Developmentally regulated caspase activities play a role in organelle loss during normal lens fiber cell differentiation

To study the potential role of caspases in organelle loss during lens fiber cell differentiation (FCD), lenses were examined for expression and activity of caspase family members. Caspases 1, 2, 3, 6, 7, 8 and 9, and many of their regulatory genes were found expressed in neonatal mouse lenses at the RNA level. Neonatal mouse lens extracts contained a strong VEID-AFC cleavage activity, and minor IETD- and LEVD-AFC cleavage activities. Reciprocal cleavage testing suggested that the IETD-AFC cleavage activity likely is due to sub-optimal cleavage by the VEIDase. Analysis of embryonic rat lens extracts indicated that VEID-AFC cleavage activity is up-regulated at embryonic day 16.5 (E16.5), shortly preceding DNA degradation, and activated caspase 6 protein is detected by E17.5. In contrast to the normal situation, transgenic mouse lenses that are undergoing apoptosis contained strong DEVD-AFC and VEID-AFC cleavage activities. To determine if caspase activity may be responsible for initiating or effecting the lens fiber organelle loss in vivo, transgenic mice were generated that express either the baculovirus caspase-inhibitory gene, p35, or a nonfunctional D87A-mutated p35, within the developing lens. Two lines of p35 transgenics developed lens primary fiber compartment, while no such defects were detected in D87A transgenics. Western blot analysis of P35 indicated that activated caspases were present in normal lenses. Histological examination showed the presence of large fragmented nuclei in both primary and secondary lens fibers. Electron microscopy showed highly disordered primary fiber and the presence of multiple organelle species in spatially inappropriate regions of the lens; these included fragmented nuclei, mitochondria, and remnants of smooth endoplasmic reticulum. These results suggest that a VEID-cleaving caspase, possibly caspase 6, is important in initiating or effecting the loss of multiple organelles during lens FCD. In contrast, apoptotic lenses activate a different set of caspases to effect total destruction of the lens. These findings suggest that the lens has modified or co-opted part of the apoptotic machinery in order to specifically target organelles for destruction while leaving other structures within the cell intact.