The Core Molecular Machinery Used for Engulfment of Apoptotic Cells Regulates the JNK Pathway Mediating Axon Regeneration in Caenorhabditis elegans
The mechanisms that govern the ability of specific neurons to regenerate their axons after injury are not well understood. In Caenorhab- ditis elegans, the initiation of axon regeneration is positively regulated by the JNK–MAPK pathway. In this study, we identify two components functioning upstream of the JNK pathway: the Ste20-related protein kinase MAX-2 and the Rac-type GTPase CED-10. CED-10, when bound by GTP, interacts with MAX-2 and functions as its upstream regulator in axon regeneration. CED-10, in turn, is
activated by axon injury via signals initiated from the integrin α-subunit INA-1 and the nonreceptor tyrosine kinase SRC-1 and trans- mitted via the signaling module CED-2/CrkII–CED-5/Dock180 –CED-12/ELMO. This module is also known to regulate the engulfment of
apoptotic cells during development. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway.
Introduction
A fundamental and conserved property of neurons is their ability to regenerate their axons after injury, an ability that is modulated by interactions between the intrinsic axon growth machinery and the local extracellular environment. In adult mammals, the PNS regenerates relatively efficiently, whereas CNS axons regenerate poorly (Chen et al., 2007). This distinction between central and peripheral axon regeneration has been attributed to the com- bined effects of extrinsic signals provided by the inhibitory glial environment and intrinsic growth capabilities (Case and Tessier- Lavigne, 2005). Because intrinsic regeneration signals can influ- ence regenerative success, they represent potential therapeutic targets to enhance regeneration. However, our understanding of the intrinsic signaling pathways that promote regeneration in the adult nervous system remains limited.The nematode Caenorhabditis elegans has recently emerged as an attractive model with which to dissect the mechanisms of axon regeneration (Yanik et al., 2004; Hammarlund et al., 2009). Its amenability to genetic analysis makes possible the discovery and elucidation of novel signaling pathways involved in regeneration. Indeed, genetic screens have identified many such genes and pathways, including those specifically required for adult axon regrowth (Chen et al., 2011; Nix et al., 2014). Recent genetic studies have shown that, in C. elegans, the JNK–MAPK pathway functions as a key intrinsic regulator of the initiation of regener- ation and may be involved in the sensing of axonal damage (Nix et al., 2011; Li et al., 2012, 2015).
The MAPK signaling pathways respond to a variety of extracellular stimuli, generating a diverse array of physiological responses within the cell (Ichijo et al., 1997; Ninomiya-Tsuji et al., 1999), and thus are involved in the regu- lation of cell proliferation, differentiation, regeneration, stress response, and apoptosis. The C. elegans JNK pathway is com- posed of the components MLK-1 MAPKKK, MEK-1 MAPKK, and KGB-1–JNK. In a typical MAPK cascade, activation of the upstream MAPKKK is a critical control point for signal specificity and amplification (English et al., 1999). In the case of axon re- generation, we showed recently that TPA-1, a C. elegans protein kinase C (PKC), functions upstream of MLK-1 in the KGB-1 pathway regulating regeneration (Pastuhov et al., 2012). TPA-1 phosphorylates MLK-1 on Ser-355 of the kinase domain activa- tion loop, leading to its activation.The C. elegans KGB-1–JNK pathway also regulates the stress response to heavy metals (Mizuno et al., 2004; Fujiki et al., 2010). In this pathway, the protein kinase MAX-2, related to yeast Ste20, also activates MLK-1 through direct phosphorylation of Ser-355. Furthermore, the Rac-type GTPase MIG-2 functions upstream of MAX-2 in the KGB-1-mediated stress response pathway. In this study, we show that MAX-2 and another Rac-type GTPase, CED- 10, regulate axon regeneration upstream of MLK-1. We also demonstrate that the integrin α-subunit INA-1 activates the JNK pathway through the signaling complex CED-2–CED-5–CED- 12, thereby activating axon regeneration. The INA-1–CED-10 signaling pathway was shown previously to regulate the engulf- ment of apoptotic cells during development (Hsu and Wu, 2010). Therefore, the conserved INA-1–CED-10 signaling unit used for engulfment of apoptotic cells is also used to promote axon regeneration max-2(nv162); tpa-1( fr1) double mu- tants showed enhanced Cu 2+ sensitivity, which was similar to that observed in kgb-1 mutants (Fig. 1B). Therefore, TPA-1 and MAX-2 regulate the response to Cu 2+ stress redundantly.
To verify the redundancy of TPA-1 with MAX-2 in the KGB-1 pathway, we exam- ined KGB-1 activation. KGB-1 is activated by MEK-1-mediated phosphorylation at the Ser-198 and Tyr-200 residues located in the KGB-1 kinase domain (Mizuno et al., 2004, 2008). The anti-pKGB-1 antibody, which recognizes the phosphorylated form of KGB-1, was used to monitor KGB-1 activa- tion in C. elegans. Western blot analysis with anti-pKGB-1 antibody clearly detected the phosphorylated form of KGB-1 in wild-type animals treated with Cu2+ ions (Fig. 1C, lane 2). In contrast, animals harboring the kgb-1(km21) deletion mutation exhibited undetectable levels of KGB-1 protein ex- pression or activation (Fig. 1C, lane 1). In the single max-2(nv162) or tpa-1(k501) mu- tants, phosphorylation of KGB-1 in re- sponse to Cu2+ treatment was somewhat lower than wild-type (Fig. 1C, lanes 3 and 4). However, in the max-2(nv162); tpa-1(k501) double mutants, KGB-1 activity was mark- edly reduced compared with either single mutant (Fig. 1C, lane 5). Together, these re- sults suggest that MAX-2 and TPA-1 acti- vate the KGB-1 response to heavy metal stress redundantly by directly phosphorylat- ing MLK-1 Ser-355 and upregulating MLK-1 kinase activity.
MAX-2 and TPA-1 regulate axon regeneration mainly in the L4 and young adult developmental stages, respectively As observed previously (Pastuhov et al., 2012), axon regeneration in tpa-1(k501) mutants was reduced only in young adult animals, not in L4 larvae (Fig. 2A, Table 2). Therefore, TPA-1 is required mainly in the adult stage for axon regeneration of D-type motor neurons after laser surgery mediated stress response pathway. The k501 allele of the tpa-1 gene contains a missense mutation causing a change of the pro- line at 544 in the kinase domain to a serine. Animals carrying the tpa-1(k501) mutation were placed on agar plates containing a high Cu 2+ concentration and their development monitored for any signs of an altered response to Cu 2+. We found that tpa- 1(k501) mutants were weakly sensitive to Cu 2+ compared with wild-type animals (Fig. 1B). To test whether TPA-1 regulates the heavy metal stress response redundantly with MAX-2, we con- structed max-2(nv162); tpa-1(k501) double mutants and found that these showed Cu 2+ sensitivity stronger than the single max- 2(nv162) mutant (Fig. 1B). Sensitivity to Cu 2+ was also observed in tpa-1( fr1) mutants (Fig. 1B). The fr1 allele contains a nonsense mutation, which results in a premature stop codon at Trp-187, suggesting that fr1 is probably a null mutation. We found that
In contrast, MLK-1 is required for axon regeneration at both stages (Fig. 2A, Table 2), suggesting that another factor may act as an activator of MLK-1 to promote axon regeneration mainly in the L4 stage. Our analysis of MAX-2 raised the possibility that it might be a candidate for such an MLK-1 activator. To test this possibility, we investigated whether MAX-2 is involved in the KGB-1-mediated axon regeneration pathway. Although the axon guidance defect was observed in max-2(nv162) mutants (Lucanic et al., 2006), we found that the D-type motor neurons of max- 2(nv162) mutants in the L4 stage were impaired in their regener- ation response (Fig. 2 A, B, Table 2). This result is consistent with a recent report of extensive screening by Nix et al. (2014). Fur- thermore, Chen et al. (2011) performed a systematic screen of mutants looking for defects in axon regeneration and identified max-2 among several genes that regulate axon regeneration pos- itively in a glutamatergic touch sensory neuron. These results support the idea that MAX-2 is generally required during regen- eration. The effect of the max-2 mutation on axon regeneration in the L4 stage was stronger than that in the young adult stage (Fig. 2A, Table 2). In addition, the max-2;tpa-1 double mutants were defective in axon regeneration in both the L4 and adult stages (Fig. 2A, Table 2). However, the axon guidance defect ob- served in max-2(nv162) mutants was not enhanced by the tpa- 1(k501) mutation. Therefore, MAX-2 and TPA-1 regulate axon regeneration mainly in the L4 and adult stages, respectively. Al- though the expression levels of the max-2 gene in whole animals are constant during various developmental stages (Spencer et al., 2011), it has been shown that max-2 is expressed in ventral cord neurons during early development, but not at the young adult stage (Lucanic et al., 2006). These results suggest that TPA-1 takes the place of MAX-2 to activate MLK-1 in axon regeneration at the adult stage.
We next investigated whether MLK-1 functions downstream of MAX-2 in axon regeneration. We generated a phosphomimetic form of MLK-1 [MLK-1(S355E)] in which Ser-355 has been mu- tated to glutamate. We found that the expression of MLK-1(S355E) by the unc-25 promoter in D-type neurons was able to suppress the regeneration defect of max-2;tpa-1 double mutants at either the L4 or adult stage (Fig. 2A, Table 2). These results suggest that MLK-1 functions cell autonomously downstream of MAX-2. C. elegans axon regeneration is regulated by the p38 and JNK– MAPK pathways, which consist of DLK-1 (MAPKKK)–MKK-4 (MAPKK)–PMK-3 (p38 MAPK), and MLK-1(MAPKKK)– MEK-1(MAPKK)–KGB-1 (JNK–MAPK), respectively (Fig. 3A) (Hammarlund et al., 2009; Yan et al., 2009; Nix et al., 2011). We have shown previously that there is cross talk between the PMK-3 and KGB-1 MAPK pathways (Nix et al., 2011). Overexpression of dlk-1 can activate the KGB-1 pathway by cross-activating MEK-1 (Fig. 3A). Consistent with this, we found that dlk-1 overexpres- sion suppressed the regeneration defect in max-2 mutants (Fig. 3B, Table 2). In addition, we have demonstrated that the DLK-1– p38 MAPK pathway activates axon injury-induced expression of svh-2 via the bZip transcription factor CEBP-1 (Fig. 3A) (Li et al., 2015). The svh-2 gene encodes a Met-like receptor tyrosine ki- nase, which activates the KGB-1–JNK pathway through tyrosine phosphorylation of MLK-1 (Li et al., 2012). Activation of the MLK-1–MEK-1 pathway requires the dual phosphorylation of MLK-1; that is, SVH-2-mediated tyrosine phosphorylation and MAX-2-mediated serine phosphorylation. In dlk-1 mutants, svh-2 is not induced by axon injury (Li et al., 2015). Therefore, it can be expected that tyrosine phosphorylation of MLK-1 may not occur in dlk-1 mutants overexpressing MAX-2. As expected, we found that overexpression of max-2 by the unc-25 promoter was unable to suppress the regeneration defect in dlk-1 mutants (Fig. 3B, Table 2). Axons in wild-type animals overexpressing MAX-2 regenerated normally (Fig. 3B, Table 2).
CED-10 functions upstream of MAX-2 in axon regeneration MAX-2 is a member of the PAK (p21-activated kinase) family, which is regulated by small GTPases (Fujiki et al., 2010). These GTPases act as molecular switches that interconvert between two states: the active, GTP-bound state and the inactive, GDP-bound state. PAKs have been shown to bind to the GTP-bound form of Cdc42/Rac family GTPases through an N-terminal Cdc42/Rac- interactive binding (CRIB) domain (Dan et al., 2001) and MAX-2 also contains this CRIB motif (Fujiki et al., 2010). In the stress response pathway, the GTP-bound form of the Rac-type GTPase MIG-2 interacts with and activates MAX-2 (Fujiki et al., 2010). We therefore investigated whether MIG-2 would also be involved in the axon regeneration pathway, but found that the mig- 2(mu28) mutation had only a weak effect on axon regeneration (Fig. 4A, Table 2). However, we found that another Rac-type GTPase, CED-10, was able to bind to MAX-2. Using a yeast two- hybrid assay, we observed that MAX-2 interacted through its CRIB domain with the activated, GTP-bound form of CED-10, but not with the GDP-bound form (Fig. 4B).We next investigated whether CED-10 functions in axon regen- eration. We observed that animals harboring the ced-10(n3246 ) mu- tation exhibited a decrease in the axon regeneration of D-type motor neurons (Fig. 4A, Table 2). The n3246 allele replaces a glycine at 60 with arginine. Because Nix et al. (2014) reported that the ced-10 mutation had no effect on axon regeneration, we examined a differ- ent allele of the ced-10 gene, n1993. The n1993 allele replaces a valine at 128 with glycine. The ced-10(n1993) mutant was also defective in axon regeneration (Fig. 4A, Table 2). It can be expected that CED-10 cycles between the GTP-bound active and the GDP-bound inactive forms. Consistent with this, we found that expression of the GTP- bound active CED-10(G12V) by the unc-25 promoter in D-type neuron, but not of the GDP-bound inactive CED-10(T17N), was able to rescue the ced-10 defect in axon regeneration (Fig. 4A, Table 2). Furthermore, the ced-10(n1993 or n3246 ) mutation had no effect on the morphology of D-type motor neurons. These results suggest that CED-10 acts to promote regeneration in the damaged neuron in a cell-autonomous manner.
To address whether ced-10 and max-2 function in the same path- way, we constructed max-2(nv162);ced-10(n3246 ) double mutants. The double mutants did not show any enhanced defect in axon regeneration compared with the single max-2(nv162) or ced- 10(n3246 ) mutants (Fig. 4A, Table 2), suggesting that CED-10 and MAX-2 function in the same pathway. Furthermore, we found that overexpression of the max-2 gene from the unc-25 promoter in D- type motor neurons was able to suppress the ced-10 defect, whereas expression of GTP-bound active CED-10(G12V) from the unc-25 promoter failed to suppress the max-2 defect (Fig. 4A, Table 2). These results suggest that MAX-2 functions cell autonomously downstream of CED-10 in the axon regeneration pathway.Previous genetic analyses have shown that CED-10 is im- portant for the engulfment of apoptotic cells during develop- ment (Hsu and Wu, 2010). We therefore investigated whether MAX-2 and MLK-1 would be required for the removal of apoptotic cells. A time course analysis of the number of cell corpses present during embryogenesis showed no obvious dif- ference between mlk-1(km19) mutants and wild-type animals at both the 1.5-fold and 2-fold stages (Table 3). In contrast, both the ced-10(n3246 ) and max-2(nv162) mutations caused an increase in the number of cell corpses (Table 3). However, we found that the max-2 mutation enhanced the defect in engulfment of apoptotic cells in ced-10 mutants (Table 3). Furthermore, overexpression of max-2 by its own promoter failed to suppress the ced-10 defect in engulfment of apoptotic cells (Table 3). These results suggest that MAX-2 and CED-10 function in independent pathways for cell corpse engulfment. Therefore, in the engulfment pathway, the CED-10 GTPase does not transduce signals to the JNK pathway.
In the engulfment pathway, CED-10 GTPase is activated by the intracellular signaling proteins CED-2, CED-5, and CED-12 (Fig. 5A) (Hsu and Wu, 2010). CED-2, CED-5, andCED-12 are similar to the SH2–SH3 domain-containing adap- tor protein CrkII, Dock180, and ELMO, respectively (Wu and Horvitz, 1998; Reddien and Horvitz, 2000, 2004; Gumienny et al., 2001). In mammals, Dock180 and ELMO form a complex that functions as a guanine nucleotide exchange factor (GEF) for Rac-GTPases (Brugnera et al., 2002). It has been proposed that CED-5/Dock180 and CED-12/ELMO may interact with CED-2/CrkII to form a trimeric complex and activate the CED-10/Rac (Gumienny et al., 2001; Wu et al., 2001). We therefore next investigated whether CED-2, CED-5, and CED-12 function in axon regeneration. We used two differentmutant alleles of each gene, which are all nonsense mutations. The e1752 and n1994 alleles encode CED-2 proteins that ter- minate at Trp-153 and Arg-102, respectively. The n1812 and n2002 alleles terminate at Glu-28 and Arg-962, respectively, of the CED-5 protein. The k149 and k156 alleles terminate at Arg-38 and Trp-195, respectively, of the CED-12 protein. We observed that all of these mutants showed a lower frequency of axon regeneration after neuronal injury compared with wild- type animals (Fig. 5B, Table 2). Furthermore, none of the ced-2, ced-5, or ced-12 mutations had any apparent effect on the morphology of D-type motor neurons.If the CED-2–CED-5–CED-12 complex functions in axon regeneration as a GEF to activate CED-10, then we would expect that the expression of a constitutively active mutant of CED-10 would suppress the ced-5 phenotype. We expressed CED-10(G12V), a mutant CED-10 locked in the GTP-bound state, in D-type motor neurons using the unc-25 promoter and found that it indeed suppressed the ced-5 defect in axon re- generation (Fig. 5C, Table 2). In contrast, similar expression of CED-10(T17N), a GDP-bound inactive form, did not sup- press ced-5 (Fig. 5C, Table 2).
Furthermore, we found that the max-2(nv162);ced-5(n1812) double mutants were no more de- fective in axon regeneration than the single ced-5(n1812) mu- tant. In addition, this ced-5 defect was suppressed by overexpression of the max-2 gene from the unc-25 promoter in D-type neurons (Fig. 5C, Table 2). These results suggest that the CED-2–CED-5–CED-12 complex acts as a GEF for theIn the apoptotic cell engulfment pathway in C. elegans, the CED-1 receptor or the integrin α-subunit INA-1 functions up- stream of CED-10 (Fig. 6A) (Zhou et al., 2001; Hsu and Wu, 2010). To investigate which receptor acts in the CED-10 path- way leading to axon regeneration, we examined ced-1(e1754 ) and the ina- 1( gm39) mutants. The e1754 allele of theced-1 gene contains a nonsense mutation, which causes termination of the protein at Gln-32. The gm39 allele is the ina-1 weak mutant allele, which encodes a substitu- tion of the amino acid from glycine to glu- tamic acid at 119 (Baum and Garriga, 1997). Although the ced-1 mutation had no effect on axon regeneration, we found that the frequency of axon regeneration after axon injury was lower in ina-1 ( gm39) mutants compared with wild- type animals (Fig. 6B, Table 2). The ina- 1( gm39) mutation did not affect the morphology of D-type motor neurons. To determine whether INA-1 can act in a cell- autonomous manner, we expressed the ina-1 cDNA from the unc-25 promoter in ina-1( gm39) mutants. The ina-1 defect was rescued by expression of ina-1 in D-type motor neurons (Fig. 6B, Table 2). These results demonstrate that INA-1functions cell autonomously.To determine whether ina-1 acts upstream of the JNK path- way in axon regeneration, we overexpressed MAX-2 in D-type motor neurons from the unc-25 promoter. Overexpression of max-2 significantly rescued the axon regeneration defect seen in ina-1( gm39) mutants (Fig. 6B, Table 2).
In addition, we found that expression of a phosphomimetic form of MLK-1 [MLK- 1(S355E)] in D-type motor neurons by the unc-25 promoter also suppressed the ina-1 defect (Fig. 6B, Table 2). These observations suggest that INA-1 likely acts upstream of MAX-2 and MLK-1 in axon regeneration.We next examined INA-1 localization in D-type motor neu- rons during axon regeneration. For this purpose, we expressed the Punc-25::ina-1::gfp gene in wild-type animals and performed fluorescent imaging analysis in D-type neurons. In the absence of axon injury, we observed that INA-1::GFP distributed uniformly throughout D-type motor axons (Fig. 7A). At 6 h after laser ab- lation of the axons, significant amounts of INA-1::GFP accumu- lated at the proximal ends of the injured axons (Fig. 7 A, B).Previous genetic and biochemical analyses have shown that the nonreceptor tyrosine kinase SRC-1 mediates apoptotic cell engulfment signaling from the INA-1 cytoplasmic domain to CED-2, a component of the CED-2–CED-5–CED-12 module that activates CED-10 (Fig. 6A) (Hsu and Wu, 2010). We there- fore investigated whether src-1 participates in axon regeneration. The cj293 allele of the src-1 gene encodes a protein that is deletedbetween aa 136 and 337, essentially a truncation at 137. There-fore, this mutation lacks the SH2 and kinase domains, suggesting that the cj293 allele is null. The src-1(cj293) mutant had a slight defect in axonal guidance during development. We found that the src-1(cj293) mutation was indeed defective in axon regenera- tion (Fig. 6B, Table 2). Similar to ina-1 mutants, the src-1 defect in axon regeneration was also suppressed by overexpression of MAX-2 or expression of MLK-1(S355E) in D-type motor neu- rons from the unc-25 promoter (Fig. 6B, Table 2). Therefore, SRC-1 regulates axon regeneration upstream of MAX-2 and MLK-1. Together, these results suggest that INA-1 and SRC-1 act upstream of the JNK pathway in axon regeneration. The src- 1(cj293) mutation caused a greater reduction in regeneration than the ina-1( gm39) mutation. Although this could be due to a difference in allele strength, it also raises the possibility that SRC-1 may mediate signals from sources other than INA-1.
Discussion
The C. elegans JNK pathway is composed of MLK-1 MAPKKK, MEK-1 MAPKK, and KGB-1–JNK and regulates both the stress response to heavy metals and axon regeneration (Mizuno et al., 2004; Nix et al., 2011). MLK-1 is activated by phosphorylation of Ser-355 in the activation loop of its kinase domain. In the stress response to heavy metals, phosphorylation of MLK-1 on Ser-355 is catalyzed by the Ste20-related kinase MAX-2 (Fujiki et al.,2010), whereas in the axon regeneration pathway, MLK-1 Ser- 355 phosphorylation is catalyzed by the PKC homolog TPA-1 (Pastuhov et al., 2012). Therefore, two different kinases, MAX-2 and TPA-1, can activate MLK-1 by phosphorylating the same Ser-355 site. Do these kinases function redundantly or indepen- dently? We have demonstrated previously that max-2-null mu- tants, unlike animals harboring mutations in kgb-1, mek-1, or mlk-1, remained partially resistant to heavy metal stress (Fujiki et al., 2010). Furthermore, the max-2 mutation caused a smaller decrease in KGB-1 activity than the mlk-1 mutation. These observations raised the possibility that another kinase may function redundantly with MAX-2 in transducing the heavy metal stress signal to MLK-1. In this study, we show that combining the tpa-1 mutation with the max-2 mutation resulted in greater sensitivity to heavy metal stress and significantly lower KGB-1 activity. Therefore, TPA-1 functions redundantly with MAX-2 in the heavy metal stress response. TPA-1 is necessary for axon regeneration of D-type motor neurons in the young adult stage, but not in the L4 stage (Pastuhov et al., 2012). Given that MLK-1 is required for axon regeneration in both the L4 and adult stages, we hypothesized that MAX-2 may activate MLK-1 mainly in the L4 stage to promote axon regeneration, and indeed we observed this to be the case.
Ste20-like protein kinases are regulated by small GTPases (Dan et al., 2001). These GTPases are active when bound to GTP and inactive after hydrolysis of the bound GTP to GDP. One family of Ste20-like kinases, PAK, has been shown to bind the GTP-bound form of the Cdc42/Rac family of GTPases through a CRIB domain found in PAK kinases (Bagrodia and Cerione, 1999). The PAK family member MAX-2 contains a CRIB motif in its N-terminal domain and is regulated in the stress response pathway by MIG-2, a member of the Rac family of small GTPases (Fujiki et al., 2010). Conversely, in the axon regeneration path- way, MAX-2 is regulated by another Rac-type small GTPase, CED-10. The finding that MAX-2 interacted with the GTP- bound form of CED-10, but not with the GDP-bound form, suggests that CED-10 may be involved in transmitting axon in- jury signals to MAX-2. This model is further supported by genetic analysis showing that the defect in axon regeneration observed in ced-10 mutants was not enhanced by the max-2 mutation, but was suppressed by overexpression of the max-2 gene. Therefore, CED-10 and MAX-2 appear to act in the same pathway and func- tion to promote axon regeneration by activating the KGB-1–JNK pathway (Fig. 8). Previous studies have revealed that CED-10 is required for the UNC-40/DCC-dependent developmental axon attraction and outgrowth, as well as the ventral guidance of re- generating adult axons, of AVM neurons (Gitai et al., 2003; Gabel et al., 2008). However, they are distinct processes because the unc-40 mutation has no effect on axon regeneration in D-type motor neurons (Nix et al., 2014).
In C. elegans, CED-10 is involved in the engulfment of apo- ptotic cells (Hsu and Wu, 2010). The intracellular signaling pro- teins CED-2, CED-5, and CED-12 function as a GEF to activate CED-10 (Gumienny et al., 2001; Wu et al., 2001). The integrin α-subunit, INA-1, recognizes apoptotic cells and mediates sig- naling through the CED-2–CED-5–CED-12 complex in the en- gulfing cells to promote cell corpse removal. Furthermore, the nonreceptor tyrosine kinase SRC-1 provides a link between INA-1 and the downstream signaling module CED-2–CED-5– CED-12, leading to CED-10 activation (Hsu and Wu, 2010). In this study, we demonstrate that these intracellular signaling com- ponents also function upstream of MAX-2 in the axon regenera- tion pathway. Although the max-2 mutation also affected the engulfment of apoptotic cells, MAX-2 and CED-10 function in parallel pathways for cell corpse engulfment. Therefore, CED-10/ Rac-GTPase activates two separate signaling pathways, one that leads to axon regeneration and involves the JNK pathway and another that leads to the engulfment of apoptotic cells, but does not involve the JNK pathway (Fig. 8).
More recently, Nichols et al. (2016) demonstrated that the apoptotic machinery, including CED-2–CED-5–CED-12 and CED-10, regulates PLM axon
degeneration. Therefore, the core molecular machinery used for the engulfment of apoptotic cells regulates both axon regeneration and degeneration. In the pro- cess of axon degeneration, the transmembrane receptor CED-1 functions non-cell autonomously in the surrounding hypoder- mis, which acts as the engulfing tissue for the severed axon. Conversely, CED-1 does not participate in axon regeneration, although INA-1 is involved in this process. INA-1 recognizes apoptotic cells through its extracellular domain and mediates signaling via its intracellular domain to promote apoptotic cell internalization (Hsu and Wu, 2010). Apoptotic cells display “eat me” signals, consisting primarily of phosphatidylserine (PS) on the outer leaflet of the plasma membrane that are recognized by engulfment receptors (Segawa and Nagata, 2015). Mammalian integrins can bind to externalized PS on apoptotic cells indirectly via bridging molecules such as the secreted MFG-E8 (Hanayama et al., 2002; Akakura et al., 2004). However, C. elegans does not appear to have an MFG-E8 homolog and it remains to be deter- mined whether INA-1 recognizes externalized PS on the surface of apoptotic cells. Previous studies have identified the C. elegans secreted, transthyretin-like protein TTR-52 as a bridging mole- cule between PS on the surface of the apoptotic cell and the CED-1 receptor on the membrane of the engulfing cell (Wang et al., 2010). A recent study indicated that PS is exposed by axon severing and promotes axonal fusion in PLM neuron through a TTR-52–CED-1-dependent signaling pathway (Neumann et al., 2015). Our results suggest that the INA-1-mediated signaling pathway regulating the engulfment of apoptotic cells has been evolutionarily coopted for the regulation of axon regeneration. These findings raise the possibility that the externalized PS gen- erated by axon severing is also recognized by INA-1 indirectly via a bridging molecule to activate the CED-2–CED-5–CED-12 pathway. In this model, axonal injury induces the presentation of PS on the surface of the vesicles generated from the distal frag- ment of the severed axon. INA-1 may then recognize PS via a bridging molecule and activate the CED-2–CED-5–CED-12 pathway. To verify this possibility, it would be necessary to iden- tify the nature of the signal that promotes axon regeneration and identify which cells generate TBOPP the signal.