Riassunto analitico
The aim of this thesis is to characterize the target of a cGMP analogue (CN03) in a cellular model for retinal degeneration. The in vitro model was based on the 661W cell line differentiated into rod photoreceptors-like cells with a medium previously determined in our laboratory and on the inhibition of Phosphodiesterase 6 (PDE6) with Zaprinast. PDE6 inhibition causes an accumulation of intracellular cGMP that activates pathways responsible for photoreceptors degeneration. The molecular mechanisms behind the degeneration have already been defined in retinae bearing a mutation in PDE6 in the rd1 mouse model.
cGMP has two main targets in photoreceptor cells: cyclic GMP/protein kinases G (PKG) and cyclic nucleotide-gated ion channels (CNGC). Since it is known from the literature that PKG activation is sufficient to trigger photoreceptors death while PKG inhibition is protective, we started a study to define which of the two PKG isoforms mediated the deadly effect. To answer this question, the expression of PKG1 and PKG2 was down-regulated with shRNA in differentiated 661W cells, and either PKG1 or PKG2 were pharmacologically activated by treating the cells with increasing concentrations of two cGMP analogues, PA5 and PA6, targeting PKG2 and PKG1, respectively. Cell viability and cell death were investigated with MTT assay and Ethidium homodimer staining. For further investigations, 661W cells downregulated for PKG1 or PKG2 were treated with CN03. CN03 is a cGMP analogue with an inhibitory effect previously demonstrated to have protective capacity on degenerating photoreceptors in vitro and in vivo. By evaluating the changes in EC50 in cells not expressing either PKG1 or PKG2, it was possible to understand which PKG type is responsible for the protective effect. Moreover, it is proved that the opening of CNGC is followed by a calcium influx that activates cell death pathways. During the two months of a Erasmus+ per Traineeship in Tübingen (Germany) a project aiming the characterization of the expression of CNGb1 in rd1 mouse model was started.
Understanding which is CN03 specific target and which PKG type is responsible for the degeneration of photoreceptors in RD type disease is the first step for the synthesis of more specific and effective therapeutic compounds, that can reduce potential off-target effects.
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