Purpose: We have previously demonstrated that in RPE-deprived retinas, lactose, galactose, and st... more Purpose: We have previously demonstrated that in RPE-deprived retinas, lactose, galactose, and structurally related glycans support the proper assembly of nascent photoreceptor outer segment membranes. Other glycans such as mannose and glucose have no effect on this process. While the ability to support outer segment assembly is highly specific, all of the permissive glycans we have tested are able to enter metabolic pathways within the retinal cells, thus the mechanism by which the glycans are functioning is still ambiguous. The present study was undertaken to determine if permissive glycan-mediated support of photoreceptor outer segment assembly occurred via a non-metabolic mechanism and if the phenomenon was reversible. Methods: The RPE was removed from isolated Xenopus laevis embryonic eyes that were allowed to complete differentiation in (1) Niu-Twitty medium, (2) Niu-Twitty supplemented with 5x10-3 M lactose or mannose, (3) Niu-Twitty supplemented with lactose and 3H-galactose, (4) Niu-Twitty medium supplemented with isopropyl beta-D-thiogalactopyranoside (IPTG) at concentrations spanning five orders of magnitude, or (5) Niu-Twitty medium containing lactose or IPTG to which 3H-leucine was added for 2 days followed by an additional 2 days in Niu-Twitty alone. Control RPE-deprived and RPE-supported retinas were included for comparison. Under all experimental conditions, retinal photoreceptors were evaluated to determine the level of organized folding of outer segment membranes. Results: Outer segment membranes of RPE-deprived retinas exposed to lactose were significantly more organized than both control RPE-deprived retinas and those exposed to mannose. In eyes exposed to the radiolabeled metabolizable glycan, the majority of the sugar was incorporated into Müller cells. IPTG, a non-metabolizable form of galactose, promoted the formation of organized outer segment assembly similar to lactose although at a 100 fold reduced concentration compared to metabolizable permissive glycans. Both IPTG and lactose supported outer segment assembly in a step-wise fashion with maximal support at 5x10(-5) M and 5x10(-3) M, respectively. Removal of the permissive glycans resulted in loss of support of outer segment assembly. Conclusions: The ability of both lactose and IPTG to support outer segment assembly in the absence of the RPE is dose-dependent and the effect of these sugars upon membrane folding is reversible. Moreover, this effect is supported by a non-metabolic mechanism and is therefore not accounted for by simple provision of an energy source.
To investigate 20 patients affected with Bardet-Biedl (BB) syndrome and compare them to an age-ma... more To investigate 20 patients affected with Bardet-Biedl (BB) syndrome and compare them to an age-matched group of 70 non-syndromic patients with retinitis pigmentosa (RP) to identify hallmarks peculiar to the BB phenotype. Patients were examined clinically and with functional tests (color vision, kinetic perimetry, electroretinography, ocular motility tests). Fundus findings were numerically graded for statistical purposes. Recurrent ocular features in BB patients were early and severe reduction of visual acuity, constantly altered color vision, high incidence of strabismus and nystagmus, mild-to-severe atrophic changes of the optic disc, and frequently absent or minimal pigmentary retinal changes. Visual acuity was more closely correlated to optic disc than to macular conditions. These findings were remarkably different from non-syndromic RP. This investigation further suggests that retinopathy in BB syndrome has features distinctive from those in non-syndromic RP. The early occurrence of optic disc atrophy in the BB syndrome, even in those patients with healthy maculas, suggests that optic atrophy could often be primary in nature and might play a major role in decreasing central vision in BB patients. Variability of some findings is in line with the documented heterogeneity of the BB syndrome.
Investigative Ophthalmology & Visual Science, Oct 1, 2006
PURPOSE. To develop and characterize a heterozygous knock-in mouse model carrying the 5-bp deleti... more PURPOSE. To develop and characterize a heterozygous knock-in mouse model carrying the 5-bp deletion in Elovl4 (E_mut ϩ/Ϫ) and to study the pathology underlying Stargardt-like macular degeneration (STGD3). METHODS. E_mut ϩ/Ϫ mice were generated by targeting a 5-bp deletion (AACTT) in the Elovl4 gene by homologous recombination. E_mut ϩ/Ϫ mice of age 2 to 18 months and age-matched wild-type (Wt) littermate control animals were analyzed for the expression of Elovl4 transcript, ELOVL4 protein, photoreceptor-specific genes, and retinal fatty acid composition. Functional retinal changes were evaluated by electroretinography (ERG) and by morphologic and ultrastructural criteria. RESULTS. E_mut ϩ/Ϫ mice retinas showed the presence of both Wt and mutant Elovl4 transcripts and proteins. Morphologic evaluation revealed cone photoreceptor ultrastructural abnormalities as early as 2 months of age, accumulation of lipofuscin in retinal pigment epithelium (RPE), and subretinal deposits at later ages. Shortening of rod outer segments (OS) was observed at ϳ10 months of age. Both cone and rod changes progressed with age. Unlike rod-specific genes, expression of selected cone specific genes was significantly reduced by 7 months of age. Mixed rod-cone and light-adapted b-waves were higher than normal at both 8 and 15 months. Levels of the fatty acids 20:5 (P ϭ 0.027), 22:5 (P ϭ 0.040) and 24:6 (P ϭ 0.005) were found to be significantly lower in the retinas of E_mut ϩ/Ϫ mice than in retinas of control subjects. CONCLUSIONS. E_mut ϩ/Ϫ animals display characteristic features associated with Stargardt-like macular degeneration and serve as a model for the study of the mechanism underlying STGD3.
Investigative Ophthalmology & Visual Science, Dec 13, 2012
PURPOSE. To determine the proportion of male patients presenting simplex retinal degenerative dis... more PURPOSE. To determine the proportion of male patients presenting simplex retinal degenerative disease (RD: retinitis pigmentosa [RP] or cone/cone-rod dystrophy [COD/CORD]) with mutations in the X-linked retinal degeneration genes RPGR and RP2.
Purpose: We have previously demonstrated that in RPE-deprived retinas, lactose, galactose, and st... more Purpose: We have previously demonstrated that in RPE-deprived retinas, lactose, galactose, and structurally related glycans support the proper assembly of nascent photoreceptor outer segment membranes. Other glycans such as mannose and glucose have no effect on this process. While the ability to support outer segment assembly is highly specific, all of the permissive glycans we have tested are able to enter metabolic pathways within the retinal cells, thus the mechanism by which the glycans are functioning is still ambiguous. The present study was undertaken to determine if permissive glycan-mediated support of photoreceptor outer segment assembly occurred via a non-metabolic mechanism and if the phenomenon was reversible. Methods: The RPE was removed from isolated Xenopus laevis embryonic eyes that were allowed to complete differentiation in (1) Niu-Twitty medium, (2) Niu-Twitty supplemented with 5x10-3 M lactose or mannose, (3) Niu-Twitty supplemented with lactose and 3H-galactose, (4) Niu-Twitty medium supplemented with isopropyl beta-D-thiogalactopyranoside (IPTG) at concentrations spanning five orders of magnitude, or (5) Niu-Twitty medium containing lactose or IPTG to which 3H-leucine was added for 2 days followed by an additional 2 days in Niu-Twitty alone. Control RPE-deprived and RPE-supported retinas were included for comparison. Under all experimental conditions, retinal photoreceptors were evaluated to determine the level of organized folding of outer segment membranes. Results: Outer segment membranes of RPE-deprived retinas exposed to lactose were significantly more organized than both control RPE-deprived retinas and those exposed to mannose. In eyes exposed to the radiolabeled metabolizable glycan, the majority of the sugar was incorporated into Müller cells. IPTG, a non-metabolizable form of galactose, promoted the formation of organized outer segment assembly similar to lactose although at a 100 fold reduced concentration compared to metabolizable permissive glycans. Both IPTG and lactose supported outer segment assembly in a step-wise fashion with maximal support at 5x10(-5) M and 5x10(-3) M, respectively. Removal of the permissive glycans resulted in loss of support of outer segment assembly. Conclusions: The ability of both lactose and IPTG to support outer segment assembly in the absence of the RPE is dose-dependent and the effect of these sugars upon membrane folding is reversible. Moreover, this effect is supported by a non-metabolic mechanism and is therefore not accounted for by simple provision of an energy source.
To investigate 20 patients affected with Bardet-Biedl (BB) syndrome and compare them to an age-ma... more To investigate 20 patients affected with Bardet-Biedl (BB) syndrome and compare them to an age-matched group of 70 non-syndromic patients with retinitis pigmentosa (RP) to identify hallmarks peculiar to the BB phenotype. Patients were examined clinically and with functional tests (color vision, kinetic perimetry, electroretinography, ocular motility tests). Fundus findings were numerically graded for statistical purposes. Recurrent ocular features in BB patients were early and severe reduction of visual acuity, constantly altered color vision, high incidence of strabismus and nystagmus, mild-to-severe atrophic changes of the optic disc, and frequently absent or minimal pigmentary retinal changes. Visual acuity was more closely correlated to optic disc than to macular conditions. These findings were remarkably different from non-syndromic RP. This investigation further suggests that retinopathy in BB syndrome has features distinctive from those in non-syndromic RP. The early occurrence of optic disc atrophy in the BB syndrome, even in those patients with healthy maculas, suggests that optic atrophy could often be primary in nature and might play a major role in decreasing central vision in BB patients. Variability of some findings is in line with the documented heterogeneity of the BB syndrome.
Investigative Ophthalmology & Visual Science, Oct 1, 2006
PURPOSE. To develop and characterize a heterozygous knock-in mouse model carrying the 5-bp deleti... more PURPOSE. To develop and characterize a heterozygous knock-in mouse model carrying the 5-bp deletion in Elovl4 (E_mut ϩ/Ϫ) and to study the pathology underlying Stargardt-like macular degeneration (STGD3). METHODS. E_mut ϩ/Ϫ mice were generated by targeting a 5-bp deletion (AACTT) in the Elovl4 gene by homologous recombination. E_mut ϩ/Ϫ mice of age 2 to 18 months and age-matched wild-type (Wt) littermate control animals were analyzed for the expression of Elovl4 transcript, ELOVL4 protein, photoreceptor-specific genes, and retinal fatty acid composition. Functional retinal changes were evaluated by electroretinography (ERG) and by morphologic and ultrastructural criteria. RESULTS. E_mut ϩ/Ϫ mice retinas showed the presence of both Wt and mutant Elovl4 transcripts and proteins. Morphologic evaluation revealed cone photoreceptor ultrastructural abnormalities as early as 2 months of age, accumulation of lipofuscin in retinal pigment epithelium (RPE), and subretinal deposits at later ages. Shortening of rod outer segments (OS) was observed at ϳ10 months of age. Both cone and rod changes progressed with age. Unlike rod-specific genes, expression of selected cone specific genes was significantly reduced by 7 months of age. Mixed rod-cone and light-adapted b-waves were higher than normal at both 8 and 15 months. Levels of the fatty acids 20:5 (P ϭ 0.027), 22:5 (P ϭ 0.040) and 24:6 (P ϭ 0.005) were found to be significantly lower in the retinas of E_mut ϩ/Ϫ mice than in retinas of control subjects. CONCLUSIONS. E_mut ϩ/Ϫ animals display characteristic features associated with Stargardt-like macular degeneration and serve as a model for the study of the mechanism underlying STGD3.
Investigative Ophthalmology & Visual Science, Dec 13, 2012
PURPOSE. To determine the proportion of male patients presenting simplex retinal degenerative dis... more PURPOSE. To determine the proportion of male patients presenting simplex retinal degenerative disease (RD: retinitis pigmentosa [RP] or cone/cone-rod dystrophy [COD/CORD]) with mutations in the X-linked retinal degeneration genes RPGR and RP2.
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