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. 2015 May 19;4:e05196. doi: 10.7554/eLife.05196

Dysregulated Dscam levels act through Abelson tyrosine kinase to enlarge presynaptic arbors

Gabriella R Sterne 1,2,†, Jung Hwan Kim 1,2,†, Bing Ye 1,2,*
Editor: Eunjoon Kim3
PMCID: PMC4434255  PMID: 25988807

Abstract

Increased expression of Down Syndrome Cell Adhesion Molecule (Dscam) is implicated in the pathogenesis of brain disorders such as Down syndrome (DS) and fragile X syndrome (FXS). Here, we show that the cellular defects caused by dysregulated Dscam levels can be ameliorated by genetic and pharmacological inhibition of Abelson kinase (Abl) both in Dscam-overexpressing neurons and in a Drosophila model of fragile X syndrome. This study offers Abl as a potential therapeutic target for treating brain disorders associated with dysregulated Dscam expression.

DOI: http://dx.doi.org/10.7554/eLife.05196.001

Research organism: D. melanogaster

eLife digest

Information is transmitted through the brain by cells called neurons, which are connected into specific circuits and networks. As the brain develops, several different signaling molecules control how the connections between neurons develop. If these signals occur at the wrong time or wrong place, or in the wrong amount, the neurons may not connect in the right way; this is the cause of several brain disorders.

One of the signaling molecules that helps neural circuits to form in the developing brain is the Dscam protein. Having too much Dscam has been linked to neurons with enlarged presynaptic terminals. Presynaptic terminals are the parts of each neuron that send information on to the next cell, and when they are enlarged it results in the neuron not being able to communicate with other neurons in a targeted way. People with brain disorders including Down syndrome, epilepsy and possibly fragile X syndrome often have excessive amounts of Dscam.

It was not known precisely how Dscam signals within neurons. Sterne, Kim and Ye have now investigated this by exploring the effects of Dscam on a group of well-known neurons in the larvae of the fruit fly species Drosophila. The presynaptic terminals of single neurons in this group were labeled in the larvae using a genetic marker. This revealed that the neurons of larvae that had been engineered to produce too much Dscam had larger presynaptic terminals than normal.

Further investigation showed that for Dscam to influence how a presynaptic terminal grows, it must interact with another signaling protein called Abelson tyrosine kinase (or Abl for short). Therefore, the larger presynaptic terminals seen in larvae that produce too much Dscam are a result of the Dscam protein activating too much Abl.

There are several drugs that are approved for use in humans that suppress the activity of Abl. Sterne, Kim and Ye used two of these to treat fruit fly larvae, and found that they reversed the detrimental effects of extra Dscam on the larvae's neural circuit. Furthermore, the drugs fixed neural defects in a fruit fly model designed to reproduce the symptoms of fragile X syndrome.

Overall, the results presented by Sterne, Kim and Ye suggest that suppressing the abnormally high activity of the Abl protein could be a way of treating the brain disorders caused by having excessive amounts of the Dscam protein. The next step is to test whether Dscam and Abl interact in the same way in mammals and whether the proposed treatment is effective in treating mammalian models of disorders that involve dysregulated Dscam signaling.

DOI: http://dx.doi.org/10.7554/eLife.05196.002

Introduction

Dscam levels are increased in the brains of human patients with DS and in mouse models of DS (Saito et al., 2000; Alves-Sampaio et al., 2010). Recent research also suggests that fragile X mental retardation protein (FMRP) binds directly to the mRNAs of Dscam from mouse brain (Brown et al., 2001; Darnell et al., 2011), and studies in Drosophila neurons further confirmed that FMRP suppresses Dscam translation (Cvetkovska et al., 2013; Kim et al., 2013). In the dendritic arborization (da) neurons in Drosophila larva, Dscam expression level is instructive for presynaptic terminal growth (Kim et al., 2013). Consistent with this, increased Dscam in Drosophila FXS models results in enlarged presynaptic arbors (Kim et al., 2013). These findings indicate the importance of proper Dscam levels in normal development and in the pathogenesis of brain disorders.

Because of the link between increased Dscam expression and neuronal defects in DS and FXS models, targeting Dscam or its signaling mechanism might prove therapeutic for these disorders. Currently, neither methods for targeting Dscam proteins nor those for targeting the signaling pathway activated by dysregulated Dscam are available, impeding the development of such therapies. In fact, very little is known about how Dscam signaling is transduced in vivo. In Drosophila, Dscam has previously been shown to bind to Dock (Schmucker et al., 2000), while in mammals it has been shown to associate with Uncoordinated-5C, Focal adhesion kinase (FAK), Fyn kinase, and PAK1 (Li and Guan, 2004; Purohit et al., 2012). In addition, studies suggest possible genetic interactions between Dscam and the Abelson tyrosine kinase (Abl) in neurite development in the central nervous system (CNS) of Drosophila embryos (Andrews et al., 2008; Yu et al., 2009). However, evidence demonstrating the requirement of these potential interactors for the defects that arise from increased Dscam expression is lacking. Moreover, whether pharmacologically targeting these molecules in vivo might alleviate the effects of increased Dscam expression is unknown.

The evolutionarily conserved Abl kinase transduces extracellular cues into cytoskeletal rearrangements that affect cell motility and shape (Bradley and Koleske, 2009) and is implicated in axonal development, including axon guidance and extension (Wills et al., 1999a; Wills et al., 1999b; Wills et al., 2002; Hsouna et al., 2003; Lee et al., 2004; Forsthoefel et al., 2005). Overexpression of Abl causes increased axon growth in the Drosophila CNS (Leyssen et al., 2005), which is reminiscent of the effect caused by Dscam overexpression in C4da neurons (Kim et al., 2013). In addition, previous studies in Drosophila have indicated that abl mutations have an additive effect with Dscam mutations, such that abl/Dscam double mutant embryos have more severe axon midline crossing defects than either abl or Dscam mutants alone (Andrews et al., 2008; Yu et al., 2009). However, the molecular nature of this interaction, that is, whether or not Dscam acts through Abl, and particularly whether inhibition of Abl mitigates neuronal defects caused by dysregulated Dscam, is unknown.

Here we show that Dscam activates Abl through its cytoplasmic domain, which is required for the presynaptic arbor enlargement caused by dysregulated Dscam expression in vivo. Importantly, we demonstrate that the pharmacological inhibition of Abl ameliorates exuberant presynaptic arbor growth both in flies overexpressing Dscam and in a fly model of FXS.

Results and discussion

We took advantage of the Drosophila larval class IV dendritic arborization (C4da) neurons to delineate the molecular mechanism of Dscam signaling in presynaptic arbor development, because the presynaptic terminal growth of these neurons is highly sensitive to Dscam levels in a linear fashion (Kim et al., 2013). For example, loss of Dscam causes C4da presynaptic terminals to fail to grow while increased Dscam levels lead to increased presynaptic terminal growth (Kim et al., 2013). From tests of candidate genes that potentially mediate Dscam function, including FAK, Fyn, PAK, RhoA, and Abl, we identified Abl as a key molecule mediating Dscam's functions in presynaptic terminal growth. We first asked whether Abl is sufficient to promote presynaptic terminal growth in C4da neurons. Consistent with a previous study performed in Drosophila adult CNS neurons (Leyssen et al., 2005), overexpression of Abl in C4da neurons caused significant overgrowth of the presynaptic terminals (Figure 1A,B,E). Since Abl is known to have both kinase-dependent and kinase-independent functions (Henkemeyer et al., 1990; Schwartzberg et al., 1991; Tybulewicz et al., 1991), we tested whether expression of a kinase-dead form of Abl, Abl-K417N (Henkemeyer et al., 1990; Wills et al., 1999b), could promote presynaptic terminal growth. We found that C4da presynaptic terminals overexpressing Abl-K417N were indistinguishable from wild-type (Figure 1D,E), indicating that Abl kinase activity is required. Consistent with the idea that Abl kinase activation is important, expression of a constitutively active form of Abl, BCR-Abl, led to extremely exuberant overgrowth (Figure 1C,E). Taken together, these results suggest that Abl is sufficient to promote presynaptic terminal growth and that the extent to which Abl instructs presynaptic terminal growth is related to Abl kinase activation.

Figure 1. Dscam requires Abl to promote presynaptic terminal growth.

(A–E) Abl is sufficient to cause presynaptic terminal overgrowth in C4da neurons. Transgenes were expressed with a C4da neuron-specific Gal4 driver, ppk-Gal4, and presynaptic terminals were visualized with a membrane monomeric RFP (mCD8-mRFP) transgene. Overexpression of Abl (B) leads to a modest increase in presynaptic terminal growth as compared to control (A). Overexpression of the constitutively active BCR-Abl (C) leads to robustly increased presynaptic terminal growth, while overexpression of kinase-dead Abl-K417N (D) is indistinguishable from control. Quantification of the number of axon connectives is shown in (E). Scale bar is 10 μm. (F–K) Abl is required in C4da neurons for Dscam to instruct presynaptic terminal growth. The arrowhead in each panel points to the location where an axon elaborates the presynaptic terminal arbor. The MARCM technique was used to generate and visualize single mutant C4da neurons. While overexpression of Dscam::GFP (G) in single C4da presynaptic terminals leads to increased length when compared to control (F), overexpression of Dscam in abl1 mutant neurons (H) leads to presynaptic terminal lengths that are indistinguishable from abl1 mutant neurons (I). Similarly, overexpression of Dscam in abl4 mutant neurons (J) does not significantly change presynaptic terminal length when compared to abl4 mutant neurons (K). (L–N) Abl is required to instruct presynaptic terminal growth in dFMRP mutants. (M) Loss of dFMRP leads to increased presynaptic terminal growth, which has previously been shown to require Dscam. Loss of one copy of abl in dFMRPΔ50M mutant neurons (N) leads to presynaptic terminal lengths that are indistinguishable from control (L). Scale bar is 10 μm. (O and P) Quantification of the presynaptic terminal length in C4da neurons of indicated genotypes. Sample number is shown in white within each bar.

DOI: http://dx.doi.org/10.7554/eLife.05196.003

Figure 1.

Figure 1—figure supplement 1. Loss of abl does not affect Dscam::GFP expression level.

Figure 1—figure supplement 1.

(A and B) Loss of abl does not affect Dscam::GFP expression in C4da cell bodies. (A) Example images of C4da neuron cell bodies (white arrowheads) in control (left) or abl1 homozygous mutant (right) animals. Upper images show merged signals of mCD8::mRFP and Dscam::GFP, while lower images show Dscam::GFP alone. Scale bar is 10 µm. (B) Quantification of the relative intensity of Dscam::GFP fluorescence normalized to mCD8::mRFP. Sample number is shown inside each bar. (C and D) Loss of abl does not affect Dscam::GFP expression in C4da presynaptic terminals. The MARCM technique was used to generate and visualize single mutant C4da neurons. (C) Example images of C4da presynaptic terminals in control (left) and abl1 mutant clones. Upper images show merged signals of mCD8::mRFP and Dscam::GFP, lower images show Dscam::GFP alone. Scale bar is 10 µm. (D) Quantification of the relative intensity of Dscam::GFP normalized to mCD8::mRFP. Sample number is shown inside each bar.
DOI: http://dx.doi.org/10.7554/eLife.05196.004

Figure 1—figure supplement 2. Loss of abl does not affect C4da dendritic length or morphology.

Figure 1—figure supplement 2.

Representative images of control (A) and abl1 mutant C4da neuron clones (B). The average total dendritic length is not significantly different between these two conditions (C). Scale bar is 50 µm.
DOI: http://dx.doi.org/10.7554/eLife.05196.005

Figure 1—figure supplement 3. Single Dscam isoform-induced ectopic repulsion between class I and class III dendrites does not require abl.

Figure 1—figure supplement 3.

The dendritic field of the class I da neuron vpda (traced in magenta) normally overlaps extensively with that of the class III da neuron v'pda (traced in cyan) (A). When a transgene expressing a single Dscam isoform is overexpressed in both neurons, their dendritic fields segregate (B), exhibiting an ectopic repulsion. The expression of the same Dscam transgene in abl1 neurons also leads to ectopic repulsion (C). Original background images show the pan-neuronal marker labeled with anti-horseradish-peroxidase antibody (red) and Dscam::GFP transgene expression (green). (D) Quantification of the number of dendritic branch crossing. Sample number is shown in white inside each bar. Scale bar is 25 µm.
DOI: http://dx.doi.org/10.7554/eLife.05196.006
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Since overexpression of Abl increases presynaptic terminal growth, similar to Dscam, we next tested whether Dscam requires Abl to instruct presynaptic terminal growth. For this, we used the mosaic analysis with a repressible cell marker (MARCM) technique to overexpress Dscam in abl1 mutant C4da neurons (Lee and Luo, 2001) and assessed presynaptic terminal length. We found that although Dscam overexpression led to significantly (150%) longer presynaptic terminals than wild-type clones (Figure 1F,G,O), abl1 mutant clones that overexpressed Dscam did not differ in length from abl1 mutant clones (Figure 1H,I,O). Presynaptic terminal length was also subtly but significantly shorter in abl1 mutant clones compared to wild-type controls (Figure 1I,O). A different loss-of-function allele of abl, abl4, exhibited similar effects on the presynaptic overgrowth caused by Dscam overexpression (Figure 1J,K,O), confirming that loss of abl function is responsible for blocking the presynaptic phenotypes caused by increased Dscam levels. As a control, abl loss-of-function mutations did not affect the expression of the Dscam transgenes in the C4da cell body or presynaptic terminals (Figure 1—figure supplement 1).

As a further proof-of-concept, we asked whether loss of abl could mitigate the effects of dysregulated Dscam levels without utilizing Dscam transgenes. FXS is caused by an absence of FMRP (Kremer et al., 1991), and is modeled in Drosophila using loss-of-function mutants for the Drosophila homolog of FMR1, dFMRP (Zhang et al., 2001; Dockendorff et al., 2002). It has previously been shown that FMRP binds to Dscam mRNA in both mammals and Drosophila (Darnell et al., 2011; Cvetkovska et al., 2013; Kim et al., 2013) and that dFMRP represses Dscam expression to control presynaptic terminal growth, so that dFMRP mutants exhibit increased presynaptic terminal length in C4da neurons (Kim et al., 2013). Strikingly, loss of only a single copy of abl significantly rescued presynaptic terminal length to wild-type levels (Figure 1L–N,P). These results suggest that Abl is required for Dscam to instruct presynaptic terminal growth.

An important function of Dscam in neuronal development is to mediate self-avoidance between neurites of the same neuron (Zipursky and Grueber, 2013). Abl does not seem to be required by Dscam for either dendrite growth (Figure 1—figure supplement 2) or for dendritic self-avoidance in C4da neurons. Loss of abl did not compromise the ectopic avoidance caused by overexpressing Dscam in distinct types of da neurons (Hattori et al., 2007; Hughes et al., 2007; Matthews et al., 2007) (Figure 1—figure supplement 3). This suggests a divergence in Dscam signaling for the development of presynaptic terminals and dendritic branches. Taken together, these results indicate that Abl is specifically required for Dscam-mediated presynaptic terminal growth.

Next, we asked how Abl might mediate Dscam signaling. Abl can be activated by binding to specific proteins, such as the cytoplasmic domains of membrane receptors (Bradley and Koleske, 2009). In contrast to the exuberant presynaptic terminal overgrowth caused by Dscam overexpression in C4da neurons (Figure 2A, middle), overexpressing a mutant form of Dscam that lacked most of the cytoplasmic domain (DscamΔCyto) did not cause presynaptic terminal overgrowth (Figure 2A, bottom). DscamΔCyto was trafficked to the axon terminals and expressed at a similar level to full-length Dscam (Figure 2—figure supplement 1). These results suggest that the cytoplasmic domain is required for Dscam to instruct presynaptic terminal growth.

Figure 2. Dscam binds to Abl through its cytoplasmic domain.

(A) The cytoplasmic domain of Dscam is required for instructing presynaptic terminal growth. Overexpression of full-length Dscam under the control of ppk-Gal4 (A, middle) leads to exuberant presynaptic terminal overgrowth when compared to control (A, top). However, overexpression of DscamΔCyto (A, bottom) fails to increase presynaptic terminal growth. Scale bar is 10 μm. (B) Dscam binds Abl via its cytoplasmic domain. S2 cells were co-transfected with Abl::Myc along with either Dscam::GFP, DscamΔCyto::GFP, or an empty vector. Dscam::GFP was immunoprecipitated with anti-GFP antibody and bound Abl::Myc was examined with anti-Myc antibody (top). Immunoprecipitated Dscam::GFP and input Dscam::GFP was examined with anti-GFP (bottom). (C) Abl colocalizes and redistributes with Dscam but not with DscamΔCyto in presynaptic terminals in vivo. When expressed alone, Abl::Myc shows a diffuse pattern (bottom). When expressed along with Dscam::GFP (top), Abl::Myc redistributes into punctate structures that colocalize with Dscam::GFP. When expressed along with DscamΔCyto::GFP (middle), Abl::Myc does not redistribute, displaying a similar pattern to when Abl::Myc is expressed alone (bottom). This is quantified using Manders' Correlation Coefficient. M1 presents a measure of the fraction of Abl::Myc that overlaps with Dscam(ΔCyto)::GFP, while M2 presents a measure of the fraction of Dscam(ΔCyto)::GFP that overlaps with Abl::Myc. Both M1 and M2 are significantly increased in Abl-Dscam coexpression when compared to Abl-DscamΔCyto coexpression. Scale bar is 5 µm.

DOI: http://dx.doi.org/10.7554/eLife.05196.007

Figure 2.

Figure 2—figure supplement 1. DscamΔCyto::GFP is trafficked to presynaptic terminals at a similar level to Dscam::GFP.

Figure 2—figure supplement 1.

Both Dscam::GFP (left) and DscamΔCyto::GFP (right) are trafficked to presynaptic terminals. In addition, presynaptic terminal overgrowth is observed 100% of the time when Dscam::GFP is overexpressed, while presynaptic terminal overgrowth is never observed when DscamΔCyto::GFP is overexpressed. Top image shows merged images mCD8::mRFP (red) and either Dscam::GFP or DscamΔCyto::GFP (green). Bottom images show Dscam::GFP or DscamΔCyto::GFP only. Scale bar is 10 µm.
DOI: http://dx.doi.org/10.7554/eLife.05196.008
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We then asked whether Dscam and Abl physically interact through the Dscam cytoplasmic domain. We found that Dscam and Abl proteins co-immunoprecipitated from transfected Drosophila Schneider 2 (S2) cells expressing these two proteins (Figure 2B, second lane from right). In contrast, DscamΔCyto did not co-immunoprecipitate Abl (Figure 2B, furthest right lane). These results suggest that Dscam and Abl proteins form a complex through Dscam's cytoplasmic domain. Next, to test the in vivo interaction of Dscam and Abl in presynaptic terminals specifically, we determined whether Abl localization in presynaptic terminals was altered by the expression of Dscam or DscamΔCyto (Figure 2C). When expressed alone or with DscamΔCyto::GFP, Abl::Myc was diffusely distributed in the presynaptic terminals, with little colocalization with DscamΔCyto::GFP (Figure 2C, middle and bottom). However, when expressed with Dscam::GFP, Abl::Myc became more punctate and clearly colocalized with Dscam::GFP (Figure 2C, top). We used Manders' Correlation Coefficients to quantify the colocalization of Dscam::GFP and Abl::Myc. Colocalization analysis revealed a significant increase in both M1 and M2 (Figure 2C, bottom right) when Abl::Myc was coexpressed with Dscam::GFP as compared to when Abl::Myc was coexpressed with DscamΔCyto::GFP, where M1 represents the fraction of Abl that overlaps with Dscam, and M2 represents the fraction of Dscam that overlaps with Abl. These findings support the idea that Abl and Dscam interact in presynaptic terminals in vivo.

Do increased Dscam levels activate Abl kinase? In mammals, autophosphorylation of Abl at tyrosines 245 and 412 (Y245 and Y412) stabilizes the active conformation of the kinase (Brasher and Van Etten, 2000; Tanis et al., 2003). As a result, phospho-specific antibodies raised against Y412 have been employed to detect active Abl kinases (Brasher and Van Etten, 2000). This approach has been used successfully to recognize the phosphorylation of the corresponding tyrosines (Y539/522) in Drosophila as an assay for Abl kinase activation (Stevens et al., 2008). Since the ability of Abl to instruct presynaptic terminal growth relies on Abl kinase activity, we tested whether Dscam activates Abl using a phosho-Y412-Abl (p-Abl) antibody. We found that Abl kinase activation was significantly increased (2.6 fold) when Abl and Dscam were co-expressed in S2 cells (Figure 3A). Furthermore, unlike wild-type Dscam, DscamΔCyto did not increase Abl kinase activation. In fact, it appears to act as a dominant-negative, as Abl activity was significantly decreased from control (Figure 3A, right). As a negative control, no signal was detected when the kinase-dead Abl-K417N was blotted with p-Abl antibody in the same assay, suggesting that our assay specifically reported Abl activation (Figure 3—figure supplement 1). These results suggest that Dscam enhances Abl kinase activity. To investigate whether the same is true in presynaptic terminals in vivo, we devised a novel method of reporting Abl activation specifically in C4da presynaptic terminals. To achieve this, we used a previously reported probe that reports Abl activity, Pickles2.31 (Mizutani et al., 2010). Pickles2.31 is composed of a fragment of a characteristic Abl substrate, CrkL, sandwiched between the fluorescent proteins Venus and enhanced CFP (ECFP) (Figure 3B). It has previously been reported that activated Abl phosphorylates Pickles2.31 on the Y207 residue of the CrkL fragment, which can be detected with an antibody against CrkL-phospho-Y207 (p-CrkL) (Mizutani et al., 2010). After expressing Pickles2.31 specifically in C4da neurons with the ppk-Gal4 driver, we dissected the larval CNS and immunoprecipitated Pickles2.31 from the lysates. Since the cell bodies of C4da neurons reside in the body wall, using only the larval CNS allowed us to monitor Pickles2.31 phosphorylation only in the C4da neuron presynaptic terminals (Figure 3C). We found that overexpression of Dscam in C4da neurons led to an increase in Y207 phosphorylation of Pickles2.31 in the presynaptic terminals, while overexpression of DscamΔCyto was indistinguishable from control (mCD8-mRFP) (Figure 3D). Consistent with the notion that Pickles2.31 is an Abl activity indicator, overexpression of BCR-Abl led to a robust increase in phospho-Y207 levels as compared to the control. These results suggest that Dscam activates Abl both in culture and in C4da presynaptic terminals in vivo, and that this activation requires the cytoplasmic domain of Dscam.

Figure 3. Dscam activates Abl kinase in culture and in vivo.

(A) Dscam activates Abl in cultured S2 cells. Abl activation was examined in S2 cell lysates transfected with indicated constructs by using anti-phospho-Y412-Abl antibody. The intensity of phospho-Abl was quantified, normalized to total Abl::Myc, and presented as bar graph (n = 3) (A, right). (B) Schematic of Pickles2.31, an Abl activity reporter that uses phosphorylation of CrkL to report Abl kinase activity. Pickles2.31 is composed of a fragment of human CrkL that contains an Abl phosphorylation site, Y207, sandwiched between ECFP and Venus. Phosphorylation of Pickles2.31 by Abl can be detected with an anti-phospho-Y207-CrkL (p-CrkL) antibody. (C) Schematic of in vivo assay for detecting Abl activity in C4da presynaptic terminals. Pickles2.31 is specifically expressed in C4da neurons. As can be appreciated from the larval fillet diagram (left), the cell bodies and dendrites of C4da neurons reside in the larval body wall while their presynaptic terminals reside in the CNS. To assay Abl activity only in presynaptic terminals, larval CNS are dissected out and solubilized into lysates. Pickles2.31 in the presynaptic terminals is then immunoprecipitated with an anti-Venus antibody (left). After running on an SDS-PAGE gel, Pickles2.31 expression level can be assayed using an anti-Venus antibody, while the phosphorylation of Y207, a proxy for Abl activity level, can be ascertained by western blotting with a p-CrkL antibody. (D) Dscam activates Abl in presynaptic terminals in vivo. Overexpression of BCR-Abl leads to a robust increase in p-CrkL staining of Pickles2.31 when compared to the mCD8-mRFP control. Similarly, overexpression of Dscam leads to consistent, though less extreme, increase in p-CrkL when compared to control. In contrast, overexpression of DscamΔCyto is indistinguishable from the mCD8-mRFP control. This is a representative blot of three experimental repeats.

DOI: http://dx.doi.org/10.7554/eLife.05196.009

Figure 3.

Figure 3—figure supplement 1. Phospho-Y412-Abl antibody specifically reports Abl activation.

Figure 3—figure supplement 1.

S2 cells were transfected with either Abl::Myc or Abl-K417N::Myc. Myc was blotted to report total Abl::Myc or Abl-K417N::Myc level (middle), while phosho-Y412-Abl (p-Abl) was blotted to report Abl kinase activation (top). While Abl::Myc displays a characteristic two-band pattern at the correct molecular weight when blotted for p-Abl, no signal is detected for Abl-K417N. This demonstrates that p-Abl specifically reports Abl activation.
DOI: http://dx.doi.org/10.7554/eLife.05196.010
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These results raised the interesting possibility that targeting Abl might be a viable therapy for brain disorders caused by increased Dscam expression. Abl is a well-established target for treating chronic myeloid leukemia, and there are multiple Abl inhibitors that are approved by the US Food and Drug Administration (FDA). As a proof-of-concept experiment, we attempted to rescue the developmental defects caused by Dscam overexpression using Abl inhibitors. We first tested nilotinib, which is a FDA-approved second-generation Abl kinase inhibitor that can cross the blood–brain barrier (Weisberg et al., 2005; Hebron et al., 2013). Using cultured S2 cells overexpressing Abl, we found that nilotinib robustly inhibited Drosophila Abl (Figure 4A). Based on these results, we tested whether administration of nilotinib to developing larvae could rescue the effects of increased Dscam expression in C4da presynaptic terminals in vivo. To do this, we performed MARCM to visualize single C4da neurons in animals fed nilotinib or vehicle and assessed presynaptic terminal length. While overexpression of Dscam caused increased (152%) presynaptic terminal length in animals fed vehicle (Figure 4B–D), the effect was significantly rescued (to 115% of control) by feeding the animals with nilotinib (Figure 4B,E). Consistent with the idea that these effects were due to inhibition of Abl activity rather than a reduction in Dscam expression, nilotinib did not change the expression of the Dscam transgene (Figure 4—figure supplement 1A).

Figure 4. Pharmacological inhibition of Abl mitigates the neuronal defects caused by increased Dscam expression in vivo.

(A) Nilotinib inhibits Drosophila Abl kinase. S2 cells were transfected with either Myc-vector or Abl::Myc, and then treated with either vehicle (DMSO) or 5 μM nilotinib for 6 hr. Total lysates were subjected to western blot analysis with phospho-Y412-Abl (p-Abl) (top) and Myc antibodies (bottom). (B) Quantification of the presynaptic terminal length of the indicated genotypes and drug treatment. Sample number is shown inside each bar. (C–H) Nilotinib treatment mitigates presynaptic arbor enlargement caused by Dscam overexpression (OE Dscam, D and E) and by dFMRP mutations (dFMRPΔ50M, G and H). Nilotinib treatment alone does not affect presynaptic terminal growth (F). The arrowhead in each panel points to the location where an axon elaborates the presynaptic terminal arbor. The MARCM technique was used to generate and visualize single presynaptic terminals of mutant C4da neurons. Drosophila larvae were raised in the presence of either 380 μM nilotinib or vehicle (DMSO) for 4 days before the analysis. Scale bar is 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.05196.011

Figure 4.

Figure 4—figure supplement 1. Nilotinib and bafatinib do not reduce Dscam transgene expression.

Figure 4—figure supplement 1.

Example images of C4da presynaptic terminals expressing Dscam::GFP in animals fed either vehicle (A and B, top), 380 µM nilotinib (A, bottom), or 125 µM bafetinib (B, bottom) throughout larval development. Images of mCD8::mRFP are shown to indicate the neuropil regions used for the quantifications (white dotted line). Scale bar is 10 μm. Quantification of the fluorescence of the Dscam::GFP transgene in neuropil region is shown on the right. Sample number is shown inside each bar.
DOI: http://dx.doi.org/10.7554/eLife.05196.012

Figure 4—figure supplement 2. Nilotinib treatment does not cause defects in dendritic development or adult viability.

Figure 4—figure supplement 2.

(A and B) Nilotinib does not affect dendritic development. After egg collection, the animals were raised on food containing either vehicle (DMSO) or 380 µM nilotinib for 4 days. C4da dendrites were visualized by expressing mCD8::GFP with ppk-Gal4 (A). Total dendritic length was measured, quantified, and presented in the bar graph (B). Sample number is shown inside each bar. Scale bar is 50 µm. (C and D) Nilotinib does not affect the development of the flies. After egg collection, the animals were raised on food containing either vehicle (DMSO) or 380 µM nilotinib. Eclosed adults were counted on a daily basis. Total number and cumulative number of adults are shown in (C) and (D) respectively.
DOI: http://dx.doi.org/10.7554/eLife.05196.013

Figure 4—figure supplement 3. Nilotinib and bafetinib act through Abl inhibition to mitigate Dscam-induced presynaptic arbor enlargement in vivo.

Figure 4—figure supplement 3.

The MARCM technique was used to generate and visualize single presynaptic terminals of mutant C4da neurons. Drosophila larvae were raised in the presence of 380 µM nilotinib, 125 µM bafetinib, or vehicle (DMSO) for 4 days before the analysis. Scale bar is 10 µm. (A–D) Nilotinib acts through Abl inhibition to mitigate presynaptic arbor enlargement in Dscam overexpressing neurons. Wt (wild-type, FRT2A), OE Dscam (overexpression of Dscam), OE Dscam, abl1 (overexpression of Dscam in abl1 homozygous mutations). Note that nilotinib does not further decrease the size of presynaptic arbors in abl1 neurons overexpressing Dscam(C and D). (E and F) Bafetinib mitigates presynaptic arbor enlargement in Dscam overexpressing neurons. (G) Quantification of the presynaptic terminal length of the indicated genotype and drug treatment. Sample number is shown below the x-axis.
DOI: http://dx.doi.org/10.7554/eLife.05196.014
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Administration of nilotinib to developing larvae did not lead to adverse effects on overall development and neuronal growth. At the dose we used, nilotinib did not cause a change in presynaptic terminal growth (Figure 4F) or dendritic growth (Figure 4—figure supplement 2A,B) in wild-type larvae. Moreover, it did not impact the number of adults that eclosed or the dynamics of eclosion when compared to vehicle-fed flies (Figure 4—figure supplement 2C,D).

Although frequently used to inhibit pathological increases in Abl activity in patients, nilotinib is known to have several off-targets, including c-Kit, PDGFR, Arg, NQ02, and DDR1 (Hantschel et al., 2008). Consistent with the idea that nilotinib acts on Abl rather than on an off-target molecule to rescue presynaptic terminal growth, administering nilotinib to larvae overexpressing Dscam in abl1 clones did not lead to a further decrease in presynaptic terminal length when compared to vehicle-fed control (Figure 4—figure supplement 3C,D,G). To further rule out the possibility that the observed rescue of presynaptic terminal length by nilotinib was the result of an off-target effect, we tested bafetinib, another Abl inhibitor with non-overlapping off-targets, Fyn and Lyn (Kimura et al., 2005). Bafetinib has also been shown to cross the blood brain barrier (Santos et al., 2010). Like nilotinib, administration of bafetinib to Dscam-overexpressing larvae led to a significant decrease in presynaptic terminal length (Figure 4—figure supplement 3A,B,F,G) without changing the expression of the Dscam transgene (Figure 4—figure supplement 1B). Bafetinib alone did not change presynaptic terminal length in wild-type larvae when compared to wild-type larvae fed vehicle (Figure 4—figure supplement 3E,G). Taken together, these results suggest that pharmacological inhibition of Abl mitigates the consequences of increased Dscam signaling in vivo.

We next sought to test the efficacy of nilotinib treatment in a model of a disease associated with dysregulated Dscam expression, FXS. Thus, we tested whether administration of nilotinib could rescue the presynaptic overgrowth caused by increased Dscam expression in dFMRP mutants. We found that, while dFMRP mutants fed vehicle showed a significant increase (130%) in presynaptic terminal length (Figure 4B,G), administration of nilotinib to dFMRP mutants almost completely rescued (to 103% of control) the exuberant presynaptic terminal growth to wild-type levels (Figure 4B,H). These results suggest that pharmacological inhibition of Abl kinase is effective for mitigating the effects of increased Dscam level in an in vivo model of FXS.

In this study, we show that Dscam requires Abl to promote presynaptic terminal growth in vivo and that the binding of Abl to the Dscam cytoplasmic domain leads to Abl kinase activation. Furthermore, we show that treating larvae with Abl inhibitors rescues the developmental defects caused by increased Dscam levels in vivo in both Dscam-overexpressing neurons and disease-relevant models. Taken together, these results suggest that Abl is a potential drug target for the treatment of brain disorders associated with dysregulated Dscam expression, including DS and FXS.

Materials and methods

Fly strains

abl1 (Gertler et al., 1989), abl4 (Bennett and Hoffmann, 1992), ppk-Gal4 (Kuo et al., 2005), UAS-Dscam[3.36.25.2]::GFP (Yu et al., 2009), UAS-Abl, UAS-BCR-Abl, UAS-Abl-K417N (Wills et al., 1999b), and dFMRPΔ50M (Zhang et al., 2001) were used in this study.

DNA constructs and generation of transgenic flies

To generate pUASTattB-Abl::Myc for expression in S2 cells, the coding region of Abl was recovered from UAS-Abl transgenic flies by PCR, subcloned into pUASTattB-Myc by using the InFusion cloning system following manufacturer's protocol (Clontech, Mountain View, California). We generated pUASTattB-Abl-K417N::Myc by PCR mutagenesis as previously described (O'Donnell and Bashaw, 2013) from pUASTattB-Abl::Myc. UAS-Dscam[3.36.25.2]::GFP was previously generated as described (Kim et al., 2013). To generate UAS-DscamΔCyto, the Dscam coding region was digested with SstI and ligated with the GFP cDNA. Pickles2.31 was generously provided by Dr Yusuke Ohba at RIKEN Brain Science Institute (Mizutani et al., 2010). To generate UAS-Pickles2.31, the Pickles2.31 coding region was subcloned from pCAGGS-Pickles2.31 into pUASTattB using the InFusion cloning system following the manufacturer's protocol (Clontech). Transgenic flies carrying UAS-DscamΔCyto, UAS-Abl::Myc, and UAS-Pickles2.31 were generated by germline transformation with support from BestGene, Inc.

Labeling presynaptic terminals using MARCM

The MARCM technique was used to visualize single neurons homozygous for abl1, abl4, or dFMRPΔ50, and overexpressing Dscam[3.36.25.2]::GFP as previously described (Kim et al., 2013).

Immunostaining and imaging

Immunostaining of third-instar larvae was accomplished as previously described (Ye et al., 2011). Antibodies used include chicken anti-GFP (Aves, Tigard, Oregon) and rabbit anti-RFP (Rockland, Limerick, Pennsylvania). Samples were dehydrated and mounted with DPX mounting media (Electron Microscopy Sciences, Hatfield, Pennsylvania). Confocal imaging was completed with a Leica SP5 confocal system equipped with a resonant scanner and 63× oil-immersion lens (NA = 1.40). Images were collected and quantified as previously described (Kim et al., 2013).

S2 cell culture and transfection

Drosophila S2 cells were maintained in Drosophila Schneider's medium supplemented with 10% fetal bovine serum at 25°C in a humidified chamber. Cells were transfected with indicated DNA constructs together with tubulin-Gal4 (Lee and Luo, 2001) by using Lipofectamine 2000 (Life Technologies, Grand Island, New York) according to manufacturer's protocol.

Co-immunoprecipitation and Western blotting

To perform co-immunoprecipitation, transfected S2 cells were harvested and lysed on ice with lysis buffer (50 mM Tris-HCl/pH 7.4, 150 mM NaCl, 2 mM sodium vanadate, 10 mM sodium fluoride, 1% Triton X-100, 10% glycerol, 10 mM imidazole and 0.5 mM phenylmethylsulfonyl fluoride). Lysates were centrifuged for 15 min at 20,000×g, 4°C and the resulting supernatant was incubated with Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology, Paso Robles, California) conjugated to mouse monoclonal anti-GFP clone 20 (Sigma-Aldrich, St. Louis, Missouri) for 4 hr at 4°C. After washing once with lysis buffer, twice with lysis buffer containing 0.1% deoxycholate, and 3 times with lysis buffer lacking Triton X-100, the immunoprecipitates and total lysates were resolved on 7.5% SDS-PAGE gels followed by western blot analysis as previously described (Kim et al., 2013).

Primary antibodies used in western blotting were mouse monoclonal anti-tubulin (Sigma), mouse anti-Myc (Sigma-Aldrich), mouse monoclonal anti-Aequorea Victoria GFP JL-8 (Clontech), and rabbit anti-phospho-Tyr412-c-Abl (Cell Signaling, Beverly, Massachusetts).

In vivo Abl activity assay with Pickles2.31

To assay in vivo Abl activation, UAS-Pickles2.31 was expressed specifically in C4da neurons using ppk-Gal4 along with other UAS transgenes. The CNS was dissected from third-instar larvae into ice-cold PBS with 2 mM sodium vanadate (∼100 per experimental condition). After a brief centrifugation, larval CNSs were transferred into lysis buffer as described above in immunoprecipitation and western blotting. Cells were disrupted using a pestle followed by brief sonication. Immunoprecipitation and western blotting of Pickles2.31 was then accomplished as described above. Primary antibodies used were rabbit anti-eGFP (a gift from Dr Yang Hong) and rabbit anti-phospho-Tyr 207-CrkL (Cell Signaling).

Drug treatment of Drosophila larvae and S2 cells

Nilotinib (Abcam, United Kingdom) and bafetinib (ApexBio Technology, Houston, Texas) were dissolved in dimethyl sulfoxide (DMSO) at 94 mM and 50 mM, respectively, as stock solutions before adding to S2 cells or fly food. S2 cells transfected with Abl::Myc were treated with either 5 μM nilotinib or the same volume of DMSO as a vehicle control for 6 hr before harvested and subjected to western blot analysis.

Nilotinib and bafetinib were administered to larvae by rearing the larvae on standard corn meal food containing different concentrations of the drugs. The highest concentrations that did not affect overall larval development were used. Fly viability on nilotinib treatment was performed by counting the number of adult flies. Seven virgin female and seven male flies were crossed and transferred to standard corn meal food containing either 380 μM nilotinib or the same volume of DMSO (0.4% final concentration). Embryos were collected for 24 hr and allowed to develop. Eclosed adult flies were counted on a daily basis.

The MARCM technique was used to generate and visualize mutant single C4da neurons as described above except that Drosophila embryos were collected and raised for 4 days on standard corn meal food containing either 380 μM nilotinib, 125 μM Bafetinib, or 0.4% DMSO. Sample preparation, imaging, and quantification were then completed as described above.

Colocalization analysis

Colocalization of Dscam and Abl was quantified with Manders' Correlation Coefficients using the Just Another Colocalization Plugin (JACoP) (Bolte and Cordelieres, 2006) in ImageJ. Images were analyzed in three dimensions. Manders' Correlation Coefficients vary between 0 and 1, with 0 representing no overlap between images and 1 representing complete colocalization. M1 and M2 describe the overlap of each channel with the other (Bolte and Cordelieres, 2006). M1 presents a measure of the fraction of Abl::Myc that overlaps Dscam(ΔCyto)::GFP, while M2 presents a measure of the fraction of Dscam(ΔCyto)::GFP that overlaps Abl::myc.

Statistical analysis

Two-way student's t- test was used for statistical analysis. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; ns: not significant.

Acknowledgements

We thank Dr Tzumin Lee for Dscam fly stocks, Dr Yusuke Ohba for the Pickles2.31 cDNA, Dr Yang Hong for the rabbit anti-GFP antibody, and Drs Cheng-yu Lee and Hideyuki Komori for the pUASTattB-Myc vector. This work was supported by grants from NIH (R01MH091186), Protein Folding Disease Initiative of the University of Michigan, and the Pew Scholars Program in the Biological Sciences to BY.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Funding Information

This paper was supported by the following grants:

  • National Institutes of Health (NIH) R01MH091186 to Bing Ye.

  • Pew Charitable Trusts 2009-000359-016 to Bing Ye.

  • University of Michigan (U-M) Protein Folding Disease Initiative to Bing Ye.

Additional information

Competing interests

GRS: have a patent application on the use of Abl inhibitors as a therapeutic approach for treating brain disorders associated with dysregulated Dscam levels (Application number: PCT/US2014/072083).

JHK: have a patent application on the use of Abl inhibitors as a therapeutic approach for treating brain disorders associated with dysregulated Dscam levels (Application number: PCT/US2014/072083).

BY: have a patent application on the use of Abl inhibitors as a therapeutic approach for treating brain disorders associated with dysregulated Dscam levels (Application number: PCT/US2014/072083).

Author contributions

GRS, Conceived the project and designed the experiments, Performed the Abl and Dscam overexpression experiments, the mosaic Abl loss-of-function experiments, the dendritic crossing experiments, the colocalization experiments, and the Abl binding and activation biochemistry experiments, Wrote the paper.

JHK, Conceived the project and designed the experiments, Performed the pilot experiments on Abl overexpression, and performed the pharmacological studies, Wrote the paper.

BY, Conceived the project and designed the experiments, Supervised the project, Wrote the paper.

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eLife. 2015 May 19;4:e05196. doi: 10.7554/eLife.05196.015

Decision letter

Editor: Eunjoon Kim1

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “Dysregulated Dscam levels act through Abelson tyrosine kinase to enlarge presynaptic arbors” for consideration at eLife. Your article has been favorably evaluated by K VijayRaghavan (Senior editor), a Reviewing editor, and three reviewers.

The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

We note key findings of the paper which show that loss of Abl kinase can reduce presynaptic terminal overgrowth caused by DsCAM overexpression, DsCAM intracellular domain can interact and activate Abl kinase and in particular that an FDA approved Abl inhibitor nilotinib can reduce presynaptic overgrowth caused by DsCAM overexpression or in fly dFMRP mutants, which also show increased Dscam expression. An added significance of the work therefore is the potential translational application of Abl inhibitors in treatment of neurological disorders.

General assessment:

CNS phenotypes for Dscam loss or misexpression had been explored in prior work. Andrews et al. (2008) showed that loss of both Dscam and its paralog Dscam3 result in CNS midline crossing defects, and that overexpression of Dscam results in ectopic midline crossings. Yu et al. (2009) showed that RNAi knockdown of Dscam resulted in morphogenesis defects as well as commissureless phenotypes. However, the effects of Dscam loss at axon terminals growth does not appear to have been demonstrated. Overall, this is a well-written and interesting study with some exciting results linking Dscam to a key axonal signalling molecule, even if some aspects of the work are at a preliminary stage.

The conclusion that Abl kinase activity is a relevant output of Dscam in determining presynaptic arbor size is supported by a convincing set of experiments in Figure 1 (and supplements), and this is a real strength of the manuscript. Given how little is known about signal transduction downstream of Dscam, this is significant. Further, the observation that Dscam signaling in regulating presynaptic arbor size and dendrite self-avoidance involves different molecular mechanisms is an interesting discovery. The studies demonstrating that Dscam can interact with Abl and promote Abl activation in S2 cells support the model that Dscam modulates Abl activity in neurons, but demonstrating the interaction in neurons would substantially improve the manuscript. Finally, the authors convincingly show that the Abl pharmacological inhibitor nilotinib can suppress presynaptic arbor overgrowth induced by Dscam or FMRP mutation, but the mechanism of action is unclear. The following substantive concerns need to be addressed.

Substantive concerns:

1a) A principal concern is that data for in vivo specificity of nilotinib on Abl are not convincing. Figure 3B is not interpretable without controls, such as abl(lf) and abl(CA) or DsCAM overexpression. Figure 3C-H, the analyses on the neuronal phenotypes in flies fed with nilotinib are surprisingly limited or selective. The value of using a fly model to test potential drug targets is that we can gain a full understanding of any possible off-target or side effects. Thus, it is equally important that they show other aspects of the neurons and animal health etc. are, or are not, affected by the drug treatment. Moreover, one can't find any data showing how the dose of nilotinib treatment, which would be highly relevant for any further interests in translational application, was decided.

1b) The specificity of the inhibitor is not discussed (or examined). Nilotinib inhibits a number of tyrosine kinases and appears to broadly inhibit tyrosine phosphorylation in larvae. The assertion that the effects on presynaptic arbor size reflect inhibition of Abl activation is not convincing. Do other small molecule inhibitors of Abl exist that have a more targeted effect (i.e., they don't broadly block tyrosine kinases) and if so, do they similarly affect presynaptic arbor growth?

1c) Figure 3. The specificity of nilotinib to Abl in Drosophila is not verified. The decrease in phospho-tyrosine in 3b demonstrates a general decrease in the CNS, and does not necessarily show a specific targeting of Abl. This must be addressed. To demonstrate specificity, a possible experiment is to repeat the experiment shown in 3B in flies that are mutated in the Abl phosphorylation site Tyr412, or simply null for Abl. If nilotinib is specific to blocking phospho-Abl, there should be no difference in p-Tyr levels whether or not nilotinib is added, if Abl phospho-sites are blocked or Abl is missing. However, if addition of nilotinib still results in a decrease in p-Tyr even when Abl phospho-sites are blocked, this suggests nilotinib is having an effect on the catalytic activity of other kinases.

2a) Figure 3. What is the effect of nilotinib treatment in wild-type flies? The results of wild type + nilotinib can be shown in addition to the experiments in 3c-g. It would be very reassuring to see that nilotinib elicits abl-like phenotypes in other parts of the nervous system or elsewhere (e.g. epithelial development in the embryo)—if the drug is selectively blocking Abl, it should phenocopy abl mutants, but not mutants in other kinases.

2b) What is the effect of drug treatment on presynaptic arbor size in a wild type background? It's impossible to evaluate the experimental results without this control.

3a) The data presented suggest that Dscam can associate with Abl and promote Abl phosphorylation, but this is not demonstrated in vivo.

3b) Figure 2. The results shown in panel 2B and 2C demonstrate binding of Abl to the cytoplasmic domain of Dscam in S2 cells using transfected constructs and levels of expression that may contribute to detection of non-physiological interaction. This is important because the fraction of the input pool that IPs with Dscam in cells overexpressing both proteins is rather low. More importantly, they do not show that this binding occurs in vivo, and that even if it does occur in vivo, that this binding is important to axon terminal growth in particular. To determine the role of Abl-Dscam binding in vivo the group should consider performing fluorescence tagging and/or immunolocalization of Abl in the three genetic backgrounds shown in panel 2A (WT, Dscam::GFP OE, Dscam cyto::GFP OE). If there is a bona fide interaction with Dscam at the terminal, co-localization of Abl with Dscam::GFP should be observed. Moreover, if this interaction requires Dscam cytoplasmic domain, expression of Dscam cyto::GFP instead of full- length Dscam should result in a re-distribution of Abl.

4) Figure 1B. The text (second paragraph of the Results section) and the figure legend state that Abl overexpression leads to a pre-synaptic overgrowth phenotype. However, while a defect in the axons is apparent in panel 1B, it is not clear whether this is due to increased growth necessarily. For example, the gross defects could result from fasciculation defects between axons as they enter the CNS neuropil. Indeed, the apparent decrease in mRFP signal intensity in the Abl OE flies raise the possibility that it is not overgrowth, but defasiculation, that is observed in 1B. This is important because Abl has been linked to many receptors/adhesion molecules that may also contribute to the normal neuropil structure for sensory terminals. Abl nulls are not shown to confirm that the phenotypes are only selective to overexpression.

5) Figure 1, general comment. All axon terminal phenotypes shown are for Dscam OE. While Dscam loss phenotypes have been shown in other systems (e.g. midline crossing), it has not been demonstrated for terminal growth. It would be useful to include data on the effects of Dscam loss at the synaptic terminal, as well as genetic experiments on the interaction between Dscam and abl loss of function alleles (as has been done in other systems). If the two genes are in one pathway for sensory terminal growth, then the double null should be indistinguishable from the single mutants. It is curious that Abl mutant terminals look relatively normal. Does this mean that Abl is not required for other receptors or DSCAM under normal expression levels?

6) Figure 1–figure supplement 1A. Dscam-GFP levels in the neuronal soma is shown to be unchanged in abl mutants. However, the observations in the cell body do not rule out the possibility that at the synapse, abl loss could have effects on Dscam levels, perhaps due to changes in Dscam protein trafficking (Abl has been implicated in axon transport by Bill Saxton). Just because abl nulls revert the Dscam OE phenotype, this doesn't prove a downstream function. Abl can be required upstream of Dscam at the synapse. To test this, a parallel analysis of Dscam-GFP could be done at axon terminals to verify that the effect of abl is the same both in the soma and at the presynaptic terminal.

7) The abl C4da dendritic phenotype analysis should be added to the supplemental figures.

8) Figure 2C. There are no data confirming the stability of the Dscam cytoplasmic domain deletion. In fact the recovery in the IP (Figure 2B) is very poor and raises the concern that this receptor mutant is not capable of accumulating to the same concentration in the OE experiment, which could explain the lack of an in vivo OE phenotype in a way that does not support requirement of the Cyto domain. Moreover, why is full-length Dscam detected as a doublet? Can this be explained by cleavage, phosphorylation, etc.?

eLife. 2015 May 19;4:e05196. doi: 10.7554/eLife.05196.016

Author response

1a) A principal concern is that data for in vivo specificity of nilotinib on Abl are not convincing. Figure 3B is not interpretable without controls, such as abl(lf) and abl(CA) or DsCAM overexpression. Figure 3C-H, the analyses on the neuronal phenotypes in flies fed with nilotinib are surprisingly limited or selective. The value of using a fly model to test potential drug targets is that we can gain a full understanding of any possible off-target or side effects. Thus, it is equally important that they show other aspects of the neurons and animal health etc. are, or are not, affected by the drug treatment. Moreover, one can't find any data showing how the dose of nilotinib treatment, which would be highly relevant for any further interests in translational application, was decided.

We appreciate the reviewers’ critiques. Following up on the reviewers’ suggestions, we have investigated the in vivo specificity of nilotinib on Abl with several experiments. The results strongly support that the effect of nilotinib on Dscam-induced presynaptic arbor growth is through Abl. In addition, we have addressed the issue of off-targets in our response to comment #1b). In the initial submission, we presented Figure 3B to show the accessibility of nilotinib into central nervous system in fly, rather than to show the specificity of nilotinib. We apologize for the confusion, and have removed the original Figure 3B to avoid the confusion.

In the revised manuscript, we have included the analysis of the viability of flies fed either vehicle (DMSO) or nilotinib (new Figure 4–figure supplement 2), which shows no difference between the two groups. Furthermore, we have analyzed C4da dendrite development in flies treated with nilotinib and found no difference between DMSO- and nilotinib-treated larvae (new Figure 4–figure supplement 2). These results suggest that the nilotinib concentration required for rescuing axonal phenotypes does not cause any severe side effects. Together with known clinical safety of nilotinib (Kantarjian et al 2011. nilotinib is effective in patients with chronic myeloid leukemia in chronic phase after imatinib resistance orintolerance: 24-month follow-up results Blood 117 (4), 1141-1145), these results show the therapeutic potential of nilotinib.

In the revised manuscript (in the subsection headed “Drug treatment of Drosophila larvae and S2 cells”), we have explained how the dose of nilotinib (and bafetinib) for treating larvae was selected. The dose was the highest concentration (among various concentrations tested) that was tolerated by the larvae without displaying any toxic responses.

1b) The specificity of the inhibitor is not discussed (or examined). Nilotinib inhibits a number of tyrosine kinases and appears to broadly inhibit tyrosine phosphorylation in larvae. The assertion that the effects on presynaptic arbor size reflect inhibition of Abl activation is not convincing. Do other small molecule inhibitors of Abl exist that have a more targeted effect (i.e., they don't broadly block tyrosine kinases) and if so, do they similarly affect presynaptic arbor growth?

The reviewers are correct that, depending on the concentration, nilotinib also inhibits several other targets. We have added a discussion on nilotinib specificity in the revised manuscript (pleas see the Results and Discussion section). In addition, we tested another Abl inhibitor, bafetinib, which has a chemical scaffold different from nilotinib. This compound is also known to cross blood-brain barrier in mice. We found that the administration of bafetinib, like nilotinib, was able to significantly rescue axon terminal length in larvae overexpressing Dscam (Figure 4–figure supplement 3). The known off-targets of nilotinib in mammals are c-Kit, PDGFR, Arg, NQ02, and DDR1, while those of bafetinib are Fyn and Lyn. Since the off-targets of these two drugs are different, their common effect on Dscam-induced axon terminal growth is likely through Abl inhibition. Furthermore, we treated the larvae overexpressing Dscam in homozygous abl-/- neurons and found that the nilotinib treatment did not further decrease size of presynaptic arbors (Figure 4–figure supplement 3). Taken together, even though nilotinib also inhibits several other tyrosine kinases, its effect of rescuing enlarged presynaptic arbors is through Abl inhibition.

1c) Figure 3. The specificity of nilotinib to Abl in Drosophila is not verified. The decrease in phospho-tyrosine in 3b demonstrates a general decrease in the CNS, and does not necessarily show a specific targeting of Abl. This must be addressed. To demonstrate specificity, a possible experiment is to repeat the experiment shown in 3B in flies that are mutated in the Abl phosphorylation site Tyr412, or simply null for Abl. If nilotinib is specific to blocking phospho-Abl, there should be no difference in p-Tyr levels whether or not nilotinib is added, if Abl phospho-sites are blocked or Abl is missing. However, if addition of nilotinib still results in a decrease in p-Tyr even when Abl phospho-sites are blocked, this suggests nilotinib is having an effect on the catalytic activity of other kinases.

We again apologize for the confusion caused by previous Figure 3B, which was to evaluate the accessibility of nilotinib into central nervous system in fly rather than the specificity of nilotinib treatment. In agreement with that nilotinib has off-targets, we found that Abl inhibition is not the main cause of the decrease in overall tyrosine phosphorylation in larval brain upon nilotinib treatment. The extent of the decrease in overall tyrosine phosphorylation persists even when nilotinib was fed to homozygous Abl-/- mutants (data not shown).

Nevertheless, as we explained in the response to comment #1b), we have performed additional experiments to demonstrate the rescuing effect of nilotinib on Dscam-induced overgrowth of presynaptic arbors is due to the inhibition of Abl. We have also added a discussion of nilotinib specificity in the revised manuscript (please see the Results and Discussion section).

2a) Figure 3. What is the effect of nilotinib treatment in wild-type flies? The results of wild type + nilotinib can be shown in addition to the experiments in 3c-g. It would be very reassuring to see that nilotinib elicits abl-like phenotypes in other parts of the nervous system or elsewhere (e.g. epithelial development in the embryo)—if the drug is selectively blocking Abl, it should phenocopy abl mutants, but not mutants in other kinases.

Although Abl is involved in multiple processes in embryonic development, we do not expect to observe similar severity of phenotypes in nilotinib-treated animals as abl mutant. This is because nilotinib was administrated through food and was thus not accessible to the animal until embryonic development is completed. Moreover, the nilotinib treatment in our experiments likely partially inhibited Abl activity for three reasons. First, the efficacy of presynaptic arbor rescue in Dscam overexpressing neurons was partial as compared to the complete rescue achieved by homozygous Abl-/- mutations (compare new Figure 4B-G with new Figure 1F-K and O). Second, the same dose of nilotinib almost completely rescued presynaptic arbor enlargement in FMRP-/- mutants while reducing only 50% of Abl gene dosage was sufficient for the same extent of rescue (compare new Figure 4B-C and 4G-H with new Figure 1L-N and 1P). Third, homozygous mutation of abl causes pupal lethality (or embryonic lethality when abl is mutated maternally) while nilotinib treatment at the concentration used does not affect fly development through adulthood (Figure 4–figure supplement 2). Finally, Abl is known to have kinase-independent functions (Henkemeyer et al. 1990. A novel tyrosine kinase-independent function of Drosophila abl correlates with proper subcellular localization. Cell 63:949-960), so even if nilotinib were capable of inhibiting all Abl kinase activity, we would not expect to see identical phenotypes as abl mutants.

We have performed additional experiments whose results support the conclusion that the effects of nilotinib on presynaptic arbor growth is through Abl inhibition (see the response to comment #1b). Our new results also demonstrate that the nilotinib concentration used in this study does not affect neuronal and animal development (see the response to comment #1a). Therefore, although nilotinib does not only inhibit Abl, its inhibition of Abl kinase is responsible for mitigating the presynaptic terminal overgrowth caused by dysregulated Dscam.

2b) What is the effect of drug treatment on presynaptic arbor size in a wild type background? It's impossible to evaluate the experimental results without this control.

We have performed the experiment and added in new Figure 4F. There is no difference in axon terminal length between wild-type flies treated with vehicle (DMSO) and with nilotinib (for the statistics see new Figure 4B).

3a) The data presented suggest that Dscam can associate with Abl and promote Abl phosphorylation, but this is not demonstrated in vivo.

We have performed two additional experiments to address this concern. The new results are presented in the new Figures 2 and 3.

First, to test if Dscam and Abl associate in vivo we expressed Dscam::GFP or DscamΔCyto::GFP along with Abl::Myc in C4da neurons, and then analyzed the extent that Dscam and Abl::Myc colocalized in C4da axon terminals (new Figure 2C). These results support the notion that Abl binds to Dscam in vivo in axon terminals.

Second, to determine whether Dscam promotes Abl phosphorylation in vivo, we developed a novel method that reports Abl activation only in the axon terminals of C4da neurons in vivo (new Figure 3B and C). We specifically expressed a probe that reports Abl activity in C4da neurons. Since the cell bodies of C4da neurons reside in the body wall, in the larval CNS this probe is only present in the axon terminals of C4da neurons. The Abl activity probe was then immunoprecipitated from the larval CNS after dissection. The phosphorylation state of the Abl target site in the probe was monitored with western blotting by using a phospho-specific antibody. Compared to a negative control transgene, expression of Dscam led to an increase in probe phosphorylation while DscamΔCyto did not (new Figure 3D), suggesting that Dscam activates Abl in vivo and that this activation requires the Dscam cytoplasmic domain.

3b) Figure 2. The results shown in panel 2B and 2C demonstrate binding of Abl to
the cytoplasmic domain of Dscam in S2 cells using transfected constructs and levels of expression that may contribute to detection of non-physiological interaction. This is important because the fraction of the input pool that IPs with Dscam in cells overexpressing both proteins is rather low. More importantly, they do not show that this binding occurs in vivo, and that even if it does occur in vivo, that this binding is important to axon terminal growth in particular. To determine the role of Abl-Dscam binding in vivo the group should consider performing fluorescence tagging and/or immunolocalization of Abl in the three genetic backgrounds shown in panel 2A (WT, Dscam::GFP OE, Dscam cyto::GFP OE). If there is a bona fide interaction with Dscam at the terminal, co-localization of Abl with Dscam::GFP should be observed. Moreover, if this interaction requires Dscam cytoplasmic domain, expression of Dscam cyto::GFP instead of full- length Dscam should result in a re-distribution of Abl.

Please refer to our response to comment #3a) above.

4) Figure 1B. The text (second paragraph of the Results section) and the figure legend state that Abl overexpression leads to a pre-synaptic overgrowth phenotype. However, while a defect in the axons is apparent in panel 1B, it is not clear whether this is due to increased growth necessarily. For example, the gross defects could result from fasciculation defects between axons as they enter the CNS neuropil. Indeed, the apparent decrease in mRFP signal intensity in the Abl OE flies raise the possibility that it is not overgrowth, but defasiculation, that is observed in 1B. This is important because Abl has been linked to many receptors/adhesion molecules that may also contribute to the normal neuropil structure for sensory terminals. Abl nulls are not shown to confirm that the phenotypes are only selective to overexpression.

In the abdominal segments 5-8 (A5-A8), the axon terminals of the three C4da neurons in each hemi-segment consist of an anterior projection that extends within one segment length. These axon terminals form a fascicle that connects two adjacent neuropils. Between the axon entry points of abdominal segment 5 and 6, fewer than three connectives were typically observed (new Figure 1E) (Wang, et al., 2014, PLOS Biology 11 (6). e1001572). Both BCR-Abl and Abl overexpression caused thickened bundle of connectives in the C4da neuropil ladder (new Figure 1B and C). We have quantified the number of connectives between the neuropil of abdominal segment 5(A5) and that of A6 (new Figure 1E). The results clearly showed an overgrowth phenotype as opposed to a defasciculation phenotype because overexpression of Abl/BCR-Abl led to a significant increase in the number of connectives when compared to wild-type. The increase in the number of connectives could either arise from increased axon branches from neurons in the same segment or overextended axons from other segments.

This particular assay is only useful for analyzing striking changes in axon growth, as single axon terminals cannot be distinguished. Abl null mutants were not shown since the decrease in axon terminal growth is subtle and would not be visible when all C4da axon terminals are imaged together. We have demonstrated the effects of Abl null mutations using single-cell genetic mosaic analysis (new Figure 1F-K), and the statistical analysis of the results is shown in the new Figure 1O.

5) Figure 1, general comment. All axon terminal phenotypes shown are for Dscam OE. While Dscam loss phenotypes have been shown in other systems (e.g. midline crossing), it has not been demonstrated for terminal growth. It would be useful to include data on the effects of Dscam loss at the synaptic terminal, as well as genetic experiments on the interaction between Dscam and abl loss of function alleles (as has been done in other systems). If the two genes are in one pathway for sensory terminal growth, then the double null should be indistinguishable from the single mutants. It is curious that Abl mutant terminals look relatively normal. Does this mean that Abl is not required for other receptors or DSCAM under normal expression levels?

We have demonstrated the consequences of Dscam loss of function in C4da axon terminal growth in a previous paper (Kim et al. 2013. Dscam expression levels determine presynaptic arbor sizes in Drosophila sensory neurons. Neuron 78:827-38). Null alleles of Dscam almost completely block presynaptic arbor growth. However, the reviewers’ comments made us aware that a discussion of the results of our previous work would be helpful to understanding the current manuscript and we have added this in the Results and Discussion section. Analysis of Dscam and Abl double loss of function larvae is technically challenging since Dscam mutations are embryonic lethal. Moreover, MARCM analysis of two genes located on different chromosomes is extremely difficult and in fact has not been demonstrated before.

Abl is known to be maternally contributed to the embryonic development, and thus there is likely to be some Abl even in abl null embryos. We think that it is likely that Abl is required for normal presynaptic arbor growth. In fact, we observed a subtle but significant reduction in presynaptic arbor size abl MARCM clones (Figure 1O). Nevertheless, our results demonstrate that the maternally contributed Abl is insufficient for the Dscam-induced presynaptic overgrowth caused by Dscam overexpression or genetic mutations of FMRP.

6) Figure 1–figure supplement 1A. Dscam-GFP levels in the neuronal soma is shown to be unchanged in abl mutants. However, the observations in the cell body do not rule out the possibility that at the synapse, abl loss could have effects on Dscam levels, perhaps due to changes in Dscam protein trafficking (Abl has been implicated in axon transport by Bill Saxton). Just because abl nulls revert the Dscam OE phenotype, this doesn't prove a downstream function. Abl can be required upstream of Dscam at the synapse. To test this, a parallel analysis of Dscam-GFP could be done at axon terminals to verify that the effect of abl is the same both in the soma and at the presynaptic terminal.

We agree that genetic epistasis does not prove an upstream-downstream relationship. In addition to the in vitro biochemistry studies suggesting that Dscam activates Abl, in the revised manuscript we show new results demonstrating that Dscam activates Abl in C4da axon terminals (new Figure 3B-D). Prompted by the reviewers’ suggestion, we have analyzed the effects of abl loss of function on Dscam expression in axon terminals. Using single-cell genetic mosaic analysis, we found that Dscam expression was unchanged in abl loss of function clones (new Figure 1–figure supplement 1C,D).

7) The abl C4da dendritic phenotype analysis should be added to the supplemental figures.

As requested, we have added supplemental figures to Figure 1 (Figure 1–figure supplement 2) showing that there is no difference in dendritic length between wild-type and abl mutant neurons using MARCM. We have also added supplemental figures to Figure 4 (Figure 4–figure supplement 2) showing that nilotinib does not affect dendritic development.

8) Figure 2C. There are no data confirming the stability of the Dscam cytoplasmic domain deletion. In fact the recovery in the IP (Figure 2B) is very poor and raises the concern that this receptor mutant is not capable of accumulating to the same concentration in the OE experiment, which could explain the lack of an in vivo OE phenotype in a way that does not support requirement of the Cyto domain. Moreover, why is full-length Dscam detected as a doublet? Can this be explained by cleavage, phosphorylation, etc.?

Thank you for making us aware that this point needed clarification. We have analyzed the expression and presence of this Dscam mutant, which is GFP-tagged, in axon terminals. The new result is shown in the Figure 2–figure supplement 1. When stained and imaged simultaneously, Dscam-GFP and DscamΔCyto-GFP are present in the axon terminals at comparable levels. In addition, we saw robust axon terminal overgrowth in the Dscam condition but never saw any change in axon terminal growth in the DscamΔCyto condition.

We believe that the lower molecular weight band of the doublet in old Figure 2C is a degradation product. This is supported by the lack of this doublet under essentially identical transfection conditions, as shown in Figure 2B.

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