Molecular Biology of Apoptosis

Apoptosis is a morphologically distinct form of programmed cell death that plays important roles in development, tissue homoestasis and a wide variety of diseases, including cancer, AIDS, stroke, myopathies and various neurodegenerative disorders. The overall objective of our research is to elucidate the mechanism by which cells undergo apoptosis, and how this process is regulated by diverse death-inducing signals. Most of our work utilizes a highly accessible model organism, the fruitfly Drosophila, which offers unique advantages for the discovery of novel cell death genes. Since the mechanism of apoptosis has been conserved in evolution from worms to insects to man, knowledge gained from studying cell death in Drosophila is likely to apply to mammalian systems as well. In order to test this directly, we are studying the activity of Drosophila cell death genes and their mouse or human homologs in cultured mammalian cells.

Regulation of neuronal death: In Drosophila, the size of the optic ganglia, the visual centers of the brain, is always perfectly matched to the variable size of the eye. We have previously shown that this matching in the number of cells is accomplished by adjusting both the rate of cell death and cell proliferation through competitive interactions between retina and optic ganglia. This situation is reminiscent of trophic interactions that have been widely described in vertebrates. Neurons also die in response to various insults and external stress. We have studied a neurodegenerative disease model in Drosophila, the late-onset retinal degeneration that is observed in certain phototransduction. We have found that retinal degeneration due to mutations in either rhodopsin or a phospholipase C gene occurs by apoptosis and can be blocked by cystein protease inhibitors. Significantly, this suppression of apoptosis restors visual function to otherwise blind flies. These findings demonstrate that stressed or damaged cells that are rescued from death can serve a useful purpose for the organism. These findings provides a strong rationale to further explore anti-apoptotic therapies in mammalian models for retinitis pigmentosa, as well as other neuro-degenerative disorders.

Molecular Genetics of Programmed Cell Death: During Drosophila development, large numbers of cells undergo natural cell death. Even though the onset of these deaths is controlled by many different signals, most of the dying cells display common morphological and biochemical changes that are characteristic of apoptosis in vertebrates. From surveying a large fraction of the Drosophila genome for genes that are required for programmed cell, three apoptotic activators, named reaper, head involution defective (hid), and grim were identified. All three genes are contained within a 300 kb interval, and their deletion blocks essentially all programmed cell deaths. These genes encode novel proteins of 65 amino acids (REAPER), 138 amino acids (GRIM) and 410 amino acids (HID) respectively. Significantly, reaper is specifically expressed in cells that are doomed to die in response to many different death-inducing signals and its expression precedes the first morphological signs of apoptosis. This indicates that the integration of multiple distinct death inducing signalling pathways occurs, at least in part, by a transcriptional mechanism. Ectopic expression of either reaper, hid or grim triggers the induction of apoptosis in cells that would normally survive. These ectopic deaths can be completely blocked with inhibitors of ced-3/ICE-like cysteine proteases, named caspases (for cysteine aspase), suggesting that expression of reaper and hid leads to the activation of a caspase pathway. We have isolated Drosophila caspase genes, and we have initiated detailed genetic and biochemical studies of one of them, named dcp-1. DCP-1 can induce apoptosis in a range of cell types, and apoptotis like events in a cell free system. Consistent with its function as an apoptotic effector, DCP-1 is widely expressed in Drosophila embryos. Loss of zygotic DCP-1 function causes larval lethality, melanotic tumors and female sterility, demonstrating that this gene is essential for normal development. Interestingly, females lacking dcp-1 activity in their germ line are sterile because of defects in oogenesis. Significantly, this is due to a failure of nurse cells to transfer ("dump") their cytoplasm into the oocyte. Normally during Drosophila oogenesis, nurse cells transfer their cytoplasmic contents and organelles through cytoplasmic bridges into the oocyte and then die. In the absence of dcp-1 function, this transfer is defective and nurse cell death is delayed, indicating that apoptosis of the nurse cells is a necessary event for oocyte development.

The pathway of death: One of the major advantages for using Drosophila for apoptosis research is the ability to employ the powerful genetic methods available in this organism for identifying new cell death genes. When expressed under the control of the eye-specific GMR promoter, reaper, hid or grim cause cell death, resulting in eye ablation. These eye phenotypes depend on transgene dosage, so that at intermediate levels of expression reduced and roughened eyes are obtained. Under conditions of partial eye ablation, cells are highly sensitive to alterations in the dosage of cell death genes acting downstream of reaper, hid and grim. This permits very simple and efficient F1 screens for genetic modifiers of reaper, hid and grim mediated cell killing: mutations that promote apoptosis can be identified as enhancers of eye defects, while mutations that inhibit death suppress this phenotype. Since the Drosophila eye is a non-essential and easily visible tissue, it is possible to screen large numbers of mutagenized flies and isolate not only inactivating alleles, but also rare gain-of-function mutations in cell death genes. Because mutants are recovered as heterozygotes in F1 screens, mutations that are homozygous lethal or sterile can be readily recovered. A large number of modifier mutations have been isolated, and we are in the process of characterizing several of the corresponding genes. This work has already led to the identification of components of the Ras/MAP-kinase (MAPK) pathway as negative regulators of hid. Apparently, the killing activity of the HID protein is inactivated through phosphorylation by MAPK. These findings offer an explanation of how survival signals acting through the Ras/MAPK pathway suppress the induction of apoptosis. We have also isolated loss-of-function and gain-of-function mutations in Drosophila homologs of the baculovirus inhibitor of apoptosis (IAP) genes (dIAPs). Interstingly, several of these mutations correspond to single amino acid changes that define domains that are critical for IAP protein function and regulation. Since both the IAP proteins and the Ras/MAPK pathway have been highly conserved in evolution, we are now in position to exploit the kowledge gained from studying these pathways in Drosophila to manipulate apoptosis in mammalian cells. More generally, we expect that the systematic characterization of our modifier mutants will fundamentally contribute to elucidate the molecular basis of apoptosis not only in Drosophila, but also in mammalian systems.

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