ARR3

Abstract: Many aspects of retinal physiology are modulated by circadian clocks, but it is unclear whether clock malfunction impinges directly on photoreceptor survival, differentiation or function. Eyes from wild-type (WT) and Period1 (Per1) and Period2 (Per2) mutant mice (Per1(Brdm1) Per2(Brdm1) ) were examined for structural (histology, in vivo imaging), phenotypical (RNA expression, immunohistochemistry) and functional characteristics. Transcriptional levels of selected cone genes [red/green opsin (Opn1mw), blue cone opsin (Opn1sw) and cone arrestin (Arr3)] and one circadian clock gene (RORb) were quantified by real-time polymerase chain reaction. Although there were no changes in general retinal histology or visual responses (electroretinograms) between WT and Per1(Brdm1) Per2(Brdm1) mice, compared with age-matched controls, Per1(Brdm1) Per2(Brdm1) mice showed scattered retinal deformations by fundus inspection. Also, mRNA expression levels and immunostaining of blue cone opsin were significantly reduced in mutant mice. Especially, there was an alteration in the dorsal-ventral patterning of blue cones. Decreased blue cone opsin immunoreactivity was present by early postnatal stages, and remained throughout maturation. General photoreceptor differentiation was retarded in young mutant mice. In conclusion, deletion of both Per1 and Per2 clock genes leads to multiple discrete changes in retina, notably patchy tissue disorganization, reductions in cone opsin mRNA and protein levels, and altered distribution. These data represent the first direct link between Per1 and Per2 clock genes, and cone photoreceptor differentiation and function.

Abstract: Arrestin 1 (Arr1) was initially named either retinal S-antigen, which referred to the soluble fraction in the retina causing uveitis,1,2or the 48-kDa (its molecular weight) protein.3,4 In the rod phototransduction cascade, Arr1 has an essential recovery role in “arresting” light-activated, phosphorylated rhodopsin.5 When dark-adapted mice are subjected to light, Arr1 translocates from the synapse and rod inner segments to the rod outer segments.6–8 Based on the molecular discovery of Arr1,9 three other homologues were later identified: two ubiquitously expressed β-arrestins (β-arrestin 1 and 2 [Arr2, Arr3])10and cone arrestin or X-arrestin (Arrestin 4 [Arr4]), which is expressed in cones and a subpopulation of pinealocytes.11–14 Subsequently, Arr1 and Arr4 were shown to be coexpressed in mouse cones, with the concentration of Arr1 expression in dark-reared mice 50-fold higher (1.7 × 108 molecules/cone) than that of Arr4 (3.3 × 106 molecules/ cone).15 This Arr1 concentration in cones even exceeds its reported concentration in rods (∼4.5 × 107 molecules/rod).8,16 A role for Arr4 in cone phototransduction has yet to be fully elucidated17; however, electrophysiological measurements from isolated cones indicate that S- and M-opsins require at least one visual arrestin (Arr1 or Arr4) for normal recovery and inactivation of phototransduction.15 Arr1 null mice (Arr1−/−) reared in cyclic light (164 lux) for at least 100 days or exposed to constant bright light (1250–1640 lux) for 1 week develop rod degeneration typified by the loss of photoreceptor nuclei and a decrease in outer nuclear layer (ONL) thickness.18 Partial rescue of this light-dependent degeneration in Arr1−/− mouse rods and its recovery function occurs in transgenic mice when either Arr1 (p48)19 or Arr420 is expressed on the Arr1 null background. While investigating the cone function of visual arrestins in retinas of Arr1−/− mice, we observed a light-independent cone dystrophy phenotype depicted by an increase in apoptosis and cone photoreceptor cell loss (Brown BM, et al. IOVS 2007;48:ARVO E-Abstract 4644). An earlier report using Drosophila also described a light-independent photoreceptor degeneration that was accelerated by light when Arr1 was absent.21 Other mouse cone-specific degenerations have been reported, including knockout of the genes CNG3,22Rpe65,23 Lrat,23 and Gnat2 (Cpfl3).24 In addition to a light-independent cone dystrophy phenotype in retinas of Arr1−/− mice, we observed a photopic ERG phenotype associated with cone light adaptation. A documented result of cone light adaptation, characterized by a b-wave amplitude increase of a dark-adapted subject during the first 15 minutes of exposure to a rod-saturating background light, has been observed in human, monkey, rat, and mouse.25–33 When cone b-wave responses to a bright light stimuli were measured over 15-minute exposure to a rod-saturating background light, we observed that wild-type (WT) and Arr4−/− mice exhibited similar b-wave amplitude increases though Arr1−/− and Arr-DKO did not. Before our discovery of Arr1 expression in cones, Arr1−/− was used as a model for a functionally rodless mouse.34In their detailed study, Lyubarsky et al.34 reported no differences in the cone b-wave amplitudes of WT and Arr1−/− mice; however, they did not evaluate whether any progressive changes took place over their light-adapting period. In this report, we present three observations related to Arr1 and Arr4 expression and function in mouse cones: light-independent cone degeneration, photopic ERG b-wave phenotype related to light adaptation resulting from the loss of Arr1 expression, and photopic ERG flicker phenotype when both visual arrestins were absent.

Abstract: Most retinoblastomas initiate in response to the inactivation of the RB1 gene and loss of functional RB protein. The tumors may form without additional genomic changes and develop after a pre-malignant retinoma phase. Despite this seemingly straightforward etiology, mouse models have not recapitulated the genetic, cellular, and stage-specific features of human retinoblastoma genesis. For example, whereas human retinoblastomas appear to derive from cone photoreceptor precursors, current mouse models develop tumors that derive from other retinal cell types. To investigate the basis of the human cone-specific oncogenesis, we compared developmental-stage-specific cone precursor responses to RB loss in human and murine retina cultures and in cone-specific Rb1 knockout mice. We report that RB-depleted maturing (ARR3+) but not immature (ARR3-) human cone precursors enter the cell cycle, proliferate, and form retinoblastoma-like lesions characterized by Flexner-Wintersteiner rosettes, then form low or non-proliferative pre-malignant retinoma-like lesions with fleurettes and high p16INK4A and p130 expression, and finally form highly proliferative retinoblastoma-like masses. In contrast, in murine retina, only RB-depleted immature (Arr3-) cone precursors entered the cell cycle and they failed to progress from S to M phase. Moreover, whereas the intrinsically highly expressed MDM2 and MYCN contribute to RB-depleted maturing (ARR3+) human cone precursor proliferation, ectopic MDM2 and Mycn promoted only immature (Arr3-) murine cone precursor cell cycle entry. These findings demonstrate that developmental-stage-specific as well as species- and cell-type-specific features sensitize to RB1 inactivation and reveal the human cone precursors capacity to model retinoblastoma initiation, proliferation, pre-malignant arrest, and tumor growth.