The role of impaired cGMP homeostasis in cone photoreceptor degeneration

Achromatopsia (ACHM) is a currently untreatable inherited eye disease that severely impairs daylight vision. The clinical manifestation results from cone photoreceptor dysfunction and is characterized by poor visual acuity, lack of colour discrimination and photophobia. In addition, a progressive degeneration and loss of macular cone photoreceptors is observed. In total six disease genes are known, but the majority of patients carries mutations in CNGA3 (29% of cases) or CNGB3 (48% of cases); the two genes encoding the cone cyclic nucleotide-gated (CNG) channel. This ion channel is directly gated by the second messenger cyclic GMP (cGMP) and is an integral part of the so called phototransduction cascade that translates light signals into electrical stimuli. Interestingly, genetic deletion of the CNG channel subunit CNGA3 in mice leads to high cellular levels of cGMP in affected cone photoreceptors. The aim of this thesis was to characterize the role of impaired cGMP signalling during cone photoreceptor degeneration in the Cnga3 knockout (KO) mouse model for ACHM. First, the developmental time course of cGMP accumulation was investigated using a cGMP-specific antibody. One point worthy of note is that elevated cGMP levels could be observed as early as four days after birth. Using a virus-mediated in vivo gene knockdown (KD) approach it was possible to identify guanylate cyclase 2E (Gucy2e) as the main source of cGMP. Moreover, Gucy2e KD resulted in prolonged survival of cones in Cnga3 KO mice suggesting a toxic role of sustained cGMP overload in affected photoreceptors. Given the absence of CNG channels in Cnga3 KO cones this toxic effect has to be mediated by other cGMP targets. Promising candidates that were further investigated were the cGMP-dependent kinases (cGKs). To investigate the impact of elevated cGK signalling for photoreceptor viability a constitutively active cGK was overexpressed in wildtype mouse photoreceptors using viral vectors. This induced a dramatic decrease of the photoreceptor layer and demonstrated that these kinases can induce cell death. To verify this and to define the effect of the two kinase types, Cnga3 KO mice were cross-bread with cGKI and cGKII KO mice and further examined morphologically. Global loss of cGKII but not cGKI significantly protected cone photoreceptors resulting in lower levels of degeneration and cell death. This finding was confirmed using viral vector-mediated delivery of a cGKII specific shRNA into cone photoreceptors of Cnga3 KO mice. Expression of cGKII-shRNA in adult Cnga3 KO mice again showed a decrease of degeneration suggesting cGKII as a novel target for neuroprotection in cone degeneration. Finally, to further unravel the cGMP signalling triggering cell death in cones two proteomic approaches were performed to identify potential downstream targets of cGKII. These screens could identify a link between a cGMP-cGK-signalling pathway and the serine/threonine kinase Atr. In conclusion this thesis presents strong evidence that cGMP-signalling accelerates retinal degeneration and identifies cGMP/cGKII signalling as a novel target for neuroprotective therapies to prolong cone survival in ACHM and other cone degenerations.