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Research fields
Team 01:
Genetic and therapy of retinal blindness
Inherited retinal dystrophies
and the visual cycle in the retinal pigment epithelium
Research Group : Tel :
+33 (0) 499 636 052 Fax
: +33 (0) 499 636 020
Philippe Brabet (CR1 INSERM) Mail : brabet@montp.inserm.fr
Carl Arndt (PH) Mail
: carl.arndt@chu-montpellier.fr
Marie Pequignot (Post-doctoral fellow) Mail
: pequignot@montp.inserm.fr
Thomas Guignard (Ph.D. student) Mail
: thomG@montp.inserm.fr
Karim Chekroud (Ph.D. student) Mail
: chekroud@montp.inserm.fr
Laurent Guillou (TR INSERM) Mail
: lguillou@montp.inserm.fr
Collaborators :
Marcel Mersel (DR2 INSERM, INM) Mail: mersel@univ-montp1.fr
Yvan Boublik (IFR122, CNRS, CRBM) Mail: yvan.boublik@crbm.cnrs.fr
Andreas Stahl (Palo Alto, CA, USA) Mail: astahl@stanford.edu
Claire Rogel-Gaillard (UMR INRA CEA 314) Mail : rogel@jouy.inra.fr
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Our objective
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Methodologies
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References
- Lab
members
Our
objective
| Our main goal is to identify new genes regulating
key steps of the visual cycle in the RPE cells, to get
insights into the molecular mechanisms which
contribute to the retinal dystrophies, and to propose therapeutic
strategies to slow the progression of diseases
involving the visual cycle. |
| Vitamin A and its derivatives (retinoids)
are essential components in vision. In the retina of
vertebrates, the Visual Cycle, which
is an enzymatic pathway of retinoids derived from beta-carotene,
is essential to light perception. Mutations of enzymes
and binding proteins involved in this process lead to
retinal degenerations and phenotypically heterogeneous
forms of blindness named pigmentary retinopathies
and macular dystrophies. Besides, visual cycle
promotes lipofuscin accumulation in the RPE, a phototoxic
deposit involved in several retinal dystrophies including Stargardt
disease, Best macular dystrophy and possibly
in age-related retinal degenerations. |
In the rod and cone
photoreceptors, G protein-coupled receptors
called opsins combine with a chromophore 11-cis retinal
to constitute the visual pigments that mediate photon
absorption. This capture generates 11-cis to all-trans
isomerization and leads to the removal of all-trans
retinal. The visual cycle aims to restore the 11-cis
retinal chromophore and so the sensitivity to light.
The retinal pigment epithelium (RPE) is a monolayer of cells
adjacent to the photoreceptors that supports many physiological functions of
the retina and notably most of the rod visual cycle steps. Key steps of the retinoid
cycle are fulfilled by three major enzymes located to endoplasmic reticulum of
the RPE. First, LRAT (lecithin retinol acyl transferase) adds
a fatty acid to photo-generated all-trans retinol forming all-trans retinyl ester.
Second RPE65, a very abundant RPE-specific protein that has
been recently demonstrated to endow the isomerohydrolase activity, triggers the
conversion of all-trans retinyl ester to 11-cis retinol. The lack of this essential
enzyme is responsible for early onset forms of retinal dystrophies such as Leber
congenital amaurosis, and childhood early onset retinal dystrophies
(Marlhens et al., 1997; Gu et al., 1997). At last, 11-cis retinal is
formed by 11-cis RDH and goes back to the photoreceptors to
regenerate visual pigments. |
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The visual cycle in vertebrate |
Methodologies
Initial
two-hybrid approach
In order to identify novel RPE genes and to enlighten
the regulation mechanisms of the visual cycle and RPE65-dependent
isomerization step, we have investigated RPE65-interacting
proteins through a yeast two-hybrid approach
using full-length human RPE65 as a bait. Our screening
has revealed 12 positive interactions with RPE65 in yeast
among the 1.2x106 clones tested from a porcine RPE mRNA
library. |
Among them, we found:
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energetic metabolism proteins
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scaffolding proteins
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hypothetical proteins
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background proteins
Molecular and
cellular biology
We apply tools and technologies developed for the
exploration of the specificity, dynamic and functional
impact of the protein interactions:
- cDNA collection encoding the main isomerisation
partners (RPE65, LRAT, RDH5, CRALBP) and some of
the newly discovered interacting proteins
- Recombinant protein production systems (in
vitro translation, bacteria, yeast, insect
cells, human cell lines) in collaboration with
the recombinant protein production platform directed
by Y. Boublik (UFR122, CNRS, CRBM
UMR 5237, Montpellier).
These
cellular systems will allow us to describe the localization
of the various partners (fluorescence confocal microscopy,
Hassan Boukhaddaoui and the staff of the Montpellier
RIO Imaging, MRI, facility), to measure
their own activity and the effects of the substrates
and products of all enzymatic partners (retinoid dosage,
fatty acid uptake, glycolysis) and to study the role
of the folding proteins in the recruitment of RPE65
to the membranes (subcellular fragmentation, immunocytochemistry). |
Biochemistry
of the visual cycle
In order to identify modulators of the visual cycle
in the RPE, Laurent Guillou is using analytic HPLC
to follow the conversion of vitamin A (all-trans retinol)
into 11-cis retinol and to dose the various retinoid
species. We apply this technique in vitro onto
porcine RPE microsomes and Sf9 insect cells over-expressing
the recombinant human proteins, and in vivo onto
the mouse RPE. |
Animal models
Behavioural
and pathophysiological studies in normal and deficient animal
The physiological impact of the interacting proteins
with a known function will be evaluated on normal and
genetically modified mouse models (knockout or transgenic),
either available or to be developed (double-knockout). |
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Innovating methodology to developped
animal models
In the team, G. Lenaers group developed
a novel approach of injection of small interfering
RNA (siRNA) directly in the vitreous of young mice
(post-natal day 11: P11, Chen-Kuo-Chang M.,
Abstract #3119, ARVO 2005) and in adults (Depeyre
C., Chen-Kuo-Chang M. and Arndt C., J Fr Ophtalmol.
2006 29(8):875-80). The histochemical and functional
validation of this approach at the two stages of development
will provide a very interesting and novel tool to explore
the function of RPE genes. |
The Drosophila model
As in vertebrates, vitamin A derivatives serve as
the visual chromophores in Drosophila. A β,β-carotene-15,15’-oxygenase
(BCO) catalyzes the conversion of β-carotene to
vitamin A. Only one BCO family member encoded by the ninaB gene
is found in the entire Drosophila genome.
The high degree of overall sequence identity (40%)
shared by RPE65 and BCO in vertebrates points to a
related biochemical function and the existence of a
common ancestor. However,the RPE65 function does not
include β-carotene cleaving activity. The protein
required for the conversion of vitamin A to chromophore
in Drosophila retinal pigment cells has still
to be identified. |
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Phylogenetic tree calculation of
BCO |
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Genetic
The genes encoding the interacting proteins are
potential candidates for the mutation screening in
patients with retinal dystrophies. |
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Two truncating RPE65 mutations in individuals with Leber congenital amaurosis. Fundus examination of patients 2 and 3, aged 20 years and 13 years, respectively, showed numerous yellowish spots in the outer layers of the retina.
From Marlhens F et al. (1997). Mutations in RPE65 cause Leber's congenital amaurosis.
Nat Genet. 1997;17(2):139-41. |
References
Rando RR. The biochemistry of the visual cycle.
Chem Rev. 2001; 101(7):1881-96.
Thompson DA, Gal A. Vitamin A metabolism in the retinal
pigment epithelium: genes, mutations, and diseases.
Prog Retin Eye Res. 2003;22(5):683-703.
Redmond TM, Yu S, Lee E, Bok D, Hamasaki D, Chen N,
Goletz P, Ma JX, Crouch RK, Pfeifer K. Rpe65 is necessary
for production of 11-cis-vitamin A in the retinal visual
cycle. Nat Genet. 1998; 20(4):344-51.
M. Jin, S. Li, W. Moghrabi, H. Sun, G. Travis. Rpe65
Is the Retinoid Isomerase in Bovine Retinal Pigment
Epithelium. Cell 2005; Volume 122, Issue 3, Pages 449-459.
Moiseyev G, Takahashi Y, Chen Y, Gentleman S, Redmond
TM, Crouch RK, Ma JX. RPE65 is an iron(II)-dependent
isomerohydrolase in the retinoid visual cycle. J Biol
Chem. 2006; 281(5):2835-40.
Guignard T, Pequignot MO, Ayoub B, Weber A, Ripoll
C, Hamel C, Brabet P. Energetic metabolism proteins
interact with RPE65 in vitro. ABSTRACT # 2030 - ARVO
2006
P. Brabet, Y. Chassigneux, T. Guignard, and C. Hamel.
Search for new partners of RPE65 in retinal pigment
epithelium. Invest. Ophthalmol. Vis. Sci. 2004 45:
E-Abstract 1257. |
Lab members
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