Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

dimmer the background, the stronger the negative feedback. As background is increased, feedback is reduced. Thus, ambient illumination produces an eCB tone that maintains transmitter release from cones within narrow limits. In this way, the ability of the cone to respond to increases and decreases of light intensity is maintained regardless of background. Retrograde transmission occurs even though Mb bipolar cells were hyperpolarized by glutamate acting on either the excitatory amino acid transporter (EAAT) or mGluR6 receptor. The retrograde effect was suppression of currents and not biphasic as expected from data obtained with low concentrations of WIN 55,212-2. It was this finding that led to the idea that the enhancing effect of WIN 55,212-2 on goldfish cones and salamander rods was due to agonist specific trafficking.

WIN 55,212-2 Affects the Cone Light Response

WIN 55,212-2 affects not only cone membrane currents, indicative of presynaptic modulation, but also the response of cones to light. Goldfish cones in an isolated retina preparation were stimulated by light in combination with voltageand current-clamp protocols (Figure 4). WIN 55,212-2 (10 mM) has no effect on the absolute sensitivity of the cones or the kinetics of the onset response. However, the light offset response is faster and the depolarizing overshoot is enhanced. This effect is seen at all but dim intensities (Figure 4(a)) and is independent of holding potential (Figure 4(b)). This is found under current-clamp as well as under voltage-clamp conditions, indicating modulation of the cyclic guanosine monophosphate (cGMP)-gated channels in the cone outer segment rather than by voltagedependent currents. The effects of WIN 55,212-2 are not blocked by SR141716A, indicating that CB1Rs are not involved. Given a train of flashes, the photocurrent recovers more quickly with WIN 55,212-2, such that the peak-to- peak response to succeeding flashes is increased. This effect, combined with the shortened recovery time to the offset of bright flashes, could increase contrast detection or critical flicker frequency. A concern is whether the effect of WIN 55,212-2 on the photoresponse would be observed with other CB1 agonists or eCBs because the effect of WIN 55,212-2 is not mediated by CB1 receptors.

In summary, cannabinoids presynaptically suppress the synaptic output of photoreceptors and on-bipolar cells. The effect is subtle as might be expected since smoking marijuana does not produce blindness. Evidence for effects of cannabinoids on amacrine cells is strongest for their suppression of dopamine release. Dopamine, a signal for light adaptation in the retina, antagonizes the action of cannabinoids in onbipolar cells. eCBs are critically involved in neuronal plasticity. This also appears to include light and dark adaptation, processes of neuronal plasticity that occur in the retina.

Cannabinoids – Development and

Neuroprotection

Studies regarding the effects of prenatal-marijuana use on children show deficits on visual habituation, tremors, and startle responses in neonates of 4–30 days old, but no effects on children of 1–6 years old. Problems with behavior, visual perceptual tasks, language comprehension, attention, and memory in 9-year-olds are attributed to effects on the prefrontal cortex, an area enriched in CB1Rs. Although CB1R localization and effects on GABA release have been studied in embryonic rat and chick retinas, no studies have investigated or commented on the effects of manipulating eCBs on retinal development.

The end point of glaucoma is ganglion cell death by apoptosis that may be caused by optic nerve injury following compression or ischemia. CB1 agonists (THC and cannabidiol) as well as inhibition of FAAH protect ganglion cells from glutamate excitotoxicity and ischemia caused by increased IOP. In contrast, COX-2 contributes to neuronal cell death following ischemia or NMDAtoxicity in glial cells, retinal pigment epithelium (RPE), and ganglion cells, while COX-2 blockers prevent ganglion cell apoptosis. Despite progress on the interaction of eCBs, COX-2 metabolites, and EP2 receptors in neuroprotection in the brain, such information is lacking in the retina.

Conclusion

The cannabinergic system is concentrated in the through pathway of the retina. Cannabinoids suppress dopamine release from amacrine cells and presynaptically inhibit potassium currents and glutamate release from cones and on-bipolar cells. How this relates to light and dark adaptation, receptive field formation, temporal properties of ganglion cell responses, and ultimately visual behavior needs to be addressed. eCBs are the most recently described neuromodulators to be studied extensively in neural and non-neural tissues. The existence of multiple eCBs, degradative enzymes, and receptors paints a picture of great complexity. They are important for their role in neuroplasticity and neuroprotection. Further study will verify the importance of eCBs in the retina as well.

See also: Information Processing: Amacrine Cells; Information Processing: Bipolar Cells; Information Processing: Ganglion Cells; Information Processing: Horizontal Cells; Neurotransmitters and Receptors: Dopamine Receptors; Phototransduction: Adaptation in Cones.

Further Reading

Fan, S. F. and Yazulla, S. (2007). Retrograde endocannabinoid inhibition of goldfish retinal cones is mediated by 2-arachidonoyl glycerol.

Visual Neuroscience 24: 257–267.

Glaser, S. T., Deutsch, D. D., Studholme, K. M., Zimov, S., and Yazulla, S. (2005). Endocannabinoids in the intact retina: 3H-anandamide uptake, fatty acid amide hydrolase immunoreactivity and hydrolysis of anandamide. Visual Neuroscience 22: 693–705.

Iversen, L. L. (2000). The Science of Marijuana. New York: Oxford University Press.

Nucci, C., Gasperi, V., Tartaglione, R., et al. (2007). Involvement of the endocannabinoid system in retinal damage after high intracellular pressure-induced ischemia in rats. Investigative Ophthalmology Visual Science 48: 2997–3004.

Onaivi, E. S., Sugiura, T., and Di Marzo, V. (eds.) (2006).

Endocannabinoids: The Brain and Body’s Marijuana and Beyond.

Boca Raton: CRC Press.

Straiker, A. and Sullivan, J. M. (2003). Cannabinoid receptor activation differentially modulates ion channels in photoreceptors of the tiger salamander. Journal of Neurophysiology 89: 2647–2654.

Straiker, A., Stella, N., Piomelli, D., et al. (1999). Cannabinoid CB1 receptors and ligands in vertebrate retina: Localization and function of an endogenous signaling system. Proceedings of the National Academy of Sciences of the United State of America 96: 14565–14570.

Struik, M., Yazulla, S., and Kamermans, M. (2006). Cannabinoid agonist WIN 55212-2 speeds up the cone light offset response in goldfish.

Visual Neuroscience 23: 285–293.

Tomida, I., Pertwee, R. G., and Azuara-Blanco, A. (2004). Cannabinoids and glaucoma. British Journal of Ophthalmology 88: 708–713.

Yazulla, S. (2008). Endocannabinoids in the retina: From marijuana to neuroprotection. Progress in Retinal and Eye Research 27(5): 501–526.

Yazulla, S., Studholme, K. M., McIntosh, H. H., and Deutsch, D. G. (1999). Immunocytochemical localization of cannabinoid CB1 receptor and fatty acid amide hydrolase in rat retina. Journal of Comparative Neurology 415: 80–90.

Yazulla, S., Studholme, K. M., McIntosh, H. H., and Fan, S. F. (2000). Cannabinoid receptors on goldfish retinal bipolar cells: Electronmicroscope immunocytochemistry and whole-cell recordings. Visual Neuroscience 17: 391–401.

Relevant Websites

http://cannabinoidsociety.org/ – This is the official website of the International Cannabinoid Research Society. It provides updates and background information on all aspect of the endocannabinoid field.

http://webvision.med.utah.edu/ – This website from the University of Utah provides extensive coverage of retinal anatomy and physiology, particularly mammals.

N J Colley, University of Wisconsin, Madison, WI, USA

ã 2010 Elsevier Ltd. All rights reserved.

Glossary

ABC-type multidrug transporter – A family of adenosine-triphosphate-binding cassette (ABC) transmembrane proteins that transports various molecules, including proteins, ions, sugars, and lipids, across extracellular and intracellular membranes using energy derived from adenosine triphosphate (ATP).

Allele – One member of a pair of genes occupying a specific location on a chromosome (locus) that controls the same trait, for example, eye color. cGMP phosphodiesterase (PDE) – This enzyme is found in several tissues, including the rod and cone photoreceptor cells, and it belongs to a large family of cyclic nucleotide PDEs that catalyze the hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) into AMP and GMP, respectively.

Class B scavenger receptors – A family of proteins, which includes the scavenger receptor class B type I (SR-BI), and CD36, which are cell surface receptors that mediate lipid uptake. They are also thought to play an important role in vitamin A metabolism by mediating the uptake of carotenoids into cells. Cyclophilin – A protein that binds the immunosuppressant drug, cyclosporin, which is often used to suppress tissue rejection following an organ transplantation. The protein displays peptidyl prolyl cis–trans isomerase activity, which catalyzes the cis/trans isomerization of peptide bonds on proline residues and is thought to play a role in protein folding.

Flippases – The enzymes located in the membrane that aid in the movement of phospholipid molecules between the two leaflets that comprise a cell’s membrane. The process requires energy derived from ATP.

Homeodomain-containing transcription factors –

A homeobox is a DNA sequence found within genes that regulates developmental processes in animals, fungi, and plants. A homeobox is about 183-DNA-bp long and it encodes a 61-amino-acid protein domain, called the homeodomain, which binds DNA and plays a key role in the regulation of gene expression.

Paired domain – A conserved domain found in a set of transcription factor proteins which are important in regulating gene expression during development.

Paired box (PAX) genes belong to the PAX family of transcription factors.

Rab-GTPase – These are small guanine-nucleotide- binding proteins (G proteins) conserved from yeast to humans and are members of the Ras superfamily of small GTPases. They function in distinct steps in membrane trafficking pathways, including vesicle formation, actinand tubulin-dependent vesicle movement, and membrane fusion events.

Retinitis pigmentosa (RP) – A heterogeneous group of genetically inherited retinal degeneration disorders leading to progressive loss in vision. Many people with RP retain some sight all their life, others become legally blind in childhood, and some become legally blind in their 40s or 50s. The progression of RP is different in each case.

Rhabdomere – The light-sensing organelle of the Drosophila photoreceptor cell. It is the functional equivalent of the outer segment of vertebrate rod and cone cells. A rhabdomere is made up of 60 000 tightly packed microvilli, and each microvillus is 50 nm in diameter and about 1–2 mm in length.

Second-site modifier screens – The genetic screens that are designed to detect a mutation in a second locus (gene) that enhances or suppresses the effect of an existing mutation.

‘Prized pest’

You hover over the soft, brown bananas like a floater, in and out of my vision, you whose eyes are so much like my own.

Who am I, dear one,

to swat at you, send you swirling toward the ceiling when, really, we share

the same humble beginnings. And you in your simplicity hold the key to my complexity.

Let me set out a plate of the sweetest peaches, invite you to rest

on the arm of my chair, however late it is.

Marilyn Annucci

Each model organism used to study retinal degenerative diseases has the advantage that others lack. Frogs, fish, rats, and mice have all provided great insights, but it is the tiny fruit fly, Drosophila melanogaster, that has played a central role in elucidating the molecular genetics of eye development and the early identification of mutations that cause retinal degeneration. The first mutations in Drosophila known to cause retinal degeneration were identified in the 1960s by the pioneering studies of Bill Pak and co-workers. At that time, these findings were only of interest to a few investigators. It was thought that animals as different as flies and humans could not share a similar genetic makeup and, therefore, the amount of transferable knowledge would be limited.

The revolutionary finding that put flies into the spotlight was the one showing that genes controlling pattern formation and development in flies could also do so in humans. In the 1980s, homeodomain-containing transcription factors were found to be essential during development in Drosophila for directing the production of appendages, such as the legs and the antennae. Almost identical homeodomain-containing genes were found in the genomes of a wide range of organisms, including humans and mice. This knowledge led to the conclusion that organisms as different as flies and humans contain nearly identical genes. A few years later, working on eye development, Walter Gehring’s lab cloned the eyeless gene in Drosophila. They discovered that eyeless is a transcription factor, containing a paired domain and a homeodomain, that directs eye formation. The eyeless gene is related to the mouse and human Pax6 genes (paired box), and the eyeless/Pax6 genes regulate a cascade of genetic processes involved in eye development. Mutations in these genes result in aniridia in humans, a Small eye (Sey) phenotype in mice and an eyeless phenotype in Drosophila. Aniridia is a congenital condition that is characterized by incomplete iris formation. Further, when expressed in flies, both the Drosophila eyeless gene and the mouse Pax6 gene (Small eye, or Sey) were able to direct the production of ectopic compound eyes. That Sey induced the formation of compound eyes and not mouse structures revealed that mice and flies share signaling components that are interchangeable. The proteins encoded by these genes share 94% identity in the paired domain and 90% identity in the homeodomain. It is remarkable that eyeless is not only essential for eye formation, but also its ectopic expression can override other developmental processes in a variety of tissues. For example, eyeless is capable of directing leg, wing, and antennal tissues to form eyes. As a result of these findings, eyeless/Pax6 was dubbed a master regulatory gene for eye formation during development. These landmark discoveries led to an explosion of exciting work in the 1990s that prompted a reassessment of the evolution of eyes. A complex network of eye determination genes direct eye formation, including another Pax6, twin

of eyeless (toy), sine oculis (so), eyes absent (eya), and dacshund (dac). Counterparts of these genes play a role in mammalian eye development and have been implicated in a variety of human diseases. These studies reveal that even though the compound eye of the fly looks very different from mammalian eyes, both share similar signaling pathways that are able to substitute for each other to form an eye.

Due to these elegant findings, it is now widely accepted that many genes are functionally equivalent between flies and humans. In addition, the same (or similar) mutations cause disease in both species. In fact, nearly three-fourths of all human disease genes have related sequences in Drosophila. Examples include gene mutations involved in retinal degeneration, deafness, skeletal malformations, cognitive impairment, cancer, immunity, alcoholism, cocaine and nicotine addiction, heart disease, metabolic and storage diseases, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease.

At the turn of the twentieth century in the hands of Thomas Hunt Morgan, the founding father of Drosophila research, Drosophila emerged as a powerful genetic workhorse. In 1910, Morgan identified the first mutation in Drosophila, which was a spontaneous white eye-pigment mutation that caused a normally red-eyed fly to be white eyed (Figure 1(a)). This first allele transformed our understanding of genetics and heredity. The white gene encodes a membrane-associated, adenosine triphosphate (ATP)-binding, ATP-binding cassette (ABC)-type multidrug transporter required for the transport of pigment precursors involved in eye pigment biosynthesis. In humans, mutations in the ABCA4 gene (also ABCR) account for approximately 3% of autosomal recessive retinitis pigmentosa (RP) and are linked to both recessive cone–rod dystrophy and recessive Stargardt macular dystrophy. Similar to the Drosophila white gene, the ABCA4 gene encodes a membrane-bound, ATP-binding transporter that in humans localizes to the rims of rod and cone outer segment disks. The ABCA4 transporter serves as a flippase in the retinoid cycle. When the ABC4 gene is mutated, toxic detergent-like by-products accumulate in the retinal pigment epithelium (RPE) leading to severe pathology. Therefore, the white gene, discovered at the turn of the century, was subsequently found to encode an ABC-type transporter required for eye pigment biosynthesis in Drosophila, and is related to another ABC-type transporter in the human eye that is involved in the retinoid cycle and several types of retinal diseases.

Not only do we share many genes in common with flies, but we also share a great deal of the same metabolic and signaling pathways. Flies are now being used as genetic models for the National Aeronautics and Space Administration (NASA) astronauts and are providing vital information on how space travel and gravitational changes alter gene expression. Work on flies continues to reveal

general principles that are fundamental to a wide spectrum of biological processes. Studies in Drosophila have led to conceptual and technical breakthroughs in the areas of development, gene expression, learning and memory, sleep, alcoholism, cocaine and nicotine addiction, ecology and evolution, olfaction, taste, mechanotransduction, vision, hearing, aging, pigmentation, biological clocks and circadian rhythms, courtship and mating behaviors, and human disease and the development of new pharmaceuticals.

In addition to sharing genes and signaling pathways with humans, flies are a powerful model for providing insights into human health and disease for other reasons. In spite of their small size, flies display complex rituals such as courtship behavior, so questions related to the genetic basis of complex behavior are tractable in the fly.

The fruit fly uses the same or similar genes to develop from a fertilized egg to an adult, but they do it in the short time of about 11 days. A female will lay hundreds of eggs, allowing large numbers of genetically identical offspring to be obtained. Flies have a short life span of about 2 months, so the onset and progression of age-related retinal degeneration disorders or any other age-related degenerative process can be studied quite rapidly.

The eye is not essential for viability or fertility of the flies, therefore genes encoding proteins that are uniquely required for visual function may be easily manipulated and studied. Large-scale mutagenesis screens have been carried out, producing hundreds of thousands of mutant flies whose phenotypes can be analyzed to identify genes required for vision. In addition, second-site modifier screens have been used to identify novel genes in

signaling pathways. Fly models can be used to dissect the cell biological basis and physiological basis of retinal degeneration, and therefore can be used to obtain insights into mechanisms of degenerative disorders. Just like in humans, electroretinogram (ERG) recordings can be carried out, and they have proved to be an indispensable means for uncovering visual system defective phenotypes that would otherwise have remained unnoticed.

Transgenic flies can be easily produced and, as a result, mutant genes may be introduced and mutant phenotypes may be complemented with wild-type transgenes. Using specialized promoters, genes may be targeted to specific tissues and may also be overexpressed. The fly has a relatively small genome, made up of about 13 600 genes in four pairs of chromosomes. However, despite the dramatic differences in size and apparent complexity between humans and flies – we have less than twice as many genes as a fly – our genome is estimated to be made up of only 20 000–25 000 genes contained in 23 pairs of chromosomes. Therefore, despite the fly’s perceived simplicity, or our perceived complexity, our genetic makeup may not be all that different. Its versatility for genetic manipulation and convenience for unraveling fundamental biological processes continue to guarantee the fly a place in the spotlight for unraveling the basis of and therapeutic treatments for human disease.

The Compound Eye and

Phototransduction

The Drosophila compound eye is composed of approximately 800 individual eye units called ommatidia, each containing the outer, R1-6 photoreceptor cells that extend the full length of the retina and express the major rhodopsin in the eye, the blue-sensitive rhodopsin, Rh1 (Figure 2). Rh1 is encoded by the ninaE gene, and it displays 22% amino acid identity with human rhodopsin. The inner photoreceptor cells, R7 and R8, are arranged such that a subset of the R7 cells expresses the ultraviolet (UV)-opsin, Rh3. They pair with the R8 cells expressing the blue-sensitive opsin Rh5 (p-ommatidia), while the R7 cells expressing the UV-opsin Rh4 pair with the R8 cells expressing the green-sensitive opsin Rh6 (y-ommatidia). The R7 cells are located above their partner R8 cells (Figures 1(b) and 2). Above the photoreceptor cells are four cone cells and two lens components; the pseudocone (also called the crystalline cone) and the corneal lens. Two primary pigment cells surround the cones and each ommatidium is optically isolated by a sheath of secondary and tertiary pigment cells (Figure 2). The adult visual system also contains three simple eyes, ocelli, located on the top of the head (Figure 1(c)). The ocelli express the violet-sensitive, Rh2 opsin. Drosophila photoreceptor cells contain specialized portions of the plasma membrane,

CL

PSC

PP

CC

TP

Figure 2 Schematic of an ommatidium. CL, corneal lens; PSC, pseudocone; PP, primary pigment cells; CC, cone cells; R1-6, R7, and R8 photoreceptor cells; SP and TP, secondary and tertiary pigment cells. Adapted from Tomlinson, A. and Ready, D. F. (1987). Cell fate in the Drosophila ommatidium.

Developmental Biology 123: 264–275.

called rhabdomeres, which comprises approximately 60 000 tightly packed microvilli containing rhodopsin photopigments and other components of the phototransduction cascade (Figure 1(b) and 1(d)). The microvillar processes of the rhabdomeres are functionally similar to the phototransducing disk membranes present in the vertebrate photoreceptor outer segments (Figure 3).

Phototransduction in Drosophila utilizes a signaling cascade in which light stimulation of rhodopsin leads to the activation of the heterotrimeric guanine- nucleotide-binding G protein (Gq) and the stimulation of phospholipase C beta (PLC-b), leading to the opening of the cation-selective transient receptor potential (TRP) and TRP-like (TRPL) channels. The photoreceptors depolarize as intracellular calcium dramatically rises from about 100 nm to about 10 mM. In the rhabdomeres, calcium rises even higher, to about 1 mM, and it is required for amplification, rapid response kinetics, and light adaptation in Drosophila. Calcium is subsequently removed from the rhabdomeres by a combination of sodium/calcium exchange and diffusion into the cell body where calcium increases to about 10 mM. Calcium in the cell body is buffered by calcium-binding proteins and is removed by uptake into intracellular stores by the sarco/endoplasmic reticulum (ER) calcium ATPase. The Drosophila phototransduction cascade shares some similarities with the phototransduction cascade in mammalian rod and cone photoreceptor cells. Both cascades are initiated by light-activation of rhodopsin that in turn leads to the stimulation of heterotrimeric G proteins. Phototransduction in Drosophila as well as in humans is terminated when the protein arrestin binds to lightstimulated rhodopsin and blocks the binding of rhodopsin

to Gq. However, notable differences are that rod and cone channels are gated by cyclic nucleotides and they close in response to light, leading to a hyperpolarizing response.

Although certain features of phototransduction in Drosophila differ from rod and cone phototransduction, Drosophila phototransduction shares many common features with the cascade in intrinsically photosensitive retinal ganglion cells (ipRGCs). These cells function in circadian rhythm entrainment and pupil constriction. The light response in ipRGCs is initiated with absorption of light by melanopsin, which is more similar to Drosophila rhodopsins than to the photopigments in rods and cones. Light-stimulated melanopsin is thought to activate a phophoinositide cascade leading to the opening of channels that display similar properties to TRP channels. Therefore, Drosophila photoreceptor cells and ipRGCs share similar phototransduction cascades, and studies in flies will continue to provide insights into ipRGC function.

Genetic Screens Identify Retinal

Degeneration Loci

Several forward genetic screens in Drosophila led to an explosion in the identification of many genes involved in retinal degeneration, and their counterparts in human disease. The approach has been to chemically mutagenize flies to disrupt photoreceptor cell function. The mutagenized flies are tested for function by ERG analysis, morphology by a deep pseudopupil (DPP), or Western blotting for the loss of candidate gene expression (such as arrestin). The ERG approach was pioneered by Bill Pak and co-workers in the 1960s and led to the isolation of over 200 ERG-defective mutants. In the 1980s, the development of gene-cloning techniques for Drosophila made it

possible to isolate the corresponding genes. For example, neither inactivation nor after potential E (ninaE), a mutant isolated in the original ERG screen was later found to harbor a mutation in the structural gene for the major rhodopsin in Drosophila, Rh1. In 1985, almost a decade prior to the cloning of eyeless, Drosophila rhodopsin, Rh1, was cloned and sequenced by two groups and found to display 22% amino acid identity with bovine rhodopsin. In addition, the first evidence that mutations in a rhodopsin gene led to retinal degeneration came from elegant studies in the 1980s in Drosophila. The use of the ERG in Drosophila was an effective strategy to identify mutants in phototransduction and also in retinal degeneration.

The DPP is a sensitive phenotype in the eye that can be easily assessed in live flies. It is based on the precise packing of the photoreceptor cells. Any mutation leading to, even subtle, structural alterations in photoreceptor cells will cause attenuation in the DPP. For example, a reduction in rhodopsin levels in the R1-6 photoreceptor cells in ninaE mutants, leads to structural alterations in the photoreceptor cells (Figures 1(e) and 1(f )) and attenuation in the DPP. A variety of mutants were isolated by this method, including dominant alleles of ninaE (rhodopsin), and alleles of two chaperone proteins, ninaA (cyclophilin) and calnexin. Both the ERG and the DPP screens accelerated the pace of identifying mutations that cause retinal degeneration in Drosophila.

Retinal Degenerations in Flies and

Humans

Mutations in rhodopsin are the leading cause of blinding disease in RP. RP is a heterogeneous group of inherited disorders that is characterized by progressive retinal

degeneration and eventual blindness. RP may be inherited as an X-linked (about 5–15% of cases), autosomal recessive (50–60%), and autosomal dominant trait (30–40%). It affects one person in 4000 worldwide and is often restricted to the eye, but not always. In about 20–30% of the cases of RP, the genetic defects are not eye specific. There are approximately 30 syndromes that involve RP. One of the most common syndromes is Usher’s syndrome. This syndrome is characterized by vision and hearing impairment, and mutations in myosin VIIA are responsible for one form, Usher 1B syndrome. Interestingly, loss of myosin VIIA function leads to deafness in Drosophila. In flies, like in humans, there are many examples of mutations in which the phenotype caused by the mutation is restricted to the eye, whereas there are others that are not. For example, mutations in the gene encoding rhodopsin (ninaE) and the arrestin gene cause defects that are restricted to the eye. Mutations in genes such as retinal degeneration B (rdgB, encoding a phosphatidylinositol transport protein), involve olfactory as well as visual defects.

Since the initial findings, in 1983, that mutations in Drosophila rhodopsin lead to retinal degeneration, over 100 mutations in human rhodopsin have been found to cause autosomal dominant RP (adRP). The first mutation identified in adRP patients, published by Dryja and coworkers in 1990, was a mutation that caused a proline residue located near the N-terminus of rhodopsin to be replaced by a histidine residue (Pro23His). A great majority of these mutants, including Pro23His, produce misfolded rhodopsin that is improperly transported through the secretory pathway. However, the mechanism by which the mutant rhodopsins cause dominant retinal degeneration was not known. In 1995, studies in Drosophila on rhodopsin mutations that act dominantly to cause retinal degeneration revealed that the retinal degeneration results from the interference in the maturation of normal rhodopsin by the mutant protein. These studies in Drosophila provided a mechanistic explanation for the cause of certain forms of adRP.

Mechanisms of Retinal Degenerations

Light-Dependent Retinal Degenerations

It is now widely appreciated that retinal defects and retinal degeneration can be triggered by mutations in almost every component of the photoreceptor cells. These mutations can be divided into two distinct classes. One class pertains to the unregulated activities of phototransduction and/or calcium toxicity. Mutations in this class lead to retinal degenerations that are dependent on or influenced by light stimulation of the cascade and the opening of the TRP and TRPL channels, and these are termed light dependent.

For example, some mutations in rhodopsin itself or mutations in the arrestin gene lead to light-dependent retinal degeneration. Arrestin is required for deactivating rhodopsin, and loss of arrestin causes unregulated rhodopsin and hence excessive activation of phototransduction. It is also thought that the loss of arrestin causes decreased endocytosis of Rh1 and all of these defects lead to retinal degeneration.

The precise spatial and temporal regulation of calcium is also essential for photoreceptor survival in flies and people. Prolonged elevation of cytosolic calcium or low levels of calcium can be toxic, leading to cell death and retinal degeneration. In Drosophila, mutations in arrestin, the Naþ/Ca2þ exchanger (calx ), the diacylglycerol kinase (retinal degeneration A, rdgA), and constitutively active TRP channels are all thought to trigger cell death by causing abnormally high levels of calcium. In humans, a lack of cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE), caused by mutations in PDE6A and PDE6B, leads to an elevation in the cGMP concentration in the outer segments, which in turn causes cGMP channels to be open, resulting in excessive levels of calcium. Defects in PDE6A and PDE6B cause recessive RP.

Light-Independent Retinal Degenerations

A second class of retinal degenerations involves defects in rhodopsin maturation and does not require activation of phototransduction by light. These are termed light independent. In Drosophila, as in humans, Rh1 is synthesized and glycosylated in the ER, binds its vitamin-A-derived chromophore (11-cis 3-hydroxyretinal), at a lysine residue in the seventh transmembrane domain, is transported through the various compartments of the Golgi, and is delivered to its final destination for phototransduction. The mechanisms that regulate rhodopsin maturation, such as its folding, glycosylation, chaperone interaction, chromophore attachment, and transport, are key to photoreceptor survival in flies and humans.

In flies, the transport of Rh1 from the ER to the rhabdomere requires the cyclophilin, NinaA. Cyclophilins are known to display peptidyl-prolyl cis–trans isomerase and are thought to play a role in protein folding during biosynthesis. Consistent with a role in protein folding, NinaA resides in the ER. In addition, NinaA is detected in secretory transport vesicles together with Rh1, and forms a specific and stable complex with Rh1, consistent with a broader role as a chaperone in the secretory pathway. Similarly, in mammals a cyclophilin-like protein (RanBP2/ Nup358) modulates protein biogenesis. The Drosophila cyclophilin, NinaA, is a chaperone that is specifically required for Rh1 biosynthesis and maturation. Another chaperone required for Rh1 biosynthesis in Drosophila is calnexin and mutations in ninaA (cyclophilin), ninaE (Rh1), and calnexin

all lead to severe retinal pathology in flies. In mammalian photoreceptors, calnexin is also expressed in the ER. Although calnexin is not required for the expression of rod rhodopsin, cone M-opsin, or melanopsin (in the ipRGCs) in the mouse, it is required for proper retinal morphology.

Rhodopsin in both mammals and flies undergoes N-linked glycosylation during biosynthesis, and in flies, elimination of the glycosylation site, asparagine 20 (N20I), results in the retention of rhodopsin in the secretory pathway. Moreover, in both mammals and flies, genes involved in rhodopsin chromophore biosynthesis and transport are critical to rhodopsin maturation and expression as well as photoreceptor function. Defects in chromophore production in the Drosophila mutants ninaB, ninaD, ninaG, and santa maria, cause a failure in Rh1 transport from the ER to the rhabdomere, resulting in a severe reduction in Rh1 and retinal pathology. The ninaB gene encodes an enzyme that catalyzes the conversion of carotenoids to retinal (b, b0 – carotene-15, 150-monoxygenase) and ninaG encodes an enzyme that acts to convert retinal to 3-hydroxyretinal (oxidoreductase). Two additional Drosophila loci, ninaD and santa maria, are both similar to the mammalian class B scavenger receptors and play a role in transporting b-carotene to cells. In flies, b-carotene is required in the diet for the production of all-trans retinol, which is in turn converted to 11-cis 3-hydroxyretinal. Upon light stimulation 11-cis 3-hydroxyretinal is photoconverted to all-trans 3-hydroxyretinal. Mutations in chromophore biosythesis result in defective Rh1 maturation, low levels of Rh1, and retinal pathology, establishing the importance of vitamin A in the fly.

Once rhodopsin exits the ER, it requires several Rabguanosine triphosphate (GTP)ases for vesicular transport through the secretory pathway in flies and in mammals. Rab-GTPases are conserved from yeast to humans and are members of the Ras superfamily of small GTPases. They function in distinct steps in membrane trafficking pathways including vesicle formation, actinand tubulin-dependent vesicle movement, and membrane fusion events. In Drosophila, Rab1, Rab6, and Rab11 mediate vesicular fusion between the ER and the Golgi (Rab1), intra-Golgi (Rab6), and post-Golgi (Rab11) transport of rhodopsin in Drosophila. Defects in Rab function cause inadequate Rh1 transport and retinal pathology. Therefore, the mechanisms that regulate Rh1 maturation, such as its folding, chaperone interaction, and chromophore binding and transport are essential for photoreceptor health in flies and humans.

Retinal Degenerations Caused by Mutations in Dual-Role Proteins

Although most retinal degenerations are classified as either light dependent or light independent, there is a growing list of retinal degenerations that fall into both

classes. In these cases, the corresponding mutant proteins play dual roles. For example, as was described above, calnexin is a chaperone required for rhodopsin maturation. In addition, it is a calcium-binding protein for regulating calcium in photoreceptor cells. Mutations in calnexin lead to defects in Rh1 maturation and retinal degeneration. The degeneration due to defects in rhodopsin maturation is light independent, but calnexin mutants also display prolonged and elevated levels of calcium, following light stimulation. In the calnexin mutants, the retinal degeneration is enhanced by the stimulation of phototransduction by light. Therefore, calnexin plays a dual role: one in rhodopsin maturation and another in calcium modulation.

Summary

In the 1980s, Drosophila took on a surprising new role, as an animal model for retinal disease, when the genetic similarities and fundamental processes between flies and humans became apparent. It became clear that information obtained in flies was transferable to human blinding diseases. As a result, and since then, there has been an explosion in the use of Drosophila as an animal model for unraveling the molecular genetic basis of retinal degeneration disorders. Despite its perceived simplicity, the fruit fly is, indeed, a remarkably complex creature with a genetic makeup that is surprisingly similar to our own. Investigators continue to capitalize on a whole host of versatile genetic techniques together with the accessibility of the fly to dissect fundamental photoreceptor cell mechanisms in vivo. The short life span of the fly, only 2 months, allows for monitoring the onset and progression of retinal degeneration in a short time. These advantageous features place Drosophila at the forefront of current research efforts, aimed at unraveling the basis of and therapeutic treatments for retinal degenerative disorders.

Acknowledgments

Our research, on retinal degeneration in Drosophila, is supported by funding from the National Eye Institute, the Retina Research Foundation, and the Retina Research Foundation/Walter H. Helmerich Research Chair. I gratefully acknowledge C. Vang, E. Rosenbaum and B. Larson for assistance with preparing the figures. For the poem, I thank M. Annucci, author of Luck (Parallel Press) and member of the Department of Languages and Literatures at the University of Wisconsin-Whitewater.

See also: Circadian Photoreception; Circadian Rhythms in the Fly’s Visual System; Coordinating Division and Differentiation in Retinal Development; Embryology and Early Patterning; Evolution of Opsins; Ganglion Cell Development: Early Steps/Fate; Genetic Dissection of

Invertebrate Phototransduction; Histogenesis: Cell Fate: Signaling Factors; Photoreceptor Development: Early Steps/Fate; The Photoreceptor Outer Segment as a Sensory Cilium; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Primary Photoreceptor Degenerations: Retinitis Pigmentosa; Primary Photoreceptor Degenerations: Terminology; Retinal Histogenesis; Secondary Photoreceptor Degenerations: Age-Related Macular Degeneration; Secondary Photoreceptor Degenerations; Xenopus laevis as a Model for Understanding Retinal Diseases; Zebra Fish as a Model for Understanding Retinal Diseases; Zebra Fish–Retinal Development and Regeneration.

Further Reading

Bok, D. (2007). Contributions of genetics to our understanding of inherited monogenic retinal diseases and age-related macular degeneration. Archives of Ophthalmology 125: 160–164.

Colley, N. J., Baker, E. K., Stamnes, M. A., et al. (1991). The cyclophilin homolog ninaA is required in the secretory pathway. Cell 67: 255–263.

Colley, N. J., Cassill, J. A., Baker, E. K., et al. (1995). Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration. Proceedings of the National Academy of Sciences of the United States of America 92: 3070–3074.

Graham, D. M., Wong, K. Y., Shapiro, P., et al. (2008). Melanopsin ganglion cells use a membrane-associated rhabdomeric phototransduction cascade. Journal of Neurophysiology 99: 2522–2532.

Greenspan, R. J. and Dierick, H. A. (2004). ‘Am not I a fly like thee?’ From genes in fruit flies to behavior in humans. Human Molecular Genetics 13(2): R267–R273.

Halder, G., Callaerts, P., and Gehring, W. J. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267: 1788–1792.

Hardie, R. C. and Postma, M. (2008). Phototransduction in microvillar photoreceptors of Drosophila and other invertebrates. In: Allan, A. K., Basbaum, I., Shepherd, G. M., and Westheimer, G. (eds.) The Senses: A Comprehensive Reference vol. 1, pp. 77–130. San Diego, CA: Academic Press.

Hartong, D. T., Berson, E. L., and Dryja, T. P. (2006). Retinitis pigmentosa. Lancet 368: 1795–1809.

Pak, W. L. (1995). Drosophila in vision research. The Friedenwald lecture. Investigative Ophthalmology and Visual Science 36: 2340–2357.

Reiter, L. T., Potocki, L., Chien, S., et al. (2001). A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster. Genome Research 11: 1114–1125.

Rosenbaum, E. E., Hardie, R. C., and Colley, N. J. (2006). Calnexin is essential for rhodopsin maturation, Ca2þ regulation, and photoreceptor cell survival. Neuron 49: 229–241.

Rubin, G. M. and Lewis, E. B. (2000). A brief history of Drosophila’s contributions to genome research. Science 287: 2216–2218.

Tomlinson, A. and Ready, D. F. (1987). Cell fate in the Drosophila ommatidium. Developmental Biology 123: 264–275.

Wang, T. and Montell, C. (2007). Phototransduction and

retinal degeneration in Drosophila. Pflugers Archiv 454: 821–847. Wernet, M. F., Celik, A., Mikeladze-Dvali, T., et al. (2007). Generation of

uniform fly retinas. Current Biology 17: R1002–R1003.

Relevant Website

http://www.sph.uth.tmc.edu – Genes and mapped loci causing retinal diseases: Homepage.