Mary A. Logan

dSarm/Sarm1 Is Required for Activation of an Injury-Induced Axon Death Pathway

Sarm-Assisted Suicide Neurodegenerative disease or nerve lesions cause axons and synapses to disintegrate through a process known as Wallerian degeneration, which may involve an active “axon death program.” Osterloh et al. (p. 481, published online 7 June; see the Perspective by Yu and Luo) identify loss-of-function mutations in Drosophila dSarm that are capable of blocking the degeneration of severed axons for the fly life span. Deletion of mouse Sarm1 provides similar protection to severed axons for weeks after injury, which suggests that Sarm is part of an ancient axonal death signaling cascade. Mutations in a scaffold protein block the Wallerian degeneration of axons in flies and mice. Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Here, we show that loss of the Drosophila Toll receptor adaptor dSarm (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway.

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.

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.

Glia and Muscle Sculpt Neuromuscular Arbors by Engulfing Destabilized Synaptic Boutons and Shed Presynaptic Debris

As synapses grow at the Drosophila neuromuscular junction, they shed membrane material in an activity-dependent manner. Glia and postsynaptic muscle cells are required to engulf this debris to ensure new synaptic growth.

Negative regulation of glial engulfment activity by Draper terminates glial responses to axon injury

Neuronal injury elicits potent cellular responses from glia, but molecular pathways modulating glial activation, phagocytic function and termination of reactive responses remain poorly defined. Here we show that positive or negative regulation of glial responses to axon injury is molecularly encoded by unique isoforms of the Drosophila melanogaster engulfment receptor Draper. Draper-I promotes engulfment of axonal debris through an immunoreceptor tyrosine–based activation motif (ITAM). In contrast, Draper-II, an alternative splice variant, potently inhibits glial engulfment function. Draper-II suppresses Draper-I signaling through a previously undescribed immunoreceptor tyrosine–based inhibitory motif (ITIM)-like domain and the tyrosine phosphatase Corkscrew (Csw). Intriguingly, loss of Draper-II–Csw signaling prolongs expression of glial engulfment genes after axotomy and reduces the ability of glia to respond to secondary axotomy. Our work highlights a novel role for Draper-II in inhibiting glial responses to neurodegeneration, and indicates that a balance of opposing Draper-I and Draper-II signaling events is essential to maintain glial sensitivity to brain injury.

The scoop on the fly brain: glial engulfment functions in Drosophila

NASA’s Hubble Space Telescope has given the world amazing images of the distant stars, planets, and galaxies. The cutting-edge imaging technology that enhances the Hubble images also extends its benefits to life here on Earth, from deciphering previously unreadable portions of the Dead Sea Scrolls to improving digital mammographies for advanced cancer detection. This imaging technology is now helping physicians to perform micro-invasive arthroscopic surgery, which is the visual examination of an interior joint such as the knee.Glial cells provide support and protection for neurons in the embryonic and adult brain, mediated in part through the phagocytic activity of glia. Glial cells engulf apoptotic cells and pruned neurites from the developing nervous system, and also clear degenerating neuronal debris from the adult brain after neural trauma. Studies indicate that Drosophila melanogaster is an ideal model system to elucidate the mechanisms of engulfment by glia. The recent studies reviewed here show that many features of glial engulfment are conserved across species and argue that work in Drosophila will provide valuable cellular and molecular insight into glial engulfment activity in mammals.

Axon degeneration induces glial responses through Draper-TRAF4-JNK signalling

Draper/Ced-1/MEGF-10 is an engulfment receptor that promotes clearance of cellular debris in C. elegans, Drosophila and mammals. Draper signals through an evolutionarily conserved Src family kinase cascade to drive cytoskeletal rearrangements and target engulfment through Rac1. Glia also alter gene expression patterns in response to axonal injury but pathways mediating these responses are poorly defined. We show Draper is cell autonomously required for glial activation of transcriptional reporters after axonal injury. We identify TNF receptor associated factor 4 (TRAF4) as a novel Draper binding partner that is required for reporter activation and phagocytosis of axonal debris. TRAF4 and misshapen (MSN) act downstream of Draper to activate c-Jun N-terminal kinase (JNK) signalling in glia, resulting in changes in transcriptional reporters that are dependent on Drosophila AP-1 (dAP-1) and STAT92E. Our data argue injury signals received by Draper at the membrane are important regulators of downstream transcriptional responses in reactive glia.

Delayed glial clearance of degenerating axons in aged Drosophila is due to reduced PI3K/Draper activity

Advanced age is the greatest risk factor for neurodegenerative disorders, but the mechanisms that render the senescent brain vulnerable to disease are unclear. Glial immune responses provide neuroprotection in a variety of contexts. Thus, we explored how glial responses to neurodegeneration are altered with age. Here we show that glia–axon phagocytic interactions change dramatically in the aged Drosophila brain. Aged glia clear degenerating axons slowly due to low phosphoinositide-3-kinase (PI3K) signalling and, subsequently, reduced expression of the conserved phagocytic receptor Draper/MEGF10. Importantly, boosting PI3K/Draper activity in aged glia significantly reverses slow phagocytic responses. Moreover, several hours post axotomy, early hallmarks of Wallerian degeneration (WD) are delayed in aged flies. We propose that slow clearance of degenerating axons is mechanistically twofold, resulting from deferred initiation of axonal WD and reduced PI3K/Draper-dependent glial phagocytic function. Interventions that boost glial engulfment activity, however, can substantially reverse delayed clearance of damaged neuronal debris.

Whole Genome Sequencing and a New Bioinformatics Platform Allow for Rapid Gene Identification in D. melanogaster EMS Screens

Forward genetic screens in Drosophila melanogaster using ethyl methanesulfonate (EMS) mutagenesis are a powerful approach for identifying genes that modulate specific biological processes in an in vivo setting. The mapping of genes that contain randomly-induced point mutations has become more efficient in Drosophila thanks to the maturation and availability of many types of genetic tools. However, classic approaches to gene mapping are relatively slow and ultimately require extensive Sanger sequencing of candidate chromosomal loci. With the advent of new high-throughput sequencing techniques, it is increasingly efficient to directly re-sequence the whole genome of model organisms. This approach, in combination with traditional chromosomal mapping, has the potential to greatly simplify and accelerate mutation identification in mutants generated in EMS screens. Here we show that next-generation sequencing (NGS) is an accurate and efficient tool for high-throughput sequencing and mutation discovery in Drosophila melanogaster. As a test case, mutant strains of Drosophila that exhibited long-term survival of severed peripheral axons were identified in a forward EMS mutagenesis. All mutants were recessive and fell into a single lethal complementation group, which suggested that a single gene was responsible for the protective axon degenerative phenotype. Whole genome sequencing of these genomes identified the underlying gene ect4. To improve the process of genome wide mutation identification, we developed Genomes Management Application (GEM.app, https://genomics.med.miami.edu), a graphical online user interface to a custom query framework. Using a custom GEM.app query, we were able to identify that each mutant carried a unique non-sense mutation in the gene ect4 (dSarm), which was recently shown by Osterloh et al. to be essential for the activation of axonal degeneration. Our results demonstrate the current advantages and limitations of NGS in Drosophila and we introduce GEM.app as a simple yet powerful genomics analysis tool for the Drosophila community. At a current cost of <

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

1,000 per genome, NGS should thus become a standard gene discovery tool in EMS induced genetic forward screens.