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.

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.

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.

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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.

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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.

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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).

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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.

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Author details

1. Gabriella R Sterne

   1. Life Sciences Institute, University of Michigan, Ann Arbor, United States
   2. Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, United States

   Contribution

   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

   Contributed equally with

   Jung Hwan Kim

   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).

2. Jung Hwan Kim

   1. Life Sciences Institute, University of Michigan, Ann Arbor, United States
   2. Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, United States

   Contribution

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

   Contributed equally with

   Gabriella R Sterne

   Competing interests

   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).

3. Bing Ye

   1. Life Sciences Institute, University of Michigan, Ann Arbor, United States
   2. Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, United States

   Contribution

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

   For correspondence

   bingye@umich.edu

   Competing interests

   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).

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