InteractiveFly: GeneBrief

draper: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - draper

Synonyms -

Cytological map position- 62B1

Function - receptor

Keywords - developmental axon degeneration, Wallerian degeneration, phagocytosis, glia, CNS

Symbol - drpr

FlyBase ID: FBgn0027594

Genetic map position - 3L

Classification - EGF-repeat, transmembrane

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Purice, M. D., Speese, S. D. and Logan, M. A. (2016). Delayed glial clearance of degenerating axons in aged Drosophila is due to reduced PI3K/Draper activity. Nat Commun 7: 12871. PubMed ID: 27647497
Summary:
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, this study explored how glial responses to neurodegeneration are altered with age. Glia-axon phagocytic interactions were shown to 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. It is proposed 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.
Timmons, A.K., Mondragon, A.A., Meehan, T.L. and McCall, K. (2016). Control of non-apoptotic nurse cell death by engulfment genes in Drosophila. Fly (Austin) [Epub ahead of print]. PubMed ID: 27686122
Summary:
Programmed cell death occurs as a normal part of oocyte development in Drosophila. For each egg that is formed, fifteen 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. It has been 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. This study shows that 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, it was 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.

Purice, M. D., Speese, S. D. and Logan, M. A. (2016). Delayed glial clearance of degenerating axons in aged Drosophila is due to reduced PI3K/Draper activity. Nat Commun 7: 12871. PubMed ID: 27647497
Summary:
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, this study explored how glial responses to neurodegeneration are altered with age. Glia-axon phagocytic interactions were shown to 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. It is proposed 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.
Lu, T.Y., MacDonald, J.M., Neukomm, L.J., Sheehan, A.E., Bradshaw, R., Logan, M.A. and Freeman, M.R. (2017). Axon degeneration induces glial responses through Draper-TRAF4-JNK signalling. Nat Commun 8: 14355. PubMed ID: 28165006
Summary:
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. This study shows that Draper is cell autonomously required for glial activation of transcriptional reporters after axonal injury. The TNF receptor associated factor 4 (TRAF4) was identified 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. These data argue injury signals received by Draper at the membrane are important regulators of downstream transcriptional responses in reactive glia.

Winfree, L. M., Speese, S. D. and Logan, M. A. (2017). Protein phosphatase 4 coordinates glial membrane recruitment and phagocytic clearance of degenerating axons in Drosophila. Cell Death Dis 8(2): e2623. PubMed ID: 28230857
Summary:
Neuronal damage induced by injury, stroke, or neurodegenerative disease elicits swift immune responses from glial cells, including altered gene expression, directed migration to injury sites, and glial clearance of damaged neurons through phagocytic engulfment. Collectively, these responses hinder further cellular damage, but the mechanisms that underlie these important protective glial reactions are still unclear. This study shows that the evolutionarily conserved trimeric protein phosphatase 4 (PP4) serine/threonine phosphatase complex is a novel set of factors required for proper glial responses to nerve injury in the adult Drosophila brain. Glial-specific knockdown of PP4 results in reduced recruitment of glia to severed axons and delayed glial clearance of degenerating axonal debris. This study shows that PP4 functions downstream of the the glial engulfment receptor Draper to drive glial morphogenesis through the guanine nucleotide exchange factor SOS and the Rho GTPase Rac1, revealing that PP4 molecularly couples Draper to Rac1-mediated cytoskeletal remodeling to ensure glial infiltration of injury sites and timely removal of damaged neurons from the CNS.
Ray, A., Speese, S. D. and Logan, M. A. (2017). Glial Draper rescues Abeta toxicity in a Drosophila model of Alzheimer's Disease. J Neurosci 37(49):11881-11893. PubMed ID: 29109235
Summary:
Pathological hallmarks of Alzheimer's disease (AD) include amyloid-beta (Abeta) plaques, neurofibrillary tangles, and reactive gliosis. Glial cells offer protection against AD by engulfing extracellular Abeta peptides, but the repertoire of molecules required for glial recognition and destruction of Abeta are still unclear. This study shows that the highly conserved glial engulfment receptor Draper/MEGF10 provides neuroprotection in an AD model of Drosophila (both sexes). Neuronal expression of human Abeta42arc in adult flies results in robust Abeta accumulation, neurodegeneration, locomotor dysfunction, and reduced lifespan. Notably, all of these phenotypes are more severe in draper mutant animals, while enhanced expression of glial Draper reverses Abeta accumulation, as well as behavioral phenotypes. 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 Abeta42arc exposure. Furthermore, Abeta42-induced upregulation of the phagolysosomal markers Atg8 and p62 was notably reduced in draper mutant flies. Based on these findings, it is proposed that glia clear neurotoxic Abeta 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.
Sigle, L. T. and Hillyer, J. F. (2018). Eater and draper are involved in the periostial haemocyte immune response in the mosquito Anopheles gambiae. Insect Mol Biol. PubMed ID: 29520896
Summary:
Haemocytes respond to infection by phagocytosing pathogens, producing the enzymes that drive the phenoloxidase-based melanization cascade, secreting lytic factors, and producing other humoral proteins. A subset of haemocytes, called periostial haemocytes, aggregate on the surface of the heart of mosquitoes and kill pathogens in areas of high haemolymph flow. Periostial haemocytes are always present, but an infection induces the recruitment of additional haemocytes to these regions. This study tested whether members of the Nimrod gene family are involved in the periostial immune response of the African malaria mosquito, Anopheles gambiae. Using organismal manipulations, RNA interference (RNAi) and microscopy, it was shown that, following an infection with Escherichia coli, nimrod - the orthologue of Drosophila NimB2 - is not involved in periostial responses. At 4 h postinfection, however, RNAi-based knockdown of draper results in a marginal increase in the number of periostial haemocytes and a doubling of E. coli accumulation at the periostial regions. Finally, at 24 h postinfection, knockdown of eater decreases the number of periostial haemocytes and decreases the phagocytosis of E. coli on the surface of the heart. Phagocytosis of bacteria is more prevalent in the periostial regions of the mid abdominal segments, and knockdown of draper, nimrod or eater does not alter this distribution. Finally, knockdown of Nimrod family genes did not have a meaningful effect on the accumulation of melanin at the periostial regions. This study identifies roles for eater and draper in the functional integration of the mosquito immune and circulatory systems.
Hilu-Dadia, R., Hakim-Mishnaevski, K., Levy-Adam, F. and Kurant, E. (2018). Draper-mediated JNK signaling is required for glial phagocytosis of apoptotic neurons during Drosophila metamorphosis. Glia [Epub ahead of print]. PubMed ID: 29520845
Summary:
Development of the central nervous system involves elimination of superfluous neurons through apoptosis and subsequent phagocytosis. In Drosophila, this occurs mainly during three developmental stages: embryogenesis, metamorphosis and emerging adult. Two transmembrane glial phagocytic receptors, SIMU (homolog of the mammalian Stabilin-2) and Draper (homolog of the mammalian MEGF10 and Jedi), mediate glial phagocytosis of apoptotic neurons during embryogenesis. However, less is known about the removal of apoptotic neurons during later stages of development. This study shows that during metamorphosis, Draper plays a critical role in apoptotic cell clearance by glia, whereas SIMU, which is mostly expressed in pupal macrophages outside the brain, is not involved in glial phagocytosis. Draper was found to activate Drosophila c-Jun N-terminal kinase (dJNK) signaling predominantly in the ensheathing glia and astrocytes, where it is required for efficient removal of apoptotic neurons. These data suggest that besides the dJNK pathway, Draper also triggers an additional signaling pathway capable of removing apoptotic neurons in the pupal brain. This study thus reveals that SIMU unexpectedly is not involved in glial phagocytosis of apoptotic neurons during metamorphosis and highlights the novel role of dJNK signaling in developmental apoptotic cell clearance downstream of Draper.
BIOLOGICAL OVERVIEW

Axon pruning is a common phenomenon in neural circuit development. Previous studies demonstrate that the engulfing action of glial cells is essential in this process. The underlying molecular mechanisms, however, remain unknown. draper (drpr), encoding an EGF-repeat single-pass transmembrane domain receptor, and ced-6, a phosphotyrosine-binding (PTB) domain protein each of which are essential for the clearance of apoptotic cells in C. elegans, function in the glial engulfment of larval axons during Drosophila metamorphosis. The drpr mutation and glia-specific knockdown of drpr and ced-6 by RNA interference suppress glial engulfment, resulting in the inhibition of axon pruning. drpr and ced-6 interact genetically in the glial action. Disruption of the microtubule cytoskeleton in the axons to be pruned occurs via ecdysone signaling, independent of glial engulfment. These findings suggest that glial cells engulf degenerating axons through drpr and ced-6. It is proposed that apoptotic cells and degenerating axons of living neurons are removed by a similar molecular mechanism (Awasaki, 2006).

Local modification and refinement of neuronal connections are essential for the development of neural circuits. Neurons often form excess axon branches, dendritic arbors, and synapses during the early phase of development. To refine the functional neural circuit, the unnecessary neural processes and synapses are selectively removed in a later phase without loss of the other parts of the axons, dendrites, and parental neurons. Selective elimination of neural processes and synapses is also required for the plasticity of synaptic connectivity. Among these reorganization processes, the elimination of axons, known as axon pruning, has been studied extensively (Awasaki, 2006 and references therein).

The pruning of Drosophila mushroom body (MB) γ neurons during metamorphosis is an important model system for investigating the mechanisms underlying axon pruning mediated by local degeneration. In larvae, the γ neurons have bifurcated axon branches. These axon branches are pruned by local degeneration during early metamorphosis, and most of them disappear within 18h after puparium formation (APF). The γ neurons then re-extend their axons to form the adult neural circuits. The pruning of the γ neurons is triggered by ecdysone stimulation and requires cell-autonomous action of the ubiquitin-proteasome system. In contrast, it has been demonstrated that the engulfing action of glial cells is essential for the proper pruning of these γ neurons (Awasaki, 2004). The larval axon branches are engulfed by glial processes that infiltrate from the outer surface of the MB lobes (Awasaki, 2004; Watts, 2004). Glial cells also have important roles in the axon pruning of motor neurons in the neuromuscular junction in mice, where distal axon tips called axosomes, which contain a high density of synaptic organelles, are engulfed by Schwann cells (Bishop, 2004). Thus, glial cells are deeply involved in the pruning of both insect and vertebrate neurons. The molecular mechanisms regulating this process, however, remain essentially unknown (Awasaki, 2006).

Once cells are subjected to apoptosis, phagocytic cells engulf and clear the apoptotic cells quickly and efficiently. Phagocytes recognize the cells to be engulfed or not by sensing various cues, which are called 'findme,' 'don’t-eat-me,' and 'eat-me' signals. In C. elegans, engulfing cells recognize and phagocytose apoptotic cells via two partially redundant genetic pathways. The first pathway involves the genes encoding CED-2, CED-5, CED-10, and CED-12, which are orthologs of mammalian CrKII, Dock180, Rac1, and ELMO, respectively. These genes regulate the cytoskeletal rearrangement of the engulfing cells, which is required for phagocytosis. The second pathway involves the genes encoding CED-1, CED-6, and CED-7, which are a scavenger receptor-like molecule (CD91/ LRP/SREC), an adaptor protein (hCED6/GLUP), and an ATP binding transporter (ABCA1), respectively (Liu, 1998; Wu, 1998a; Wu, 1998b; Zhou, 2001). It is likely that these proteins participate in the recognition of apoptotic cells (Awasaki, 2006).

In the pruning of Drosophila γ neurons, glial infiltration and engulfment are induced extrinsically by the MB γ neurons (Awasaki, 2004). Furthermore, the engulfing glial cells express Draper (Drpr), which is the Drosophila ortholog of the C. elegans CED-1. The expression of drpr in glial cells is essential for the clearance of apoptotic neurons in the Drosophila embryonic central nervous system (Freeman, 2003). These findings led to a hypothesis that engulfment of degenerating axon branches by glial cells share common molecular mechanisms with the clearance of apoptotic cells by phagocytes. This study demonstrates that drpr and the Drosophila ortholog of nematode ced-6 are essential for the glial engulfment of larval axons during pruning of MB neurons. In addition, evidence is provided that disruption of the microtubule cytoskeleton in the larval axons of MB γ neurons is induced by ecdysone stimulation independent from glial engulfment. These findings suggest that glial cells recognize and engulf degenerating axon branches of living neurons through the function of Drpr and Ced-6 in developmentally programmed axon pruning (Awasaki, 2006).

Expression of drpr and ced-6, which are essential for the clearance of apoptotic cells in C. elegans, are functionally required for the glial engulfment of larval axons during Drosophila metamorphosis. It is highly likely that these proteins interact with each other in the engulfing action of glial cells. The disruption of the microtubule cytoskeleton in the larval axons is induced by ecdysone independently from glial engulfment. These results suggest that developmentally programmed axon pruning is achieved by the phagocyte-like action of glial cells that recognize and engulf degenerating axon branches of living neurons through Drpr and Ced-6 (Awasaki, 2006).

The present study demonstrated that elevated expression of drpr and ced-6 is induced in a specific subset of glial cells that surround the MB lobe. The essential function of drpr in the glial engulfing action was demonstrated using drpr mutants and a glia-specific RNAi system. In contrast, inhibition of the ecdysone receptor in the MB γ neurons suppresses glial engulfment, even though drpr is expressed normally in the glial cells (Awasaki, 2004). The drpr gene encodes a scavenger receptor-like molecule (Freeman, 2003). These findings strongly suggest that the glial engulfment is regulated by extracellular signals from the MB axons that are recognized by glial cells through the drpr receptor (Awasaki, 2006).

It was also demonstrated that ced-6 is essential for glial engulfment. The ced-6 gene encodes an adaptor molecule that potentially interacts with the intracellular domain of drpr (Smits, 1999; Freeman, 2003). Thus, Ced-6 might function in glial engulfment by mediating signals from the drpr receptor to the cytoplasm (Awasaki, 2006).

The engulfing action of glia in MB axon pruning consists of at least two different phases: infiltration of glial processes into the MB lobe, and engulfment of larval axons of γ neurons (Awasaki, 2004). Engulfment of the larval axons by infiltrating glial processes is demonstrated by electron microscopy analysis (Watts, 2004). In which phase does drpr function? The results indicated that glial infiltration is severely suppressed in the drpr mutant and drpr RNAi pupae. Although the glial processes in these pupae still surrounded the MB lobe and contacted the larval γ neuron axons in the periphery, only a very few glial lumps, most of which were small, formed. This suggests that glial infiltration as well as lump formation were suppressed by the loss of drpr function. Thus, drpr is essential for recognizing signals for both infiltration of glial processes and engulfment of the larval axons (Awasaki, 2006).

In C. elegans, genetic analysis indicated partial redundancy in the involvement of two groups of genes in the engulfment and clearance of apoptotic cells. The first group (ced-2, ced-5, ced-10, and ced-12) functions in reorganizing the actin cytoskeleton, whereas the second group (ced-1, ced-6, and ced-7) functions in recognizing apoptotic cells (Liu, 1998; Wu, 1998a; Wu, 1998b; Zhou, 2001). A recent study reported that CED-1 functions cooperatively with CED-6 early in the engulfment process, either before or during actin cytoskeleton rearrangement (Kinchen, 2005). In addition, a biochemical interaction between CED-1 and CED-6 has been demonstrated using a yeast two-hybrid assay (Su, 2002). The present study demonstrated a genetic interaction between drpr and ced-6 in the glial engulfment. Furthermore, none of the MB γ neurons underwent apoptosis during metamorphosis. Therefore, Drpr/CED-1 and Ced-6/CED-6 are involved in the recognition of not only apoptotic cells, but also degenerating axons of the living neurons (Awasaki, 2006).

Of note, the effect of the glial knockdown of ced-6 on the engulfing action of glial cells was weaker than that of drpr knockdown. There are two possible explanations for this result. First, the suppression of ced-6 using RNAi might have been incomplete. A very weak expression of Ced-6, which could not be detected with anti- Ced-6 antibody, might affect the glial engulfing action. Second, Drpr might activate not only Ced-6, but also other molecules that function partially redundantly. The cytoplasmic tails of Drosophila Drpr and C. elegans CED-1 contain two conserved putative tyrosine phosphorylation sites, the NPXY and YXXL motifs, which potentially interact with proteins containing PTB and SH2 domains, respectively (Zhou, 2001; Freeman, 2003). Functional analyses of NPXY and YXXL motifs demonstrated that they are partially redundant in the function of CED-1 (Zhou, 2001). These raise the possibility that the YXXL motif-associated and Ced-6-independent intracellular signaling pathway might be simultaneously activated by Drpr (Awasaki, 2006).

In apoptotic cells, caspase action is essential for both the induction of nuclear DNA degradation and the induction of engulfment by phagocytes. Apoptotic cells secrete a chemotactic signal that attracts phagocytes in a caspase-3-dependent manner. Overexpression of caspase inhibitors in MB neurons, however, has no effect on the pruning of their axons (Watts, 2003) or on the engulfing action of glia. Thus, caspases are not likely to be involved in the engulfing process of the MB neuron axons. Whereas phagocytes and glial cells commonly use Drpr/CED-1 and Ced-6/CED-6 to recognize their engulfing targets, the intracellular mechanisms in the target to be engulfed are likely to be different between apoptotic cells and axons to be pruned (Awasaki, 2006).

Once cells undergo apoptosis, phagocytes engulf and clear them quickly and efficiently. When apoptotic cells fail to be cleared, they undergo postapoptotic necrosis, which causes harmful inflammatory responses by releasing intracellular contents. Inhibition of the clearance or engulfment of apoptotic cells affects development directly or indirectly and causes ectopic survival of cells that are programmed to die during development. Thus, apoptotic cells must be removed in a timely manner to ensure the proper development of organisms (Awasaki, 2006).

Similarly, prompt and efficient removal of dysfunctional or degenerating axons might be essential for avoiding their harmful influence on the proper development of the neural circuit. Indeed, inhibition of glial engulfment of larval axon branches caused defects in the development of the adult MB: larval axon branches of g neurons survived abnormally and the medial β lobes of both hemispheres fused in the drprD5 mutant adults. The abnormally remaining larval axon branches might disturb the normal development of newly extending, adult-specific axons (Awasaki, 2006).

When axons are transected, the distal parts of these axons are degenerated or fragmented, which is known as 'Wallerian-type degeneration.' Glial cells participate in the removal of transected axons in such cases. The mechanisms underlying the recognition and engulfment of transected axons by phagocytes, however, are unclear. The axon pruning of the Drosophila MB γ neurons shares similarities with Wallerian-type degeneration (Luo, 2005). In both cases, microtubule breakdown is induced in the early stage of axon degeneration, and the ubiquitin-proteasome system is involved in these processes. In addition, axon pruning and Wallerian- type degeneration are not associated with apoptosis and activation of caspases. Thus, Drpr/CED-1 and Ced-6/CED-6 might also be involved in the glial engulfment of axons that cause Wallerian-type degeneration. In fact, drpr is involved (MacDonald, 2006) in the clearance of severed Drosophila axons (Awasaki, 2006).

Disruption of the microtubule cytoskeleton occurs in the early phase of pruning of the larval MB γ axons (Watts, 2003). Ectopic expression of yeast ubiquitin protease UBP2 in γ neurons suppresses the disruption of the microtubule cytoskeleton and the engulfment of degenerating axons by extrinsic cells (Watts et al., 2004). In the dendrite pruning of Drosophila sensory neurons, phagocytes attack dendrites in which microtubule destabilization is induced. These studies show a correlation between the disruption of the microtubule cytoskeleton and phagocyte engulfment. The present study provides evidence that disruption of the microtubule cytoskeleton in the larval axons of γ neurons is induced by ecdysone, even when glial engulfment is disturbed. It has been demonstrated that ecdysone receptor inhibition in these neurons suppresses glial engulfment extrinsically. Taken together, the disruption of the microtubule cytoskeleton in the target neurons might be involved in the induction of the engulfing action of glial cells. Apoptotic cells induce phagocytes to engulf them with various signals, including find-me and eat-me signals. The best-characterized eat-me signal is the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane. A potential candidate find-me signal is the lipid lysophosphatidylcholine, which is released from apoptotic cells. Although it remains unknown whether similar signals are involved in the induction of glial engulfment of degenerating axons, disruption of the microtubule cytoskeleton might be necessary for the secretion or presentation of a putative ligand or ligand complex for Drpr/CED-1 (Awasaki, 2006 and references therein).

The present study demonstrated that the pruning of unnecessary axon branches and the clearance of apoptotic cells share common molecular mechanisms. This provides a systematic perspective for understanding the reorganizing process of neural circuits. Further comparative analyses of pruning and apoptosis, including the identification of the ligand or ligand complex of the Drpr/CED-1 receptor, and analyses of the intracellular molecular mechanisms downstream of the receptor will provide important clues to elucidate how precise and efficient removal of unwanted cells and neural processes is achieved in developing organisms (Awasaki, 2006).

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. This study investigated the mechanism of corpse clearance in the Drosophila 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. This study shows that the JNK pathway is activated and required in engulfing follicle cells. 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 (Etchegaray, 2012).

How non-professional phagocytes respond to dying cells and modulate their phagocytic capabilities is unclear. This study has used the Drosophila ovary as a model to study engulfment by non-professional phagocytes. In this system, the germline can be induced to undergo PCD upon starvation (Etchegaray, 2012).

Following the initiation of PCD, a layer of epithelial FCs synchronously engulfs the dying germline. The engulfment genes drpr, shark and Rac1 are required for engulfment by FCs. The JNK pathway is specifically activated during engulfment and is required for proper engulfment by FCs. This analysis suggests that drpr and JNK are involved in a circuit, where the dying germline activates Drpr, which activates JNK, and JNK signaling leads to an increase in Drpr and likely other engulfment genes. Surprisingly, activation of JNK is sufficient to rescue drpr engulfment defects, indicating that other pathways can carry out engulfment in the absence of drpr. A likely candidate pathway is CED-2, CED-5, CED-12, which can promote engulfment in the absence of ced-1 in C. elegans (Etchegaray, 2012).

In other systems, such as C. elegans and mammalian macrophages, there is redundancy among engulfment pathways. In Drosophila embryos lacking drpr, unprocessed apoptotic particles are detected within glia, suggesting that other pathways can facilitate engulfment of corpses. However, in FCs, drpr is essential for corpse removal. This may be because FCs die if they are engulfment-defective, and there may not be time to activate redundant pathways prior to FC death. It is important to note that drpr (and JNK pathway) mutant FCs survive in healthy egg chambers under starvation conditions; it is only during terminal phases of egg chamber degeneration that they die. Why do the FCs die if they are engulfment defective? Perhaps they have a metabolic requirement, and starve if they cannot obtain nutrients from the germline. Another possibility is that wild-type FCs are programmed to die after completing engulfment, and this PCD may be activated prematurely if engulfment is defective. Mammalian macrophages eliminate themselves after engulfment of specific pathogens or following efferocytosis in ABC transporter mutants. Alternatively, FCs may die because of death 'by confusion', where disruption of the proper signaling network culminates in PCD. Attempts were made to block FC death by expression of caspase inhibitors p35 and Diap1, but FC death was still observed in control, drpr5- and bskDN-expressing egg chambers, indicating that FCs die via a caspase-independent pathway (Etchegaray, 2012).

In mammals, JNK is activated in engulfing professional and non-professional phagocytes, although it remains to be determined whether JNK is required for engulfment. Recently in Drosophila, JNK has been found to be required for the removal of imaginal disc cells succumbing to cell competition, and for the removal of severed axons. These findings suggest that JNK may play a conserved role in engulfment. A role for JNK in engulfment has not been explored in C. elegans and no transcription factor has been shown to activate engulfment genes. This is surprising as levels of CED-1 increase in engulfing cells (Etchegaray, 2012).

How does JNK become activated during engulfment? It may occur via Shark, a kinase that has been shown to interact with both Drpr and JNK in Drosophila. Another candidate is Rac1, which can act upstream of JNK and may act downstream of Drpr. Interestingly, JNK activity is sufficient to restore engulfment in drpr-null egg chambers, suggesting that the primary role of Drpr is to activate JNK. Thus, functions attributed to Drpr such as actin reorganization, Ca2+ signaling, the formation of junctional complexes and autophagy, may depend on JNK activity. Indeed, JNK has been shown to induce autophagy genes in Drosophila (Etchegaray, 2012).

Remarkably, drpr or hepCA overexpression in FCs promoted death of egg chambers even when flies were not starved. This is the first time that overexpression of an engulfment gene has been shown to induce non-autonomous cell death. In other systems, engulfment can promote the death of cells that are weakened, perhaps on the brink of death. For example, mutations in engulfment genes can lead to the survival of cells fated to die in C. elegans ced-3 hypomorphs, and to the survival of 'loser' cells in Drosophila imaginal discs. In mammals, neuronal exposure to amyloid Aβ peptide or LPS leads to cell death, which can be inhibited by blocking phagocytosis. Interestingly, treated neurons transiently expose phosphatidylserine, perhaps to announce their vulnerability. These findings differ from scenarios in that the egg chambers are healthy. However, mid-stage egg chambers are more susceptible to death stimuli than egg chambers at other stages of oogenesis. Overexpression of Drpr in early oogenesis did not lead to egg chamber death, but death was observed later in mid-oogenesis. Thus, it may be that Drpr is not sufficient to kill the germline until mid-oogenesis, when it is more vulnerable. The factors that contribute to this vulnerability are unknown (Etchegaray, 2012).

Overexpression of drpr in FCs led to death of the underlying NCs before there was any engulfment by the FCs, suggesting that drpr produces a death signal that is sent to the germline. Overexpression of the JNKK hepCA led to destruction of egg chambers earlier in oogenesis than overexpression of drpr, suggesting that JNK did not require the vulnerability at midoogenesis. Furthermore, hepCA-expressing FCs engulfed intact NCs ('hyper-engulfment'), rather than inducing death first. This phenotype resembles the process of entosis, where living cells are engulfed by their neighbors (Etchegaray, 2012).

Germline PCD in mid-oogenesis requires caspases, and the current results indicate that caspase activity is required to stimulate FCs to engulf the germline. Surprisingly, germline caspase activity was not necessary or sufficient to activate JNK or induce Drpr in the FCs. This suggests that a caspase-dependent pathway, distinct from the pathway(s) that activate Drpr-JNK, is required for engulfment in mid-oogenesis. The caspase-dependent signal and the responding pathway in the FCs remain to be elucidated. Another open issue is how Drpr, and thereby JNK, becomes activated in response to the dying germline. The complexity of cell surface modifications that occur during apoptosis will make this a challenge to determine. Draper and JNK may become activated directly in the FCs in response to starvation; however, this scenario seems less likely than activation by the dying germline for two reasons. First, many egg chambers do not die immediately upon starvation, and activation of Draper and JNK was observed only in egg chambers that had begun to die. Second, germline death triggered by overexpression of dcp-1 could lead to Draper and JNK activation in FCs in the absence of starvation. The activation of JNK and Drpr illustrate ways in which non-professional phagocytes change in response to apoptotic cells. Future work will reveal the network of pathways activated in non-professional phagocytes to enhance apoptotic cell clearance (Etchegaray, 2012).


REGULATION

Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis

Phagocytosis is important during development and in the immune response for the removal of apoptotic cells and pathogens, yet its molecular mechanisms are poorly understood. In Caenorhabditis elegans, the CED2/5/10/12 pathway regulates actin during phagocytosis of apoptotic cells, whereas the role of the CED1/6/7 pathway in phagocytosis is unclear. This study reports that Undertaker (UTA), a Drosophila Junctophilin protein, is required for Draper (CED-1 homolog)-mediated phagocytosis. Junctophilins couple Ca2+ channels at the plasma membrane to those of the endoplasmic reticulum (ER), the Ryanodine receptors. This study places Draper, its adaptor drCed-6, UTA, the Ryanodine receptor Rya-r44F, the ER Ca2+ sensor dSTIM, and the Ca2+-release-activated Ca2+ channel dOrai in the same pathway that promotes calcium homeostasis and phagocytosis. Thus, these results implicate a Junctophilin in phagocytosis and link Draper-mediated phagocytosis to Ca2+ homeostasis, highlighting a previously uncharacterized role for the CED1/6/7 pathway (Cuttell, 2008).

In a deficiency screen, a mutant was characterized in which embryonic macrophages poorly engulfed apoptotic cells. In an RNAi screen using S2 cells, undertaker/retinophilin (uta) was identified as being responsible for this phenotype. uta encodes a membrane occupational and recognition nexus (MORN) repeat-containing protein with homology to mammalian Junctophilins (JPs). JPs form junctional complexes between the plasma membrane (PM) and the endoplasmic/sarcoplasmic reticulum (ER/SR) Ca2+ storage compartment (Takeshima 2000). These complexes allow for crosstalk between Ca2+ channels at the PM and the ER/SR Ca2+ channels, or Ryanodine receptors (RyRs), thus regulating Ca2+ homeostasis and functions of excitable cells. Although a role for Ca2+ in phagocytosis of various particles by mammalian phagocytes has been previously described, the molecular mechanisms underlying Ca2+ fluxes associated with these events are not known (Cuttell, 2008).

This study reports that, as for UTA, the Drosophila Ryanodine receptor, Rya-r44F, plays a role in phagocytosis of apoptotic cells in vivo. A requirement in phagocytosis was found for store-operated Ca2+ entry (SOCE) via dSTIM, a Ca2+ sensor of the ER/SR lumen, and CRACM1/dOrai, a Ca2+-release-activated Ca2+ channel (CRAC). uta and rya-r44F genetically interact with drced-6 and drpr, and uta, drced6, and drpr are required for SOCE in S2 cells. Thus, these genes act in the same pathway that plays a general role in phagocytosis; uta, dstim, dorai, drced-6, and drpr are also required for efficient phagocytosis of bacteria. These results provide a link between SOCE and phagocytosis, imply that UTA plays a similar role in macrophages to that of JPs in excitable cells, and shed light on a role for the CED1/6/7 pathway in Ca2+ homeostasis during phagocytosis (Cuttell, 2008).

Binding of various particles induces a rise in [Ca2+]i in mammalian phagocytes. In dendritic cells, [Ca2+]i increases upon apoptotic cell binding via integrin, and inhibition studies have suggested that both Ca2+ release from the ER/SR storage pool and extracellular Ca2+ entry into the cytosol are required for this process. Neutrophils also rely on such changes to promote particle engulfment. Yet, the molecular mechanisms underlying this rise in [Ca2+]i and what role it plays in phagocytes are poorly understood (Cuttell, 2008).

This study found that uta, a Drosophila gene encoding a JP-related protein, is required for phagocytosis of apoptotic cells. Genetic evidence of a role for a Ryanodine receptor, Rya-r44F is provided, and uta and rya-44F were genetically linked. It was also found that SOCE via dstim and dorai promotes efficient apoptotic cell clearance. uta and rya-44F were genetically linked to drpr and drced-6, and a role was found for uta, drpr, and drced-6 in SOCE, thus demonstrating a functional link between the DRPR/drCed-6 pathway and SOCE during phagocytosis (Cuttell, 2008).

A model is proposed whereby apoptotic cell binding via DRPR (and possibly other receptors, such as CRQ) leads to ER Ca2+ release via Rya-r44F. DRPR, which bears an immunoreceptor tyrosine-based activation motif (ITAM) that is phosphorylated via Src and Syk family kinase-mediated signaling, appears to behave like an immunoreceptor. In B and T lymphocytes, engagement of Fc immunoreceptors (the signaling of which also relies on phosphorylation on ITAMs) leads to a rise in [Ca2+]i. Thus, DRPR might play a similar role to that of Fc receptors in the signaling, leading to a rise in [Ca2+]i in macrophages (Cuttell, 2008).

UTA is localized in the ER and at the PM. Thus, it is proposed that, like JPs, UTA forms junctional complexes that link the PM events to the ER and trigger Ca2+ release from ER stores. These studies, however, did not address whether the formation of UTA junctional complexes is required to trigger ER Ca2+ release via Rya-r44F, nor what triggers ER Ca2+ release. The resting membrane potential of mammalian phagocytes is depolarized upon contact with apoptotic cells. As in mammalian muscle cells, such changes in fly phagocytes might initiate ER Ca2+ release (Cuttell, 2008).

In S2 cells, Ca2+ imaging results with drpr and drced-6 RNAi and the results of drced-6 RNAi suggest that drpr and drced-6 are required for dOrai-mediated Ca2+ entry upon TG treatment (which bypasses the need for particle binding to the receptor). It is proposed that ER Ca2+ release feeds back onto DRPR and drCed-6 to activate their downstream signaling cascade. Although further studies will be required to test the validity of this proposal, several reports already support it: DRPR-mediated phagocytosis depends on Src and Syk family kinase signaling, and the activity of such kinases can be Ca2+ dependent in mammalian cells (Cuttell, 2008).

It is then proposed that signaling downstream of DRPR and drCed-6 promotes and/or maintains the formation of UTA junctional complexes, thereby linking ER Ca2+ release to SOCE. dSTIM is indeed likely to act as an ER Ca2+ sensor that oligomerizes and redistributes to ER-PM junctions upon ER Ca2+ depletion, as for its mammalian counterparts. It is proposed that UTA junctional complexes are needed to maintain a close proximity between the ER Ca2+ stores and the PM and to juxtapose dSTIM oligomers and dOrai, thereby promoting conformational changes and opening of dOrai. DRPR- and drCed-6-dependent signaling and/or UTA may also be required for dSTIM oligomerization. The resulting increase in [Ca2+]i then promotes engulfment of the particle (Cuttell, 2008).

Ca2+ may promote phagocytosis via several ways. It can enhance scavenger receptor (SR) activity: adhesion of mouse macrophages to a fibronectin-coated surface via integrin binding results in an increase in the number of SRs at their PM, which enhances their binding activity. This enrichment in SRs is dependent on extracellular Ca2+ influx, arguing in favor of a role for Ca2+ in SR trafficking and/or recycling. Several SRs or related receptors play a role in phagocytosis of apoptotic corpses, including the mammalian CD36 and its Drosophila homolog CRQ. Although no change in CRQ expression is seen in uta mutant macrophages, CRQ and UTA colocalize and genetically interact. One possible model is that CRQ is recruited to the phagocytic cup upon apoptotic cell binding after SOCE that depends on UTA, DRPR, and drCed-6, and that this might strengthen the binding and uptake of the corpse (Cuttell, 2008).

In C. elegans, CED-1 (DRPR homolog) is related to the endothelial scavenger receptor SREC. Its recruitment to the phagocytic cup depends on functional CED-7, and may occur by exocytosis (Yu, 2006). Components of the exocyst were implicated in phagocytosis (Stuart, 2007). Moreover, Orai1 is required for degranulation of mast cells, which occurs by exocytosis (Vig, 2008). Thus, like Orai1, dOrai may be required for exocytosis and, whereas DRPR appears to always be present at the PM, CRQ may be recruited from its intracellular vesicular pool to the phagocytic cup by exocytosis, as previously proposed for CED-1, to promote apoptotic cell uptake (Cuttell, 2008).

A rise in Ca2+ was observed in mammalian neutrophils upon particle binding, and Ca2+ participates in phagocytosis by promoting F-actin breakdown and phagosome maturation. Mycobacterium tuberculosis is able to invade human macrophages without triggering an increase in [Ca2+]i: in the absence of Ca2+ signaling, phagosomes containing M. tuberculosis fail to mature, perhaps explaining the survival of this bacterium in the cell. A role for Ca2+ in particle binding and phagosome maturation in macrophages, however, was once discounted. uta, dstim, dorai, drCed-6, and drpr are required to trigger SOCE. Yet, although they are poorly phagocytic, macrophages in drced-6 hypomorphs and drpr null mutants engulf bacteria into fully matured phagosomes, arguing against Ca2+ being involved in phagosome maturation. This maturation, however, might still occur with lower efficiency when SOCE fails, as RNAi-treated S2 cells for all genes in this pathway poorly engulfed bacteria (Cuttell, 2008).

The findings that UTA links DRPR-mediated phagocytosis and Ca2+ homeostasis provide the opportunity to pursue the dissection of the DRPR pathway in Drosophila. DRPR is homologous to CED-1, which belongs to the CED1/6/7 pathway where CED-7 is an ABC transporter. Interestingly, an ABC transporter can modulate Ca2+ channel activity in plants, further supporting a link between the CED1/6/7-like pathways and Ca2+ homeostasis, which appears to have been conserved throughout evolution. Furthermore, a mutation in human Orai1 was found in some patients with severe combined immune deficiency. Thus, pursuing such studies might be relevant to mammalian systems and to human health (Cuttell, 2008).

A modifier screen in Drosophila melanogaster implicates cytoskeletal regulators, Jun N-terminal kinase, and Yorkie in Draper signaling

The Drosophila melanogaster homolog of the ced-1 gene from Caenorhabditis elegans is draper, which encodes a cell surface receptor required for the recognition and engulfment of apoptotic cells, glial clearance of axon fragments and dendritic pruning, and salivary gland autophagy. To further elucidate mechanisms of Draper signaling, a genetic screen of chromosomal deficiencies was performed to identify loci that dominantly modify the phenotype of over-expression of Draper isoform II, which suppresses differentiation of the posterior crossvein in the wing. The existence of 43 genetic modifiers of Draper II was deduced. 24 of the 37 suppressor loci and 3 of the 6 enhancer loci have been identified. A further 5 suppressors and 2 enhancers were identified from mutations in functionally related genes. These studies indicated positive contributions to Drpr signaling for the Jun N-terminal Kinase pathway, supported by genetic interactions with hemipterous, basket, jun, and puckered, and for cytoskeleton regulation as indicated by genetic interactions with rac1, rac2, RhoA, myoblast city, Wiskcott-Aldrich syndrome protein, and the formin CG32138, and for yorkie and expanded. These findings indicate that Jun N-terminal Kinase activation and cytoskeletal remodeling collaborate in the engulfment process downstream of Draper activation. The relationships between Draper signaling and Decapentaplegic signaling, insulin signaling, Salvador-Warts-Hippo signaling, apical-basal cell polarity, and cellular responses to mechanical forces are further investigated and discussed (Fullard, 2014).

PI3K signaling and Stat92E converge to modulate glial responsiveness to axonal injury

Glial cells are exquisitely sensitive to neuronal injury but mechanisms by which glia establish competence to respond to injury, continuously gauge neuronal health, and rapidly activate reactive responses remain poorly defined. This study shows glial PI3K signaling in the uninjured brain regulates baseline levels of Draper, a receptor essential for Drosophila glia to sense and respond to axonal injury. After injury, Draper levels are up-regulated through a Stat92E-modulated, injury-responsive enhancer element within the draper gene. Surprisingly, canonical JAK/STAT signaling does not regulate draper expression. Rather, injury-induced draper activation is downstream of the Draper/Src42a/Shark/Rac1 engulfment signaling pathway. Thus, PI3K signaling and Stat92E are critical in vivo regulators of glial responsiveness to axonal injury. Evidence is provided for a positive auto-regulatory mechanism whereby signaling through the injury-responsive Draper receptor leads to Stat92E-dependent, transcriptional activation of the draper gene. It is proposed that Drosophila glia use this auto-regulatory loop as a mechanism to adjust their reactive state following injury (Doherty, 2014: PubMed).

Corpse engulfment generates a molecular memory that primes the macrophage inflammatory response

Macrophages are multifunctional cells that perform diverse roles in health and disease. Emerging evidence has suggested that these innate immune cells might also be capable of developing immunological memory, a trait previously associated with the adaptive system alone. While recent studies have focused on the dramatic macrophage reprogramming that follows infection and protects against secondary microbial attack, can macrophages also develop memory in response to other cues? This study shows that apoptotic corpse engulfment by Drosophila macrophages is an essential primer for their inflammatory response to tissue damage and infection in vivo. Priming is triggered via calcium-induced JNK signaling, which leads to upregulation of the damage receptor Draper, thus providing a molecular memory that allows the cell to rapidly respond to subsequent injury or infection. This remarkable plasticity and capacity for memory places macrophages as key therapeutic targets for treatment of inflammatory disorders (Weaverss, 2016).

Transcriptional Regulation

How are the expression of drpr and ced-6 regulated in the specific subset of glial cells? Because the elevated expression of these genes occurred shortly after the onset of the pupal metamorphosis, a most likely candidate mechanism would be ecdysone signal-mediated regulation. Inhibition of the ecdysone receptor in γ neurons by ectopic expression of the dominant negative form of the ecdysone receptor (EcR-DN), however, does not affect the expression of drpr in glial cells (Awasaki, 2004). In situ hybridization confirmed the elevated expression of drpr and ced-6 at 6h APF, as in the control animal. These observations exclude the possibility that the expression of drpr and ced-6 in glia is induced extrinsically by γ neurons (Awasaki, 2006).

Therefore the cell-autonomous role of ecdysone signals in glia were examined using GAL4-repo-driven ectopic expression of EcR-DN in glial cells. Although development of the repo>EcR-DN pupae was arrested in the late prepupal stage, the pupae developed normally until at least 6h APF. In these pupae, neither intense expression of drpr transcripts nor the Drpr protein was detected in the glial cells at 6h APF. In addition, no glial processes infiltrated the MB lobe. Quantification of the expression level indicated that although the amount of drpr transcripts increased by approximately 30% in 6h APF pupae of wild-type and 201Y>EcR-DN compared with that of L3 wild-type larvae, there was no increase of drpr transcripts in the pupae of repo>EcR-DN (Awasaki, 2006).

Interestingly, Ced-6 expression was not affected by EcR-DN expression: Ced-6 was expressed in the same number of glial cells as those that coexpressed Drpr and Ced-6 in the control pupae at 6h APF (between one and three cells, an average of 2.1 cells per lobe). These results suggest that ecdysone-induced stimulation in glia is essential for the elevated expression of drpr, but not ced-6 (Awasaki, 2006).

Insulin-like signaling promotes glial phagocytic clearance of degenerating axons through regulation of Draper

Neuronal injury triggers robust responses from glial cells, including altered gene expression and enhanced phagocytic activity to ensure prompt removal of damaged neurons. The molecular underpinnings of glial responses to trauma remain unclear. This study shows that the evolutionarily conserved insulin-like signaling (ILS) pathway promotes glial phagocytic clearance of degenerating axons in adult Drosophila. It was found that the insulin-like receptor (InR) and downstream effector Akt1 are acutely activated in local ensheathing glia after axotomy and are required for proper clearance of axonal debris. InR/Akt1 activity is also essential for injury-induced activation of STAT92E and its transcriptional target draper, which encodes a conserved receptor essential for glial engulfment of degenerating axons. Increasing Draper levels in adult glia partially rescues delayed clearance of severed axons in glial InR-inhibited flies. The study proposes that ILS functions as a key post-injury communication relay to activate glial responses, including phagocytic activity (Musashe, 2016).

Protein Interactions

The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 and its Drosophila orthologue, Draper. The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells, developmentally pruned axons and dendrites, and axons undergoing Wallerian degeneration. This study shows that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70 (Ferrante, 1995), binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. The Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. It is proposed that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells. Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade (Ziegenfuss, 2008).

Developing tissues produce excessive numbers of cells and selectively destroy a subpopulation through programmed cell death to regulate growth. Rapid clearance of cell corpses is essential for maintaining tissue homeostasis and preventing the release of potentially cytotoxic or antigenic molecules from dying cells, and defects in cell corpse clearance are closely associated with autoimmune and inflammatory diseases. In C. elegans the CED-1 receptor is expressed in engulfing cells, where it acts to recognize cell corpses and drive their phagocytosis. CED-1 promotes engulfment through an intracellular NPXY motif, a binding site for proteins containing a phosphotyrosine-binding (PTB) domain, and a YXXL motif, a potential interaction site for proteins containing SH2 domains. The PTB domain adaptor protein CED-6 can bind the NPXY motif of CED-1, is required for cell corpse engulfment and acts in the same genetic pathway as CED-1. CED-1 ultimately mediates actin-dependent cytoskeletal reorganization through the Rac1 GTPase, and Dynamin modulates vesicle dynamics downstream of CED-1 during engulfment, but the molecular signalling cascade that allows CED-1 to execute phagocytic events remains poorly defined (Ziegenfuss, 2008).

Glia are the primary phagocytic cell type in the developing and mature brain. Glia rapidly engulf neuronal cell corpses produced during development, as well as neuronal debris generated during axon pruning or during Wallerian degeneration in the adult brain. In Drosophila, glial phagocytosis of these engulfment targets requires Draper, the fly orthologue of CED-1. Draper, like CED-1, contains 15 extracellular atypical epidermal growth factor (EGF) repeats, a single transmembrane domain, and NPXY and YXXL motifs in its intracellular domain. Drosophila Ced-6 is also required for the clearance of pruned axons, indicating possible conservation of the interaction between CED-1 and CED-6 in flies, but additional signalling molecules acting downstream of Draper have not been identified (Ziegenfuss, 2008).

Draper was identified in a yeast two-hybrid screen for molecules interacting with the regulatory region of Shark. When LexA-Shark, constitutively active Src kinase and AD-Draper are present, Shark and Draper interact physically. In the absence of Src kinase, Shark and Draper fail to interact, indicating that phosphorylation of Draper by Src may be essential for Shark-Draper interactions. The Draper intracellular domain contains an ITAM (YXXI/L-X6-12-YXXL), a key domain found in many mammalian immunoreceptors including Fc, T-cell and B-cell receptors. SFKs phosphorylate the tyrosines in ITAM domains, thereby allowing ITAM association with SH2-domain-containing signal transduction proteins including Syk and Zap-70. Y-->F substitutions of the tyrosine residues were generated within or near the Draper ITAM, and it was found that Tyr 949 and Tyr 934 are critical for robust Draper-Shark binding. These correspond to the consensus tyrosine residues in the predicted Draper ITAM. Plasmids were transfected with carboxy-terminally haemagglutinin-tagged Draper (Draper-HA) or with Draper-HA and Shark with an amino-terminal Myc tag (Myc-Shark) into Drosophila S2 cells, immunoprecipitated with anti-HA antibodies, and western blots were performed with anti-phosphotyrosine, anti-Myc and anti-HA antibodies. Myc-Shark was found to co-immunoprecipitate with Draper-HA, and that anti-phosphotyrosine antibodies label a band corresponding to the position of Draper-HA that is absent in empty vector controls. Further, it was found that a Y949F substitution markedly reduces Draper-Shark association. Taken together, these data indicate Draper and Shark can associate physically through the Draper ITAM domain (Ziegenfuss, 2008).

Attempts were made to determine whether Shark is required for glial phagocytic activity in vivo. Severing adult Drosophila olfactory receptor neurons (ORNs) initiates Wallerian degeneration of ORN axons. Antennal lobe glia surrounding these severed axons respond to this injury by extending membranes towards severed axons and engulfing degenerating axonal debris. These glia express high levels of Draper, and in draperδ5 null mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the central nervous system (CNS). Thus, both the extension of glial membranes to severed axons and the phagocytosis of degenerating axonal debris require Draper signalling (Ziegenfuss, 2008).

Whether Shark function in glia is essential for glial responses to axon injury was explored by driving a UAS-regulated double-stranded RNAi construct designed to target shark (sharkRNAi) with the glial-specific repo-Gal4 driver, severing ORN axons, and assaying the recruitment of Draper and green fluorescent protein (GFP)-labelled glial membranes to severed axons. Maxillary palp-derived ORN axons project to 6 of the roughly 50 glomeruli in the antennal lobe. Within hours after maxillary palps have been ablated in control animals, Draper immunoreactivity decorates severed axons projecting to and within maxillary palp ORN-innervated glomeruli, and GFP-labelled glial membranes are recruited to these severed axons. Strikingly, knocking down Shark in glia completely suppresses these events. Next, antennal ORN axons were severed; these axons project to about 44 of the 50 antennal lobe glomeruli. Antennal ablation therefore injures nearly all glomeruli in the antennal lobe and results in the majority of antennal lobe glia in control animals upregulating Draper and undergoing hypertrophy. It was found that knocking down Shark in glia also blocks this glial response to axon injury. Thus, Shark is essential for all axon-injury-induced changes in glial morphology and Draper expression (Ziegenfuss, 2008).

To determine whether Shark is required for glial phagocytosis of severed axons, a subset of maxillary palp ORN axons were labelled with mCD8::GFP, Shark function was knocked down in glia, and the clearance of severed axons was assayed. In control animals severed GFP-labelled ORN axonal debris was cleared from the CNS within 5 days. In contrast, glial-specific sharkRNAi potently suppresses the clearance of degenerating axons, with severed axons lingering in the CNS for at least 5 days. Then whether mutations in the shark gene affected the glial clearance of degenerating axons was examined. The null allele of shark, shark1, is pupal lethal (Fernandez, 2000). Therefore glial responses to axon injury were assayed in shark1 heterozygous mutants, and dominant genetic interactions between draperδ5 and shark1 were tested. It was found that both draperδ5/+ and shark1/+ animals showed defects in glial phagocytic function: 5 days after injury, significant amounts of axonal debris remained within OR85e-innervated glomeruli. Moreover, shark1/+; draperδ5/+ animals showed a striking suppression of glial clearance of severed axons almost equivalent to that of draperδ5 mutants. Thus, shark mutations dominantly suppress the glial clearance of degenerating ORN axons, and this phenotype is strongly enhanced by removing one copy of draper. These data, taken together with sharkRNAi data, show that Shark is essential for the clearance of degenerating axons by glia (Ziegenfuss, 2008).

Is Shark required for the glial clearance of neuronal cell corpses? In embryonic stage 14-15 control animals, 24.4 cell corpses were found per hemisegment. In contrast, it was found that shark1 null mutants showed a marked increase in CNS cell corpses, with null mutants containing almost twice as many corpses per hemisegment. shark1/Df(2R)6063 mutants accumulate cell corpses at levels similar to those in shark1, indicating that this phenotype maps to shark. These cell corpse engulfment phenotypes are indistinguishable from that of draperδ5 mutants. It is concluded that Shark, like Draper, is also essential for the efficient clearance of embryonic neuronal cell corpses by glia (Ziegenfuss, 2008).

Because it was found that Shark binds Draper only in the presence of an active Src kinase in two-hybrid assays, Drosophila Src kinases were screened for roles in glial phagocytic activity. Interestingly, it was found that glia-specific knockdown of Src42A (src42ARNAi) potently suppress glial phagocytic activity: in src42ARNAi animals, Draper is not recruited to severed maxillary palp axons; glial hypertrophy and upregulation of Draper after antennal ablation was blocked; and GFP-labelled severed axons lingered in the CNS for 5 days. Knockdown of two other Drosophila Src kinases, Btk29A and Src64B, had no effect on the glial phagocytosis of severed axons. Thus, Src42A seems to be essential for all morphological responses of glia to axon injury and for the efficient clearance of degenerating axonal debris from the CNS (Ziegenfuss, 2008).

It was predicted that Draper phosphorylation status should be sensitive to the SFK inhibitor PP2. Indeed, addition of PP2 to S2 cultures led to a decrease in the phosphorylation of Draper and Draper-Shark association. Strikingly, co-transfection of Draper and Src42A led to a marked increase in Draper phosphorylation, which was PP2-sensitive and Draper-specific. Draper-Shark interactions are not dependent on Shark kinase activity because kinase-dead Shark (Shark K698R) associates with Draper. These data indicate that Src42A may phosphorylate the Draper intracellular domain, thereby increasing the association of Shark with Draper and the activation of downstream glial phagocytic signalling (Ziegenfuss, 2008).

This study has identified Shark and Src42A as novel components of the Draper pathway. One potential model for Draper-Shark-Src42A interactions is that Shark and Src42A drive the recruitment of Draper to engulfment targets. However, CED-1 has been shown to cluster around cell corpses even in the absence of its intracellular domain. Moreover, Zap-70 and Syk bind phosphorylated ITAM domains in mammalian immunoreceptors when ITAM domains are phosphorylated by Src after ligand-dependent receptor. Therefore a model is favoured in which the engagement of Draper with its ligand (presumably presented by engulfment targets) promotes receptor clustering, tyrosine phosphorylation of Draper by Src42A, association of Shark, and activation of downstream phagocytic signalling events. This work suggests that Draper is an ancient immunoreceptor in which the extracellular domain is tuned to recognize modified self and the intracellular domain signals through ITAM-Src-Syk-mediated mechanisms. This is the first identification of ITAM-Src-Syk signalling in invertebrates, and it suggests that a pathway similar to Draper-Ced-1 may ultimately have given rise to ITAM-based signalling cascades in mammalian myeloid and lymphoid cells, including those regulated by Fc, B-cell and T-cell receptors (Ziegenfuss, 2008).

Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells

Phagocytic removal of cells undergoing apoptosis is necessary for animal development and tissue homeostasis. Draper, a homologue of the C. elegans phagocytosis receptor CED-1, is responsible for the phagocytosis of apoptotic cells in Drosophila, but its ligand presumably present on apoptotic cells remains unknown. An endoplasmic reticulum protein that binds to the extracellular region of Draper was isolated. Loss of this protein, which has been named Pretaporter, led to a reduced level of apoptotic cell clearance in embryos, and the overexpression of Pretaporter in the mutant flies rescued this defect. Results from genetic analyses suggested that Pretaporter functionally interacts with Draper and the corresponding signal mediators. Pretaporter was exposed at the cell surface after the induction of apoptosis, and cells artificially expressing Pretaporter at their surface became susceptible to Draper-mediated phagocytosis. Finally, the incubation with Pretaporter augmented the tyrosine-phosphorylation of Draper in phagocytic cells. These results collectively suggest that Pretaporter relocates from the endoplasmic reticulum to the cell surface during apoptosis to serve as a ligand for Draper in the phagocytosis of apoptotic cells (Kuraishi, 2009).

Throughout the life of multi-cellular organisms, cells of particular types die and are eventually eliminated in certain places in the body and at certain developmental stages under physiological and sub-physiological conditions. Such 'unwanted cells' are mostly induced to undergo apoptosis and subjected to phagocytic elimination. Prompt and selective phagocytosis of apoptotic cells is prerequisite for morphogenesis, establishment of tissue functions, tissue renewal, avoidance of diseases, and effective progress of tissue functions (Kuraishi, 2009 and references therein).

There are two genetically identified signalling pathways that lead to the induction of phagocytosis of dying cells in C. elegans: one is made up of the proteins CED-2, CED-5, and CED-12, and the other consists of CED-1, CED-6, and CED-7. The pathways converge at CED-10, a small G-protein responsible for the rearrangement of cytoskeleton in phagocytes (Kinchen, 2005). The fact that all these C. elegans proteins possess counterparts in Drosophila and mammals (Lettre, 2006; Kinchen, 2007) suggests that these two partially redundant signalling pathways are evolutionally conserved. However, the mode of action of those proteins still remains to be clarified, and there are missing components in the pathways. The selectivity in the recognition of apoptotic cells by phagocytes is due to the specific interaction between receptors of phagocytes and their ligands present at the surface of target cells. Presumably, there are two phagocytosis receptors in C. elegans, and CED-1 is likely the one located upstream of CED-6 and CED-7 (Zhou, 2001; Yu, 2006; Venegas, 2007) whereas the other has not been found in genetic studies. Draper (Drpr), a Drosophila homologue of CED-1, has been shown to act as a receptor in the phagocytosis of apoptotic cells and more recently molecules that act with Drpr to accomplish the engulfment and subsequent processing of apoptotic cells in Drosophila phagocytes were reported (Cuttell, 2008; Kurant, 2008; Ziegenfuss, 2008). However, a molecule(s) present at the surface of apoptotic cells and recognized by Drpr is yet to be identified. The study by Venegas (2007) has implied that the membrane phospholipid phosphatidylserine, the best-characterized phagocytosis marker in mammals, could be a ligand for CED-1. In contrast, this study showed that phosphatidylserine is not required in the Drpr-mediated phagocytosis of apoptotic cells. In this study, a phagocytosis marker(s) recognized by Drpr was sought, and an endoplasmic reticulum (ER) protein was identified as a strong candidate (Kuraishi, 2009).

This study reports the identification of a Drosophila protein, Prtp, serving as a ligand for Drpr in the phagocytosis of apoptotic cells. The level of phagocytosis in embryos of the null mutants for prtp and drpr was almost the same and about half of that in controls. This suggests that Prtp is solely responsible for the Drpr-mediated phagocytosis by embryonic haemocytes, and that there are additional ligands and receptors for phagocytosis. The following pathway is proposed for the induction of the Prtp-dependent, Drpr-mediated phagocytosis of apoptotic cells: Prtp relocates from the ER to the cell surface during apoptosis; Prtp binds to Drpr of haemocytes and induces tyrosine phosphorylation; Ced-6 binds to phosphorylated Drpr and further transmits the signal leading to the activation of Rac1 and/or Rac2; and cytoskeletons are reorganized for engulfment. Additional molecules could participate in this pathway: a membrane protein named Six-microns-under reportedly acts upstream of Drpr in the phagocytosis of apoptotic neurons by glia (Kurant, 2008}; another signal mediator, named Shark, located proximal downstream of Drpr has been reported (Ziegenfuss, 2008); and Undertaker, an intracellular membrane protein involved in the regulation of calcium homeostasis, was shown to function downstream of Drpr (Cuttell, 2008}. In particular, Six-microns-under has been suggested to bridge glia and apoptotic neurons (Kurant, 2008}. The results suggested that Prtp still serves as a ligand for Drpr in the phagocytosis by glia that express Six-microns-under. Further investigation is necessary before a complete picture is obtained of the Prtp/Drpr-initiated signalling pathway for the phagocytosis of apoptotic cells. More molecules are involved in the phagocytosis of apoptotic cells in Drosophila: the receptor Croquemort, the phagocytosis marker Calreticulin, and Pallbearer, a component of ubiquitin ligase, which might be integrated into the other engulfment pathway (Kuraishi, 2009).

Prtp normally resides in the ER, most likely in its lumen, and a portion of it seems to relocate to the cell surface during apoptosis. The ER and plasma membranes exchange their components, and this might be enhanced upon the induction of apoptosis. In fact, a variety of proteins and lipids of the ER are exposed at the surface of apoptotic human cells (Franz, 2007). More recently, it was reported that the exposure of ER proteins at the surface of apoptotic mammalian cells occurs by SNARE-dependent exocytosis (Panaretakis, 2009). It is thus probable that together with other ER components Prtp moves to the cell surface during apoptosis and serves as a ligand for the phagocytosis receptor Drpr. Prtp seemed dispensable for the removal of degenerated neural axons during metamorphosis, which requires the action of Drpr. It is speculated that Prtp is not exposed on the surface of degenerated axons because the removal of these axons occurs independently of caspases, and that another ligand for Drpr exists at the surface of degenerated axons. In contrast, Drpr appears to serve as a receptor in the phagocytosis of bacteria (Cuttell, 2008; Hashimoto, 2009). Taken together, it is likely that Drpr is a multi-ligand receptor for phagocytosis responsible for the maintenance of tissue homeostasis through the removal of degenerated own cells and invading microbial pathogens (Kuraishi, 2009).

A counterpart for Prtp in C. elegans could be a ligand for CED-1, but there seems no complete homologue of prtp in its genome. The current analysis has suggested that the presence of two thioredoxin-like domains is sufficient for the binding of Prtp to Drpr. Therefore, a C. elegans protein containing such a structure, PDI for example, is a candidate for the CED-1 ligand. In contrast, the mammalian homologue of Prtp seems to exist; a mouse protein called ERp46 and human proteins belonging to the TXNDC5 family possess a domain composition similar to that of Prtp. It is important to examine if C. elegans PDI and mammalian ERp46 and TXNDC5 act as ligands for CED-1 and its mammalian homologue MEGF10, respectively, in the phagocytosis of apoptotic cells (Kuraishi, 2009).

Identification of lipoteichoic acid as a ligand for draper in the phagocytosis of Staphylococcus aureus by Drosophila hemocytes

Phagocytosis is central to cellular immunity against bacterial infections. As in mammals, both opsonin-dependent and -independent mechanisms of phagocytosis seemingly exist in Drosophila. Although candidate Drosophila receptors for phagocytosis have been reported, how they recognize bacteria, either directly or indirectly, remains to be elucidated. Staphylococcus aureus genes required for phagocytosis by Drosophila hemocytes were sought in a screening of mutant strains with defects in the structure of the cell wall. The genes identified included ltaS, which encodes an enzyme responsible for the synthesis of lipoteichoic acid. ltaS-dependent phagocytosis of S. aureus required the receptor Draper but not Eater or Nimrod C1, and Draper-lacking flies showed reduced resistance to a septic infection of S. aureus without a change in a humoral immune response. Finally, lipoteichoic acid bound to the extracellular region of Draper. It is proposed that lipoteichoic acid serves as a ligand for Draper in the phagocytosis of S. aureus by Drosophila hemocytes and that the phagocytic elimination of invading bacteria is required for flies to survive the infection (Hashimoto, 2009).

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. This 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 (Logan, 2012).

This study has shown how a single receptor, Draper, can positively or negatively influence glial responses to axonal injury. The work reveals a crucial role for ITAM and ITIM-like signaling events in regulating the activation, termination and maintenance of engulfment signal transduction cascades during glial responses to axonal injury. In addition, direct evidence is provided that negative regulation of glial responses to neurodegeneration is essential for glia to reset their responses after an initial injury, and thereby remain competent to respond efficiently to subsequent brain trauma (Logan, 2012).

The unique intracellular domains of Draper-I and Draper-II determine their effects on glial responses to axonal injury. Whereas Draper-I promotes engulfment of axonal debris, Draper-II completely inhibits glial clearance of degenerating axons, and the inhibitory activity mapped to a Draper-II-specific intracellular motif that contains an ITIM-like domain. It is noted that this insertion also produces two ITAMs in Draper-II, raising the possibility that one or both of these may function as an inhibitory ITAM (ITAMi). Recent work has shown that some ITAMs function in a dual manner, recruiting activating or inhibitory effectors in response to changes in receptor configuration. However, a model is favored in which Draper-II acts exclusively as an inhibitory ITIM-like receptor, as the ITIM-like Draper-II domain is not a functional activator in any context examined in vivo (Logan, 2012).

There are two unique Draper extracellular domains that are likely to be involved in recognition of engulfment targets. These are fully interchangeable in the engulfment assay, indicating that neither extracellular domain contains inherent inhibitory activity. It is possible that both domains recognize the same molecule, perhaps a ligand presented by degenerating axons. Alternatively, each extracellular domain may recognize a unique ligand. Identifying specific factors that associate with the extracellular region of each Draper isoform after axotomy will provide key insight into these post-injury neuron-glia communication events (Logan, 2012).

Following axotomy, engulfment molecules (Draper and Ced-6) are robustly upregulated in responding glia and return to baseline levels once axonal debris has been cleared. Notably, it was found that Csw signaling is essential to restore Draper and Ced-6 to basal levels as glia terminate responses to axotomy and that glia lacking Draper-II-Csw signaling fail to respond to secondary injuries in the brain. These data highlight previously unknown in vivo requirements for Draper-I and Draper-II-Csw signaling in coordinating the activation, termination and maintenance of glial cellular responses to axonal injury (Logan, 2012).

It is proposed that Draper-I, acting via Src42A and Shark, promotes the expression of engulfment genes after axotomy and phagocytosis of degenerating axons; such upregulation of engulfment genes is probably essential for rapid clearance of degenerating axons. It is also proposed that Draper-II and Csw then negatively regulate Draper-I signaling to terminate reactive glial responses and allow glia to return to a resting state. The data showing differential regulation of Draper-I and Draper-II transcripts, with Draper-I preceding Draper-II by several hours, supports the idea that Draper-II-Csw signaling may be delayed relative to Draper-I-ITAM signaling, thereby allowing for potent activation and temporal regulation of responses. Curiously, csw transcript levels seem to decrease ~4 h after injury, suggesting that although Draper-II and Csw are both influenced by post-injury signaling, they are differentially regulated (Logan, 2012).

It will be important in the future to identify additional factors functioning within the Draper-II-Csw inhibitory signaling pathway. Following engagement of mammalian ITIM receptors, activated SHP-1 or SHP-2 can target the Src-family kinases that phosphorylate the crucial ITAM tyrosine residues required for ITAM receptor signaling. Thus, Src42A, the kinase that phosphorylates the Draper-I ITAM, may also be an in vivo Csw target. SHP phosphatases can also influence gene transcription within immune cells, although it is largely unclear how this occurs at the molecular level (Logan, 2012).

The discovery that unique Draper isoforms execute opposing functions during the engulfment of degenerating axons raises interesting questions regarding the putative function of Draper homologs in other species. The related mammalian receptors Jedi-1 and MEGF10 are expressed in glia and play a conserved role in glial phagocytic activity (Cahoy, 2008; Singh, 2010; Wu, 2009). Jedi and MEGF10 drive engulfment in peripheral glia that clear large numbers of apoptotic neurons in the developing dorsal root ganglia. It is unknown whether splice variants of Jedi or MEGF10 exist. Notably, the role of Draper-II may be specific to adults; Draper-II transcript was not detected at embryonic or larval stages, which may indicate that the exuberant phagocytic activity of glia that occurs during nervous system development does not require negative regulation (Logan, 2012).

It is predicted that inhibitory constraints on reactive gliosis will be common to many organisms, and that these constraints will be particularly important in the context of neuropathological conditions. It is well known that reactive microglia can actively destroy neurons through phagocytic activity and reactive glia release a number of factors to promote neuronal death or inhibit neuronal function. Maintaining glial cells in an activated state might exacerbate these effects. The roles of MEGF10 and/or Jedi in glial response to adult neural injury have not yet been examined, but there is evidence that ITIM-mediated pathways may provide neuroprotection. For example, in the SHP-1 knockout mouse motheaten there is striking evidence of enhanced microglial activation and neurodegeneration after acute neuronal damage (Logan, 2012).

Promoting glial termination of reactive responses may be particularly important in neurodegenerative disease, which amounts to a continuous series of neural injuries. Reactive gliosis is certainly a hallmark of nearly all neurodegenerative diseases and there is growing evidence that glia help to promote disease pathology in mouse models. The Draper-II-Csw signaling pathway is remarkably specific in its negative regulation of glial responses to axonal injury, and it provides an exciting molecular entry point to understanding how glial cells terminate cellular and molecular responses to neural trauma (Logan, 2012).

DRK/DOS/SOS converge with Crk/Mbc/dCed-12 to activate Rac1 during glial engulfment of axonal debris

Nervous system injury or disease leads to activation of glia, which govern postinjury responses in the nervous system. Axonal injury in Drosophila results in transcriptional upregulation of the glial engulfment receptor Draper; there is extension of glial membranes to the injury site (termed activation), and then axonal debris is internalized and degraded. Loss of the small GTPase Rac1 from glia completely suppresses glial responses to injury, but upstream activators remain poorly defined. Loss of the Rac guanine nucleotide exchange factor (GEF) Crk/myoblast city (Mbc)/dCed-12 has no effect on glial activation, but blocks internalization and degradation of debris. This study shows that the signaling molecules Downstream of receptor kinase (DRK) and Daughter of sevenless (DOS) (mammalian homologs, Grb2 and Gab2, respectively) and the GEF Son of sevenless (SOS) (mammalian homolog, mSOS) are required for efficient activation of glia after axotomy and internalization/degradation of axonal debris. At the earliest steps of glial activation, DRK/DOS/SOS function in a partially redundant manner with Crk/Mbc/dCed-12, with blockade of both complexes strongly suppressing all glial responses, similar to loss of Rac1. This work identifies DRK/DOS/SOS as the upstream Rac GEF complex required for glial responses to axonal injury, and demonstrates a critical requirement for multiple GEFs in efficient glial activation after injury and internalization/degradation of axonal debris (Lu, 2014).

Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain

The brain has a limited capacity to self-protect against protein aggregate-associated pathology, and mounting evidence supports a role for phagocytic glia in this process. This study establishes a Drosophila model to investigate the role of phagocytic glia in clearance of neuronal mutant huntingtin (Htt) aggregates associated with Huntington disease. It was found that glia regulates steady-state numbers of Htt aggregates expressed in neurons through a clearance mechanism that requires the glial scavenger receptor Draper and downstream phagocytic engulfment machinery. Remarkably, some of these engulfed neuronal Htt aggregates effect prion-like conversion of soluble, wild-type Htt in the glial cytoplasm. The study provided genetic evidence that this conversion depends strictly on the Draper signalling pathway, unveiling a previously unanticipated role for phagocytosis in transfer of pathogenic protein aggregates in an intact brain. These results suggest a potential mechanism by which phagocytic glia contribute to both protein aggregate-related neuroprotection and pathogenesis in neurodegenerative disease (Pearce, 2015).

This study establishes a Drosophila model that demonstrates an essential role for phagocytic glia in clearance of Htt aggregates from ORN axons undergoing Wallerian degeneration after antennal axotomy or from non-severed axons. It was shown that HttQ91 aggregate clearance is mediated by a pathway that requires the glial engulfment receptor Draper and downstream genes, and is genetically indistinguishable from the pathway that operates in other glial phagocytic processes, including clearance of axonal or cellular debris following injury and axon pruning during development. Surprisingly, it was found that HttQ91 aggregates taken up by this Draper-dependent process are able to access and initiate a prion-like conversion of normally soluble, cytoplasmic HttQ25 in glia. These two findings have important implications for the potential role of glia in the suppression and/or progression of neurodegenerative diseases (Pearce, 2015).

Glial phagocytosis plays an important neuroprotective role in response to many types of brain injury, including insults associated with the production of neurodegenerative disease-linked insoluble protein aggregates. Secretion of pro-inflammatory cytokines and opsonins by activated glia promotes phagocytic clearance of damaged neurons, neuronal processes and cellular debris. In vitro, mouse astrocytes can bind to and degrade extracellular Aβ aggregates in cell culture and in brain slices. In vivo, pharmacological activation of microglia promotes clearance of Aβ deposits in a transgenic mouse model of Alzheimer disease (AD), and antibodies against Aβ or α-synuclein promote microglial-mediated phagocytic removal of the corresponding extracellular aggregates. Compelling evidence for a neuroprotective role for glial phagocytosis has come from recent studies linking missense mutations in TREM2, which encodes a microglial phagocytic receptor, to several neurodegenerative diseases including AD, Parkinson disease (PD) and amyotrophic lateral sclerosis (ALS) (Pearce, 2015).

In this study, an essential role for phagocytic glia in clearance of HttQ91 aggregates from ORN axons was established, but whether this phagocytosis is initiated as a specific response to the presence of aggregates or a collateral result of constitutive axon turnover and remodelling is unclear. This strict dependence on phagocytosis contrasts with previous work showing that extracellular aggregates can enter the cytoplasms of many different types of cultured cells, and cell surface proteins are only partially responsible for this entry. It is likely that the discrepancy between these previous studies and the current one at least in part reflects differences in the extracellular environment in cell culture, which is homogeneous and effectively infinite, and in the intact brain, where extracellular space is severely restricted in volume and is continuously patrolled by phagocytic glia. This view is supported by observations that, in the fly brain, aggregate uptake by glia occured only in close proximity to ORN axons containing HttQ91 aggregates and that detergent solubilization was required to immunolabel the aggregates. This study therefore favors a mechanism in which HttQ91 aggregates are phagocytosed by glia together with surrounding axonal membrane, analogous to the process by which supernumerary synapses are eliminated by Draper in Drosophila development and by the mammalian Draper orthologue, MEGF10, in the adult mouse brain (Pearce, 2015).

The observation that phagocytosed neuronal HttQ91 aggregates could nucleate aggregation of cytoplasmic HttQ25 in glia is surprising because phagocytosis normally leads to encapsulation and degradation of internalized debris within the membrane-enclosed phagolysosomal system. However, two independent lines of evidence were provided to support the conclusion that ORN-derived HttQ91 aggregates encounter HttQ25 in the glial cytoplasm. First, because HttQ25 is a highly soluble protein that does not aggregate in cells unless seeded by a pre-existing aggregate, the strict dependence of HttQ25 puncta formation on HttQ91 expression in ORNs indicates that these two Htt species must have physically interacted with one another. In principle, it is possible that, instead of HttQ91 aggregates being internalized by glia, this transfer could occur in the opposite direction, namely by the transfer of soluble HttQ25 into ORNs. However, the finding that HttQ25 aggregation was blocked by glial-specific knockdown of Draper and enhanced by antennal injury indicates an absolute requirement for phagocytic uptake of HttQ91, supporting ORN-to-glia transfer (Pearce, 2015).

Second, the majority of HttQ91 and HttQ25 aggregates co-localized with the cytoplasmic chaperones Hsp70/Hsc70 and Hsp90, indicating that aggregated HttQ25 is in direct contact with the cytoplasm. It is conceivable that merging of the phagocytic and autophaghic pathways could provide an opportunity for HttQ91 aggregates and soluble HttQ25 to encounter one another. However, substantial (<15%) co-localization of either HttQ91 or HttQ25 puncta with the autophagy markers, Atg8 and p62, or with the early endosome marker, Rab5 was not detected. Moreover, the amount of soluble HttQ25 that could be captured within an autophagosome was miniscule compared with the total cytoplasmic pool that would be available to be nucleated by an internalized HttQ91 seed. Altogether, these data strongly support the conclusion that phagocytosed HttQ91 aggregates are able to access the glial cytoplasm, thereby affording the opportunity to effect prion-like spreading of disease pathology (Pearce, 2015).

How do these engulfed HttQ91 aggregates breach the membrane barrier that separates the phagolysosomal lumen from the cytoplasm? The dependence of this process on Draper, shark and the actin-remodelling complex indicates that aggregates must access the glial cytoplasm at a step during or subsequent to phagocytic engulfment. It is possible that cytoplasmic entry can be facilitated by interference of phagocytosed aggregates with membrane fusion events during phagosome maturation. However, this ‘foot-in-the-door’ mechanism would be favoured by larger aggregates, which is in opposition to the finding that smaller HttQ91 aggregates were more strongly associated with cytoplasmic nucleation of glial HttQ25. It is proposed instead that slow or inefficient completion of membrane fusion events could provide a temporary conduit to the cytoplasm for HttQ91 aggregates. Such a mechanism would predict an upper size limit for cytoplasmic entry that could be exploited by small, newly formed aggregates. This prediction was supported by the finding that neither the induced HttQ25 aggregates nor the co-localized nucleating HttQ91 puncta in glia were labelled with antibodies to ubiquitin, a marker previously identified with more mature, larger Htt puncta in both cell culture and transgenic mouse models of HD. (Pearce, 2015).

The findings described in this study have broader implications for how potentially toxic protein aggregates are dispersed throughout the diseased brains. Phagocytic removal of aggregates is neuroprotective, but it is likely that phagocytes become impaired in their ability to clear debris as the disease worsens and that chronic glial activation becomes detrimental to the health of nearby neurons. The finding that phagocytosed neuronal Htt aggregates can enter the glial cytoplasm suggests that this transfer process could generate a reservoir of prion-like species inside glia, possibly facilitating their spread to other cells. A growing body of evidence supports the view that glial dysfunction exacerbates neurodegenerative disease pathogenesis by influencing the survival of neurons, and determining the mechanism(s) by which glia contribute to toxicity will be of great value to the development of therapeutic strategies to combat these devastating disorders (Pearce, 2015).


DEVELOPMENTAL BIOLOGY

Larval

The lobes of the larval MB are formed by a bundle of axon branches of the MB γ neurons. The distal tip of the MB dorsal lobe is surrounded by a few glial cells, which extend glial processes that cover the tip and shaft region of the lobe. Shortly after the onset of pupal metamorphosis, these glial processes infiltrate the lobes to engulf the axon branches (Awasaki, 2006).

Infiltrating glial processes are labeled with anti-Drpr antibody (Awasaki, 2004). To identify the glial cells that extend these Drpr-positive processes, the expression of drpr was examined by in situ hybridization, focusing on the area around the MB dorsal lobe. The drpr transcript was observed in a small number of cells adjacent to the dorsal lobe. Whereas the expression was weak in wandering larvae (L3) and pupae at 12h APF, it was elevated in pupae at 6h APF. The cells that express drpr were also labeled with anti-Repo antibody and expressed the nuclear-localizing signal LacZ fusion reporter (nLacZ), which was driven by GAL4-repo (repo>nLacZ), indicating that they were glia (Awasaki, 2006).

In C. elegans, the nematode ortholog of drpr, ced-1, interacts genetically with ced-6 in the engulfment of apoptotic cells, and CED-1 and CED-6 are colocalized in the cells around early apoptotic cells (Ellis, 1991; Liu, 1998; Kinchen, 2005). These proteins also interact biochemically (Su, 2002). Therefore, the expression of Drosophila ced-6 was examined in larval and pupal brains. In situ hybridization revealed weak expression of ced-6 in the cells adjacent to the MB dorsal lobe at L3 and 12h APF and elevated expression at 6h APF. The ced-6-expressing cells were glial cells, because they were labeled with anti-Repo antibody and repo>nLacZ (Awasaki, 2006).

Although both drpr and ced-6 transcripts are expressed in the glial cells adjacent to the MB dorsal lobes with a similar pattern, only a specific subset of the glial cells in this area expressed these genes. To determine whether they are expressed in the same glial cells, the distribution of the Drpr and Ced-6 proteins were examined using anti-Drpr and anti-Ced-6 antibodies. Drpr and Ced-6 were detected in the same glial cells around the MB lobe. Between one and three glial cells in this area coexpressed Drpr and Ced-6 at 6h APF (an average of 2.2 glial cells per lobe). Other glial cells expressed neither Drpr nor Ced-6. Drpr and Ced-6 were, however, detected in all of the infiltrating glial processes. This finding strongly suggests that only a specific subset of the glial cells in which these two proteins are expressed extend their processes into the MB lobes. In addition to the MB dorsal lobe, glial processes infiltrating the medial lobe were labeled with both anti-Drpr and anti-Ced-6 antibodies. These processes derived from the glial cells that were arranged in the posterior region of the medial lobe (Awasaki, 2006).

These findings indicate that elevated expression of both drpr and ced-6 was transiently induced in the specific subset of glial cells that send their processes into the MB lobes early in metamorphosis. Although more than eight glial cells are arranged around the MB dorsal lobe at 6h APF, only a subset of them contribute to infiltration by glial processes (Awasaki, 2006).

Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons

Precise neural circuit assembly is achieved by initial overproduction of neurons and synapses, followed by refinement through elimination of exuberant neurons and synapses. Glial cells are the primary cells responsible for clearing neuronal debris, but the cellular and molecular basis of glial pruning is poorly defined. This study shows that Drosophila larval astrocytes transform into phagocytes through activation of a cell-autonomous, steroid-dependent program at the initiation of metamorphosis and are the primary phagocytic cell type in the pupal neuropil. The developmental elimination was examined of two neuron populations-mushroom body (MB) gamma neurons and vCrz+ neurons (expressing Corazonin [Crz] neuropeptide in the ventral nerve cord [VNC])-where only neurites are pruned or entire cells are eliminated, respectively. MB gamma axons were found to be engulfed by astrocytes using the Draper and Crk/Mbc/dCed-12 signaling pathways in a partially redundant manner. In contrast, while elimination of vCrz+ cell bodies requires Draper, elimination of vCrz+ neurites is mediated by Crk/Mbc/dCed-12 but not Draper. Intriguingly, it was also found that elimination of Draper delayed vCrz+ neurite degeneration, suggesting that glia promote neurite destruction through engulfment signaling. This study identifies a novel role for astrocytes in the clearance of synaptic and neuronal debris and for Crk/Mbc/dCed-12 as a new glial pathway mediating pruning and reveals, unexpectedly, that the engulfment signaling pathways engaged by glia depend on whether neuronal debris was generated through cell death or local pruning (Tasdemir-Yilmaz, 2013).

Astrocytes serve as regulators of synapse formation and function and are generally supportive of neural circuits. Based on several lines of evidence, this study showed that astrocytes transform morphologically and functionally into phagocytes at pupariation and engulf significant amounts of neural debris. Phagocytic astrocytes take on a highly vacuolated appearance, up-regulate the engulfment molecule Draper, contain cytoplasmic vacuoles filled with debris that stains for synaptic or axonal markers, and exhibit high levels of lysosomal activity. Activation of this phagocytic program depends on cell-autonomous signaling through the EcR, since blockade of EcR in even single astrocytes suppressed their transformation by all morphological and molecular criteria. Blockade of astrocyte phagocytic function by multiple methods (e.g., EcRDN or Shits) suppressed the clearance of synapses throughout the CNS, axons of MB γ neurons in the central brain, and the neurites of vCrz+ cells in the VNC. This latter observation defines vCrz+ neurons as a new system to explore astrocyte-neuron interactions during neuronal apoptosis and neurite or synapse elimination in the CNS (Tasdemir-Yilmaz, 2013).

While roles for Drosophila glia in the engulfment of pruned MB γ axons have been previously described, it was unexpected that astrocytes would be the phagocytic cell type. In the adult Drosophila brain, a second type of neuropil glial cells, ensheathing glia, is responsible for engulfing injured axonal debris, while astrocytes fail to respond in a detectable way morphologically or molecularly (e.g., by Draper up-regulation). The stark difference in glial subtypes executing phagocytic function in the pupa versus the adult could result from differences in glial genetic programs during development versus in mature glia or indicate a key difference in the molecular nature of pruned neurites compared with those undergoing injury-induced axon degeneration (Tasdemir-Yilmaz, 2013).

The extent to which mammalian astrocytes are phagocytes in vivo has remained unclear until recently. In culture, astrocytes can be phagocytic and engulf apoptotic cells or amyloid-β. In vivo, the post-laminar optic nerve head myelination transition zone (MTZ) astrocytes express Mac-2, a molecule implicated in phagocytic activity, and internalize axonal evulsions, and this event appears to be increased in frequency in glaucoma models. Most impressively, it has been recently demonstrated that astrocytes engulf synaptic material in vivo and that this event is mediated by MEGF10 (mouse Draper) and the engulfment receptor MERTK. Thus, Draper/MEGF10-dependent engulfment of pruned synapses appears to be an ancient astrocytic mechanism; whether the same is true for the Crk/Mbc/dCed-12 pathway remains to be determined. Transcriptome data from purified mouse forebrain astrocytes support this notion, since molecular pathways for engulfment are highly enriched, including, in addition to MEGF10, Gulp1 (dCed-6), and in addition to Crk, Dock1 (Mbc) and Elmo (dCed-12). It will be important to explore whether these additional pathways have similar roles in astrocytes for the pruning of mammalian neural circuits during development and whether astrocyte engulfment activity is modified in neurological diseases involving axonal, dendritic, or synaptic loss (Tasdemir-Yilmaz, 2013).

Two types of neurons that undergo different types of developmental reorganization were examined. MB γ neurons prune their medial and dorsal axon branches and dendrites, their cell bodies remain viable, and, at midpupal stages, they re-extend medial axon branches to establish adult-specific connectivity. In contrast, vCrz+ neurons exhibit complete neurite degeneration, and cell bodies undergo apoptotic death and eventually are completely eliminated. In addition to there being remarkable differences in the patterns of fragmentation exhibited by these subsets of neurons, there are also critical differences in the engulfment signaling pathways used to promote their initial destruction and clearance (Tasdemir-Yilmaz, 2013).

Previous work has revealed a role for Draper in MB γ neuron pruning, with draper-null mutants exhibiting a delay of ~2 d in the clearance of pruned MB γ neuron axonal debris. This study provides evidence supporting a key role for the Crk/Mbc/dCed-12 complex in the clearance of MB γ neuron axonal debris and demonstrates that Crk/Mbc/dCed-12 knockdown and draper mutants have additive phenotypes, indicating that these signaling pathways act in a partially redundant fashion in astrocytes to promote clearance of pruned MB γ neuron axons. While these phenotypes are additive, it is noted that draper mutants exhibit a stronger delay in MB γ neuron pruning, suggesting that Draper signaling plays a more prominent role in this brain region than signaling through the Crk/Mbc/dCed-12 complex. However, the possibility that a Crk, mbc, or dCed-12 mutant might have a stronger phenotype than the RNAi lines used in this study cannot be excluded (Tasdemir-Yilmaz, 2013).

vCrz+ neuronal clearance appears to involve both Draper signaling and the Crk/Mbc/dCed-12 complex, with the former primarily promoting clearance of vCrz+ cell bodies, and the latter driving clearance of degenerating vCrz+ neurites. In draper-null mutants at 18 h APF, the majority of neuronal cell bodies remained at the edge of the neuropil, while neurite debris was largely cleared. Reciprocally, astrocytic knockdown of dCed-12 suppressed neurite clearance, while vCrz+ cell bodies were promptly eliminated. The lack of additivity of the phenotypes for either neurite or cell body clearance in draper-null mutants with astrocytic dCed-12RNAi suggests that elimination of cell bodies is primarily driven by the Draper pathway, while neurite clearance is largely accomplished by signaling through the Crk/Mbc/dCed-12 complex (Tasdemir-Yilmaz, 2013).

This study reveals that the genetic pathways engaged by glia to engulf pruned neuronal material versus apoptotic neurons are context-dependent and correlate with the type of destructive program. Interestingly, the molecular pathways that mediate axonal degeneration during axon pruning versus apoptosis are also distinct in mammalian cultured neurons. Local withdrawal of NGF in the axon activates Caspase 6-dependent axon degeneration, which is not sensitive to XIAP1 inhibition. In contrast, whole-cell NGF withdrawal leads to Caspase 6-independent apoptotic cell death and degeneration of axons, which is then sensitive to XIAP1. These observations argue that context matters when neurites and cell bodies are being destroyed. It is speculated that neuron-glia signaling during engulfment events might also be compartmentalized, with neurites and cell bodies generating different types of 'eat me' cues for clearance by glia (Tasdemir-Yilmaz, 2013).

Do engulfing glial cells actively promote the destruction of target neurons? The intriguing observation was made that loss of a single copy of draper is sufficient to dominantly suppress the elimination of vCrz+ cell bodies and neurites at 6 h APF. Moreover, loss of two copies results in the retention of nearly all vCrz+ cell bodies and significant parts of the vCrz+ scaffold, and many regions of the scaffold appeared morphologically intact, suggesting a delay of neurite fragmentation. Previous work has shown that expression of the anti-apoptotic molecule P35 is sufficient to suppress the pruning of vCrz+ neurites for at least 1 d; this study extended this observation and found this to be true for at least 2 d. Thus, blocking apoptosis in vCrz+ is sufficient to significantly delay neurite degeneration. This observation, coupled with the known role for CED-1 in actively promoting the death of engulfment targets, suggests that engulfing nonastrocyte glia may promote neuronal apoptosis through Draper signaling. This in turn would promote degeneration of the neurite scaffold after cell body death. If this underlies the delay that was observed in neurite degeneration in draper mutants, it would argue that glia actively sculpt neural networks by promoting the destruction of selected subsets of neurons. Subsequent work exploring the survival of cell bodies preserved in draper-null animals will be essential to explore this exciting possibility (Tasdemir-Yilmaz, 2013).

Astrocytes play a key role in Drosophila mushroom body axon pruning

Axon pruning is an evolutionarily conserved strategy used to remodel neuronal connections during development. The Drosophila mushroom body (MB) undergoes neuronal remodeling in a highly stereotypical and tightly regulated manner, however many open questions remain. Although it has been previously shown that glia instruct pruning by secreting a TGF-beta ligand, myoglianin, which primes MB neurons for fragmentation and also later engulf the axonal debris once fragmentation has been completed, which glia subtypes participate in these processes as well as the molecular details are unknown. This study shows that, unexpectedly, astrocytes are the major glial subtype that is responsible for the clearance of MB axon debris following fragmentation, even though they represent only a minority of glia in the MB area during remodeling. Furthermore, astrocytes both promote fragmentation of MB axons as well as clear axonal debris and that this process is mediated by ecdysone signaling in the astrocytes themselves. In addition, this study found that blocking the expression of the cell engulfment receptor Draper in astrocytes affects only axonal debris clearance. Thereby this study uncoupled the function of astrocytes in promoting axon fragmentation to that of clearing axonal debris after fragmentation has been completed. This study finds a novel role for astrocytes in the MB and suggests two separate pathways in which they affect developmental axon pruning (Hakim 2014).

Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila

During developmental remodeling, neurites destined for pruning often degenerate on-site. Physical injury also induces degeneration of neurites distal to the injury site. Prompt clearance of degenerating neurites is important for maintaining tissue homeostasis and preventing inflammatory responses. This study shows that in both dendrite pruning and dendrite injury of Drosophila sensory neurons, epidermal cells rather than hemocytes are the primary phagocytes in clearing degenerating dendrites. Epidermal cells act via Draper-mediated recognition to facilitate dendrite degeneration and to engulf and degrade degenerating dendrites. Using multiple dendritic membrane markers to trace phagocytosis, it was shown that two members of the CD36 family, croquemort (crq) and debris buster (dsb), act at distinct stages of phagosome maturation for dendrite clearance. These findings reveals the physiological importance of coordination between neurons and their surrounding epidermis, for both dendrite fragmentation and clearance (Han, 2014).

Removal of nonfunctional or damaged tissues is an important biological process during tissue remodeling or repair. This study shows that, for Drosophila class IV da neurons in the periphery, degenerating dendrites in both dendrite pruning and injury models are removed by neighboring epithelial cells rather than professional phagocytes. By developing multiple dendritic markers that label phagosomes differentially, the clearance of degenerating dendrites was established as an in vivo model to study phagocytosis. With these tools, key players in engulfment and phagosome maturation were analyzed, and roles of the CD36 family members Crq and Dsb were elucidated. This study further reveals that, as phagocytes, epidermal cells actively participate in not only the removal but also the fragmentation of degenerating dendrites (Han, 2014).

Professional phagocytes such as macrophages in vertebrates and plasmatocytes in Drosophila dispose the majority of apoptotic cells in development, as well as invading microorganisms during infection. However, nonprofessional phagocytes may take charge when macrophages or other professional phagocytes are absent or cannot easily access cell corpses, as in apoptosis of rat lens cells, follicular atresia, and degeneration of Drosophila egg chambers induced by protein deprivation. This scenario does not apply to Drosophila da neuronal dendrites, which are exposed to circulating plasmatocytes in the hemolymph and sessile plasmatocytes clustered around da neuron somas. Indeed, previous observation of dendrite debris engulfment by plasmatocytes during dendrite pruning has led to the conclusion that plasmatocytes clear pruned dendrites. The current finding that clearance of degenerating dendrites is mainly carried out by epidermal epithelial cells demonstrates that nonprofessional phagocytes are not just a substitute for professional phagocytes in their absence. Rather, plasmatocytes and epidermal cells probably carry out different functions reflecting specialization of cellular functions. The removal of pruned dendrites by Drosophila epidermal cells perhaps can be seen as a parallel to the clearance of photoreceptor outer segments by retinal pigment epithelial cells; in both cases epithelial cells maintain homeostasis of the nervous system as part of their physiological functions. The observation that epidermal cells are also responsible for clearing injured dendrites indicates that the same cellular mechanism is also used to cope with perturbations in the peripheral nervous system (Han, 2014).

Epithelial cells may profoundly influence the development of dendritic arbors of da neurons. During larval development, growing epithelial cells signal to the dendritic arbors so they can grow proportionally to epithelial cells in order to maintain the same coverage of receptive fields of the sensory neurons, a phenomenon known as dendritic scaling. Epithelial cells also contribute to the patterning of dendritic arbors of da neurons by tethering dendrites to the 2D space of the extracellular matrix so that dendrites have to avoid sister dendrites from the same neuron (self-avoidance) or dendrites from neighboring like-neurons (tiling). The finding that epithelial cells mediate the clearance of degenerating dendrites substantially adds to the growing list of dendrite properties regulated by epithelial cells (Han, 2014).

The vertebrate CD36 family members CD36 and scavenger receptor class B type I (SR-BI) mediate phagocytosis of apoptotic cells and microbial pathogens in vitro. The Drosophila CD36 family member Crq is required for efficient phagocytosis of cell corpses in embryos and mediates binding of apoptotic cells by in vitro cultured cells, leading to its proposed role as a receptor for apoptotic cells. This study shows that in epithelial cells crq is required for phagosome maturation but not for the engulfment of degenerating dendrites. As loss of crq does not completely abolish the engulfment of apoptotic cells in the embryo, it is possible that the cell-corpse clearance defect in crq mutant embryos may be a consequence of blocked phagosome maturation. An alternative possibility is that Crq may be required for engulfment and/or phagosome maturation of apoptotic cells by embryonic macrophages but only required for phagosome maturation of pruned or injured dendrites by epithelial cells. This could be due to the fact that macrophages have to actively search for and bind apoptotic cells, while epithelial cells engulf neighboring debris. Further experiments will be needed to determine whether Crq also plays a role in phagosome maturation during phagocytosis by macrophages (Han, 2014).

Loss of Crq function resulted in the fusion of dendrite-derived phagosomes accompanied with a failure of degradation of phagosome contents, most likely due to inefficient delivery of degradation machineries to late phagosomes. As phagosomes normally acquire hydrolases and other phagolysosomal components by fusing with endosomes and lysosomes, it is hypothesized that Crq suppresses homotypic phagosome fusion to promote fusion between phagosomes and late endosomes/lysosomes. Homotypic phagosome fusion rarely happens during normal phagocytosis but is induced by infection of bacterial pathogens such as Helicobacter pylori and Chlamydia trachomatis; the ability of different strains of H. pylori to induce phagosome fusion correlates with the virulence and intracellular survival of these bacteria. Therefore, regulation of the balance between homotypic phagosome fusion and heterotypic fusion between phagosomes and late endosomes/lysosomes is probably critical for the degradation of internalized materials (Han, 2014).

The Drosophila genome encodes fourteen CD36 family members. Besides the involvement of Crq in phagocytosis, another member Pes mediates mycobacteria infection. This study found that the CD36 family member Dsb regulates late stages of phagosome maturation. Interestingly, LIMP-2, the mammalian CD36 family member with the highest homology to Dsb, is an intrinsic lysosomal protein required for the degradation of Listeria in phagosomes. Dsb and LIMP-2 thus appear to have evolutionarily conserved functions in phagosome maturation (Han, 2014).

Phagocytes not only clear cell corpses but may also engulf still-living cells and promote cellular degeneration in many contexts. This study shows that efficient degeneration of dendrites requires the coordination with phagocytic epithelial cells. One mechanism for such coordination is the Drpr-mediated recognition of degenerating dendrites by epidermal phagocytes that form actin-rich membrane structures wrapping around the dendrites to facilitate their fragmentation. In the postnatal mouse brain, microglia actively induce apoptosis of Purkinje cells by producing superoxide ions. It remains to be determined whether nonprofessional phagocytes such as epidermal cells also promote neurite degeneration by emitting diffusible agents (Han, 2014).

Adult

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. This study defines 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. Beuropil regions were found to 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, it was found that 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 (Doherty, 2009).

In an effort to identify distinct morphological subtypes of glial cells in the mature Drosophila brain, the MARCM system was used to generate small clones of glial cells labeled with membrane-tethered GFP. Larvae containing a hs-flipase allele and a wild-type chromosome arm for recombination were subjected to a short heat shock (37°C) and glial cells within clones were visualized by use of the pan-glial driver, repo-Gal4. The analysis of glial subtype morphology was focused mainly on the adult antennal lobe region owing to its well defined histology and accessibility to genetic manipulation. In the adult brain clones were identified resembling each of the three main types of glial cells found in embryos and larvae: (1) cortex glia, which resided outside the neuropil in regions housing neuronal cell bodies, ramified dramatically to surround individual cell bodies; (2) surface glia, which appeared as large flat cells enveloping the surface of the brain, did not extend any processes into the brain; and (3) neuropil glia, which were closely associated with the neuropil, extended membranes into synaptic regions, and surrounded large bundles of axons. As in the embryo and larva, glial cell bodies were not found within the neuropil, rather they resided at the edge of the neuropil (neuropil glia), in the cortex (cortex glia), or at the surface of the brain (surface glia) (Doherty, 2009).

Interestingly, the single-cell resolution provided by MARCM analysis allowed further subdivision of neuropil glia into two distinct morphological classes, 'ensheathing glia' and 'astrocytic glia.' Ensheathing glia appeared as flattened cells that lined the borders of the neuropil and subdivided regions of the brain by isolating neuropil from the surrounding cortex. Within the antennal lobe, ensheathing glial membranes surrounded individual glomeruli (the functional units of the antennal lobe) but did not extend into the synaptic regions of the glomeruli. In addition, an astrocyte-like cell type was identified that extended membrane processes deeply into the neuropil and ramified profusely in synaptic-rich regions. This latter cell type is referred to as the fly 'astrocyte', based on its striking morphological similarity to mammalian astrocytes, as well as the conserved expression of a number of molecular markers used to identify astrocytes in the mammalian brain. Mammalian astrocytes remove excess amounts of extracellular glutamate through the high-affinity excitatory amino acid transporters (EAATs), GLAST and GLT-1, which transports the glutamate into glial cells where it is then converted into glutamine by glutamine synthetase. It was found that Drosophila astrocytes also express the transporter EAAT1. While the adult antennal lobe was used as the primary model tissue in this study, the morphological glial subtypes described above were observed in all brain regions examined, suggesting that the results are generally applicable to glial populations throughout the adult Drosophila brain (Doherty, 2009).

Attempts were made to identify Gal4 driver lines that would allow unique labelling and and manipulation of these glial populations, with a major focus being to genetically subdivide neuropil glia (i.e., ensheathing glia versus astrocytes). To accomplish this, UAS-mCD8::GFP was crossed to a previously described collection of embryonic and larval glial drivers (Ito, 1995), as well as a number of drivers generated for this study. The adult antennal lobe was studied to examine the morphology and spatial distribution of cell types marked by these drivers in a background with glial nuclei ({alpha}-Repo) and the neuropil ({alpha}-nc82) also labeled. The repo-Gal4 driver labeled all Repo+ glial subtypes in the adult brain, as evidence by {alpha}-Repo immunostaining in the nuclei of GFP+ cells. Membrane processes from Repo+ cells are found throughout the adult brain, and together they constitute the diverse collection of glial subtypes identified in single-cell MARCM analysis. Upon examination of a single glomerulus within the antennal lobe, membranes from Repo+ cells were found to both surround and invade glomeruli. All GFP expression in the adult brain driven by the repo-Gal4 driver can be suppressed by coexpression of Gal80 (a Gal4 inhibitor) under control of the repo promoter (repo-Gal80), arguing that repo-Gal80 can efficiently block Gal4-mediated activation of UAS-reporters in all adult brain glia (Doherty, 2009).

Two drivers, mz0709-Gal4 and alrm-Gal4, appeared to show very specific expression in ensheathing glia and astrocytes, respectively. Glial processes labeled by mz0709-Gal4 were found at the edge of the antennal lobe and extended deeply into the neuropil region. These flattened glial processes surrounded, but did not invade, individual glomeruli, and did not extend into the cortex region. With the exception of variable expression in a small number of neurons, all mz0709-Gal4-induced expression was suppressed by repo-Gal80, indicating that mz0709-Gal4 is largely specific to ensheathing glia. The generation of MARCM clones labeled with the mz0709-Gal4 driver resulted in the consistent labeling of ensheathing glia, but not astrocytes, within the antennal lobes. Reciprocally, alrm-Gal4 was found to be expressed exclusively in astrocytes. All cellular processes from cells labeled with alrm-Gal4 extended into the neuropil, showed a highly branched or tufted morphology, invaded individual glomeruli, and all alrm-Gal4-driven expression was suppressed by repo-Gal80. Additionally, single cell MARCM clones were found labeled with the alrm-Gal4 driver resulted in the consistent labeling of astrocytes, but not ensheathing glia. Together, these drivers are excellent tools to manipulate and functionally distinguish different subtypes of glia in the adult Drosophila brain (Doherty, 2009).

What are the functional roles for each glial subtype in the adult brain? Is each subtype responsible for a unique collection of tasks, or are all glial subtypes functionally equivalent? As a first step to determining the in vivo functional differences between adult brain glial subtypes the cell autonomy of glial phagocytic function was explored. Severing olfactory receptor neuron (ORN) axons by surgical ablation of maxillary palps leads to axon degeneration (termed Wallerian degeneration), recruitment of glial membranes to fragmenting axons, and glial engulfment of axonal debris. These glial responses are mediated by Draper, the Drosophila ortholog of the C. elegans cell corpse engulfment receptor cell death defective-1 (CED-1). In draper null mutants, glia fail to extend membranes to degenerating ORN axons and axonal debris is not removed from the CNS. Thus, Draper function should be autonomously required in phagocytic glial subtypes and Draper expression is predicted to act as a molecular marker for glial cells capable of performing engulfment functions (Doherty, 2009).

To define the precise cell types that express Draper, all glial membranes were labeled with mCD8::GFP driven by repo-Gal4, stained with α-Draper antibodies, and assayed for colocalization of Draper and GFP. Extensive overlap of Draper and GFP was found in this background. Draper and GFP signals overlapped at the edge of the neuropil, in membranes surrounding antennal lobe glomeruli, and in all cortex glia. This labeling was specific to Draper since expression of a UAS-draperRNAi construct with repo-Gal4 led to the elimination of all Draper immunoreactivity in the adult brain. Thus, the entire population of cortex glia appear to express Draper and are likely to be phagocytic. However, cortex glia do not extend membranes into the antennal lobe neuropil, even after ORN axon injury. Therefore, cortex glia are not likely responsible for clearing severed ORN axonal debris from the antennal lobe neuropil (Doherty, 2009).

Interestingly, when mCD8::GFP was driven by mz0709-Gal4 extensive overlap of Draper and GFP was observed in neuropil-associated ensheathing glia. A high-magnification view of the antennal lobe revealed Draper and mz0709-Gal4 labeled membranes colocalizing and surrounding, but not innervating individual glomeruli. Moreover, expression of UAS-draperRNAi in ensheathing glia with mz0709-Gal4 led to a dramatic reduction in Draper immunoreactivity in the neuropil, but the weaker Draper immunoreactivity in the cortex remained unchanged. Conversely, no overlap was observed between Draper and GFP when astrocytic membranes were labeled using the alrm-Gal4 driver. Furthermore, driving the expression of UAS-draperRNAi in astrocytes had no obvious effect on Draper expression in the brain. These results indicate that Draper is expressed in cortex glia and ensheathing glia but not in astrocytes (Doherty, 2009).

The specific expression of Draper in antennal lobe ensheathing glia suggests that this glial subset is the phagocytic cell type responsible for engulfing degenerating axonal debris after ORN axotomy. To explore this possibility, it was asked whether ensheathing glia or astrocytes extend membranes to severed axons after injury, and in which cell type Draper was required for clearance of axonal debris from the CNS. To assay extension of glial membranes to severed axons, glial membranes were labeled with mCD8::GFP, maxillary palp axons were severed, and colocalization of Draper and GFP was assayed in glomeruli housing severed ORN axons. Within 1 d after injury, Repo+ glial membranes were found to localize to glomeruli housing severed maxillary palp axons and these membranes were decorated with Draper immunoreactivity. Similarly, mz0709+ glial membranes also localized to severed axons and colocalized with intense Draper immunoreactivity 1 d after injury. Knockdown of Draper with UAS-draperRNAi using repo-Gal4 or mz0709-Gal4 completely suppressed the recruitment of both Draper and glial membranes to severed axons. In contrast, when astrocyte membranes were labeled with GFP no colocalization of GFP and Draper immunoreactivity was observed 1 d after axotomy. In addition, knockdown of Draper in astrocytes with UAS-draperRNAi did not suppress the recruitment of Draper to severed axons. In an effort to identify any indirect role for astrocytes during the injury response, the morphology of astrocytes was examined both before and after injury to determine whether they exhibited any overt changes in morphology or retracted their membranes from the site of injury to accommodate the recruitment of ensheathing glial membranes. However, no obvious changes were detected in morphology or in the positions of the astrocyte glial cells in response to axon injury. Together, these data indicate that Draper is required in ensheathing glia for recruitment of glial membranes and accumulation of Draper on severed ORN axons, and suggest that Drosophila astrocytic glia do not undergo any dramatic changes in morphology in response to ORN axotomy (Doherty, 2009).

From the above data it is predicted that ensheathing glia would act as phagocytes to engulf degenerating ORN axonal debris from the CNS. To test this a subset of maxillary palp ORN axons was labeled with mCD8::GFP using the OR85e-mCD8::GFP transgene, Draper function was knocked down in glial subsets using subset-specific driver lines, maxillary palp ORN axons were severed, and clearance of axons was assayed 5 d after injury. GFP-labeled axons were first severed in control animals with each driver and GFP+ axonal debris was found to be efficiently cleared from the CNS within 5 d after injury, confirming that glial phagocytic function is not affected in the driver lines. Strikingly, RNA interference (RNAi) knockdown of Draper using UAS-draperRNAi in a background with repo-Gal4 or mz0709-Gal4 completely blocked clearance of GFP-labeled axonal debris from the CNS, while RNAi knockdown of Draper in astrocytes with alrm-Gal4 had no effect on axon clearance. Thus, Draper is required autonomously in ensheathing glia for the clearance of degenerating ORN axonal debris from the CNS. In addition, knockdown of Draper in ensheathing glia with mz0709-Gal4 had no measurable effect on Draper expression in cortex glia, arguing that cortex glia are not capable of compensating for the loss of phagocytic activity in ensheathing glia during the clearance of axonal debris from the antennal lobe neuropil after axotomy. From these data on morphogenic responses to injury and phagocytic function, it is concluded that astrocytic, cortex, and ensheathing glia represent functionally distinct subsets of glial cells in the adult Drosophila brain (Doherty, 2009).

The Draper signaling pathway is a central mediator driving glial engulfment of neuronal cell corpses, and axons undergoing Wallerian degeneration in Drosophila. These studies show that within the adult brain neuropil, ensheathing glial cells are the only cell type that expresses Draper. Draper expression overlaps precisely with ensheathing glial membranes (when labeled with GFP driven by the ensheathing glia-specific driver mz0709-Gal4), and RNAi for draper in ensheathing glia leads to the elimination of Draper immunoreactivity in the neuropil. Similarly, it was found that dCed-6, the fly ortholog of C. elegans CED-6, a PTB-domain binding protein that functions genetically downstream of worm CED-1, is expressed in the adult brain in a pattern indistinguishable from Draper. dCed-6 immunoreactivity is strongly reduced in the neuropil through mz0709-Gal4-mediated knockdown (with UAS-dCed-6RNAi), and eliminated from the entire brain when dCed-6RNAi treatment is performed with the pan-glial driver repo-Gal4. Thus, in the adult brain dCed-6 appears to be expressed exclusively in glia, including cortex glia and ensheathing glia of the neuropil (Doherty, 2009).

Components of the Draper signaling pathway are required specifically in ensheathing glia for glial membrane recruitment to severed ORN axons, and clearance of degenerating axonal debris from the brain. Knocking down either draper or shark in ensheathing glia is sufficient to block recruitment of glial membranes and Draper to severed axons and clearance of degenerating axonal debris from the CNS. It is suspected that dCed-6 is also required in ensheathing glia for a number of reasons. First, dCed-6 and Draper show perfect overlap in expression in the neuropil, and Draper is only expressed in ensheathing glia. Second, ensheathing glia are recruited to degenerating axon injury, and dCed-6, like Draper, is specifically recruited to degenerating maxillary palp ORN axons after maxillary palp ablation. Third, dCed-6 expression is dramatically increased in ensheathing glia surrounding the antennal lobe after antennal ablation, similar to what has been found for Draper. Surprisingly, RNAi knock down of dced-6 in ensheathing glia (with mz0709-Gal4) failed to suppress the recruitment of Draper to severed ORN axons or the clearance of axonal debris from the brain. It is suspected that this is because mz0709-Gal4-mediated knockdown of dced-6 is incomplete in ensheathing glia, based on reduced but not eliminated staining in this background. Nevertheless, the observations that dCed-6 is localized with Draper in immunostains, is required in glia by RNAi knockdown with repo-Gal4, and that a null allele of dced-6 genetically interacts with draper null mutations in axon engulfment, argues strongly for a role for dCed-6 in ensheathing glia (Doherty, 2009).

Moreover, knockdown of draper in ensheathing glia, or suppressing glial engulfing activity by blocking endocytic function with Shibirets fully suppresses the recruitment of Draper to severed axons and clearance of degenerating axonal debris from the brain. In contrast, astrocytes do not express Draper (or dCed-6), fail to respond morphologically to axon injury, and knockdown of draper in astrocytes has no effect on the recruitment of Draper to severed axons or clearance of axonal debris from the CNS. Such a separation of phagocytic function in neuropil glial cells suggests similarities to the assigned functional roles in mammalian glia, with ensheathing glia as resident phagocytes, engulfing the majority or all axonal debris and astrocytes perhaps playing a less important role in phagocytosis of degenerating axons (Doherty, 2009).

It is concluded that ensheathing glia are the phagocytes of the central brain, responsible for engulfing degenerating ORN axons after axotomy. Based on their expression of Draper and dCed-6 it is proposed that cortex glia play a similar role in the cortex, perhaps engulfing degenerating axons in this tissue, or cell corpses generated during neuronal development or after brain injury. Drosophila astrocytes, in contrast, are in close association with synapse rich regions of the brain and it is speculated they likely play an important role in neural circuit and synapse physiology. This work represents the first functional dissection of glial subtypes in the central brain of adult Drosophila, and lays the foundation for future functional studies of these diverse classes of glia (Doherty, 2009).

Draper/CED-1 mediates an ancient damage response to control inflammatory blood cell migration in vivo

Recent studies in zebrafish have shown that wound-induced H2O2 is detected by the redox-sensitive Src family kinase (SFK) Lyn within responding blood cells. This study shows the same signaling occurs in Drosophila inflammatory cells in response to wound-induced H2O2 with mutants for the Lyn homolog Src42A displaying impaired inflammatory migration to wounds. Activation of Src42A is necessary to trigger a signaling cascade within the inflammatory cells involving the ITAM domain-containing protein Draper-I (a member of the CED-1 family of apoptotic cell clearance receptors) and a downstream kinase, Shark, that is required for migration to wounds. The Src42A-Draper-Shark-mediated signaling axis is homologous to the well-established SFK-ITAM-Syk-signaling pathway used in vertebrate adaptive immune responses. Consequently, the results suggest that adaptive immunoreceptor-signaling pathways important in distinguishing self from non-self appear to have evolved from a more-ancient damage response. Furthermore, this changes the role of H2O2 from an inflammatory chemoattractant to an activator signal that primes immune cells to respond to damage cues via the activation of damage receptors such as Draper (Evans, 2015).


EFFECTS OF MUTATION

Glia are the most abundant cell type in the mammalian brain. They regulate neuronal development and function, CNS immune surveillance, and stem cell biology, surprisingly little is known about glia in any organism. This study identified over 40 new Drosophila glial genes. glial cells missing (gcm) mutants and misexpression were used to verify they are Gcm regulated in vivo. Many genes show unique spatiotemporal responsiveness to Gcm in the CNS, and thus glial subtype diversification requires spatially or temporally restricted Gcm cofactors. These genes provide insights into glial biology: unc-5 (a repulsive netrin receptor) is shown to orient glial migrations and the draper gene mediates glial engulfment of apoptotic neurons and larval locomotion. Many identified Drosophila glial genes have homologs expressed in mammalian glia, revealing conserved molecular features of glial cells. 80% of these Drosophila glial genes have mammalian homologs; these are now excellent candidates for regulating human glial development, function, or disease (Freeman, 2003).

An antibody was generated to the Draper intracellular domain, which is predicted to recognize all known protein isoforms. Draper protein was detected on the plasma membrane of all glia, including glial membranes ensheathing axon tracts, and outside the CNS on the membranes of macrophages. All Drpr immunoreactivity is absent in drprΔ5 mutant embryos. Several draper mutations were generated: a predicted null allele (drprΔ5) that is embryonic lethal and two hypomorphic alleles (drprEP(3)522 and drprΔ19) that are larval lethal. Embryos homozygous for the drprΔ5 null allele showed no defects in early CNS development (e.g., glial and neuronal specification, migration, proliferation), but did show clear defects in CNS cell corpse engulfment. Wild-type embryos had 25.9 ± 0.8 cell corpses per hemisegment, while drprΔ19 had 31.0 ± 0.9, and drprΔ5 had 43.6 ± 1.7. Consistent with this phenotype, strong Draper immunoreactivity was found on vesicles within glia that contain neuronal cell corpses. Together, these data indicate that draper is a downstream target of Gcm in glia and macrophages, it encodes a Ced-1-like transmembrane domain receptor expressed on glial and macrophage membranes, and it is required for cell corpse removal in the CNS (Freeman, 2003).

Draper protein is detected at high levels on glial membranes that ensheath motor nerves, so its role in motor neuron function was tested. Flies homozygous for hypomorphic draper alleles died as first instar larvae, which allowed assay of the role of draper in larval locomotion. It was found that drprEP(3)522 and drprΔ19 larvae were profoundly uncoordinated as judged by quantitative assays or by time lapse recordings. This phenotype is characteristic of a failure in glial ensheathment of neurons and suggests that Draper may be required for maintaining proper motor axonal physiology (Freeman, 2003).

Essential role of the apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning during Drosophila metamorphosis

Inhibition of glial engulfment suppresses pruning of the larval axon branches of γ neurons (Awasaki, 2004). Thus, if drpr has an essential role in glial engulfment, the loss of drpr function would affect pruning. To test this, the axon pruning of γ neurons using a drprD5 (Freeman, 2003) null mutant (Awasaki, 2006).

The axon branches of γ neurons were labelled specifically with cytoplasmic green fluorescent protein (cGFP) driven by GAL4-201Y (201Y>cGFP). In the control pupae, most of the larval axons in the MB dorsal lobe were pruned at 18h APF. Only a very small amount of larval γ axons were labeled with GFP at this stage. In comparison, however, most larval axons appeared to remain in the lobes of the drprD5 mutant. To quantitatively examine the amount of remaining larval axons, the number of pixels labeled with the GFP signal were compared within the volume of the dorsal lobe using stacks of confocal serial sections. In the control animals, the number of labeled pixels at 18 hr decreased to less than 5% of that of L3, while approximately 60% of the pixels remain labeled in the drprD5 mutant pupae at 18h APF. Thus, drpr mutation strongly suppressed axon pruning of the γ neurons. The development of the unpruned γ neurons was further examined in the drprD5 mutant in later stages (Awasaki, 2006).

The 201Y>cGFP labels γ neurons intensely through larval to adult stages. In the drprD5 mutants, the larval axon branches of the γ neurons remained in both the dorsal and medial lobes at 24h and 48h APF, though their total volume appeared to decrease. From 48h APF onward, 201Y>cGFP labeled a subset of newly emerging α/β neurons. To distinguish these neurons from the γ neurons, two more molecular markers were combined. In the adult brain, anti-FasII antibody labeled γand α/β neurons, but not α'/β' neurons. Anti-Trio antibody, in contrast, labeled γ and α'/β' neurons, but not α/β neurons. In the drprD5 young adult within 2 days after eclosionand also in 1 week old mature adults, there were abnormal axon branches outside of the α/β lobes. Because these axon branches were labeled by 201Y>cGFP, anti- FasII, and anti-Trio antibodies, they are likely to be the remnants of larval γ neurons. This finding indicates that without drpr function, at least some of the larval axon branches persist in the adult brain (Awasaki, 2006).

Of note, whereas the axons of the left and right β lobes never cross the midline in the control animals, these lobes were frequently fused in the midline in the drprD5 mutants. Fusion of the β lobes was observed after 48h APF, and was likely due to an overshoot of the medial axons of the α/β neurons. Thus, the drprD5 mutation affects not only the pruning of the larval axons, but also the precise formation of the adult neural circuits (Awasaki, 2006).

Next, the effect of the drprD5 mutation on the engulfing action of glia was examined. Once glial processes infiltrate the MB lobes, they form lumps to engulf and degrade clusters of γ neuron axon branches (Awasaki, 2004). To evaluate this process quantitatively, the amount of the infiltrating glial processes in the distal tip region of the MB dorsal lobe was measured. The glial processes and γ neurons were labeled with cGFP driven by GAL4-repo (repo>cGFP) and anti-FasII antibody, respectively. In the control pupae at 6h APF, the number of pixels occupied by the infiltrating glial processes corresponded to approximately 25% of the whole volume of the distal tip region. In the homozygous drprD5 mutant, less than 4% of the pixels were labeled by infiltrating glia. Though infiltrating glial processes were observed in a slightly larger number of pixels at 18h APF, it was still less than 7% of the whole volume of the distal tip. At this stage only a small amount of glial infiltration was observed in the control pupae. In the drprD5 mutant, the dorsal lobes were occupied by unpruned axon branches of the larval γ neurons. In contrast, the lobe structure was essentially collapsed in the control pupae, because most of the larval axon branches had disappeared (Awasaki, 2006).

In the early phase of axon pruning, glial processes form lumps that engulf clusters of varicosities on the axon branches (Awasaki, 2004). Because the diameter of the largest varicosities was less than 2 mm, the number of glial lumps that were larger than 2 mm were counted. In the control pupae at 6h APF, an average of 10.2 lumps was observed in the distal tip region. Several glial lumps were very large in size; an average of 2.0 large glial lumps were over 5 mm in diameter (Awasaki, 2006).

In the homozygous drprD5 mutant pupae, the formation of the glial lumps was greatly suppressed. There were very few glial lumps over 2 mm and no lumps over 5 mm at both 6h and 18h APF. There were a few small lumps that were less than 2 mm in diameter in the MB lobe at these stages. Although such small lumps were frequently observed in the control pupae, it was difficult to count their number precisely, because some of them were fused with larger lumps (Awasaki, 2006).

The engulfing action of glia, however, was not completely suppressed; there was an average of 0.2 and 0.5 glial lumps over 2 mm per MB lobe in the drprD5 pupae at 6h and 18h APF, respectively. In the drprD5 mutant, however, glial lumps were observed only near the periphery of the MB lobes in the vicinity of the glial cell bodies. In contrast, in the control pupae, engulfing glial lumps formed deep inside the lobes. In the control MB lobes, several clusters of regions were observed where FasII-positive axons were absent, apparently because of glial cell engulfment. Such FasII-negative clusters were not observed in the lobes of the drprD5. These findings suggest that both the infiltration of glial processes and the engulfment of larval axon branches are severely impaired in the drprD5 mutants (Awasaki, 2006).

It is possible that glial infiltration does occur, but that these processes become so thin that the GFP fluorescence is below the limits of detection. In fact, cGFP tends to visualize thicker processes strongly but thinner ones relatively weakly. To visualize thin glial processes more specifically, expression of the cytoskeleton-targeted Actin::GFP and membrane-tagged mCD8::GFP reporters were induced in glial cells using GAL4-repo in the drprD5 mutant. Although these reporters should label thin cellular processes very well, no signal of the infiltrating glial processes was detected in the lobes. Thus, the infiltration and engulfing action of glia is suppressed in drprD5 mutants (Awasaki, 2006).

Compared to Drpr, functional analysis of Ced-6 was difficult, because there are no ced-6 loss-of-function mutants available. Moreover, careful examination of confocal serial sections revealed that the anti-Drpr antibody labeled not only glial cells, but also weakly labeled the cell bodies of most neurons. It is therefore possible that some of the drprD5 mutant phenotypes observed might actually be due to the loss of neuronal drpr expression. Weak neuronal expression was also observed with the anti-Ced-6 antibody. To disturb drpr and ced-6 function specifically in the glial cells, RNA interference (RNAi) was induced in the glial cells by expressing double-stranded RNA of each gene with GAL4-repo. The effect of RNAi was examined by quantitative RT-PCR of the RNA extracted from the whole brain of 6h APF pupae. Whereas little drpr transcript was detected in the homozygous drprD5 mutant, the expression level of the drpr and ced-6 mRNA was reduced by approximately 25% and 65% with respective glial RNAi. Antibody staining no longer detected Drpr and Ced-6 proteins in the glial cells surrounding the MB dorsal lobe, whereas the weak staining in neurons was not affected. Therefore, it was concluded that glial RNAi effectively suppressed the specific expression of each gene in the glial cells. In glial RNAi of drpr, infiltration of the glial processes was severely suppressed, and there were only a few glial lumps over 2 mm and no lumps over 5 mm at 6h and 18h APF. There were only a few small glial lumps smaller than 2 mm. The phenotypes caused by glial RNAi were essentially the same as that of the homozygous drprD5 mutant. These results strongly suggest that the expression of drpr in the specific subset of the glial cells, but not in neurons, is essential for the engulfing action of glia (Awasaki, 2006).

The infiltration of glial processes and the formation of glial lumps were also severely suppressed in glial RNAi of ced-6 at 6h APF. At 18h APF, the amount of glial infiltration and the number of observed glial lumps somewhat increased. The number of observed glial lumps, however, was less than half of that observed in the control pupae at 6h APF (Awasaki, 2006).

Both Drosophila drpr and C. elegans ced-1 are essential for the engulfment of apoptotic cells (Ellis, 1991; Zhou, 2001; Freeman, 2003; Manaka, 2004). Thus, the glial cells might infiltrate the MB lobes to engulf the axons of the apoptotic neurons rather than to prune the axons of living neurons. Because γ neurons re-extend their axons after pruning of their larval axon branches, it is likely that most of them do not undergo apoptosis. This, however, has not been confirmed (Awasaki, 2006).

To test this, the existence of apoptotic cells was examined with anti-active caspase-3 antibody, which detects activated drICE and DCP1 caspases, in the cell body cluster of γ neurons at 0h, 6h, 12h, 18h, and 24h APF. Although there was a small number of cells labeled around the cluster of the MB neuron cell bodies, no cells were colabeled with 201Y>cGFP at the stages examined. This suggests that most of the γ neurons did not undergo apoptosis during early pupal stages. Furthermore, no axons of γ neurons in the MB lobes were labeled with the antibody, suggesting that few cells contained activated caspases. To further confirm the independence between glial engulfment and apoptosis, two caspase inhibitors, virus-derived p35 and Drosophila DIAP1, were expressed in the γ neurons. This did not affect the pruning of the axon branches and the engulfing action of glia. These results strongly suggest that glial engulfment of MB axon branches is independent of apoptosis and activation of caspases in the MB γ neurons. Therefore, glial cells do not engulf apoptotic neurons, but rather axons of the living neurons (Awasaki, 2006).

Drpr and Ced-6 were colocalized in the infiltrating glial processes, and the loss of either protein caused similar phenotypes. It was therefore asked whether there is a genetic interaction between drpr and ced-6 by examining the effect of the heterozygous drprD5 mutation on glial RNAi of ced-6. In the heterozygous drprD5 mutant, glial infiltration and lump formation were not affected. In ced-6 RNAi pupae at 18h APF, the suppression of the glial actions was weaker than that at 6h APF. When ced-6 RNAi and heterozygous drprD5 mutation coincided in the pupae, however, both glial infiltration and lump formation were significantly reduced. These findings suggest that drpr genetically interacted with ced-6 in the glial engulfment of the larval axon branches. Ced-6, an adaptor molecule, contains the phosphotyrosine binding (PTB) domain, which potentially interacts with the intracellular domain of Drpr, a scavenger receptor-like molecule. The purified intracellular region of Drpr was retained more effectively by Sepharose containing the GST-fused N-terminal half of Ced-6 than Sepharose containing either GST alone or the GST-fused C-terminal half of Ced-6. These suggest that the N-terminal half of Ced-6 might bind the intracellular region of drpr (Awasaki, 2006).

Even when the glial engulfing action was disrupted in the drprD5 mutant, the amount of the residual larval axon branches decreased only gradually during metamorphosis. This suggests that some of the larval axon branches can be pruned, albeit very slowly, even when the engulfing action of the glial cells is impaired (Awasaki, 2006).

Disruption of the microtubule cytoskeleton occurs in the early phase of larval axon pruning. Therefore whether this disruption is induced by glial engulfment or occurs independently from the glial action was examined. To visualize the microtubule cytoskeleton, Myc::α-tubulin fusion protein was expressed together with cGFP in γ neurons. In the control animals, Myc::α-tubulin was detected abundantly in the γ neuron axons in L3 but diminished at 6h APF. When Myc::α-tubulin was expressed in the drprD5 mutant, it was detected abundantly at L3 and diminished at 6h APF, just as in controls. There were no significant differences in the ratio of Myc::α-tubulin and cGFP signals in the MB dorsal lobe between control and drprD5 mutant pupae at L3 or 6h APF, indicating that disruption of the cytoskeleton occurs normally in the drprD5 mutant. This finding suggests that microtubule disruption occurs independent of glial engulfment (Awasaki, 2006).

If disruption of the microtubule cytoskeleton is not regulated by glial cells, it might be cell-autonomously regulated. Thus, Myc::α-tubulin and EcR-DN were coexpressed in γ neurons. The Myc::α-tubulin level was not decreased in the MB lobe at 6h APF. The ratio of Myc::α-tubulin and GFP signals in the MB dorsal lobe was not significantly different between L3 and 6h APF. This suggests that disruption of the microtubule cytoskeleton is cell-autonomously regulated by ecdysone through its receptors in γ neurons. These findings demonstrate that ecdysone orchestrates the disruption of the microtubule cytoskeleton in larval axons and the expression of drpr in a specific subset of glial cells in the same period to enable the pruning of unwanted larval axon branches with precise developmental timing. Glial cells therefore engulf degenerating axons in which the microtubule cytoskeleton has already been disrupted (Awasaki, 2006).

The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons

Neuron-glia communication is central to all nervous system responses to trauma, yet neural injury signaling pathways remain poorly understood. This study explores cellular and molecular aspects of neural injury signaling in Drosophila. Transected Drosophila axons undergo injury-induced degeneration that is morphologically similar to Wallerian degeneration in mammals and can be suppressed by the neuroprotective mouse Wallerian degeneration slow (Wlds) protein. Axonal injury elicits potent morphological and molecular responses from Drosophila glia: glia upregulate expression of the engulfment receptor Draper, undergo dramatic changes in morphology, and rapidly recruit cellular processes toward severed axons. In draper mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the CNS. Thus Draper appears to act as a glial receptor for severed axon-derived molecular cues that drive recruitment of glial processes to injured axons for engulfment (MacDonald, 2006).

Whether severed Drosophila axons undergo Wallerian degeneration and whether Drosophila glia respond to axon injury were tested. This study used the adult olfactory system to study neuron-glia interactions following nerve transection. This tissue is well-defined histologically and a number of useful reagents are available to label and manipulate olfactory receptor neurons (ORNs) and glia (MacDonald, 2006).

ORN cell bodies are housed in the third antennal segments or maxillary palps of adult Drosophila with axons projecting to the antennal lobe of the brain via the antennal or maxillary nerve, respectively. Axon projections from ORNs expressing the same odorant receptor (OR) gene converge on common, spatially discrete glomerular targets in the antennal lobe. Conveniently, these subsets of ORNs and their axons can be labeled and genetically manipulated using a number of available OR gene promoter-Gal4 driver lines, which drive UAS-regulated expression in highly reproducible subsets of ORNs. Glial cells in the antennal lobe can be identified based on their expression of the reversed polarity (repo) gene and labeled or manipulated using the repo-Gal4 driver. Glial cell nuclei were found at the edge of the antennal lobe neuropil; glial membranes delineate the boundaries of the antennal lobe and extend into the neuropil where they ensheath individual glomeruli (MacDonald, 2006).

Axon injury was induced by nonlethal surgical ablation of third antennal segments or maxillary palps. This ablation completely removed ORN cell bodies and fully transected the antennal or maxillary nerve, respectively. Degeneration of severed ORN axons and glial responses to axonal injury were monitored in vivo for several weeks after injury. For the majority of the experiments the OR22a-Gal4 driver was used to label subsets of antennal ORNs, or OR85e-Gal4 was used to label subsets of maxillary palp ORNs; however, similar results were obtained using additional OR-Gal4 driver lines (MacDonald, 2006).

Attempts were made to determine the time course and morphology of ORN axon degeneration after nerve transection. Three markers were used to visualize axons: UAS-mCD8::GFP (axonal membranes); UAS-α-tubulin::GFP (axonal microtubule cytoskeleton); and UAS-n-Synaptobrevin::YFP::YFP::YFP (UAS-n-Syb::3XYFP) (axon terminals). In control uninjured animals, OR22a+ axons (labeled with mCD8::GFP) had a smooth morphology as they projected across the antennal lobe, and GFP intensity in glomeruli was very strong. However, 1 day after ablation of third antennal segments, axon fibers outside glomeruli appeared highly fragmented and were undetectable at 3 or 5 days after injury. GFP signals in the OR22a-innervated glomeruli (termed DM2) remained near control levels 1 day after injury and were reduced to ~30% of control levels 3 days after injury, and nearly all GFP+ material was cleared from the CNS within 5 days. A similar profile of axon removal was observed with α-tubulin::GFP-labeled axons. It was found that axonal fibers were largely fragmented 1 day after injury and were absent by 3 days after injury. Interestingly, 1 day after injury, GFP signals within DM2 glomeruli were already reduced to ~50% of control levels. This observation suggests that the microtubule cytoskeleton may degenerate more rapidly than axonal membranes. GFP signals within DM2 glomeruli were near 15% of control levels; by 5 days after injury nearly all α-tub::GFP was cleared from the CNS. Finally, when OR22a+ axon terminals were marked with n-Syb::3XYFP, it was found that clearance of YFP+ axonal material occurred within 9 days after injury. Thus, axonal membranes and the microtubule cytoskeleton rapidly degenerate when Drosophila ORN axons are severed, clearance of axonal material within the DM2 glomerulus (i.e., membrane and cytoskeletal elements) was slightly delayed relative to axons outside glomeruli, and nearly all degenerating GFP+/YFP+-labeled axonal material was cleared from the antennal lobe within 5–9 days after axons were severed. A similar time course and morphology of axon fragmentation was observed when ORN axons were marked with the OR85e-Gal4 driver and ablated maxillary palps (MacDonald, 2006).

Axon degeneration appears to occur simultaneously in all portions of the severed axon rather than in a linear fashion along the length of the axon. For example, maxillary palp ORN axons in the maxillary nerve are closest to the site of injury (i.e., the ablated maxillary palp), while midline-crossing ORN axons within the antennal lobe commissure are the most distal to the site of injury. OR85e+ maxillary palp ORNs were labeled with mCD8::GFP, ablated maxillary palps, and it was found that bright GFP+ puncta (indicative of fragmentation) first appeared ~4 hr after injury. This occurred coincidently in the maxillary nerve and in midline crossing axons, suggesting that axon fragmentation can be initiated at any point along the axon. It was also found that axon destruction was specific to severed ORN axons. For example, if OR85e+ maxillary palp ORNs were labeled with mCD8::GFP and then ablated third antennal segments, OR85e+ ORN axons showed normal morphology even 9 days after injury. At this time point all antennal ORN axons were removed from the antennal lobe. Thus the cellular mechanisms mediating clearance of severed axons can discriminate between healthy and injured neurons, which suggests that severed axons are autonomously tagged for clearance from the CNS (MacDonald, 2006).

Degeneration of Severed ORN Axons is Suppressed by Wlds or Nmnat

The above data show severed Drosophila axons undergo injury-induced fragmentation that is morphologically similar to mammalian Wallerian degeneration. How similar are these events at the molecular level? Wallerian degeneration had long been thought to represent a simple wasting away of the severed axons due to a lack of nutrients supplied by the cell body. However, when nerves are transected in the spontaneous mutant mouse line C57BL/Wlds (for Wallerian degeneration slow) severed axons survive in a functionally competent state for weeks after injury (Glass, 1993; Lunn, 1989). This observation suggests that severed axons, rather than simply wasting away, may use an active autodestruction program to drive degeneration. Axon-sparing activity in the C57Bl/Wlds line has recently been found to map to a novel chimeric protein termed Wlds, which is generated from the fusion of two genes: Ube4b, an E4 ubiquitin ligase, and Nmnat1, a nicotinamide mononucleotide adenylyltransferase, a NAD+ biosynthetic and salvaging enzyme (Conforti, 2000; Mack, 2001). To determine whether Wlds-modulated mechanisms regulate axon degeneration in Drosophila, the ability of mouse Wlds to protect severed Drosophila axons from injury-induced degeneration was tested (MacDonald, 2006).

UAS-Wlds and UAS-mCD8::GFP were coexpressed in antennal ORNs using OR22a-Gal4, ablated third antennal segments, and subsequently scored axon morphology. Control animals (OR22a-Gal4 driving only UAS-mCD8::GFP) exhibited a normal rate of axon degeneration with all mCD8::GFP being absent by 5 days after injury. In striking contrast, Wlds+ axons did not undergo Wallerian degeneration: animals bearing a single copy of the UAS-Wlds transgene exhibited normal mCD8::GFP fluorescence in severed ORN axons 5 days after injury, and Wlds+ axons maintained normal morphology. Neuroprotective effects of Wlds was further explored by labeling axon terminals (with n-Syb::3XYFP) and the axonal microtubule cytoskeleton (with α-tubulin::GFP) and inducing injury. Impressively, in severed Wlds+ axons, n-Syb::3XYFP signals appeared morphologically normal and YFP intensity remained at control levels, even 9 days after injury. Similarly, in Wlds+ axons α-tubulin::GFP labeling appeared morphologically normal 5 days after injury, and GFP intensities in glomeruli remained at levels comparable to uninjured animals. Similar levels of protection of axons and their terminals were found with an independent UAS-Wlds insertion line (MacDonald, 2006).

How long can Wlds protect severed Drosophila axons? Severed axon morphology was examined in Wlds+ ORNs at 20, 30, and 50 days after injury. Remarkably, even 20 or 30 days after antennal ablation, Wlds+ ORN axons maintained largely normal morphology, and GFP intensity within glomeruli remained close to control levels. By 50 days after injury, the axons of Wlds+ neurons had degenerated significantly; only ~20% of antennal lobes contained detectable GFP+ axonal fibers, but mCD8::GFP signals within glomeruli remained at ~35% of control levels. These observations indicate that mouse Wlds can protect severed Drosophila axons from Wallerian degeneration for weeks, but severed Drosophila axons ultimately degenerate between 30–50 days after injury. It was also noted that these data show that mCD8::GFP is stable in axons for up to 50 days in the absence of transcription (MacDonald, 2006).

In vitro studies with mammalian DRG neurons suggest that (Nicotinamide mononucleotide adenylyltransferase 1) Nmnat1 provides the activity essential for Wlds-mediated neuroprotection (Araki, 2004; Wang, 2005). To determine whether Drosophila Nmnat (dNmnat) could protect severed axons from Wallerian degeneration in vivo, a UAS-dNmnat transgenic line was generated, dNmnat was coexpressed in OR22a+ ORNs with mCD8::GFP, ablated third antennal segments, and axon morphology was assayed 5 days after injury. dNmnat was found to protect severed axons from Wallerian degeneration 5 days after injury. The efficacy of protection may be reduced compared to Wlds, since 5 days after injury axon degeneration occurs at some level in dNmnat+, but not Wlds+, axons. Nevertheless, these data argue that Wlds-mediated axon protection in vivo occurs through dNmnat-dependent mechanisms. Together these data show that severed Drosophila axons undergo Wallerian degeneration, and the observation that Wlds potently suppresses this process in Drosophila argues strongly that Wlds-modulated mechanisms of severed axon autodestruction are conserved in Drosophila and mammals (MacDonald, 2006).

Drosophila Glia Rapidly Respond to ORN Axon Injury: Requirements for draper in glial morphological responses to antennal ORN injury

Glial cells are responsible for mediating postinjury events in the mammalian nervous system, but glial responses to injury have not been explored in Drosophila. This study explored morphological and molecular responses of Drosophila antennal lobe glia to axon injury. In control, uninjured animals, glial membranes were found to delineate the borders of the antennal lobe and were intimately associated with antennal lobe glomeruli. However, 1 day after antennal ablation, glia exhibited dramatic changes in morphology and appeared to significantly increase their membrane surface area. This expansion of glial membranes appeared to be a local response since antennal lobe glia, but not glia in surrounding brain regions, responded to ablation of third antennal segments in this way. During this injury response glial nuclei consistently remained at the periphery of the antennal lobe, indicating that glia responded to ORN injury by extending membranes toward severed axons rather than by migrating as an entire cell into the antennal lobe (MacDonald, 2006).

Mammalian microglia and Schwann cells, but not astrocytes, normally proliferate in response to neuronal injury; therefore Drosophila glia were assayed for injury-induced proliferative events. Third antennal segments were ablated, adult brains were costained with anti-Repo antibodies and the mitotic marker anti-phosphohistone H3 (PHH3), and Repo+/PHH3+ cells were assayed at 1, 3, 5, 7, and 9 days after ORN injury. No examples of mitotic glia were found at any of these time points after ORN injury, no gross increase in glial numbers surrounding the antennal lobe, nor evidence for proliferation in any other cell types in the brain after injury at these time points. Peripheral glial responses along the antennal nerve were assayed. In response to antennal ablation these peripheral glia do not proliferate, and no gross changes were observe in glial numbers along the antennal nerve (MacDonald, 2006).

To gain insight into how Drosophila glia might be responding to severed axons at the molecular level, a collection of embryonically expressed glial genes were assayed for those specifically enriched in antennal lobe glia 1 day after antennal ablation. Among these candidates draper, the Drosophila ortholog of the C. elegans cell corpse engulfment gene ced-1 was detected; draper has been shown to be required for glial engulfment of apoptotic neuronal cell corpses during embryonic development (Freeman, 2003). Such an engulfment receptor is an excellent candidate for driving glial removal of severed axons, though it would be somewhat surprising in light of the fact that CED-1/Draper is thought to play a role in the recognition of cell corpses, and severed axons degenerate via mechanisms that are molecularly distinct from apoptosis (MacDonald, 2006).

Using an antibody specific to Draper it was found that Draper is expressed in all Repo+ adult brain glia, including antennal lobe glia. Interestingly, 1 day after ablation of third antennal segments a dramatic increase was observed in Draper immunoreactivity in antennal lobe glia. Similar to the expansion of antennal lobe glial membranes after antennal ablation, increased glial Draper was found to be a local response to injury as only antennal lobe glia exhibited increased Draper after antennal ORN injury. All observed staining represents glial expression of Draper: anti-Draper immunoreactivity was absent in drprΔ5 null mutants, and glial specific knockdown of draper mRNA by dsRNAi removes all detectable Draper immunoreactivity in adult brains even after antennal ablation. Together these data indicate that Drosophila glia rapidly respond to ORN axon injury with changes in morphology and Draper protein levels. How glial membranes specifically interact with severed axons has been further explored, and the Draper receptor is shown to be essential for all glia responses to ORN axon injury (MacDonald, 2006).

The third antennal segments each house ~600 ORNs, and their ablation severs axons that project to ~44/50 antennal lobe glomeruli. Such ablations resulted in a robust upregulation of Draper and a dramatic expansion of antennal lobe glial membranes, but precisely how antennal lobe glia were interacting with severed axons under this conditions was unclear. For example, were glial membranes invading the antennal lobe and extending specifically toward severed axons? To explore glial membrane dynamics after ORN axon injury with higher resolution a second ORN injury assay was used: maxillary palp ablation. The maxillary palp houses only ~60 ORNs which collectively innervate ~6 glomeruli, all positioned in the ventro-medial region of the antennal lobe. Ablation of maxillary palps should therefore result in the injury of only this small subset of ORNs. If glial processes are recruited to severed ORNs, it is predicted that glial Draper and membranes would be specifically targeted to this subset of maxillary palp-innervated antennal lobe glomeruli after maxillary palp ablation (MacDonald, 2006).

Prior to injury Draper immunoreactivity was only weakly detectable within antennal lobe glomeruli and along the length of the maxillary nerve. However, 1 day after maxillary palp ablation, Draper immunoreactivity was found at high levels in ~6 ventro-medially positioned antennal lobe glomeruli—one of these was positively identified as maxillary palp-innervated with the OR85e-Gal4 driver—and along the entire length of the maxillary nerve that was visible in preparations. Draper localization to severed axons was observed beginning as early as 4 hr after maxillary palp ablation, colocalizing coincidently with the appearance of GFP+ puncta from degenerating axons, and Draper levels on severed axons appeared to be strongest between 12 hr and 1 day after injury. Draper protein was maintained at elevated levels on all injured axonal elements while they were still detectable (by GFP signals) and returned to control levels after axons had been cleared from the CNS. Interestingly, maxillary palp ablation did not lead to a dramatic upregulation of Draper protein in all antennal lobe glia as was seen after ablation of the third antennal segment. Thus, injuring a smaller number of ORN axons through maxillary palp ablation leads to a qualitatively different response by glia in the antennal lobe (MacDonald, 2006).

Glial membranes showed a similar pattern of rapid and specific localization to severed maxillary palp ORN axons after injury. Prior to injury GFP-labeled glial membranes were detectable at low levels around (but not within) glomeruli and at low levels along the maxillary nerve. However, 1 day after ablation of maxillary palps, glial membranes were found to be enriched within ~6 ventro-medially positioned antennal lobe glomeruli and along the entire maxillary nerve. Glial membrane-decorated glomeruli perfectly overlapped with those showing high-level Draper immunoreactivity and presumably represent glomeruli housing severed maxillary palp ORN axons (MacDonald, 2006).

In summary, glial-expressed Draper and glial membranes are rapidly and specifically recruited to severed ORN axons; their localization is coincident with the initiation of severed axon fragmentation; and Draper protein remains associated with degenerating axons until they are cleared from the CNS. These data further demonstrate that severed Drosophila axons generate molecular cues that elicit potent responses from glia and that these neuron→glia injury signals are sufficient to drive the selective recruitment of glial processes to severed axons (MacDonald, 2006).

In C. elegans, CED-1 has been shown to be essential for the engulfment of cell corpses and is believed to act as a recognition receptor for molecular cues presented by corpses (Zhou, 2001). Additional cellular targets for CED-1 have not been identified. This study shows that Draper is required to drive injury-induced changes in glial morphology and for glial clearance of severed axons from the CNS (MacDonald, 2006).

The requirements for draper in glial morphological responses to antennal ORN injury were determined. In control animals 1 day after antennal ablation a widespread expansion of antennal lobe glial membranes was observed. In contrast, when third antennal segments were ablated in drprΔ5 mutants, antennal lobe glia showed no noticeable changes in morphology or GFP intensity. It is noted that antennal lobe morphology was grossly normal in drprΔ5 mutants: ORN axons projected to the correct glomeruli, glomeruli appeared well-defined morphologically, and glial membrane processes were found in their normal positions ensheathing antennal lobe glomeruli. Together these data indicate that Draper is not required for antennal lobe development, but that glial morphological responses to antennal ORN axon injury require Draper signaling (MacDonald, 2006).

Maxillary palps were ablated and glial recruitment to the severed maxillary palp ORN axons was assayed. In control animals glial membranes were found to be highly enriched in maxillary palp-innervated glomeruli and on the maxillary nerve 1 day after injury. However, these responses were blocked in drprΔ5 mutants: glial processes did not accumulate in glomeruli housing severed maxillary palp ORN axons, nor were they recruited at high levels to the maxillary nerve. The autonomy of Draper function was further assayed by glial-specific RNAi knockdown of Draper. It was found that blocking Draper function in glia also fully suppressed glial responses to antennal or maxillary palp ablation, consistent with a requirement for Draper in glia. Thus all morphological changes exhibited by glia after injury (i.e., membrane expansion and process extension toward severed axons) require Draper signaling. These observations suggest that Draper may act as a glial receptor for severed axon-derived cues that drive glial responses to axon injury (MacDonald, 2006).

CED-1/Draper encodes a receptor essential for engulfment of cell corpses in both C. elegans (Zhou et al., 2001) and Drosophila (Freeman, 2003). Is Draper required for glial clearance of degenerating axons from the CNS? To test this the OR22a+ subset of antennal ORN axons in the drprΔ5 null mutant background was marked with mCD8::GFP and removal of injured axons was assayed. In control animals severed ORN axons outside glomeruli were cleared from the CNS by 3 days after injury (20/20 antennal lobes), and GFP+ axonal material was cleared from the CNS within 5 days. In striking contrast, the majority of axonal debris lingered in the CNS of drprΔ5 mutants after ablation of the third antennal segments. For example, 3 days after injury there was an abundance of GFP-labeled OR22a+ axon fibers remaining in the antennal lobe (20/20 antennal lobes). Similarly, 5 days after injury in drprΔ5 mutants, ~65% of antennal lobes retained GFP+ axonal fibers and GFP levels in DM2 glomeruli remained at levels comparable to control uninjured animals. Severed axon fragmentation was not blocked in drprΔ5 mutants. Severed axons showed signs of fragmentation as early as 5 hr after injury in drprΔ5 mutants—this approximates the earliest time points at which axons fragmented in control animals—and axon fibers were highly fragmented in these animals 3 and 5 days after injury. These data indicate that Draper is essential for glial clearance of degenerating axons from the Drosophila CNS, and axons thus represent a new engulfment target for this receptor. These observations also indicate that severed axons actively communicate with glia (via Draper) to promote their clearance from the CNS.

It is proposed that Draper acts as a glial receptor for severed axon-derived molecular cues, and that a Draper ligand encodes a neuron→glia injury signal that drives glial responses to axon injury. The ligand on cell corpses recognized by CED-1 remains to be identified, but is thought to encode the “eat-me” signal that initiates engulfment. Do severed axons, developmentally pruned axons, and cell corpses present similar molecular cues for engulfment? This may indeed be the case since CED-1/Draper is required for the clearance of each of these engulfment targets. However, Wallerian degeneration, developmental axon pruning, and apoptotic cell death are clearly distinct at the molecular level. For example, Wlds expression in neurons does not block apoptosis in neuronal cell bodies, nor the developmental pruning of Drosophila mushroom body γ neurons. Reciprocally, inhibition of canonical cell death pathways is not sufficient to block Wallerian degeneration. Nevertheless, it is possible that Wallerian degeneration, developmental axon pruning, and apoptotic cell death, though promoted by different molecular mechanisms, lead to the production of the same engulfment cue that acts as a ligand for CED-1/Draper (MacDonald, 2006).

Degenerating axons and cell corpses could also generate distinct engulfment cues that are each recognized by CED-1/Draper. One model would be that CED-1/Draper would not discriminate these targets, but only drive their nonspecific engulfment. Alternatively, distinct Draper receptor isoforms might recognize specific ligands presented by a cell corpse or a severed axon and potentially play an important role in discriminating these engulfment targets. Intriguingly, at least two isoforms of Draper have been identified in Drosophila that vary significantly in their extracellular domains; perhaps these bind distinct ligands on engulfment targets in vivo. Future studies aimed at defining the functional requirements of specific Draper receptor isoforms in the engulfment of cell corpses, developmentally pruned axons, and severed axons, as well as studies aimed at identifying the CED-1/Draper ligand(s) presented by these engulfment targets, should help resolve these issues (MacDonald, 2006).

Glial Responses to Severed Axons Are Suppressed by Wlds and dNmnat

Draper becomes localized to severed axons ~4 hr after injury, coincident with the initiation of axon fragmentation. Is axon degeneration essential to recruit glial processes in Drosophila? Mouse Wlds and dNmnat were used as tools to block ORN axon degeneration and glial responses were assayed to these severed, but nondegenerating, axons. UAS-Wlds or UAS-dNmnat were coexpressed with UAS-n-Syb::3XYFP in OR85e+ maxillary palp ORNs, ablated maxillary palps, and subsequently Draper localization to severed axons was assayed. Interestingly, while Draper was detected at high levels 1 day after maxillary palp ablation in 5/6 maxillary palp ORN-innervated glomeruli, Wlds-expressing axons showed control levels of Draper immunoreactivity. Thus Wlds expression is sufficient to potently suppress glial recruitment to severed axons. dNmnat was also found to suppress glial recruitment to severed axons, though, as was found in severed axon protection experiments, dNmnat was not as efficient at suppressing glial recruitment to severed axons. Nevertheless, this observation suggests that dNmnat activity is an important requirement for Wlds to block glial responses to severed axons. Together these data indicate that production of the molecular cues in severed axons that elicit glial responses is genetically downstream of Wlds and are consistent with axon fragmentation being essential for robust glial responses to injury. In addition, the results indicate that Wlds acts in a cell-autonomous fashion in Drosophila, as the recruitment of glial processes to severed axons is suppressed in Wlds-expressing axons but not in adjacent wild-type injured axons (MacDonald, 2006).

The mechanism by which Wlds exerts its neuroprotective effects remains unclear. One model proposes that Wlds acts prior to injury in the nucleus through the NAD binding histone deacetylase Sirt1 to effect neuroprotective changes in gene expression. In contrast, a second model proposes that Wlds acts locally in axons after injury to maintain high NAD levels which ultimately block axonal degeneration. Although transfection of Nmnat1 into DRG explant cultures indeed suppressed Wallerian degeneration in vitro, it was not known whether Nmnat1 could protect severed axons in vivo. This study has shown that dNmnat can suppress severed axon autodestruction in Drosophila; however, dNmnat appears less efficient than Wlds in protecting axons and suppressing glial responses to axon injury. The results therefore support the notion that Nmnat activity is an important component of Wlds neuroprotective function in vivo, but indicate that Wlds somehow provides more effective neuroprotection than dNmnat (MacDonald, 2006).

Wlds protection distinguishes axon degeneration following injury from naturally occurring developmental pruning

Axon pruning by degeneration remodels exuberant axonal connections and is widely required for the development of proper circuitry in the nervous system from insects to mammals. Developmental axon degeneration morphologically resembles injury-induced Wallerian degeneration, suggesting similar underlying mechanisms. As previously reported for mice, this study shows that Wlds protein substantially delays Wallerian degeneration in flies. Surprisingly, Wlds has no effect on naturally occurring developmental axon degeneration in flies or mice, although it protects against injury-induced degeneration of the same axons at the same developmental age. By contrast, the ubiquitin-proteasome system is intrinsically required for both developmental and injury-induced axon degeneration. The glial cell surface receptor Draper is required for efficient clearance of axon fragments during developmental axon degeneration, similar to its function in injury-induced degeneration. Thus, mechanistically, naturally occurring developmental axon pruning by degeneration and injury-induced axon degeneration differ significantly in early steps, but may converge onto a common execution pathway (Hoopfer, 2005).

Selective pruning of exuberant axons and axon branches occurs widely from insects to mammals and plays an essential role in the proper wiring of the nervous system. A major form of developmental axon pruning occurs via degeneration, qualitatively characterized by axon fragmentation and the subsequent removal of fragments by other cells. Axon degeneration was first reported in the large-scale remodeling of the retinotopic map in the optic tectum/superior colliculus (SC). Recently, pruning of Drosophila mushroom body (MB) γ neuron axons was demonstrated to occur via degeneration, as shown by axon fragmentation and glial engulfment of axon fragments. Developmental axon pruning can also be achieved by a distal-to-proximal retraction of the axon. Axon retraction is used in the stereotyped pruning of the infrapyramidal bundle of hippocampal mossy fibers, and has been directly observed for short branches of Cajal-Retzius and thalamocortical axons in the developing cerebral cortex. In the same study, pruning of long branches of thalamocortical axons was found to occur via degeneration, supporting the general trend that pruning of long portions of primary axons or their collateral branches occurs via degeneration. Additionally, synapse elimination at the developing neuromuscular junction in mammals occurs via an “axosome shedding” mechanism: motor axon terminals appear to undergo a distal-to-proximal retraction, but shed membrane-enclosed axosomes at the distal ends, which are engulfed by surrounding Schwann cells. Thus, although multiple mechanisms exist, axon degeneration is commonly used for developmental pruning, particularly for longer axon segments (Hoopfer, 2005 and references therein).

Axon degeneration during developmental pruning in flies and mammals occurs within a short period of time along the entire length of the axon segments to be pruned. This resembles fragmentation of distal axons in response to axon severing, a process known as Wallerian degeneration. Wallerian degeneration is characterized by the rapid breakdown of the cytoskeleton and fragmentation along the entire length of axon segments distal to the injury, usually occurring after a variable waiting period after the initial insult. Indeed, the progression of axonal fragmentation occurs with such rapidity that most axons appear either fully intact or completely fragmented at a given time postinjury. Axon fragmentation is followed by a series of responses from surrounding cells, including recruitment of microglia and macrophages to the severed axons. These morphological similarities between axon degeneration during developmental pruning and after injury raise the possibility that they share similar mechanisms (Hoopfer, 2005 and references therein).

Studies of Wallerian degeneration slow (Wlds) mice have provided major mechanistic insights into Wallerian degeneration. Compared to the rapid degeneration of severed axons within days in wild-type (wt) mice, Wlds mice exhibit much slower Wallerian degeneration: distal parts of severed axons persist for one to two weeks without obvious signs of fragmentation. Wlds is a dominant mutation resulting in the overexpression of a fusion protein of the first 70 amino acids of UFD2/E4, an evolutionarily conserved protein used in protein polyubiquitination, and the full-length nicotinamide mononucleotide adenylyltransferase (Nmnat), an enzyme that facilitates NAD synthesis. The effect of Wlds is autonomous to injured neurons, and neuronal expression of Wlds protein protects against the degeneration of severed distal axons in a dose-dependent manner. In vitro studies suggest that the Nmnat part of the fusion protein is responsible for its protective effect (Araki, 2004; Wang, 2005). Remarkably, Wlds also has protective effects in a variety of disease models, including motor axon “dying back” caused by a tubulin chaperone mutation, axon atrophy caused by demyelination, axonal spheroid pathology in gracile axonal dystrophy mice, and axon degeneration in a Parkinson's disease model (Hoopfer, 2005 and references therein).

Given its widespread protective effect against injury- and disease-induced axon degeneration, can Wlds interfere with developmental axon pruning that occurs by degeneration? How similar are the mechanisms involved in axon degeneration during developmental pruning and after injury? This study reports that in Wlds mice, pruning of both the retinocollicular and Layer 5 subcortical projections occur normally, indistinguishable from wt mice in the extent and timing. By contrast, Wlds protects axons of developing retinal ganglion cells (RGCs) from degeneration following complete transection of the optic nerve in the first postnatal week, the same developmental age at which Wlds has no effect on the naturally occurring developmental degeneration of these same axon projections. It is further shown that the expression of mouse Wlds protein has no effect on developmental Drosophila MB γ axon pruning, despite its potent effect in protecting fragmentation of severed axons in a Drosophila injury model (see also MacDonald, 2006. By comparing axon degeneration during developmental pruning and after injury, it was found that they share similarities in the neuron-intrinsic requirement of the ubiquitin-proteasome system (UPS) and Draper-mediated clearance of degenerating axon fragments by glia, but differ substantially in early steps that trigger axon fragmentation. These data provide insights into both developmental axon pruning and Wallerian degeneration (Hoopfer, 2005).

Therefore naturally occurring developmental pruning of axons resembles injury-induced Wallerian degeneration of axons in adults. These two processes share some molecular mechanisms: they both depend on UPS action in neurons and axon fragment clearance by the cell corpse engulfment receptor Draper in glia. However, this analysis also revealed fundamental differences between developmental and injury-induced axon degeneration. Whereas expression of Wlds protein potently delays axon degeneration after injury in adult flies and in both developing and adult mice, it does not affect axon degeneration in three developmental axon-pruning paradigms in either organism. It is proposed that axon degeneration during developmental pruning and after injury differ in the early steps leading to axon fragmentation but may later converge onto a common pathway (Hoopfer, 2005).

Beyond the morphological features of axon fragmentation and engulfment of fragments by extrinsic cells, axon degeneration during developmental pruning and after injury share additional similarities. Both processes require cell-autonomous programs, neither requires caspase-dependent apoptotic machinery, and microtubule fragmentation/disappearance is an early sign of axon degeneration in both cases. By systematically comparing developmental and injury-induced axon degeneration in Drosophila, additional mechanistic similarities were found that likely extend to vertebrate systems (Hoopfer, 2005).

MB γ axon pruning requires the intrinsic activity of the UPS (Watts, 2003). UPS inhibition significantly delays injury-induced axon degeneration. At least at early stages, UPS inhibition appears to delay axon fragmentation. This is similar to an in vitro Wallerian degeneration model in mammals, in which inhibition of UPS by application of a proteasome inhibitor or expression of UBP2 delays axon fragmentation. These studies suggest that UPS action in facilitating axon fragmentation after injury is a conserved feature from flies to mammals (Hoopfer, 2005).

Although UPS inhibition can block developmental axon pruning completely under appropriate conditions (Watts, 2003), it cannot prevent injured axons from eventual degeneration. One possible explanation for this difference could be because of the short window of time in which developmental pruning occurs: if pruning is delayed beyond this “critical period,” neurons may no longer be in a competent state to carry out the degenerative pruning program. Consistent with this notion is the recent finding of a critical period for large-scale axon degeneration of the retinocollicular projection coincident with the brief time window during which the projection normally undergoes pruning. In the case of Wallerian degeneration after complete axon transection, since the injury physically separates the axons from their cell bodies, the most that can be done is to prolong the inevitable onset of fragmentation of the already severed axon. However, many neurodegenerative disease models exhibit Wallerian-like axon degeneration, which can be ameliorated by Wlds expression. Thus, understanding the mechanisms that trigger axon fragmentation may lead to therapeutic strategies to stop axon degeneration before it begins (Hoopfer, 2005).

MacDonald (2006) has demonstrate a role for glia in the clearance of axon fragments in a Drosophila model of Wallerian degeneration. In particular, a requirement of the cell corpse engulfment receptor Draper in glia for this function was demonstrated. This study finds that removal of Draper in glia markedly delays clearance of axon fragments during MB axon pruning. In addition, ultrastructural analysis of degenerating axons after injury reveals degenerating profiles similar to what have been found during MB axon pruning (Watts, 2004). These similarities strongly suggest that mechanisms for this late phase of axon degeneration and clearance are shared between developmental pruning and Wallerian degeneration (Hoopfer, 2005).

Glial function has not been systematically explored in developmental axon pruning in mammals. However, the transient appearance in early postnatal cats of clusters of macrophages contiguous to the corpus callosum during the period of callosal axon pruning suggests that these cells might serve a phagocytic function in vertebrate developmental axon pruning analogous to that of Draper-expressing glia in flies (Hoopfer, 2005 and references therein).

An important finding of this study is that developmental axon degeneration and Wallerian degeneration can be clearly distinguished mechanistically by their differential sensitivity to the protective effect of Wlds. Remarkably, this mouse fusion protein can also potently protect axon degeneration after injury in flies. Despite the phylogenetically conserved function of Wlds in protecting injured axons, this study shows in three diverse developmental degeneration paradigms in mice and flies that Wlds expression has no effect on naturally occurring developmental axon pruning. In particular, the injury-induced degeneration of transected RGC axons is markedly protected in Wlds mice at the same age when naturally occurring degenerative pruning of the same developing RGC axons is not protected. This finding demonstrates unequivocally the fundamental difference between axon degeneration during developmental pruning and after injury in the same type of neuron at the same developmental stage. Similarly, Wlds protects young motor axons from degeneration after injury while having no effect in the remodeling of neuromuscular junction, although such remodeling uses a distinct mechanism (Hoopfer, 2005).

What might be the reasons behind this difference between developmental and injury-induced axon degeneration? The trigger for naturally occurring developmental axon degeneration and injury-induced Wallerian degeneration differs, as do their respective roles. Wallerian degeneration is triggered extrinsically by axonal insult. Because degeneration is restricted to the portion of the axon distal to the injury site, it must be executed without new transcription or transport from the cell body. These properties suggest that the effectors of the program are continuously present along the entire length of axons. The function of Wallerian degeneration could be to remove the damaged axons as a step in facilitating regeneration and repair (Hoopfer, 2005).

In contrast to Wallerian degeneration, the role of developmental axon degeneration is to remodel initially exuberant neuronal connections into a functionally appropriate adult circuit. It is likely triggered by diverse mechanisms including cell-autonomous transcriptional regulation, patterned neuronal activity, the local environment of the axon, or their combination. For example, MB γ axon pruning is triggered by activation of the steroid hormone receptor EcR to cell-autonomously regulate gene expression. In the case of the retinocollicular projection, spatial control of pruning is instructed by the distribution and levels of EphA receptors along the length of RGC axons, which is controlled in part by transcriptional regulation autonomous to the RGC. Non-RGC autonomous mechanisms, including the level of ephrin-As to which an RGC axon segment is exposed within the target as well as correlated patterns of RGC activity generated by networks of RGCs and cholinergic amacrine cells that produce spontaneous retinal waves, are also essential factors for the pruning of inappropriate axon segments (Hoopfer, 2005).

It remains a future challenge to investigate mechanistically how diverse triggers in developmental pruning, injury, and disease lead to similar late stage execution of axon degeneration. The findings of both a differential effect of Wlds in these processes and similar roles for the intrinsic activity of UPS and glial clearing of fragmented axons provide an essential step toward this goal (Hoopfer, 2005).

Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages

The mechanism of phagocytic elimination of dying cells in Drosophila is poorly understood. This study was undertaken to examine the recognition and engulfment of apoptotic cells by Drosophila hemocytes/macrophages in vitro and in vivo. In the in vitro analysis, l(2)mbn cells (a cell line established from larval hemocytes of a tumorous Drosophila mutant) were used as phagocytes. When l(2)mbn cells were treated with the molting hormone 20-hydroxyecdysone, the cells acquired the ability to phagocytose apoptotic S2 cells, another Drosophila cell line. S2 cells undergoing cycloheximide-induced apoptosis exposed phosphatidylserine on their surface, but their engulfment by l(2)mbn cells did not seem to be mediated by phosphatidylserine. The level of Croquemort, a candidate phagocytosis receptor of Drosophila hemocytes/macrophages, increased in l(2)mbn cells after treatment with 20-hydroxyecdysone, whereas that of Draper, another candidate phagocytosis receptor, remained unchanged. However, apoptotic cell phagocytosis was reduced when the expression of Draper, but not of Croquemort, was inhibited by RNA interference in hormone-treated l(2)mbn cells. Whether Draper is responsible for the phagocytosis of apoptotic cells in vivo was examined using an assay for engulfment based on assessing DNA degradation of apoptotic cells in dICAD mutant embryos (which only occurred after ingestion by the phagocytes). RNA interference-mediated decrease in the level of Draper in embryos of mutant flies was accompanied by a decrease in the number of cells containing fragmented DNA. Furthermore, histochemical analyses of dispersed embryonic cells revealed that the level of phagocytosis of apoptotic cells by hemocytes/macrophages was reduced when Draper expression was inhibited. These results indicate that Drosophila hemocytes/macrophages execute Draper-mediated phagocytosis to eliminate apoptotic cells (Manaka, 2004).

It has been suggested that there are two signaling pathways that lead to the induction of engulfment in phagocytes. These pathways are most likely to be triggered by distinct ligands, to be mediated by distinct receptors, and to involve partially overlapping signal mediators and downstream uptake mechanisms. The membrane receptors called CED-1 and phosphatidylserine receptor PSR-1 (Wang, 2003) have been genetically shown to act farthest upstream in the pathways in C. elegans (Manaka, 2004 and references therein).

In Drosophila, Croquemort, a member of the CD36 family of proteins, is the only protein known to be involved in phagocytosis of apoptotic cells by hemocytes/macrophages. However, Croquemort appears to be structurally unrelated to either CED-1 or PSR-1, and the identity of its ligand or how it transduces signal(s) for phagocytosis remains unknown. A Drosophila homologue of C. elegans CED-1 has been suggested to play a role in phagocytosis by glia (i.e. loss of Draper expression caused an increase in the number of apoptotic neurons in the central nervous system of Drosophila embryos). This study confirms the role of Draper in glia in a more direct analysis and, more importantly, found that Draper is also involved in the phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages. This indicates that at least one of the two signaling pathways for phagocytosis of apoptotic cells in C. elegans exists in Drosophila. In contrast, the existence of a Drosophila homologue of the mammalian PS receptor has been noted, but its role in the phagocytosis of apoptotic cells in Drosophila remains to be determined. The finding that Draper was not solely responsible for the phagocytosis by larval hemocyte-derived cell line l(2)mbn, embryonic hemocytes/macrophages, or embryonic glia suggests the presence of other phagocytosis receptors in those phagocytes. It is, however, unlikely, at least for l(2)mbn cells, that a homologue of the PS receptor is a putative additional phagocytosis receptor, because phagocytosis by 20-hydroxyecdysone-treated l(2)mbn cells was shown not to be mediated by PS present on the surface of target apoptotic cells. At this point, it is not clear whether or not PS serves as a phagocytosis marker at all in Drosophila (Manaka, 2004).

It is most probable that Draper induces engulfment of apoptotic cells in phagocytes by recognizing a specific ligand (i.e. a 'phagocytosis marker' molecule present on the surface of target cells). The ligand for Draper/CED-1, however, remains to be identified. PS appears not to be recognized by this receptor; this study that phagocytosis of PS-exposing apoptotic S2 cells by 20-hydroxyecdysone-treated l(2)mbn cells is mediated by Draper with no dependence on PS. A mammalian membrane protein called low density lipoprotein receptor-like protein (LRP, also known as CD91) shares some structural similarity with CED-1 and is considered to be its mammalian homologue (Su, 2002). Ogden (2001) has suggested that CD91 acts as a phagocytosis receptor due to its association with calreticulin, a molecular chaperone present in the endoplasmic reticulum. In addition, the involvement of calreticulin in the phagocytosis of yeast by hemocytes of another insect, Pieris rapae, has been reported. A Dictyostelium mutant lacking both calreticulin and calnexin, another molecular chaperone in the endoplasmic reticulum, shows a reduced level of phagocytosis of yeast, although both of these proteins appear to act not at the recognition step but at some downstream events during phagocytosis by Dictyostelium. Furthermore, like calreticulin, calnexin has been shown to translocate to the surface of mammalian cells under certain circumstances. Calreticulin and calnexin, which both exist in Drosophila, are thus good candidates for the ligand of Draper/CED-1 (Manaka, 2004 and references therein).

Treatment with 20-hydroxyecdysone does not alter the general phagocytic activity of l(2)mbn cells but augments the level of phagocytosis of apoptotic cells or zymosan particles. This suggests that the hormone causes quantitative and/or qualitative changes in l(2)mbn cells that are specific, if not solely responsible, for phagocytosis of apoptotic cells. A simple explanation would be an increase in the amount of Draper present at the surface of the phagocyte, but this was shown not to be the case. In contrast, the amount of Croquemort in l(2)mbn cells increases upon treatment with the hormone, but the results of an RNA interference experiment showed that this receptor was not involved in the phagocytosis of apoptotic cells by l(2)mbn cells. Another possibility is that Draper is functionally activated in hormone-treated l(2)mbn cells. This might be true, because it was found that migration of Draper protein in a polyacrylamide gel becomes a little bit faster after treatment with 20-hydroxyecdysone. Although no evidence of modification of CED-1 or Draper has been reported so far, the results suggest that Draper is structurally modified, through for example dephosphorylation or deglycosylation, and activated by 20-hydroxyecdysone (Manaka, 2004).

The innate immune system, like adaptive immunity, consists of humoral and cellular reactions. In Drosophila, the humoral responses, most of which are conducted through the action of the fat body (a functional equivalent of the mammalian liver), include production of antimicrobial peptides and melanization at wound sites or around microorganisms. In contrast, the cellular responses in Drosophila consist of phagocytosis and encapsulation of microorganisms by hemocytes, both of which events lead to killing of the invaders. Although the mechanism of the humoral responses has been well characterized, how Drosophila hemocytes act against invading microorganisms remains to be clarified. Phagocytosis of apoptotic cells by hemocytes is considered part of the cellular immune response of Drosophila. The data showed that Draper is involved in hemocyte phagocytosis of apoptotic cells but not of zymosans. It is thus anticipated that distinct systems are utilized by Drosophila hemocytes for the recognition of endogenous apoptotic cells and invading microorganisms. To clarify these recognition systems is necessary for a better understanding of cellular innate immunity in Drosophila (Manaka, 2004).

Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons

The removal of apoptotic cells by phagocytic neighbors is essential for metazoan development but remains poorly characterized. This study reports the discovery of a Drosophila phagocytosis receptor, Six-microns-under (SIMU), which is expressed in highly phagocytic cell types during development and required for efficient apoptotic cell clearance by glia in the nervous system and by macrophages elsewhere. SIMU is part of a conserved family of proteins that includes CED-1 and Draper (DRPR). Phenotypic analysis reveals that simu acts upstream of drpr in the same pathway and affects the recognition and engulfment of apoptotic cells, while drpr affects their subsequent degradation. SIMU strongly binds to apoptotic cells, presumably through its EMILIN-like domain, but requires no membrane anchoring, suggesting that it can function as a bridging molecule. This study introduces an important factor in tissue-resident apoptotic clearance and underscores the prominent role of glia as 'semiprofessional' phagocytes in the nervous system (Kurant, 2008).

The elimination of superfluous or damaged cells through programmed cell death plays an essential role in metazoan development and tissue homeostasis; its critical final stage is the clearance of the apoptotic cells through phagocytosis. The proper recognition, uptake, and degradation of dying cells is accomplished either by 'professional' phagocytes, such as macrophages and immature dendritic cells, or by 'nonprofessional' tissue-resident neighboring cells. While professional phagocytes have been studied extensively, relatively little is known about the biological significance and the molecular underpinnings of tissue-resident phagocytosis (Kurant, 2008 and references therein).

Apoptotic cell clearance is a complex process, involving recognition, engulfment, phagosome formation and maturation as distinct steps. The apoptotic cell displays distress ('eat me') signals that are recognized by the phagocyte, either directly by phagocytic receptors or indirectly through bridging molecules (opsonins), supplied systemically through the serum or secreted locally by the phagocyte. Two types of phagocytic receptors have been implicated in this recognition process: tethering receptors without a significant intracellular domain, such as CD36 or SRA, and docking receptors with noncatalytic intracellular domains permitting interaction with other proteins, such as CED-1 and its homolog Draper (DRPR), or LRP. The clustering of both types of receptors is thought to be required for the recruitment of the downstream machinery to the docking sites, which leads to cytoskeletal reorganization and engulfment of the apoptotic cell. The phagocytosis process is completed by the formation of a phagosome and its maturation to a phagolysosome, effecting the degradation of the apoptotic particle (Kurant, 2008).

Insight into the molecular mechanisms by which nonprofessional tissue-resident cells effect apoptotic clearance came initially from the worm, which does not have professional phagocytes. Genetic screens identified several phagocytosis genes that fall into two partially redundant pathways: one consists of a phagocytic docking receptor (CED-1), its adapter (CED-6), an ABC transporter (CED-7), and dynamin; the other pathway consists of an actin-regulating protein complex (CED-2/5/10/12) that presumably acts downstream of an unknown phagocytic receptor. Recently, the phosphatidylserine receptor BAI1 was found to act upstream of the homologous complex in mouse. In Drosophila, only three factors have been implicated in apoptotic cell clearance to date: the macrophage-specific CD36 homolog Croquemort (CRQ), the F Box protein Pallbearer, and the broadly expressed CED-1 homolog DRPR, which plays a role in glial phagocytosis of apoptotic neurons. A recent study (Hamon, 2006) suggests that the apoptotic clearance function of CED-1/DRPR is conserved in vertebrates (Kurant, 2008).

Thus, most of the players involved in tissue-resident clearance are still unknown, in particular the phagocytic receptors and their cognate ligands on the apoptotic cell. More generally, a better understanding of the cellular and molecular underpinnings of apoptotic clearance in vivo is highly desirable. In the work presented in this study, it is demonstrated that glia are the main phagocytes in the late developing nervous system of the fly. A new tethering receptor named Six-microns-under (SIMU), is described that functions in apoptotic clearance by both glia and macrophages; it acts upstream of DRPR in the same pathway. This study reveals an evolutionary expansion and concomitant specialization of the repertoire of CED-1-like receptors, with SIMU required for the recognition and engulfment, and DRPR for the degradation of corpses (Kurant, 2008).

The simu gene is predicted to encode a 377 aa transmembrane protein, comprising an N-terminal signal peptide, a large extracellular portion, a single putative transmembrane (TM) domain, and a short cytoplasmic tail at the C terminus. The extracellular portion of the protein consists of an N-terminal EMILIN (EMI)-like domain (Callebaut, 2003) and four EGF domains with highly stereotyped spacing of Cys residues, recently named Nimrod (NIM) repeats (Kurucz, 2007). Intriguingly, the combination of an EMI(-like) domain, with a highly conserved CC×GY motif at its C-terminal end and immediately followed by a NIM repeat, is found across the vertebrate/invertebrate divide in a small number of both secreted and transmembrane factors, some of which have been shown to function in phagocytosis. They include the CED-1 homologs in all species (DRPR, human MEGF10, mouse Jedi), the fly bacterial phagocytosis factor Eater (Kocks, 2005), and the large Nimrod gene cluster located at 34E (Kurucz, 2007), the most proximal member of which is simu (= NimC4), as well as human SREC (Kurant, 2008).

This study has demonstrated that tissue-resident clearance by neighboring glia plays a major role in the removal of apoptotic neurons during nervous system development and has characterized the function of the new tethering receptor SIMU in this important process. Using several different markers, it was shown that, once the nerve cord is ensheathed and macrophages no longer have access, astrocytic glia avidly phagocytose apoptotic cells in their vicinity. This pronounced phagocytic activity is reflected on a molecular level by a strong differential expression of phagocytosis genes in the glia. In vivo time-lapse recordings indicate that the stationary glia are as efficient and fast in corpse uptake as the highly motile macrophages, and differences in clearance are largely a reflection of differential motility. Notably, glia also phagocytose axon branches during neuronal remodeling and injury-induced Wallerian degeneration. Collectively, these findings indicate that glia are potent 'semiprofessional' phagocytes and that, at least during development, tissue-resident apoptotic clearance equals macrophage-mediated clearance in importance. In vertebrates, tissue-resident phagocytosis by neighbors has been demonstrated in vivo in some contexts including glia, but its overall contribution to apoptotic clearance and the underlying molecular mechanisms have not been established (Kurant, 2008).

The investigation of the gene simu was originally motivated by two intriguing features: its expression during all major epochs of developmental apoptosis and in the major phagocytic cell types -- in particular the glia -- and a protein domain structure that is similar to but distinct from that of the CED-1 homolog DRPR. The two proteins are expressed in strongly overlapping patterns throughout embryogenesis and largely colocalize at the plasma membrane of glia and macrophages. Genetic analysis now reveals that they are components of the same phagocytic pathway, with simu acting upstream of drpr. Observations both in fixed material and in time-lapse recordings show simu affecting the early steps of recognition and engulfment of apoptotic particles, while drpr affects their subsequent degradation; the double-mutant phenotype closely resembles that of simu alone, indicating epistasy of simu over drpr. Unlike DRPR, SIMU lacks a large cytoplasmic domain with docking sites for downstream effectors, and the results suggest that its molecular function is to recognize the apoptotic cell and tether it to the phagocyte. Purified SIMU protein strongly binds to apoptotic cells in vitro; the in vivo analysis indicates that the N-terminal EMI-like domain, which is essential for SIMU function, is likely involved in the recognition and binding. To date, EMI domains have only been implicated in protein-protein interaction, suggesting that SIMU may recognize a protein rather than a lipid or carbohydrate component on the apoptotic cell. The lack of a cytoplasmic domain and the dispensability of membrane anchoring for in vivo function imply that SIMU must interact, directly or indirectly, with docking receptors on the phagocyte surface to effect engulfment and subsequent degradation. Given its phenotype and action downstream of simu, drpr is the prime candidate for mediating degradation. However, the difference in the simu and drpr phenotypes (in particular the essentially unimpaired engulfment of apoptotic particles observed in drpr mutants) strongly argues for the existence of an intermediary factor that controls the cytoskeletal reorganization necessary for engulfment downstream of simu. The existence of a SIMU binding partner other than DRPR is supported by the finding that, in drpr null mutants, secreted SIMUDeltaTM still accumulates on macrophage cell surfaces, and by the fact that no physical interaction was observed between SIMU and DRPR in coimmunoprecipitation experiments. Thus, the following model seems most likely: As part of a linear pathway, SIMU interacts with a factor X, leading to the recruitment of cytoskeletal components for engulfment and phagosome formation, followed by X interacting with DRPR, which leads to the recruitment of degradation components for phagosome maturation. The identification of factor X and its extra- and intracellular binding partners will be the subject of future investigation, and understanding the mechanisms by which the different phagocytic receptors cluster and coordinate their function will be of particular interest (Kurant, 2008).

The existence of the SIMU protein was unexpected both genomically and genetically: the C. elegans genome does not contain a SIMU homolog and in fact no other EMI(-like)+NIM domain protein. Moreover, in contrast to drpr, CED-1 is crucial for recognition, engulfment, and the degradation of corpses in the worm, suggesting that biological functions that are performed by a single factor in the worm become distributed among more specialized proteins in the fly. Intriguingly, when the worm CED-1 mutant is rescued by a transgene that lacks the intracellular docking domain, recognition and engulfment, but not degradation, are restored, resulting in a drpr-like phenotype. Several recent papers have suggested that DRPR acts as an engulfment receptor in different developmental contexts in the fly. Most observed an increase in the number of apoptotic cells or axon fragments in drpr mutants, which is consistent with the current findings. Unlike the current analysis, however, none of these studies established whether the excess apoptotic particles accumulate outside or inside the respective phagocytes. It is also noteworthy that apoptotic clearance is much faster (minutes versus hours or days) than axon pruning/Wallerian degeneration and possibly involves distinct signals from the degrading cells (caspase versus non-caspase-mediated mechanism), making it likely that differences exist at the level of the recognition factors (Kurant, 2008).

The comparison of apoptotic clearance in worm, fly, and vertebrate provides interesting evolutionary perspectives. Between worm and fly, the demand for apoptotic clearance increases both in quantitative and qualitative terms: During worm development, only 10% of cells are fated to die, and they are removed shortly after their birth through phagocytosis by immediate neighbors, apparently without need for dedicated phagocytes. During fly development, a much larger proportion of cells die through apoptosis (>30%), and their death is spread out over a wide range of stages in their life cycle: immediately after their birth, during fating and differentiation, and even after long-term function. To meet this challenge, macrophages emerge as professional phagocytes, and in addition, several cell types differentially increase their phagocytic capacity and become semiprofessional phagocytes, including the ectoderm and the glia. In vertebrates, the apoptotic load is presumably even greater, and the cellular complexity of the innate immune system is of course greatly increased. Strikingly, these changes are accompanied by an increase in complexity at the molecular level, both in terms of a greater structural diversity of phagocytic receptors generally and of CED-1-like factors in particular. While C. elegans seems to have only one CED-1 gene encoding a single protein form, Drosophila has two different DRPR isoforms with distinct extracellular domains and the entire Nimrod gene family, including SIMU and Eater (Kurucz, 2007). This family contains both secreted and transmembrane proteins, and receptors for the clearance of apoptotic cells (SIMU) and pathogens (Eater, NIM C1), but the function of most family members is not known. The in vivo function of the vertebrate CED-1-like factors is also unknown, but cell culture experiments suggest that MEGF10 is a phagocytic receptor for apoptotic cells and SREC for (oxidized) LDL. The molecular comparisons indicate that these phagocytic factors all share an N-terminal EMI(-like)+NIM core, presumably for recognition, and that invertebrates and vertebrates amplified different EGF-type repeats present in the ancestral CED-1, either to achieve appropriate spacing or to facilitate homotypic cis interaction in factor clustering (Kurant, 2008).

This molecular expansion is likely accompanied by an increase in functional redundancy between phagocytic pathways: already in the worm, at least two (CED-1/6/7 and CED-2/5/10/12) and likely a third (currently unknown) pathway participate in the phagocytosis process. As shown in this study for the fly, joint removal of simu and drpr results in viable adults whose apoptotic clearance is impaired but not abrogated, indicating the existence of additional as-yet-unidentified pathways. Finally, in vertebrates, a large number of different types of macrophage receptors have been identified, which under single-mutant conditions either only reduce apoptotic clearance or have no effect at all (Kurant, 2008).

Overall, this study has shown that the differences between professional and nonprofessional phagocytes are perhaps more fluid than originally thought. Not only can tissue-resident phagocytic cells differentially upregulate a similar molecular repertoire of phagocytic pathways, but they also can use it as quickly and efficiently as the macrophages when confronted with apoptotic neighbors. This suggests a simple division of labor -- in freely accessible spaces, actively patrolling macrophages clear the corpses, while in sequestered spaces (such as the late nervous system) the task falls to resident neighbors like the glia. In addition to its role in development, the efficient clearance of apoptotic cells is essential for avoiding secondary necrosis and exposure of cytoplasmic components to the immune system, acutely causing inflammation and, chronically, autoimmune disease. While apoptotic clearance by tissue-resident cells has received relatively little attention in vertebrate research, its comprehensive analysis in genetic models such as the fly will continue to provide new and interesting insight into the underlying cellular and molecular mechanisms (Kurant, 2008).

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. 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, the widespread appearance of presynaptically derived 'debris' was observed 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, it was found 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 (Fuentes-Medel, 2009. Full text of article).

Previous studies have shown that the PTB-domain protein dCed-6 functions downstream of Draper. Therefore, RNAi knockdown of dCed-6 in muscle or glia was used as a second approach to blocking glial and muscle engulfment activity. As in draper mutants, downregulating dCed-6 in either muscle or peripheral glia resulted in significant decrease in the number of synaptic boutons. In contrast, no effect was observed when dCed-6-RNAi was expressed in motorneurons. Similar to Draper RNAi knockdown, expressing dCed-6-RNAi in muscles or glia had differential consequences for the appearance of presynaptic debris versus ghost boutons. Decreased levels of dCed-6 in muscles led to an increase in the number of ghost boutons, but had no influence in the deposition of presynaptic debris. Downregulating dCed-6 in glia, on the other hand, led to a significant increase in presynaptic debris deposition, but the number of ghost boutons remained unaltered. These results are consistent with the notion that dCed-6 functions downstream of Draper during the development of the NMJ. Further, they support the model that both muscle and glia contribute differentially to the clearance of debris versus ghost boutons at the NMJ (Fuentes-Medel, 2009).

Interestingly, it was found that in draper mutants both disconnected ghost boutons and presynaptic debris accumulated, and this accumulation had a negative effect on NMJ expansion and bouton morphology. Moreover, synaptic growth appeared to be highly sensitive to both types of shed presynaptic material since the accumulation of either ghost boutons or presynaptic debris (when engulfment activity was blocked in muscles or glia, respectively) led to reductions in bouton growth similar to that seen in draper null mutants. As mentioned above, shed material might contain important signaling factors that potently stimulate or inhibit new synapse formation. If, for example, presynaptic debris contains molecules that inhibit synaptogenesis, the accumulation of such material would be expected to negatively regulate synaptic growth. Perhaps a similar type of inappropriate modulation of synaptogenesis by the membrane fragments of pruned terminals also accounts for their rapid clearance from the central nervous system after degeneration (Fuentes-Medel, 2009).

Drosophila glial cells also engulf neuronal cell corpses and pruned or degenerating axons. Each of these targets is generated by a unique degenerative molecular cascade: cell corpses are produced by canonical apoptotic cell death pathways, pruned axons undergo degeneration through a ubiquitin proteasome-dependent mechanism, and severed axons undergo Wallerian degeneration via Wlds-modulated mechanisms (Wldsis a dominant mutation resulting in the overexpression of a fusion protein of the first 70 amino acids of UFD2/E4, an evolutionarily conserved protein used in protein polyubiquitination, and the full-length nicotinamide mononucleotide adenylyltransferase, an enzyme that facilitates NAD synthesis). Both corpses and degenerating axons are engulfed by glia through Draper-dependent mechanisms, implying that these engulfment targets autonomously tag themselves with molecularly similar 'eat me' cues. The observations that mutations in draper led to accumulation of presynaptic debris and detached ghost boutons suggests that these new glial/muscle engulfment targets also produce similar cues for phagocytic cells to promote their destruction. If so, these data argue that all the necessary machinery essential for tagging membrane fragments for engulfment are present in a ghost bouton or fragment of presynaptic membrane. Importantly, while a lack of glial-mediated clearance of several targets has been observed in vivo -- cell corpses, pruned axons or dendrites, and axons undergoing Wallerian degeneration -- almost nothing is known about phenotypic consequences of a lack of glial engulfment function in the nervous system. This study demonstrates that failure of glia and muscle to clear presynaptically derived material negatively regulates synaptic growth (Fuentes-Medel, 2009).

In conclusion these studies demonstrate that the process of synaptic growth includes a significant degree of membrane/synaptic instability, and that growing terminals are constantly sloughing off undifferentiated boutons and fragments of membrane. These observations demonstrate that growing NMJs generate an excess number of undifferentiated synaptic boutons and that only a fraction becomes stabilized and drive the assembly of the postsynaptic apparatus. Exuberant synapses that have failed to form successful postsynaptic contacts are shed, and cleared from the NMJ by glia and muscle cells. The presence of such a pool ensures a continuous supply of nascent synapses available for use to rapidly increase input into the muscle if dictated by dynamic changes in signaling at the NMJ (Fuentes-Medel, 2009).

Silencing of Drpr leads to muscle and brain Degeneration in Adult Drosophila

Mutations in the gene encoding the single transmembrane receptor multiple epidermal growth factor-like domain (MEGF) 10 cause an autosomal recessive congenital muscle disease in humans. Although mammalian MEGF10 is expressed in the central nervous system as well as in skeletal muscle, patients carrying mutations in MEGF10 do not show symptoms of central nervous system dysfunction. Draper (Drpr) is the sole Drosophila homolog of the human genes MEGF10, MEGF11, and MEGF12 (JEDI, PEAR). The functional domains of MEGF10 and Drpr bear striking similarities, and residues affected by MEGF10 mutations in humans are conserved in Drpr. This analysis of drpr mutant flies revealed muscle degeneration with fiber size variability and vacuolization, as well as reduced motor performance, features that have been observed in human MEGF10 myopathy. Vacuolization was also seen in the brain. Tissue-specific RNAi experiments demonstrated that drpr deficiency in muscle, but not in the brain, leads to locomotor defects. The histological and behavioral abnormalities seen in the affected flies set the stage for further studies examining the signaling pathway modulated by MEGF10/Drpr in muscle, as well as assessing the effects of genetic and/or pharmacological manipulations on the observed muscle defects. In addition, the absence of functional redundancy for Drpr in Drosophila may help elucidate whether paralogs of MEGF10 in humans (eg, MEGF11) contribute to maintaining wild-type function in the human brain (Draper, 2014).

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. This study demonstrates 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 (Timmons, 2016).

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. This study reports 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. The 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. Previously, defects in dead cell clearance were linked to neurodegeneration, but the exact mechanisms are not well understood. This study reports 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. The data indicate that corpses persist because of defective degradation of cells rather than recognition of dead cells (Etchegaray, 2016).

Components of the engulfment machinery have distinct roles in corpse processing

This study used the Drosophila ovarian follicle cells as a model for engulfment of apoptotic cells by epithelial cells. Engulfed material was shown to be 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, the roles of Draper, integrins, and downstream signaling components in corpse processing were also investigated. It was found that some proteins are required for internalization only, while others have defects in corpse processing as well. This suggests that several of the core engulfment proteins are required for distinct steps of engulfment. By performing double mutant analysis, it was found that combined loss of draper and αPS3 still results in a small number of engulfed vesicles. Next, another known engulfment receptor, Crq, was investigated. It was found that loss of all three receptors does 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 (Meehan, 2016).


EVOLUTIONARY HOMOLOGS

A subset of panglial genes are likely to play a role in glial function, rather than early developmental events, due to their broad and later expression patterns. the expression and function of one of these novel genes was characterized, CG2086 (named draper; drpr), which is expressed in all Gcm+ glia and macrophages. Seven cDNAs representing transcripts from the draper locus were obtained and sequenced. Each is predicted to encode one of three different splice variants of an EGF-repeat single-pass transmembrane domain receptor molecule. BLAST homology searches reveal that draper appears to be the sequence homolog of the C. elegans cell corpse engulfment gene ced-1. draper also shares strong homology with the mouse jedi-1 gene (Carninci, 1996) and the human MEGF10 and MEGF11 genes. In C. elegans, Ced-1 is required for the engulfment of apoptotic cell corpses, though this pathway has not been described in other organisms. Both mammalian and Drosophila glia are responsible for removing apoptotic neuronal cell corpses from the CNS; however, the molecular pathways involved have not been identified (Freeman, 2003).

C. elegans gene ced-1 is required for the engulfment of cells undergoing programmed cell death. ced-1 encodes a transmembrane protein similar to human SREC (Scavenger Receptor from Endothelial Cells). ced-1 is expressed in and functions in engulfing cells. The CED-1 protein localizes to cell membranes and clusters around neighboring cell corpses. CED-1 failed to cluster around cell corpses in mutants defective in the engulfment gene ced-7. Motifs in the intracellular domain of CED-1 known to interact with PTB and SH2 domains were necessary for engulfment but not for clustering. These results indicate that CED-1 is a cell surface phagocytic receptor that recognizes cell corpses. It is suggested that the ABC transporter CED-7 promotes cell corpse recognition by CED-1, possibly by exposing a phospholipid ligand on the surfaces of cell corpses (Zhou, 2001).

The removal of apoptotic cells is essential for the physiological well being of the organism. In C. elegans, two conserved, partially redundant genetic pathways regulate this process. In the first pathway, the proteins CED-2, CED-5 and CED-12 (mammalian homologues CrkII, Dock180 and ELMO, respectively) function to activate CED-10 (Rac1). In the second group, the candidate receptor CED-1 (CD91/LRP/SREC) probably recognizes an unknown ligand on the apoptotic cell and signals via its cytoplasmic tail to the adaptor protein CED-6 (hCED-6/GULP), whereas CED-7 (ABCA1) is thought to play a role in membrane dynamics. Molecular understanding of how the second pathway promotes engulfment of the apoptotic cell is lacking. This study shows that CED-1, CED-6 and CED-7 are required for actin reorganization around the apoptotic cell corpse, and that CED-1 and CED-6 colocalize with each other and with actin around the dead cell. Furthermore, it was found that the CED-10(Rac) GTPase acts genetically downstream of these proteins to mediate corpse removal, functionally linking the two engulfment pathways and identifying the CED-1, -6 and -7 signalling module as upstream regulators of Rac activation (Kinchen, 2005).

Dynamins are large GTPases that act in multiple vesicular trafficking events. Fourteen loss-of-function alleles of the C. elegans dynamin gene, dyn-1, were identified that are defective in the removal of apoptotic cells. dyn-1 functions in engulfing cells to control the internalization and degradation of apoptotic cells. dyn-1 acts in the genetic pathway composed of ced-7 (ABC transporter), ced-1 (phagocytic receptor), and ced-6 (CED-1's adaptor). DYN-1 transiently accumulates to the surface of pseudopods in a manner dependent on ced-1, ced-6, and ced-7, but not on ced-5, ced-10, or ced-12. Abnormal vesicle structures accumulate in engulfing cells upon dyn-1 inactivation. dyn-1 and ced-1 mutations block the recruitment of intracellular vesicles to pseudopods and phagosomes. It is proposed that DYN-1 mediates the signaling of the CED-1 pathway by organizing an intracellular vesicle pool and promoting vesicle delivery to phagocytic cups and phagosomes to support pseudopod extension and apoptotic cell degradation (Yu, 2006).

MEGF10 and MEGF11 mediate homotypic interactions required for mosaic spacing of retinal neurons

In many parts of the nervous system, neuronal somata display orderly spatial arrangements. In the retina, neurons of numerous individual subtypes form regular arrays called mosaics: they are less likely to be near neighbours of the same subtype than would occur by chance, resulting in 'exclusion zones' that separate them. Mosaic arrangements provide a mechanism to distribute each cell type evenly across the retina, ensuring that all parts of the visual field have access to a full set of processing elements. Remarkably, mosaics are independent of each other: although a neuron of one subtype is unlikely to be adjacent to another of the same subtype, there is no restriction on its spatial relationship to neighbouring neurons of other subtypes. This independence has led to the hypothesis that molecular cues expressed by specific subtypes pattern mosaics by mediating homotypic (within-subtype) short-range repulsive interactions. So far, however, no molecules have been identified that show such activity, so this hypothesis remains untested. This study demonstrates in mouse that two related transmembrane proteins, MEGF10 and MEGF11, have critical roles in the formation of mosaics by two retinal interneuron subtypes, starburst amacrine cells and horizontal cells. MEGF10 and 11 and their invertebrate relatives Caenorhabditis elegans CED-1 and Drosophila Draper have hitherto been studied primarily as receptors necessary for engulfment of debris following apoptosis or axonal injury. The current results demonstrate that members of this gene family can also serve as subtype-specific ligands that pattern neuronal arrays (Kay, 2012).


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date revised: 20 May 2017

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