Molecular Dissection of Neurobeachin Function at Excitatory Synapses

Abstract

Spines are small protrusions from dendrites where most excitatory synapses reside. Changes in number, shape, and size of dendritic spines often reflect changes of neural activity in entire circuits or at individual synapses, making spines key structures of synaptic plasticity. Neurobeachin is a multidomain protein with roles in spine formation, postsynaptic neurotransmitter receptor targeting and actin distribution. However, the contributions of individual domains of Neurobeachin to these functions is poorly understood. Here, we used mostly live cell imaging and patch-clamp electrophysiology to monitor morphology and function of spinous synapses in primary hippocampal neurons. We demonstrate that a recombinant full-length Neurobeachin from humans can restore mushroom spine density and excitatory postsynaptic currents in neurons of Neurobeachin-deficient mice. We then probed the role of individual domains of Neurobeachin by comparing them to the full-length molecule in rescue experiments of knockout neurons. We show that the combined PH-BEACH domain complex is highly localized in spine heads, and that it is sufficient to restore normal spine density and surface targeting of postsynaptic AMPA receptors. In addition, we report that the Armadillo domain facilitates the formation of filopodia, long dendritic protrusions which often precede the development of mature spines, whereas the PKA-binding site appears as a negative regulator of filopodial extension. Thus, our results indicate that individual domains of Neurobeachin sustain important and specific roles in the regulation of spinous synapses. Since heterozygous mutations in Neurobeachin occur in autistic patients, the results will also improve our understanding of pathomechanism in neuropsychiatric disorders associated with impairments of spine function.

Keywords: AMPA receptor; autism; dendritic spine; hippocampus; neurobeachin; synaptic transmission.

FIGURE 1

Full-length human Nbea restores EPSC defects in null-mutant neurons. (A) Representative traces of continuous recordings of mEPSCs from cultured hippocampal neurons of WT, Nbea KO (KO), KO transfected with human full-length Nbea (KO + Nbea FL) and KO transfected with NbeaΔPKA (KO + NbeaΔPKA). (B) Averaged traces of mEPSCs recorded from WT (red line), KO (black), and KO + Nbea FL (dashed) neurons indicate faster kinetics in Nbea KO that can be rescued by Nbea FL. (C–F) Quantitative analysis of rise time (C), decay time (D), inter-event interval (E) and amplitude (F) of mEPSCs in WT, KO, KO + Nbea FL and KO + NbeaΔPKA. Digits in bars give the number of recorded neurons; from each neuron more than 100 consecutive mEPSCs were analyzed for evaluation. Data are means ± SEM; ^∗P < 0.05, n.s., not significant; by one-way ANOVA with Tukey’s multiple comparisons test.

FIGURE 2

Evoked EPSCs at autaptic synapses are restored by full-length Nbea and NbeaΔPKA. (A) Sample traces of evoked EPSCs recorded from individual hippocampal neurons grown on micro islands that form autapses. The eEPSCs shown are from untransfected WT, from Nbea KO neurons mock transfected with GFP alone, and transfected with Nbea FL or NbeaΔPKA constructs. (B) Average values of eEPSC amplitudes from WT, KO/GFP, Nbea FL, and NbeaΔPKA. Data are means ± SEM; ^∗∗∗P < 0.001, n.s., not significant; by one-way ANOVA with Tukey’s multiple comparisons test. (C) Overlay of representative traces of eEPSCs during AP stimulation trains (10 Hz) from WT, KO/GFP, KO + Nbea FL and KO + NbeaΔPKA; insets depict magnifications of the response to the first three stimuli. (D) Change in EPSC amplitudes during a 10 Hz stimulation train in WT, KO, KO + Nbea FL and KO + NbeaΔPKA autaptic glutamatergic neurons. N_WT = 5 neurons, N_KO = 7 neurons, N_KO+NbeaFL = 11 neurons, N_KO+NbeaΔPKA = 6 neurons.

FIGURE 3

Individual Nbea domains are able to localize to dendrites and spines. (A) Nbea motif architecture depicting its multidomain composition. GFP, N-terminally fused EGFP tag; Lectin, concanavalin A-like lectin domain; Armadillo, armadillo repeat; AKAP, A-kinase anchoring protein; DUF1088, domain-of-unknown-function; PH, pleckstrin homology-like domain; BEACH, Beige and Chediak-Higashi domain; WD40, tryptophan-aspartic acid repeats. (B) Predicted structural model of Nbea with domains color coded as in (A). See “Materials and Methods” section for details on templates and modeling procedure. (C) Representative images of dendrites from primary hippocampal neurons transfected with cytosolic marker t-dimer-RFP and isolated GFP-tagged Nbea domains as indicated. Scale bar: 2.5 μm. (D) Quantification of fluorescence intensity of individual Nbea domains in dendritic spine (DS) heads. Data are means ± SEM (arbitrary units, A.U.). N, number of neurons (in bars); quantification was carried out on at least 50 spines per neuron. One-way ANOVA with Tukey’s multiple comparisons test; n.s., not significant, ^∗P < 0.05, ^∗∗∗P < 0.001. (E) Quantification of fluorescence intensity of individual Nbea domains in dendritic shaft. Data are means ± SEM (arbitrary units, A.U.). N, number of neurons (in bars); quantification was carried out on at least four 20 μm-long dendritic windows per neuron. One-way ANOVA with Tukey’s multiple comparisons test; n.s., not significant, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 4

PH-BEACH domain restores surface targeting of GluA2 receptors in Nbea-deficient neurons. (A) Representative dendrites of primary hippocampal neurons from wild-type (WT) and Nbea null-mutant (KO) mice transfected with cytosolic marker t-dimer-RFP alone and in combination with GFP-tagged full-length Nbea (KO + Nbea FL) or the PH-BEACH domain (KO + PH-BEACH). Right panels, surface populations of GluA2 receptor subunits visualized by live labeling of neurons with an antibody directed against an extracellular epitope of the GluA2 subunit. Scale bar: 2.5 μm. (B) Quantification of surface GluA2 fluorescence intensity measured on dendrites of WT, KO, KO + Nbea FL and KO + PH-BEACH neurons. Data are means ± SEM. N, number of dendritic window/neurons (in bars); ^∗∗P < 0.01, ^∗∗∗P < 0.001, by one-way ANOVA with Tukey’s multiple comparisons test.

FIGURE 5

PH-BEACH domain of Nbea is sufficient to rescue the defect on mushroom spines. (A) Representative dendrites of primary hippocampal neurons from wild-type (WT) and Nbea null-mutant (KO) mice transfected with cytosolic marker t-dimer-RFP alone and in combination with GFP-tagged full-length Nbea (KO + Nbea FL) or PH-BEACH domain (KO + PH-BEACH). As additional control, KO neurons were mock transfected with GFP alone (KO + GFP). Scale bar: 2.5 μm. (B) Quantification of the number of mature mushroom-like DSs in different genotypes and upon expression of different constructs as indicated. Data are means ± SEM; N, number of neurons (in bars). Quantification was carried out analyzing the whole dendritic tree for each neuron. ^∗P < 0.05 ^∗∗∗P < 0.001, n.s., not significant by one-way ANOVA with Tukey’s multiple comparisons test.

FIGURE 6

Armadillo domain and AKAP motif of Nbea affect filopodia formation. (A) Scheme depicting major categories of DS morphology, including mature mushroom spines, long thin spines, stubby spines, and filopodia. (B) Structural model of Arm-Long sequences (left) including the AKAP helix (magenta) and Arm-Core (right). An Armadillo domain consists of eight repeats of three helices H1 (blue), H2 (green), and H3 (red). The AKAP domain of Nbea is present in the Arm-Long construct but deleted in the Arm-Core ΔPKA construct (C–E). (C) Representative dendrites of primary hippocampal neurons from wild-type (WT) and Nbea null-mutant (KO) mice transfected with cytosolic marker t-dimer-RFP alone and in combination with GFP-tagged full-length Nbea (KO + Nbea FL), Armadillo domain with AKAP motif (KO + Arm-Long), Armadillo domain without AKAP (KO + Arm-CoreΔPKA) and full-length Nbea lacking the AKAP (KO + NbeaΔPKA). As additional control, KO neurons were mock transfected with GFP alone (KO + GFP). Scale bar: 2.5 μm. (D) Quantification of number of mushroom-shaped DS in WT and Nbea KO neurons and upon expression of Nbea domains as detailed in (C). Values for WT, KO, KO + FL, and KO + GFP are the same as in Figure 4B and displayed here again to allow for easy comparison with individual domains. Data are means ± SEM, N, number of neurons (in bars). Quantification was carried out by analyzing the whole dendritic tree for each t-dimer-RFP transfected neuron. ^∗P < 0.05, n.s., not significant by one-way ANOVA with Tukey’s multiple comparisons test. (E) Quantification of filopodial protrusions in WT and Nbea KO neurons and upon expression of Nbea domains as detailed in (C,D). Data are means ± SEM, N, number of neurons (in bars). Quantification was carried out analyzing the whole dendritic tree for each t-dimer-RFP transfected neuron. ^∗∗∗P < 0.001, n.s., not significant by one-way ANOVA with Tukey’s multiple comparisons test.

FIGURE 7

Impaired basal motility of Nbea-deficient DSs. (A) Representative image frames of WT and Nbea KO spines at different time points and transfected with Lifeact fused to RFP. The fluorescent signal of Lifeact-RFP highlights specifically polymerized filamentous F-Actin. White areas represent regions taken in consideration for center of mass (COM) displacement analysis in each time point. Red crosses depict the position of COM at each time point. (B) Quantification of COM displacement in WT and Nbea KO single spine. Data are means ± SEM, N, number of spines/neurons (in bars); ^∗∗∗P < 0.001 by unpaired t-test. (C,D) Similar analysis to (A,B) using an alternative label of the spine volume, cytosolic GFP expressed in WT and Nbea KO neurons. White areas represent regions taken in consideration for COM displacement analysis in each time point. Red crosses depict the position of COM at each time point. Data are means ± SEM, N, number of spines/neurons (in bars); ^∗∗P < 0.01 by unpaired t-test. Scale bar: 1 μm; pixel size = 0.211 μm.

FIGURE 8

Normal actin polymerization and turnover in Nbea-deficient DSs. (A) Representative images of hippocampal WT and Nbea KO neurons stained with fluorescent phalloidin. Red arrowheads in images point to ectopic actin clusters mostly in the soma and soma-near large dendrites. Scale bar: 5 μm. (B) Quantification of actin cluster areas detected by phalloidin staining. Data are means ± SEM, N, number of neurons (in bars); ^∗∗∗P < 0.001 by unpaired t-test. (C) Quantification of the percentage of cells with visible phalloidin-stained actin clusters in WT and KO cultures. Data are means ± SEM, N, number of visual fields counted (in bars); ^∗∗∗P < 0.001 by unpaired t-test. (D) Representative images of FRAP experiments of wild-type (WT) and Nbea-deficient (KO) spines at different time points. Samples show GFP-actin transfected dendrites at the indicated time points after bleaching, pre-Bleach indicates spines before quenching. Green circles, region selected for bleaching around spine heads. Scale bar: 2.5 μm. (E,F) Quantification of FRAP data with first order exponential equation fitting of relative fluorescence intensity of GFP-Actin values over time (E). Halftime recovery of GFP-Actin was derived from exponential curves (F). Data are means ± SEM, N, number of spines/neurons (in bars); n.s., not significant by unpaired t-test.