Drosophila adult glia express three Draper isoforms
Three Draper receptor isoforms are generated through alternative splicing of the draper
gene (Draper-I, Draper-II, and Draper-III) and each contains a unique combination of extracellular and intracellular sequences () 19
. To determine which isoforms are present in the adult brain, we prepared head protein lysates from control yw
null mutant flies and performed anti-Draper Western immunoblots using an antibody that recognizes all isoforms 19
. We detected bands corresponding to at least two Draper isoforms (): a larger predominant band representing Draper-I (113kD) and a smaller band at the predicted sizes of Draper-II and/or Draper-III (65kD and 59kD, respectively). Since it was difficult to resolve Draper-II and Draper-III proteins, we performed RT-PCR and detected transcripts for all three isoforms in whole adult heads and dissected brains. draper-I
are also expressed in embryos, larval brains, and larval body wall tissue. Notably, draper-II
is selectively expressed in adults (Supplemental Fig. 2
Draper-I is sufficient for glial clearance of degenerating axons in the adult Drosophila CNS
Draper-I is required for glial engulfment of severed axons
Surgical ablation of the antennae or maxillary palps severs olfactory receptor neuron (ORN) axons that project into the brain, triggering their destruction through Wallerian degeneration. Glia infiltrate the injury site and engulf ORN axonal debris, but in the absence of Draper, glia fail to react and axonal debris lingers in the brain 8,18
. To determine how each Draper isoform modulates glial responses to injury in vivo
, we used the pan-glial driver repo-Gal4
to drive expression of UAS-Draper-I
, or UAS-Draper-III
null mutants, severed ORN axons, and assayed glial clearance of axon debris (). We confirmed expression of each UAS-Draper
transgene by anti-Draper immunostaining () and immunoblots (). Next, we quantified clearance of degenerating GFP+
axons (labeled with OR85e-mCD8::GFP
) three days after axotomy. In control animals, GFP+
axonal debris was cleared from the brain, while virtually all GFP+
degenerating ORN axons persisted in draperΔ5
mutants () 8
. Strikingly, glial expression of Draper-I rescued the engulfment defect of draper
mutants to near control levels. In contrast, expression of Draper-II or Draper-III failed to rescue draper
engulfment defects (). Glia cleared severed axons normally in heterozygous UAS-Draper
flies (Supplemental Fig. 3
), indicating these phenotypes were not due to insertional transgene effects. Thus, Draper-I, but not Draper-II or Draper-III, is sufficient for glial phagocytic engulfment of degenerating axons in the adult brain.
To determine if loss of Draper-I was sufficient to suppress engulfment activity, we generated an RNA interference (RNAi) construct (UAS-Draper-IRNAi
) that targets the unique extracellular domain region in Draper-I (depicted by black ovals in ) 20
. We first confirmed specificity by ubiquitously driving UAS-Draper-IRNAi
and performing an anti-Draper immunoblot (). Next, we used repo-Gal4
to drive UAS-DraperIRNAi
and assessed glial clearance of severed axons. Clearance of degenerating axons was significantly inhibited to levels indistinguishable from those observed by knockdown of all Draper isoforms or in draper
null mutants (). We also noted that glial expression of UAS-Draper-IRNAi
resulted in a loss of the vast majority of Draper staining (), suggesting that Draper-I is the predominant isoform in adult glia. These clearance phenotypes were not an artifact of activating the RNAi machinery as repo-Gal4
driven RNAi targeting other genes did not affect clearance of severed axons (see Supplemental Fig. 4
). Since glial expression of Draper-II and –III is retained in DraperIRNAi
animals, we conclude these isoforms are not sufficient to drive glial engulfment of degenerating axons. Together, our findings suggest that Draper-I is necessary and sufficient for glial engulfment of axonal debris after axotomy.
Draper-I is required for glial clearance of severed axons
The Draper-I intracellular domain activates engulfment
Since Draper-I is structurally unique in both its extracellular and intracellular domain compared to Draper-II and –III, engulfment-promoting activity could map to either region. To examine this, we generated chimeric receptors that contained every combination of extracellular (ex) and intracellular (int) domains from each Draper isoform () and tested the ability of each to promote glial engulfment of severed axons. We first confirmed that each construct was stably expressed in adult brains (). Next, we examined the ability of each chimeric receptor to drive glial clearance of axonal debris in draper a mutant background. Surprisingly, glial expression of Draper-II/III(ex)-I(int) resulted in normal clearance of degenerating axons after axotomy (). Thus, the shorter extracellular domain of Draper-II and -III can functionally replace that of Draper-I during glial clearance of severed axons.
Either Draper ECD with the ICD of Draper-I can trigger glial phagocytic activity
We next asked whether the intracellular domain of Draper-I contains unique engulfment-promoting activity. All three isoforms contain an NPXY motif proposed to bind Ced-621–25
, but because only Draper-I rescued the draper
engulfment defect, this domain is not sufficient for clearance of degenerating axons. Draper-I also contains an essential intracellular ITAM motif () 18
, while a C-terminal truncation in Draper-III deletes half of this ITAM sequence, including a key tyrosine residue. We overexpressed Draper-I(ex)
mutant glia and found that this provided no rescue of the engulfment defect (), supporting the notion that a complete ITAM domain is required for Draper-I-mediated engulfment of severed axons.
The Draper-II intracellular domain contains an 11 amino acid insertion in the middle of the ITAM present in Draper-I (). When we expressed Draper-I(ex)-II(int) in draper mutant glia, we found that this chimeric isoform failed to rescue clearance of axonal debris (). Thus, although the extracellular domains of Draper are fully interchangeable, only the Draper-I intracellular domain is sufficient to drive glial engulfment activity. Remarkably, the 11 amino acid insertion in the intracellular domain of Draper-II renders this isoform incapable of promoting glial engulfment activity.
Draper-II inhibits engulfment through an ITIM-like domain
Upon closer inspection of Draper-II, we identified a motif that is reminiscent of an immunoreceptor tyrosine-based inhibitory motif (ITIM; box in (VKIYXXI)). Mammalian ITIM-bearing receptors inhibit ITAM signaling cascades 14,15,26
, which raised the intriguing possibility that Draper-II negatively regulates Draper-I. To test this idea, we first overexpressed Draper-II in glia of wild type flies and found that glial clearance of axonal debris was completely blocked (). Likewise, glial overexpression of Draper-I(ex)
blocked glial engulfment of GFP+
debris (). In contrast, we overexpressed Draper-I or Draper-III in wild type glia and discovered that engulfment activity was comparable to that of control animals (). Thus, the inhibitory activity of Draper-II maps to the intracellular domain, and since the unique feature of this domain is the ITIM-like motif, we conclude this motif confers inhibitory signaling activity.
Draper-II contains an inhibitory motif in the ICD that inhibits glial engulfment activity
Finally, since Gal4/UAS-driven Draper-II levels were much higher than endogenous Draper levels, we reasoned that providing a high level of Draper-I might overcome the inhibitory activity of Draper-II. We expressed Draper-I and Draper-II together in glia using repo-Gal4 but found that glial engulfment of axonal debris was still potently suppressed (). Thus, providing roughly equivalent levels of Draper-I and Draper-II is insufficient to overcome the inhibitory activity of Draper-II, which may explain the predominance of Draper-I in wild type brains.
Corkscrew preferentially binds Draper-II
In vertebrates, the tyrosine phosphatases SHP-1 and SHP-2 typically bind ITIMs 14,27
. We explored the possibility that Draper-II might associate with Csw, the Drosophila
homolog of SHP-1/SHP-2. We transfected Drosophila
S2 cells with plasmids for wild type Csw (CswWT
), HA-tagged versions of Draper-I or Draper-II, immunoprecipitated with anti-Csw antibody, and performed anti-HA and anti-Csw Western blots on these samples. We consistently detected robust association of Csw with Draper-II, and, notably, significantly more HA-tagged Draper-II co-immunoprecipitated with Csw compared to Draper-I (, lane 4 versus lane 5, 24 ± 1.7-fold increase, p<0.0001), indicating that Csw preferentially associates with Draper-II.
Corkscrew associates preferentially with and dephosphorylates Draper-II
Catalytically inactive phosphatases can bind target substrates but fail to release because they cannot dephosphorylate the phosphotyrosine residue required for dissociation, thereby “trapping” the substrate 28–30
. As an alternative strategy to show Draper-II/Csw association, we used a substrate-trapping Csw, CswC583S
. We transfected plasmid for CswCS
, and/or Draper-II::HA, immunoprecipitated with anti-Csw, and performed a Western immunoblot with anti-HA to visualize Draper-II::HA. We found that more Draper-II::HA co-immunoprecipitated with the catalytically inactive CswCS
as compared to CswWT
(, lane 4 versus lane 5, 1.7 ± 0.15-fold increase, p<0.05), suggesting that Draper-II is a Csw substrate requiring Csw catalytic activity for dissociation. These results, together with the presence of an ITIM-like sequence in Draper-II, and the ability of Draper-II to inhibit ITAM-mediated signaling during glial engulfment in vivo
, strongly implicate Draper-II as a functional ITIM-like receptor that negatively regulates glial engulfment activity.
Draper-II is dephosphorylated by Corkscrew
To determine if Csw alters the tyrosine phosphorylation status of Draper, we transfected S2 cells with CswWT, CswCS and HA-tagged Draper-I or Draper-II and performed anti-HA immunoprecipitations followed by anti-phosphotyrosine immunoblots. Co-transfection of CswWT caused a striking reduction in the intensity of the anti-phosphotyrosine band for Draper-II::HA (, lane 7 versus lane 8, 28 ± 4% decrease, p<0.01). In contrast, Draper-II phosphorylation was significantly higher when co-expressed with CswCS (, lane 6 versus lane 7, 330 ± 90% increase, p<0.01), arguing that Draper-II is a phosphatase substrate for Csw. Tyrosine phosphorylation of Draper-I was unaffected by Csw, as we found no significant changes in the phosphorylation status of Draper-I::HA when co-transfected with CswWT (, lane 5 versus lane 6, 6 ± 3% decrease, p=0.13) or CswCS (, lane 4 versus lane 5, 40 ± 60% increase with CswCS, p= 0.85). We note that it remains possible Draper-I is a Csw substrate in vivo since expression of constructs in cultured cells does not fully mimic an engulfment event. Nevertheless, these results suggest that Draper-II is a physiological substrate for Csw, whereby Csw dephosphorylates the intracellular domain of Draper-II, perhaps to facilitate the dissociation of Csw or other signaling molecules.
We wondered whether DraperII::HA/Csw signaling might affect the tyrosine phosphorylation status of Shark, which is required for Draper-mediated engulfment in vivo
. We therefore transfected Draper-I::HA or Draper-II::HA plus N-terminal myc-tagged Shark (Myc::Shark) with or without CswWT
into S2 cells and performed anti-HA immunoprecipitation. Interestingly, we found that CswWT
significantly reduced the intensity of the Shark anti-phosphotyrosine band in the presence of Draper-II::HA (, lane 7 versus lane 8, 32 ± 2.6% decrease, p<0.01), but not in the presence of Draper-I::HA (, lane 5 versus lane 6, 7 ± 12% decrease with CswWT, p=0.59). Anti-phosphotyrosine Shark levels were higher in the presence of CswCS
as compared to CswWT
(, lane 6 versus lane 7, 520 ± 140%-fold greater for Shark, p<0.05) when co-transfected with Draper-II::HA. Importantly, tyrosine phosphorylation of Shark was not significantly altered by CswCS in the presence of Draper-I::HA (, lane 5 versus lane 4, 9± 40% increase, p=0.82,). We also performed reciprocal experiments in which we transfected DraperII::HA and Myc::Shark plus CswWT
(or no Csw), immunoprecipitated Shark using anti-myc, and then analyzed the tyrosine phosphorylation status of Shark. Again, we found that phosphorylation of Myc::Shark was consistently reduced when co-transfected with CswWT
(, lane 2 versus lane 3, 10% ± 3 % decrease, p<0.05), but increased in the presence of CswCS
(, lane 2 versus 4, 21% ± 8% increase, p<0.05). Finally, we observed that Myc::Shark co-immunoprecipitated equally with Draper-I::HA and Draper-II::HA, regardless of the phosphorylation status of Myc::Shark (, lanes 4–7). Together, these data support a model whereby Draper-II/Csw inhibits glial engulfment activity by reducing the activity of Draper-I signaling effectors, including Shark, through targeted dephosphorylation.
Corkscrew is required for Draper-II inhibitory activity
To determine whether Csw was required for Draper-II signaling in vivo,
we assayed the effects of csw
mutants and RNAi on Draper-II-dependent inhibition of glial engulfment. Glial overexpression of Draper-II completely blocked engulfment of severed axons, but this phenotype was significantly suppressed in heterozygous mutant cswva199
(). Glial engulfment activity was normal in cswva199
/+ animals, indicating that Csw is not a positive regulator of glial engulfment. To determine the cell autonomy of Csw function, we used repo-Gal4
to drive UAS-cswRNAi
in glia (Supplemental Fig. 5
) and found that this significantly suppressed the inhibitory activity of Draper-II (). Thus, Csw, like Draper-II, functions in glia to inhibit engulfment activity.
Draper-II inhibition of glial engulfment of severed axons is mediated through Corkscrew
Draper-II/Csw terminates glial responses to axon injury
Our findings suggest that Draper-I and Draper-II each play important, but opposing, roles in glial responses to axotomy. Previous work has shown that Draper immunoreactivity is robustly upregulated in antennal lobe glia within 24 hours after ORN axon injury 8
. We performed real time quantitative PCR on central brains at 0, 1.5, 3, 4.5, 6, 12, and 24 hours, and 7 days after axon injury. We found that draper-I
mRNA abundance was increased within 1.5 hours after axotomy (). Interestingly, draper-II
transcripts also increased, although these levels did not peak until 4.5 hours after ablation (). csw
levels also appear to be modulated by injury and were significantly decreased at 4.5 hours, coordinate with Draper-II increases (Supplemental Fig. 6
). Thus, draper-I
transcript levels are regulated in response to injury, with up-regulation of the pro-engulfment isoform preceding that of the inhibitory Draper-II isoform.
Csw signaling is required for normal termination of glial responses to axon degeneration
Since Draper-II/Csw negatively regulates glial engulfment activity, we suspected Draper-II/Csw signaling might function to downregulate glial responses to injury after axon clearance. We therefore assayed the efficiency of initiation and termination of glial responses to antennal nerve axotomy in wild type and CswRNAi animals. We first asked whether the expression of key glial engulfment molecules (i.e. Draper and Ced-6) were regulated normally before and after injury. Glial knockdown of Csw had no effect on the basal expression of Ced-6 or Draper protein in uninjured animals (). Both control and CswRNAi animals exhibited a robust increase in Ced-6 and Draper protein in antennal lobe glia 1 and 5 days after ORN axotomy (), indicating glial engulfment responses were activated normally. Furthermore, we observed no difference in clearance of degenerating axons five days maxillary nerve injury in control versus CswRNAi animals (“single injury” ), indicating that glial engulfment of severed axons occurred normally in CswRNAi backgrounds. In controls, at 7 and 10 days after injury, while Ced-6 remained elevated, Draper levels returned to baseline, indicating that glia were terminating responses. However, in CswRNAi animals, Ced-6 levels were further elevated at 10 days, and Draper levels remained high at 7 and 10 days (). These data argue that loss of Draper-II/Csw signaling results in a prolonged glial response and in turn a failure to properly terminate glial responses to axotomy.
Csw signaling is required for proper glial clearance of severed axons
Sustained expression of engulfment molecules after a primary injury might enhance glial responses to subsequent injuries. Alternatively, a failure to terminate glial responses to axotomy might negatively affect the responsiveness of glial cells to further injury events. To discriminate between these possibilities we developed a serial axotomy assay (Supplemental Fig. 7
). Briefly, in animals expressing GFP in maxillary palp neurons (OR85e-mCD8::GFP
) we generated a primary antennal nerve lesion to activate glial injury responses. Five days later, we performed a second (maxillary nerve) axotomy, and then assayed clearance of OR85e+
axonal debris. Interestingly, while control animals cleared most axonal debris within 5 days after the secondary injury, CswRNAi
animals failed to efficiently clear GFP+
axonal debris at this time point () and we observed nearly twice as many brains that contained visible axonal tracts of fragmented GFP+
material (; 42% in controls versus 83% in CswRNAi
animals). Uninjured maxillary palp ORNs appeared morphologically normally after the initial injury in control and CswRNAi
animals (), arguing that activated wild type and Csw−
glia do not promote the indiscriminant overt destruction of uninjured axons in the area of injury. Together these data argue strongly that Draper-II/Csw form a functional ITIM-like signaling cascade that suppresses Draper-I/ITAM signaling, which is essential for the proper termination of glial responses to axon injury in vivo
. Furthermore, this work reveals a novel role for Draper and ITIM-like signaling in maintaining glial responsiveness to neural trauma, since efficient termination of an initial glial response appears critical for glia to respond subsequent neural injury.