1. Nephropathic cystinosis: 10th anniversary of the CTNS gene Elena Levtchenko, MD, PhD University 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
incidence ~1:200,000
clustering in some populations
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. Treatment with cysteamine
cystinosin
cytoplasm
cytoplasm
cysteine –cysteamine
cystine
cysteamine
cysteine
NH COOH CH
CH2 S
NH COOH
lysosome
NH COOH CH
CH2 S
NH2 SH
CH2 CH
NH COOH HS
CH2
NH2
CAT-
transporter
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 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

9. CTNS gene structure (17p13, 23 kb)
ATG
TAG 1 2 3c 44 5 6 7 8 9 10 11 12
Cystinosin: predicted structure
GYDQL
YFPQA
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

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

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

13. 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

14. - LKG - LKG cystine interstitium lumen cystine cysteine  cystine
nucleus
cytosole
lysosome
- LKG
cystine
influence on gene expression
cysteine
 cystine
cystinosin
 oxidative redox state
cystine reductase
GCS
   cystine
- LKG
-glut-cys
cystine
Cysteinylation of PKC-
 GSSG/GSH GS
H2O2
 GSSG
 GSH
oxoglutarate carrier
dicarboxilate carrier
glucose
GSH reductase
mitochonrion
glycolytic pathway
GSH
 GSSG
GSH
H2O2
GSH peroxidase
citric acid cycle
O2-.
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
 lipid peroxides
H2O2
O2-.
Disturbed ATP metabolism 14

15. 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

16. Obstacles of gene therapy in cystinosis:
Molecular therapy, vol.16, No.8, , 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

17. 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.

18. Cystinosis research group Nijmegen/Leuven
Thanks to
Bambino Gesù Children 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

19. QUESTIONS?

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

21. 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 ceroid lipofiscinosis (CLN7)

22. 22
corneal cystine crystals
cystinotic kerathopathy
cystinotic retinopathy

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

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

26. Cystine accumulation in cystinosis
Stokes et al. 2008
Wilmer et al. 2008
Kidney: x normal
Liver: x normal
Muscle: x normal
Brain: x normal