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Caspase-dependent secondary lens fiber cell disintegration in alphaA-/alphaB-crystallin double-knockout mice
Viktor Morozov1, Eric F Wawrousek
1Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Building 7, 7 Memorial Drive, MSC 0704, Bethesda, MD 20892, USA. morozovv@nei.nih.gov
Abstract:
alphaB-crystallin has been demonstrated, in tissue culture experiments, to be a caspase 3 inhibitor; however, no animal model studies have yet been described. Here, we show that morphological abnormalities in lens secondary fiber cells of alphaA-/alphaB-crystallin gene double knockout (DKO) mice are consistent with, and probably result from, elevated DEVDase and VEIDase activities, corresponding to caspase 3 and caspase 6, respectively. Immunofluorescence microscopy revealed an increased amount of caspase 6, and the active form of caspase 3, in specific regions of the DKO lens, coincident with the site of cell disintegration. TUNEL labeling illustrated a higher level of DNA fragmentation in the secondary fiber lens cells of DKO mice, compared with wild-type mice. Using a pull-down assay, we show interaction between caspase 6 and alphaA- but not alphaB-crystallin. These studies suggest that alpha-crystallin plays a role in suppressing caspase activity, resulting in retention of lens fiber cell integrity following degradation of mitochondria and other organelles, which occurs during the apoptosis-like pathway of lens cell terminal differentiation.
Insights
Alpha-crystallin proteins inhibit caspase activity, preventing lens cell disintegration. Studies in knockout mice reveal elevated caspase activity and DNA fragmentation, highlighting alpha-crystallin
Area of Science:
- Ophthalmology
- Molecular Biology
- Cell Biology
Background:
- Alpha-crystallins are known to inhibit caspase 3 in cell cultures.
- No prior animal studies have investigated the role of alpha-crystallins in caspase inhibition in vivo.
- Lens fiber cell integrity is crucial for vision and maintained through regulated cellular processes.
Purpose of the Study:
- To investigate the in vivo role of alphaA- and alphaB-crystallin in regulating caspase activity in the lens.
- To determine the impact of alpha-crystallin deficiency on lens cell integrity and apoptosis-like pathways.
- To explore the interaction between alpha-crystallins and specific caspases in the lens.
Main Methods:
- Utilized alphaA-/alphaB-crystallin gene double knockout (DKO) mice model.
- Assessed lens morphology and caspase activities (DEVDase/VEIDase).
- Employed immunofluorescence microscopy for caspase localization and TUNEL assay for DNA fragmentation.
Main Results:
- DKO mice exhibited morphological abnormalities in lens secondary fiber cells.
- Elevated caspase 3 and caspase 6 activities were observed in DKO lenses.
- Increased active caspase 3 and caspase 6, along with DNA fragmentation, were localized to disintegrating cells in DKO lenses.
- Caspase 6 interacted with alphaA-crystallin, but not alphaB-crystallin.
Conclusions:
- Alpha-crystallins play a significant role in suppressing caspase activity within the lens.
- Loss of alpha-crystallins leads to increased caspase activity, DNA fragmentation, and lens cell disintegration.
- Alpha-crystallins are essential for maintaining lens fiber cell integrity during the apoptosis-like differentiation process.
