[Neuroprotection of photoreceptor cells in rod-cone dystrophies: from cell therapy to cell signalling].
ABSTRACT Neuroprotection of photoreceptor cells in rod-cone dystrophies: from cell therapy to cell signalling. Neuroprotection of photoreceptor cells in rod-cone degenerations is primarily targeted at preventing the loss of function. Strategies for protecting rod cells should therefore aim not only at structural preservation but also must be assessed using functional parameters (e.g., electroretinogram). Given the number of mutations leading to an impaired visual response of rods, the preservation of cones is a realistic approach since (1) numerous mutations do not affect proteins expressed by cones; (2) the secondary degeneration of cones is the main event leading to profound visual impairment; (3) even a small proportion of functional cones is sufficient for major visual functions. Our group has (1) established and confirmed the existence of non cell autonomous mechanisms promoting cone cell viability; (2) shown that rod cell protection or replacement provides a mean to extend the survival of cones; (3) demonstrated that rod-cone trophic interactions are mediated by diffusible proteins; (4) identified by expression cloning a protein mediating such interactions: RdCVF (Rod-derived Cone Viability Factor). These studies provide clues for broad neuroprotective therapies of rod-cone dystrophies.
Article: Expression of rod-derived cone viability factor: dual role of CRX in regulating promoter activity and cell-type specificity.[show abstract] [hide abstract]
ABSTRACT: RdCVF and RdCVF2, encoded by the nucleoredoxin-like genes NXNL1 and NXNL2, are trophic factors with therapeutic potential that are involved in cone photoreceptor survival. Studying how their expression is regulated in the retina has implications for understanding both their activity and the mechanisms determining cell-type specificity within the retina. In order to define and characterize their promoters, a series of luciferase/GFP reporter constructs that contain various fragments of the 5'-upstream region of each gene, both murine and human, were tested in photoreceptor-like and non-photoreceptor cell lines and also in a biologically more relevant mouse retinal explant system. For NXNL1, 5'-deletion analysis identified the human -205/+57 bp and murine -351/+51 bp regions as having promoter activity. Moreover, in the retinal explants these constructs drove expression specifically to photoreceptor cells. For NXNL2, the human -393/+27 bp and murine -195/+70 bp regions were found to be sufficient for promoter activity. However, despite the fact that endogenous NXNL2 expression is photoreceptor-specific within the retina, neither of these DNA sequences nor larger upstream regions demonstrated photoreceptor-specific expression. Further analysis showed that a 79 bp NXNL2 positive regulatory sequence (-393 to 315 bp) combined with a 134 bp inactive minimal NXNL1 promoter fragment (-77 to +57 bp) was able to drive photoreceptor-specific expression, suggesting that the minimal NXNL1 fragment contains latent elements that encode cell-type specificity. Finally, based on bioinformatic analysis that suggested the importance of a CRX binding site within the minimal NXNL1 fragment, we found by mutation analysis that, depending on the context, the CRX site can play a dual role. The regulation of the Nucleoredoxin-like genes involves a CRX responsive element that can act as both as a positive regulator of promoter activity and as a modulator of cell-type specificity.PLoS ONE 01/2010; 5(10):e13075. · 4.09 Impact Factor
Neuroprotection of photoreceptor cells in rod-cone dystrophies: from cell therapy to cell
Authors: José-Alain Sahel and Thierry Léveillard
Inserm Unit 592 : Physiopathologie Cellulaire et Moléculaire de la Rétine, Paris / Université
Pierre et Marie Curie, Paris / Institute of Ophthalmology- University College London
Departments of Ophthalmology : Centre Hospitalier National d’Ophtalmologie des Quinze-
Vingts / Fondation Ophtalmologique Rothschild
Neuroprotection of photoreceptor cells in rod-cone degenerations is primarily targeted at
preventing the loss of function. Strategies for protecting rod cells should therefore aim not
only at structural preservation but also must be assessed using functional parameters (e.g.
ERG). Given the number of mutations leading to an impaired visual response of rods, the
preservation of cones is a realistic approach since 1) numerous mutations do not affect
proteins expressed by cones; 2) the secondary degeneration of cones is the main event leading
to profound visual impairment; 3) even a small proportion of functional cones is sufficient for
major visual functions.
Our group has 1) established and confirmed the existence of non cell autonomous
mechanisms promoting cone cell viability; 2) shown that rod cell protection or replacement
provides a mean to extend the survival of cones; 3) demonstrated that rod-cone trophic
interactions are mediated by diffusible proteins; 4) identified by expression cloning a protein
mediating such interactions: RdCVF (Rod-derived Cone Viability Factor).
1) The rd1 mouse is a model of rod-cone degeneration. A recessive mutation carried by the
gene encoding the beta subunit of the rod-phophodiesterase is leading to the rapid
degeneration of rod photoreceptors through apoptosis and a secondary degeneration of cones,
while these neurons to not express the mutated gene and are not directly suffering form the
enzymatic deficit. This model does mimick the sequence of evets in patients affected with
Rod-Cone dystrophies i.e. primary loss of dark-adapted vision followed with loss of central
and light-adapted vision. By transplanting normal photoreceptors in the subretinal space of the
rd1 mouse immediately after rod death, we have demonstrated that the cones from the grafted
animal are surviving significatively longer than the shamed animals (Mohand-Saïd et al.,
2000). This confirms the existence of non cell autonomous mechanisms promoting cone
viability (Mohand-Saïd et al., 1998).
2) The most straightforward interpretation of our data is that cone degenerate when rods are
missing. We have further validated this model by showing that the trophic factor GDNF, and
the pharmacological treatment of the rd1 mouse with the calcium channel diltiazem are
generating rod protection that is translated by the persistence of cone function (Frasson et al.,
1999a; 1999b). It should be noticed that future therapeutic approaches aimed at preserving
cone function would be optimal.
3) In order to study the effect of rods on cones, we have used a co-culture system. Rod-less
retinal explants from the rd1 mouse co-cultured with rod-enriched retina show a 40% rescue
of cones. Not only this experiment demonstrated that rod are exerting a trophic activity
toward cones in vitro, but it also established that the trophic activity is diffusible (Mohand-
Saïd et al.,1998). The activity is present in the conditioned media prepared from rod-enriched
retina. Using a partial purification scheme, we have shown that the activity is carried by
molecules with the physical properties of proteins (Fintz et al., 2003).
4) We have adopted a systematic high content screening approach based of a cone-enriched
cultures system and an expression cloning protocol to identify from an expression library
clones that encode for cone viability factor. By screening 210,000 clones, we have identified a
factor Rod-derived Cone Viability Factor (Léveillard et al., 2004). RdCVF is expressed in a
rod dependant manner and the activity of rod-enriched conditioned medium is largely
decreased by RdCVF antibodies. The current model is that cones degenerate in the rd1 mouse
by trophic support withdrawal after rod degeneration and RdCVF loss of expression. RdCVF
is a truncated thioredoxin produced by a novel gene Txnl6. Our laboratory is currently
working on RdCVF delivery and signaling.
These studies provide a rationale for protecting non functional rods and clues for broad
neuroprotective therapies of rod-cone dystrophies. Therefore, planning for clinical trials
implies the identification of genotyped patients with symmetrical progressive disease and the
implementation of outcome measurements that allow early and reproducible detection of both
rod and cone function and structure. Such facilities and programs (from fine matrix mapping,
autofluorescence confocal imaging, optical coherence tomography and adaptative optics are
currently implemented at the Clinical Investigation Center of the Centre Hospitalier National
d’Ophtalmologie des Quinze-Vingts.
FINTZ AC, AUDO I, HICKS D, MOHAND-SAID S, LEVEILLARD T, SAHEL JA. Partial characterization of retina-
derived cone neuroprotection in two culture models of photoreceptor degeneration. Invest Ophthalmol Vis Sci.
FRASSON M., PICAUD S., LÉVEILLARD T., MOHAND-SAÏD S., DREYFUS H., HICKS D., SAHEL J-A. - Glial cell-line-
Derived Neurotrophic Factor induces structural and functional protection of rod photoreceptors in the retinal
degeneration (rd/rd) mouse. Invest Ophthalmol Vis Sci 1999a, 40 : 2724-2734.
FRASSON M., SAHEL J-A., SIMONUTTI M., DREYFUS H., PICAUD S. - Retinitis pigmentosa : rod
photoreceptor rescue by a Ca2+ channel blocker in the rd mouse. Nature Medicine, 1999b, 5 : 1183-1187.
LEVEILLARD T, MOHAND-SAID S, FINTZ AC, LAMBROU G, SAHEL JA. The search for rod-dependent cone
viability factors, secreted factors promoting cone viability. Novartis Found Symp. 2004;255:117-27; discussion
LEVEILLARD T, MOHAND-SAID S, LORENTZ O, HICKS D, FINTZ AC, CLERIN E, SIMONUTTI M, FORSTER V,
CAVUSOGLU N, CHALMEL F, DOLLE P, POCH O, LAMBROU G, SAHEL JA. Identification and characterization of
rod-derived cone viability factor. Nat Genet. 2004 Jul;36(7):755-9.
MOHAND-SAID S., DEUDON-COMBE A., HICKS D., FINTZ A.C., SIMONUTTI M., FORSTER V.,
LEVEILLARD T., DREYFUS H., SAHEL J-A. - Normal rod photoreceptors increase cone survival in the
retinal degeneration (rd) mouse. Proc. Natl. Acad. Sci. 1998, Vol 95 (14), 8357-8362.
MOHAND-SAID S., HICKS D., DREYFUS H., SAHEL J.A.. – Selective transplantation of rods delays cone
loss in a retinitis pigmentosa model. Arch. Ophthalmol. 2000 ; 118 :807-811.
SAHEL J.A., MOHAND-SAID S., LEVEILLARD T., HICKS D., PICAUD S., DREYFUS H. – Rod-cone
interdependence : implications for therapy of photoreceptor cell diseases. Prog Brain Res 2001;131:649-61