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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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date revised: 15 July 2008
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