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,' 'donít-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 drpr^D5 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).

GENE STRUCTURE

cDNA clone length - 3193 bases

Bases in 5' UTR - 702

Exons - 8

Bases in 3' UTR - 706

PROTEIN STRUCTURE AND EVOLUTIONARY HOMOLOGS

Amino Acids - 594 (isoform A)

Structural Domains

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).

date revised: 10 December 2006

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