Complement-Related Regulates Autophagy in Neighboring Cells.

Autophagy degrades cytoplasmic components and is important for development and human health. Although autophagy is known to be influenced by systemic intercellular signals, the proteins that control autophagy are largely thought to function within individual cells. Here, we report that Drosophila macroglobulin complement-related (Mcr), a complement ortholog, plays an essential role during developmental cell death and inflammation by influencing autophagy in neighboring cells. This function of Mcr involves the immune receptor Draper, suggesting a relationship between autophagy and the control of inflammation. Interestingly, Mcr function in epithelial cells is required for macrophage autophagy and migration to epithelial wounds, a Draper-dependent process. This study reveals, unexpectedly, that complement-related from one cell regulates autophagy in neighboring cells via an ancient immune signaling program.

Engulfed by Glia: Glial Pruning in Development, Function, and Injury across Species.

Phagocytic activity of glial cells is essential for proper nervous system sculpting, maintenance of circuitry, and long-term brain health. Glial engulfment of apoptotic cells and superfluous connections ensures that neuronal connections are appropriately refined, while clearance of damaged projections and neurotoxic proteins in the mature brain protects against inflammatory insults. Comparative work across species and cell types in recent years highlights the striking conservation of pathways that govern glial engulfment. Many signaling cascades used during developmental pruning are re-employed in the mature brain to "fine tune" synaptic architecture and even clear neuronal debris following traumatic events. Moreover, the neuron-glia signaling events required to trigger and perform phagocytic responses are impressively conserved between invertebrates and vertebrates. This review offers a compare-and-contrast portrayal of recent findings that underscore the value of investigating glial engulfment mechanisms in a wide range of species and contexts.

C8orf46 homolog encodes a novel protein Vexin that is required for neurogenesis in Xenopus laevis.

Neural basic helix-loop helix (bHLH) transcription factors promote progenitor cell differentiation by activation of downstream target genes that coordinate neuronal differentiation. Here we characterize a neural bHLH target gene in Xenopus laevis, vexin (vxn; previously sbt1), that is homologous to human c8orf46 and is conserved across vertebrate species. C8orf46 has been implicated in cancer progression, but its function is unknown. Vxn is transiently expressed in differentiating progenitors in the developing central nervous system (CNS), and is required for neurogenesis in the neural plate and retina. Its function is conserved, since overexpression of either Xenopus or mouse vxn expands primary neurogenesis and promotes early retinal cell differentiation in cooperation with neural bHLH factors. Vxn protein is localized to the cell membrane and the nucleus, but functions in the nucleus to promote neural differentiation. Vxn inhibits cell proliferation, and works with the cyclin-dependent kinase inhibitor p27Xic1 (cdkn1b) to enhance neurogenesis and increase levels of the proneural protein Neurog2. We propose that vxn provides a key link between neural bHLH activity and execution of the neurogenic program.

Glial Draper Rescues Aß Toxicity in a Drosophila Model of Alzheimer's Disease.

Pathological hallmarks of Alzheimer's disease (AD) include amyloid-ß (Aß) plaques, neurofibrillary tangles, and reactive gliosis. Glial cells offer protection against AD by engulfing extracellular Aß peptides, but the repertoire of molecules required for glial recognition and destruction of Aß are still unclear. Here, we show that the highly conserved glial engulfment receptor Draper/MEGF10 provides neuroprotection in an AD model of Drosophila (both sexes). Neuronal expression of human Aß42arc in adult flies results in robust Aß accumulation, neurodegeneration, locomotor dysfunction, and reduced lifespan. Notably, all of these phenotypes are more severe in draper mutant animals, whereas enhanced expression of glial Draper reverses Aß accumulation, as well as behavioral phenotypes. We also show that the signal transducer and activator of transcription (Stat92E), c-Jun N-terminal kinase (JNK)/AP-1 signaling, and expression of matrix metalloproteinase-1 (Mmp1) are activated downstream of Draper in glia in response to Aß42arc exposure. Furthermore, Aß42-induced upregulation of the phagolysosomal markers Atg8 and p62 was notably reduced in draper mutant flies. Based on our findings, we propose that glia clear neurotoxic Aß peptides in the AD model Drosophila brain through a Draper/STAT92E/JNK cascade that may be coupled to protein degradation pathways such as autophagy or more traditional phagolysosomal destruction methods.SIGNIFICANCE STATEMENT Alzheimer's disease (AD) and similar dementias are common incurable neurodegenerative disorders in the aging population. As the primary immune responders in the brain, glial cells are implicated as key players in the onset and progression of AD and related disorders. Here we show that the glial engulfment receptor Draper is protective in a Drosophila model of AD, reducing levels of amyloid ß (Aß) peptides, reversing locomotor defects, and extending lifespan. We further show that protein degradation pathways are induced downstream of Draper in AD model flies, supporting a model in which glia engulf and destroy Aß peptides to reduce amyloid-associated toxicity.

Activation of autophagy during cell death requires the engulfment receptor Draper.

Autophagy degrades cytoplasmic components that are required for cell survival in response to starvation. Autophagy has also been associated with cell death, but it is unclear how this is distinguished from autophagy during cell survival. Drosophila salivary glands undergo programmed cell death that requires autophagy genes, and engulfment of salivary gland cells by phagocytes does not appear to occur. Here we show that Draper (Drpr), the Drosophila melanogaster orthologue of the Caenorhabditis elegans engulfment receptor CED-1, is required for autophagy during cell death. Null mutations in, and salivary gland-specific knockdown of, drpr inhibit salivary gland degradation. Knockdown of drpr prevents the induction of autophagy in dying salivary glands, and expression of the Atg1 autophagy regulator in drpr mutants suppresses the failure in degradation of salivary glands. Surprisingly, drpr is required in the same dying salivary gland cells in which it regulates autophagy induction, but drpr knockdown does not prevent starvation-induced autophagy in the fat body, which is associated with survival. In addition, components of the conserved engulfment pathway are required for clearance of dying salivary glands. To our knowledge, this is the first example of an engulfment factor that is required for self-clearance of cells. Further, Drpr is the first factor that distinguishes autophagy that is associated with cell death from autophagy associated with cell survival.

A roundtable discussion: home healthcare-not a hospital in the home.

Home healthcare is vital for a large percentage of the population. According to data from the U.S. Food and Drug Administration (FDA) and the Centers for Disease Control (CDC), 7 million people in the United States receive home healthcare annually. The use of medical devices in the home and other nonclinical environments is increasing dramatically. By the year 2050, an estimated 27 million people will need continuing care in the home or in the community and not in a controlled clinical environment. 1 The FDA recently announced its Home Use Devices Initiative and issued the document, "Draft Guidance for Industry and FDA Staff-Design Considerations for Devices Intended for Home Use" on Dec. 12, 2012. 2 The Center for Devices and Radiological Health (CDRH) regulates medical devices, but that regulatory authority alone is not enough to ensure safe and effective use of devices in the home. To address these and other issues, AAMI and FDA will co-host a summit on healthcare technology in nonclinical settings Oct. 9-10, 2013.

Glia and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons and shed presynaptic debris.

Synapse remodeling is an extremely dynamic process, often regulated by neural activity. Here we show during activity-dependent synaptic growth at the Drosophila NMJ many immature synaptic boutons fail to form stable postsynaptic contacts, are selectively shed from the parent arbor, and degenerate or disappear from the neuromuscular junction (NMJ). Surprisingly, we also observe the widespread appearance of presynaptically derived "debris" during normal synaptic growth. The shedding of both immature boutons and presynaptic debris is enhanced by high-frequency stimulation of motorneurons, indicating that their formation is modulated by neural activity. Interestingly, we find that glia dynamically invade the NMJ and, working together with muscle cells, phagocytose shed presynaptic material. Suppressing engulfment activity in glia or muscle by disrupting the Draper/Ced-6 pathway results in a dramatic accumulation of presynaptic debris, and synaptic growth in turn is severely compromised. Thus actively growing NMJ arbors appear to constitutively generate an excessive number of immature boutons, eliminate those that are not stabilized through a shedding process, and normal synaptic expansion requires the continuous clearance of this material by both glia and muscle cells.

Brain inflammation triggers macrophage invasion across the blood-brain barrier in Drosophila during pupal stages.

The nervous system is shielded from circulating immune cells by the blood-brain barrier (BBB). During infections and autoimmune diseases, macrophages can enter the brain where they participate in pathogen elimination but can also cause tissue damage. Here, we establish a Drosophila model to study macrophage invasion into the inflamed brain. We show that the immune deficiency (Imd) pathway, but not the Toll pathway, is responsible for attraction and invasion of hemolymph-borne macrophages across the BBB during pupal stages. Macrophage recruitment is mediated by glial, but not neuronal, induction of the Imd pathway through expression of Pvf2. Within the brain, macrophages can phagocytose synaptic material and reduce locomotor abilities and longevity. Similarly, we show that central nervous system infection by group B Streptococcus elicits macrophage recruitment in an Imd-dependent manner. This suggests that evolutionarily conserved inflammatory responses require a delicate balance between beneficial and detrimental activities.

Perceptions on undertaking regular asymptomatic self-testing for COVID-19 using lateral flow tests: a qualitative study of university students and staff.

OBJECTIVES: Successful implementation of asymptomatic testing programmes using lateral flow tests (LFTs) depends on several factors, including feasibility, acceptability and how people act on test results. We aimed to examine experiences of university students and staff of regular asymptomatic self-testing using LFTs, and their subsequent behaviours. DESIGN AND SETTING: A qualitative study using semistructured remote interviews and qualitative survey responses, which were analysed thematically. PARTICIPANTS: People who were participating in weekly testing feasibility study, between October 2020 and January 2021, at the University of Oxford. RESULTS: We interviewed 18 and surveyed 214 participants. Participants were motivated to regularly self-test as they wanted to know whether or not they were infected with SARS-CoV-2. Most reported that a negative test result did not change their behaviour, but it did provide them with reassurance to engage with permitted activities. In contrast, some participants reported making decisions about visiting other people because they felt reassured by a negative test result. Participants valued the training but some still doubted their ability to carry out the test. Participants were concerned about safety of attending test sites with lots of people and reported home testing was most convenient. CONCLUSIONS: Clear messages highlighting the benefits of regular testing for family, friends and society in identifying asymptomatic cases are needed. This should be coupled with transparent communication about the accuracy of LFTs and how to act on either a positive or negative result. Concerns about safety, convenience of testing and ability to do tests need to be addressed to ensure successful scaling up of asymptomatic testing.

Feasibility and Acceptability of Community Coronavirus Disease 2019 Testing Strategies (FACTS) in a University Setting.

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic in 2020, the UK government began a mass severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing program. This study aimed to determine the feasibility and acceptability of organized regular self-testing for SARS-CoV-2. METHODS: This was a mixed-methods observational cohort study in asymptomatic students and staff at University of Oxford, who performed SARS-CoV-2 antigen lateral flow self-testing. Data on uptake and adherence, acceptability, and test interpretation were collected via a smartphone app, an online survey, and qualitative interviews. RESULTS: Across 3 main sites, 551 participants (25% of those invited) performed 2728 tests during a follow-up of 5.6 weeks; 447 participants (81%) completed at least 2 tests, and 340 (62%) completed at least 4. The survey, completed by 214 participants (39%), found that 98% of people were confident to self-test and believed self-testing to be beneficial. Acceptability of self-testing was high, with 91% of ratings being acceptable or very acceptable. A total of 2711 (99.4%) test results were negative, 9 were positive, and 8 were inconclusive. Results from 18 qualitative interviews with students and staff revealed that participants valued regular testing, but there were concerns about test accuracy that impacted uptake and adherence. CONCLUSIONS: This is the first study to assess feasibility and acceptability of regular SARS-CoV-2 self-testing. It provides evidence to inform recruitment for, adherence to, and acceptability of regular SARS-CoV-2 self-testing programs for asymptomatic individuals using lateral flow tests. We found that self-testing is acceptable and people were able to interpret results accurately.

Rapid community point-of-care testing for COVID-19 (RAPTOR-C19): protocol for a platform diagnostic study.

BACKGROUND: The aim of RApid community Point-of-care Testing fOR COVID-19 (RAPTOR-C19) is to assess the diagnostic accuracy of multiple current and emerging point-of-care tests (POCTs) for active and past SARS-CoV2 infection in the community setting. RAPTOR-C19 will provide the community testbed to the COVID-19 National DiagnOstic Research and Evaluation Platform (CONDOR). METHODS: RAPTOR-C19 incorporates a series of prospective observational parallel diagnostic accuracy studies of SARS-CoV2 POCTs against laboratory and composite reference standards in patients with suspected current or past SARS-CoV2 infection attending community settings. Adults and children with suspected current SARS-CoV2 infection who are having an oropharyngeal/nasopharyngeal (OP/NP) swab for laboratory SARS-CoV2 reverse transcriptase Digital/Real-Time Polymerase Chain Reaction (d/rRT-PCR) as part of clinical care or community-based testing will be invited to participate. Adults (≥ 16 years) with suspected past symptomatic infection will also be recruited. Asymptomatic individuals will not be eligible. At the baseline visit, all participants will be asked to submit samples for at least one candidate point-of-care test (POCT) being evaluated (index test/s) as well as an OP/NP swab for laboratory SARS-CoV2 RT-PCR performed by Public Health England (PHE) (reference standard for current infection). Adults will also be asked for a blood sample for laboratory SARS-CoV-2 antibody testing by PHE (reference standard for past infection), where feasible adults will be invited to attend a second visit at 28 days for repeat antibody testing. Additional study data (e.g. demographics, symptoms, observations, household contacts) will be captured electronically. Sensitivity, specificity, positive, and negative predictive values for each POCT will be calculated with exact 95% confidence intervals when compared to the reference standard. POCTs will also be compared to composite reference standards constructed using paired antibody test results, patient reported outcomes, linked electronic health records for outcomes related to COVID-19 such as hospitalisation or death, and other test results. DISCUSSION: High-performing POCTs for community use could be transformational. Real-time results could lead to personal and public health impacts such as reducing onward household transmission of SARS-CoV2 infection, improving surveillance of health and social care staff, contributing to accurate prevalence estimates, and understanding of SARS-CoV2 transmission dynamics in the population. In contrast, poorly performing POCTs could have negative effects, so it is necessary to undertake community-based diagnostic accuracy evaluations before rolling these out. TRIAL REGISTRATION: ISRCTN, ISRCTN14226970.

Ensheathing glia function as phagocytes in the adult Drosophila brain.

The mammalian brain contains many subtypes of glia that vary in their morphologies, gene expression profiles, and functional roles; however, the functional diversity of glia in the adult Drosophila brain remains poorly defined. Here we define the diversity of glial subtypes that exist in the adult Drosophila brain, show they bear striking similarity to mammalian brain glia, and identify the major phagocytic cell type responsible for engulfing degenerating axons after acute axotomy. We find that neuropil regions contain two different populations of glia: ensheathing glia and astrocytes. Ensheathing glia enwrap major structures in the adult brain, but are not closely associated with synapses. Interestingly, we find these glia uniquely express key components of the glial phagocytic machinery (e.g., the engulfment receptor Draper, and dCed-6), respond morphologically to axon injury, and autonomously require components of the Draper signaling pathway for successful clearance of degenerating axons from the injured brain. Astrocytic glia, in contrast, do not express Draper or dCed-6, fail to respond morphologically to axon injury, and appear to play no role in clearance of degenerating axons from the brain. However, astrocytic glia are closely associated with synaptic regions in neuropil, and express excitatory amino acid transporters, which are presumably required for the clearance of excess neurotransmitters at the synaptic cleft. Together these results argue that ensheathing glia and astrocytes are preprogrammed cell types in the adult Drosophila brain, with ensheathing glia acting as phagocytes after axotomy, and astrocytes potentially modulating synapse formation and signaling.

In Vivo Analysis of Glial Immune Responses to Axon Degeneration in Drosophila melanogaster.

Axon degeneration elicits a range of immune responses from local glial cells, including striking changes in glial gene expression, morphology, and phagocytic activity. Here, we describe a detailed set of protocols to assess discrete components of the glial reaction to axotomy in the adult nervous system of Drosophila melanogaster. These methods allow one to visualize and quantify transcriptional, morphological, and functional responses of glia to degenerating axons in a model system that is highly amenable to genetic manipulation.