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.

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.

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.

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.

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.

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.

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.

Employment outcomes from VA vocational services involving transitional work for veterans with a diagnosis of posttraumatic stress disorder.

OBJECTIVE: Transitional work (TW) for veterans with psychiatric disabilities is the predominant model of vocational rehabilitation in the Veterans Health Administration (VA). Although, on average, TW employment outcomes have been demonstrated to be inferior to supported employment, little is known about the potential subgroup of veterans for which TW may be most effective. This study of veterans with posttraumatic stress disorder (PTSD) examines differences in competitive employment outcomes and identifies characteristics of veterans who chose to engage in TW compared with those who did not. METHOD: A post hoc comparative subgroup analysis of veterans with PTSD randomly assigned to TW as part of a randomized controlled trial was conducted. Veterans were divided into 2 subgroups: those who engaged in TW (n = 141) and nonengagers (n = 129). Differences in baseline characteristics were examined and 18-month employment outcomes were compared. RESULTS: There were no differences in 18-month employment outcomes between TW engagers and nonengagers. Compared with TW engagers, those that did not engage in TW were 2.5 times more likely to get a competitive job within the first 6 months and were less likely to obtain lower skilled jobs. Younger age, adequate housing, personal means of transportation, and recent work history factor into the odds of gaining and maintaining competitive work. CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: Consistent with past research, engagement in TW did not result in improved long-term competitive employment outcomes for veterans with PTSD. Those who did not engage in TW were more likely to gain a competitive job within the first 6 months. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

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.

I Spy in the Developing Fly a Multitude of Ways to Die.

Cell proliferation and cell death are two opposing, yet complementary fundamental processes in development. Cell proliferation provides new cells, while developmental programmed cell death adjusts cell numbers and refines structures as an organism grows. Apoptosis is the best-characterized form of programmed cell death; however, there are many other non-apoptotic forms of cell death that occur throughout development. Drosophila is an excellent model for studying these varied forms of cell death given the array of cellular, molecular, and genetic techniques available. In this review, we discuss select examples of apoptotic and non-apoptotic cell death that occur in different tissues and at different stages of Drosophila development. For example, apoptosis occurs throughout the nervous system to achieve an appropriate number of neurons. Elsewhere in the fly, non-apoptotic modes of developmental cell death are employed, such as in the elimination of larval salivary glands and midgut during metamorphosis. These and other examples discussed here demonstrate the versatility of Drosophila as a model organism for elucidating the diverse modes of programmed cell death.

The ABC Transporter Eato Promotes Cell Clearance in the Drosophila melanogaster Ovary.

The clearance of dead cells is a fundamental process in the maintenance of tissue homeostasis. Genetic studies in Drosophila melanogaster, Caenorhabditis elegans, and mammals have identified two evolutionarily conserved signaling pathways that act redundantly to regulate this engulfment process: the ced-1/-6/-7 and ced-2/-5/-12 pathways. Of these engulfment genes, only the ced-7/ABCA1 ortholog remains to be identified in D. melanogaster Homology searches have revealed a family of putative ced-7/ABCA1 homologs encoding ATP-binding cassette (ABC) transporters in D. melanogaster To determine which of these genes functions similarly to ced-7/ABCA1, we analyzed mutants for engulfment phenotypes in oogenesis, during which nurse cells (NCs) in each egg chamber undergo programmed cell death (PCD) and are removed by neighboring phagocytic follicle cells (FCs). Our genetic analyses indicate that one of the ABC transporter genes, which we have named Eato (Engulfment ABC Transporter in the ovary), is required for NC clearance in the ovary and acts in the same pathways as drpr, the ced-1 ortholog, and in parallel to Ced-12 in the FCs. Additionally, we show that Eato acts in the FCs to promote accumulation of the transmembrane receptor Drpr, and promote membrane extensions around the NCs for their clearance. Since ABCA class transporters, such as CED-7 and ABCA1, are known to be involved in lipid trafficking, we propose that Eato acts to transport membrane material to the growing phagocytic cup for cell corpse clearance. Our work presented here identifies Eato as the ced-7/ABCA1 ortholog in D. melanogaster, and demonstrates a role for Eato in Drpr accumulation and phagocytic membrane extensions during NC clearance in the ovary.

Veterans individual placement and support towards advancing recovery: Methods and baseline clinical characteristics of a multisite study.

OBJECTIVE: This article describes the methodology and the baseline characteristics of veterans with posttraumatic stress disorder (PTSD) enrolled in a multisite trial comparing supported employment individual placement and support (IPS) to a stepwise vocational transitional work program (TWP). METHOD: The Veterans Affairs Cooperative Studies Program randomized 541 veterans with PTSD across 12 Veterans Affairs Medical Centers to either IPS or TWP. Demographic and clinical characteristics were evaluated at baseline. RESULTS: Participants averaged 42 (SD ± 11) years of age and had PTSD for 13 (SD ± 11) years. The group was comprised of 18% female, 42% African Americans, and 16% Latino participants. Approximately 60% of participants served in the military since 2001, 89% were receiving or applying for service-connected disability, 60% had PTSD from nonsexual combat-related trauma, and 17% had PTSD from military sexual trauma. One third had not held a competitive job in the past 3 years; the average length of unemployment was 2.8 (SD ± 4) years. Unique study features included the focus on veterans with PTSD, a comparison of a promising practice with a usual-care practice, and the outcome criterion of achieving steady competitive employment. Conclusions and Implication for Practice: This study is the first large-scale randomized trial of IPS in a PTSD population. These baseline findings illustrate the characteristics of the study sample, which are representative of a veteran population in need of vocational rehabilitation services and can be used to help guide the implementation of tailored veteran-centered programs. (PsycINFO Database Record

Scrambled Eggs: Apoptotic Cell Clearance by Non-Professional Phagocytes in the Drosophila Ovary.

For half of a century, it has been known that non-professional phagocytes, such as fibroblasts, endothelial, and epithelial cells, are capable of efferocytosis (engulfment of apoptotic cells). Non-professional phagocytes differ from professional phagocytes in the range and efficiency of engulfment. Much of the recognition and underlying signaling machinery between non-professional and professional phagocytes is the same, but it is not known how the engulfment capacity of non-professional phagocytes is controlled. Moreover, the signaling networks involved in cell corpse recognition, engulfment, and phagosome maturation are only partially understood. The Drosophila ovary provides an excellent system to investigate the regulation of phagocytic activity by epithelial cells, a major class of non-professional phagocytes. During Drosophila oogenesis, mid-stage egg chambers undergo apoptosis of the germline in response to nutrient deprivation. Epithelial follicle cells then undergo major cell shape changes and concomitantly engulf the germline material. Our previous work has established that Draper and the integrin α-PS3/ß-PS heterodimer are required in follicle cells for germline cell clearance. In addition, we have characterized phagosome maturation pathways, and found that the JNK pathway amplifies the engulfment response. In this review, we discuss recent advances on the interplay between engulfment pathways in the follicular epithelium for cell clearance in the Drosophila ovary. We also provide a comparison to apoptotic cell clearance mechanisms in C. elegans and mammals, illustrating strong conservation of efferocytosis mechanisms by non-professional phagocytes.

Control of non-apoptotic nurse cell death by engulfment genes in Drosophila.

Programmed cell death occurs as a normal part of oocyte development in Drosophila. For each egg that is formed, 15 germline-derived nurse cells transfer their cytoplasmic contents into the oocyte and die. Disruption of apoptosis or autophagy only partially inhibits the death of the nurse cells, indicating that other mechanisms significantly contribute to nurse cell death. Recently, we demonstrated that the surrounding stretch follicle cells non-autonomously promote nurse cell death during late oogenesis and that phagocytosis genes including draper, ced-12, and the JNK pathway are crucial for this process. When phagocytosis genes are inhibited in the follicle cells, events specifically associated with death of the nurse cells are impaired. Death of the nurse cells is not completely blocked in draper mutants, suggesting that other engulfment receptors are involved. Indeed, we found that the integrin subunit, αPS3, is enriched on stretch follicle cells during late oogenesis and is required for elimination of the nurse cells. Moreover, double mutant analysis revealed that integrins act in parallel to draper. Death of nurse cells in the Drosophila ovary is a unique example of programmed cell death that is both non-apoptotic and non-cell autonomously controlled.

Analysis of Phagocytosis in the Drosophila Ovary.

Programmed cell death (PCD) is essential for health and development. Generally, the last step of PCD is clearance, or engulfment, by phagocytes. Engulfment can be broken down into five basic steps: attraction of the phagocyte, recognition of the dying cell, internalization, phagosome maturation, and acidification of the engulfed material. The Drosophila melanogaster ovary serves as an excellent model to study diverse types of PCD and engulfment by epithelial cells. Here, we describe several methods to detect and analyze multiple steps of engulfment in the Drosophila ovary: recognition, vesicle uptake, phagosome maturation, and acidification. Annexin V detects phosphatidylserine, which is flipped to the outer leaflet of the plasma membrane of apoptotic cells, serving as an "eat me" signal. Several germline markers including tral-GFP, Orb, and cleaved Dcp-1 can all be used to label the germline and visualize its uptake into engulfing follicle cells. Drosophila strains expressing GFP and mCherry protein fusions can enable a detailed analysis of phagosome maturation. LysoTracker labels highly acidified compartments, marking phagolysosomes. Together these labels can be used to mark the progression of engulfment in Drosophila follicle cells.

Components of the Engulfment Machinery Have Distinct Roles in Corpse Processing.

Billions of cells die in our bodies on a daily basis and are engulfed by phagocytes. Engulfment, or phagocytosis, can be broken down into five basic steps: attraction of the phagocyte, recognition of the dying cell, internalization, phagosome maturation, and acidification. In this study, we focus on the last two steps, which can collectively be considered corpse processing, in which the engulfed material is degraded. We use the Drosophila ovarian follicle cells as a model for engulfment of apoptotic cells by epithelial cells. We show that engulfed material is processed using the canonical corpse processing pathway involving the small GTPases Rab5 and Rab7. The phagocytic receptor Draper is present on the phagocytic cup and on nascent, phosphatidylinositol 3-phosphate (PI(3)P)- and Rab7-positive phagosomes, whereas integrins are maintained on the cell surface during engulfment. Due to the difference in subcellular localization, we investigated the role of Draper, integrins, and downstream signaling components in corpse processing. We found that some proteins were required for internalization only, while others had defects in corpse processing as well. This suggests that several of the core engulfment proteins are required for distinct steps of engulfment. We also performed double mutant analysis and found that combined loss of draper and αPS3 still resulted in a small number of engulfed vesicles. Therefore, we investigated another known engulfment receptor, Crq. We found that loss of all three receptors did not inhibit engulfment any further, suggesting that Crq does not play a role in engulfment by the follicle cells. A more complete understanding of how the engulfment and corpse processing machinery interact may enable better understanding and treatment of diseases associated with defects in engulfment by epithelial cells.

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.

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.