Chaperones

The retinal loop indicates that chaperones play an important role at each locus in the cycle, guiding retinoids to their specific site.

PHOTOTRANSDUCTION

Activation

The light activation of rhodopsin generates an amplification cascade that leads to changes in the resting potential of the photoreceptor. There is ongoing outward potassium current through nongated K+-selective channels. This outward current tends to hyperpolarize the photoreceptor at around −70 mV (the equilibrium potential for K+). There is also inward sodium current carried by cyclic guanosine monophosphate (cGMP)-gated sodium channels. This so-called “dark current” depolarizes the cell to around −40 mV.

Light activation causes metarhodopsin II to activate transducin, which in turn activates a photoreceptor-specific cGMP phosphodiesterase, resulting in a decrease in intracellular cGMP. It reduces the permeability of plasma membrane cGMP-gated cation channels, leading to hyperpolarization of the photoreceptor cell membrane and decreased neurotransmitter release at synapses with bipolar cells. While in the dark, as cGMP levels are high, cGMP-gated sodium channels remain open, allowing a steady inward current which keeps the cell depolarized at about −40 mV. The depolarization of the cell membrane in the dark allows sodium to exit and calcium to enter and opens voltagegated calcium channels, releasing glutamate into the synaptic cleft. This excitatory neurotransmitter hyperpolarizes on-center bipolar cells and depolarizes the off-center bipolar cells.

Inactivation

Removing the light stimulus leads to inactivation of the visual cascade. This involves several steps:

●  The rhodopsin is phosphorylated by rhodopsin kinase. Arrestin (S-antigen) binds to the phosphorylated proteins, preventing continued activation of transducin.

●  GTPase-activating protein (RGS9) interacts with the alpha subunit of transducin, and causes it to hydrolyze its bound guanosine triphosphate (GTP) to guanosine diphosphate (GDP), and thus halts the action of phosphodiesterase, stopping the transformation of cGMP to GMP.

●  Guanylate cyclase activating protein (GCAP) is a calcium-binding protein, and as the calcium levels in the cell decrease, GCAP dissociates from its bound calcium ions, and interacts with guanylate cyclase, activating it. Guanylate cyclase then proceeds to transform GTP to cGMP, replenishing the cell’s cGMP levels and thus reopening the sodium channels that were closed during phototransduction.

●  Metarhodopsin II is deactivated. Recoverin, a calcium-binding protein, is normally bound to rhodopsin kinase. When the calcium levels fall during phototransduction, the calcium dissociates from recoverin, and rhodopsin kinase is released. It phosphorylates metarhodopsin II, which decreases its affinity for transducin.

RETINAL PIGMENT EPITHELIUM AND LIPOFUSCIN

RETINAL PIGMENT EPITHELIUM

An intact RPE is essential for the proper functioning of the visual process. This postmitotic, multifunctional layer is involved in the degradation of photoreceptor outer segments, vitamin A cycle, and support of retinal metabolism and maintenance of the outer BRB. As a conse-

quence of excessive metabolism, high oxygen levels, exposure to light of short wavelength, and free radical formation, the RPE is constantly exposed to insults and cumulative RPE damage.

LIPOFUSCIN

Cells of the RPE phagocytose the membranous discs of the rods and cones. These discs are made of rhodopsin, proteins, and lipids. Rhodopsin consists of 11 cis-retinal and opsin. When light-activated, 11-cis retinal is converted to all-trans retinal and then reduced to all- trans retinol to proceed through the visual cycle. However, the excess of all-trans retinal reacts with the lipids and proteins of the discs to form lipofuscin.

Formation of lipofuscin

The most widely studied lipofuscin pigments in the RPE are the bisretinoid compound A2E. A2E formation begins in photoreceptor outer segments from nonenzymatic reaction between the membrane lipid, phosphatidylethanolamine, and all-trans retinal to form N-retinylidene phosphatidylethanolamine (NRPE). After tautomerization, NRPE reacts with a second molecule of all-trans retinal to form a pyridinium ring to generate A2PE-H2. This compound autooxidizes and yields A2-PE. The A2-PE is deposited in RPE cells during the normal process of outer-segment phagocytosis and subsequent hydrolysis by phospholipase D generates A2E. A2E is taken up by the lysosomes. When A2E is photoactivated, it fragments and generates a number of photo-oxida- tion products, including epoxides, furanoid oxides, and cyclic peroxides. Other products of reactions of all-trans retinal vary in structure and absorbance spectra. Little is known of these products. The accumulation of lipofuscin increases with age to approximately 70 years. With age, the RPE cells become less efficient in coping with A2E and the associated burden. The lipofuscin fluorescence decreases or reaches a plateau in the eighth decade, probably due to death of lipofuscin-laden RPE cells and the change in ratio of A2E and A2E photo-oxidation products within the cells.

Lipofuscin and RPE atrophy

A healthy RPE is essential for the proper functioning of the rods and cones. Lipofuscin may induce RPE cell death and atrophy in several ways. Blue light (wavelength 435 nm) has been shown to cause damage to RPE cells in vivo and in vitro. The propensity for RPE cells to be susceptible to blue light-induced damage or death is related to the proportion of A2E accumulation in the cells. This insult is not seen with green light (wavelength 514–532 nm) or in RPE cells devoid of A2E. The lightinduced damage may be caused by the direct reaction of the photoactivation molecule with the cellular constituents and through the formation of reactive oxygen species. Antioxidants such as vitamins C and E and Bilberry have been shown to suppress photo-oxidation of A2E in RPE. The RPE cell death pathway involves the activation of caspases and is modulated by the mitochondrial protein Bcl-2. The JNK signaling pathway may have a protective role of photo-oxidation of A2E.

Another hypothesis that explains A2E-induced RPE damage is lysosomal dysfunction. Studies have shown that lipofuscin also causes instability of lysosmal membranes due to membrane blebbing. It also reduces the acidity of the cell organelles, resulting in failure of proteolytic digestion.

Lipofuscin also interferes with the metabolism of phagocytosed lipids of the outer segments within the RPE cells. The A2E-induced alterations in cholesterol homeostasis within the RPE may have several bearings on the aging process as well as in hereditary retinal degeneration and age-related macular degeneration. It is postulated that free cholesterol and the esters extruded from the RPE may contribute to age-related lipid accumulation in the BM and the lipid constituents of drusen and other subretinal deposits. In addition, it may act as a nidus for chronic inflammatory reaction, interfere with the visual cycle, and inhibit other degradative processes and functions of other lipiddependent receptors and transporters.

Retina in Sciences Basic • 1 section