Janis Lem

Rapid and Reproducible Deactivation of Rhodopsin Requires Multiple Phosphorylation Sites

Efficient single-photon detection by retinal rod photoreceptors requires timely and reproducible deactivation of rhodopsin. Like other G protein-coupled receptors, rhodopsin contains multiple sites for phosphorylation at its COOH-terminal domain. Transgenic and electrophysiological methods were used to functionally dissect the role of the multiple phosphorylation sites during deactivation of rhodopsin in intact mouse rods. Mutant rhodopsins bearing zero, one (S338), or two (S334/S338) phosphorylation sites generated single-photon responses with greatly prolonged, exponentially distributed durations. Responses from rods expressing mutant rhodopsins bearing more than two phosphorylation sites declined along smooth, reproducible time courses; the rate of recovery increased with increasing numbers of phosphorylation sites. We conclude that multiple phosphorylation of rhodopsin is necessary for rapid and reproducible deactivation.

Evidence for two apoptotic pathways in light-induced retinal degeneration

Excessive phototransduction signaling is thought to be involved in light-induced and inherited retinal degeneration. Using knockout mice with defects in rhodopsin shut-off and transducin signaling, we show that two different pathways of photoreceptor-cell apoptosis are induced by light. Bright light induces apoptosis that is independent of transducin and accompanied by induction of the transcription factor AP-1. By contrast, low light induces an apoptotic pathway that requires transducin. We also provide evidence that additional genetic factors regulate sensitivity to light-induced damage. Our use of defined mouse mutants resolves some of the complexity underlying the mechanisms that regulate susceptibility to retinal degeneration.

Physiological features of the S- and M-cone photoreceptors of wild-type mice from single-cell recordings.

Cone cells constitute only 3% of the photoreceptors of the wild-type (WT) mouse. While mouse rods have been thoroughly investigated with suction pipette recordings of their outer segment membrane currents, to date no recordings from WT cones have been published, likely because of the rarity of cones and the fragility of their outer segments. Recently, we characterized the photoreceptors of Nrl −/− mice, using suction pipette recordings from their “inner segments” (perinuclear region), and found them to be cones. Here we report the use of this same method to record for the first time the responses of single cones of WT mice, and of mice lacking the α-subunit of the G-protein transducin (G tα−/−), a loss that renders them functionally rodless. Most cones were found to functionally co-express both S- (λmax = 360 nm) and M- (λmax = 508 nm) cone opsins and to be maximally sensitive at 360 nm (“S-cones”); nonetheless, all cones from the dorsal retina were found to be maximally sensitive at 508 nm (“M-cones”). The dim-flash response kinetics and absolute sensitivity of S- and M-cones were very similar and not dependent on which of the coexpressed cone opsins drove transduction; the time to peak of the dim-flash response was ∼70 ms, and ∼0.2% of the circulating current was suppressed per photoisomerization. Amplification in WT cones (A ∼4 s−2) was found to be about twofold lower than in rods (A ∼8 s−2). Mouse M-cones maintained their circulating current at very nearly the dark adapted level even when >90% of their M-opsin was bleached. S-cones were less tolerant to bleached S-opsin than M-cones to bleached M-opsin, but still far more tolerant than mouse rods to bleached rhodopsin, which exhibit persistent suppression of nearly 50% of their circulating current following a 20% bleach. Thus, the three types of mouse opsin appear distinctive in the degree to which their bleached, unregenerated opsins generate “dark light.”

Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice

A 10 μm spot of argon laser light was focused onto the outer segments of intact mouse rods loaded with fluo‐3, fluo‐4 or fluo‐5F, to estimate dark, resting free Ca2+ concentration ([Ca2+]i) and changes in [Ca2+]i upon illumination. Dye concentration was adjusted to preserve the normal physiology of the rod, and the laser intensity was selected to minimise bleaching of the fluorescent dye. Wild‐type mouse rods illuminated continuously with laser light showed a progressive decrease in fluorescence well fitted by two exponentials with mean time constants of 154 and 540 ms. Rods from transducin α‐subunit knock‐out (Trα–/–) animals showed no light‐dependent decline in fluorescence but exhibited an initial rapid component of fluorescence increase which could be fitted with a single exponential (τ∼1–4 ms). This fluorescence increase was triggered by rhodopsin bleaching, since its amplitude was reduced by pre‐exposure to bright bleaching light and its time constant decreased with increasing laser intensity. The rapid component was however unaffected by incorporation of the calcium chelator BAPTA and seemed therefore not to reflect an actual increase in [Ca2+]i. A similar rapid increase in fluorescence was also seen in the rods of wild‐type mice just preceding the fall in fluorescence produced by the light‐dependent decrease in [Ca2+]i. Dissociation constants were measured in vitro for fluo‐3, fluo‐4 and fluo‐5F with and without 1 mm Mg2+ from 20 to 37 °C. All three dyes showed a strong temperature dependence, with the dissociation constant changing by a factor of 3–4 over this range. Values at 37 °C were used to estimate absolute levels of rod [Ca2+]i. All three dyes gave similar values for [Ca2+]i in wild‐type rods of 250 ± 20 nm in darkness and 23 ± 2 nm after exposure to saturating light. There was no significant difference in dark [Ca2+]i between wild‐type and Trα–/– animals.

Spontaneous activity of opsin apoprotein is a cause of Leber congenital amaurosis

Mutations in Rpe65 disrupt synthesis of the opsin chromophore ligand 11-cis-retinal and cause Leber congenital amaurosis (LCA), a severe, early-onset retinal dystrophy. To test whether light-independent signaling by unliganded opsin causes the degeneration, we used Rpe65-null mice, a model of LCA. Dark-adapted Rpe65−/− mice behaved as if light adapted, exhibiting reduced circulating current, accelerated response turn-off, and diminished intracellular calcium. A genetic block of transducin signaling completely rescued degeneration irrespective of an elevated level of retinyl ester. These studies clearly show that activation of sensory transduction by unliganded opsin, and not the accumulation of retinyl esters, causes light-independent retinal degeneration in LCA. A similar mechanism may also be responsible for degeneration induced by vitamin A deprivation.

Membrane protein diffusion sets the speed of rod phototransduction

Retinal rods signal the activation of a single receptor molecule by a photon. To ensure efficient photon capture, rods maintain about 109 copies of rhodopsin densely packed into membranous disks. But a high packing density of rhodopsin may impede other steps in phototransduction that take place on the disk membrane, by restricting the lateral movement of, and hence the rate of encounters between, the molecules involved. Although it has been suggested that lateral diffusion of proteins on the membrane sets the rate of onset of the photoresponse, it was later argued that the subsequent processing of the complexes was the main determinant of this rate. The effects of protein density on response shut-off have not been reported. Here we show that a roughly 50% reduction in protein crowding achieved by the hemizygous knockout of rhodopsin in transgenic mice accelerates the rising phases and recoveries of flash responses by about 1.7-fold in vivo. Thus, in rods the rates of both response onset and recovery are set by the diffusional encounter frequency between proteins on the disk membrane.

Umami Taste Responses Are Mediated by α-Transducin and α-Gustducin

The sense of taste comprises at least five distinct qualities: sweet, bitter, sour, salty, and umami, the taste of glutamate. For bitter, sweet, and umami compounds, taste signaling is initiated by binding of tastants to G-protein-coupled receptors in specialized epithelial cells located in the taste buds, leading to the activation of signal transduction cascades. α-Gustducin, a taste cell-expressed G-protein α subunit closely related to the α-transducins, is a key mediator of sweet and bitter tastes. α-Gustducin knock-out (KO) mice have greatly diminished, but not entirely abolished, responses to many bitter and sweet compounds. We set out to determine whether α-gustducin also mediates umami taste and whether rod α-transducin (αt-rod), which is also expressed in taste receptor cells, plays a role in any of the taste responses that remain in α-gustducin KO mice. Behavioral tests and taste nerve recordings of single and double KO mice lacking α-gustducin and/or αt-rod confirmed the involvement of α-gustducin in bitter (quinine and denatonium) and sweet (sucrose and SC45647) taste and demonstrated the involvement ofα-gustducin in umami [monosodium glutamate (MSG), monopotassium glutamate (MPG), and inosine monophosphate (IMP)] taste as well. We found that αt-rod played no role in taste responses to the salty, bitter, and sweet compounds tested or to IMP but was involved in the umami taste of MSG and MPG. Umami detection involving α-gustducin and αt-rod occurs in anteriorly placed taste buds, however taste cells at the back of the tongue respond to umami compounds independently of these two G-protein subunits.

Lentiviral gene transfer of RPE65 rescues survival and function of cones in a mouse model of Leber congenital amaurosis.

Background RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice. Methods and Findings Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease. Conclusions By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65 −/− mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose.

Subunit Dissociation and Diffusion Determine the Subcellular Localization of Rod and Cone Transducins

Activation of rod photoreceptors by light induces a massive redistribution of the heterotrimeric G-protein transducin. In darkness, transducin is sequestered within the membrane-enriched outer segments of the rod cell. In light, it disperses throughout the entire neuron. We show here that redistribution of rod transducin by light requires activation, but it does not require ATP. This observation rules out participation of molecular motors in the redistribution process. In contrast to the light-stimulated redistribution of rod transducin in rods, cone transducin in cones does not redistribute during activation. Remarkably, when cone transducin is expressed in rods, it does undergo light-stimulated redistribution. We show here that the difference in subcellular localization of activated rod and cone G-proteins correlates with their affinity for membranes. Activated rod transducin releases from membranes, whereas activated cone transducin remains bound to membranes. A synthetic peptide that dissociates G-protein complexes independently of activation facilitates dispersion of both rod and cone transducins within the cells. This peptide also facilitates detachment of both G-proteins from the membranes. Together, these results show that it is the dissociation state of transducin that determines its localization in photoreceptors. When rod transducin is stimulated, its subunits dissociate, leave outer segment membranes, and equilibrate throughout the cell. Cone transducin subunits do not dissociate during activation and remain sequestered within the outer segment. These findings indicate that the subunits of some heterotrimeric G-proteins remain associated during activation in their native environments.

Genetic Dissection of Rod and Cone Pathways in the Dark-Adapted Mouse Retina

A monumental task of the mammalian retina is to encode an enormous range (>10(9)-fold) of light intensities experienced by the animal in natural environments. Retinal neurons carry out this task by dividing labor into many parallel rod and cone synaptic pathways. Here we study the operational plan of various rod- and cone-mediated pathways by analyzing electroretinograms (ERGs), primarily b-wave responses, in dark-adapted wildtype, connexin36 knockout, depolarizing rod-bipolar cell (DBCR) knockout, and rod transducin alpha-subunit knockout mice [WT, Cx36(-/-), Bhlhb4(-/-), and Tralpha(-/-)]. To provide additional insight into the cellular origins of various components of the ERG, we compared dark-adapted ERG responses with response dynamic ranges of individual retinal cells recorded with patch electrodes from dark-adapted mouse retinas published from other studies. Our results suggest that the connexin36-mediated rod-cone coupling is weak when light stimulation is weak and becomes stronger as light stimulation increases in strength and that rod signals may be transmitted to some DBCCs via direct chemical synapses. Moreover, our analysis indicates that DBCR responses contribute about 80% of the overall DBC response to scotopic light and that rod and cone signals contribute almost equally to the overall DBC responses when stimuli are strong enough to saturate the rod bipolar cell response. Furthermore, our study demonstrates that analysis of ERG b-wave of dark-adapted, pathway-specific mutants can be used as an in vivo tool for dissecting rod and cone synaptic pathways and for studying the functions of pathway-specific gene products in the retina.