Rod Transduction Research Articles

Novel AAV44.9-Based Vectors Display Exceptional Characteristics for Retinal Gene Therapy.

The majority of inherited retinal diseases (IRDs) are caused by mutations in genes expressed in photoreceptors (PRs). The ideal vector to address these conditions is one that transduces PRs in large areas of retina with the smallest volume/lowest titer possible, and efficiently transduces foveal cones, the cells responsible for acute, daylight vision that are often the only remaining area of functional retina in IRDs. The purpose of our study was to evaluate the retinal tropism and potency of a novel capsid, AAV44.9, and rationally designed derivatives thereof. We found that AAV44.9 and AAV44.9(E531D) transduced retinas of subretinally injected (SRI) mice with higher efficiency than did benchmark AAV5- and AAV8-based vectors. In macaques, highly efficient cone and rod transduction was observed following submacular and peripheral SRI. AAV44.9- and AAV44.9(E531D)-mediated GFP fluorescence extended laterally well beyond SRI bleb margins. Notably, extrafoveal injection (i.e., fovea not detached during surgery) led to transduction of up to 98% of foveal cones. AAV44.9(E531D) efficiently transduced parafoveal and perifoveal cones, whereas AAV44.9 did not. AAV44.9(E531D) was also capable of restoring retinal function to a mouse model of IRD. These novel capsids will be useful for addressing IRDs that would benefit from an expansive treatment area.

Rod Outer Segment Development Influences AAV-Mediated Photoreceptor Transduction After Subretinal Injection.

Vectors based on the adeno-associated virus (AAV) are currently the preferred tools for delivering genes to photoreceptors (PR) in small and large animals. AAVs have been applied successfully in various models of PR dystrophies. However, unknown barriers still limit AAV's efficient application in several forms of severe PR degenerations due to insufficient transgene expression and/or treated cells at the time of injection. Optimizations of PR gene therapy strategies will likely benefit from the identification of the cellular factors that influence PR transduction. Interestingly, recent studies have shown that the AAV transduction profile of PRs differs significantly between neonatal and adult mouse retinas after subretinal injection. This phenomenon may provide clues to identify host factors that influence the efficiency of AAV-mediated PR transduction. This study demonstrates that rod outer segments are critical modulators of efficient AAV-mediated rod transduction. During retinal development, rod transduction correlated temporally and spatially with the differentiation order of PRs when vectors were introduced subretinally but not when introduced intravitreally. All subretinally injected vectors had an initial preference to transduce cones in the absence of formed rod outer segments and then displayed a preference for rods as the cells matured, independently of the expression cassette or AAV serotype. Consistent with this observation, altered development of rod outer segments was associated with a strong reduction of rod transduction and an increase in the percentage of transduced cones by 2- to 2.8-fold. A similar increase of cone transduction was observed in the adult retinal degeneration 1 (rd1) retina compared to wild-type mice. These results suggest that the loss of rod outer segments in diseased retinas could markedly affect gene transfer efficiency of AAV vectors by limiting the ability of AAVs to infect dying rods efficiently. This information could be exploited for the development of more efficient AAV-based PR gene delivery procedures.

Contributions of Rod and Cone Pathways to Retinal Direction Selectivity Through Development.

Direction selectivity is a robust computation across a broad stimulus space that is mediated by activity of both rod and cone photoreceptors through the ON and OFF pathways. However, rods, S-cones, and M-cones activate the ON and OFF circuits via distinct pathways and the relative contribution of each to direction selectivity is unknown. Using a variety of stimulation paradigms, pharmacological agents, and knockout mice that lack rod transduction, we found that inputs from the ON pathway were critical for strong direction-selective (DS) tuning in the OFF pathway. For UV light stimulation, the ON pathway inputs to the OFF pathway originated with rod signaling, whereas for visible stimulation, the ON pathway inputs to the OFF pathway originated with both rod and M-cone signaling. Whole-cell voltage-clamp recordings revealed that blocking the ON pathway reduced directional tuning in the OFF pathway via a reduction in null-side inhibition, which is provided by OFF starburst amacrine cells (SACs). Consistent with this, our recordings from OFF SACs confirmed that signals originating in the ON pathway contribute to their excitation. Finally, we observed that, for UV stimulation, ON contributions to OFF DS tuning matured earlier than direct signaling via the OFF pathway. These data indicate that the retina uses multiple strategies for computing DS responses across different colors and stages of development. The retina uses parallel pathways to encode different features of the visual scene. In some cases, these distinct pathways converge on circuits that mediate a distinct computation. For example, rod and cone pathways enable direction-selective (DS) ganglion cells to encode motion over a wide range of light intensities. Here, we show that although direction selectivity is robust across light intensities, motion discrimination for OFF signals is dependent upon ON signaling. At eye opening, ON directional tuning is mature, whereas OFF DS tuning is significantly reduced due to a delayed maturation of S-cone to OFF cone bipolar signaling. These results provide evidence that the retina uses multiple strategies for computing DS responses across different stimulus conditions.

A novel Ca2+-feedback mechanism extends the operating range of mammalian rods to brighter light.

Sensory cells adjust their sensitivity to incoming signals, such as odor or light, in response to changes in background stimulation, thereby extending the range over which they operate. For instance, rod photoreceptors are extremely sensitive in darkness, so that they are able to detect individual photons, but remain responsive to visual stimuli under conditions of bright ambient light, which would be expected to saturate their response given the high gain of the rod transduction cascade in darkness. These photoreceptors regulate their sensitivity to light rapidly and reversibly in response to changes in ambient illumination, thereby avoiding saturation. Calcium ions (Ca2+) play a major role in mediating the rapid, subsecond adaptation to light, and the Ca2+-binding proteins GCAP1 and GCAP2 (or guanylyl cyclase-activating proteins [GCAPs]) have been identified as important mediators of the photoreceptor response to changes in intracellular Ca2+. However, mouse rods lacking both GCAP1 and GCAP2 (GCAP-/-) still show substantial light adaptation. Here, we determined the Ca2+ dependency of this residual light adaptation and, by combining pharmacological, genetic, and electrophysiological tools, showed that an unknown Ca2+-dependent mechanism contributes to light adaptation in GCAP-/- mouse rods. We found that mimicking the light-induced decrease in intracellular [Ca2+] accelerated recovery of the response to visual stimuli and caused a fourfold decrease of sensitivity in GCAP-/- rods. About half of this Ca2+-dependent regulation of sensitivity could be attributed to the recoverin-mediated pathway, whereas half of it was caused by the unknown mechanism. Furthermore, our data demonstrate that the feedback mechanisms regulating the sensitivity of mammalian rods on the second and subsecond time scales are all Ca2+ dependent and that, unlike salamander rods, Ca2+-independent background-induced acceleration of flash response kinetics is rather weak in mouse rods.

Single-Photon Sensitivity of Lamprey Rods with Cone-like Outer Segments

Most vertebrates have a duplex retina containing rods for dim light vision and cones for bright lights and color detection. Photoreceptors like cones are present in many invertebrate phyla as well as in chordata, and rods evolved from cones, but the sequence of events is not well understood. Since duplex retinas are present in both agnatha and gnathostomata, which diverged more than 400 million years ago, some properties of ancestral rods may be inferred from a comparison of cells in these two groups. Lamprey have two kinds of photoreceptors, called "short" and "long", which seem to be rods and cones; however, the outer segments of both have an identical cone-like morphology of stacks of lamellae without a continuous surrounding plasma membrane. This observation and other aspects of the cellular and molecular biology of the photoreceptors have convinced several investigators that "the features of 'true' rod transduction in jawed vertebrates, which permit the reliable detection of single photons of light, evolved after the separation of gnathostomes from lampreys". To test this hypothesis, we recorded from photoreceptors of the sea lamprey Petromyzon marinus and show that their rods have a single-photon sensitivity similar to that of rods in other vertebrates. Thus, photoreceptors with many of the features of rods emerged before the split between agnatha and gnathostomata, and a rod-like outer segment with cytosolic disks surrounded by a plasma membrane is not necessary for high-sensitivity visual detection.

Variation in Rhodopsin Kinase Expression Alters the Dim Flash Response Shut Off and the Light Adaptation in Rod Photoreceptors

Rod photoreceptors are exquisitely sensitive light detectors that function in dim light. The timely inactivation of their light responses is critical for the ability of rods to reliably detect and count photons. A key step in the inactivation of the rod transduction is the phosphorylation of the rod visual pigment, rhodopsin, catalyzed by G-protein-dependent receptor kinase 1 (GRK1). Absence of GRK1 greatly prolongs the photoreceptors' light response and enhances their susceptibility to degeneration. This study examined the light responses from mouse rods expressing various levels of GRK1 to evaluate how their function is modulated by rhodopsin inactivation. Transretinal and single-cell rod electrophysiological recordings were obtained from several strains of mice expressing GRK1 at 0.3- to 3-fold the wild-type levels. The effect of GRK1 expression level on the function of mouse rods was examined in darkness and during background adaptation. Altering the expression of GRK1 from 0.3- to 3-fold that in wild-type rods had little effect on the single photon response amplitude. Notably, increasing the expression level of GRK1 accelerated the dim flash response shut off but had no effect on the saturated response shut off. Additionally, GRK1 excess abolished the acceleration of saturated responses shut off during light adaptation. These results demonstrate that rhodopsin inactivation can modulate the kinetics of recovery from dim light stimulation. More importantly, the ratio of rhodopsin kinase to its modulator recoverin appears critical for the proper adaptation of rods and the acceleration of their response shut off in background light.

Function of the asparagine 74 residue of the inhibitory γ-subunit of retinal rod cGMP-phophodiesterase (PDE) in vivo

The inhibitory subunit of rod cyclic guanosine monophosphate (cGMP) phosphodiesterase, PDE6γ, is a major component of rod transduction and is required to support photoreceptor integrity. The N74A allele of PDE6γ has previously been shown in experiments carried out in vitro to reduce the regulatory inhibition on the PDE6 catalytic core subunits, PDE6αβ. This should, in intact rods, lead to an increase in basal (dark) PDE6 activity producing a state equivalent to light adaptation in the rods and we have examined this possibility using ERG and suction-electrode measurements. The murine opsin promoter was used to drive the expression of a mutant N74A and a wild-type PDE6γ control transgene in the photoreceptors of +/ Pde6g tm1 mice. This transgenic line was crossed with Pde6g tm1 / Pde6g tm1 mice to generate animals able to synthesize only the transgenic mutant PDE6γ. We find that the N74A mutation did not produce a significant decrease in circulating current, a decrease in sensitivity or affect the kinetics of the light response, all hallmarks of the light-adapted state. In an in vitro assay of the PDE purified from the N74A transgenic mice and control mice we could find no increase in basal activity of the mutant PDE6. Both the results from the physiology and the biochemistry experiments are consistent with the interpretation that the mutation causes a much milder phenotype in vivo than was predicted from observations made using a cell-free assay system. The in vivo regulation of PDE6γ on PDE6αβ may be more dynamic and context-dependent than was replicated in vitro.

RAAV2/5 gene-targeting to rods:dose-dependent efficiency and complications associated with different promoters

A prerequisite for using corrective gene therapy to treat humans with inherited retinal degenerative diseases that affect primarily rods is to develop viral vectors that target specifically this population of photoreceptors. The delivery of a viral vector with photoreceptor tropism coupled with a rod-specific promoter is likely to be the safest and most efficient approach to target expression of the therapeutic gene to rods. Three promoters that included a fragment of the proximal mouse opsin promoter (mOP), the human G-protein coupled receptor protein kinase 1 promoter (hGRK1), or the cytomegalovirus immediate early enhancer combined with the chicken beta actin proximal promoter CBA) were evaluated for their specificity and robustness in driving GFP reporter gene expression in rods, when packaged in a recombinant adeno-associated viral vector of serotype 2/5 (AAV2/5), and delivered via subretinal injection to the normal canine retina. Photoreceptor specific promoters (mOP, hGRK1) targeted robust GFP expression to rods, while the ubiquitously expressed CBA promoter led to transgene expression in the retinal pigment epithelium, rods, cones and rare Müller, horizontal and ganglion cells. Late onset inflammation was frequently observed both clinically and histologically with all three constructs when the highest viral titers were injected. Cone loss in the injected regions of the retinas that received the highest titers occurred with both the hGRK1 and CBA promoters. Efficient and specific rod transduction, together with preservation of retinal structure was achieved with both mOP and hGRK1 promoters when viral titers in the order of 1011 vg/ml were used.

Highlights From The Literature

Highlights From The Literature

Physiological evidence that light-mediated decrease in cyclic GMP is an intermediary process in retinal rod transduction.

Brief, intracellularly injected pulses of cyclic GMP transiently depolarized toad retinal rod outer segments (ROS). The depolarization is antagonized by light, perhaps by the activation of phosphodiesterase (PDE), as shown in the biochemical studies of others. As measured by the antagonism of cyclic GMP pulses by light, PDE activity peaks after the peak of the receptor potential and has approximately the same recovery time as the membrane voltage after weak illumination, but recovers more slowly than the membrane potential after strong illumination, as sensitivity does in other preparations. A cyclic GMP pulse delivered just after the hyperpolarizing phase of the receptor potential tends to turn off the light response. The kinetics of recovery from this turnoff are similar to those of the initial phase of the receptor potential. This similarity suggests that the initial phase of the receptor potential is controlled by light-activated PDE. Both EGTA and saturating doses of cyclic GMP block the light response, but only cyclic GMP increases response latency, which suggests that if calcium is involved in transduction, it is controlled by the hydrolysis of cyclic GMP. After brief pulses of cyclic AMP, a new steady state of increased depolarization occasionally develops. The effects described above also occur under these conditions. The results are consistent with the hypothesis that light-activated hydrolysis of cGMP is an intermediary process in transduction.

Studies on the effects of bleaching amphibian rod pigments: IV. Photoresponses recorded intracellularly from axolotl red rods following bleaching flashes

Intracellular recordings from axolotl red rods following flashes bleaching 50–80% of their visual pigments have been compared with estimates of their vitamin A concentration, to investigate whether the growth in maximum responses to test stimuli correlates with vitamin A formation. No straightforward correlation was observed: most of the growth occurred after more than 60% of the vitamin A had already appeared. Examination of the phenomena associated with growth revealed a number of complexities: the bleaching flashes gave rise to hyperpolarizing responses which persisted for 20–30 min; in the early period after the flashes intense test stimuli could not drive the rod membrane voltage to the peak negative values seen in dark adapted receptors; there was a change in the amplitude vs. intensity relationship of photoresponses during the post-flash period; there was also a change in the waveform of maximum responses. It is argued that the complexities in the rod transduction mechanism implied by these observations may obscure the dependence of intracellularly recorded rod responses on biochemical processes in the rod outer limbs.