Figure 1.
Presynaptic Nrg is essential for synapse stability.
(A–C) NMJs on muscle 4 stained for the presynaptic motoneuron membrane (Hrp, white), the presynaptic active zone marker Brp (green), and postsynaptic glutamate receptors (DGluRIII, red). (A) A stable wild-type NMJ indicated by perfect apposition of pre- and postsynaptic markers. (B) Knockdown of presynaptic Nrg resulted in severe synaptic retraction indicated by the loss of presynaptic Brp despite the presence of postsynaptic glutamate receptors and by a fragmentation of the presynaptic membrane. Synaptic retractions caused a characteristic fusion of postsynaptic glutamate receptor clusters. (C) Knockdown of postsynaptic Nrg did not impair synapse stability. (D–F) NMJs on muscle 4 stained for the presynaptic motoneuron membrane (Hrp, white), presynaptic vesicles (Syn, green), and postsynaptic Dlg (red). Identical phenotypes were observed when using an independent Nrg RNAi line and independent pre- and postsynaptic markers. Only presynaptic knockdown of Nrg resulted in synaptic retractions indicated by a selective loss of synaptic vesicles and a fragmentation of the presynaptic membrane. (G) Quantification of synaptic retractions. Neuronal but not muscle-specific knockdown of Nrg using different Gal4 driver combinations or independent RNAi constructs resulted in a significant increase in synaptic retractions on muscle 4. The synaptic retraction frequency was significantly rescued (p≤0.001) by co-expression of UAS–nrg180 but not when we co-expressed either UAS–mCD8–GFP or UAS–fasII. Neuronal expression of UAS–fasII in a wild-type background (neu2 FasII) did not result in a significant increase in synaptic retractions (genotypes: neu1 = elavC155–Gal4; neu2 = elavC155–Gal4; ok371–Gal4; neu3 = elavC155–Gal4; UAS-dcr2; neu4 = elavC155–Gal4; sca–Gal4 UAS–dcr2; mus1 = UAS–dcr2; mef2–Gal4; RNAi1 = VDRC6688; RNAi2 = VDRC107991; GFP, Nrg, and FasII indicate co-expression of the corresponding UAS construct; the number of analyzed animals is indicated). (H) Western blot analysis of the genotypes in (G) probed with an antibody against Nrg180 (Nrg180BP104). Neuronal but not muscle-specific Nrg RNAi resulted in efficient knockdown of Nrg180 in larval brains. Nrg180 levels could be rescued by co-expression of Nrg180 but not by co-expression of mCD8-GFP. (I–K) Characterization of multiple presynaptic markers after knockdown of presynaptic Nrg. (I) In wild-type animals presynaptic Ank2-L (green) and Brp (red) were present in all synaptic boutons. In the absence of Nrg, Ank2-L was lost prior to Brp at distal parts of an NMJ that was still stable as indicated by the continuous membrane staining. (J) Similarly, Ank2-L was lost prior to the presynaptic vesicle marker DvGlut (red) at a semistable NMJ. (K) In wild-type animals the microtubule-associated protein Futsch (green) and DvGlut (red) were present in all boutons. Knockdown of Nrg resulted in a loss of Futsch prior to the disassembly of DvGlut at early stages of synapse retraction. Scale bar in (A) corresponds to (A–F), 10 µm, inset 5 µm. Scale bar in (I) corresponds to (I–K), 5 µm. Error bars represent SEM.
Figure 2.
MARCM analysis demonstrates requirement of extra- and intracellular domains of Nrg180 for synapse stability.
(A) Overview of the genomic locus of nrg. The Pacman construct spans 92 kb including the endogenous enhancer elements up- and downstream of nrg. The Nrg167- and Nrg180-specific exons and the relevant amino acid sequences are depicted. The position of the common Ig-domains 3 and 4 is indicated. The isoform-specific FIGQY sequences are highlighted in red and the PDZ protein-binding motif of Nrg180 isoform is underlined. (B) A nrg14 MARCM clone rescued by a wild-type nrg Pacman construct. The motoneuron clone was marked by the expression of mCD8–GFP (green). Synaptic vesicles (DvGlut, red) were found opposite postsynaptic Dlg (blue), indicating a stable NMJ. (C) A nrg14 MARCM clone showing a synaptic retraction. Only fragmented remnants of the membrane GFP marker were still present at a nerve terminal that almost completely lacked the presynaptic active zone marker Brp (red). Postsynaptic glutamate receptor clusters were still present. In addition, the axonal membrane prior to the NMJ was also fragmented. (D) A nrg14 MARCM clone expressing only Nrg180 lacking the FIGQY motif. The mutant (GFP-positive) NMJ was retracted while a neighboring control NMJ (asterisk) remained stable. (E) Composite image overview of an nrg14 MARCM clone expressing a mutated form of Nrg180 lacking the extracellular Ig3–4 domains. Three areas are shown at larger magnification in 1–3. (E1) At the NMJ no presynaptic vesicles were present opposite postsynaptic Dlg. The Dlg staining was no longer interconnected and only remnants of the presynaptic membrane marker mCD8–GFP were visible, indicating a complete elimination. (E2) Approximately 150 µm proximal from the NMJ, the axon ended in a “bulb-like” structure. Between the “bulb-like” axon ending and the NMJ, only punctate staining of the membrane marker was visible. (E3) At a significant distance from the “bulb,” a large axonal swelling was visible that contained aggregates of the synaptic vesicle marker DvGlut. (F) Quantification of mCD8-marked axons that were not connected to target muscles in the indicated genetic background. The nrg14 mutant phenotype was significantly rescued by the presence of a wild-type nrg Pacman construct, a nrg construct lacking the FIGQY domain of Nrg167, and only partially rescued by P[nrg180ΔFIGQY] and not rescued by P[nrg180ΔIg3–4]. (G) Analysis of muscle innervation pattern of motoneuron MARCM clones that were connected to postsynaptic muscles. In all genotypes we observed normal innervation patterns for all four major classes of motoneurons. Scale bar in (B) corresponds to (B-D), 10 µm. Scale bar in (E), 20 µm. Error bars represent SEM.
Figure 3.
Mutations in the FIGQY–Ankyrin binding motif alter the Nrg–Ank2 interaction and increase Nrg mobility in vivo.
(A) Co-immunoprecipition (Co-IP) of Ank2-L from larval brain by Nrg180. IP of Nrg180 using the neuronal-specific antibody NrgBP104 co-precipitates the large Ank2-L isoform (450 kDa) from larval brain extracts. No Ank2-L signal is observed when using empty beads. (B) IP of Nrg180-HA proteins using Ank2-S–GFP from co-transfected S2-cells. Ank2-S pulled down wild-type Nrg180-HA efficiently. Mutations in the FIGQY domain differentially affected binding efficiency. Western blots show IPs and input controls. (C) Quantification of four independent IP experiments demonstrated reduced Ank2 binding due to the specific mutations within the FIGQY motif. (D–G) Fluorescence recovery after photobleaching (FRAP) experiments using GFP-tagged versions of wild-type and mutant forms of Nrg180 at the NMJ. (D) Representative recoveries of FRAP in motoneurons for Nrg180wt, Nrg180Y-F, and Nrg180ΔFIGQY. (E) Equal levels of all GFP-tagged constructs were expressed in motoneurons (ok371–Gal4) in wild-type animals as demonstrated by Western blot analysis. The Nrg180cyto antibody recognizes both wild-type Nrg180 and Nrg180–GFP. In contrast, Nrg180BP104 recognizes endogenous Nrg180 but only the wild-type version of Nrg180–GFP. This indicates that NrgBP104 binds specifically to the Nrg180 FIGQY motif and any alteration of the tyrosine will abolish protein recognition. (F and G) Recovery curves of multiple independent FRAP experiments were fitted to a double exponential curve and used to calculate the mobile fraction of Nrg180. Wild-type Nrg180 recovered to about 40% within the 200 s time frame. The mobility of Nrg180 was significantly increased (more than 1.5×) when the FIGQY motif was mutated (Nrg180Y-F, Nrg180Y-A, Nrg180Y-D). An almost 2-fold increase in mobility was observed after deletion of the Ankyrin-binding motif (Nrg180ΔFIGQY). Numbers in F represent number of independent experiments analyzed. Scale bar in (D) represents 5 µm. Error bars represent SEM.
Figure 4.
Impairment of Ank2 binding results in synaptic retractions.
(A–C) Analysis of synapse stability in nrg14 null mutant animals expressing different nrg Pacman constructs using the presynaptic marker Brp (green), postsynaptic DGluRII (red), and the presynaptic membrane Hrp (white). (A) Animals carrying the wild-type nrg Pacman in the nrg14 mutant background had stable NMJs. (B) Presence of the Nrg180Y-A mutation resulted in increased synaptic retraction rates. (C) Animals lacking the Ank2 binding motif FIGQY showed synaptic retractions including complete eliminations. (D and E) Quantification of retraction frequency and severity demonstrated increasing levels of synaptic retractions correlating with the gradual loss of Ank2 binding capacities of Nrg180. Deletion of the Nrg167–FIGQY motif or the PDZ protein binding domain of Nrg180 did not result in a significant increase in retraction frequency or severity (n = 12–22 animals). Asterisks indicate p≤0.01 for ** and p≤0.001 for ***. Scale bar in (A) corresponds to (A–C), 10 µm, inset 5 µm. Error bars represent SEM.
Figure 5.
Impairment of Ank2 binding of Nrg180 increases NMJ growth.
Analysis of NMJ growth in nrg14 mutant animals expressing different mutated nrg Pacman constructs using the presynaptic vesicle marker Synapsin (Syn, green), the postsynaptic marker Dlg (red), and a marker for the presynaptic membrane (Hrp, blue). (A) Presence of the nrg Pacman wild-type construct resulted in wild-type muscle 4 NMJs. The inset shows individual presynaptic boutons at higher magnification. (B) The Nrg180Y-F mutation resulted only in small alterations of NMJ growth. (C) The Nrg180Y-A mutation led to a significant increase in NMJ length. (D) Deletion of the Nrg180–FIGQY motif resulted in a significant, almost 2-fold overgrowth and a corresponding reduction in the area of individual boutons. (E and F) Quantification of bouton number, NMJ length, and bouton area. NMJ growth defects correlated with an increasing loss of Ank2 binding capacities. No alterations were observed for mutations affecting the PDZ protein binding site of Nrg180 or the Nrg167–FIGQY motif. Values were normalized to wild-type rescue. Asterisks indicate highly significant changes (p≤0.001) for bouton number in (E) and for bouton area in (F) (n = 69–176 NMJs for bouton number, n = 20 NMJs for NMJ length, and n = 10 NMJs for bouton area quantifications). Scale bar in (A) corresponds to (A–D), 10 µm, inset 5 µm. Error bars represent SEM.
Figure 6.
Electrophysiological phenotypes of nrg mutants in the giant fiber circuit.
(A and B) Sample traces of different nrg mutants. (A) TTM responses in nrg mutants (asterisks) upon GF stimulation in the brain (solid grey line). The average response latency in wild-type flies is 0.8 ms (dashed grey line). Sample traces of nrg14; P[nrgwt], nrg14; P[nrg180Y-F], nrg14; P[nrg180Y-A], and nrg14; P[nrg180ΔFIGQY] are shown. Mutations in the Nrg180-FIGQY motif led to a delay or absence of responses at the TTM. (B) As a measure for synaptic reliability, the ability to follow stimuli at 100 Hz was determined. In contrast to nrg14; P[nrgwt], the GF–TTM pathway in nrg14; P[nrg180Y-F], nrg14; P[nrg180Y-A], and nrg14; P[nrg180ΔFIGQY] mutants was not able to follow stimuli at 100 Hz upon GF stimulation in the brain; only rare responses were observed (asterisks). (C and D) Quantifications of electrophysiological phenotypes of nrg mutants. (C) Average latency of wild-type and nrg mutants. There was no significant difference (p = 0.681, Mann–Whitney Rank Sum Test) in the average response latency between control (w1118) and nrg14; P[nrgwt], nrg14; P[nrg167ΔFIGQY], or nrg14; P[nrg180ΔPDZ] flies. In contrast, the response latency was significantly increased in all nrg180 mutants with a mutated FIGQY motif (Mann–Whitney Rank sum test, p≤0.001). (D) Average following frequencies at 100 Hz in wild-type and nrg mutants. There was no significant difference (p = 0.841, Mann–Whitney Rank Sum Test) in the average of following frequencies at 100 Hz between control flies (w1118) and nrg14; P[nrgwt], nrg14; P[nrg167ΔFIGQY], and nrg14; P[nrg180ΔFIGQY]. In contrast, following frequencies were significantly reduced in all nrg180 mutants with a missense mutation in or deletion of the FIGQY motif (Mann–Whitney Rank sum test, p≤0.001). Error bars represent SEM.
Figure 7.
Anatomical phenotypes of the giant fiber synaptic terminals in nrg mutants.
(A) GF synaptic terminals were visualized by injection of Rhodamine-dextran (red) into the GF. Dye-coupling of the GF to its target neurons, the Tergo Trochanteral motoneuron (TTMn), and the peripheral synapsing interneuron (PSI) via co-injection of Biotin (green) allows the detection of gap junctions between these neurons. In w1118, nrg14; P[nrgwt] and nrg14; P[nrg167ΔFIGQY], a normal, large GF terminal was present and we observed dye-coupling with the TTMn and the PSI. In nrg14; P[nrg180Y-F], nrg14; P[nrg180Y-A], and nrg14; P[nrg180ΔFIGQY] mutants, the presynaptic terminal of the GF exhibited variable abnormal morphologies. They were thinner or swollen and contained large vacuole-like structures. However, in most cases the GF still dye-coupled with the postsynaptic target, the TTMn, and the PSI. Scale bar, 15 µm. (B) Quantification of morphological defects in w1118 flies and nrg mutants. Only mutations affecting the Nrg180–FIGQY motif resulted in severe GF terminal aberrations. (C) Quantification of GF-to-TTMn dye-coupling (black bars) and comparison to animals with no electrophysiological responses (red bars) of the TTM with GF stimulation in the brain. A large percentage of animals expressing mutant versions of Nrg180–FIGQY proteins completely lacked electrophysiological responses despite the presence of dye-coupling, demonstrating a severe functional defect in these animals.
Figure 8.
Temporal and spatial requirements of transsynaptic Nrg signaling.
(A) Schematic of GF to TTMn synapse development. First dye-coupling between the GF (red) and the TTMn (blue) can be demonstrated at 40% of pupal development [54]. Positions of stimulating and recording electrodes are indicated. Brain stimulation was used to test the GF–TTMn synapse, while thoracic stimulation bypasses the GF and allowed testing of the TTMn NMJ directly. Expression profiles of the different Gal4 lines are indicated. (B) Rescue of nrg14; P[nrg180Y-F] phenotypes using Gal4/UAS-mediated expression of wild-type Nrg180. Both average response latency and the ability to follow high-frequency stimulation could be rescued significantly by simultaneous expression of Nrg180 pre- and postsynaptically or on either side of the synapse alone (Mann–Whitney Rank sum test, p≤0.001). Less than 20% of animals showed an electrophysiological impairment even when a late presynaptic Gal4 driver line was used for rescue (right). (C and D) Rescue of nrg14; P[nrg180Y-A] and nrg14; P[nrg180ΔFIGQY] animals using cell autonomous expression of Nrg180. Simultaneous expression of Nrg180 pre- and postsynaptically or only on one side of the synapse throughout development significantly rescued the response latency (left) and following frequencies (middle) of these mutations. More than 80% of all GF–TTMn synapses showed wild-type properties (right, Mann–Whitney Rank sum test, p≤0.001). In contrast to nrg14;P [nrg180Y-F], late expression of UAS–nrg180 in the GF alone in nrg14; P[nrg180Y-A] and nrg14; P[nrg180ΔFIGQY] animals did not significantly improve the average response latency (left, Mann–Whitney Rank sum test, p = 0.061 and p = 0.057, respectively) or the following frequencies (middle, Mann–Whitney Rank sum test, p = 0.9 and p = 0.081, respectively). (E) Presynaptic GF terminal morphology was rescued by either pre- or postsynaptic expression of Nrg180 in nrg14; P[nrg180ΔFIGQY] mutant animals. Scale bar, 15 µm. Error bars represent SEM.