Nephropathic cystinosis:

10th 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 – 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)

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

5

Treatment with cysteamine

cystine

cytoplasm

cystinosin

lysosomeNH2 COOH

CH

CH2

S

S

CH2

CH

NH2 COOH

+

HS

CH2

CH2

NH2

SH

CH2

CH

NH2 COOH

NH2 COOH

CH

CH2

S

S

CH2

CH2

NH2

+

cystine cysteamine cysteinecysteine –cysteamine

cysteine transporter

CAT-

transporter

cytoplasm

lysosome

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)

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)

1 2 3c

44

5 6 7 8 9 10

11 12

ATG TAG

Cystinosin: predicted structure

GYDQLYFPQA

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)

Functional assay of cystinosin

C

N

C

N

[35S] - C-S-S-C

lysosome

cytoplasm

Kalatzis et al. 2001, 2004

+ H+

Expression of Cystinosin-LKG is not restricted to lysosomes

Taranta et al. 2008

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

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lysosome

mitochonrion

nucleus

cystine

cystine

cysteinecystine reductase

cystinosin

GCS

-glut-cys

GS

GSHoxoglutarate carrier

I II III IV V

ATPO2-. O2-.H2O2

SOD

GSSGGSH peroxidase

GSH

GSH reductase

dicarboxilate carrier

GSSG

oxidative redox state

GSSG/GSH

influence on gene expression

Disturbed ATP metabolismO2-.

glucose

pyruvate

glycolytic pathwayGSH

citric acid cycle

NADH

FADH2

Stimulation of apoptosis

H2O2

SOD

lipid peroxides

H+

e-

e-

H+

ADP

+

P

O2-.

pyruvate carrier

H+

H2O2

lumen interstitium

cytosole

cystine

Cysteinylation of PKC-

- LKG

- LKG

cystine

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

Molecular therapy, vol.16, No.8, 1372-1381, aug.2008

Liver from 3-month old ctns -/- mice

Liver from 6-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

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.

Thanks to

Cystinosis research group Nijmegen/Leuven

L.Monnens M. Wilmer M. Besouw B. Van den Heuvel

Bambino Gesù Children hospital, Rome

F. Emma

A. Taranta

VUMC, Amsterdam

H. Blom

M. Wamelink

QUESTIONS?

Lysosomal membrane transport

lysosome

H+

H+

ATPADP

Acid hydrolases:ProteasesGlycosidasesNucleasesPhosphatasesSulphatases

H+

Substrates:Amino acidsMonosaccharidesNucleosidesDi/tripeptidesInorganic ionsVitamins

?

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)

22corneal cystine crystals cystinotic kerathopathy cystinotic retinopathy

23

nucleus

H+

H+

cystine

cystine

cysteine

lysosome

cytoplasma

cystinosin

protein degradation

cystine

cystine

cysteine

cysteine

24

Markello et al. 1993

n= 67

n =

32

n = 17

Cystine accumulation in cystinosis

Kidney: 200 - 400 x normal

Liver: 80 - 1000 x normal

Muscle: 40 - 70 x normal

Brain: 5 - 20 x normal Stokes et al. 2008

Wilmer et al. 2008