Further understanding of the molecular basis of cystinosis and development of novel
therapeutics for the disease
C. Antignac, Inserm U574, Necker Hospital, Paris, France.
Progress report - December 2004
The gene underlying cystinosis, CTNS, was identified in 1998 by our laboratory, in collaboration with
William van’t Hoff and Margaret Town in London and encodes a novel lysosomal membrane protein.
Part of the project described here focuses on the pathophysiology of the renal anomalies associated
with infantile cystinosis. The other part of the project is aimed at further characterising the phenotype
of the cystinosis mouse model we have generated, and to use this animal model to test novel
pharmacological components. The whole project is aimed at better understanding the role of cystinosin
and the pathogenesis of cytinosis, and, in the long term, at attempting to develop a therapy, which is
more efficient and better tolerated than the treatment actually available today.
This project is currently being carried out by a research assistant, Nathalie Nevo (“Assistant
Ingénieur” at Inserm), a post-doctoral researcher Anne Bailleux (position funded by the Cystinosis
Foundation from October 2004) and a PhD student, Marie Chol (grant from AIRG) under the
supervision of Dr C. Antignac.
Results obtained in 2003-2004 are indicated in bold green.
Background
Cystinosis (MIM 21980) is an autosomal recessive disorder characterized by an accumulation of intralysosomal cystine. The infantile form appears at 6 to 12 months of age with a Fanconi syndrome and
progresses, if untreated, to terminal renal failure before the age of 10 (Gahl et al. 1995). Other clinical
signs (severe growth retardation, ocular anomalies, diabetes, portal hypertension, hypothyroidism,
neurological deterioration) are due to the accumulation of cystine in different organs. Allelic forms
with a later onset, or characterized exclusively by ocular anomalies, have also been described.
Treatment by the drug cysteamine, which reduces the concentration of intracellular cystine, delays the
progression towards renal failure and the appearance of other clinical anomalies if used early in the
disease and in high doses. This treatment, however, is not free of side effects, such as digestive
intolerance and a persistent nauseating odor, and requires regularly-spaced doses, thereby rendering its
administration difficult. The diagnosis of cystinosis can be confirmed by assaying intra-leukocyte
cystine levels, a test that also allows the identification of heterozygous carriers. It has been suggested
that the underlying metabolic defect of cystinosis is a defective transport of cystine across the
lysosomal membrane (Gahl et al. 1982). Lysosomal cystine transport has been studied biochemically
and it has been observed that ATP indirectly stimulates this transporter and that it is distinct from the
plasma membrane cystine transporter (Gahl et al. 1983). The gene for cystinosis was initially mapped
to the short arm of chromosome 17 in 1995 (The Cystinosis Collaborative Research Group 1995).
Work performed in the laboratory to date
- Cloning the causative gene and characterization of cystinosin, the CTNS gene product
We subsequently confirmed the chromosomal localization, reduced the genetic interval to 1 cM (Jean
et al, 1995) and, in collaboration with Margaret Town (Guy's Hospital) and William van't Hoff
(Institute of Child Health) in London, we identified the causative gene, CTNS, using a postional
cloning strategy. CTNS encodes a novel protein of 367 amino acids, which we named cystinosin.
Computer-aided sequence analysis of cystinosin predicted that the protein has 7 transmembrane
domains (TM) preceded by 7 potential glycosylation sites at the amino-terminal end, and followed by
a classic tyrosine-based lysosomal targeting signal (GY-XX-hydrophobic residue) at the carboxyterminal end. Taken together these elements were indicative of a lysosomal membrane protein, which
we confirmed by confocal microscopy, using constructs encoding cystinosin-GFP or mutant
cystinosin-GFP fusion proteins transfected in MDCK cells. We also demonstrated that cystinosin is
targeted to the lysosome by two lysosomal sorting signals, one classical tyrosine-based GYDQL
lysosomal sorting motif in its C-terminal tail, and a novel conformational lysosomal sorting motif in
the 5th inter-TM loop, both of which are oriented toward the cytoplasm (Cherqui et al., 2001). We have
also shown that cystinosin is a lysosomal cystine transporter, and that this transporter is highly specific
for L-cystine and is proton-driven (Kalatzis et al., 2001a), which explains the early observations on
whole lysosomes that ATP indirectly stimulates lysosomal cystine transport (Gahl et al. 1983). The
work on the subcellular localization and biochemical characterization of cystinosin has been
performed in collaboration with G. Trugnan (Inserm U538, Paris) and B. Gasnier (IBCP, Paris),
respectively.
- CTNS mutation screening and phenotype-genotype correlation
Since we identified the gene, we conducted a search for CTNS mutations in more than 100 patients
with cystinosis and detected the causative mutations, either deletions or point mutations, in 95% of
patients (Attard et al., 2000; Kalatzis et al., 2002). We characterized the junction breakpoints of a 57kb deletion and showed that it is due to a founder effect, which arose in a Caucasian individual around
the middle of the first millenium. In addition, we developed a rapid and reliable PCR-based assay for
the detection of this mutation, which is found in 76% of patients of European origin, either in the
homozygous or the heterozygous states (Forestier et al., 1999). Furthermore, we detected a small
deletion, 898-900+24del27, which affects the correct splicing of exon 8, in half of the families
originating from Brittany (Kalatzis et al., 2001b). We searched for point mutations in the CTNS gene
of individuals with nephropathic cystinosis for whom we had not detected homozygous deletions, as
well as of patients with late onset or atypical cystinosis. This led to the detection of either loss-offunction mutations or mutations resulting in amino acid substitutions or in-frame deletions/insertions
mostly located in the carboxy-terminal part of the protein or in the transmembrane domains for the
former group. In contrast, individuals with late onset or atypical cystinosis carried either one “severe”
mutation (known to be associated with infantile cystinosis) and one mild mutation, or two mild
mutations. These mild mutations resulted in amino acid substitutions or in-frame deletions/insertions
mostly located in the amino-terminal part of the protein or in the lysosomal loops (Attard et al., 1999
and unpublished results), in agreement with other reported mutation studies (Shotelersuk et al., 1998;
Anikster et al., 1999b). Furthermore, in a recent study by our group, thirty-one pathogenic
mutations (24 missense mutations, 7 in-frame deletions or insertions) were analyzed for the
transport activity and intracellular localization of the resulting mutant proteins. Most mutations
did not alter the lysosomal localization of cystinosin. Sixteen of 19 mutations associated with
infantile cystinosis abolished transport, whereas three of 5 mutations associated with juvenile or
ocular forms strongly reduced transport, consistent with the milder clinical phenotype. Five out
of 7 atypical, unclassified or misclassified mutations could be clarified using the transport data
and additional genetic information. Overall, our data demonstrate that, excluding premature
termination of cystinosin, impaired transport is the most frequent cause of pathogenicity, with
infantile cystinosis generally resulting from a total loss of activity (Kalatzis et al. 2004).
- Generation of a mouse model for cystinosin (collaboration with G. Hamard, Institut Cochin, Paris)
We cloned the murine homologue of CTNS, Ctns (Cherqui et al, 2000) and generated a knock-out
mouse model using a "promoter trap" approach, by replacing the last four Ctns exons with an IRES-neo cassette. This lead to the production of a truncated protein that, if stable, is mislocalized and
nonfunctional. The Ctns-/- mice show an accumulation of cystine in all tissues tested, as compared to
the controls, and this accumulation is present from birth and increases with age. The highest cystine
levels were consistently seen in the liver and the kidney, and the lowest in the brain. Moreover,
cystine accumulation was also observed histologically, in the form of cystine crystals. Cystine crystals
were detected in most tissues of 8 month-old Ctns-/- mice, though at a lower frequency than seen in
humans, most notably in the Kupffer cells of the liver, proximal tubular cells of the kidney, in skeletal
muscle and in the cornea by slit lamp examination. However, despite the significant cystine
accumulation in the kidney, the oldest mice do not present with any evidence of a proximal
tubulopathy or generalized renal dysfunction, as assayed by plasma and urine analyses. In contrast,
from 6-8 months of age, they show decreased activity and osteoporosis, muscular abnormalities in
some mice, as well as ocular lesions similar to those observed in patients (Cherqui et al., 2002).
Research project
The research project is aimed at understanding the pathogenesis of cystinosis and comprises the
following sections:
• Characterization of the role of cystinosin
The second motif we have identified does not resemble the lysosomal sorting motifs so far defined,
given its localization in a cytoplasmic inter-TM loop and not in the carboxy-terminal tail of the
protein. Furthermore, experiments in which cystinosin-GFP and cystinosin-GFP constructs deleted for
each of the lysosomal targeting signals were overexpressed in different cell lines, suggest that this
novel lysosomal targeting signal might play a role in lysosomal fusion. This hypothesis is further
strengthened by the recent identification of mutations in the VPS33B gene encoding a lysosomal
protein involved in vesicle membrane fusion in patients with ARC syndrome characterized by
arthrogryposis, cholestasis and renal tubular dysfunction (Gissen et al., 2004). In order to verify this
hypothesis and to better understand this novel signal, we will study lysosome formation and fusion in
cell lines already available in the laboratory, stably overexpressing cystinosin-GFP, either the
wild-type cystinosin or cystinosin deleted of one or both lysosomal targeting signals, by timelapse fluorescence in live cells using the FRAP (Fluorescence Recovery After Photobleaching)
technique. Depending upon the results, we will repeat the experiments in the presence of nocodazole,
to verify if microtubules are involved in the clustering processes, and of dominant negative mutants of
proteins known to regulate lysosome fusion (such as Rab7). In addition, to better assess the trafficking
of cystinosin within the cell, we will identify which adapter protein subunits interact with the GYDQL
-1 to AP-4 – reviewed in Bonifacino and Traub, 2003) and study the
consequences of their inactivation, using RNAi, on the subcellular localization of cystinosin.
This part will be performed in collaboration with the laboratory of G. Trugnan, in which the
techniques of live cell imaging are currently being performed.
• Refined characterization of the phenotype of the Ctns-/- mice on various genetic backgrounds
In order to verify whether the Ctns-/- mouse phenotype may be dependent upon genetic background,
we have transferred the Ctns mutation onto the C57BL/6 and FVB/N strains. In these congenic Ctns /- mice, we hypothesized, for example, that the tissue cystine content would be less variable. We are
now planning to better characterize the phenotypes observed in these strains. Wild-type and KO mice
are being sacrificed at various ages (3, 6, 9 and 12 months) and urine and blood samples are collected
as well as tissues for dosage of cystine content and histopathological studies.
The neurological phenotype of the Ctns-/- vs +/+ mice will be studied by Dr D. Trauner, who is
planning to spend a six-month sabbatical period in the laboratory. She will perform both behavioral
studies and brain histological studies in mice treated or not by cysteamine.
In addition, we will set up a controlled cysteamine trial in the strain with the more severe phenotype.
The treatment will be started early (either during gestation or after weaning) and will continue
throughout the lifetime of the mice. During the time of treatment, mice will be assayed for behavioral,
bone and ocular changes and compared with both Ctns+/+ and untreated Ctns-/- mice. In this way, we
will be able to determine the optimal dosage and the age at which to begin drug administration which
lead to optimal cystine clearance from various tissues. Additionally, the long-term effects of
cysteamine treatment, as yet unknown, will be studied.
Moreover, this work will also provide us with a direct comparative model to evaluate other therapeutic
agents. We are planning to set up a collaboration with Biomarin Pharmaceutical Inc (California)
to test S-palmytoyl cysteamine derivatives for the treatment of cystinosis.
• Study of the pathophysiology of the Fanconi syndrome (in collaboration with P. Rustin, Inserm
U393, Paris, France and M. Sarwal, Stanford University Medical School, Stanford, CA).
To perform part of these studies and to learn the cDNA microarray technology, Marie Chol has
made a two-week visit in the laboratory of Dr M. Sarwal at Stanford University, in October
2004.
Fanconi syndrome is the first symptom observed in infantile cystinosis in humans and appears as early
as six months of age (and even earlier), in association with diffuse proximal tubule alterations.
However, cystine crystals are rarely reported in tubular cells in patients and cysteamine is usually
inefficient against the Fanconi syndrome. These observations suggest that the proximal tubulopathy
seen in humans may be a secondary metabolic consequence of cystine accumulation rather than a
direct effect of cystine storage.Along this line, previous studies have shown that the lysosomes of
cystine-loaded rabbit proximal tubules display a significant reduction in intracellular ATP
concentrations, leading to an inhibition of Na/K-ATPase activity (Coor et al., 1991). These data
suggest that the mitochondrial oxidative phosphorylation process responsible for ATP synthesis is
impaired in cystinosis proximal tubular cells. Because these studies have been performed in vitro
using normal proximal tubular cells, the use of patient or KO mouse cells will be a useful tool for
verifying the above-mentioned hypothesis.
Despite cystine accumulation, the Ctns-/- mice that we generated do not present with either signs of a
proximal tubulopathy, even at two years of age, or with the severe and diffuse proximal tubule
alterations seen in humans. Nevertheless, they have focal cystine crystal deposits within proximal
tubular cells, and, large mitochondria are focally observed in their proximal tubular cells. Thus, the
lack of tubulopathy in Ctns/- mice might be accounted for by the existence of an alternative pathway
that rescues ATP depletion in murine proximal tubular cells. Another hypothesis could be the
existence of passive efflux of cystine from mouse lysosomes. The lysosomal cystine transporter
studied in mouse L-929 fibroblasts has the same characteristics as human cystinosin (Green et al.,
1990). However, it has been postulated that a non-saturable pathway for cystine efflux may exist in
mouse lysosomes (ibid). If such a pathway exists in mouse and not in humans, it may act to keep
cystine levels below the critical threshold for the appearance of the clinical signs associated with
cystinosis. Finally, the absence of the tubulopathy may be linked to the genetic background of the
Ctns-/- mice.
Nevertheless, these mice will be a valuable tool for studying oxidative phosphorylation in tubular cell,
under several experimental conditions. Futhermore, an alternative strategy is to exploit the cDNA
microarray technology to screen gene expression in the kidneys of Ctns-/- vs Ctns+/+ mice with the
goal of detecting, by gene expression profiling, compensatory changes in and/or activation of cellular
pathways. Alternatively, subtle changes in gene expression not sufficient to reach the threshold level
necessary to lead to clinical manifestations, might also be detected. This part of the project will be
conducted in collaboration with Dr Minnie Sarwal (Stanford University Medical School, Stanford,
CA), thereby providing us access to the Stanford mouse cDNA array which bears 40,000 cDNAs.
Experimental protocol
- Oxidative phosphorylation
The activity of oxidative stress response enzymes, as determined by assaying superoxide
dismutase (SOD) activity and gluthatione levels, has been performed in three untreated normal
and three patient fibroblast cell lines available in the laboratory. Indeed, a consistent although
limited reduction of glutathione content was noted in cystinotic cell lines as compared to
controls, as well as a moderate but significant induction of SOD activity (Chol et al., 2004). In
addition, we showed that a compensation of the gluthatione deficiency can be obtained by
adding a series of exogenous precursors of cyteine, including mercaptopyruvate, ornithine and
N-acetyl cysteine.
The experiments will now be done using conditionally immortalized Ctns-/- and Ctns+/+ mouse
tubular cell lines that we have generated in the laboratory by isolating proximal tubular cells of
Ctns-/- mice crossed with the heterozygous Immortomouse (Jat et al., 1991).
-Mouse cDNA microarrays
Hybridization of the mouse arrays will be performed at the mouse array facility of Stanford
University. However, RNA preparation and quality control, as well as data analysis, using the
bioinformatic tools generated at Stanford, will be performed in our laboratory. Significant variations in
the expression of genes will be validated by Real Time quantitative RT-PCR or by immunochemistry,
and will be further studied under other experimental conditions.
The first set of experiments will consist of comparing the gene expression levels of a limited number
of Ctns-/- whole kidneys vs littermate Ctns +/+ kidneys. The mice will be 6 to 8 months of age when
the cystine crystals and the mitochondrial abnormalities in proximal tubular cells are present.
So far, in a preliminary experiment comparing total kidney RNA from 4 Ctns-/- mice and their
control littermates, ~170 genes have been found that are upregulated in the Ctns-/- strain.
Interestingly, a several fold, highly significant, increase in the expression of genes involved in
apoptosis has been observed in Ctns-/- kidneys. This is in agreement with the recent work of
Park et al. (2002), which showed that lysosomal cystine accumulation augments apoptosis in
cultured human fibroblasts and in renal tubular epithelial cells. We are now analyzing the data in
more detail and are verifying the differential expression of several genes of interest by RT-PCR and
real time PCR in various tissues.
Given the results obtained in these preliminary experiments, several other experimental conditions will
be tested, in particular by using proximal tubular cell lines (cf supra) or a suspension of renal proximal
tubules, as a source of RNA.
• CTNS mutation screening
We provide CTNS mutation screening in patients with all forms of cystinosis. During 2003 and
2004, 8 patients and their families from four countries including Germany, Belgium, Morocco
and France, have been tested allowing the detection of new mutations both in infantile and
juvenile-ocular forms.
In conclusion, since we cloned the gene underlying cystinosis in 1998, we have successfully
elucidated the subcellular localisation, targeting and function of cystinosin, as well as generated a
mouse model of the disease. This mouse model now represents a unique tool for studying the
pathophysiology of cystinosis and for developing novel therapeutics. The funding of this current
proposal will allow us to continue our work in this direction, in the hope of developing a treatment that
is more efficient and better tolerated than the current cysteamine treatment.
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Financial aspects - Funding requested to the Cystinosis Foundation
In addition to the salaries paid by Inserm and the Cystinosis Foundation for Nathalie Nevo and Anne
Bailleux respectively and the grants from AIRG and, the laboratory received funding from Inserm and
from two French associations (VML – Vaincre les Maladies Lysosomales and AURA – Association
pour l’Utilisation du Rein Artificiel) which allows to cover most of the running costs.
Thus, the funding requested to the Cystinosis Foundation concerns:
- in absolute priority, the salary of the second and third years of the salary of Anne Bailleux
(48 000 euros a year including taxes, from October 2005)
- the cDNA microarrays which will be evaluated separately but would need ~20 000 euros.