Kimberly McCall - US grants

DESCRIPTION (provided by applicant): Programmed cell death plays a central role in development and in multiple diseases, notably cancer and neurodegenerative disorders. -The long-term objective of this proposed research is to further understand the mechanisms of programmed cell death and its role in development. The model being used is the ovary of the fruitfly, Drosophila melanogaster, a system with unique advantages for the study of cell death. Programmed cell death of ovarian nurse cells is essential for proper oocyte development. Nurse cells possess distinct traits which make them an ideal model for analyzing cell death in vivo. These cells are abundant, die synchronously in clusters, and are large, making them amenable to subcellular analyses. Furthermore, disruption of nurse cell death results in sterility, a phenotype that permits straightforward genetic analysis. This proposal addresses multiple aspects of the nurse cell death pathway. The specific aims are to 1) further address the requirement for caspases in this pathway by the expression of constitutively active Drosophila caspase- 1 (Dcp- 1) in the germline, the characterization of new alleles of dcp-1, and the characterization of the caspase Drice; 2) isolate additional genes involved in nurse cell death by histological examination of mutants previously shown to have defects in oogenesis; and 3) test the role of individual caspase targets during cell death in vivo by the expression of cleavage-defective molecules. These experiments should yield an understanding of the molecules that are critical for nurse cell death, from the initial signal to the final proteolyzed targets. This knowledge is likely to have implications for other cell death pathways in Drosophila and other organisms.

DESCRIPTION (provided by applicant): Programmed cell death plays a central role in many diseases, including cancer and neurodegenerative disorders. Programmed cell death is also a critical process for proper patterning during development. The long-term goal of this project is to understand how programmed cell death is regulated during development. IAPs, inhibitor of apoptosis proteins, are a family of cellular inhibitors of apoptotic cell death. Drosophila IAP1 (Diap1) is a critical regulator of apoptosis during Drosophila development. Null mutations in diap1 result in extensive cell death during early embryogenesis, indicating that Diap1 must be present in most, if not all cells, to prevent apoptosis. The diap1 gene was originally called thread, named for the first mutation identified in the gene. The thread1 mutation is homozygous viable, and the only phenotypic effect is a disruption of branching in the antennal arista. The mechanisms controlling branch formation in the arista are not well understood. Preliminary studies indicate that thread1 mutants show excessive cell death restricted to the antennal imaginal disc during larval development. This suggests that there is a narrow window of development in which regulation of programmed cell death is essential to proper branch formation in the arista. The arista-specific phenotype seen in the thread1 mutant suggests that the mutation may disrupt thread expression only in the developing arista. Preliminary data indicate that the mutation is not in a protein-coding region. The goal of this proposed research is to determine the molecular nature of the thread1 allele and to determine how this mutation affects expression of the thread gene. The cis-regulatory elements affected by the mutation and the upstream signaling pathways controlling thread expression will be investigated. These findings will lead to a better understanding of the transcriptional regulation of IAPs, which may alter the threshold for induction of apoptosis. Furthermore, this work will provide insight into the role of programmed cell death in the development of branched organs.

DESCRIPTION (provided by applicant): The efficient removal of dead cells is an important process in animal development and homeostasis. Cell corpses are often engulfed by professional phagocytes such as macrophages. However, some tissues have limited accessibility to circulating cells, and engulfment is carried out by neighboring non-professional phagocytes such as epithelial cells. The mechanisms of cell corpse recognition, engulfment and phagosome breakdown are only partially understood. The Drosophila ovary provides an excellent system for the study of engulfment by non-professional phagocytes. The ovary is closed to circulating cells, and degeneration of entire egg chambers can be induced easily by starvation. In such degenerating egg chambers, the germline nurse cells are engulfed by the surrounding somatic follicle cells. This engulfment process happens synchronously and rapidly at the onset of cell death, however the genetic requirements for follicle cell engulfment are completely unknown. This proposal aims to identify the genes and pathways required for engulfment by follicle cells. First, known engulfment genes and candidate signaling molecules will be investigated for their role in engulfment by follicle cells. Second, engulfing follicle cells will be isolated and their expression profiles will be compared to non-engulfing follicle cells using DNA microarray and proteomic approaches. Third, genetic screens will be conducted to identify new engulfment genes. Genes identified through these approaches will be characterized for their roles in engulfment in follicle cells and other tissues. These findings are expected to reveal the signaling mechanisms that are required for an epithelial cell to switch to an engulfing cell state. As the mechanisms of engulfment are highly conserved, these findings will have implications for the understanding and treatment of human diseases, including neurodegenerative and auto-immune disorders.

Project Summary Neurodegenerative disorders, such as Alzheimer's Disease and Parkinson's Disease, are major health issues world-wide. The role of glia in neurodegeneration is not well understood, and the role may differ depending on the specific type and stage of disease. A major function of glia during brain homeostasis is the phagocytosis of pruned axons, dead or injured cells, and debris. Several lines of evidence suggest that glial phagocytosis plays an important role in the prevention or progression of brain disorders, including neurodegenerative diseases. In this proposal, we investigate the role of phagocytosis in neurodegenerative disorders using the model organism Drosophila melanogaster. Drosophila provides a wealth of genetic and cell biological tools for the study of glial phagocytosis and neurodegeneration. Several human neurodegenerative disorders have been modeled in Drosophila, providing a powerful platform for screening for genetic interactors. Moreover, the genes controlling phagocytosis are largely conserved between flies and mammals. Recently, we have shown that a disruption of the phagocytic receptor Draper in glia leads to persisting neuronal corpses and neurodegeneration in Drosophila. Here we will examine whether persistent corpses or debris in the draper mutant brain lead to neurodegeneration by triggering inflammation. We will additionally determine how loss of Draper interacts with genetic models of Alzheimer's disease, and investigate the role of inflammation in these disease models. Phenotypes will be analyzed by molecular, cellular and behavioral assays. These findings will provide insight into the impact of defective phagocytosis in neurodegenerative disorders.

Project Summary Cell death is a fundamental process in animal development and homeostasis, and misregulation of cell death is associated with a large number of human diseases. Our research program aims to understand the diverse mechanisms of cell death, how dead cells are efficiently removed, and the physiological effects on organisms when these processes go awry. We study these questions in Drosophila, a model organism with exceptional genetic, genomic and cell biological tools. The research in this proposal focuses on four key questions. The first project investigates non-apoptotic cell death, which contributes significantly to development and disease, but is poorly understood. In the Drosophila ovary, germline-derived nurse cells undergo non-apoptotic programmed cell death as part of normal development. We have found that nurse cell death is controlled largely non-autonomously by the surrounding somatic follicle cells, and current work investigates the role of lysosomes and cell signaling in this process. A second major project investigates how dead cells are removed, particularly in tissues without access to circulating phagocytes such as macrophages. In many mammalian tissues, dead cells can be cleared by epithelial cells. In a Drosophila model for engulfment by epithelial cells, starvation induces degeneration of egg chambers, where the germline is engulfed by the surrounding epithelial follicle cells. This engulfment process happens synchronously and rapidly at the onset of cell death; however the genetic requirements for engulfment by epithelial cells are not well understood. In particular, how such non-professional phagocytes can improve their phagocytic capacity is not known, and our research program will reveal the requirements for engulfment by epithelial cells. A third project addresses how phagocytic cells can promote the death of their neighbors, through a newly described type of cell death called phagoptosis. In the fourth project, we investigate the consequences of persisting cell corpses in the ovary and the brain, and how defective phagocytosis can affect disease progression. We will examine the global changes to organisms with defective cell death or phagocytosis and how defective phagocytosis interacts with genetic models of human disease. Given the high degree of conservation of cell death mechanisms between Drosophila and mammals identified thus far, we expect that pathways that we uncover in the fly will provide insight into the diversity of cell death mechanisms and consequences of defective cell removal in humans. These studies may reveal new therapeutic targets for diseases with excessive or insufficient cell death such as neurodegenerative disorders and cancer.