Comprehensive analysis of rod-cone photoreceptor degeneration associated with rhodop

The Austrian partners will be mainly contributing to Workpackages 4 (Rod and cone function in animal models of RCD carrying rod-specific gene mutations) and 5 (Assess the effect of potential novel therapies on rod and cone degeneration in animal model of RCD). Workpackage 4: We will study rod and cone structure and function in two animal models with rod-specific mutations, (rd10 mouse, P23H rat) which are key actors in preclinical studies, and share similarities with RCD in humans. Imaging abnormalities and functional tests will be correlated with histology and immunological markers at different time points of interest in sections or in whole mount retinas. Variance of topographic relationships and proportions of rods and cone spectral subtypes as well as of morpho- functional remodelling of cone organelles and degradation patterns in associated interneurons. As there are indications for regional differences in damage susceptibility (Vaughan et al., 2003), particular attention will be drawn on the topographic and temporal variation of opsins based on dorsoventral retinal co-expression gradients present in mouse and rat strains (Glösmann & Ahnelt 2002, Glösmann et al., 2009). Photoreceptor cell death will be molecularly tracked by using fluorescent substrates or immunostaining in retinal sections from the two murine models. These phenotypic data will be of great value while testing therapeutic strategies. These data will be not only crucial for the assessment of degenerative events but may also be useful to identify specific effects of disease related events as well as of treatment strategies onto opsin/cone photoreceptor subtypes.

To investigate fractalkine (CX3CL1) protein expression in wild type (wt) retina and its alterations during retinal degeneration in mouse model (rd10) of retinitis pigmentosa.Approximately one among 4000 EU citizens is affected by genetic defects in the low light sensitive photoreceptor cells the so-called rods and the associated signal pathways. This leads to progressive night blindness. Unfortunately, also the photoreceptors for high quality daylight vision the cone photoreceptors (not impaired by the mutations themselves), may also be affected by the degeneration process, for reasons not yet well understood. Eventually this may lead to severe deficits in vision and to complete blindness.The EU (e-rare) funded project RHORCOD (2010-2014) focused on the expansion of knowledge about the underlying genetic defects, as well as on the investigation of resulting cellular degradation processes and the associated functional consequences. This knowledge is a prerequisite for finding starting-points to therapies for slowing down or even preventing these disease outcomes. In animal models with the same genetic defects crucial elements of the involved processes can be investigated in fast motion.The Austrian RHORCOD sub project at the Centre for Physiology and Pharmacology focused on signal molecules which actual initiate the degeneration of affected sensory cells. Mice with the same rd10 mutations as occurring people provide a time lapse model. They develop similar retinitis symptoms soon after birth and lose a large part of the photoreceptors after 3 months. This depletion is carried out by so-called microglia, which occurs in inactive forms anywhere in central nervous tissues. This "regulatory mode of microglia is maintained as their chemokine receptors (CX3CR1) are acting as docking stations for the specific CX3CL1 (Fractalkine) ligand types from the Chemokine family of signaling molecules. These CX3C ligands are formed on the cell membrane of neurons and may be delivered in different subforms (100, 95 and 85 kiloDalton). The Austrian working group has compared the proportions of these Fractalkine subforms during the first months of normal (wild-type) mice with those occurring in rd10 mice at different stages of retinal disease progression.Already at the 10th postnatal day, the rd10 mice 85 kDa CX3CL1 form of Fractalkine showed a relative increase while in the retinas of wild type mice still the 95 kDa and 100 kDa forms dominated. This shift occured well before other changes in glial cells and photoreceptors could be detected with immune histological methods. Such pathologic alterations become detectable at about the 3rd week of life and then proceed towards a stage of widespread depletion of the photoreceptor layer after 60 days. In situ hybridization and transgenic mCherry reporter fluorescence methods allowed identifying that not the photoreceptors themselves but rather certain neuron types in the inner retinal layers are places of synthesis for CX3CL1.Apparently a conversion of the ligands, from a membrane-associated (100 kDa) or intracellular (95kDa) form into a soluble (85 kDa) form results in activating the microglia. As corroborated by other recent studies, the present work suggests, that - by modulating this pathway - an extended conservation of light cells, or at least a deceleration of their degeneration could be achievable.