Alternative programs of cell death in developing retinal tissue.

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.

Gap junctions mediate bystander cell death in developing retina.

During development of the retina, programmed cell death helps to establish the final size and distribution of various cell classes in distinct layers of the tissue. Here we show that dying cells in the developing ganglion and inner nuclear layers are clustered spatially and that gap junction inhibitors decrease the clustering of dying cells. To confirm the role of gap junctions in cell death, we induced targeted cell death via intracellular cytochrome c (Cc) and examined the induced cells and their neighbors for apoptotic morphology or caspase-3 cleavage. These studies indicate that bystander killing extends to coupled cells. Quantitative studies of bystander killing were performed by scrape-loading retinas with Cc in the presence of rhodamine dextran (RD; to identify Cc-loaded cells) and by counting pyknotic cells in cryosections. Although only 1.5% of control scrape-loaded cells (RD alone) showed apoptotic morphology, 97% of Cc scrape-loaded cells were pyknotic. Moreover, bystander killing extended to neighboring cells, not labeled with RD, and was reduced significantly by the gap junction inhibitors octanol and carbenoxolone. We hypothesize that dying cells in the retina generate a gap junction-permeant apoptotic signal that mediates bystander killing. This novel finding of naturally occurring bystander cell death may have important implications in the histogenesis and pathology of the nervous system.

Hydrogen peroxide induces caspase activation and programmed cell death in the amitochondrial Tritrichomonas foetus.

Tritrichomonas foetus is an amitochondrial parasite protist which lacks typical eukaryote organelles such as mitochondria and peroxisomes, but possesses the hydrogenosome, a double-membrane-bound organelle that produces ATP. The cell death of amitochondrial organisms is poorly studied. In the present work, the cytotoxic effects of hydrogen peroxide on T. foetus and its participation on cell death were analyzed. We took advantage of several microscopy techniques, including videomicroscopy, light microscopy immunocytochemistry for detection of caspase activation, and scanning and transmission electron microscopy. We report here that in T. foetus: (1) H(2)O(2) leads to loss of motility and induces cell death, (2) the dying cells exhibit some characteristics similar to those found during the death of other organisms, and (3) a caspase-like protein seems to be activated during the death process. Thus, we propose that, although T. foetus does not present mitochondria nor any known pathways of cell death, it is likely that it bears mechanisms of cell demise. T. foetus exhibits morphological and physiological alterations in response to H(2)O(2) treatment. The hydrogenosome, a unique organelle which is supposed to share a common ancestral origin with mitochondria and has an important role in oxidative responses in trichomonads, is a candidate for participating in this event.