG has probably other functions in the cell additionally to lysosomal cystine transport: • Cystine-binding in the cytosole: regulate cystine - cysteine pool in the cell and prevent the formation of disulfide compounds • Alternative plasma membrane cystine transporter

  14. Molecular therapy, vol.16, No.8, 1372-1381, aug.2008 Liver from 3-month old ctns -/- mice • Obstacles of gene therapy in cystinosis: • Gene should be delivered to all organs • Use of viral vectors • Immune response • Short duration of cystine-lowering effect • Gene transfer should be administered at early age Liver from 6-month old ctns -/- mice

  15. Summary • Discovery of cystinosin in 1998 opened a new chapter of diseases caused by defects of lysosomal transport proteins. • New data indicate that the expression of cystinosin is not restricted to the lysosomal membrane. Ongoing studies will identify new functions and regulators of cystinosin activity  new insights into the pathogenesis of cystinosis. • Gene therapy in cystinosis is feasible and is a subject of intensive research. Many obstacles should be overcome.

  16. Thanks to Bambino GesùChildren hospital, Rome F. Emma A. Taranta Cystinosis research group Nijmegen/Leuven VUMC, Amsterdam H. Blom M. Wamelink L.Monnens M. Wilmer M. Besouw B. Van den Heuvel

  17. QUESTIONS?

  18. Lysosomal membrane transport H+ ADP ATP ? lysosome H+ H+ Acid hydrolases: Proteases Glycosidases Nucleases Phosphatases Sulphatases Substrates: Amino acids Monosaccharides Nucleosides Di/tripeptides Inorganic ions Vitamins

  19. New insights into the lysosomal membrane transport • Competition experiments in isolated lysosomes predicted ~20 lysosomal transporters (Pisoni, Thoene 1991) • Analogous to cystinosin: functional assays of lysosomal membrane proteins (genotype-phenotype correlations in sialic acid storage disease (Morin et al. 2004) • Proteomics studies of lysosomal membrane (Schroder et al. 2007) • Identification of new transporters (glucose (GLUT8), myo-inisitol (HMIT), Zn transporter, KCC1…) • New genes responsible for lysosomal transport disorders (mucolipidosis type IV (MCOLN1), infantile malignant osteopetrosis (ClC7), late infantile neuronal ceroidlipofiscinosis (CLN7)

  20. 22 corneal cystine crystals cystinotic kerathopathy cystinotic retinopathy

  21. nucleus cystine cystine H+ cystine cysteine cystinosin cysteine H+ cysteine cystine lysosome protein degradation cytoplasma 23

  22. n= 67 n =32 n = 17 24 Markello et al. 1993

  23. Stokes et al. 2008 Wilmer et al. 2008 Cystine accumulation in cystinosis Kidney: 200 - 400 x normal Liver: 80 - 1000 x normal Muscle: 40 - 70 x normal Brain: 5 - 20 x normal

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