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AlphaA-crystallin R49Cneo mutation influences the architecture of lens fiber cell membranes and causes posterior and nuclear cataracts in mice.

Andley UP - BMC Ophthalmol (2009)

Bottom Line: AlphaA-crystallin (CRYAA/HSPB4), a major component of all vertebrate eye lenses, is a small heat shock protein responsible for maintaining lens transparency.By 3 weeks, WT/R49Cneo mice exhibited large vacuoles in the cortical region 100 mum from the lens surface, and by 3 months posterior and nuclear cataracts had developed.It is apparent that modification of membrane and cell-cell interactions occurs in the presence of the alphaA-crystallin mutation and rapidly leads to lens cell pathology in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St Louis, Missouri, USA. andley@vision.wustl.edu

ABSTRACT

Background: AlphaA-crystallin (CRYAA/HSPB4), a major component of all vertebrate eye lenses, is a small heat shock protein responsible for maintaining lens transparency. The R49C mutation in the alphaA-crystallin protein is linked with non-syndromic, hereditary human cataracts in a four-generation Caucasian family.

Methods: This study describes a mouse cataract model generated by insertion of a neomycin-resistant (neor) gene into an intron of the gene encoding mutant R49C alphaA-crystallin. Mice carrying the neor gene and wild-type Cryaa were also generated as controls. Heterozygous knock-in mice containing one wild type gene and one mutated gene for alphaA-crystallin (WT/R49Cneo) and homozygous knock-in mice containing two mutated genes (R49Cneo/R49Cneo) were compared.

Results: By 3 weeks, WT/R49Cneo mice exhibited large vacuoles in the cortical region 100 mum from the lens surface, and by 3 months posterior and nuclear cataracts had developed. WT/R49Cneo mice demonstrated severe posterior cataracts at 9 months of age, with considerable posterior nuclear migration evident in histological sections. R49Cneo/R49Cneo mice demonstrated nearly complete lens opacities by 5 months of age. In contrast, R49C mice in which the neor gene was deleted by breeding with CreEIIa mice developed lens abnormalities at birth, suggesting that the neor gene may suppress expression of mutant R49C alphaA-crystallin protein.

Conclusion: It is apparent that modification of membrane and cell-cell interactions occurs in the presence of the alphaA-crystallin mutation and rapidly leads to lens cell pathology in vivo.

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Related in: MedlinePlus

Immunofluorescence analysis of fiber cells in wild type and R49Cneo/R49Cneo knock-in lenses. Immunofluorescence staining of lens sections with an antibody to membrane intrinsic protein MIP and an Alexa568-conjugated secondary antibody (red) revealed a normal fiber cell morphology in wild type lenses (A), whereas the R49Cneo/R49Cneo lenses (B) showed disorganized fiber cells and swollen cells (arrows) enclosed by membranes. Nuclei are shown by arrowheads.
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Figure 9: Immunofluorescence analysis of fiber cells in wild type and R49Cneo/R49Cneo knock-in lenses. Immunofluorescence staining of lens sections with an antibody to membrane intrinsic protein MIP and an Alexa568-conjugated secondary antibody (red) revealed a normal fiber cell morphology in wild type lenses (A), whereas the R49Cneo/R49Cneo lenses (B) showed disorganized fiber cells and swollen cells (arrows) enclosed by membranes. Nuclei are shown by arrowheads.

Mentions: Immunofluorescence analysis with an antibody to membrane intrinsic protein (MIP) revealed that the swollen cells are enclosed by fiber cell membranes (Figure 9). Swollen cells were not found in wild type littermates or in WTneo mice. Opacification was related to defects in membrane structure of cortical lens fiber cells. Lenses were further examined by cryoimmuno electron microscopy with an antibody to αA-crystallin. Wild type lenses showed smooth and linear plasma membranes between the fiber cells, and αA-crystallin was restricted to the cytoplasm (Figure 10A). In contrast, fiber cell membranes of heterozygous WT/R49Cneo lenses were non-linear with extensive undulations, and significant gaps between the cells. A greater proportion of the αA-crystallin immunoreactivity was associated with fiber cell membranes in the heterozygous lenses (Figure 10B) than in wild type lenses.


AlphaA-crystallin R49Cneo mutation influences the architecture of lens fiber cell membranes and causes posterior and nuclear cataracts in mice.

Andley UP - BMC Ophthalmol (2009)

Immunofluorescence analysis of fiber cells in wild type and R49Cneo/R49Cneo knock-in lenses. Immunofluorescence staining of lens sections with an antibody to membrane intrinsic protein MIP and an Alexa568-conjugated secondary antibody (red) revealed a normal fiber cell morphology in wild type lenses (A), whereas the R49Cneo/R49Cneo lenses (B) showed disorganized fiber cells and swollen cells (arrows) enclosed by membranes. Nuclei are shown by arrowheads.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724435&req=5

Figure 9: Immunofluorescence analysis of fiber cells in wild type and R49Cneo/R49Cneo knock-in lenses. Immunofluorescence staining of lens sections with an antibody to membrane intrinsic protein MIP and an Alexa568-conjugated secondary antibody (red) revealed a normal fiber cell morphology in wild type lenses (A), whereas the R49Cneo/R49Cneo lenses (B) showed disorganized fiber cells and swollen cells (arrows) enclosed by membranes. Nuclei are shown by arrowheads.
Mentions: Immunofluorescence analysis with an antibody to membrane intrinsic protein (MIP) revealed that the swollen cells are enclosed by fiber cell membranes (Figure 9). Swollen cells were not found in wild type littermates or in WTneo mice. Opacification was related to defects in membrane structure of cortical lens fiber cells. Lenses were further examined by cryoimmuno electron microscopy with an antibody to αA-crystallin. Wild type lenses showed smooth and linear plasma membranes between the fiber cells, and αA-crystallin was restricted to the cytoplasm (Figure 10A). In contrast, fiber cell membranes of heterozygous WT/R49Cneo lenses were non-linear with extensive undulations, and significant gaps between the cells. A greater proportion of the αA-crystallin immunoreactivity was associated with fiber cell membranes in the heterozygous lenses (Figure 10B) than in wild type lenses.

Bottom Line: AlphaA-crystallin (CRYAA/HSPB4), a major component of all vertebrate eye lenses, is a small heat shock protein responsible for maintaining lens transparency.By 3 weeks, WT/R49Cneo mice exhibited large vacuoles in the cortical region 100 mum from the lens surface, and by 3 months posterior and nuclear cataracts had developed.It is apparent that modification of membrane and cell-cell interactions occurs in the presence of the alphaA-crystallin mutation and rapidly leads to lens cell pathology in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St Louis, Missouri, USA. andley@vision.wustl.edu

ABSTRACT

Background: AlphaA-crystallin (CRYAA/HSPB4), a major component of all vertebrate eye lenses, is a small heat shock protein responsible for maintaining lens transparency. The R49C mutation in the alphaA-crystallin protein is linked with non-syndromic, hereditary human cataracts in a four-generation Caucasian family.

Methods: This study describes a mouse cataract model generated by insertion of a neomycin-resistant (neor) gene into an intron of the gene encoding mutant R49C alphaA-crystallin. Mice carrying the neor gene and wild-type Cryaa were also generated as controls. Heterozygous knock-in mice containing one wild type gene and one mutated gene for alphaA-crystallin (WT/R49Cneo) and homozygous knock-in mice containing two mutated genes (R49Cneo/R49Cneo) were compared.

Results: By 3 weeks, WT/R49Cneo mice exhibited large vacuoles in the cortical region 100 mum from the lens surface, and by 3 months posterior and nuclear cataracts had developed. WT/R49Cneo mice demonstrated severe posterior cataracts at 9 months of age, with considerable posterior nuclear migration evident in histological sections. R49Cneo/R49Cneo mice demonstrated nearly complete lens opacities by 5 months of age. In contrast, R49C mice in which the neor gene was deleted by breeding with CreEIIa mice developed lens abnormalities at birth, suggesting that the neor gene may suppress expression of mutant R49C alphaA-crystallin protein.

Conclusion: It is apparent that modification of membrane and cell-cell interactions occurs in the presence of the alphaA-crystallin mutation and rapidly leads to lens cell pathology in vivo.

Show MeSH
Related in: MedlinePlus