Volume 96, Issue 6, Pages 1303-1316.e6 (December 2017) NRAP-1 Is a Presynaptically Released NMDA Receptor Auxiliary Protein that Modifies Synaptic Strength  Ning Lei, Jerry E. Mellem, Penelope J. Brockie, David M. Madsen, Andres V. Maricq  Neuron  Volume 96, Issue 6, Pages 1303-1316.e6 (December 2017) DOI: 10.1016/j.neuron.2017.11.019 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 nrap-1 Encodes an LDLa-Domain Protein Expressed in Presynaptic Cells and Required for NMDAR-Dependent Behavior (A–D) Average forward duration (mean ± SEM). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by Mann-Whitney U test (A, C, and D) or Welch’s t test (B). n = 8 for all genotypes in (A). For genotypes in (B), wild-type, n = 21; ak365, n = 20; gk874318, n = 20; ak365; Pnrap-1::nrap-1::mCherry, n = 13. n = 9 for all genotypes in (C). For genotypes in (D), wild-type, n = 11; ky176, n = 12; ok3474, n = 7; ky176; ok3474, n = 12. (E) NRAP-1 amino acid sequence. Signal peptide (boxed), conserved cysteines (red), residues mutated in nrap-1(gk874318) (asterisks) and nrap-1(ak365) (red arrowhead), region deleted in nrap-1(ok3474) (blue), and predicted N-linked glycosylation (black arrowhead). (F and G) Images of anterior (head) and posterior (tail) neuronal cell bodies in transgenic worms that co-expressed Pnrap-1::nrap-1::SL2::gfp with either Peat-4::mCherry (F) or Pnmr-1::mCherry (G). GFP was tagged with a nuclear localization sequence. Scale bars, 10 μm. (H and I) Images of the VNC in transgenic worms that co-expressed Pnrap-1::gfp::nrap-1 with either Peat-4::eat-4::mCherry (H) or Pflp-18::mCherry::nmr-2 (I). Scale bars, 5 μm. (J) Images of a transgenic worm that expressed Pnrap-1::nrap-1::mCherry. Arrowheads indicate coelomocytes. Scale bar, 5 μm. See also Figures S1 and S2. Neuron 2017 96, 1303-1316.e6DOI: (10.1016/j.neuron.2017.11.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 nrap-1 Is Specifically Required for NMDA-Gated Current In Vivo (A and B) Currents in AVA in response to glutamate (A) or NMDA (B). Dashed gray line in (A) indicates baseline before glutamate application. (C) Average peak current (mean ± SEM). ∗∗p < 0.01, significantly different from wild-type by Mann-Whitney U test. ∗p < 0.05, ∗∗∗p < 0.001, significantly different from nrap-1 mutants by one-sample, one-tailed Student’s t test comparing the mean for each genotype with zero (i.e., no current). Wild-type, n = 10; nrap-1 mutants, n = 12; ak365 + Pnrap-1::nrap-1::mCh, n = 4. ND, no current detected. (D) NMDA-gated currents in AVA. (E) Average peak current (mean ± SEM). Significantly different from nrap-1(ak365) mutants where no current was detected (data not shown); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by one-sample, one-tailed Student’s t test. Wild-type, n = 10; Peat-4, Pnmr-1, and Pflp-18, n = 4; Pmyo-3, n = 5. (F) Images of mCherry and GFP fluorescence in the VNC of a transgenic worm that expressed Pflp-18::nrap-1::gfp and Prig-3::mCherry::nmr-2 in AVA. Scale bar, 5 μm. (G) NMDA-gated currents in AVA in wild-type, mutant, and transgenic mutants. Wild-type average peak current shown in (C) and (E) was from pooled data. See also Figure S2. Neuron 2017 96, 1303-1316.e6DOI: (10.1016/j.neuron.2017.11.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 nrap-1 Is Not Required for NMDAR Localization or Surface Expression (A) Images of AVA processes in the VNC in a transgenic wild-type worm (left) or nrap-1 mutant (right) that expressed SEP::mCherry::nmr-2. Scale bar, 5 μm. (B) Images of neural processes in the VNC in a transgenic wild-type worm (left) or nmr-1; nmr-2 mutant (right) that expressed SEP::mCherry::nrap-1 in presynaptic neurons. Scale bar, 5 μm. (C and D) Images of the VNC in transgenic nmr-1; nmr-2 nrap-1 triple mutants that co-expressed Pmyo-3::nrap-1::gfp with either Pflp-18::mCherry::nmr-1 and Prig-3::mCherry::nmr-2 (C) or Prig-3::mCherry (D) (two examples from each strain). Scale bars, 2 μm. See also Figures S2 and S3. Neuron 2017 96, 1303-1316.e6DOI: (10.1016/j.neuron.2017.11.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 NRAP-1 Modifies the Function of NMDARs (A and B) Currents in response to 1 mM glutamate (A) or 1 mM NMDA (B) application to Xenopus oocytes that expressed various combinations of NMR-1, NMR-2, and NRAP-1 (Ca2+ Ringer’s solution). (C–E) Currents in response to NMDA or glutamate (200 μM) in oocytes that expressed NMR-1, NMR-2, and NRAP-1. (C) Current responses in oocytes bathed in either Ca2+ or Ba2+ containing Ringer’s solution. (D and E) Current responses before, during, and after washout of MK-801 (D), or before and during incubation in Con-A (E). (F) Current responses to 200 μM glutamate in an oocyte that expressed NMR-1 and NMR-2 (top). Current was not detected in uninjected, control oocytes (bottom). Neuron 2017 96, 1303-1316.e6DOI: (10.1016/j.neuron.2017.11.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Recombinant NRAP-1 Restores Current in nrap-1 Mutants (A and B) Currents in AVA neurons in response to either NMDA (A) or glutamate (B) in the presence or absence of recombinant NRAP-1 or control media. (C) Currents in AVA neurons in response to NMDA following selective knockdown of NRAP-1 in presynaptic glutamatergic neurons. Neurons were held at +40 mV. (D and E) Model showing NRAP-1 secretion from presynaptic glutamatergic neurons followed by diffusion and binding to postsynaptic NMDARs (D), or NRAP-1 anchored to the presynaptic membrane via an unknown factor (Protein X), thereby stabilizing its association with postsynaptic NMDARs (E). Neuron 2017 96, 1303-1316.e6DOI: (10.1016/j.neuron.2017.11.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Recombinant NRAP-1 Reveals the Presence of Silent NMDARs (A–C) Currents in AVA neurons in response to NMDA. Cells held at +40 mV. (A) The initial response in a wild-type worm and that following 2 and 5 min of bath solution exchange. (B) Current in an nrap-1 mutant in the presence of recombinant NRAP-1 and after 1 and 2 min of washout. (C) Currents in a wild-type worm in the absence and presence of recombinant NRAP-1 and after 5 min of washout. (D–F) Currents in response to NMDA in muscle cells of transgenic worms that ectopically express NMR-1 and NMR-2 (D) or NMR-1, NMR-2, and NRAP-1 (E and F) specifically in muscle cells. Shown are currents before (D and E) and after (D–F) recombinant NRAP-1 application, and after 8 min of washout (D and F). See also Figure S4. Neuron 2017 96, 1303-1316.e6DOI: (10.1016/j.neuron.2017.11.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 NRAP-1 Is Required for the Gating of NMDARs Currents in response to rapid uncaging of glutamate. Cells were held at +40 mV. Gray arrowheads indicate a 1 ms pulse of UV light. Gray record shows the response to light in the absence of MNI-glutamate. (A) Shown are the averages of ten uncaging events per genotype. (B–D) Individual current records (two per genotype) in a glr-1 mutant (B), nmr-1; glr-1 mutant (C), or glr-1; nrap-1 mutant (D). (E) Individual current records in the presence of recombinant NRAP-1 and following 2, 4, 8, and 10 min of washout. Neuron 2017 96, 1303-1316.e6DOI: (10.1016/j.neuron.2017.11.019) Copyright © 2017 Elsevier Inc. Terms and Conditions