Abstract:
Visual signalling initiates in the rod and cone photoreceptors in the retina. The first steps include a multi-step amplification cascade starting in the outer segments upon light exposure and activating the phosphodiesterase 6 (PDE6). PDE6 reduces the level of the second messenger cyclic guanosine monophosphate (cGMP) by hydrolysis. Low levels of cGMP in turn mediate the closure of cyclic nucleotide-gated (CNG) channels as the final step of phototransduction causing a voltage change, the first electrical signal in rods. At downstream photoreceptor synaptic terminals this leads to a reduction in neurotransmitter release which activates the bipolar cells, retinal second order neurons. Thus, the focus of this thesis was to investigate rod signalling precisely by means of rod-specific mutations in rodent models and electroretinography (ERG) which measures the electrical activity of the retina including photoreceptor outer segment function and signal transmission to bipolar. This work concentrates on the role of four distinctive components located in different compartments of rods from the outer segment to the synaptic terminal.
The first part of the thesis addresses the contribution of the PDE6 and the CNG channels, located in rod outer segments, to signal generation. The role of PDE6 was studied in Pde6a mutants with differently compromising missense mutations in the alpha-subunit of rod PDE6 (Pde6a) resulting in a gradually reduced PDE6 activity in each Pde6a variant. This causes a continuously elevated level of cGMP which triggers a premature degeneration of rods and secondary cone cell death. In this regard, we have found that functionally the generation of rod-driven electrical signals are prevented, resulting in a mostly cone-driven vision in Pde6a variants. The remaining cone signal in each Pde6a line is ultimately determined by the speed of photoreceptor degeneration.
The role of CNG channels was addressed in the scope of AAV-mediated gene replacement therapy in the Cngb1-/- knockout model characterized by a lack of the beta subunit of the CNG channel (CNGB1) and a respective functional silencing of rod signalling. Functional assessment revealed that genetic restoration of Cngb1 established rod electrical signals which were even translated to second and third order neurons in the retina.
The second part concerns the role of the hyperpolarization-activated and cyclic nucleotide-gated channels 1 (HCN1) situated functionally downstream at the inner segments of rod photoreceptors. This work illustrates that a loss of HCN1 channels prolonged rod responses and subsequently saturate rod pathway. Consequently, under regular conditions, HCN1 mediates an inward current which reduces outer segment activity during bright light and enhances rod responsivity. We show here that HCN1 channels are important components of early signal processing within the photoreceptor.
The final part of the thesis describes the role of voltage-gated calcium (CaV1.4) channels. These channels control transmitter release at synaptic terminals, the final step of rod signal processing. Our functional studies describe the consequence of two mutations in the CaV1.4 channels on the synaptic activity. A complete loss of the Cacna1f causes a complete failure of signal transmission from photoreceptors to second-order neurons.
Experimental Ophthalmology
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