Author response: Restraint of presynaptic protein levels by Wnd/DLK signaling mediates synaptic defects associated with the kinesin-3 motor Unc-104
2017-04-05

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The kinesin-3 family member Unc-104/KIF1A is required for axonal transport of many presynaptic components to synapses, and mutation of this gene results in synaptic dysfunction in mice, flies and worms. Our studies at the Drosophila neuromuscular junction indicate that many synaptic defects in unc-104-null mutants are mediated independently of Unc-104’s transport function, via the Wallenda (Wnd)/DLK MAP kinase axonal damage signaling pathway. Wnd signaling becomes activated when Unc-104’s function is disrupted, and leads to impairment of synaptic structure and function by restraining the expression level of active zone (AZ) and synaptic vesicle (SV) components. This action concomitantly suppresses the buildup of synaptic proteins in neuronal cell bodies, hence may play an adaptive role to stresses that impair axonal transport. Wnd signaling also becomes activated when pre-synaptic proteins are over-expressed, suggesting the existence of a feedback circuit to match synaptic protein levels to the transport capacity of the axon. https://doi.org/10.7554/eLife.24271.001 eLife digest Each nerve cell, or neuron, has a long nerve fiber – called an axon – that forms specialized sites for information exchange – called synapses – with other cells. Many molecules work at synapses to coordinate the exchange of information. These molecules are largely made in the central part of the neuron – known as the cell body – and are then transported along the axon to the synapses. The transport of these molecules is carried out by proteins known as molecular motors. One molecular motor, called KIF1A in humans and Unc-104 in fruit flies, is thought to be a major transporter of synaptic molecules. Mutations that hinder this molecular motor result in neurons failing to form synapses and, instead, synaptic components accumulate in the cell body. However, it was not clearif Unc-104 does actually carry all of the components needed to assemble synapses along axons, or if it influences synapse formation in another way. Now, Li, Zhang et al. report new evidence that supports the second of these two hypotheses. The experiments made use of fruit flies in which the gene for Unc-104 had been deleted, and revealed that inhibiting enzymes in a specific signaling pathway could reverse the synaptic problems caused by the loss of Unc-104. The signaling pathway, which is conserved between flies and humans, involves an enzyme that is called Wnd in flies and DLK in humans. The Wnd/DLK signaling pathway was previously known to regulate how neurons respond when their axons are damaged (either by growing new axons or dying, depending on the context). Further investigation by Li, Zhang et al. revealed that signaling via the Wnd enzyme becomes triggered whenever the Unc-104 molecular motor is impaired. This activation correlates with the build-up of synaptic proteins in the cell body. Once activated, the pathway then reduces the total amount of synaptic proteins that the cell makes. This reduction matches the neuron’s reduced ability to transport them along the axon, and may help the neuron to adapt when axonal transport is impaired. However, the reduction in synaptic proteins also impaired the exchange of information at the synapses. These findings suggest how DLK could be behind problems with synapses in diseases in which transport along axons is impaired. These diseases include hereditary spastic paraplegia, which has been linked to mutations in human KIF1A, and may also include ALS and Alzheimer’s disease, which have recently been linked to DLK. DLK has received recent attention as a candidate drug target because it contributes to the deterioration of damaged neurons. These new findings further expand that interest by suggesting that inhibiting DLK may help neurons to maintain working synapses, which is more useful than simply preventing damaged neurons from dying. https://doi.org/10.7554/eLife.24271.002 Introduction Synapse development, maintenance and plasticity involve highly orchestrated trafficking events in both pre and postsynaptic cells. In contrast to postsynaptic receptors, whose trafficking and organization has been studied extensively in many different synapse types (Choquet and Triller, 2013), much less is known about the mechanisms that regulate the assembly and maintenance of the neurotransmitter release machinery in the presynaptic neuron. This machinery includes the active zone (AZ), an electron-dense complex of structural proteins that scaffold both calcium channels and synaptic vesicles (SV) for the coordination of calcium-regulated exocytosis (Südhof, 2012). The protein components of the AZ are synthesized in cell bodies and trafficked together in association with vesicles (known as piccolo-bassoon transport vesicles (PTVs) (Ahmari et al., 2000; Maas et al., 2012; Shapira et al., 2003). SV precursors are also synthesized in cell bodies, and carried by kinesin motors to synapses (Hall and Hedgecock, 1991; Okada et al., 1995). Regulation of synapse development likely involves a global coordination of the synthesis and transport of both AZ and SV components. However the mechanisms that regulate these important steps in synapse development and maintenance are poorly understood. A critical role in synapse development has been assigned to the kinesin-3 family of motor proteins (Hall and Hedgecock, 1991; Kern et al., 2013; Niwa et al., 2016; Pack-Chung et al., 2007; Yonekawa et al., 1998). Mutations in mammalian Kif1a and its unc-104 orthologues in C. elegans and Drosophila (also known as imac, Klp53D and bris in Drosophila) cause severe defects in synapse development. In Drosophila unc-104-null mutants, synaptic boutons fail to form, SV and AZ components fail to traffic to nascent synapses, and concomitantly, SV and AZ associated proteins accumulate in the cell body (Pack-Chung et al., 2007). It is broadly accepted that Unc-104 protein functions as a molecular motor to physically deliver presynaptic components to their destinations in the synaptic terminal (Goldstein et al., 2008). However, while there is biochemical evidence that KIF1A can interact with and ‘carry’ SV precursors (Okada et al., 1995), there is little evidence that KIF1A (or Unc-104) carries AZ components. The mechanistic role of Unc-104 in AZ transport and assembly remains unclear. In this study we found that synaptic defects in embryonic unc-104-null mutants, including the failure to form synaptic boutons and AZs, arise not from direct loss of Unc-104 transport function, but via an indirect mechanism, which involves activation of the Wnd/DLK axonal damage signaling pathway. The Wnd/DLK mixed lineage kinase has recently received intense interest for its roles in regulating both regenerative and degenerative responses to axonal damage in vertebrate and invertebrate neurons (Gerdts et al., 2016; Hao and Collins, 2017; Li and Collins, 2017; Tedeschi and Bradke, 2013). We found that the Wnd/DLK signaling pathway becomes activated when Unc-104’s function is impaired, and then promotes synaptic dysfunction by restraining expression of multiple pre-synaptic AZ and SV protein components. This restraint concomitantly reduces protein buildup in cell bodies, which may play an adaptive role to stresses that disrupt intracellular transport, and contribute to pathologies that arise when transport is disrupted. Results Roles for the Unc-104 kinesin in AZ transport and synaptic bouton growth can be functionally separated from SV transport Previous studies of NMJ development in unc-104-null mutant animals have revealed essential roles for this kinesin in synaptic maturation (Hall and Hedgecock, 1991; Kern et al., 2013; Pack-Chung et al., 2007; Yonekawa et al., 1998). At the Drosophila neuromuscular junction (NMJ), unc-104-null mutants are severely defective in the formation of presynaptic boutons, fail to localize SVs to NMJ terminals and show strong reductions in AZ localization (Pack-Chung et al., 2007 and Figure 1). We found that disrupting the axonal damage signaling kinase Wnd (wnd3 single mutants) had no significant effect on bouton formation, AZ number, or presynaptic protein localization, but double mutants with unc-104-null alleles (unc-104P350, unc-104170, and unc-10452), gave a very informative phenotype: in unc-104null;wnd3/3 double mutants the synaptic bouton formation (Figure 1A and C), AZ number (Figure 1A and D), and synaptic levels of the AZ protein Brp (Figure 1A and E) were restored to a wild type phenotype. In contrast, the synaptic levels of SV proteins (VGlut, SytI and CSP) remained negligible, as in the unc-104null single mutants (Figure 1B and E). These results are consistent with previous findings that Unc-104 functions as an essential molecular motor to transport SV precursors to synaptic terminals. However our findings indicate that transport of AZ precursors and bouton formation can occur independently of Unc-104. These defects are mediated by a second and separable mechanism, which depends upon the function of the Wnd kinase. Figure 1 Download asset Open asset AZ transport and Synaptic bouton formation but not SVP transport defects in unc-104-null mutants are rescued by mutations in wnd. Representative images of ISNb neuromuscular junction (NMJ) terminals at muscles 6, 7, 12 and 13 at embryonic stage 17 (20–21 hr AEL). We observed similar results with multiple independently generated unc-104-null alleles including unc-104P350, unc-104170and unc-10452. Representative images with unc-104P350 are shown here. (A) Images of ISNb NMJ terminals immunostained for a neuronal membrane marker to show axons and terminal boutons (HRP, green in upper panel) and the AZ component Brp (magenta in upper panel, white in lower panel). Boutons failed to form and AZs failed to localize to NMJ in unc-104null mutants. Both defects were restored in unc-104null(P350);wnd3 mutants. (B) Images of ISNb NMJ terminals immunostained for presynaptic vesicle proteins (VGlut, upper panels; SytI, lower panel). The insets show HRP staining of the nerve terminals. Note VGlut and SytI failed to localize to NMJ in both unc-104null(P350) and unc-104null(P350); wnd3 mutants. (C) The bouton formation defect was quantified as the percentage of ISNb NMJ terminals that contained at least one presynaptic varicosity, identified by HRP staining (and scored while blind to genotype). This method over-estimates the actual number of boutons in unc-104null mutants, since any varicosity within any of the ISNb terminals (on muscles 6, 7, 12 or 13) was counted. (D) The number of AZs (identified as Brp punctae) formed at ISNb NMJ terminals (on muscles 6, 7, 12 and 13). (E) The total (sum) intensity of Brp, VGlut, SytI and CSP measured across the ISNb NMJ terminals. All data are represented as mean ±SEM; At least 9 animals and 20 ISNb NMJ terminals were examined per genotype; N.S., not significant; ****p<0.0001, *p<0.05; Tukey test for multiple comparison; Scale bar, 10 μm. https://doi.org/10.7554/eLife.24271.003 The Wnd signaling pathway mediates presynaptic defects in unc-104-hypomorph mutants To further study the effect of Wnd upon AZ assembly we utilized several hypomorphic loss-of-function mutations of unc-104, whose ability to survive to the third instar larval stage has allowed for extensive characterization of synaptic defects associated with Unc-104 (Barkus et al., 2008; Cao et al., 2014; Kern et al., 2013; Zhang et al., 2016; 2017). We used the EMS-generated alleles bris/null (Kern et al., 2013) and O3.1/null (Barkus et al., 2008), and knockdown in motoneurons via independent RNAi lines. All of these mutants share similar characteristics: Post Synaptic Densities (PSDs) form (identified by the presence of a core receptor subunit GluRIII), however ~50% lack apposing presynaptic AZ components, as identified by Brp (Figure 2A and C), Liprin-α (Figure 2B and D) and the voltage gated calcium channel Cac-GFP (Figure 2—figure supplement 2). This defect is accompanied by a reduction in the levels of Brp within individual synapses and also across entire NMJ terminals (Figure 2E). Similarly to the unc-104-null mutants (Figure 1), mutations in wnd fully rescued these presynaptic AZ assembly defects (Figure 2A–E). RNAi knockdown of unc-104 using either pan-motoneuron or single motoneuron driver lines led to AZ defects and concomitant knockdown of wnd in neurons led to rescue, (Figure 2—figure supplement 1), indicating a cell-autonomous role for both Unc-104 and Wnd in the synaptic defects. Figure 2 with 2 supplements see all Download asset Open asset Wnd signaling pathway is required for the presynaptic defects in unc-104-hypomorph mutants. (A–B) Representative confocal images of third instar larval neuromuscular junctions (NMJ) at muscle 4. Postsynaptic densities (PSDs) identified by GluRIII staining (Green) that lacked apposing AZ components Brp (magenta in A), or Liprin-α (magenta in B) are highlighted by arrowheads. Note that when determining the absence of AZ components, the pixel saturation threshold was reduced to ensure detection ofweak signals (this resulted in saturation of certain pixels in wt controls. For more details, see Experimental Procedures.). (A) Alignment of postsynaptic GluRIII (green) with presynaptic AZ component Brp (magenta) in Canton-S (wt), unc-104bris/P350, unc-104bris/P350;wnd3 and wnd3. Unc-104bris is a partial loss of function mutation (Kern et al., 2013), while unc-104P350 is a null allele (Barkus et al., 2008). wnd3 is a presumptive null mutation in wnd (Collins et al., 2006). (B) Alignment of postsynaptic GluRIII (green) with presynaptic AZ component Liprin-α (magenta). Liprin-α-GFP was driven by rab7 promoter and UAS-unc-104 RNAi and UAS-wnd RNAi were driven by neuronal elav-Gal4. UAS-mCD8-RFP was used as a control for UAS dosage. (C–D) The percentage of PSDs which lack any trace of apposing presynaptic AZ protein, based on (C) Brp or (D) Liprin-α-GFP. (E) Signal intensity for Brp staining at individual synapses or summed across the entire NMJ terminal (at muscle 4), normalized to that in wt (Canton S) animals. (F) The total intensity of Vglut protein measured across the entire synaptic NMJ terminal at the muscle 4, normalized to that in wt (Canton S). All data are represented as mean ±SEM; At least 6 animals and 12 muscle 4 NMJ terminals were examined per genotype; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Scale bar, 5 μm. For additional data, see Figure 2—figure supplements 1–2. https://doi.org/10.7554/eLife.24271.004 In axonal regeneration and synaptic overgrowth, Wnd has been shown to act in a signaling pathway consisting of a cascade of MAP kinases and transcription factors (Collins et al., 2006; Nakata et al., 2005; Xiong et al., 2010). We found that inhibition of the downstream MAP Kinase JNK (Bsk) and Fos transcription factor, via expression of dominant-negative (DN) isoforms in neurons, could also rescue the presynaptic defect shown in the unc-104 single mutants (Figure 2C and Figure 2—figure supplement 2). These results suggest that a signaling pathway consisting of Wnd, JNK and Fos mediates synaptic defects observed in unc-104 mutants. Suppression of unc-104-hypomorph defects reveals additional roles for the Wnd signaling pathway in restraining neurotransmission In contrast with unc-104-null mutants, we observed that wnd mutations caused a striking increase in quantity of VGlut at unc-104-hypomorphic mutant NMJs (Figure 2F). The dramatic rescue of structural defects as well as SV protein localization at NMJs suggested that Wnd pathway mutations may also rescue synaptic transmission defects in unc-104-hypomorph mutants. Unc-104-hypomorph mutant NMJs have severely reduced mini frequency, and modestly reduced mEJP amplitude, EJP amplitude and quantal content (Zhang et al., 2017). We observed that mutations in wnd, jnk (bsk) or fos fully rescued the mEJP frequency defects (Figure 3A and C and Figure 3—figure supplement 1). mEJP amplitude, EJP amplitude and quantal content were also rescued by wnd mutations (Figure 3), while Bsk (JNK) and Fos inhibition rescued some aspects of these defects (Figure 3—figure supplement 1 and Figure 3—source datas 1–3). Through distribution analysis, we further confirmed the change in mEJP amplitude and validated the coverage of mEJP events for frequency analysis (Figure 3—figure supplement 2). The suppression of these phenotypes by mutations in wnd suggests that the Wnd pathway impairs multiple aspects of synaptic structure and transmission. Figure 3 with 4 supplements see all Download asset Open asset The synaptic transmission defect in unc-104 mutants is suppressed by wnd mutations. (A–B) Representative electrophysiological traces of (A) miniature Excitatory Junctional Potentials (mEJP) and (B) Evoked Excitatory Junctional Potentials (EJP) recorded from muscle 6 of third instar larvae. (C) Quantification of impaired mEJP frequency for unc-104-hypomorph mutants and their rescue by wnd mutations or expression of dominant negative (DN) bsk or fos (driven by elav-Gal4 and BG380-Gal4, respectively). Representative traces for bskDN and fosDN are shown in Figure 3—figure supplement 1. (D–F) Quantification of (D) mEJP amplitude, (E) EJP amplitude and (F) quantal content (corrected for nonlinear summation). See also Figure 3—figure supplement 1 for additional quantification of bskDN and fosDN data. Unc-10403.1 and unc-104bris are hypomorphic alleles, while unc-104P350 and unc-104d11204 are null alleles. wnd3 is a presumptive null mutation in wnd (Collins et al., 2006). wt animals are Canton S. All data are represented as mean ±SEM; N.S., not significant; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Tukey test for multiple comparison. For additional data, see Figure 3—figure supplements 1–4. https://doi.org/10.7554/eLife.24271.007 Figure 3—source data 1 Measurements of EJP and mEJP. The file contains data from 3 independent experiments and 6 source data sheets: EJP amplitude, corrected EJP amplitude and Quantal content, mEJP amplitude and frequency of CS, unc-104, unc-104;wnd and wnd. This is corresponding to Figure 3. https://doi.org/10.7554/eLife.24271.012 Download elife-24271-fig3-data1-v1.xlsx Figure 3—source data 2 Measurements of EJP and mEJP. The file contains EJP amplitude, corrected EJP amplitude and Quantal content, mEJP amplitude and frequency of CS, unc-104bris/d11204, unc-104bris/d11204;bskDN and bskDN. This is corresponding to Figure 3C and Figure 3—figure supplement 1. https://doi.org/10.7554/eLife.24271.013 Download elife-24271-fig3-data2-v1.xlsx Figure 3—source data 3 Measurements of EJP and mEJP. The file contains EJP amplitude, corrected EJP amplitude and Quantal content, mEJP amplitude and frequency of CS, unc-104bris/O3.1, unc-104bris/O3.1;fosDN and fosDN. This is corresponding to Figure 3C and Figure 3—figure supplement 1. https://doi.org/10.7554/eLife.24271.014 Download elife-24271-fig3-data3-v1.xlsx The total levels of Unc-104 protein remained low in the unc-104hypomorph;wnd double mutants (Figure 3—figure supplement 3). Moreover, defects in larval motility and lethality of unc-104-hypomorph mutants largely remained in unc-104hypomorph;wnd double mutants, implying the persistence of major defects from the loss of Unc-104’s function (Figure 3—figure supplement 4). Hence the extensive suppression of the synaptic defects at the larval NMJ is unlikely to be the result of increased stability of the residual Unc-104 protein. Instead, as indicated by the suppression of unc-104-null defects (Figure 1), the synaptic phenotypes reflect an activity of the Wnd pathway rather than a direct consequence of impaired Unc-104 driven transport. Wnd signaling becomes activated when Unc-104’s function is lost To test for Wnd signaling activation in unc-104 mutants, we utilized a transcriptional reporter of JNK signaling, the puckered (puc)-lacZ enhancer trap (Martín-Blanco et al., 1998), which has been previously shown to report Wnd signaling activity in motoneurons (Valakh et al., 2013; Xiong et al., 2010). The basal expression of puc-lacZ is very low in wild type animals but was significantly increased in all unc-104 LOF mutant backgrounds (including bris/null, O3.1/null, and 2 independent RNAi lines) (Figure 4A and B). This increase was abolished when wnd was concomitantly knocked-down by RNAi (Figure 4B), hence reflects activation of a Wnd-mediated nuclear signaling cascade. Figure 4 Download asset Open asset The Wnd signaling pathway is activated in unc-104 mutants. (A) Images of motoneuron cell bodies near the midline of larval ventral nerve cord, immunostained for motoneuron nuclear marker phospho-SMAD (magenta) and the Wnd/JNK nuclear reporter puc-LacZ (green). UAS-RNAi lines (including UAS-moody RNAi as a control) were driven by pan-neuronal BG380-Gal4. At least 3 dorsal abdominal clusters of motoneurons per VNC from 6 animals per genotype were examined. (B) Quantification of puc-lacZ expression in multiple unc-104 mutant backgrounds. Unc-10403.1 and unc-104bris are hypomorphic alleles, while unc-104170 is a null allele. In addition we tested two independent unc-104 RNAi lines (vdrc 23465, I and TRiP BL43264, II). Unc-104 RNAi II was accompanied with Dicer2 expression to facilitate the knock-down. Co-expression of wnd-RNAi, compared to UAS-mCD8-ChRFP reduced the effect of unc-104 knockdown. The ‘control’ genotype has UAS-mCD8-ChRFP, (which as a control for of UAS Quantification methods are in Experimental (C) bouton and at the via for HRP at muscle 4. The presynaptic was in unc-104 bris mutants compared to wt (Canton S) control and this was rescued in double mutants. (D) Quantification of presynaptic was by the total NMJ the to the bouton of the presynaptic nerve terminal at muscle 4, via The data shown used unc-104bris and while and were driven by elav-Gal4. wt control animals were Canton S. (E) axonal of axons 10 9 hr or hr nerve from wt compared to mutant animals. the and the of the cell body. 9 hr nerve axons in wild type new growth via from the axonal At a are and either the axon or the cell body. In unc-104 mutants a increase in new axonal at 9 and at hr these new axonal similar but as as wild type (F) 9 hr the percentage of axons which more than 5 per axon. The of the per nerve at hr mutant and 2 independent unc-104 RNAi lines and control RNAi (driven by were examined. All data are represented as mean ±SEM; N.S., not significant; ****p<0.0001, Tukey test for multiple comparison; Scale bar, 20 μm. evidence for Wnd signaling activation was revealed by the presynaptic nerve terminal of unc-104 hypomorphic mutant which show increased of synaptic and boutons which are in and and Kern et These of presynaptic nerve terminal have previously been for Wnd pathway activation et al., 2014; et al., 2006; et al., 2013; et al., 2007). We confirmed that this in unc-104 mutants the function of Wnd, JNK and Fos in presynaptic motoneurons (Figure and we observed that unc-104-hypomorph mutants an ability to axons (Figure A similar role in restraining axonal growth has recently been for unc-104 development in C. elegans et al., previous studies have shown that activation of Wnd/DLK signaling the ability of axons to regenerative axonal growth et al., et al., 2012; Xiong et al., et al., the activation of Wnd signaling in unc-104 mutants may this function in restraining rather than axonal Previous studies have suggested between JNK signaling and the of axonal transport and 2014; and It is that other mutations that disrupt axonal transport, including mutations which and not significantly the expression of puc-lacZ (Figure and Xiong et al., 2010). our results suggest a between Wnd pathway and the Unc-104 and that the Wnd signaling pathway becomes activated when Unc-104’s function is Wnd protein is to the cell body in unc-104 mutants Previous studies have that the protein levels of Wnd and its DLK in and C. elegans are and with the activity of Wnd signaling pathway and 2016; Hao et al., 2016; et al., 2013). A highly conserved protein the levels of Wnd/DLK protein and Wnd/DLK signaling activation in wild type animals et al., 2013; et al., 2006; Nakata et al., 2005; Xiong et al., 2010). Mutations in to levels of Wnd protein whose localization can be in and at synapses (Collins et al., 2006). We examined Wnd protein in unc-104 mutants, for to the previous in mutants. In contrast to mutations in unc-104 not to a increase in global levels of Wnd protein by (Figure or in synaptic Wnd protein level (Figure In unc-104 mutants not share the previously of mutants of axonal et al., and data not These suggest that the of Wnd signaling activation in unc-104 mutants is unlikely to occur via Figure 5 with 3 supplements see all Download asset Open asset Wnd signaling is activated via an in unc-104 mutants. (A) Representative of larval for Wnd and and quantification of Wnd levels normalized to intensity 3). examined include wnd3 and (B) Images of larval ventral nerve from animals immunostained for Note the increased in mutants, but not control or unc-104 mutants. This also that the Wnd (Collins et al., to by (C) Images of motoneuron cell bodies in animals immunostained with to these images are in a and to the motoneuron cell cell bodies are by green At least 6 animals were examined per (D) Images of motoneuron cell bodies via in (which is kinase to pathway driven by A cell body is by a green At least 6 animals were examined per Quantification of intensity from (C–D) in cell bodies, normalized to wt animals. Note that kinase protein has a basal expression level which for increased in to Wnd protein. Representative images of presynaptic Brp and postsynaptic GluRIII from and and and PSDs are highlighted by arrowheads. is Canton of PSDs from and All data are represented as mean ±SEM; N.S., not Tukey test for multiple comparison; Scale 20 and 5 μm. For additional data, see Figure supplements We then further at localization using with Wnd protein remained the detection threshold in both wild type and unc-104 mutant animals not to further upon Wnd protein we utilized two different a et al., in which a