ANIRET (Dr. Arpad Palfi) Scientific Results:

Degenerative retinopathies, prominent among which is retinitis pigmentosa, probably represent the most genetically heterogeneous of any hereditary condition for which molecular pathologies have so far been explored, such mutational heterogeneity representing a significant challenge in the development of economically viable therapeutics.

During the course of the research programme, animal models have either been developed for the purposes of workup of novel therapeutic approaches (see McNally et al, 2002) or have been used in the identification of novel retinopathy genes (see Kennan et al, 2002). In respect to the validation of mutation-independent approaches to therapy for retinitis pigmentosa based upon mutations within the rhodopsin gene, a transgenic murine model (Rho-/-) with a modified replacement human rhodopsin gene with therapeutic potential has been developed (see O'Reilly et al, 2004). In addition, RNAi-mediated suppression of transcripts derived from both the rhodopsin and rds-peripherin genes have been optimized (see Palfi, 2004; Kaing and Palfi, 2004).

These data provide support for the development and deployment of genetically-based therapeutic approaches for autosomal dominant forms of retinal degeneration representing a major cause of registered visual handicap among those of working age in all member states of the European Union.

CLINRET (Dr. Barbara Innocenti) Scientific Results:

Apoptosis is a widespread normal phenomenon occurring during development of the nervous system. During the first week of rat retina development, almost 50 % of RGCs die during the first two weeks of rat retinal development, before definitive connections to the brain are established. In addition, at that time, microglial cells invade in high number the retina, removing the bodies of damaged and dead cells.

Several ATP receptors (P2 and P2 subunits) are expressed in mammalian retina. P2X7 is a receptor-operated channel permeable to cations such as Na2+, Ca2+ and K+. Under prolonged or repetitive stimulation, its activation induces the opening of large pores permeable to molecule up to 900 Da. P2X7 activation could lead to cell permeabilization and thus cell death.

If P2X7 is involved in cell death events, the expectation is that young retinas express P2X7 receptor at high levels. Actually we discovered that P2X7 is expressed as early as post-natal day 2 and it is highly expressed in the GCL and the inner part of the neuroblastic (NB) layer during the first week of retinal development. Furthermore, YO-PRO-1 (indicator for cell permeabilization) uptake experiments showed that ATP permeabilizes a significantly higher number of retinal cells during the first week of retinal development compared to later stages.

The results of the studies will help us to better understand the function of the neurotransmitter ATP in mammalian retina. In addition, the discovery that ATP signaling plays a role in the induction and/or maintenance of retinal cellular degeneration may leave major consequences for developing new therapeutic strategies.

GENERET (Dr. Christina Chakarova) Scientific Results:

During the three years of RETRAINET project our work has focused on the molecular genetic aspects of retinal degeneration. A new splicing factor gene PRP3) has been identified to cause a dominant form of retinitis pigmentosa (RP18) see Chakarova et al, 2002) on chromosome 1q. Two recurring mutations (Pro493Ser and Thr494Met) were identified in adjacent codons and were used for site-directed mutagenesis of the wild type (WT) cDNA and cloned into an expression vector. The WT and mutant constructs have been used to investigate its cellular localization, protein function and possible protein-protein interactions. It was noted that the mutations did not interfere with the nuclear localisation of the protein.

A new gene causing RP17 on chromosome 17q has been cloned (see Yang et al, 2004 in press). The gene is carbonic anhydrase 4 (CA4) and is thought to impair pH regulation that eventually results in photoreceptor degeneration Three mutations have been identified in two different exons and at the 3'UTR of the CA4 gene.

We have collected DNAs from two new Roma (Gypsy) families from Bulgaria with very severe form dominant RP (adRP). Preliminary linkage analysis using microsatellite markers show that these families are excluded from all known loci for adRP. Therefore these families represent a potential source for novel RP genes.

THERET (Dr. Kudasiewicz-Kardaszewska) Scientific Results:

The studies aim at analyzing the significance of the host gliotic activity on the process of graft-host integration following subretinal transplantation of retinal cells. Green fluorescent protein-expressing (GFP+) donor retinas (6 days-old) were subretinally transplanted to adult rd1 mice deficient in glial fibrillary acidic protein (GFAP) and vimentin (vim). The number of GFP+ bridging fibers and of GFP+ cells within the host rd1 retina was verified after 7-21 days after surgery.

The same analysis was carried out in vitro, by co-culturing retinas of GFP mice with retinas of adult rd1, GFAP-/-vim-/- mice. As controls, retinas from rd1 mice expressing GFAP and vim were used both in vivo and in vitro. Integration was observed in all groups analyzed. However, no significant difference was found between transplantation to GFAP-/-vim-/- deficient mice and to their congenic controls. In both groups, integration was limited to places where the outer limiting membrane (OLM) of the rd1 retina appeared disrupted. The same observations were made in vitro.

The results indicate that the OLM plays a major role in limiting graft-host integration and that decreased gliotic activity within the host retina does not have a significant effect. However, it is still possible that lack of GFAP and vim may not be sufficient to effectively reduce the inhibitory influences of reactive gliosis on regeneration.

PRORET (Dr. Carolina Valera-Rodriguez) Scientific Results:

The main aim of the project was to test from a functional point of view the visual function of the mouse models of the Duchenne Muscular Dystrophy. Specially the mdx3cv, that has a severe reduction of the expression of all types of Dystrophins. Oriented to this purpose we planned two types of approaches, the patch-clamp recording and a behavioural test that based in the optokinetic reflex try to find out a measure of the visual acuity. The use of the optokinetic nistagmus as a measure of the "visual acuity" is done counting the number of head movements of each mouse in response to nine different optotypes that turn around it.

Patch-clamp recording in isolated retinal cells: To perform the experiments of patch-clamp in isolated cells is necessary to kill one mouse par day and it was not possible until now because we had not enough number of mutant and transgenic mice. Nowadays we have got a sufficient quantity of mice to perform a rational study. However, we have already notice that the mutant mice mdx seems to have a GABAergic problem at the amacrine cells level.

Optokinetic reflex: With this test we have already notice that the mdx3cv mutants have an impairment of the "visual acuity" statistically significant (n = 6; p< 0,05) in scotopic conditions and compared with a control group of littermates that have obviously the same genetic background. In photopic condition it seems to do not exist any difference between both groups. The mutant mdx seems to have behavioural difference but not easily quantifiable. So, the "visual acuity" can be considered unchanged but the number of movement of the head are surprisingly reduced in both scotopic and photopic conditions. Always compared with a control group with the same genetic background. The Knock-Out for the Dp71 has been test but we have not already some control experiments to compare. In comparison with control mice of a different genetic background it seems to do not exist any difference in young ones but in old mice. However this transgenic mice developed a very small cataract that can justified this results.

Different molecules including ionic channel blockers were shown to limit rod photoreceptor degeneration in different animal models of retinitis pigmentosa. To further understand the molecular mechanisms of this neuroprotection, it became crucial to develop a preparation allowing to record mammalian rod photoreceptors with the patch clamp technique. The photoreceptor physiology was indeed mainly investigated in low vertebrate species which have large photoreceptor cells easily recordable. We have here developed a preparation from the pig retina allowing stable recording from rod photoreceptors with the patch clamp technique. Rod physiological features and the effect of some calcium channels were investigated on this preparation. Rod cells expressed:

(1) a hyperpolarization-activated inward-rectifying conductance [Ih] sensitive to external Cs+,
(2) a sustained outward K+ current [IK] sensitive to tetraethylammonium,
(3) a sustained voltage-gated Ca2+ current [ICa] sensitive to benzothiazepine (diltiazem) and phenylalkylamine (verapamil) derivatives,
(4) a Ca2+-activated Cl- current [ICl(Ca)],
(5) a plasma membrane Ca2+-ATPase.

By contrast to other L-type Ca2+ channels, rod Ca2+ channels were blocked at relatively high concentrations by the diltiazem isomers and verapamil. The Ca2+-ATPase, which was localized at the axon terminal, was found to contribute to Ca2+ extrusion. These results indicated that rod physiological features were preserved during evolution. They showed further that rod neuroprotection by ionic channel blockers could be attributed to the block of cGMP-gated channels rather than that of Ca2+ channels.