Dissection of visual signalling based on functionally specific rod photoreceptor mutants

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

Electroretinographic assessment of rod- and cone-mediated bipolar cell pathways using flicker stimuli in mice

Mouse full-field electroretinograms (ERGs) are dominated by responses of photoreceptors and depolarizing (ON-) bipolar cells, but not much of hyperpolarizing (OFF-) bipolar cells under conventional recording conditions. Here we investigate a novel ERG protocol in mice for functional assessment of the major ON-and OFF- bipolar cell pathways using flicker stimuli for a high luminance with varying frequency up to 30 Hz. Wild- type (WT) and functionally specific transgenic mice (Cnga3(-/-),no cone photoreceptor function;rho(-/-),no rod photoreceptor function;mGluR6(-/-),no ON-bipolar cell function) were examined. The Cnga3(-/-) flicker ERG was similar to the WT flicker ERG at very low stimulus frequencies, whereas ERGs were comparable between WT and rho(-/-) mice at 5 Hz and above. Between 5 and 15 Hz, ERGs in mGluR6(-/-) mice differed in configuration and amplitude from those in WT and rho(-/-) mice;in contrast, response amplitudes above 15 Hz were comparable among WT, rho(-/-) and mGluR6(-/-) mice. In summary, we found three frequency ranges with these conditions that are dominated by activity in the rod pathways (below 5 Hz),cone ON-pathway (between 5 and 15 Hz),and cone OFF-pathway (above 15 Hz) that enables a quick overview of the functionality of the major bipolar cell pathways

Lack of the Sodium-Driven Chloride Bicarbonate Exchanger NCBE Impairs Visual Function in the Mouse Retina

Regulation of ion and pH homeostasis is essential for normal neuronal function. The sodium-driven chloride bicarbonate exchanger NCBE (Slc4a10), a member of the SLC4 family of bicarbonate transporters, uses the transmembrane gradient of sodium to drive cellular net uptake of bicarbonate and to extrude chloride, thereby modulating both intracellular pH (pHi) and chloride concentration ([Cl-]i) in neurons. Here we show that NCBE is strongly expressed in the retina. As GABAA receptors conduct both chloride and bicarbonate, we hypothesized that NCBE may be relevant for GABAergic transmission in the retina. Importantly, we found a differential expression of NCBE in bipolar cells: whereas NCBE was expressed on ON and OFF bipolar cell axon terminals, it only localized to dendrites of OFF bipolar cells. On these compartments, NCBE colocalized with the main neuronal chloride extruder KCC2, which renders GABA hyperpolarizing. NCBE was also expressed in starburst amacrine cells, but was absent from neurons known to depolarize in response to GABA, like horizontal cells. Mice lacking NCBE showed decreased visual acuity and contrast sensitivity in behavioral experiments and smaller b-wave amplitudes and longer latencies in electroretinograms. Ganglion cells from NCBE-deficient mice also showed altered temporal response properties. In summary, our data suggest that NCBE may serve to maintain intracellular chloride and bicarbonate concentration in retinal neurons. Consequently, lack of NCBE in the retina may result in changes in pHi regulation and chloride-dependent inhibition, leading to altered signal transmission and impaired visual function

PGC-1α Determines Light Damage Susceptibility of the Murine Retina

The peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) proteins are key regulators of cellular bioenergetics and are accordingly expressed in tissues with a high energetic demand. For example, PGC-1α and PGC-1β control organ function of brown adipose tissue, heart, brain, liver and skeletal muscle. Surprisingly, despite their prominent role in the control of mitochondrial biogenesis and oxidative metabolism, expression and function of the PGC-1 coactivators in the retina, an organ with one of the highest energy demands per tissue weight, are completely unknown. Moreover, the molecular mechanisms that coordinate energy production with repair processes in the damaged retina remain enigmatic. In the present study, we thus investigated the expression and function of the PGC-1 coactivators in the healthy and the damaged retina. We show that PGC-1α and PGC-1β are found at high levels in different structures of the mouse retina, most prominently in the photoreceptors. Furthermore, PGC-1α knockout mice suffer from a striking deterioration in retinal morphology and function upon detrimental light exposure. Gene expression studies revealed dysregulation of all major pathways involved in retinal damage and apoptosis, repair and renewal in the PGC-1α knockouts. The light-induced increase in apoptosis in vivo in the absence of PGC-1α was substantiated in vitro, where overexpression of PGC-1α evoked strong anti-apoptotic effects. Finally, we found that retinal levels of PGC-1 expression are reduced in different mouse models for retinitis pigmentosa. We demonstrate that PGC-1α is a central coordinator of energy production and, importantly, all of the major processes involved in retinal damage and subsequent repair. Together with the observed dysregulation of PGC-1α and PGC-1β in retinitis pigmentosa mouse models, these findings thus imply that PGC-1α might be an attractive target for therapeutic approaches aimed at retinal degeneration diseases

AAV-Mediated Gene Supplementation Therapy in Achromatopsia Type 2: Preclinical Data on Therapeutic Time Window and Long-Term Effects

Achromatopsia type 2 (ACHM2) is a severe, inherited eye disease caused by mutations in the CNGA3 gene encoding the a subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel. Patients suffer from strongly impaired daylight vision, photophobia, nystagmus, and lack of color discrimination. We have previously shown in the Cnga3 knockout (KO) mouse model of ACHM2 that gene supplementation therapy is effective in rescuing cone function and morphology and delaying cone degeneration. In our preclinical approach, we use recombinant adeno-associated virus (AAV) vector-mediated gene transfer to express the murine Cnga3 gene under control of the mouse blue opsin promoter. Here, we provide novel data on the efficiency and permanence of such gene supplementation therapy in Cnga3 KO mice. Specifically, we compare the influence of two different AAV vector capsids, AAV2/5 (Y719F) and AAV2/8 (Y733F), on restoration of cone function, and assess the effect of age at time of treatment on the long-term outcome. The evaluation included in vivo analysis of retinal function using electroretinography (ERG) and immunohistochemical analysis of vector-driven Cnga3 transgene expression. We found that both vector capsid serotypes led to a comparable rescue of cone function over the observation period between 4 weeks and 3 months post treatment. In addition, a clear therapeutic effect was present in mice treated at 2 weeks of age as well as in mice treated at 3 months of age at the first assessment at 4 weeks after treatment. Importantly, the effect extended in both cases over the entire observation period of 12 months post treatment. However, the average ERG amplitude levels differed between the two groups, suggesting a role of the absolute age, or possibly, the associated state of the degeneration, on the achievable outcome. In summary, we found that the therapeutic time window of opportunity for AAV-mediated Cnga3 gene supplementation therapy in the Cnga3 KO mouse model extends at least to an age of 3 months, but is presumably limited by the condition, number and topographical distribution of remaining cones at the time of treatment. No impact of the choice of capsid on the therapeutic success was detected

HCN1 Channels Enhance Rod System Responsivity in the Retina under Conditions of Light Exposure.

Vision originates in rods and cones at the outer retina. Already at these early stages, diverse processing schemes shape and enhance image information to permit perception over a wide range of lighting conditions. In this work, we address the role of hyperpolarization-activated and cyclic nucleotide-gated channels 1 (HCN1) in rod photoreceptors for the enhancement of rod system responsivity under conditions of light exposure.To isolate HCN1 channel actions in rod system responses, we generated double mutant mice by crossbreeding Hcn1-/- mice with Cnga3-/- mice in which cones are non-functional. Retinal function in the resulting Hcn1-/- Cnga3-/- animals was followed by means of electroretinography (ERG) up to the age of four month. Retinal imaging via scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) was also performed to exclude potential morphological alterations.This study on Hcn1-/- Cnga3-/- mutant mice complements our previous work on HCN1 channel function in the retina. We show here in a functional rod-only setting that rod responses following bright light exposure terminate without the counteraction of HCN channels much later than normal. The resulting sustained signal elevation does saturate the retinal network due to an intensity-dependent reduction in the dynamic range. In addition, the lack of rapid adaptational feedback modulation of rod photoreceptor output via HCN1 in this double mutant limits the ability to follow repetitive (flicker) stimuli, particularly under mesopic conditions.This work corroborates the hypothesis that, in the absence of HCN1-mediated feedback, the amplitude of rod signals remains at high levels for a prolonged period of time, leading to saturation of the retinal pathways. Our results demonstrate the importance of HCN1 channels for regular vision