Transcript Studying the pathogenesis of renal disease in cystinosis

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)
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
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
4
Gahl et al. 2002
Treatment with cysteamine
cystinosin
cytoplasm
cytoplasm
cystine
NH2
cysteamine
cysteine –
cysteamine
cysteine
COOH
CH
lysosome
CH
CH2
CH2
CH2
CH2
CH
HS
S
+
CH2
NH2
NH2
CH
COOH
COOH
SH
CH2
S
NH2
lysosome
NH2
cystine
COOH
+
S
S
CH2
CH2
NH2
cysteine
transporter
5
CAT-
transporter
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
CTNS gene structure (17p13, 23 kb)
TAG
ATG
1
2
3
c
4
4
5
6
7
8
9
10
11
12
Cystinosin: predicted structure
YFPQA
GYDQL
Chergui et al. 2001
9
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
[35S] - C-S-S-C + H+
N
C
C
cytoplasm
lysosome
N
Kalatzis et al. 2001, 2004
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
al. 1991, Ben-Nun et al. 1993, Foreman et al. 1995, Wilmer et al. 2006)
– No prove in humans or in ctns -/- mice model
(Coor et
interstitium
lumen
nucleus
cytosole
lysosome
- LKG
influence on gene expression
cystine
cysteine
 oxidative redox
state
- LKG
-glut-cys
 GSSG
cystinosin
cystine reductase
GCS
 GSSG/GSH
Cysteinylation
of PKC-
 cystine
cystine
cystine
GS
H2O2

GSH

oxoglutarate carrier
dicarboxilate carrier
glucose
GSH reductase
mitochonrion
GSH
 GSSG
glycolytic
pathway
GSH
GSH peroxidase
Stimulation of
apoptosis
.
O2-
14
SOD
O2-. ADP
eI
H+
 lipid peroxides
H2O2
H2O2
II
III
H+
SOD
IV
O2-.
.
O2-
ATP
+
P V
e-
citric acid
cycle
pyruvate
NADH
FADH2
pyruvate carrier
H+
Disturbed ATP metabolism
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
Obstacles of gene
therapy in cystinosis:
• Gene should be delivered to all
organs
• Use of viral vectors
Liver from 6-month
old ctns -/- mice
• 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
Bambino Gesù
Children hospital,
Rome
F. Emma
A. Taranta
VUMC,
Amsterdam
L.Monnens
M. Wilmer M. Besouw B. Van den Heuvel
H. Blom
M. Wamelink
QUESTIONS?
Lysosomal membrane transport
H+
ATP
ADP
lysosome
Acid hydrolases:
Proteases
Glycosidases
Nucleases
Phosphatases
Sulphatases
H+
?
H+
Substrates:
Amino acids
Monosaccharides
Nucleosides
Di/tripeptides
Inorganic ions
Vitamins
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)
corneal 22
cystine crystals
cystinotic kerathopathy
cystinotic retinopathy
nucleus
cystine
cystine
cysteine
H+
cystinosin
cystine
cysteine
H+
cystine
cysteine
lysosome
protein degradation
cytoplasma
23
n= 67
n = 32
n = 17
24
Markello et al. 1993
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