James A. Kuchenbecker

Gene therapy for red–green colour blindness in adult primates

Red–green colour blindness, which results from the absence of either the long- (L) or the middle- (M) wavelength-sensitive visual photopigments, is the most common single locus genetic disorder. Here we explore the possibility of curing colour blindness using gene therapy in experiments on adult monkeys that had been colour blind since birth. A third type of cone pigment was added to dichromatic retinas, providing the receptoral basis for trichromatic colour vision. This opened a new avenue to explore the requirements for establishing the neural circuits for a new dimension of colour sensation. Classic visual deprivation experiments have led to the expectation that neural connections established during development would not appropriately process an input that was not present from birth. Therefore, it was believed that the treatment of congenital vision disorders would be ineffective unless administered to the very young. However, here we show that the addition of a third opsin in adult red–green colour-deficient primates was sufficient to produce trichromatic colour vision behaviour. Thus, trichromacy can arise from a single addition of a third cone class and it does not require an early developmental process. This provides a positive outlook for the potential of gene therapy to cure adult vision disorders.

Changes in the colour of light cue circadian activity

The discovery of melanopsin, the non-visual opsin present in intrinsically photosensitive retinal ganglion cells (ipRGCs), has created great excitement in the field of circadian biology. Now, researchers have emphasized melanopsin as the main photopigment governing circadian activity in vertebrates. Circadian biologists have tested this idea under standard laboratory, 12h Light: 12h Dark, lighting conditions that lack the dramatic daily colour changes of natural skylight. Here we used a stimulus paradigm in which the colour of the illumination changed throughout the day, thus mimicking natural skylight, but luminance, sensed intrinsically by melanopsin containing ganglion cells, was kept constant. We show in two species of cichlid, Aequidens pulcher and Labeotropheus fuelleborni, that changes in light colour, not intensity, are the primary determinants of natural circadian activity. Moreover, opponent-cone photoreceptor inputs to ipRGCs mediate the sensation of wavelength change, and not the intrinsic photopigment, melanopsin. These results have implications for understanding the evolutionary biology of non-visual photosensory pathways and answer long-standing questions about the nature and distribution of photopigments in organisms, including providing a solution to the mystery of why nocturnal animals routinely have mutations that interrupt the function of their short wavelength sensitive photopigment gene.

Topography of the long- to middle-wavelength sensitive cone ratio in the human retina assessed with a wide-field color multifocal electroretinogram.

The topographical distribution of relative sensitivity to red and green lights across the retina was assayed using a custom-made wide-field color multifocal electroretinogram apparatus. There were increases in the relative sensitivity to red compared to green light in the periphery that correlate with observed increases in the relative amount of long (L) compared to middle (M) wavelength sensitive opsin mRNA. These results provide electrophysiological evidence that there is a dramatic increase in the ratio of L to M cones in the far periphery of the human retina. The central to far peripheral homogeneity in cone proportions has implications for understanding the developmental mechanisms that determine the identity of a cone as L or M and for understanding the circuitry for color vision in the peripheral retina.

S-opsin knockout mice with the endogenous M-opsin gene replaced by an L-opsin variant

Specific variants of human long-wavelength (L) and middle-wavelength (M) cone opsin genes have recently been associated with a variety of vision disorders caused by cone malfunction, including red-green color vision deficiency, blue cone monochromacy, myopia, and cone dystrophy. Strikingly, unlike disease-causing mutations in rhodopsin, most of the cone opsin alleles that are associated with vision disorders do not have deleterious point mutations. Instead, specific combinations of normal polymorphisms that arose by genetic recombination between the genes encoding L and M opsins appear to cause disease. Knockout/knock-in mice promise to make it possible to study how these deleterious cone opsin variants affect the structure, function, and viability of the cone photoreceptors. Ideally, we would like to evaluate different variants that cause vision disorders in humans against a control pigment that is not associated with vision disorders, and each variant should be expressed as the sole photopigment in each mouse cone, as is the case in humans. To evaluate the feasibility of this approach, we created a line of mice to serve as the control in the analysis of disease-causing mutations by replacing exon 2 through 6 of the mouse M-opsin gene with the corresponding cDNA for a human L-opsin variant that is associated with normal vision. Experiments reported here establish that the resulting pigment, which differs from the endogenous mouse M opsin at 35 amino acid positions, functions normally in mouse cones. This pigment was evaluated in mice with and without coexpression of the mouse short wavelength (S) opsin. Here, the creation and validation of two lines of genetically engineered mice that can be used to study disease-causing variants of human L/M-opsins, in vivo, are described.

Longitudinal evaluation of expression of virally delivered transgenes in gerbil cone photoreceptors

Delivery of foreign opsin genes to cone photoreceptors using recombinant adeno-associated virus (rAAV) is a potential tool for studying the basic mechanisms underlying cone based vision and for treating vision disorders. We used an in vivo retinal imaging system to monitor, over time, expression of virally-delivered genes targeted to cone photoreceptors in the Mongolian gerbil (Meriones unguiculatus). Gerbils have a well-developed photopic visual system, with 11-14% of their photoreceptors being cones. We used replication deficient serotype 5 rAAV to deliver a gene for green fluorescent protein (GFP). In an effort to direct expression of the gene specifically to either S or M cones, the transgene was under the control of either the human X-chromosome opsin gene regulatory elements, i.e., an enhancer termed the locus control region (LCR) and L promoter, or the human S-opsin promoter. Longitudinal fluorescence images reveal that gene expression is first detectable about 14 days post-injection, reaches a peak after about 3 months, and is observed more than a year post-injection if the initial viral concentration is sufficiently high. The regulatory elements are able to direct expression to a subpopulation of cones while excluding expression in rods and non-photoreceptor retinal cells. When the same viral constructs are used to deliver a human long-wavelength opsin gene to gerbil cones, stimulation of the introduced human photopigment with long-wavelength light produces robust cone responses.

Cone-isolating ON–OFF electroretinogram for studying chromatic pathways in the retina

The electroretinogram (ERG) provides information about outer retina function in both clinical and research applications. ERG components elicited by light increments and decrements can be separated using a long-flash paradigm in which periods of light ON and OFF are alternated. Here, the ON-OFF ERG is combined with a silent substitution technique to elicit responses from individual cone photoreceptor classes by modulating the intensities of three color lights between the two periods. The results focus on the short wavelength (S) cone pathways since they are vulnerable to disease and because there are many unanswered questions about S-cone ON and OFF circuitry.

Role of a Dual Splicing and Amino Acid Code in Myopia, Cone Dysfunction and Cone Dystrophy Associated with L/M Opsin Interchange Mutations

Purpose Human long (L) and middle (M) wavelength cone opsin genes are highly variable due to intermixing. Two L/M cone opsin interchange mutants, designated LIAVA and LVAVA, are associated with clinical diagnoses, including red-green color vision deficiency, blue cone monochromacy, cone degeneration, myopia, and Bornholm Eye Disease. Because the protein and splicing codes are carried by the same nucleotides, intermixing L and M genes can cause disease by affecting protein structure and splicing. Methods Genetically engineered mice were created to allow investigation of the consequences of altered protein structure alone, and the effects on cone morphology were examined using immunohistochemistry. In humans and mice, cone function was evaluated using the electroretinogram (ERG) under L/M- or short (S) wavelength cone isolating conditions. Effects of LIAVA and LVAVA genes on splicing were evaluated using a minigene assay. Results ERGs and histology in mice revealed protein toxicity for the LVAVA but not for the LIAVA opsin. Minigene assays showed that the dominant messenger RNA (mRNA) was aberrantly spliced for both variants; however, the LVAVA gene produced a small but significant amount of full-length mRNA and LVAVA subjects had correspondingly reduced ERG amplitudes. In contrast, the LIAVA subject had no L/M cone ERG. Conclusions Dramatic differences in phenotype can result from seemingly minor differences in genotype through divergent effects on the dual amino acid and splicing codes. Translational Relevance The mechanism by which individual mutations contribute to clinical phenotypes provides valuable information for diagnosis and prognosis of vision disorders associated with L/M interchange mutations, and it informs strategies for developing therapies.

Correlated Evolution of Short Wavelength Sensitive Photoreceptor Sensitivity and Color Pattern in Lake Malawi Cichlids

For evolutionary ecologists, the “holy grail” of visual ecology is to establish an unambiguous link between photoreceptor sensitivity, the spectral environment, and the perception of specific visual stimuli (e.g., mates, food, predators, etc.). Due to the bright nuptial colors of the males, and the role female mate choice plays in their evolution, the haplochromine cichlid fishes of the African great lakes are favorite research subjects for such investigations. Despite this attention, current evidence is equivocal; while distinct correlations among photoreceptor sensitivity, photic environment, and male coloration exist in Lake Victorian haplochromines, attempts to find such correlations in Lake Malawian cichlids have failed. Lake Malawi haplochromines have a wide variability in their short-wavelength-sensitive photoreceptors, especially compared to their mid- and long-wavelength-sensitive photoreceptors; these cichlids also vary in the degree to which they express one of three basic color patterns (vertical bars, horizontal stripes, and solid patches of colors), each of which is likely used in a different form of communication. Thus, we hypothesize that, in these fishes, spectral sensitivity and color pattern have evolved in a correlated fashion to maximize visual communication; specifically, ultraviolet sensitivity should be found in vertically-barred species to promote ‘private’ communication, while striped species should be less likely to have ultraviolet sensitivity, since their color pattern carries ‘public’ information. Using phylogenetic independent contrasts, we found that barred species had strong sensitivity to ultraviolet wavelengths, but that striped species typically lacked sensitivity to ultraviolet light. Further, the only variable, even when environmental variables were simultaneously considered, that could predict ultraviolet sensitivity was color pattern. We also found that, using models of correlated evolution, color pattern and ultraviolet sensitivity are correlated in Lake Malawi cichlid evolution, with the likely ancestor being a vertically-barred, ultraviolet-sensitive species, the descendants of which lost both ultraviolet sensitivity and a barred color pattern. These results, indicating that communication of ‘public’ and ‘private’ signals is mediated via differing perceptions of color patterns, suggest a functional connection between visual sensitivity and colour pattern, a novel finding in Lake Malawi cichlids.