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Mutations in zebrafish lrp2 result in adult-onset ocular pathogenesis that models myopia and other risk factors for glaucoma.

Veth KN, Willer JR, Collery RF, Gray MP, Willer GB, Wagner DS, Mullins MC, Udvadia AJ, Smith RS, John SW, Gregg RG, Link BA - PLoS Genet. (2011)

Bottom Line: Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes.This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis.Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

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Retinal proliferation and apoptosis.A–F Mcm5 expression in 1 (A–C) and 2-month (D–F) cryosections. Dashed white lines denote proliferative ciliary marginal zone (CMZ) of the retina; asterisks indicate autofluorescent blood vessels. G Quantitation of apoptotic cells identified on whole retina flat-mounts by activated caspase-3 (aCasp3) immunofluorescence. H Confocal images of aCasp3-positive cell in 1 year old bugeye mutant. Upper shows compressed z-stacks, lower shows 90°rotation to reveal z location of positive cell (arrow). The flat mounted retinas were orientated with retinal ganglion cell layer up. n = 15 eyes for each condition; ***p<0.001, **p<0.01, n.s., not significant (t-test).
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pgen-1001310-g006: Retinal proliferation and apoptosis.A–F Mcm5 expression in 1 (A–C) and 2-month (D–F) cryosections. Dashed white lines denote proliferative ciliary marginal zone (CMZ) of the retina; asterisks indicate autofluorescent blood vessels. G Quantitation of apoptotic cells identified on whole retina flat-mounts by activated caspase-3 (aCasp3) immunofluorescence. H Confocal images of aCasp3-positive cell in 1 year old bugeye mutant. Upper shows compressed z-stacks, lower shows 90°rotation to reveal z location of positive cell (arrow). The flat mounted retinas were orientated with retinal ganglion cell layer up. n = 15 eyes for each condition; ***p<0.001, **p<0.01, n.s., not significant (t-test).

Mentions: The altered retinal cell density in lrp2 mutants could be due to either insufficient cell generation to match scleral growth and remodeling, or through increased cell death. To address these possibilities we analyzed by immunofluorescence the number of proliferating cells within the ciliary margin zone (using Minichromosome maintenance homolog 5, Mcm5 antibodies) and the number of apoptotic cells across the retina (using activated-Caspase3 antibodies). Mcm5 is required for DNA replication and is expressed throughout the cell cycle in all proliferating cells, but the protein is rapidly lost in post-mitotic cells. Proteolytic cleavage of Caspase3, recognized by the activated-Caspase3 antibody, is one of the last steps in the apoptosis cascade and marks cells committed to die in a number of contexts, including glaucoma. At 1 month, proliferation in both wild-type and lrp2 mutant retinas was primarily confined to the ciliary marginal zone, a stem cell niche where ongoing proliferation from multipotent elongated neuroepithelial cells is known to occur in fish [17] (Figure 6A–6C). For each genotype, occasional Mcm5-positive cells were also located in the inner nuclear layer, which have previously been shown to be rod progenitor cells in teleost fish [18]–[20]. At 2 months, cell counts indicated a reduction in Mcm5-positive cells per CMZ niche in bugeye fish, suggesting maintenance of stem cells was inadequate to match eye globe growth (Figure 6D–6F). Consistent with this observation, a role for Lrp2 in maintaining neuronal stem cells of the adult mouse forebrain has been recently described [21].


Mutations in zebrafish lrp2 result in adult-onset ocular pathogenesis that models myopia and other risk factors for glaucoma.

Veth KN, Willer JR, Collery RF, Gray MP, Willer GB, Wagner DS, Mullins MC, Udvadia AJ, Smith RS, John SW, Gregg RG, Link BA - PLoS Genet. (2011)

Retinal proliferation and apoptosis.A–F Mcm5 expression in 1 (A–C) and 2-month (D–F) cryosections. Dashed white lines denote proliferative ciliary marginal zone (CMZ) of the retina; asterisks indicate autofluorescent blood vessels. G Quantitation of apoptotic cells identified on whole retina flat-mounts by activated caspase-3 (aCasp3) immunofluorescence. H Confocal images of aCasp3-positive cell in 1 year old bugeye mutant. Upper shows compressed z-stacks, lower shows 90°rotation to reveal z location of positive cell (arrow). The flat mounted retinas were orientated with retinal ganglion cell layer up. n = 15 eyes for each condition; ***p<0.001, **p<0.01, n.s., not significant (t-test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040661&req=5

pgen-1001310-g006: Retinal proliferation and apoptosis.A–F Mcm5 expression in 1 (A–C) and 2-month (D–F) cryosections. Dashed white lines denote proliferative ciliary marginal zone (CMZ) of the retina; asterisks indicate autofluorescent blood vessels. G Quantitation of apoptotic cells identified on whole retina flat-mounts by activated caspase-3 (aCasp3) immunofluorescence. H Confocal images of aCasp3-positive cell in 1 year old bugeye mutant. Upper shows compressed z-stacks, lower shows 90°rotation to reveal z location of positive cell (arrow). The flat mounted retinas were orientated with retinal ganglion cell layer up. n = 15 eyes for each condition; ***p<0.001, **p<0.01, n.s., not significant (t-test).
Mentions: The altered retinal cell density in lrp2 mutants could be due to either insufficient cell generation to match scleral growth and remodeling, or through increased cell death. To address these possibilities we analyzed by immunofluorescence the number of proliferating cells within the ciliary margin zone (using Minichromosome maintenance homolog 5, Mcm5 antibodies) and the number of apoptotic cells across the retina (using activated-Caspase3 antibodies). Mcm5 is required for DNA replication and is expressed throughout the cell cycle in all proliferating cells, but the protein is rapidly lost in post-mitotic cells. Proteolytic cleavage of Caspase3, recognized by the activated-Caspase3 antibody, is one of the last steps in the apoptosis cascade and marks cells committed to die in a number of contexts, including glaucoma. At 1 month, proliferation in both wild-type and lrp2 mutant retinas was primarily confined to the ciliary marginal zone, a stem cell niche where ongoing proliferation from multipotent elongated neuroepithelial cells is known to occur in fish [17] (Figure 6A–6C). For each genotype, occasional Mcm5-positive cells were also located in the inner nuclear layer, which have previously been shown to be rod progenitor cells in teleost fish [18]–[20]. At 2 months, cell counts indicated a reduction in Mcm5-positive cells per CMZ niche in bugeye fish, suggesting maintenance of stem cells was inadequate to match eye globe growth (Figure 6D–6F). Consistent with this observation, a role for Lrp2 in maintaining neuronal stem cells of the adult mouse forebrain has been recently described [21].

Bottom Line: Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes.This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis.Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

Show MeSH
Related in: MedlinePlus