Bruce M. Brown

Mouse Cones Require an Arrestin for Normal Inactivation of Phototransduction

Arrestins are proteins that arrest the activity of G protein-coupled receptors (GPCRs). While it is well established that normal inactivation of photoexcited rhodopsin, the GPCR of rod phototransduction, requires arrestin (Arr1), it has been controversial whether the same requirement holds for cone opsin inactivation. Mouse cone photoreceptors express two distinct visual arrestins: Arr1 and Arr4. By means of recordings from cones of mice with one or both arrestins knocked out, this investigation establishes that a visual arrestin is required for normal cone inactivation. Arrestin-independent inactivation is 70-fold more rapid in cones than in rods, however. Dual arrestin expression in cones could be a holdover from ancient genome duplication events that led to multiple isoforms of arrestin, allowing evolutionary specialization of one form while the other maintains the basic function.

GRK1-Dependent Phosphorylation of S and M Opsins and Their Binding to Cone Arrestin during Cone Phototransduction in the Mouse Retina

The shutoff mechanisms of the rod visual transduction cascade involve G-protein-coupled receptor (GPCR) kinase 1 (GRK1) phosphorylation of light-activated rhodopsin (R*) followed by rod arrestin binding. Deactivation of the cone phototransduction cascade in the mammalian retina is delineated poorly. In this study we sought to explore the potential mechanisms underlying the quenching of the phototransduction cascade in cone photoreceptors by using mouse models lacking rods and/or GRK1. Using the “pure-cone” retinas of the neural retina leucine zipper (Nrl) knock-out (KO, -/-) mice (Mears et al., 2001), we have demonstrated the light-dependent, multi-site phosphorylation of both S and M cone opsins by in situ phosphorylation and isoelectric focusing. Immunoprecipitation with affinity-purified polyclonal antibodies against either mouse cone arrestin (mCAR) or mouse S and M cone opsins revealed specific binding of mCAR to light-activated, phosphorylated cone opsins. To elucidate the potential role of GRK1 in cone opsin phosphorylation, we created Nrl and Grk1 double knock-out (Nrl-/-Grk1-/-) mice by crossing the Nrl-/- mice with Grk1-/- mice (Chen et al., 1999). We found that, in the retina of these mice, the light-activated cone opsins were neither phosphorylated nor bound with mCAR. Our results demonstrate, for the first time in a mammalian species, that cone opsins are phosphorylated and that CAR binds to phosphorylated cone opsins after light activation.

Visual Arrestin 1 Acts As a Modulator for N-Ethylmaleimide-Sensitive Factor in the Photoreceptor Synapse

In the G-protein-coupled receptor phototransduction cascade, visual Arrestin 1 (Arr1) binds to and deactivates phosphorylated light-activated opsins, a process that is critical for effective recovery and normal vision. In this report, we discovered a novel synaptic interaction between Arr1 and N-ethylmaleimide-sensitive factor (NSF) that is enhanced in a dark environment when mouse photoreceptors are depolarized and the rate of exocytosis is elevated. In the photoreceptor synapse, NSF functions to sustain a higher rate of exocytosis, in addition to the compensatory endocytosis to retrieve and to recycle vesicle membrane and synaptic proteins. Not only does Arr1 bind to the junction of NSF N-terminal and its first ATPase domains in an ATP-dependent manner in vitro, but Arr1 also enhances both NSF ATPase and NSF disassembly activities. In in vivo experiments in mouse retinas with the Arr1 gene knocked out, the expression levels of NSF and other synapse-enriched components, including vGLUT1 (vesicular glutamate transporter 1), EAAT5 (excitatory amino acid transporter 5), and VAMP2 (vesicle-associated membrane protein 2), are markedly reduced, which leads to a substantial decrease in the exocytosis rate with FM1-43. Thus, we propose that the Arr1 and NSF interaction is important for modulating normal synaptic function in mouse photoreceptors. This study demonstrates a vital alternative function for Arr1 in the photoreceptor synapse and provides key insights into the potential molecular mechanisms of inherited retinal diseases, such as Oguchi disease and Arr1-associated retinitis pigmentosa.

Visual Arrestin 1 Contributes to Cone Photoreceptor Survival and Light Adaptation

PURPOSE To evaluate morphologic and functional contributions of Arrestin 1 (Arr1) and Arrestin 4 (Arr4) in cone photoreceptors, the authors examined the phenotypes of visual arrestin knockout mice (Arr1(-/-), Arr4(-/-), Arr1(-/-)Arr4(-/-) [Arr-DKO]) reared in darkness. METHODS Retinal rods and cones were evaluated in wild-type (WT), Arr1(-/-), Arr4(-/-), and Arr-DKO mice using quantitative morphologic analysis, immunoblot, immunohistochemistry, TUNEL, and electroretinographic (ERG) techniques. RESULTS Compared with either Arr4(-/-) or WT, Arr1(-/-) and Arr-DKO mice had increased apoptotic nuclei in their retinal outer nuclear layer (ONL) at postnatal day (P) 22. By P60, cone density was significantly diminished, but the ONL appeared normal. After 1 minute of background illumination, cone ERG b-wave amplitudes were similar in WT and all Arr KO mice. However, by 3 minutes and continuing through 15 minutes of light adaptation, the cone b-wave amplitudes of WT and Arr4(-/-) mice increased significantly over those of the Arr1(-/-) and Arr-DKO mice, which demonstrated no cone b-wave amplitude increase. In contrast, ERG flicker analysis after the 15-minute light adaptation period demonstrated no loss in amplitude for either Arr1(-/-) or Arr4(-/-) mice, whereas Arr-DKO had significantly lower amplitudes. When Arr1 expression was restored in Arr1(-/-) mice (+p48(Arr1-/-)), normal cone density and light-adapted ERG b-wave amplitudes were observed. CONCLUSIONS In the adult dark-reared Arr1(-/-) and Arr-DKO mice, viable cones diminish over time. Arr1 expression is essential for cone photoreceptor survival and light adaptation, whereas either Arr1 or Arr4 is necessary for maintaining normal flicker responses.

Bovine arylalkylamine N-acetyltransferease activity correlated with mRNA expression in pineal and retina

Arylalkylamine N-acetyltransferase (AA-NAT, E. C. 2.3.1.87) is the enzyme that catalyzes the transfer of an acetyl group from acetyl-CoA to serotonin to form N-acetylserotonin (NAS) in the indoleamine biosynthetic pathway. Bovine pineal AA-NAT, partially purified on an anion exchange column, displayed an 8-fold higher enzymatic activity in pineals from animals killed in early morning (0800) compared to an afternoon group (1430). Poly A(+) mRNA was isolated from early morning bovine pineals, used to construct a mammalian expression cDNA library (lambdaZAP Express), and then screened with a rat AA-NAT cDNA to isolate a 924 basepair cDNA that encodes the bovine pineal AA-NAT. The amino acid sequence alignment reveals that bovine AA-NAT shares 94.20%, 78.54%, 76.33% and 56.3% identity to ovine, rat, human and chicken sequences, respectively. Northern blot analysis demonstrates a 0.7-fold higher mRNA level in pineal glands taken from animals from the 0800 time-point compared with mRNA from the 1430 time-point. AA-NAT mRNA was expressed at high levels in pineal and retina, but the message was undetectable in adrenal, cerebellum, cortex, small intestine, testis and thyroid. Based on the significant identity of amino acid sequence and the similar mRNA expression pattern, these data suggest that the bovine AA-NAT is more analogous to the ovine rather than either the rat, human or chicken AA-NAT.

Cone Arrestin Expression and Induction in Retinoblastoma Cells

Arrestins are regulatory proteins that down-regulate phosphorylated G-protein coupled receptors (GPCRs). The arrestin superfamily includes visual arrestins, beta-arrestins (βarrestins) and insect chemosensory arrestins. Two members of visual arrestins have been identified in vertebrate photoreceptors: rod arrestin (also known as S-antigen or Arrestin 1) and cone arrestin (X-arrestin or Arresting. Rod arrestin was the first member to be molecularly characterized,1 and its structure and function have been extensively studied in recent years. In vertebrate rod photoreceptors, rod arrestin quenches the light-induced phototransduction cascade by binding preferentially to the light-activated, phosphorylated rhodopsin.2,3 Subsequently, βarrestinl4 and βarrestin25 were identified and shown to have a ubiquitous expression pattern. βarrestins have an analogous function to rod arrestin in the termination of GPCR signaling by sterically inhibiting the coupling of phosphorylated receptors to their respective G proteins.4, 5 Recent evidence suggests additional roles for βarrestins in both termination of receptor signaling and signaling to downstream effectors by interacting with clathrin-coated pits and the tyrosine kinase c-Src.6-11

Neovascularization, Enhanced Inflammatory Response, and Age-Related Cone Dystrophy in the Nrl−/−Grk1−/− Mouse Retina

PURPOSE The effects of aging and light exposure on cone photoreceptor survival were compared between mouse retinas of neural retina leucine zipper knockout (Nrl(-/-)) mice and double-knockout mice lacking G-protein-coupled receptor kinase 1 (Nrl(-/-)Grk1(-/-)). METHODS Mice were reared in total darkness, ambient cyclic light, or constant light, and their retinas were evaluated from 1 to 9 months of age using immunohistochemistry, electroretinography, and fluorescein angiography. Retinal gene expression and statistically significant probe sets were categorized using analysis software. Select gene expression changes were confirmed with quantitative RT-PCR. RESULTS In contrast to retinas from Nrl(-/-), those from Nrl(-/-)Grk1(-/-) exhibit a progressive loss of the outer nuclear layer, retinal physiology deficits, and a higher rate of degeneration with increasing age that is independent of environmental light exposure. Changes in retinal neovascularization occur in the Nrl(-/-)Grk1(-/-) at 1 month, before the onset of significant cone functional deficits. Microarray analyses demonstrate statistically significant changes in transcript levels of more than 400 genes, of which the oncostatin M signaling pathway and the inflammatory disease response network were identified. CONCLUSIONS These data demonstrate that the loss of functional Grk1 on the enhanced S-cone Nrl(-/-) background exacerbates age-related cone dystrophy in a light-independent manner, mediated partly through the inflammatory response pathway and neovascularization. According to these findings, Grk1 helps to maintain a healthy cone environment, and the Nrl(-/-)Grk1(-/-) mouse allows examination of the alternative roles of Grk1 in cone photoreceptor homeostasis.

Effect of G Protein-Coupled Receptor Kinase 1 (Grk1) Overexpression on Rod Photoreceptor Cell Viability

PURPOSE Photoreceptor rhodopsin kinase (Rk, G protein-dependent receptor kinase 1 [Grk1]) phosphorylates light-activated opsins and channels them into an inactive complex with visual arrestins. Grk1 deficiency leads to human retinopathy and heightened susceptibility to light-induced photoreceptor cell death in the mouse. The goal of this study was to determine whether excess Grk1 activity is protective against photoreceptor cell death. METHODS Grk1-overexpressing transgenic mice (Grk1(+)) were generated by using a bacterial artificial chromosome (BAC) construct containing mouse Grk1, along with its flanking sequences. Quantitative reverse transcription-PCR, immunoblot analysis, immunostaining, and activity assays were combined with electrophysiology and morphometric analysis, to evaluate Grk1 overexpression and its effect on physiologic and morphologic retinal integrity. Morphometry and nucleosome release assays measured differences in resistance to photoreceptor cell loss between control and transgenic mice exposed to intense light. RESULTS Compared with control animals, the Grk1(+) transgenic line had approximately a threefold increase in Grk1 transcript and immunoreactive protein. Phosphorylated opsin immunochemical staining and in vitro phosphorylation assays confirmed proportionately higher Grk1 enzyme activity. Grk1(+) mice retained normal rod function, normal retinal appearance, and lacked evidence of spontaneous apoptosis when reared in cyclic light. In intense light, Grk1(+) mice showed photoreceptor damage, and their susceptibility was more pronounced than that of control mice with prolonged exposure times. CONCLUSIONS Enhancing visual pigment deactivation does not appear to protect against apoptosis; however, excess flow of opsin into the deactivation pathway may actually increase susceptibility to stress-induced cell death similar to some forms of retinal degeneration.

Visual Cone Arrestin 4 Contributes to Visual Function and Cone Health

PURPOSE Visual arrestins (ARR) play a critical role in shutoff of rod and cone phototransduction. When electrophysiological responses are measured for a single mouse cone photoreceptor, ARR1 expression can substitute for ARR4 in cone pigment desensitization; however, each arrestin may also contribute its own, unique role to modulate other cellular functions. METHODS A combination of ERG, optokinetic tracking, immunohistochemistry, and immunoblot analysis was used to investigate the retinal phenotypes of Arr4 null mice (Arr4-/-) compared with age-matched control, wild-type mice. RESULTS When 2-month-old Arr4-/- mice were compared with wild-type mice, they had diminished visual acuity and contrast sensitivity, yet enhanced ERG flicker response and higher photopic ERG b-wave amplitudes. In contrast, in older Arr4-/- mice, all ERG amplitudes were significantly reduced in magnitude compared with age-matched controls. Furthermore, in older Arr4-/- mice, the total cone numbers decreased and cone opsin protein immunoreactive expression levels were significantly reduced, while overall photoreceptor outer nuclear layer thickness was unchanged. CONCLUSIONS Our study demonstrates that Arr4-/- mice display distinct phenotypic differences when compared to controls, suggesting that ARR4 modulates essential functions in high acuity vision and downstream cellular signaling pathways that are not fulfilled or substituted by the coexpression of ARR1, despite its high expression levels in all mouse cones. Without normal ARR4 expression levels, cones slowly degenerate with increasing age, making this a new model to study age-related cone dystrophy.

SLOWED PHOTORESPONSE RECOVERY AND AGE-RELATED DEGENERATION IN CONES LACKING G PROTEIN-COUPLED RECEPTOR KINASE 1

The vertebrate retina is a specialized neural network that contains very sensitive signal transducers—the rod and cone photoreceptors. Rods function in near darkness (scotopic) and are responsible for dim light vision, while cones operate in bright light (photopic) and provide daytime, high acuity color vision. In the human retina, rods are the dominant photoreceptor cell type and comprise about 95% of all photoreceptor cells, while cones account for only 5% of the cells. Yet in a bright light environment, normal cone function is essential for visual perception since rods become saturated and are rendered nonfunctional.