Abstract
We examined cell death in developing retinal tissue, following inhibition of protein synthesis, which kills undifferentiated post-mitotic cells. Ultrastructural features were found of both apoptosis and autophagy. Only approximately half of the degenerating cells were either terminal dUTP nick-end labeling (TUNEL)-positive or reacted with antibodies specific for activated caspases-3 or -9. Bongkrekic acid completely inhibited any appearance of cell death, whereas inhibitors of autophagy, caspases-9 or -3, prevented only TUNEL-positive cell death. Interestingly, inhibition of caspase-6 blocked TUNEL-negative cell death. Simultaneous inhibition of caspases-9 and -6 prevented cell death almost completely, but degeneration dependent on autophagy/caspase-9 still occurred under inhibition of both caspases-3 and -6. Thus, inhibition of protein synthesis induces in the developing retina various post-translational, mitochondria-dependent pathways of cell death. Autophagy precedes sequential activation of caspases-9 and -3, and DNA fragmentation, whereas, in parallel, caspase-6 leads to a TUNEL-negative form of cell death. Additional mechanisms of cell death may be engaged upon selective caspase inhibition.
Highlights
Programmed cell death is a major component of both normal development and disease [1,2,3,4,5,6,7,8,9] and occurs in various forms, such as apoptosis, autophagy, and others
In retinal tissue treated with the inhibitor of protein synthesis anisomycin (ANI), ganglion cells degeneration is blocked, whereas cell death is induced in the neuroblastic layer (NBL)
2) Cell death induced by inhibition of protein synthesis depends on mitochondrial commitment, but only about half of the dying cells show signs of activation of caspases-9 and -3 and TUNELpositive DNA fragmentation
Summary
Materials—Anisomycin (Sigma), an inhibitor of protein synthesis, was used at 1 g/ml from a stock solution in Me2SO. 3-Methyladenine (3MA1; Sigma), an inhibitor of autophagy, was used at 1–10 mM from a stock solution in culture medium. The sections were washed with TBS/Triton and incubated with biotinylated secondary antibody (diluted in TBS plus 3% BSA; 1:200 for secondary antibody from Vectastain HRP-ABC kit, Vector Laboratories) for 1 h at room temperature. After washing with TBS/Triton, sections were incubated for 30 min with 0.6% H2O2 in TBS at room temperature. Sections were washed with TBS/Triton and incubated for 1 h with ABC reagent at room temperature. Immunocytochemistry for activated caspase-3 was done using the CM1 polyclonal antiserum that recognizes the p20 activated form of human, mouse, and rat caspase-3, without significant cross-reactivity with the zymogen form of 32 kDa [45] This antibody was a gift from Dr Anu Srinivasan (Idun Pharmaceuticals), and a detailed description was provided in the publication mentioned above.
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