Analysis Tools
cardiovascular system
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• at 4 weeks of age, slit-lamp analysis revealed the presence of a fibrous mass posterior to the lens, identified as hyaloid artery retention
• p62 staining confirmed persistent fetal vasculature in P30 lenses
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vision/eye
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• at 4 weeks of age, slit-lamp analysis revealed the presence of a fibrous mass posterior to the lens, identified as hyaloid artery retention
• p62 staining confirmed persistent fetal vasculature in P30 lenses
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• at 4 weeks of age, the distinct Y-shaped suture line characteristic of wild-type and heterozygous lenses is distorted
• DAPI-staining revealed accumulation of nuclei and nuclear debris in the OFZ (organelle free zone) and in the nuclear region; a 2-3 fold increase in nuclei is observed, suggesting that the programmed nuclear degradation process is impaired
• EM analysis of the lens nuclear region revealed the presence of cytoplasmic debris in the lens center, unlike in control lenses
• mutant lens homogenates show a 60% increase in calpain activity relative to wild-type controls
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• at 4 weeks of age, TEM analysis revealed an increased number of double-membraned vesicles containing undegraded organelles, mostly mitochondria, in the lens epithelium, unlike in control lenses
• in culture, mutant lens epithelium cells (LECs) exhibit increased lysosome size relative to wild-type LECs, suggesting accumulation of undigested material
• mutant LECs show reduced F-actin staining and increased aggregation in the alpha-spectrin meshwork and alpha-tubulin strands, indicating impaired cytoskeleton integrity
• mutant LECs show clear calpain staining in the nucleus whereas wild-type LECs display calpain staining around the nucleus
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• at 4 weeks of age, orientation of the lens fiber cells is altered
• DAPI-staining revealed that primary fiber cells in the center and secondary fiber cells in the outer most layers of the OFZ retain their nuclei, unlike in control lenses
• EM analysis of the lens equatorial region revealed the presence of mitochondria and other organelles in lens fiber cells, unlike in control lenses
• outer cortical fiber cells show 3-fold increase in calpain-3 expression relative to wild-type cells
• strikingly, 60-70% of cortical fiber cell nuclei exhibit positive calpain-3 staining
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• all mice develop congenital nuclear cataracts, detected as soon as mice open their eyes
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• all mice develop congenital nuclear cataracts, detected as soon as mice open their eyes
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small lens
(
J:235696
)
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• at 4 weeks of age, mutant lenses weigh 25-30% less than wild-type lenses
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• at 4 weeks of age, mutant eyeballs are 20-30% smaller than those in wild-type or heterozygous controls
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cellular
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• at 4 weeks of age, mutant lenses show increased expression and degradation of full length alpha II-spectrin
• in culture, mutant LECs show reduced F-actin staining as well as increased aggregation in the alpha-spectrin meshwork and alpha-tubulin strands, indicating impaired cytoskeleton integrity
• however, beta-actin and alpha-tubulin protein levels are not significantly altered
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• at 4 weeks of age, lenses show accumulation of residual bodies containing highly indigestible organelles, mostly consisting of partially degraded mitochondria
• cox-iv immunostaining confirmed that mitochondria are retained in the lens equatorial region
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• in culture, mutant LECs exhibit increased lysosome size relative to wild-type LECs, suggesting accumulation of undigested material
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• mutant lenses exhibit impaired autophagic cargo clearance, as shown by the retention of degraded nuclear debris in the cortical and nuclear regions of the lens, the presence of autophagic cargos suggested by LC3-II and p62/SQSTM1 and poly-ubiquitin staining in the lens epithelium and in outer cortical fiber cells, the presence of damaged mitochondria in the lens epithelium, and the presence of cytoplasmic debris in the lens nuclear region
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• in culture, mutant LECs exhibit normal autophagosome-lysosome fusion but show increased lysosome size relative to wild-type LECs, suggesting that block in autophagy is due to lysosomal dysfunction
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homeostasis/metabolism
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• mutant lenses exhibit impaired autophagic cargo clearance, as shown by the retention of degraded nuclear debris in the cortical and nuclear regions of the lens, the presence of autophagic cargos suggested by LC3-II and p62/SQSTM1 and poly-ubiquitin staining in the lens epithelium and in outer cortical fiber cells, the presence of damaged mitochondria in the lens epithelium, and the presence of cytoplasmic debris in the lens nuclear region
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• in culture, mutant LECs exhibit significantly higher Ca2+ fluorescence intensity than wild-type LECs, with more intense calcium staining localized around the nucleus relative to the diffused cytoplasmic staining observed in wild-type LECs
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• mutant lens homogenates show a 60% increase in calpain activity relative to wild-type controls
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