Non-autonomous cell death induced by the Draper phagocytosis receptor requires signaling through the JNK and SRC pathways.

The last step of cell death is cell clearance, a process critical for tissue homeostasis. For efficient cell clearance to occur, phagocytes and dead cells need to reciprocally signal to each other. One important phenomenon that is under-investigated, however, is that phagocytes not only engulf corpses but contribute to cell death progression. The aims of this study were to determine how the phagocytic receptor Draper non-autonomously induces cell death, using the Drosophila ovary as a model system. We found that Draper, expressed in epithelial follicle cells, requires its intracellular signaling domain to kill the adjacent nurse cell population. Kinases Src42A, Shark and JNK (Bsk) were required for Draper-induced nurse cell death. Signs of nurse cell death occurred prior to apparent engulfment and required the caspase Dcp-1, indicating that it uses a similar apoptotic pathway to starvation-induced cell death. These findings indicate that active signaling by Draper is required to kill nurse cells via the caspase Dcp-1, providing novel insights into mechanisms of phagoptosis driven by non-professional phagocytes.

Phagocytosis genes nonautonomously promote developmental cell death in the Drosophila ovary.

Programmed cell death (PCD) is usually considered a cell-autonomous suicide program, synonymous with apoptosis. Recent research has revealed that PCD is complex, with at least a dozen cell death modalities. Here, we demonstrate that the large-scale nonapoptotic developmental PCD in the Drosophila ovary occurs by an alternative cell death program where the surrounding follicle cells nonautonomously promote death of the germ line. The phagocytic machinery of the follicle cells, including Draper, cell death abnormality (Ced)-12, and c-Jun N-terminal kinase (JNK), is essential for the death and removal of germ-line-derived nurse cells during late oogenesis. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation of the nurse cells are impaired when phagocytosis is inhibited. Moreover, elimination of a small subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of nonapoptotic, nonautonomous PCD, providing insight on the diversity of cell death mechanisms.

Defective Phagocytic Corpse Processing Results in Neurodegeneration and Can Be Rescued by TORC1 Activation.

The removal of apoptotic cell corpses is important for maintaining homeostasis. Previously, defects in apoptotic cell clearance have been linked to neurodegeneration. However, the mechanisms underlying this are still poorly understood. In this study, we report that the absence of the phagocytic receptor Draper in glia leads to a pronounced accumulation of apoptotic neurons in the brain of Drosophila melanogaster. These dead cells persist in the brain throughout the lifespan of the organism and are associated with age-dependent neurodegeneration. Our data indicate that corpses persist because of defective phagosome maturation, rather than recognition defects. TORC1 activation, or inhibition of Atg1, in glia is sufficient to rescue corpse accumulation as well as neurodegeneration. These results suggest that phagocytosis of apoptotic neurons by glia during development is essential for brain homeostasis in adult flies. Furthermore, it suggests that TORC1 regulates Draper-mediated phagosome maturation. SIGNIFICANCE STATEMENT: Previously, defects in dead cell clearance were linked to neurodegeneration, but the exact mechanisms are not well understood. In this study, we report that the absence of an engulfment receptor leads to a pronounced accumulation of dead neurons in the brain of the fruit fly Drosophila melanogaster. These dead cells persist in the brain throughout the lifespan of the organism and are associated with age-dependent neurodegeneration. Our data indicate that corpses persist because of defective degradation of cells rather than recognition of dead cells.

Chromosomal microarray versus karyotyping for prenatal diagnosis.

BACKGROUND: Chromosomal microarray analysis has emerged as a primary diagnostic tool for the evaluation of developmental delay and structural malformations in children. We aimed to evaluate the accuracy, efficacy, and incremental yield of chromosomal microarray analysis as compared with karyotyping for routine prenatal diagnosis. METHODS: Samples from women undergoing prenatal diagnosis at 29 centers were sent to a central karyotyping laboratory. Each sample was split in two; standard karyotyping was performed on one portion and the other was sent to one of four laboratories for chromosomal microarray. RESULTS: We enrolled a total of 4406 women. Indications for prenatal diagnosis were advanced maternal age (46.6%), abnormal result on Down's syndrome screening (18.8%), structural anomalies on ultrasonography (25.2%), and other indications (9.4%). In 4340 (98.8%) of the fetal samples, microarray analysis was successful; 87.9% of samples could be used without tissue culture. Microarray analysis of the 4282 nonmosaic samples identified all the aneuploidies and unbalanced rearrangements identified on karyotyping but did not identify balanced translocations and fetal triploidy. In samples with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 6.0% with a structural anomaly and in 1.7% of those whose indications were advanced maternal age or positive screening results. CONCLUSIONS: In the context of prenatal diagnostic testing, chromosomal microarray analysis identified additional, clinically significant cytogenetic information as compared with karyotyping and was equally efficacious in identifying aneuploidies and unbalanced rearrangements but did not identify balanced translocations and triploidies. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT01279733.).

Draper acts through the JNK pathway to control synchronous engulfment of dying germline cells by follicular epithelial cells.

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, in some tissues with limited accessibility to circulating cells, engulfment is carried out by neighboring non-professional phagocytes such as epithelial cells. Here, we investigate the mechanism of corpse clearance in the Drosophila melanogaster ovary, a tissue that is closed to circulating cells. In degenerating egg chambers, dying germline cells are engulfed by the surrounding somatic follicular epithelium by unknown mechanisms. We show that the JNK pathway is activated and required in engulfing follicle cells. We find that the receptor Draper is also required in engulfing follicle cells, and activates the JNK pathway. Overexpression of Draper or the JNK pathway in follicle cells is sufficient to induce death of the underlying germline, suggesting that there is coordination between the germline and follicular epithelium to promote germline cell death. Furthermore, activation of JNK bypasses the need for Draper in engulfment. The induction of JNK and Draper in follicle cells occurs independently of caspase activity in the germline, indicating that at least two pathways are necessary to coordinate germline cell death with engulfment by the somatic epithelium.

Bcl-2 proteins and autophagy regulate mitochondrial dynamics during programmed cell death in the Drosophila ovary.

The Bcl-2 family has been shown to regulate mitochondrial dynamics during cell death in mammals and C. elegans, but evidence for this in Drosophila has been elusive. Here, we investigate the regulation of mitochondrial dynamics during germline cell death in the Drosophila melanogaster ovary. We find that mitochondria undergo a series of events during the progression of cell death, with remodeling, cluster formation and uptake of clusters by somatic follicle cells. These mitochondrial dynamics are dependent on caspases, the Bcl-2 family, the mitochondrial fission and fusion machinery, and the autophagy machinery. Furthermore, Bcl-2 family mutants show a striking defect in cell death in the ovary. These data indicate that a mitochondrial pathway is a major mechanism for activation of cell death in Drosophila oogenesis.

Protocol to analyze 3D neurodegenerative vacuoles in Drosophila melanogaster.

Vacuole formation is a key hallmark of age-dependent neurodegeneration in the Drosophila brain. Here, we present a protocol to analyze 3D neurodegenerative vacuoles in the whole-mount Drosophila melanogaster brain. We describe steps for whole-brain dissection, staining, 3D imaging, and z-stack image processing using Fiji ImageJ. We then detail procedures for annotating and 3D-reconstructing neurodegenerative vacuoles with WEBKNOSSOS and Python, and performing statistical analysis in Python. This protocol enables measurement of parameters such as the number and volume of each vacuole. For complete details on the use and execution of this protocol, please refer to Elguero et al.1.

Professional phagocytes are recruited for the clearance of obsolete nonprofessional phagocytes in the Drosophila ovary.

Cell death is an important process in the body, as it occurs throughout every tissue during development, disease, and tissue regeneration. Phagocytes are responsible for clearing away dying cells and are typically characterized as either professional or nonprofessional phagocytes. Professional phagocytes, such as macrophages, are found in nearly every part of the body while nonprofessional phagocytes, such as epithelial cells, are found in every tissue type. However, there are organs that are considered "immune-privileged" as they have little to no immune surveillance and rely on nonprofessional phagocytes to engulf dying cells. These organs are surrounded by barriers to protect the tissue from viruses, bacteria, and perhaps even immune cells. The Drosophila ovary is considered immune-privileged, however the presence of hemocytes, the macrophages of Drosophila, around the ovary suggests they may have a potential function. Here we analyze hemocyte localization and potential functions in response to starvation-induced cell death in the ovary. Hemocytes were found to accumulate in the oviduct in the vicinity of mature eggs and follicle cell debris. Genetic ablation of hemocytes revealed that the presence of hemocytes affects oogenesis and that they phagocytose ovarian cell debris and in their absence fecundity decreases. Unpaired3, an IL-6 like cytokine, was found to be required for the recruitment of hemocytes to the oviduct to clear away obsolete follicle cells. These findings demonstrate a role for hemocytes in the ovary, providing a more thorough understanding of phagocyte communication and cell clearance in a previously thought immune-privileged organ.

Characterization of female reproductive disturbances post-traumatic injury in Drosophila melanogaster.

Traumatic injuries (TIs) from intimate partner violence, vehicular collisions, high-impact sports, and even mundane activities can be fatal. However, survivors of TIs can have pathophysiological disturbances post-injury, including neurodegenerative diseases, mental illness, and metabolic disorders.Reproductive issues are a known consequence of TI especially in women, however this has remained poorly understood. Drosophila melanogaster has recently emerged as a stellar model of TI, however reproductive consequences have not been reported. Using the Drosophila model, we find reproductive consequences in the form of decreased egg laying and the retention of mature egg chambers mimicking issues in ovulation. These findings indicate that reproductive consequences of TI are conserved between Drosophila and humans.

Defective phagocytosis leads to neurodegeneration through systemic increased innate immune signaling.

In nervous system development, disease and injury, neurons undergo programmed cell death, leaving behind cell corpses that are removed by phagocytic glia. Altered glial phagocytosis has been implicated in several neurological diseases including Alzheimer's disease, Parkinson's disease, and traumatic brain injury. To untangle the links between glial phagocytosis and neurodegeneration, we investigated Drosophila mutants lacking the phagocytic receptor Draper. Loss of Draper leads to persistent neuronal cell corpses and age-dependent neurodegeneration. Here we investigate whether the phagocytic defects observed in draper mutants lead to chronic increased immune activation that promotes neurodegeneration. A major immune response in Drosophila is the activation of two NFκB signaling pathways that produce antimicrobial peptides, primarily in the fat body. We found that the antimicrobial peptide Attacin-A is highly upregulated in the fat body of aged draper mutants and that inhibition of the Immune deficiency (Imd) pathway in the glia and fat body of draper mutants led to reduced neurodegeneration, indicating that immune activation promotes neurodegeneration in draper mutants. Taken together, these findings indicate that phagocytic defects lead to neurodegeneration via increased immune signaling, both systemically and locally in the brain.

Defective phagocytosis leads to neurodegeneration through systemic increased innate immune signaling.

In nervous system development, disease, and injury, neurons undergo programmed cell death, leaving behind cell corpses that are removed by phagocytic glia. Altered glial phagocytosis has been implicated in several neurological diseases including Alzheimer's disease. To untangle the links between glial phagocytosis and neurodegeneration, we investigated Drosophila mutants lacking the phagocytic receptor Draper. Loss of Draper leads to persistent neuronal cell corpses and age-dependent neurodegeneration. Here we investigate whether the phagocytic defects observed in draper mutants lead to chronic increased immune activation that promotes neurodegeneration. We found that the antimicrobial peptide Attacin-A is highly upregulated in the fat body of aged draper mutants and that the inhibition of the Immune deficiency (Imd) pathway in the glia and fat body of draper mutants led to reduced neurodegeneration. Taken together, these findings indicate that phagocytic defects lead to neurodegeneration via increased immune signaling, both systemically and locally in the brain.

A Randomized Controlled Trial Evaluating the Effectiveness of Supported Employment Integrated in Primary Care.

OBJECTIVES: Mental health issues can cause serious problems in occupational functioning, including higher rates of unemployment. Individual placement and support (IPS) is an evidence-based supported employment intervention that is typically integrated within a mental health setting; however, many primary care patients view referral to a mental health clinic as stigmatizing. Thus, this study examined whether delivery of IPS in a primary care setting provides an effective treatment option and avoids unnecessary delays in obtaining competitive employment. METHODS: U.S. military veterans (N=119) who had a diagnosis in a broad range of nonpsychotic psychiatric disorders and who were receiving care from Veterans Health Administration (VHA) patient-aligned care teams were prospectively randomly assigned to IPS (N=58) or standard VHA non-IPS vocational rehabilitation (VR) (N=61). The primary outcome was achievement of steady worker status, defined as holding a competitive job for ≥6 months of the 12-month follow-up. RESULTS: As hypothesized, a significantly greater proportion of IPS participants achieved steady worker status (45%), compared with VR participants (25%) (p=0.02; odds ratio=2.49, 95% confidence interval=1.14-5.43). On average, the IPS participants worked significantly more weeks (p=0.003) and earned significantly more income (p=0.033) from competitive jobs, compared with VR participants. CONCLUSIONS: The results provide supporting evidence for offering IPS within primary care with the aim of restoring meaningful and sustained competitive employment for veterans living with a mental disorder. Such modifications could improve veterans' vocational outcomes, moving a significantly greater number of disabled veterans back to full and productive lives in the community.

RpL10A regulates oogenesis progression in the banana prawn Fenneropenaeus merguiensis and Drosophila melanogaster.

To develop banana prawn (Fenneropenaeus merguiensis) aquaculture, the mechanism of ovarian maturation is under investigation. In a previous study, we reported the RpL10A protein as an ovarian maturation stimulator. To further investigate the function of RpL10A, we turned to the fruit fly (Drosophila melanogaster) to take advantage of the genetic tools available. Here, we elucidate the expression and function of RpL10A in the D. melanogaster ovary. RpL10A is expressed in the cytoplasm of both nurse and follicle cells throughout oogenesis. While shrimp have one RpL10A gene, D. melanogaster has two genes, RpL10Aa and RpL10Ab. RpL10Ab displays more similarity with shrimp RpL10A and was further investigated. RpL10Ab homozygous mutants are lethal and germline clone analysis showed that RpL10Ab is an essential gene in oogenesis. Moreover, RpL10Ab(-) germline clones resulted in premature death of the follicle cells. This phenotype is reminiscent of some insulin pathway mutants, suggesting that RpL10Ab may be involved in the insulin signaling pathway. In addition, RpL10Ab(-) follicle cells showed abnormal nuclei and membranes. Shrimp RpL10A rescued RpL10Ab homozygous mutants, revealing their functional conservation. Surprisingly, we found cell death in multiple tissues when RpL10A was over-expressed, suggesting that proper RpL10A levels are important. This research reveals novel findings about the role of RpL10A during oogenesis and may, in the future, lead to new approaches to stimulate ovarian development in shrimp.

Murder on the Ovarian Express: A Tale of Non-Autonomous Cell Death in the Drosophila Ovary.

Throughout oogenesis, Drosophila egg chambers traverse the fine line between survival and death. After surviving the ten early and middle stages of oogenesis, egg chambers drastically change their size and structure to produce fully developed oocytes. The development of an oocyte comes at a cost, the price is the lives of the oocyte's 15 siblings, the nurse cells. These nurse cells do not die of their own accord. Their death is dependent upon their neighbors-the stretch follicle cells. Stretch follicle cells are nonprofessional phagocytes that spend the final stages of oogenesis surrounding the nurse cells and subsequently forcing the nurse cells to give up everything for the sake of the oocyte. In this review, we provide an overview of cell death in the ovary, with a focus on recent findings concerning this phagocyte-dependent non-autonomous cell death.

An RNAi screen of the kinome in epithelial follicle cells of the Drosophila melanogaster ovary reveals genes required for proper germline death and clearance.

Programmed cell death and cell corpse clearance are an essential part of organismal health and development. Cell corpses are often cleared away by professional phagocytes such as macrophages. However, in certain tissues, neighboring cells known as nonprofessional phagocytes can also carry out clearance functions. Here, we use the Drosophila melanogaster ovary to identify novel genes required for clearance by nonprofessional phagocytes. In the Drosophila ovary, germline cells can die at multiple time points. As death proceeds, the epithelial follicle cells act as phagocytes to facilitate the clearance of these cells. We performed an unbiased kinase screen to identify novel proteins and pathways involved in cell clearance during two death events. Of 224 genes examined, 18 demonstrated severe phenotypes during developmental death and clearance while 12 demonstrated severe phenotypes during starvation-induced cell death and clearance, representing a number of pathways not previously implicated in phagocytosis. Interestingly, it was found that several genes not only affected the clearance process in the phagocytes, but also non-autonomously affected the process by which germline cells died. This kinase screen has revealed new avenues for further exploration and investigation.

Drosophila Chk2 and p53 proteins induce stage-specific cell death independently during oogenesis.

In Drosophila, the checkpoint protein-2 kinase (DmChk2) and its downstream effector protein, Dmp53, are required for DNA damage-mediated cell cycle arrest, DNA repair and apoptosis. In this study we focus on understanding the function of these two apoptosis inducing factors during ovarian development. We found that expression of Dmp53, but not DmChk2, led to loss of ovarian stem cells. We demonstrate that expression of DmChk2, but not Dmp53, induced mid-oogenesis cell death. DmChk2 induced cell death was not suppressed by Dmp53 mutant, revealing for the first time that in Drosophila, over-expression of DmChk2 can induce cell death which is independent of Dmp53. We found that over-expression of caspase inhibitors such as DIAP1, p35 and p49 did not suppress DmChk2- and Dmp53-induced cell death. Thus, our study reveals stage-specific effects of Dmp53 and DmChk2 in oogenesis. Moreover, our results demonstrate that although DmChk2 and Dmp53 affect different stages of ovarian development, loss of ovarian stem cells by p53 expression and mid-oogenesis cell death induced by DmChk2 do not require caspase activity.

Nuclear degradation dynamics in a nonapoptotic programmed cell death.

Nuclear degradation is a major event during programmed cell death (PCD). The breakdown of nuclear components has been well characterized during apoptosis, one form of PCD. Many nonapoptotic forms of PCD have been identified, but our understanding of nuclear degradation during those events is limited. Here, we take advantage of Drosophila oogenesis to investigate nuclear degeneration during stress-induced apoptotic and developmental nonapoptotic cell death in the same cell type in vivo. We find that nuclear Lamin, a caspase substrate, dissociates from the nucleus as an early event during apoptosis, but remains associated with nuclei during nonapoptotic cell death. Lamin reveals a series of changes in nuclear architecture during nonapoptotic death, including nuclear crenellations and involutions. Stretch follicle cells contribute to these architecture changes, and phagocytic and lysosome-associated machinery in stretch follicle cells promote Lamin degradation. More specifically, we find that the lysosomal cathepsin CP1 facilitates Lamin degradation.

Cracking open cell death in the Drosophila ovary.

The Drosophila melanogaster ovary is a powerful yet simple system with only a few cell types. Cell death in the ovary can be induced in response to multiple developmental and environmental signals. These cell deaths occur at distinct stages of oogenesis and involve unique mechanisms utilizing apoptotic, autophagic and perhaps necrotic processes. In this review, we summarize recent progress characterizing cell death mechanisms in the fly ovary.

Detection of cell death in Drosophila.

Drosophila is a powerful model system for the identification of cell death genes and understanding the role of cell death in development. In this chapter, we describe three methods typically used for the detection of cell death in Drosophila. The TUNEL and acridine orange methods are used to detect dead or dying cells in a variety of tissues. We focus on methods for the embryo and the ovary, but these techniques can be used on other tissues as well. The third method is the detection of genetic interactions by expressing cell death genes in the Drosophila eye.

Lysosomal Machinery Drives Extracellular Acidification to Direct Non-apoptotic Cell Death.

Cell death is a fundamental aspect of development, homeostasis, and disease; yet, our understanding of non-apoptotic forms of cell death is limited. One such form is phagoptosis, in which one cell utilizes phagocytosis machinery to kill another cell that would otherwise continue living. We have previously identified a non-autonomous requirement of phagocytosis machinery for the developmental programmed cell death of germline nurse cells in the Drosophila ovary; however, the precise mechanism of death remained elusive. Here, we show that lysosomal machinery acting in epithelial follicle cells is used to non-autonomously induce the death of nearby germline cells. Stretch follicle cells recruit V-ATPases and chloride channels to their plasma membrane to extracellularly acidify the germline and release cathepsins that destroy the nurse cells. Our results reveal a role for lysosomal machinery acting at the plasma membrane to cause the death of neighboring cells, providing insight into mechanisms driving non-autonomous cell death.