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. 2018 Jul 31;24(5):1218-1230.
doi: 10.1016/j.celrep.2018.06.102.

Noelin1 Affects Lateral Mobility of Synaptic AMPA Receptors

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Free PMC article

Noelin1 Affects Lateral Mobility of Synaptic AMPA Receptors

Nikhil J Pandya et al. Cell Rep. .
Free PMC article

Abstract

Lateral diffusion on the neuronal plasma membrane of the AMPA-type glutamate receptor (AMPAR) serves an important role in synaptic plasticity. We investigated the role of the secreted glycoprotein Noelin1 (Olfactomedin-1 or Pancortin) in AMPAR lateral mobility and its dependence on the extracellular matrix (ECM). We found that Noelin1 interacts with the AMPAR with high affinity, however, without affecting rise- and decay time and desensitization properties. Noelin1 co-localizes with synaptic and extra-synaptic AMPARs and is expressed at synapses in an activity-dependent manner. Single-particle tracking shows that Noelin1 reduces lateral mobility of both synaptic and extra-synaptic GluA1-containing receptors and affects short-term plasticity. While the ECM does not constrain the synaptic pool of AMPARs and acts only extrasynaptically, Noelin1 contributes to synaptic potentiation by limiting AMPAR mobility at synaptic sites. This is the first evidence for the role of a secreted AMPAR-interacting protein on mobility of GluA1-containing receptors and synaptic plasticity.

Keywords: AMPAR-associated protein; glutamate receptor; receptor mobility; synapse function; synaptic plasticity.

Figures

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Figure 1
Figure 1
Noelin1 Is PSD Enriched and Interacts with AMPAR Subunits (A) Biochemical fractions—homogenate (H), microsomes (M), crude synaptic membranes (P2), synaptosomes (SS), synaptic membranes (SM), and postsynaptic density fraction (PSD; Triton X-100 insoluble)—of mature mouse hippocampus analyzed for specific markers. (B) Immunoblot (IB) analysis of native hippocampal immunoprecipitated GluA2/3 complexes versus peptide blocking (PB) control (left) and of immunoprecipitated Noelin1 complexes in the Noelin1 IP versus the PB control (right). Since Noelin1-2 runs at the same height as the antibody light chain (25 kDa), a clear enrichment remains inconclusive; this Noelin1 form was not tested in further experiments. (C) Heterologous expression (HEK293T cells) of individual Noelin1 isoforms (see Figures S1 and S2) with the GluA2 subunit. The molecular weight (in kilodaltons) is indicated. For full blots, see Figure S8. For full Noelin-1 native hippocampal IP, see Table S1. See also Figures S1, S2, and S8 and Table S1.
Figure 2
Figure 2
Noelin1-3 Interacts Directly with the AMPAR GluA2 Subunit (A) Sensorgrams (SPR analysis) from interaction of medium containing Noelin1-3 (black lines) and from medium of non-transfected HEK293T (negative control; gray lines) with surface-immobilized GluA2 (see Figure S2). A reversible 1-step model was fitted to the double-referenced experimental data (green lines). (B) Double-referenced sensorgrams from the interaction of Noelin1-3 with GluA2 under non-reducing (blue, cycles 10 and 28) and reducing (red, cycles 19 and 37) conditions. (C) Bar chart of binding levels upon injection of 7 nM Noelin1-3 over the GluA2 AMPAR surfaces in the absence (blue) and presence of 1 mM DTT (red), extracted from sensorgrams displayed in (B) (black dots), with the respective cycles (in parentheses). (D) 1D BN-PAGE IB for Noelin1 (hippocampus P2 + M fraction) extracted by 1% and 2% DDM, respectively. For SPR controls, see Figure S2; for full blots, see Figure S8. (E) Mass spectrometry identified cross-linked tryptic peptides of the GluA2 AMPAR (W277-K294) and Noelin1 (L225-K240); cross-linked lysine residues (Gria2, K283; Noelin1, K229; red). The chemical crosslink (red line) between the olfactomedin domain of Noelin1 and the AMPAR N-terminal domain (NTD) is depicted in the 3D structure models of a GluA2 homomer (blue; PDB: 5KBS) (Twomey et al., 2016) and the Noelin1 olfactomedin domain homodimer (green; PDB: 5AMO) (Pronker et al., 2015). Note: the orientation of Noelin1 at the AMPAR cannot be well predicted and is an impression. (F) Top view on the NTD GluA2 homomer (2 subunits, light blue; 2 subunits dark blue) in an occupancy analysis using the DisVis web server (van Zundert et al., 2017) showing a volume (pink) that gives a normalized indication of how frequently a grid point is occupied by Noelin1. The accessible interaction space (i.e., the space containing all possible protein complex conformations) was calculated, assuming a maximal Cα-Cα distance of 23.4 Å between K283 and K229 of Gria2 and Noelin1, respectively. See also Figures S2 and S8.
Figure 3
Figure 3
Noelin1 Has No Effect on AMPAR Channel Properties (A) Peak-scaled example traces of whole-cell recording from HEK293T cells (Figure S3) expressing heteromeric GluA1/2-containing AMPAR channels in the absence (black) or presence (red) of Noelin1. Currents were evoked by direct application of 1 mM glutamate during 1 ms. (B) Bar graphs (mean ± SEM) summarize the effect of the addition of Noelin1-3 on rise time and decay time of AMPAR currents. (C) Recovery of desensitization (two consecutive 1-ms glutamate applications with inter-pulse intervals of 20, 50, 100, 200, 300, 400, 500, 750, and 1,000 ms) from HEK293T cells expressing a heteromeric AMPAR channel in the absence (black) or presence (red) of Noelin1-3. Insets indicate rectification (left), and recovery of desensitization. See also Figure S3.
Figure 4
Figure 4
Noelin1 Is Enriched at Extrasynaptic and Postsynaptic Sites of Hippocampal Neurons, Where It Colocalizes with the AMPAR and Brevican (A) STED imaging in DIV21 cultured hippocampal neurons show overview or zoom-ins of dendrites (right) or spines (right, inset). Permeabilized stainings of Noelin1 (green), GluA2 (red), and Homer1 (blue, synaptic marker) are shown (Figure S4). Merge shows color-overlay images. Scale bars are indicated. Inset shows a 2-fold enlargement. (B and C) Line scans over dendrite (1 and 2) and spines (3) are displayed in (B) and (C), respectively. Numbered dashed white lines on the overlay image indicate locations of line scans across the three channels (x axis: distance, in microns; y axis: intensity in a.u.). Graphs illustrate co-enrichment of immunofluorescence intensities. (D and E) Confocal imaging in DIV21 cultured hippocampal neurons untreated (Con; D) or treated marker Homer1 (blue), and surface staining of the ECM marker Brevican (red), are shown (for soma and dendrites in upper panels, scale bars represent 10 μm; for zoom-in of dendrites in lower panels, scale bars represent 1 μm). Colocalization of Noelin1 and Brevican (closed arrowheads), and Noelin1 and Homer1+ puncta (open arrowheads). (F) Quantification of the total dendritic intensity of Brevican and Noelin1. (G) Quantification of perisynaptic Brevican and Noelin1 intensity. (H) Quantification of the fraction of Homer1 particles positive for Noelin1. See also Figure S4.
Figure 5
Figure 5
Chemical LTP Recruits AMPAR and Noelin1 to the Synapse (A) Representative images of permeabilized immunostaining for Noelin1 and Homer1 and surface staining for GluA2 under basal conditions and 1 hr after stimulation with 4-AP and bicuculline (4-AP/BIC) in mature DIV21 neurons (scale bar, 20 μm). (B and C) Bar graphs (mean ± SEM) show normalized total dendritic and synaptic Noelin1 (B) and GluA2 (C) under control conditions and 1 hr after a 15-min 4-AP/BIC stimulation.
Figure 6
Figure 6
Noelin1 Regulates AMPAR Mobility in HEK293T Cells and Young Neurons (A) Representative traces of SEP::GluA2 receptors expressed in HEK293T cells (upper panels; Figure S5) or in young neurons (DIV11–13; Figure S6) at extrasynaptic and synaptic sites (lower panels) without (control) (left) and with (right) (addition of) Noelin1-3. In the lower panels, the Homer1::dsRED signal is indicated. Scale bars, 2 μm. (B) Boxplots of the diffusion coefficient (Dinst) for HEK293T cell expression of single particles of GluA2 (black), GluA2 co-expressed with Noelin1-3 (GluA2_Noelin1, red), GluA2 plus control medium (GluA2 + control, gray), and GluA2 plus medium containing Noelin1-3 (GluA2 + Noelin1, orange). (C) Bar graphs indicate the proportion of immobile SEP::GluA2 particles for GluA2 versus GluA2_Noelin1. (D) Temporal dynamics of mobility (area covered) of GluA2::pHluorin receptors in cultured hippocampal neurons (DIV11–13). (E) Dinst for extrasynaptic mobile GluA2:: pHluorin AMPARs without and with Noelin1 incubation. (F) Boxplots for Dinst for extrasynaptic SEP::GluA2 AMPARs without versus with Noelin1 incubation. (G) Quantification of the immobile fraction obtained from (F). Number of cells are indicated ≥ two independent biological replicates. See also Figures S5 and S6.
Figure 7
Figure 7
Noelin1 Rescues the Effect of Hyaluronidase Treatment on GluA1-Containing AMPAR Mobility and PPR in Older Neurons (A) Representative traces of GluA1::pHluorin receptors expressed in DIV21 primary neurons at extrasynaptic and synaptic locations after control (upper) or hyaluronidase (lower) treatment and subsequent incubation without (control; left) or with Noelin1-3 conditioned medium (right) (Figure S7). Scale bars represent 4 μm (or 2 μm for the zoom-ins). (B–D) The distribution (B) and boxplot (C) of Dinst and bar graphs of the immobile fraction (D) for extrasynaptic SEP::GluA1 receptors. (E–G) The distribution (E) and boxplot (F) of Dinst and bar graphs of the immobile fraction (G) for synaptic SEP::GluA1 receptors. (H) Representative traces of electrically evoked AMPAR currents without and with hyaluronidase treatment and without and with addition of Noelin1 in DIV18 primary neurons. (I) Bar graphs of paired-pulse ratio (PPR) I1/I2. Number of cells are indicated ≥ two independent biological replicates. See also Figure S7.

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