The E3 ligase Highwire promotes synaptic transmission by targeting the NAD‐synthesizing enzyme dNmnat
2019; Springer Nature; Volume: 20; Issue: 3 Linguagem: Inglês
10.15252/embr.201846975
ISSN1469-3178
AutoresAlexandra Russo, Pragya Goel, E. J. Brace, Christopher Buser, Dion Dickman, Aaron DiAntonio,
Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle29 January 2019free access Transparent process The E3 ligase Highwire promotes synaptic transmission by targeting the NAD-synthesizing enzyme dNmnat Alexandra Russo Department of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA Search for more papers by this author Pragya Goel Department of Neurobiology, University of Southern California, Los Angeles, CA, USA Search for more papers by this author EJ Brace Department of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA Search for more papers by this author Chris Buser Oak Crest Institute of Science, Monrovia, CA, USA Search for more papers by this author Dion Dickman Department of Neurobiology, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Aaron DiAntonio Corresponding Author [email protected] orcid.org/0000-0002-7262-0968 Department of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA Search for more papers by this author Alexandra Russo Department of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA Search for more papers by this author Pragya Goel Department of Neurobiology, University of Southern California, Los Angeles, CA, USA Search for more papers by this author EJ Brace Department of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA Search for more papers by this author Chris Buser Oak Crest Institute of Science, Monrovia, CA, USA Search for more papers by this author Dion Dickman Department of Neurobiology, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Aaron DiAntonio Corresponding Author [email protected] orcid.org/0000-0002-7262-0968 Department of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA Search for more papers by this author Author Information Alexandra Russo1, Pragya Goel2, EJ Brace1, Chris Buser3, Dion Dickman2 and Aaron DiAntonio *,1 1Department of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA 2Department of Neurobiology, University of Southern California, Los Angeles, CA, USA 3Oak Crest Institute of Science, Monrovia, CA, USA *Corresponding author. Tel: +1 314 362 9925: E-mail: [email protected] EMBO Rep (2019)20:e46975https://doi.org/10.15252/embr.201846975 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The ubiquitin ligase Highwire restrains synaptic growth and promotes evoked neurotransmission at NMJ synapses in Drosophila. Highwire regulates synaptic morphology by downregulating the MAP3K Wallenda, but excess Wallenda signaling does not account for the decreased presynaptic release observed in highwire mutants. Hence, Highwire likely has a second substrate that inhibits neurotransmission. Highwire targets the NAD+ biosynthetic and axoprotective enzyme dNmnat to regulate axonal injury responses. dNmnat localizes to synapses and interacts with the active zone protein Bruchpilot, leading us to hypothesize that Highwire promotes evoked release by downregulating dNmnat. Here, we show that excess dNmnat is necessary in highwire mutants and sufficient in wild-type larvae to reduce quantal content, likely via disruption of active zone ultrastructure. Catalytically active dNmnat is required to drive defects in evoked release, and depletion of a second NAD+ synthesizing enzyme is sufficient to suppress these defects in highwire mutants, suggesting that excess NAD+ biosynthesis is the mechanism inhibiting neurotransmission. Thus, Highwire downregulates dNmnat to promote evoked synaptic release, suggesting that Highwire balances the axoprotective and synapse-inhibitory functions of dNmnat. Synopsis The E3 ubiquitin ligase Highwire targets and downregulates the NAD+ synthesizing enzyme dNmnat to promote evoked synaptic transmission at the Drosophila larval neuromuscular junction. Hiw mutants exhibit defects in evoked synaptic transmission, release probability, and active zone T-bar structure, each of which are dNmnat-dependent. Excess dNmnat in a wildtype background can depress evoked release, and this is dependent on the catalytic activity of dNmnat, suggesting that excess NAD+ synthesis drives the defects in synaptic transmission. Hiw balances the axoprotective and synapse-inhibitory functions of dNmnat by tightly regulating dNmnat levels in Drosophila. Introduction The E3 ubiquitin ligase PHR is a central regulator of neural circuit development, function, and maintenance with conserved activities in worms, flies, and mammals 1, 2. PHR is a huge, multidomain protein, but its best understood functions are to promote the turnover of target proteins via proteasomal degradation 2-4. In Drosophila, the PHR ortholog Highwire (Hiw) is a key regulator of neuromuscular junction (NMJ) morphology and physiology 5-8. The NMJs of hiw mutant larvae are massively overgrown relative to wild type (WT) with a dramatic increase in synaptic terminal branching and bouton number 5, 8. In addition, hiw mutant NMJs exhibit reduced synaptic strength due to a decrease in quantal content, the number of vesicles released following an action potential 7, 8. We wished to identify the functionally relevant protein targets of Highwire to gain insight into the molecular mechanisms controlling synaptic morphology and function. We identified the mitogen-activated protein kinase kinase kinase (MAP3K) Wallenda (Wnd), the Drosophila ortholog of Dual leucine zipper kinase (DLK), as the Highwire target responsible for synaptic terminal overgrowth in highwire mutants 8. Wallenda protein levels are increased in hiw mutants, wnd is necessary for the synaptic terminal overgrowth in hiw mutants, and overexpression of wnd is sufficient to phenocopy this overgrowth in otherwise wild-type larvae. To our surprise, however, Wallenda is not the Highwire target responsible for the defect in evoked transmitter release in hiw mutants 8. Even though hiw;wnd double mutants have NMJs that are morphologically indistinguishable from wild type, these double mutants are still defective in evoked synaptic transmission 8, 9, implying that Hiw must regulate a second substrate to promote synaptic release. While Highwire and its orthologs were first studied due to their effects on neural circuit development and function 4, more recently it was discovered that Highwire is also a key determinant of axonal survival following injury 10. In the absence of Highwire or its vertebrate ortholog Phr1, Wallerian degeneration of injured axons is dramatically delayed 10, 11. For this function, Highwire targets nicotinamide mononucleotide adenyltransferase (dNmnat), an NAD+ biosynthetic enzyme which, along with its mammalian orthologs, is a potent axonal maintenance factor 10-13. In addition, dNmnat also promotes synaptic maintenance, acting as a chaperone for the active zone scaffolding protein Bruchpilot (Brp) 14. While it is clear that dNmnat is necessary to maintain axons and synapses, we wondered whether the elevation in dNmnat levels in hiw mutants, which is apparent in both the synaptic terminal and axon 10, could also impact the synapse, inducing the defects in synaptic release in highwire mutants. To investigate whether dNmnat could be the Highwire target inhibiting evoked release, we first demonstrated that excess dNmnat is sufficient to impair evoked synaptic transmission. Moreover, dNmnat is necessary for the defective evoked release in hiw mutants. Downregulation of dNmnat in the hiw mutant fully suppresses defects in evoked release, but has no impact on NMJ terminal morphology. This excess dNmnat leads to a decrease in release probability and disrupts the architecture of T-bars at active zones. In addition, depletion of NAD+ Synthetase, the subsequent enzyme in the NAD+ biosynthesis pathway, also suppresses the defect in evoked release in hiw mutants. These findings support the model that excess NAD+ biosynthetic activity impairs evoked synaptic transmission, and suggests that Highwire locally tunes levels of dNmnat protein, and therefore local NAD+ levels, to promote efficient synaptic transmission. These findings identify an unexpected activity of dNmnat in the inhibition of evoked synaptic release and suggest that Highwire controls dNmnat levels to balance its promotion of axonal maintenance and inhibition of synaptic transmission. Results Excess presynaptic dNmnat is sufficient to reduce evoked release Hiw mutants exhibit decreased synaptic strength at the larval NMJ; however, the substrate regulated by Hiw to promote synaptic transmission remains unknown. Given that the NAD+ synthesizing enzyme dNmnat is a known target for degradation by the E3 ligase Hiw, and that in hiw mutants there is an excess of dNmnat protein throughout the nervous system including at the synaptic terminal 10, we hypothesized that increased dNmnat protein levels decrease synaptic transmission. To begin probing this hypothesis, we first asked whether an excess of neuronal dNmnat is sufficient to induce a decrease in synaptic strength. We reasoned that if excess dNmnat protein in the hiw mutant synaptic terminal is the primary driver of diminished synaptic transmission, then overexpression of dNmnat protein in an otherwise wild-type background could phenocopy mutations in hiw and cause a decrease in evoked release. To test this, we overexpressed a dNmnat transgene (UAS-dNmnat) via the glutamatergic VGlut-GAL4 driver 15 to express dNmnat in motor neurons and measured spontaneous and evoked synaptic transmission at the larval NMJ. Overexpression of dNmnat induces a decrease in EPSP amplitude, the postsynaptic response to the evoked release of many synaptic vesicles, that is not seen upon overexpression of a control transgene (UAS-RFP; Fig 1A and B). To determine whether there was a postsynaptic contribution to the decrease in EPSP amplitude, we measured mEPSP amplitudes, the postsynaptic response to the transmitter content of single vesicles and therefore a measure of postsynaptic receptor responsivity to neurotransmitter. Expression of dNmnat did not lead to a significant difference in mEPSP amplitude (Fig 1A for representative traces, 1C). The quantal content, or average number of synaptic vesicles released upon an action potential, is calculated by dividing mean EPSP amplitude by mEPSP amplitude and demonstrates a significant decrease (Fig 1B). Hence, neuronal overexpression of dNmnat can impair presynaptic transmitter release. Overexpression of the MAP3K Wnd, known to be the Hiw substrate that promotes presynaptic terminal growth 8, did not elicit defects in quantal content. While EPSP amplitudes were decreased relative to controls (Fig 1A and B), this decrease was driven by a substantial decrease in mEPSP amplitudes (Fig 1A and C), leaving quantal content unchanged (Fig 1D). These results are consistent with recent work demonstrating that excess presynaptic Wnd drives reduced postsynaptic responsivity to transmitter 9, but does not decrease neurotransmitter release from the presynaptic motor neuron. Figure 1. Excess presynaptic dNmnat is sufficient to depress evoked release without altering NMJ growth Representative EPSP and mEPSP electrophysiological traces from larval NMJs at muscle 6 in which a motoneuron-specific driver was used to overexpress either a control transgene (DVGlut-GAL4 > UAS-RFP), dNmnat (DVGlut-Gal4 > UAS-dNmnat), or Wnd (DVGlut-Gal4 > UAS-Wnd). Quantification of mean (± SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then, cell amplitudes were averaged per genotype [UAS-RFP n = 13 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n = 10 cells. One-way ANOVA with Tukey's multiple comparisons, DF = 36, F = 12.66, P < 0.0001. UAS-RFP vs. UAS-dNmnat P < 0.0001 (****), UAS-RFP vs. UAS-Wnd P = 0.0093 (**)]. Quantification of mean (± SEM) mEPSP amplitudes, in which 75 consecutive spontaneous events were averaged per cell, and then, cell amplitudes were averaged per genotype [UAS-RFP n = 15 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n = 10 cells. One-way ANOVA with Tukey's multiple comparisons, DF = 38, F = 11.66, P = 0.0001. UAS-RFP vs. UAS-dNmnat P = 0.994 (NS), UAS-RFP vs. UAS-Wnd P = 0.0003 (***)]. Quantification (mean ± SEM) of quantal content, which was calculated individually per cell by dividing the mean EPSP amplitude by the mean mEPSP amplitude and then averaged per genotype [UAS-RFP n = 15 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n = 10 cells. One-way ANOVA with Tukey's multiple comparisons, DF = 38, F = 9.286, P = 0.0006. UAS-RFP vs. UAS-dNmnat P = 0.0015 (**), UAS-RFP vs. UAS-Wnd P = 0.9892 (NS). Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in the electrophysiology experiments in (A–D). NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red). Quantification of the mean (± SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [UAS-RFP n = 23, UAS-dNmnat n = 21, UAS-Wnd n = 20 NMJs. One-way ANOVA with Tukey's multiple comparisons, DF = 63, F = 68.31, P < 0.0001. UAS-RFP vs. UAS-dNmnat P = 0.9818 (NS), UAS-RFP vs. UAS-Wnd P < 0.0001 (****)]. Download figure Download PowerPoint Given the effect of excess dNmnat on the function of NMJ synapses, we next investigated whether NMJ terminal morphology was impacted by overexpressing dNmnat. Synaptic terminal growth, as measured by counting the number Drosophila vesicular glutamate transporter (DVGlut)-positive synaptic boutons localized within HRP-positive terminal membrane 7, 8, was unchanged upon dNmnat overexpression (Fig 1E and F). In contrast, overexpressing Wnd in glutamatergic neurons was sufficient to drive synaptic overgrowth at the NMJ, consistent with previous findings (Fig 1E and F) 8, 9. Hence, overexpression of two distinct Hiw substrates in a wild-type background can each phenocopy one aspect of the hiw mutant phenotype—excess neuronal dNmnat impairs synaptic release while excess neuronal Wnd promotes synaptic terminal overgrowth. Evoked release defects in Hiw mutants are dependent on presynaptic dNmnat Having demonstrated that excess dNmnat is sufficient to reduce presynaptic release, we next investigated whether dNmnat is necessary for the observed decrease in evoked release in a hiw mutant background. We genetically depleted dNmnat in a hiw mutant, using the strong hypomorphic allele hiwND8 5, and measured synaptic transmission at the NMJ. dNmnat is an essential gene and mutants are embryonic lethal 16, so to decrease levels of dNmnat we used the pan-neuronal driver Elav to express transgenic RNAis against dNmnat. This approach limits knockdown to the neuron while leaving dNmnat function intact in the postsynaptic muscle and other non-neuronal cells. As previously published, hiw mutant NMJs have decreased synaptic strength relative to wild-type NMJs, as measured by decreased EPSP amplitudes and a decrease in quantal content (Fig 2A–C, 7, 8). We confirmed previously published data that presynaptic depletion of Wnd via a transgenic RNAi is unable to suppress defective evoked release in a hiw mutant (Fig 2A–C), despite suppressing synaptic terminal morphology and bouton number (Fig 2D and E). Depletion of dNmnat in a hiw mutant background, however, fully suppresses the decrease in both EPSP amplitude and quantal content (Fig 2A–C). mEPSP amplitudes were not significantly different among all genotypes [Fig 2A for representative traces, mean (± SEM) mEPSP amplitudes for 75 consecutive spontaneous events per cell, averaged across genotype—WT n = 10 cells, mean amplitude = 0.7424 mV; hiw n = 10 cells, mean amplitude = 0.6329 mV; hiw > Wnd-RNAi n = 10 cells, mean amplitude = 0.7685 mV; and hiw > dNmnat-RNAi n = 10 cells, mean amplitude = 0.6709 mV. One-way ANOVA with Tukey's multiple comparisons, DF = 39, F = 2.454, P = 0.0789. WT vs. hiw P = 0.2397 (NS), WT vs. hiw > Wnd-RNAi P = 0.9670 (NS), WT vs. hiw > dNmnat-RNAi P = 0.5914 (NS)]. We repeated the experiment in a second hiw mutant background (hiw∆N, which is a null allele, 7) and found that a second non-overlapping RNAi line targeting dNmnat partially suppressed decreased EPSP amplitudes and completely suppressed the decrease in quantal content in the hiw null background (Fig EV1A–D). Figure 2. Excess presynaptic dNmnat drives defects in evoked neurotransmission at the NMJ in Hiw mutants, but has no role in synaptic overgrowth Representative EPSP and mEPSP physiological traces from larval NMJs at muscle 6. Recordings were taken from WT control larvae (Elav-Gal4 > UAS-RFP), hiw mutants driving a control transgene (hiwND8;Elav-Gal4 > UAS-RFP), and hiw mutants expressing RNAis to knockdown either dNmnat (hiwND8;Elav-Gal4 > UAS-dNmnat-RNAi [#29402]) or Wnd (hiwND8;Elav-Gal4 > UAS-Wnd-RNAi [#25396]). Quantification of mean (± SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then, cell amplitudes were averaged per genotype [WT n = 10 cells, hiw n = 10 cells, hiw > Wnd-RNAi n = 10 cells, hiw > dNmnat-RNAi n = 10 cells. One-way ANOVA with Tukey's multiple comparisons, DF = 39, F = 14.02, P < 0.0001. WT vs. hiw P = 0.0012 (**), WT vs. hiw > Wnd-RNAi P < 0.0001 (****), WT vs. hiw > dNmnat-RNAi P = 0.9880 (NS)]. Quantification of quantal content (± SEM) [WT n = 9 cells, hiw n = 9 cells, hiw > Wnd-RNAi n = 10 cells, hiw > dNmnat-RNAi n = 10 cells. One-way ANOVA with Tukey's multiple comparisons, DF = 38, F = 2.508, P < 0.0001. WT vs. hiw P = 0.0361 (*), WT vs. hiw > Wnd-RNAi P = 0.0001 (***), WT vs. hiw > dNmnat-RNAi P = 0.9889 (NS)]. Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in panels (A–C). NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red). Quantification of the mean (± SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype [WT n = 23, hiw n = 21, hiw > Wnd-RNAi n = 16, hiw > dNmnat-RNAi n = 19. One-way ANOVA with Tukey's multiple comparisons, DF = 78, F = 169.1, P < 0.0001. WT vs. hiw P < 0.0001 (****), WT vs. hiw > Wnd-RNAi P = 0.9988 (NS), WT vs. hiw > dNmnat-RNAi P < 0.0001 (****), hiw vs. hiw > dNmnat-RNAi P = 0.9695 (NS)]. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. A second RNAi targeting dNmnat suppresses defects in evoked release in the hiw null background Representative EPSP and mEPSP physiological traces from larval NMJs at muscle 6. Recordings were taken from WT control larvae (Elav-Gal4 > UAS-RFP), hiw null mutants driving a control transgene (hiw∆N;Elav-Gal4 > UAS-RFP), and hiw mutants expressing an RNAi to knockdown dNmnat (hiwND8;Elav-Gal4 > UAS-dNmnat-RNAi [KK#107262]). This RNAi is unique from the one presented in Fig 2 and targets a non-overlapping portion of the dNmnat gene product (VDRC KK#107262). Quantification of mean (± SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then, cell amplitudes were averaged per genotype [control n = 7 cells, hiw∆N n = 6 cells, hiw∆N > dNmnat-RNAi n = 8 cells. One-way ANOVA with Tukey's multiple comparisons, DF = 20, F = 46.58, P < 0.0001. Control vs. hiw P < 0.0001 (****), control vs. hiw > dNmnat-RNAi P = 0.0013 (**), hiw vs. hiw > dNmnat-RNAi P < 0.0001 (****)]. Quantification of mean (± SEM) mEPSP amplitudes, in which 75 consecutive spontaneous events were averaged per cell, and then, cell amplitudes were averaged per genotype [control n = 8 cells, hiw∆N n = 6 cells, hiw∆N > dNmnat-RNAi n = 9 cells. One-way ANOVA with Tukey's multiple comparisons, DF = 22, F = 6.729, P = 0.0058. Control vs. hiw∆N P = 0.0042 (**), control vs. hiw∆N > dNmnat-RNAi P = 0.1901 (NS), hiw∆N vs. hiw∆N > dNmnat-RNAi P = 0.1185 (NS)]. Quantification (mean ± SEM) of quantal content [control n = 7, hiw∆N n = 6, hiw∆N > dNmnat-RNAi n = 8. One-way ANOVA with Tukey's multiple comparisons, DF = 20, F = 11.58, P = 0.0006. Control vs. hiw∆N P = 0.0005 (***), control vs. hiw∆N > dNmnat-RNAi P = 0.4282 (NS), hiw∆N vs. hiw∆N > dNmnat-RNAi P = 0.0057 (**).] Download figure Download PowerPoint We next examined whether this degree of dNmnat depletion influenced basal neurotransmission in an otherwise WT background (Elav-GAL4 > UAS-RFP vs. Elav-GAL4 > UAS-dNmnat-RNAi). We observed no significant difference in EPSP amplitudes or quantal content upon dNmnat knock down in wild-type motor neurons compared to a control transgene (Fig EV2A–C). These findings show that in the hiw mutant, excess dNmnat impairs evoked synaptic transmission, but in wild-type larvae, the normal level of dNmnat does not disrupt transmission. Finally, we tested whether the synaptic overgrowth observed at the hiw mutant NMJ was altered by depletion of dNmnat. As expected from the overexpression studies (Fig 1), depleting dNmnat from a hiw mutant background did not suppress the dramatic morphological overgrowth at the larval NMJ; this phenotype is entirely Wnd-dependent and can be suppressed through presynaptic expression of an RNAi against Wnd (Fig 2D and E). Thus, we conclude that Hiw targets two unique substrates to control different aspects of synaptic development; Hiw limits levels of the MAP3K Wnd to restrain synaptic growth, while limiting levels of dNmnat to promote evoked synaptic transmission. Click here to expand this figure. Figure EV2. Neither Wnd nor dNmnat depletion impact basal evoked neurotransmission in an otherwise wild-type background Representative EPSP traces from larval NMJs at muscle 6. Recordings were taken from a wild-type control expressing a control transgene (elav > UAS-RFP), wild type expressing an RNAi against Wnd (elav > Wnd-RNAi [#25396]), and wild type expressing an RNAi against dNmnat (elav > dNmnat-RNAi [#29402]). Quantification of mean (± SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then, cell amplitudes were averaged per genotype [elav > RFP n = 10, elav > wnd-RNAi n = 9, elav > dNmnat-RNAi n = 11. One-way ANOVA with Tukey's multiple comparisons, DF = 29, F = 0.4903, P = 0.6178. RFP vs. Wnd-RNAi P = 0.7995 (NS), RFP vs. dNmnat-RNAi P = 0.5992 (NS). Quantification (mean ± SEM) of quantal content [elav > RFP n = 10, elav > wnd-RNAi n = 9, elav > dNmnat-RNAi n = 11. One-way ANOVA with Tukey's multiple comparisons, DF = 29, F = 0.715, P = 0.4982. RFP vs. Wnd-RNAi P = 0.4735 (NS), RFP vs. dNmnat-RNAi P = 0.9016 (NS)]. Download figure Download PowerPoint Excess dNmnat decreases release probability in hiw mutants We next probed the physiological mechanisms by which excess dNmnat impairs synaptic release in hiw mutants. Presynaptic strength is largely defined by two key parameters: N, which is the number of synaptic release sites, and Pr, the probability of release from each site. We first explored the impact of excess dNmnat on N at the hiw mutant NMJ. At the Drosophila NMJ, active zones are marked by the presence of the scaffolding protein Bruchpilot and are apposed to clusters of postsynaptic glutamate receptors17, 18. To estimate N, we defined a “release site” as a Brp-positive puncta in close apposition to DGluRIII, an essential subunit of the glutamate receptor 19. We assessed Brp and DGluRIII at NMJs of wild-type controls expressing RFP in neurons, hiwND8 mutants expressing RFP in neurons, and hiwND8 mutants expressing dNmnat-RNAi in neurons. As expected, hiw mutant NMJs have an expanded synaptic surface area relative to control, and dNmnat knockdown does not suppress this morphological phenotype (Fig 3A and B). Interestingly, hiw mutant synaptic terminals exhibited no significant differences in the total number of Brp puncta per NMJ compared to controls (Fig 3A and C) and dNmnat knockdown did not increase the number of release sites in the hiw mutant background (Fig 3A and C). Further, neither hiw mutants nor hiw mutants in which dNmnat is knocked down exhibit a substantial misapposition defect; nearly all Brp puncta in the presynaptic terminal are apposed to DGluRIII clusters, with no significant difference from WT terminals (Fig 3A and D). Hence, we do not observe a significant change in N in hiw with or without dNmnat knockdown, suggesting that excess dNmnat is not impairing release by decreasing the total number of functional release sites. Figure 3. hiw mutants exhibit dNmnat-dependent defects in release probability (Pr) at the larval NMJ Representative images of immunostained larval NMJs and active zones from WT, hiw mutant, and hiw mutant driving an RNAi against dNmnat [#29402]. In the top three panels, grayscale images of entire terminal were acquired by staining for nerve marker HRP. Presynaptic Brp puncta (green) and postsynaptic dGluRIII clusters (magenta) were imaged to quantify N in each genotype. Quantification (mean ± SEM) of NMJ synaptic terminal surface area in each genotype [WT n = 14, hiw n = 12, hiw > dNmnat-RNAi n = 12. One-way ANOVA with Tukey's multiple comparisons, DF = 37, F = 22.427, P < 0.0001. WT vs. hiw P < 0.0001 (****), WT vs. hiw > dNmnat-RNAi P < 0.0001 (****), hiw vs. hiw > dNmnat-RNAi P = 0.2907 (NS)]. Quantification (mean ± SEM) of BRP puncta per NMJ [WT n = 14, hiw n = 12, hiw > dNmnat-RNAi n = 12. One-way ANOVA with Tukey's multiple comparisons, DF = 37, F = 2.854, P = 0.0711. WT vs. hiw P = 0.0573 (NS), WT vs. hiw > dNmnat-RNAi P = 0.4529 (NS), hiw vs. hiw > dNmnat-RNAi P = 0.5030 (NS)]. Quantification (mean ± SEM) of percentage of Brp puncta that were apposed to a dGluRIII clusters at each NMJ [WT n = 14, hiw n = 12, hiw > dNmnat-RNAi n = 12. One-way ANOVA with Tukey's multiple comparisons, DF = 37, F = 2.093, P = 0.4716. WT vs. hiw P = 0.2168 (NS), WT vs. hiw > dNmnat-RNAi P = 0.1882 (NS), hiw vs. hiw > dNmnat-RNAi P = 0.9966 (NS)]. Representative electrophysiological traces from a synaptic facilitation experiment in which trains of five pulses were used to evoke consecutive action potentials at 100 ms ISI. Genotypes for these experiments were as follows: WT (elav>UAS-RFP), hiw mutant (hiw; elav>UAS-RFP), or hiw depleted of dNmnat (hiw; elav>dNmnat-RNAi). Quantification (mean ± SEM) of the facilitation index, in which the amplitude of the 5th pulse is divided by the amplitude of the 1st pulse [100 ms ISI: WT n = 11, hiw n = 9, hiw>dNmnat-RNAi n = 12. One-way ANOVA with Tukey's multiple comparisons, DF = 31, F = 7.546, P = 0.0023. WT vs. hiw P = 0.0020 (**), hiw vs. hiw> dNmnat-RNAi P = 0.0229 (*), WT vs. hiw > dNmnat-RNAi P = 0.5141 (NS). 50 ms ISI: WT n = 11, hiw n = 9, hiw>dNmnat-RNAi n = 12. One-way ANOVA with Tukey's multiple comparisons, DF = 31, F = 5.587, P = 0.0089. WT vs. hiw P = 0.0129 (*), hiw vs. hiw> dNmnat-RNAi P = 0.0217 (*), WT vs. hiw> dNmnat-RNAi P = 0.9546 (NS)]. Download figure Download PowerPoint Since hiw mutants do not have a defect in N, we next assessed release probability. Relative changes in release probability can be estimated by examining short-term plasticity in a synaptic facilitation paradigm. There is an inverse correlation between synaptic facilitation and release probability, such that facilitation is more pronounced when probability of release is lower 20, 21. We calculated the facilitation index as the amplitude of the fifth pulse compared to the first in a train of stimuli at two different interstimulus intervals (50 and 100 ms) in wild type, hiw mutants, and hiw mutants expressing the dNmnat-RNAi. Hiw mutants exhibit enhanced short-term facilitation consistent with a decreased release probability (Fig 3E and F). Depletion of dNmnat rescues this enhanced facilitation in hiw mutants (Fig 3E and F). Hence, these data indicate that excess dNmnat in the hiw mutant impairs quantal content by decreasing release probability. Hiw mutant active zones are enriched in dNmnat Thus far, we have demonstrated that Hiw functions to keep dNmnat levels low to promote evoked synaptic release via maintenance of a normal synaptic release probability. It was previously demonstrated that dNmnat levels are elevated in axons and neuromuscular junctions of Hiw mutants 10. Given the effect of dNmnat on release probability, we investigated whether this excess dNmnat protein in hiw mutants accumulates near or at active zones, the sites controlling release probability. The UAS-dNmnat protein is HA-tagged, so we stained for HA-dNmnat at the NMJ of wild-type and hiw mutant larvae. As previously reported, dNmnat levels are higher at hiw NMJs than wild-type NMJs (10, Fig 4A and B). We also stained for the active zone protein Bruchpilot as well as the entire NMJ, so that we could assess dNmnat abundance separately in two compartments: the entire terminal minus Brp-positive puncta (Fig 4C, “Terminal regions excluding Brp-positive active zones”) and the Brp-positive active zones (Fig 4C, “Brp-positive active zones only”). By using masks to select and separate Brp-positive regions from the rest of the terminal compartment, we were able to assess whether Brp-positive regions are differentially enriched for HA-dNmnat compared to Brp-negative, “non-active zone” regions. We compared HA-dNmnat levels between these two compartments in wild type and hiw NMJs. In hiw mutant terminals, there is a significant increase in dNmnat levels exclusively in the active zone compartment (Fig 4C). Th
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