Photoreceptor deficits appear at eye opening in Rs1 mutant mouse models of X-linked retinoschisis.

X-linked retinoschisis (XLRS) is an early onset degenerative retinal disease characterized by cystic lesions in the middle layers of the retina. These structural changes are accompanied by a loss of visual acuity and decreased contrast sensitivity. XLRS is caused by mutations in the gene Rs1 which encodes the secreted protein Retinoschisin 1. Young Rs1-mutant mouse models develop key hallmarks of XLRS including intraretinal schisis and abnormal electroretinograms. The electroretinogram (ERG) comprises activity of multiple cellular generators, and it is not known how and when each of these is impacted in Rs1 mutant mice. Here we use an ex vivo ERG system and pharmacological blockade to determine how ERG components generated by photoreceptors, ON-bipolar, and Müller glial cells are impacted in Rs1 mutants and to determine the time course of these changes. We report that ERG abnormalities begin near eye-opening and that all ERG components are involved.

Lutein and Zeaxanthin Isomers Protect against Light-Induced Retinopathy via Decreasing Oxidative and Endoplasmic Reticulum Stress in BALB/cJ Mice.

Oxidative stress (OS) and endoplasmic reticulum stress (ERS) are the major factors underlying photoreceptor degeneration. Lutein, RR-zeaxanthin (3R,3’R-zeaxanthin) and RS (meso)-zeaxanthin (3R,3’S-RS- zeaxanthin) (L/Zi) could protect against cell damage by ameliorating OS in retina. In this study, we examined the effect of L/Zi supplementation in a mouse model of photoreceptor degeneration and investigated whether the treatment of L/Zi ameliorated OS and ERS. BALB/cJ mice after light exposure were used as the animal model. The protective effects of L/Zi were observed by electroretinography (ERG) and terminal deoxyuridine triphosphate nick-end labeling (TUNEL) analysis. The underlying mechanisms related to OS and ERS were explored by Western blotting. After L/Zi treatment, the ERG amplitudes were significantly higher, and the number of TUNEL-positive cells was significantly reduced compared to that of the vehicle group. Western blotting results revealed that OS was ameliorated according to the significant downregulation of phosphorylated c-Jun N-terminal kinase (p-JNK), and significant upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2). In addition, ERS was reduced according to the significant downregulation of 78 kDa glucose-regulated protein (GRP78), phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), activating transcription factor 4 (ATF4) and activating transcription factor (ATF6). Our data shows that L/Zi provided functional and morphological preservation of photoreceptors against light damage, which is probably related to its mitigation of oxidative and endoplasmic reticulum stress.

Lutein and Zeaxanthin Isomers Reduce Photoreceptor Degeneration in the Pde6b rd10 Mouse Model of Retinitis Pigmentosa.

PURPOSE: Lutein, RR-zeaxanthin, and RS-zeaxanthin (L-Z) are antioxidants which can reduce endoplasmic reticulum stress (ERS) and oxidative stress (OS), and ameliorate neurodegenerative diseases. However, their treatment effect in the Pde6b rd10 (rd10) mouse model of retinitis pigmentosa (RP) and the underlying cellular mechanisms have not been studied. ERS is an important factor which causes photoreceptor apoptosis. The aim of the current project is to test the treatment effect of L-Z in rd10 mice and to investigate the underlying molecular mechanisms of ERS. METHODS: L-Z (Lutemax 2020, 10 mg/kg) diluted in sunflower oil (SFO, 1 mg/ml) or the same volume of SFO was administrated via gavage from postnatal day 6 (P6) to P20 daily in L-Z group (n=5) or SFO group (n=6) of rd10 mice. At P21, electroretinography (ERG) was performed to show the functional change of retinas. 78 kDa glucose-regulated protein (GRP78) and endoplasmic reticulum protein 29 (ERp29) were tested by western blot and immunostaining. RESULTS: The ERG amplitudes were larger in the L-Z group than those of the SFO group in all flash luminances of dark-adapted and light-adapted ERG (all p < 0.01). Western blot revealed that GRP78 in the retinas of the L-Z group was significantly downregulated compared to that of the SFO group (p < 0.01). Meanwhile, the retinal ERp29 protein was significantly upregulated in the L-Z treatment group than that of the SFO group (p < 0.01). CONCLUSIONS: L-Z provide protection to the photoreceptors of rd10 mouse model of RP, which is probably associated with the reduction of ERS.

Carnosic acid slows photoreceptor degeneration in the Pde6b(rd10) mouse model of retinitis pigmentosa.

The photoreceptor cell death associated with the various genetic forms of retinitis pigmentosa (RP) is currently untreatable and leads to partial or complete vision loss. Carnosic acid (CA) upregulates endogenous antioxidant enzymes and has proven neuroprotective in studies of neurodegenerative models affecting the brain. In this study, we examined the potential effect of CA on photoreceptor death in the Pde6b(rd10) mouse model of RP. Our data shows that CA provided morphological and functional preservation of photoreceptors. CA appears to exert its neuroprotective effects through inhibition of oxidative stress and endoplasmic reticulum stress.

Deficiency of CC chemokine ligand 2 and decay-accelerating factor causes retinal degeneration in mice.

CC chemokine ligand 2 (CCL2) recruits macrophages to reduce inflammatory responses. Decay-accelerating factor (DAF) is a membrane regulator of the classical and alternative pathways of complement activation. In view of the link between complement genes and retinal diseases, we evaluated the retinal phenotype of C57BL/6J mice and mice lacking Ccl2 and/or Daf1 at 12 months of age, using scanning laser ophthalmoscopic imaging, electroretinography (ERG), histology, immunohistochemistry, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. In comparison to C57BL/6J mice, mutant mice had an increased number of autofluorescent foci, with the greatest number in the Ccl2(-/-)/Daf1(-/-) retina. ERG amplitudes in Ccl2(-/-)/Daf1(-/-), Ccl2(-/-) and Daf1(-/-) mice were reduced, with the greatest reduction in Ccl2(-/-)/Daf1(-/-) mice. TUNEL-positive cells were not seen in C57BL/6J retina, but were prevalent in the outer and inner nuclear layers of Ccl2(-/-)Daf1(-/-) mice and were present at reduced density in Ccl2(-/-) or Daf1(-/-) mice. Cell loss was most pronounced in the outer and inner nuclear layers of Ccl2(-/-)/Daf1(-/-) mice. The levels of the endoplasmic reticulum chaperone GPR78 and transcription factor ATF4 were significantly increased in the Ccl2(-/-)/Daf1(-/-) retina. In comparison to the C57BL/6J retina, the phosphorylation of NF-κB p65, p38, ERK and JNK was significantly upregulated while SIRT1 was significantly downregulated in the Ccl2(-/-)/Daf1(-/-) retina. Our results suggest that loss of Ccl2 and Daf1 causes retinal neuronal death and degeneration which is related to increased endoplasmic reticulum stress, oxidative stress and inflammation.

Retinal histopathology in eyes from patients with autosomal dominant retinitis pigmentosa caused by rhodopsin mutations.

PURPOSE: To evaluate the histopathology in donor eyes from patients with autosomal dominant retinitis pigmentosa (ADRP) caused by p.P23H, p.P347T and p.P347L rhodopsin ( RHO ) gene mutations. METHODS: Eyes from a 72-year-old male (donor 1), an 83-year-old female (donor 2), an 80-year-old female (donor 3), and three age-similar normal eyes were examined macroscopically, by scanning laser ophthalmoscopy and optical coherence tomography imaging. Perifoveal and peripheral pieces were processed for microscopy and immunocytochemistry with markers for photoreceptor cells. RESULTS: DNA analysis revealed RHO mutations c.68C>A (p.P23H) in donor 1, c.1040C>T (p.P347L) in donor 2 and c.1039C>A (p.P347T) in donor 3. Histology of the ADRP eyes showed retinas with little evidence of stratified nuclear layers in the periphery and a prominent inner nuclear layer present in the perifoveal region in the p.P23H and p.P347T eyes, while it was severely atrophic in the p.P347L eye. The p.P23H and p.P347T mutations cause a profound loss of rods in both the periphery and perifovea, while the p.P347L mutation displays near complete absence of rods in both regions. All three rhodopsin mutations caused a profound loss of cones in the periphery. The p.P23H and p.P347T mutations led to the presence of highly disorganized cones in the perifovea. However, the p.P347L mutation led to near complete absence of cones also in the perifovea. CONCLUSIONS: Our results support clinical findings indicating that mutations affecting residue P347 develop more severe phenotypes than those affecting P23. Furthermore, our results indicate a more severe phenotype in the p.P347L retina as compared to the p.P347T retina.

Histopathological comparison of eyes from patients with autosomal recessive retinitis pigmentosa caused by novel EYS mutations.

To evaluate the retinal histopathology in donor eyes from patients with autosomal recessive retinitis pigmentosa (arRP) caused by EYS mutations. Eyes from a 72-year-old female (donor 1, family 1), a 91-year-old female (donor 2, family 2), and her 97-year-old sister (donor 3, family 2) were evaluated with macroscopic, scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) imaging. Age-similar normal eyes and an eye donated by donor 1's asymptomatic mother (donor 4, family 1) were used as controls. The perifovea and peripheral retina were processed for microscopy and immunocytochemistry with markers for cone and rod photoreceptor cells. DNA analysis revealed EYS mutations c.2259 + 1G > A and c.2620C > T (p.Q874X) in family 1, and c.4350_4356del (p.I1451Pfs*3) and c.2739-?_3244 + ?del in family 2. Imaging studies revealed the presence of bone spicule pigment in arRP donor retinas. Histology of all three affected donor eyes showed very thin retinas with little evidence of stratified nuclear layers in the periphery. In contrast, the perifovea displayed a prominent inner nuclear layer. Immunocytochemistry analysis demonstrated advanced retinal degenerative changes in all eyes, with near-total absence of rod photoreceptors. In addition, we found that the perifoveal cones were more preserved in retinas from the donor with the midsize genomic rearrangement (c.4350_4356del (p.I1451Pfs*3) and c.2739-?_3244 + ?del) than in retinas from the donors with the truncating (c.2259 + 1G > A and c.2620C > T (p.Q874X) mutations. Advanced retinal degenerative changes with near-total absence of rods and preservation of some perifoveal cones are observed in arRP donor retinas with EYS mutations.

Interaction of tubby-like protein-1 (Tulp1) and microtubule-associated protein (MAP) 1A and MAP1B in the mouse retina.

Tubby-like protein-1 (Tulp1) is a photoreceptor-specific protein involved in the transport of specific proteins from the inner segment (IS) to the outer segment (OS) in photoreceptor cells. Mutations in the human TULP1 gene cause an early onset form of retinitis pigmentosa. Our previous work has shown an association between Tulp1 and the microtubule-associated protein, MAP1B. An allele of Mtap1a, which encodes the MAP1A protein, significantly delays photoreceptor degeneration in Tulp1 mutant mice. MAP1 proteins are important in stabilizing microtubules in neuronal cells, but their role in photoreceptors remains obscure. To investigate the relationship between Tulp1 and MAP1 proteins, we performed western blots, immunoprecipitations (IP), immunohistochemistry and proximity ligand assays (PLA) in wild-type and tulp1-/- mouse retinas. Our IP experiments provide evidence that Tulp1 and MAP1B interact while PLA experiments localize their interaction to the outer nuclear layer and IS of photoreceptors. Although MAP1A and MAP1B protein levels are not affected in the tulp1-/- retina, they are no longer localized to the OS of photoreceptors. This may be the cause for disorganized OSs in tulp1-/- mice, and indicate that their transport to the OS is Tulp1-dependent.

Identification of three ABCA4 sequence variations exclusive to African American patients in a cohort of patients with Stargardt disease.

PURPOSE: To describe the clinical and molecular findings in ten unrelated African American patients with Stargardt disease. DESIGN: Retrospective, observational case series. METHODS: We reviewed the clinical histories, examinations, and genotypes of 85 patients with molecular diagnoses of Stargardt disease. Three ABCA4 sequence variations identified exclusively in African Americans were evaluated in 300 African American controls and by in silico analysis. RESULTS: ABCA4 sequence changes were identified in 85 patients from 80 families, of which 11 patients identified themselves as African American. Of these 11 patients, 10 unrelated patients shared 1 of 3 ABCA4 sequence variations: c.3602T>G (p.L1201R); c.3899G>A (p.R1300Q); or c.6320G>A (p.R2107H). The minor allele frequencies in the African American control population for each variation were 7.5%, 6.3%, and 2%, respectively. This is comparable to the allele frequency in African Americans in the Exome Variant Server. In contrast, the allele frequency of all three of these variations was less than or equal to 0.05% in European Americans. Although both c.3602T>G and c.3899G>A have been reported as likely disease-causing variations, one of our control patients was homozygous for each variant, suggesting that these are nonpathogenic. In contrast, the absence of c.6320G>A in the control population in the homozygous state, combined with the results of bioinformatics analysis, support its pathogenicity. CONCLUSIONS: Three ABCA4 sequence variations were identified exclusively in 10 unrelated African American patients: p.L1201R and p.R1300Q likely represent nonpathogenic sequence variants, whereas the p.R2107H substitution appears to be pathogenic. Characterization of population-specific disease alleles may have important implications for the development of genetic screening algorithms.

Protein partners of dynamin-1 in the retina.

Dynamin proteins are involved in vesicle generation, providing mechanical force to excise newly formed vesicles from membranes of cellular compartments. In the brain, dynamin-1, dynamin-2, and dynamin-3 have been well studied; however, their function in the retina remains elusive. A retina-specific splice variant of dynamin-1 interacts with the photoreceptor-specific protein Tubby-like protein 1 (Tulp1), which when mutated causes an early onset form of autosomal recessive retinitis pigmentosa. Here, we investigated the role of the dynamins in the retina, using immunohistochemistry to localize dynamin-1, dynamin-2, and dynamin-3 and immunoprecipitation followed by mass spectrometry to explore dynamin-1 interacting proteins in mouse retina. Dynamin-2 is primarily confined to the inner segment compartment of photoreceptors, suggesting a role in outer segment protein transport. Dynamin-3 is present in the terminals of photoreceptors and dendrites of second-order neurons but is most pronounced in the inner plexiform layer where second-order neurons relay signals from photoreceptors. Dynamin-1 appears to be the dominant isoform in the retina and is present throughout the retina and in multiple compartments of the photoreceptor cell. This suggests that it may function in multiple cellular pathways. Surprisingly, dynamin-1 expression and localization did not appear to be disrupted in tulp1−/− mice. Immunoprecipitation experiments reveal that dynamin-1 associates primarily with proteins involved in cytoskeletal-based membrane dynamics. This finding is confirmed by western blot analysis. Results further implicate dynamin-1 in vesicular protein transport processes relevant to synaptic and post-Golgi pathways and indicate a possible role in photoreceptor stability.

Autosomal dominant retinitis pigmentosa secondary to pre-mRNA splicing-factor gene PRPF31 (RP11): review of disease mechanism and report of a family with a novel 3-base pair insertion.

Several forms of autosomal dominant retinitis pigmentosa (adRP) are caused by mutations in genes encoding proteins that are ubiquitously expressed and involved in the pre-mRNA spliceosome such as PRPF31. This paper provides an overview of the molecular genetics, pathophysiology, and mechanism for incomplete penetrance and retina-specific disease in pedigrees of families who harbor mutations in PRPF31 (RP11). The molecular and clinical features of a family with a novel 3-base insertion, c.914_915insTGT (p.Val305_Asp306insVal) in exon 9 of PRPF31 are described to illustrate the salient clinical features of mutations in this gene.

Dichogamy correlates with outcrossing rate and defines the selfing syndrome in the mixed-mating genus Collinsia.

BACKGROUND AND AIMS: How and why plants evolve to become selfing is a long-standing evolutionary puzzle. The transition from outcrossing to highly selfing is less well understood in self-compatible (SC) mixed-mating (MM) species where potentially subtle interactions between floral phenotypes and the environment are at play. We examined floral morphological and developmental traits across an entire SC MM genus, Collinsia, to determine which, if any, predict potential autonomous selfing ability when pollinators are absent (AS) and actual selfing rates in the wild, s(m), and to best define the selfing syndrome for this clade. METHODS: Using polymorphic microsatellite markers, we obtained 30 population-level estimates of s(m) across 19 Collinsia taxa. Species grand means for the timing of herkogamy (stigma-anther contact) and dichogamy (stigmatic receptivity, SR), AS, floral size, longevity and their genetic correlations were quantified for 22 taxa. KEY RESULTS: Species fell into discrete selfing and outcrossing groups based on floral traits. Loss of dichogamy defines Collinsia's selfing syndrome. Floral size, longevity and herkogamy also differ significantly between these groups. Most taxa have high AS rates (>80 %), but AS is uncorrelated with any measured trait. In contrast, s(m) is significantly correlated only with SR. High variance in s(m) was observed in the two groups. CONCLUSIONS: Collinsia species exhibit clear morphological and developmental traits diagnostic of 'selfing' or 'outcrossing' groups. However, many species in both the 'selfing' and the 'outcrossing' groups were MM, pointing to the critical influence of the pollination environment, the timing of AS and outcross pollen prepotency on s(m). Flower size is a poor predictor of Collinsia species' field selfing rates and this result may apply to many SC species. Assessment of the variation in the pollination environment, which can increase selfing rates in more 'outcrossing' species but can also decrease selfing rates in more 'selfing' species, is critical to understanding mating system evolution of SC MM taxa.